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Shu-Ting Pan,1 Yiru Qin,2 Zhi-Wei Zhou,2,3 Zhi-Xu He,3 Xueji Zhang,4 Tianxin Yang,5 Yin-Xue Yang,6 Dong Wang,7 Shu-Feng Zhou,2 Jia-Xuan Qiu1 1Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China; 4Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 5Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 7Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China Abstract: Tongue squamous cell carcinoma (TSCC) is the most common malignancy in oral and maxillofacial tumors with highly metastatic characteristics. Plumbagin (5-hydroxy-2-methyl-1, 4-naphthoquinone; PLB), a natural naphthoquinone derived from the roots of Plumbaginaceae plants, exhibits various bioactivities, including anticancer effects. However, the potential molecular targets and underlying mechanisms of PLB in the treatment of TSCC remain elusive. This study employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic approach to investigate the molecular interactome of PLB in human TSCC cell line SCC25 and elucidate the molecular mechanisms. The proteomic data indicated that PLB inhibited cell proliferation, activated death receptor-mediated apoptotic pathway, remodeled epithelial adherens junctions pathway, and manipulated nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response signaling pathway in SCC25 cells with the involvement of a number of key functional proteins. Furthermore, we verified these protein targets using Western blotting assay. The verification results showed that PLB markedly induced cell cycle arrest at G2/M phase and extrinsic apoptosis, and inhibited epithelial to mesenchymal transition (EMT) and stemness in SCC25 cells. Of note, N-acetyl-l-cysteine (NAC) and l-glutathione (GSH) abolished the effects of PLB on cell cycle arrest, apoptosis induction, EMT inhibition, and stemness attenuation in SCC25 cells. Importantly, PLB suppressed the translocation of Nrf2 from cytosol to nucleus, resulting in an inhibition in the expression of downstream targets. Taken together, these results suggest that PLB may act as a promising anticancer compound via inhibiting Nrf2-mediated oxidative stress signaling pathway in SCC25 cells. This study provides a clue to fully identify the molecular targets and decipher the underlying mechanisms of PLB in the treatment of TSCC. Keywords: PLB, SILAC, EMT, stemness, Nrf2, tongue squamous cell carcinoma

Juan-Juan Yin,1,2 Stepan P Shumyak,2 Christopher Burgess,2 Zhi-Wei Zhou,2 Zhi-Xu He,3 Xue-Ji Zhang,4 Shu-Ting Pan,2,5 Tian-Xin Yang,6 Wei Duan,7 Jia-Xuan Qiu,5 Shu-Feng Zhou21Xiaolan People’s Hospital, Southern Medical University, Zhongshan, Guangdong, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, 4Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 5Department of Oral and Maxillofacial Surgery, the First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 6Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 7School of Medicine, Deakin University, Waurn Ponds, VIC, AustraliaAbstract: Breast cancer is a leading killer of women worldwide. Cyclodextrin-based estrogen receptor-targeting drug-delivery systems represent a promising direction in cancer therapy but have rarely been investigated. To seek new targeting therapies for membrane estrogen receptor-positive breast cancer, an estrogen-anchored cyclodextrin encapsulating a doxorubicin derivative Ada-DOX (CDE1-Ada-DOX) has been synthesized and evaluated in human breast cancer MCF-7 cells. First, we synthesized estrone-conjugated cyclodextrin (CDE1), which formed the complex CDE1-Ada-DOX via molecular recognition with the derivative adamantane-doxorubicin (Ada-DOX) (Kd=1,617 M-1). The structure of the targeting vector CDE1 was fully characterized using 1H- and 13C-nuclear magnetic resonance, mass spectrometry, and electron microscopy. CDE1-Ada-DOX showed two-phase drug-release kinetics with much slower release than Ada-DOX. The fluorescence polarization analysis reveals that CDE1-Ada-DOX binds to recombinant human estrogen receptor α fragments with a Kd of 0.027 µM. Competition assay of the drug complex with estrogen ligands demonstrated that estrone and tamoxifen competed with CDE1-Ada-DOX for membrane estrogen receptor binding in MCF-7 cells. Intermolecular self-assembly of CDE1 molecules were observed, showing tail-in-bucket and wire-like structures confirmed by transmission electronic microscopy. CDE1-Ada-DOX had an unexpected lower drug uptake (when the host–guest ratio was >1) than non-targeting drugs in MCF-7 cells due to ensconced ligands in cyclodextrins cavities resulting from the intermolecular self-assembly. The uptake of CDE1-Ada-DOX was significantly increased when the host–guest ratio was adjusted to be less than half at the concentration of CDE1 over 5 µM due to the release of the estrone residues. CDE1 elicited rapid activation of mitogen-activated protein kinases (p44/42 MAPK, Erk1/2) in minutes through phosphorylation of Thr202/Tyr204 in MCF-7 cells. These results demonstrate a targeted therapeutics delivery of CDE1-Ada-DOX to breast cancer cells in a controlled manner and that the drug vector CDE1 can potentially be employed as a molecular tool to differentiate nongenomic from genomic mechanism.Keywords: breast cancer, drug vector, functionalized, membrane estrogen receptor, polysaccharide, targeted drug delivery

Jin-Ping Li,1,2 Yin-Xue Yang,3 Qi-Lun Liu,1 Shu-Ting Pan,1,4 Zhi-Xu He,5 Xueji Zhang,6 Tianxin Yang,7 Xiao-Wu Chen,8 Dong Wang,9 Jia-Xuan Qiu,4 Shu-Feng Zhou2,51Department of Surgical Oncology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia; 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 5Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, 6Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 7Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 8Department of General Surgery, The First People’s Hospital of Shunde, Southern Medical University, Shunde, Foshan, Guangdong, 9Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of ChinaAbstract: Alisertib (ALS) is an investigational potent Aurora A kinase inhibitor currently undergoing clinical trials for the treatment of hematological and non-hematological malignancies. However, its antitumor activity has not been tested in human breast cancer. This study aimed to investigate the effect of ALS on the growth, apoptosis, and autophagy, and the underlying mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. In the current study, we identified that ALS had potent growth-inhibitory, pro-apoptotic, and pro-autophagic effects in MCF7 and MDA-MB-231 cells. ALS arrested the cells in G2/M phase in MCF7 and MDA-MB-231 cells which was accompanied by the downregulation of cyclin-dependent kinase (CDK)1/cell division cycle (CDC) 2, CDK2, and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53, suggesting that ALS induces G2/M arrest through modulation of p53/p21/CDC2/cyclin B1 pathways. ALS induced mitochondria-mediated apoptosis in MCF7 and MDA-MB-231 cells; ALS significantly decreased the expression of B-cell lymphoma 2 (Bcl-2), but increased the expression of B-cell lymphoma 2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and increased the expression of cleaved caspases 3 and 9. ALS significantly increased the expression level of membrane-bound microtubule-associated protein 1 light chain 3 (LC3)-II and beclin 1 and induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways in MCF7 and MDA-MB-231 cells as indicated by their altered phosphorylation, contributing to the pro-autophagic activities of ALS. Furthermore, treatment with wortmannin markedly downregulated ALS-induced p38 MAPK activation and LC3 conversion. In addition, knockdown of the p38 MAPK gene by ribonucleic acid interference upregulated Akt activation and resulted in LC3-II accumulation. These findings indicate that ALS promotes cellular apoptosis and autophagy in breast cancer cells via modulation of p38 MAPK/Akt/ mTOR pathways. Further studies are warranted to further explore the molecular targets of ALS in the treatment of breast cancer.Keywords: ALS, breast cancer, cell cycle, apoptosis, autophagy, p38 MAPK

Zhi-Wei Zhou,1,2 Xing-Xiao Li,1 Zhi-Xu He,2 Shu-Ting Pan,1,3 Yinxue Yang,4 Xueji Zhang,5 Kevin Chow,1 Tianxin Yang,6 Jia-Xuan Qiu,3 Qingyu Zhou,1 Jun Tan,7 Dong Wang,8 Shu-Feng Zhou1,2 1Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 2Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino–US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China; 3Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 4Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan City, Ningxia, People’s Republic of China; 5Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 6Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 7Department of Psychiatry and Behavioral Neurosciences, Silver Child Development Center, Rashid Laboratory for Developmental Neurobiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA; 8Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China Abstract: Plumbagin (PLB) has been shown to have anticancer activities in animal models, but the role of PLB in prostate cancer treatment is unclear. This study aimed to investigate the effects of PLB on apoptosis and autophagy and the underlying mechanisms in human prostate cancer cell lines PC-3 and DU145. Our study has shown that PLB had potent pro-apoptotic and pro-autophagic effects on PC-3 and DU145 cells. PLB induced mitochondria-mediated apoptosis and autophagy in concentration- and time-dependent manners in both PC-3 and DU145 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways and activation of 5'-AMP-dependent kinase (AMPK) as indicated by their altered phosphorylation, contributing to the pro-autophagic activity of PLB. Modulation of autophagy altered basal and PLB-induced apoptosis in both cell lines. Furthermore, PLB downregulated sirtuin 1 (Sirt1), and inhibition of Sirt1 enhanced autophagy, whereas the induction of Sirt1 abolished PLB-induced autophagy in PC-3 and DU145 cells. In addition, PLB downregulated pre-B cell colony-enhancing factor/visfatin, and the inhibition of pre-B cell colony-enhancing factor/visfatin significantly enhanced basal and PLB-induced apoptosis and autophagy in both cell lines. Moreover, reduction of intracellular reactive oxygen species (ROS) level attenuated the apoptosis- and autophagy-inducing effects of PLB on both PC-3 and DU145 cells. These findings indicate that PLB promotes apoptosis and autophagy in prostate cancer cells via Sirt1- and PI3K/Akt/mTOR-mediated pathways with contribution from AMPK-, p38 MAPK-, visfatin-, and ROS-associated pathways. Keywords: AMPK, visfatin, PC-3, DU145, ROS

Shu-Ting Pan,1 Yiru Qin,2 Zhi-Wei Zhou,2,3 Zhi-Xu He,3 Xueji Zhang,4 Tianxin Yang,5 Yin-Xue Yang,6 Dong Wang,7 Jia-Xuan Qiu,1 Shu-Feng Zhou2 1Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino–US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China; 4Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 5Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 7Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China Abstract: Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PLB), a naturally occurring naphthoquinone isolated from the roots of Plumbaginaceae plants, has been reported to possess anticancer activities in both in vitro and in vivo studies, but the effect of PLB on tongue squamous cell carcinoma (TSCC) is not fully understood. This study aimed to investigate the effects of PLB on cell cycle distribution, apoptosis, and autophagy, and the underlying mechanisms in the human TSCC cell line SCC25. The results have revealed that PLB exerted potent inducing effects on cell cycle arrest, apoptosis, and autophagy in SCC25 cells. PLB arrested SCC25 cells at the G2/M phase in a concentration- and time-dependent manner with a decrease in the expression level of cell division cycle protein 2 homolog (Cdc2) and cyclin B1 and increase in the expression level of p21 Waf1/Cip1, p27 Kip1, and p53 in SCC25 cells. PLB markedly induced apoptosis and autophagy in SCC25 cells. PLB decreased the expression of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and B-cell ­lymphoma-extra large (Bcl-xl) while increasing the expression level of the pro-apoptotic protein ­Bcl-2-associated X protein (Bax) in SCC25 cells. Furthermore, PLB inhibited phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), glycogen synthase kinase 3β (GSK3β), and p38 mitogen-activated protein kinase (p38 MAPK) pathways as indicated by the alteration in the ratio of phosphorylation level over total protein expression level, contributing to the autophagy inducing effect. In addition, we found that wortmannin (a PI3K inhibitor) and SB202190 (a selective inhibitor of p38 MAPK) strikingly enhanced PLB-induced autophagy in SCC25 cells, suggesting the involvement of PI3K- and p38 MAPK-mediated signaling pathways. Moreover, PLB induced intracellular reactive oxygen species (ROS) generation and this effect was attenuated by L-glutathione (GSH) and N-acetyl-L-cysteine (NAC). Taken together, these results indicate that PLB promotes cellular apoptosis and autophagy in TSCC cells involving p38 MAPK- and PI3K/Akt/mTOR-mediated pathways with contribution from the GSK3ß and ROS-mediated pathways. Keywords: TSCC, cell cycle, ROS, p38 MAPK, GSK3β

Ning-Kui Niu,1–3 Zi-Li Wang,1 Shu-Ting Pan,2,4 Hui-Qiang Ding,1 Giang HT Au,2 Zhi-Xu He,5 Zhi-Wei Zhou,2,5 Guozhi Xiao,6 Yin-Xue Yang,7 Xueji Zhang,8 Tianxin Yang,9 Xiao-Wu Chen,10 Jia-Xuan Qiu,4 Shu-Feng Zhou2 1Department of Spinal Surgery, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Orthopedics, General Hospital of Tianjin Medical University, Tianjin, 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, 5Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guizhou Medical University, Guiyang, Guizhou, People’s Republic of China; 6Department of Biochemistry, Medical Center, Rush University, Chicago, IL, USA; 7Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 8Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 9Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 10Department of General Surgery, The First People’s Hospital of Shunde affiliated to Southern Medical University, Foshan, Guangdong, People’s Republic of China Abstract: Osteosarcoma (OS) is the most common malignant bone tumor occurring mostly in children and adolescents between 10 and 20 years of age with poor response to current therapeutics. Alisertib (ALS, MLN8237) is a selective Aurora kinase A inhibitor that displays anticancer effects on several types of cancer. However, the role of ALS in the treatment of OS remains unknown. This study aimed to investigate the effects of ALS on the cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) and the underlying mechanisms in two human OS cell lines U-2 OS and MG-63. The results showed that ALS had potent growth inhibitory, pro-apoptotic, pro-autophagic, and EMT inhibitory effects on U-2 OS and MG-63 cells. ALS remarkably induced G2/M arrest and down-regulated the expression levels of cyclin-dependent kinases 1 and 2 and cyclin B1 in both U-2 OS and MG-63 cells. ALS markedly induced mitochondria-mediated apoptosis with a significant increase in the expression of key pro-apoptotic proteins and a decrease in main anti-apoptotic proteins. Furthermore, ALS promoted autophagic cell death via the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways, and activation of 5'-AMP-dependent kinase (AMPK) signaling pathway. Inducers or inhibitors of apoptosis or autophagy simultaneously altered ALS-induced apoptotic and autophagic death in both U-2 OS and MG-63 cells, suggesting a crosstalk between these two primary modes of programmed cell death. Moreover, ALS suppressed EMT-like phenotypes with a marked increase in the expression of E-cadherin but a decrease in N-cadherin in U-2 OS and MG-63 cells. ALS treatment also induced reactive oxygen species (ROS) generation but inhibited the expression levels of sirtuin 1 and nuclear factor-erythroid-2-related factor 2 (Nrf2) in both cell lines. Taken together, these findings show that ALS promotes apoptosis and autophagy but inhibits EMT via PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways with involvement of ROS- and sirtuin 1-associated pathways in U-2 OS and MG-63 cells. ALS is a promising anticancer agent in OS treatment and further studies are needed to confirm its efficacy and safety in OS chemotherapy. Keywords: ALS, autophagy, apoptosis, osteosarcoma, PI3K/Akt/mTOR pathway, EMT

Yan-Yang Wang,1,2 Yin-Xue Yang,3 Ren Zhao,1 Shu-Ting Pan,2,4 Hong Zhe,1 Zhi-Xu He,5 Wei Duan,6 Xueji Zhang,7 Tianxin Yang,8 Jia-Xuan Qiu,4 Shu-Feng Zhou2,51Department of Radiation Oncology, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, 5Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, People’s Republic of China; 6School of Medicine, Deakin University, Waurn Ponds, VIC, Australia; 7Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 8Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USAAbstract: Natural and synthetic triterpenoids have been shown to kill cancer cells via multiple mechanisms. The therapeutic effect and underlying mechanism of the synthetic triterpenoid bardoxolone methyl (C-28 methyl ester of 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid; CDDO-Me) on esophageal cancer are unclear. Herein, we aimed to investigate the anticancer effects and underlying mechanisms of CDDO-Me in human esophageal squamous cell carcinoma (ESCC) cells. Our study showed that CDDO-Me suppressed the proliferation and arrested cells in G2/M phase, and induced apoptosis in human ESCC Ec109 and KYSE70 cells. The G2/M arrest was accompanied with upregulated p21Waf1/Cip1 and p53 expression. CDDO-Me significantly decreased B-cell lymphoma-extra large (Bcl-xl), B-cell lymphoma 2 (Bcl-2), cleaved caspase-9, and cleaved poly ADP ribose polymerase (PARP) levels but increased the expression level of Bcl-2-associated X (Bax). Furthermore, CDDO-Me induced autophagy in both Ec109 and KYSE70 cells via suppression of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. There were interactions between the autophagic and apoptotic pathways in Ec109 and KYSE70 cells subject to CDDO-Me treatment. CDDO-Me also scavenged reactive oxygen species through activation of the nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) pathway in Ec109 and KYSE70 cells. CDDO-Me inhibited cell invasion, epithelial–mesenchymal transition, and stemness in Ec109 and KYSE70 cells. CDDO-Me significantly downregulated E-cadherin but upregulated Snail, Slug, and zinc finger E-box-binding homeobox 1 (TCF-8/ZEB1) in Ec109 and KYSE70 cells. CDDO-Me significantly decreased the expression of octamer-4, sex determining region Y-box 2 (Sox-2), Nanog, and B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1), all markers of cancer cell stemness, in Ec109 and KYSE70 cells. Taken together, these results indicate that CDDO-Me is a promising anticancer agent against ESCC. Further studies are warranted to explore the molecular targets, efficacy and safety of CDDO-Me in the treatment of ESCC.Keywords: CDDO-Me, esophageal squamous cell carcinoma, cell cycle, apoptosis, autophagy, EMT, stemness, Akt, mTOR

Shu-Ting Pan,1,* Zhi-Wei Zhou,2,3,* Zhi-Xu He,3 Xueji Zhang,4 Tianxin Yang,5 Yin-Xue Yang,6 Dong Wang,7 Jia-Xuan Qiu,1 Shu-Feng Zhou2 1Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 4Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 5Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 7Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China *These two authors contributed equally to this work Abstract: 5,6-Dimethylxanthenone 4-acetic acid (DMXAA), also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC) and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data showed that treatment with DMXAA modulated the expression of 588 protein molecules in A549 cells, with 281 protein molecules being up regulated and 306 protein molecules being downregulated. Ingenuity pathway analysis (IPA) identified 256 signaling pathways and 184 cellular functional proteins that were regulated by DMXAA in A549 cells. These targeted molecules and signaling pathways were mostly involved in cell proliferation and survival, redox homeostasis, sugar, amino acid and nucleic acid metabolism, cell migration, and invasion and programed cell death. Subsequently, the effects of DMXAA on cell cycle distribution, apoptosis, autophagy, and reactive oxygen species (ROS) generation were experimentally verified. Flow cytometric analysis showed that DMXAA significantly induced G1 phase arrest in A549 cells. Western blotting assays demonstrated that DMXAA induced apoptosis via a mitochondria-dependent pathway and promoted autophagy, as indicated by the increased level of cytosolic cytochrome c, activation of caspase 3, and enhanced expression of beclin 1 and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) in A549 cells. Moreover, DMXAA significantly promoted intracellular ROS generation in A549 cells. Collectively, this SILAC study quantitatively evaluates the proteomic response to treatment with DMXAA that helps to globally identify the potential molecular targets and elucidate the underlying mechanism of DMXAA in the treatment of NSCLC. Keywords: DMXAA, non-small cell lung cancer, cell cycle, apoptosis, autophagy, SILAC

Jin-Ping Li,1,2 Yin-Xue Yang,3 Qi-Lun Liu,1 Zhi-Wei Zhou,2,4 Shu-Ting Pan,2,5 Zhi-Xu He,4 Xueji Zhang,6 Tianxin Yang,7 Si-Yuan Pan,8 Wei Duan,9 Shu-Ming He,10 Xiao-Wu Chen,11 Jia-Xuan Qiu,5 Shu-Feng Zhou2,41Department of Surgical Oncology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 4Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China; 5Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 6Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 7Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 8Department of Pharmacology, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 9School of Medicine, Deakin University, Waurn Ponds, VIC, Australia; 10Department of Obstetrics and Gynecology, Xiaolan People’s Hospital affiliated to Southern Medical University, Zhongshan, Guangdong, People’s Republic of China; 11Department of General Surgery, The First People’s Hospital of Shunde affiliated to Southern Medical University, Shunde, Foshan, Guangdong, People’s Republic of China Abstract: Danusertib (Danu) is a pan-inhibitor of Aurora kinases and a third-generation breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (Bcr-Abl) tyrosine kinase inhibitor, but its antitumor effect and underlying mechanisms in the treatment of human breast cancer remain elusive. This study aimed to investigate the effects of Danu on the growth, apoptosis, autophagy, and epithelial-to-mesenchymal transition (EMT) and the molecular mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. The results demonstrated that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both breast cancer cell lines. Danu arrested MCF7 and MDA-MB-231 cells in G2/M phase, accompanied by the downregulation of cyclin-dependent kinase 1 and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53. Danu significantly decreased the expression of B-cell lymphoma-extra-large (Bcl-xl) and B-cell lymphoma 2 (Bcl-2), but increased the expression of Bcl-2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and promoted the cleavage of caspases 3 and 9. Furthermore, Danu significantly increased the expression levels of the membrane-bound microtubule-associated protein 1A/1B-light chain 3 (LC3-II) and beclin 1 in breast cancer cells, two markers for autophagy. Danu induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (Erk1/2) and inhibited the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathways in breast cancer cells. Treatment with wortmannin (a phosphatidylinositol 3-kinase inhibitor) markedly inhibited Danu-induced activation of p38 MAPK and conversion of cytosolic LC3-I to membrane-bound LC3-II. Pharmacological inhibition and small interfering RNA-mediated knockdown of p38 MAPK suppressed Akt activation, resulting in LC3-II accumulation and enhanced autophagy. Pharmacological inhibition and small interfering RNA-mediated knockdown of Erk1/2 also remarkably increased the level of LC3-II in MCF7 cells. Moreover, Danu inhibited EMT in both MCF7 and MDA-MB-231 cells with upregulated E-cadherin and zona occludens protein 1 (ZO-1) but downregulated N-cadherin, zinc finger E-box-binding homeobox 1 (TCF8/ZEB1), snail, slug, vimentin, and ß-catenin. Notably, Danu showed lower cytotoxicity toward normal breast epithelial MCF10A cells. These findings indicate that Danu promotes cellular apoptosis and autophagy but inhibits EMT in human breast cancer cells via modulation of p38 MAPK/Erk1/2/Akt/mTOR signaling pathways. Danu may represent a promising anticancer agent for breast cancer treatment. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Danu in breast cancer therapy. Keywords: Danusertib, breast cancer, cell cycle, apoptosis, autophagy, EMT

Yong-Hui Ding,1,2 Zhi-Wei Zhou,2,3 Chun-Fang Ha,1 Xue-Yu Zhang,1 Shu-Ting Pan,4 Zhi-Xu He,3 Jeffrey L Edelman,2 Dong Wang,5 Yin-Xue Yang,6 Xueji Zhang,7 Wei Duan,8 Tianxin Yang,9 Jia-Xuan Qiu,4 Shu-Feng Zhou2,3 1Department of Gynecology, General Hospital of Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA ; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, 5Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, 6Department of Colorectal Surgery, General Hospital of Ningxia Medical University, Yinchuan, 7Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 8School of Medicine, Deakin University, Waurn Ponds, Australia; 9Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA Abstract: Ovarian cancer is a leading killer of women, and no cure for advanced ovarian cancer is available. Alisertib (ALS), a selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects, and is under clinical investigation for the treatment of advanced solid tumor and hematologic malignancies. However, the role of ALS in the treatment of ovarian cancer remains unclear. This study investigated the effects of ALS on cell growth, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT), and the underlying mechanisms in human epithelial ovarian cancer SKOV3 and OVCAR4 cells. Our docking study showed that ALS, MLN8054, and VX-680 preferentially bound to AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS had potent growth-inhibitory, proapoptotic, proautophagic, and EMT-inhibitory effects on SKOV3 and OVCAR4 cells. ALS arrested SKOV3 and OVCAR4 cells in G2/M phase and induced mitochondria-mediated apoptosis and autophagy in both SKOV3 and OVCAR4 cell lines in a concentration-dependent manner. ALS suppressed phosphatidylinositol 3-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase pathways but activated 5′-AMP-dependent kinase, as indicated by their altered phosphorylation, contributing to the proautophagic activity of ALS. Modulation of autophagy altered basal and ALS-induced apoptosis in SKOV3 and OVCAR4 cells. Further, ALS suppressed the EMT-like phenotype in both cell lines by restoring the balance between E-cadherin and N-cadherin. ALS downregulated sirtuin 1 and pre-B cell colony enhancing factor (PBEF/visfatin) expression levels and inhibited phosphorylation of AURKA in both cell lines. These findings indicate that ALS blocks the cell cycle by G2/M phase arrest and promotes cellular apoptosis and autophagy, but inhibits EMT via phosphatidylinositol 3-kinase/Akt/mTOR-mediated and sirtuin 1-mediated pathways in human epithelial ovarian cancer cells. Further studies are warranted to validate the efficacy and safety of ALS in the treatment of ovarian cancer. Keywords: alisertib, Aurora kinase A, epithelial ovarian cancer, cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition, sirtuin 1

Feng Wang,1,2 Hai Li,3 Xiao-Gang Yan,4 Zhi-Wei Zhou,2 Zhi-Gang Yi,5 Zhi-Xu He,6 Shu-Ting Pan,7 Yin-Xue Yang,3 Zuo-Zheng Wang,1 Xueji Zhang,8 Tianxing Yang,9 Jia-Xuan Qiu,7 Shu-Feng Zhou21Department of Hepatobiliary Surgery, General Hospital, Ningxia Medical University, Yinchuan, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Department of Colorectal Surgery, General Hospital, Ningxia Medical University, 4Department of Oncological Surgery, The First People’s Hospital of Yinchuan, 5Department of General Surgery, Changqing Yangehu Hospital, Yinchuan, 6Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, 7Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, 8Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 9Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USAAbstract: Pancreatic cancer is the most aggressive cancer worldwide with poor response to current therapeutics. Alisertib (ALS), a potent and selective Aurora kinase A inhibitor, exhibits potent anticancer effects in preclinical and clinical studies; however, the effect and underlying mechanism of ALS in the pancreatic cancer treatment remain elusive. This study aimed to examine the effects of ALS on cell growth, autophagy, and epithelial-to-mesenchymal transition (EMT) and to delineate the possible molecular mechanisms in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that ALS exerted potent cell growth inhibitory, pro-autophagic, and EMT-suppressing effects in PANC-1 and BxPC-3 cells. ALS remarkably arrested PANC-1 and BxPC-3 cells in G2/M phase via regulating the expression of cyclin-dependent kinases 1 and 2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. ALS concentration-dependently induced autophagy in PANC-1 and BxPC-3 cells, which may be attributed to the inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal-regulated kinases 1 and 2 (Erk1/2) but activation of 5'-AMP-dependent kinase signaling pathways. ALS significantly inhibited EMT in PANC-1 and BxPC-3 cells with an increase in the expression of E-cadherin and a decrease in N-cadherin. In addition, ALS suppressed the expression of sirtuin 1 (Sirt1) and pre-B cell colony-enhancing factor/visfatin in both cell lines with a rise in the level of acetylated p53. These findings show that ALS induces cell cycle arrest and promotes autophagic cell death but inhibits EMT in pancreatic cancer cells with the involvement of PI3K/Akt/mTOR, p38 MAPK, Erk1/2, and Sirt1-mediated signaling pathways. Taken together, ALS may represent a promising anticancer drug for pancreatic cancer treatment. More studies are warranted to investigate other molecular targets and mechanisms and verify the efficacy and safety of ALS in the treatment of pancreatic cancer.Keywords: alisertib, pancreatic cancer, cell cycle, autophagy, EMT, Sirt1

Feng Wang,1,2,* Qi Wang,1,* Zhi-Wei Zhou,2,3 Song-Ning Yu,1 Shu-Ting Pan,4 Zhi-Xu He,3 Xueji Zhang,5 Dong Wang,6 Yin-Xue Yang,7 Tianxing Yang,8 Tao Sun,9 Min Li,10 Jia-Xuan Qiu,4 Shu-Feng Zhou2 1Department of Hepatobiliary Surgery, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 2Department of Pharmaceutical Science, College of Pharmacy, University of South Florida, Tampa, FL, USA; 3Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, People’s Republic of China; 4Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, People’s Republic of China; 5Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China; 6Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing, People’s Republic of China; 7Department of Colorectal Surgery, General Hospital, Ningxia Medical University, Yinchuan, Ningxia, People’s Republic of China; 8Department of Internal Medicine, University of Utah and Salt Lake Veterans Affairs Medical Center, Salt Lake City, UT, USA; 9Key Lab of Craniocerebral Diseases of Ningxia, Ningxia Medical University, Yinchuan, People’s Republic of China; 10Department of Medicine and Department of Surgery, The University of Oklahoma Health Sciences Center, Stanton L Young Biomedical Research Center, Oklahoma City, OK, USA *These authors contributed equally to this work Abstract: Plumbagin (PLB), an active naphthoquinone compound, has shown potent anticancer effects in preclinical studies; however, the effect and underlying mechanism of PLB for the treatment of pancreatic cancer is unclear. This study aimed to examine the pancreatic cancer cell killing effect of PLB and investigate the underlying mechanism in human pancreatic cancer PANC-1 and BxPC-3 cells. The results showed that PLB exhibited potent inducing effects on cell cycle arrest in PANC-1 and BxPC-3 cells via the modulation of cell cycle regulators including CDK1/CDC2, cyclin B1, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53. PLB treatment concentration- and time-dependently increased the percentage of autophagic cells and significantly increased the expression level of phosphatase and tensin homolog, beclin 1, and the ratio of LC3-II over LC3-I in both PANC-1 and BxPC-3 cells. PLB induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B/mammalian target of rapamycin and p38 mitogen-activated protein kinase (p38 MAPK) pathways and activation of 5'-AMP-dependent kinase as indicated by their altered phosphorylation, contributing to the proautophagic activities of PLB in both cell lines. Furthermore, SB202190, a selective inhibitor of p38 MAPK, and wortmannin, a potent, irreversible, and selective PI3K inhibitor, remarkably enhanced PLB-induced autophagy in PANC-1 and BxPC-3 cells, indicating the roles of PI3K and p38 MAPK mediated signaling pathways in PLB-induced autophagic cell death in both cell lines. In addition, PLB significantly inhibited epithelial to mesenchymal transition phenotype in both cell lines with an increase in the expression level of E-cadherin and a decrease in N-cadherin. Moreover, PLB treatment significantly suppressed the expression of Sirt1 in both cell lines. These findings show that PLB promotes cell cycle arrest and autophagy but inhibits epithelial to mesenchymal transition phenotype in pancreatic cancer cells with the involvement of PI3K/protein kinase B/ mammalian target of rapamycin and p38 MAPK mediated pathways. Keywords: Plumbagin, pancreatic cancer, cell cycle, autophagy, EMT, Sirt1

Background: Liver transplantation is the main treatment for cholestatic liver disease and some metabolic liver diseases in children. However, no accurate prediction model to determine the survival probability of grafts prior to surgery exists. This study aimed to develop an effective prognostic model for allograft survival after pediatric liver transplantation., Methods: This retrospective cohort study included 2032 patients who underwent pediatric liver transplantation between January 1, 2006, and January 1, 2020. A nomogram was developed using Cox regression and validated based on bootstrap sampling. Predictive and discriminatory accuracies were determined using the concordance index and visualized using calibration curves; net benefits were calculated for model comparison. An online Shiny application was developed for easy access to the model., Results: Multivariable analysis demonstrated that preoperative diagnosis, recipient age, body weight, graft type, preoperative total bilirubin, interleukin-1β, portal venous blood flow direction, spleen thickness, and the presence of heart disease and cholangitis were independent factors for survival, all of which were selected in the nomogram. Calibration of the nomogram indicated that the 1-, 3-, and 5-year predicted survival rates agreed with the actual survival rate. The concordance indices for graft survival at 1, 3, and 5 years were 0.776, 0.757, and 0.753, respectively, which were significantly higher than those of the Pediatric End-Stage Liver Disease and Child-Pugh scoring systems. The allograft dysfunction risk of a recipient could be easily predicted using the following URL: https://aspelt.shinyapps.io/ASPELT/ / CONCLUSION: The allograft survival after pediatric liver transplantation (ASPELT) score model can effectively predict the graft survival rate after liver transplantation in children, providing a simple and convenient evaluation method for clinicians and patients., (© 2023. The Author(s).)

Emergency medical retrieval services (EMRS) in remote Indigenous islands is rarely investigated. We analyzed the characteristics of patients who underwent EMRS in Lanyu, an offshore island of Taiwan, from January 1, 2014 to December 31, 2021. The need for EMRS for Lanyu Indigenous residents (N=132, 3.83‰) was almost 1.5-fold and 100-fold for non-Indigenous residents (N=16, 2.64‰) and tourists (N=40, 0.04‰), respectively. The resident group had a longer hospitalization (12.0 ± 12.9 vs. 5.9 ± 11.7 days, p=.007). The tourist group had more near-drowning or decompression sickness (44.0% vs. 3.0%, p<.001) and secondary transfers (20.0% vs. 5.4%, p=.003). All the patients (N=12) that required multiple retrievals were Lanyu Indigenous residents. The Lanyu Indigenous residents, compared with the non-Indigenous residents, had fewer admissions to intensive care units (47.7% vs. 80.0%) and more in-hospital mortalities (10.6% vs. 0.0%). Multifaceted approaches should be initiated to improve the health care system in remote Indigenous islands.

The two most commonly used airway management techniques during general anaesthesia are supraglottic airway devices and tracheal tubes. In older patients undergoing elective non-cardiothoracic surgery under general anaesthesia with positive pressure ventilation, we hypothesised that a composite measure of in-hospital postoperative pulmonary complications would be less frequent when a supraglottic airway device was used compared with a tracheal tube. We studied patients aged ≥ 70 years in 17 clinical centres. Patients were allocated randomly to airway management with a supraglottic airway device or a tracheal tube. Between August 2016 and April 2020, 2900 patients were studied, of whom 2751 were included in the primary analysis (1387 with supraglottic airway device and 1364 with a tracheal tube). Pre-operatively, 2431 (88.4%) patients were estimated to have a postoperative pulmonary complication risk index of 1-2. Postoperative pulmonary complications, mostly coughing, occurred in 270 of 1387 patients (19.5%) allocated to a supraglottic airway device and 342 of 1364 patients (25.1%) assigned to a tracheal tube (absolute difference -5.6% (95%CI -8.7 to -2.5), risk ratio 0.78 (95%CI 0.67-0.89); p < 0.001). Among otherwise healthy older patients undergoing elective surgery under general anaesthesia with intra-operative positive pressure ventilation of their lungs, there were fewer postoperative pulmonary complications when the airway was managed with a supraglottic airway device compared with a tracheal tube., (© 2023 Association of Anaesthetists.)

Cisplatin plus 5-fluorouracil (PF) is used as the standard neoadjuvant chemotherapy (also called preoperative chemotherapy) in the treatment of tongue squamous cell carcinoma (TSCC). Although PF chemotherapy reduces the distant metastasis of TSCC, the five-year survival rate has not significantly improved. In recent years, components considered in traditional Chinese medicine have been researched as adjuvant drugs for radiotherapy and chemotherapy. Plumbagin (PB) is a quinone component isolated from Plumbago zeylanica L. Notably, PB demonstrates numerous anticancer properties. In order to examine the chemosensitization effect of PB on PF and its associated mechanisms, in vitro experiments using TSCC Cal27 and cisplatin (CDDP)-resistant Cal27/CDDP cells were carried out in the present study, and the results were subsequently verified using nude mice xenografts. Results of the present study demonstrated that PB enhanced the anticancer effects of PF on the proliferation, migration, and invasion of Cal27 and Cal27/CDDP cells. Cell cycle assays demonstrated that both Cal27 and Cal27/CDDP cells were arrested in the S phase following the combined treatment of PF and PB. Moreover, the PF and PB combination group induced higher levels of apoptosis in Cal27 and Cal27/CDDP cells compared with the group treated with PF alone. In addition, the results of the present study demonstrated that combined PB and PF inhibited the PI3K/AKT/mTOR/p70S6K pathway in TSCC cells. Moreover, the weight and volumes of tumors in nude mice were reduced following treatment with a combination of PF and PB. Results of the present study also demonstrated that the expression levels of Ki67 were markedly reduced in the combined treatment group compared with the group treated with PF alone. In summary, the results of the present study demonstrated that PB enhanced the PF sensitivity of TSCC through induction of S-phase arrest and apoptosis via the PI3K/AKT/mTOR/p70S6K pathway., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Shu-Ting Pan et al.)

High-grade neuroendocrine tumors (NETs) of the lung consist of small-cell lung cancer (SCLC) and large-cell neuroendocrine carcinoma (LCNEC). Both exhibit aggressive malignancy with poor prognosis. The transformation of lung adenocarcinoma (ADC) to SCLC or LCNEC also contributes to acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs). Despite initially being responsive to chemotherapy, high-grade NET patients inevitably develop drug resistance; thus, novel therapeutic targets are urgently needed for these patients. Our study reported that VGF (nerve growth factor inducible), a factor mainly expressed in neurons during neural development, is highly expressed in SCLC and LCNEC as well as in a subset of ADCs, whereas targeting VGF attenuates cancer cell growth and tumor formation. High VGF expression was associated with advanced stage SCLC and predicted poor prognosis in lung ADC. In addition, EGFR-TKI selection enriched VGF expression in TKI-resistant ADC under epigenetic control. The VGF locus possessed the HDAC1 binding site, and treatment of ADC cells with the HDAC1 inhibitor induced VGF expression. High VGF expression was associated with chemoresistance, and silencing VGF induced BMF and BCL2L11 expression and rendered lung cancer cells sensitive to chemotherapy drugs. These findings suggested the potential of VGF as a prognostic factor and therapeutic target in lung cancers with neuroendocrine feature., (© 2022 UICC.)

Primary intestinal T-cell lymphoma (PITL) is highly aggressive and includes celiac disease-related enteropathy-associated T-cell lymphoma (EATL), monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), and primary intestinal peripheral T-cell lymphoma, not otherwise specified (ITCL-NOS). MEITL is the most common PITL in Asia, comprising of monomorphic medium-sized cells typically expressing CD8, CD56, and cytotoxic granules. Occasional cases with intermediate features between MEITL and ITCL-NOS are difficult to be classified and warrant further investigation. We collected 54 surgically resected PITLs from Taiwan, with 80% presenting with bowel perforation. The overall outcome was poor with a median survival of 7 months. Based on histopathology (monomorphic vs. pleomorphic) and immunophenotype, we classified these cases into 4 groups: MEITL with typical immunophenotype (n=34), MEITL with atypical immunophenotype (n=5), pleomorphic PITL with MEITL-like immunophenotype (n=6), and ITCL-NOS (n=9). There was no EATL in our cohort. Targeted next-generation sequencing of the first 3 groups showed highly prevalent loss-of-function mutations for SETD2 (85%, 80%, and 83%, respectively) and frequent activating mutations for STAT5B (64%, 60%, and 50%, respectively) and JAK3 (38%, 20%, and 50%, respectively). In contrast, ITCL-NOS cases had less frequent mutations of SETD2 (56%) and STAT5B (11%) and rare JAK3 mutations (11%). Our results suggest that there is a wider morphologic and immunophenotypic spectrum of MEITL as currently defined in the 2017 WHO classification. MEITL with atypical immunophenotype and PITL with MEITL-like immunophenotype shared clinicopathologic and molecular features similar to MEITL but distinct from ITCL-NOS, indicating that such cases may be considered as immunophenotypic or histopathologic variants of MEITL., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the Ministry of Science and Technology, Taiwan (107-2320-B-384-002). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Physical exercise plays a role on the prevention and treatment of Alzheimer's disease (AD), but the exercise mode and the mechanism for these positive effects is still ambiguous. Here, we investigated the effect of an aerobic interval exercise, running in combination with swimming, on behavioral dysfunction and associated adult neurogenesis in a mouse model of AD. We demonstrate that 4 weeks of the exercise could ameliorate Aβ 42 oligomer-induced cognitive impairment in mice utilizing Morris water maze tests. Additionally, the exercised Aβ 42 oligomer-induced mice exhibited a significant reduction of anxiety- and depression-like behaviors compared to the sedentary Aβ 42 oligomer-induced mice utilizing an Elevated zero maze and a Tail suspension test. Moreover, by utilizing 5'-bromodeoxyuridine (BrdU) as an exogenous cell tracer, we found that the exercised Aβ 42 oligomer-induced mice displayed a significant increase in newborn cells (BrdU + cells), which differentiated into a majority of neurons (BrdU + DCX + cells or BrdU + NeuN + cells) and a few of astrocytes (BrdU + GFAP + cells). Likewise, the exercised Aβ 42 oligomer-induced mice also displayed the higher levels of NeuN, PSD95, synaptophysin, Bcl-2 and lower level of GFAP protein. Furthermore, alteration of serum metabolites in transgenic AD mice between the exercised and sedentary group were significantly associated with lipid metabolism, amino acid metabolism, and neurotransmitters. These findings suggest that combined aerobic interval exercise-mediated metabolites and proteins contributed to improving adult neurogenesis and behavioral performance after AD pathology, which might provide a promising therapeutic strategy for AD., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Single-factor intervention, such as physical exercise and auditory and visual stimulation, plays a positive role on the prevention and treatment of Alzheimer's disease (AD); however, the therapeutic effects of single-factor intervention are limited. The beneficial effects of these multifactor combinations on AD and its molecular mechanism have yet to be elucidated. Here, we investigated the effect of multifactor intervention, voluntary wheel exercise, and involuntary treadmill running in combination with acousto-optic stimulation, on adult neurogenesis and behavioral phenotypes in a mouse model of AD. We found that 4 weeks of multifactor intervention can significantly increase the production of newborn cells (BrdU + cells) and immature neurons (DCX + cells) in the hippocampus and lateral ventricle of Aβ oligomer-induced mice. Importantly, the multifactor intervention could promote BrdU + cells to differentiate into neurons (BrdU + DCX + cells or BrdU + NeuN + cells) and astrocytes (BrdU + GFAP + cells) in the hippocampus and ameliorate Aβ oligomer-induced cognitive impairment and anxiety- and depression-like behaviors in mice evaluated by novel object recognition, Morris water maze tests, elevated zero maze, forced swimming test, and tail suspension test, respectively. Moreover, multifactor intervention could lead to an increase in the protein levels of PSD-95, SYP, DCX, NeuN, GFAP, Bcl-2, BDNF, TrkB, and pSer473-Akt and a decrease in the protein levels of BAX and caspase-9 in the hippocampal lysates of Aβ oligomer-induced mice. Furthermore, sequencing analysis of serum metabolites revealed that aberrantly expressed metabolites modulated by multifactor intervention were highly enriched in the biological process associated with keeping neurons functioning and neurobehavioral function. Additionally, the intervention-mediated serum metabolites mainly participated in glutamate metabolism, glucose metabolism, and the tricarboxylic acid cycle in mice. Our findings suggest the potential of multifactor intervention as a non-invasive therapeutic strategy for AD to anti-Aβ oligomer neurotoxicity., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Abbreviations: ATG14: autophagy related 14; CDH2: cadherin 2; ChIP-qPCR: chromatin immunoprecipitation quantitative polymerase chain reaction; CQ: chloroquine; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; EPCAM: epithelial cell adhesion molecule; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NDUFV2: NADH:ubiquinone oxidoreductase core subunit V2; OCR: oxygen consumption rate; ROS: reactive oxygen species; RT-qPCR: reverse-transcriptase quantitative polymerase chain reaction; SC: scrambled control; sh RNA : short hairpin RNA; SNAI2: snail family transcriptional repressor 2; SOX2: SRY-box transcription factor 2; SQSTM1/p62: sequestosome 1; TGFB/TGF-β: transforming growth factor beta; TOMM20: translocase of outer mitochondrial membrane 20; ZEB1: zinc finger E-box binding homeobox 1.

Background/purpose: Taiwan set up disaster medical assistance teams (DMATs) after the Chi-Chi earthquake, but these teams lack unified standards., Methods: This study was divided into two phases. Phase I was a Delphi study conducted in 2019 with 26 experts who were invited to establish Taiwan's DMAT standards by modifying the World Health Organization Emergency Medical Team (WHO EMT) type I fixed standards. Phase II was a cross-sectional study conducted in 2020. A questionnaire was used to evaluate the disaster preparedness of DMATs by standards set in phase I., Results: In phase I, Taiwan's DMAT standards were established after three rounds of Delphi consensus, with a response rate of 88.5%. The major departures from the WHO EMT standards were the exclusion of obstetric care, mental health, rehabilitation, and laboratory and blood transfusion standards and the addition of an ultrasound standard. During phase II, a total of 32 teams were invited, and the response rate was 96.9%. The overall standard achievement rate was 56.9%, and the three lowest achievement rates corresponded to sanitation (22.6%), medical malpractice insurance (25.8%), and pharmacy and drug supply (25.8%). The national DMATs, official DMATs, DMATs funded by government, and DMATs with ≥10 years of experience had significantly higher achievement rates for partial or overall standards., Conclusion: Using localized standards to evaluate the disaster preparedness of each team, DMATs were found to have many shortcomings mainly due to the lack of unified government announcement standards, legal protection, and adequate financial support., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2021. Published by Elsevier B.V.)

In recent ten years, with the fast development of digital and engineering manufacturing technology, additive manufacturing has already been more and more widely used in the field of dentistry, from the first personalized surgical guides to the latest personalized restoration crowns and root implants. In particular, the bioprinting of teeth and tissue is of great potential to realize organ regeneration and finally improve the life quality. In this review paper, we firstly presented the workflow of additive manufacturing technology. Then, we summarized the main applications and recent research progresses of additive manufacturing in dentistry. Lastly, we sketched out some challenges and future directions of additive manufacturing technology in dentistry., Competing Interests: The authors declare that there are no conflicts of interests regarding the publication of this paper., (Copyright © 2022 Gan Huang et al.)

Porous scaffolds have long been regarded as optimal substitute for bone tissue repairing. In order to explore the influence of unit cell structure and inherent material characteristics on the porous scaffolds in terms of mechanical and biological performance, selective laser melting (SLM) technology was used to fabricate porous tantalum (Ta) and titanium alloy (Ti6Al4V) with diamond (Di) or rhombic dodecahedron (Do) unit cell structure. The mechanical strength of all the porous scaffolds could match that of trabecular bone, while the biological performance of each scaffold was diverse from each other. Moreover, the ILK/ERK1/2/Runx2 signaling pathway had been verified to be involved in the osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs) cultured on those porous scaffolds. Unit cell structure and material characteristics of the porous Ta and Ti6Al4V scaffolds can synergistically modulate this axis and further impact on the osteogenic effects. Our results hence illustrate that porous Ta scaffold with diamond unit cell structure possesses excellent osteogenic effects and moderate mechanical strength and porous Ti6Al4V scaffold with rhombic dodecahedron unit cell structure has the highest mechanical strength and moderate osteogenic effects. Both porous Ta and Ti6Al4V can be applied in different settings requiring either better biological performance or higher mechanical demand., (Copyright © 2021. Published by Elsevier B.V.)

Long non-coding RNAs (lncRNAs) play critical roles in the various physiological processes of insects. The wing is a successful adaptation allowing insects to escape from unfavorable environments, while information on lncRNAs related to wing development is limited. In this study, we constructed 12 libraries from two RNA-seq comparisons: 4th instar winged nymphs versus winged adults and 4th instar wingless nymphs versus wingless adults in the brown citrus aphid Aphis citricidus, to identify the wing development-associated lncRNAs. A total of 2914 lncRNAs were identified and 50 lncRNAs were differentially expressed during the 4th instar winged nymphs to winged adults transition, and 28 lncRNAs changed during the 4th instar wingless nymphs to wingless adults transition. The differentially expressed lncRNAs were grouped into six clusters according to the expression patterns in the combined two-winged morphs. lncRNA Ac&#95;lnc54106.1 was up-regulated during 4th instar winged nymphs to winged adults transition, but a lack of change during the 4th instar wingless nymphs to wingless adults transition implied a critical role in the specific regulation of wing development. RNA interference of Ac&#95;lnc54106.1 resulted in malformed wings. Targets prediction, expression patterns, and RNAi assay results showed that Ac&#95;lnc54106.1 may target the PiggyBac transposable element-derived protein 4 (PGBD4) gene, decrease expression of the canonical wing development-related genes, and finally regulate wing development. The systematic identification of lncRNAs in an aphid increases our understanding of how non-coding RNA mediates the wing plasticity of insects., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Flexible bronchoscopy is commonly used to examine patients suspected to have lung cancer. Bronchial brushing is one of the cytological technologies for lung specimens obtained through a bronchoscope. However, the accuracy of bronchial brushing cytology (BBC) for lung cancer diagnosis is still inconclusive. The aim of this study was to evaluate the diagnostic accuracy of BBC., Methods: A literature search was conducted with PubMed, Embase, the Cochrane Library, Web of Science, Biomed Central, Clinical Key, and ClinicalTrials.gov. Studies that assessed the efficacy of BBC in detecting lung cancer were included. Articles that estimated the accuracy on a per-patient basis were included. Review articles, case reports, and research that provided insufficient data to construct a 2 × 2 table were excluded. Both prospective trials and retrospective studies were included. English language studies were reviewed. Data synthesis was performed with a random-effects model., Results: Seventeen studies with 2538 patients were included in the study. The meta-analysis for BBC generated a pooled sensitivity of 0.67 (95% confidence interval [CI], 0.65-0.70) and a pooled specificity of 0.91 (95% CI, 0.89-0.93). The pooled diagnostic odds ratio for BBC was 24.55 (95% CI, 12.39-48.66). The subgroup analysis for studies using liquid-based cytology (LBC) generated a pooled sensitivity of 0.68 and a pooled specificity of 0.92. The pooled diagnostic odds ratio of studies using LBC was 114.18., Conclusions: These findings indicate that BBC is a discriminative diagnostic approach with moderate sensitivity and high specificity for diagnosing peripheral pulmonary lesions. BBC using LBC has higher diagnostic performance., (© 2021 American Cancer Society.)

This study was designed to investigate whether plumbagin (PL) could sensitize ionizing radiation (IR) in tongue squamous cell carcinoma (TSCC) cells and its possible mechanisms. Cell proliferation and combination index analysis was based on MTT and colony formation assay. Flow cytometry was applied to analyze the cell cycle distribution and apoptosis after the treatment of PL and/or IR. RT-PCR was used to examine the gene expression level of ataxia telangiegatasiata muted (ATM) and nuclear factor kappa beta (NF-κB) after various treatment groups. Western blot was used to examine the protein level of ATM and NF-κB as well as their phosphorylation level. PL enhances the cytotoxicity of IR in TSCC cells. Combination index was <1 which represents a synergistic effect. Combined PL and IR promoted G2/M arrest and apoptosis which could be reversed by ATM activator chloroquine phosphate. ATM and NF-κB were both inhibited by PL and IR combination. PL can efficiently enhance the radiosensitivity of TSCC cells by inducing G2/M arrest and apoptosis via downregulating ATM., Competing Interests: The authors report no conflicts of interest for this work., (Copyright © 2021 Shu-Ting Pan et al.)

Inhibitors of DNA binding and cell differentiation (ID) proteins regulate cellular differentiation and tumor progression. Whether ID family proteins serve as a linkage between pathological differentiation and cancer stemness in colorectal cancer is largely unknown. Here, the expression of ID4, but not other ID family proteins, was enriched in LGR5-high colon cancer stem cells. Its high expression was associated with poor pathological differentiation of colorectal tumors and shorter survival in patients. Knockdown of ID4 inhibited the growth and dissemination of colon cancer cells, while enhancing chemosensitivity. Through gene expression profiling analysis, brain-derived neurotrophic factor (BDNF) was identified as a downstream target of ID4 expression in colorectal cancer. BDNF knockdown decreased the growth and migration of colon cancer cells, and its expression enhanced dissemination, anoikis resistance and chemoresistance. ID4 silencing attenuated the epithelial-to-mesenchymal transition pattern in colon cancer cells. Gene cluster analysis revealed that ID4 and BDNF expression was clustered with mesenchymal markers and distant from epithelial genes. BDNF silencing decreased the expression of mesenchymal markers Vimentin, CDH2 and SNAI1. These findings demonstrated that ID4-BDNF signaling regulates colorectal cancer survival, with the potential to serve as a prognostic marker in colorectal cancer., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Lymphoma involving serous effusion is uncommon. The diagnosis of effusion lymphoma may be challenging, particularly when the lymphoid cells are small to medium-sized, which would be difficult for differentiating reactive effusions from low grade lymphomas. Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is an uncommon type of aggressive intestinal T cell lymphoma with a median survival of 7 months. MEITL rarely disseminates to the body cavities. To date, there are only three reported cases of MEITL with malignant effusion. Here we report two additional cases of MEITL with lymphoma cells involving the pleural effusion and the ascites, respectively. Review of the three literature cases and our two new cases of MEITL with malignant effusion, cytoplasmic azurophilic granules were identified in both the two cases with Liu stain. The median survival time was 1.5 months after the occurrence of malignant effusion, even shorter than the median survival in patients with MEITL. Although the case number is small, malignant effusion seems to be a poor prognostic factor of MEITL., (© 2021 Wiley Periodicals LLC.)

Porous tantalum (Ta) is a promising biomaterial and has been applied in orthopedics and dentistry for nearly two decades. The high porosity and interconnected pore structure of porous Ta promise fine bone ingrowth and new bone formation within the inner space, which further guarantee rapid osteointegration and bone-implant stability in the long term. Porous Ta has high wettability and surface energy that can facilitate adherence, proliferation and mineralization of osteoblasts. Meanwhile, the low elastic modulus and high friction coefficient of porous Ta allow it to effectively avoid the stress shield effect, minimize marginal bone loss and ensure primary stability. Accordingly, the satisfactory clinical application of porous Ta-based implants or prostheses is mainly derived from its excellent biological and mechanical properties. With the advent of additive manufacturing, personalized porous Ta-based implants or prostheses have shown their clinical value in the treatment of individual patients who need specially designed implants or prosthesis. In addition, many modification methods have been introduced to enhance the bioactivity and antibacterial property of porous Ta with promising in vitro and in vivo research results. In any case, choosing suitable patients is of great importance to guarantee surgical success after porous Ta insertion.

Introduction: Disaster medical assistance team (DMAT) and urban search and rescue team (USAR) need to cooperate seamlessly to save lives in disasters, but related research is limited., Objectives: To estimate the disaster preparedness of the DMAT and the barriers affecting interagency cooperation between the DMAT and the USAR team., Methods: This was an observational study of a full-scale exercise conducted in Taiwan from November 16 to 18, 2018. The exercise scenario simulated a magnitude 7 earthquake in Tainan City. DMATs from other counties were deployed and cooperated with local USAR teams to carry out disaster relief. Our study invited 7 experts to evaluate DMATs on disaster preparedness capabilities and the interagency collaboration between DMATs and USAR., Results: A total of eight DMATs, consisting of 30 physicians, 65 nurses, 74 logisticians, 5 health bureau personnel, and 85 USAR teams, participated in this exercise. During the mission, 176 patients were treated. The capabilities of each team were generally consistent with the basic technical standards for type I emergency medical teams, but the compliance rates for basic local anesthesia, cold chain equipment for medication, rapid blood test tools, and sterilization devices were only 50%, 12.5%, 12.5%, and 9%, respectively. In addition, 53% of participants reported abnormal vital signs, indicating that it was a high-stress situation. Moreover, the main barriers to interagency collaboration were differing perspectives and poor mutual understanding., Conclusion: A full-scale exercise carried out jointly with DMATs and USAR teams was valuable for disaster preparedness, particularly in terms of understanding the weaknesses of those teams and the barriers to interagency collaboration., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Ning-Ping Foo et al.)

Background: Thyroid transcription factor-1 (TTF-1) is a useful marker for identifying thyroid and lung cancers in diagnostic pathology, particularly for the investigation of unknown primary cancers. However, some other tumors such as colorectal cancer might aberrantly express TTF-1, particularly with the less specific clone SPT24. Occasional diffuse large B-cell lymphoma (DLBCL) cases have been reported to be TTF-1-positive, yet there is no information on TTF-1 expression in peripheral T-cell lymphoma (PTCL)., Methods: We investigated a series of PTCL and DLBCL by immunohistochemistry for TTF-1 expression using 2 commercially available clones., Results: We found that 33% (5/15) adult T-cell leukemia/lymphomas (ATLLs) and 25% (2/8) angioimmunoblastic T-cell lymphomas (AITLs) were positive by clone SPT24 and only 2ATLL cases were positive by clone 8G7G3/1. Overall TTF-1 expression rates of PTCL by SPT24 and 8G7G3/1 were 16% (7/43) and 5% (2/43), respectively. All DLBCLs were negative., Conclusion: Although TTF-1 is a relatively specific marker for thyroid and lung cancers, it might be expressed in some lymphomas, particularly PTCL when using clone SPT24. Pathologist should be aware of this possible diagnostic pitfall when using TTF-1 in investigating tumors of unknown origin.

Head and neck cancer is a highly genetic and metabolic heterogeneous collection of malignancies of the lip, oral cavity, salivary glands, pharynx, esophagus, paranasal sinuses, and larynx with five-year survival rates ranging from 12% to 93%. Patients with head and neck cancer typically present with advanced stage III, IVa, or IVb disease and are treated with comprehensive modality including chemotherapy, radiotherapy, and surgery. Despite advancements in treatment modality and technique, noisome recurrence, invasiveness, and resistance as well as posttreatment complications severely influence survival rate and quality of life. Thus, new therapeutic strategies are urgently needed that offer enhanced efficacy with less toxicity. ROS in cancer cells plays a vital role in regulating cell death, DNA repair, stemness maintenance, metabolic reprogramming, and tumor microenvironment, all of which have been implicated in resistance to chemo-/radiotherapy of head and neck cancer. Adjusting ROS generation and elimination to reverse the resistance of cancer cells without impairing normal cells show great hope in improving the therapeutic efficacy of chemo-/radiotherapy of head and neck cancer. In the current review, we discuss the pivotal and targetable redox-regulating system including superoxide dismutases (SODs), tripeptide glutathione (GSH), thioredoxin (Trxs), peroxiredoxins (PRXs), nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/keap1), and mitochondria electron transporter chain (ETC) complexes and their roles in regulating ROS levels and their clinical significance implicated in chemo-/radiotherapy of head and neck cancer. We also summarize several old drugs (referred to as the non-anti-cancer drugs used in other diseases for a long time) and small molecular compounds as well as natural herbs which effectively modulate cellular ROS of head and neck cancer to synergize the efficacy of conventional chemo-/radiotherapy. Emerging interdisciplinary techniques including photodynamic, nanoparticle system, and Bio-Electro-Magnetic-Energy-Regulation (BEMER) therapy are promising measures to broaden the potency of ROS modulation for the benefit of chemo-/radiotherapy in head and neck cancer., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Gan Huang and Shu-Ting Pan.)

Objective: To observe the effect of electroacupuncture (EA) preconditioning on the expressions of tyrosine kinase Lyn and spleen tyrosine kinase (Syk) in mast cells of subcutaneous loose connective tissue in the rats with urticaria and explore the potential biological mechanism of EA in the intervention of urticaria., Methods: A total of 32 SD rats were randomized into a blank group, a model group, an EA group and a positive medication group, 8 rats in each one. Except of the blank group, the passive cutaneous anaphylaxis (PCA) was adopted to prepare the model of urticaria in the rats of the rest three groups. In the EA group, EA was applied to bilateral "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36), with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity, once daily, for 20 min each time, consecutively for 7 days. In the positive medication group, loratadine (1 mg•kg -1 •d -1 ) was for intragastric administration, once daily, consecutively for 7 days. The samples were collected for index detection 30 min after PCA antigen challenge in the rats of each group. Spectrophotometer was adopted to determine the effusion quantity of Evans blue in the allergized site of skin. HE staining was used to observe the morphological changes in the allergized site of skin. Toluidine blue staining was provided to observe mast cell degranulation in subcutaneous loose connective tissue in the allergized site of skin. Immunohistochemistry was applied to determine the protein expressions of Lyn and Syk during degranulation of mast cells., Results: In the rats of the odel group, the eipdermis of allergized site was thickening, cells were disorganized in hierarchy and inflammatory cells were infiltrated largely in the dermis. In the positive medication group and the EA group, the epidermis was getting thin, cell arrangement was clear and the inflammatory cell infiltration was obviously alleviated as compared with the model group. Compared with the blank group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all increased in the model group ( P <0.01). Compared with the model group, the OD value of skin dye effusion quantity, the degranulation rate of mast cells and the positive expressions of Lyn and Syk were all reduced in the EA group and the positive medication group ( P <0.01). Compared with the positive medication group, the degranulation rate of mast cells was increased significantly in the EA group ( P <0.01)., Conclusion: Electroacupuncture at "Quchi" (LI 11), "Xuehai" (SP 10) and "Zusanli" (ST 36) reduces vascular permeability and gives play to the role of anti-allergy by the way of regulating and controlling the degranulation of mast cells in the rats with urticaria and the effect mechanism of electroacupuncture may be related to the inhibition of protein expressions of Lyn and Syk in mast cells.

Background: Drug resistance is a major cause of therapeutic failure that is often associated with elevated autophagy and apurinic/apyrimidinic endonuclease 1 (APE1) expression. Herein, we investigated the role of APE1 and autophagy in A549 cells treated with cisplatin., Methods: SILAC proteomics was applied to obtain a panoramic view of cisplatin treatment in KRAS G12S -mutant A549 cells. Quantity analysis of cellular apoptosis and autophagy was based on flow cytometry. Western blotting was used to examine the expression levels of apoptosis- and autophagy-related proteins, as well as those of APE1. Knockdown of APE1 was achieved by RNA interference. Immunoprecipitation was further employed to reveal the molecular interaction of APE1, p53, and LC3 when A549 cells were exposed to cisplatin., Results: SILAC proteomics revealed that 72 canonical pathways, including base excision repair (BER) and autophagy signalling pathways, were regulated after cisplatin treatment in A549 cells. Cisplatin markedly induced autophagy and apoptosis in A549 cells, accompanied by remarkable APE1 increase. Suppression of autophagy enhanced the inhibition effect of cisplatin on cell growth, proliferation, and colony formation; however, APE1 inhibition enhanced the expression of LC3-I/II, suggesting that APE1 and autophagy are compensatory for cell survival to evade the anticancer action of cisplatin. Immunoprecipitation results revealed the triple complex of APE1-p53-LC3 in response to cisplatin plus CQ in A549 cells. Dual inhibition of APE1 and autophagy significantly enhanced cisplatin-induced apoptosis, which eventually overcame drug resistance in cisplatin-resistant A549 cells., Conclusions: Dual inhibition of APE1 and autophagy greatly enhances apoptosis in parental KRAS G12S -mutant A549 cells and cisplatin-resistant A549 cells via regulation of APE1-p53-LC3 complex assembly, providing therapeutic vulnerability to overcome cisplatin resistance in the context of KRAS G12S -mutant lung cancer.

Cisplatin is one of the most widely used anticancer agents for patients with tongue squamous cell carcinoma (TSCC), but its efficacy is limited by chemoresistance. Accumulated evidence has demonstrated that reactive oxygen species (ROS) plays a critical role in multiple tumor chemotherapy resistance. In the present study, we aimed to investigate the role of ROS in cisplatin resistance of TSCC and explore its underlying molecular mechanism in vitro. Our results showed that pre-treatment with ROS scavenger N-acetylcysteine reduced cisplatin-induced cytotoxicity, autophagy, and apoptosis in TSCC cells. Down-regulation of intracellular ROS attenuated apoptosis and autophagy of TSCC cisplatin-resistant CAL27/CDDP cells by reversing the inhibition of p38MAPK/mTOR pathway. Taken together, these findings suggest that down-regulation of intracellular ROS reduces the cytotoxicity of cisplatin by inhibiting apoptosis and autophagy in TSCC cells involving p38MAPK/mTOR mediated pathway. Low intracellular ROS levels may be one of the main mechanisms of cisplatin resistance in TSCC., Competing Interests: Declaration of Competing Interest All authors declared no conflicts of financial interest to this study., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: To observe the effect of electroacupuncture (EA) on degranulation of intraperitoneal mast cells (MCs) and expression of mitogen-activated protein kinase (MAPK) signaling related proteins, tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) in urticaria rats, so as to reveal its mechanisms underlying improvement of urticaria., Methods: Thirty-two SD rats were randomly divided into control，model，EA and medication groups ( n ＝8 in each group). The urticaria model was established by using passive cutaneous anaphylaxis (PCA) reaction method. EA (2 Hz /15 Hz, 1 mA) was applied to bilateral "Zusanli"(ST36), "Quchi "(LI11) and "Xuehai"(SP10) for 20 min，once daily for 7 consecutive days before antigen attack. Rats of the medication group were treated by gavage of Loratadine(1 mg•kg －1 •d －1 )for 7 days. The diameter of cutaneous Evan's blue spots was measured to evaluate the severity of PCA. Intraperitoneal fluid smears were prepared to observe the degranulation state of MCs. The contents of TNF-α and IL-6 in the intraperitoneal fluid were detected by ELISA, and the expression of extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, c-Jun N-terminal kinase (JNK), p-JNK, P38MAPK and p-P38MAPK of the acquired intraperitoneal MCs was detected by Western blot., Results: The diameter of cutaneous Evan's blue spot was significantly increased in the model group than that in the control group ( P <0.01), and considerably decreased in both EA and medication groups compared with the model group( P <0.01). After modeling，the percentage of degranulated MCs, contents of TNF-α and IL-6, and expression levels of ERK, p-ERK, JNK, p-JNK, P38MAPK and p-P38MAPK were remarkably increased in the mo-del group than those in the control group ( P <0.01, P <0.05). After the treatment, the percentage of degranulated MCs, contents of TNF-α and IL-6, and expression levels of p-ERK, JNK, p-JNK and p-P38MAPK were obviously decreased in both EA and medication groups relevant to the model group ( P <0.01, P <0.05), while no significant changes were found in the expression of ERK in both EA and medication groups, and P38MAPK in the EA group. Compared with the model and EA groups, expression levels of P38MAPK were down-regulated in the medication group ( P <0.05)., Conclusion: EA can reduce skin allergic reaction in rats with urticaria, which may be related to its effects in inhibiting the degranulation of intraperitoneal MCs, down-regulating the expression of MAPK signaling-related proteins and the level of pro-inflammatory factors TNF-α and IL-6 in intraperitoneal MCs.

Cisplatin is widely used in the treatment of tongue squamous cell carcinoma (TSCC), but its clinical efficacy is limited by drug resistance and toxic side effects. Hence, a novel compound capable of enhancing the anticancer effect of cisplatin while reducing the side effects is urgently needed. We have previously shown that plumbagin (PLB), an anticancer phytochemical, is able to inhibit the growth of TSCC in vitro and in vivo . The objective of this study was to investigate the effect of PLB in reversing the resistance of TSCC to cisplatin as well as its molecular mechanisms. Here, we found that PLB enhances cisplatin-induced cytotoxicity, apoptosis, and autophagy in CAL27 and cisplatin-resistant CAL27/CDDP cells. PLB could inhibit the viability and growth of TSCC cells by increasing the production of intracellular reactive oxygen species (ROS). In addition, the combination treatment of PLB and cisplatin resulted in a synergistic inhibition of TSCC viability, apoptosis, and autophagy by increasing intracellular ROS, which may be achieved by activating JNK and inhibiting AKT/mTOR signaling pathways. Finally, the synergistic treatment was also demonstrated in vivo . Therefore, PLB combined with cisplatin is a potential therapeutic strategy against therapy TSCC cisplatin resistance., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Danfeng Xue et al.)

Background/purpose: People with different age distributions and extrication times might have distinct injury characteristics and outcomes in earthquakes., Methods: A retrospective study was conducted to analyze the casualties in the 2016 Taiwan earthquake using data from the incident registry system and the field disaster operation system. The study subjects were assigned to 4 groups by age: preschool (<5 years), school (5-17 years), adult (18-64 years), and elderly (>64 years). Classification and regression tree analysis and receiver-operating characteristic curves were utilized to examine several factors, including extrication time, age group, floor height, and structural damage, for earthquake-related mortality. A two-sided p value less than 0.05 was considered statistically significant., Results: A total of 238 enrollees were assigned to the preschool (n = 18, 7.6%), school (n = 45, 18.9%), adult (n = 169, 71.0%), or elderly (n = 6, 2.5%) groups. Among the parameters, the extrication time exhibited the strongest association with mortality. Regarding the association between the extrication time and mortality hazard in multivariate models, we found significant odds ratios (ORs) at the extrication time cutoffs of 12, 24 and 72 h (OR = 42.61, 95% confidence interval [CI]: 13.92-130.37; OR = 37.58, 95% CI: 14.77-95.60; OR = 95.16, 95% CI: 23.02-393.48, respectively, all p < 0.001). The optimal extrication time cutoff for mortality was 12 h in the preschool group and 24 h in the school and adult groups., Conclusion: Extrication time is strongly associated with earthquake-related mortality. These findings may facilitate strategic approaches for patients entrapped in damaged buildings and can contribute to future training for field search and rescues after earthquakes., (Copyright © 2019 Formosan Medical Association. Published by Elsevier B.V. All rights reserved.)

Objective: To observe the effect of deep electroacupuncture (EA) stimulation at "Huantiao"(GB30) on hindlimb motor function and expression of p38 mitogen-activated protein kinase (p38 MAPK ) and p53 proteins in dorsal root ganglia (DRG) in rats with chronic constrictive injury (CCI) of sciatic nerve., Methods: Forty-eight SD rats (half male and half female) were randomly divided into control, model, shallow EA (SEA) stimulation and deep EA (DEA) stimulation groups ( n =12 in each group). The CCI model was constructed by implanting a silicone tube close to the sciatic nerve of the left hind limb. For DEA group and SEA group, filiform acupuncture needles were inserted into GB30 about 12-14 mm deep and 5-8 mm deep (monitored by using a high-frequency ultrasound device), respectively, followed by electrical stimulation (2 Hz/100 Hz, 1 mA) using an EA stimulator. The intervention was conducted for 15 min every time, once daily for 14 days. The sciatic nerve function index (SFI) calculated to assess the motor function status. Histopathological changes of the sciatic nerve were displayed by H.E. staining. The expression levels of phosphorylated-p38 MAPK (p-p38) and phosphorylated-tumor protein p53 (p-p53) in DRGs of L4-L5 on the affected side were observed by immunohistochemical staining., Results: Following modeling, the SFI were significantly decreased ( P <0.01), and the expression levels of p-p38 and p-p53 proteins of L4-L5 DRGs were considerably increased in the model group ( P <0.05). After the intervention, the SFI were obviously increased, and the expression levels of p-p38 and p-p53 proteins notably down-regulated in both DEA and SEA groups relevant to the model group ( P <0.01, P <0.05). The therapeutic effect of DEA was significantly superior to that of SEA in raising SFI and down-regulating expression le-vels of p-p38 and p-p53 proteins ( P <0.01, P <0.05). H.E. staining showed disordered arrangement of the sciatic nerve fibers and myelin, disaggregation of the myelin and axons with deformity and vacuolation in some of them and with an increase of Schwann cells in the model group, which was relatively milder in both DEA and SEA groups., Conclusion: Both DEA and SEA at GB30 can obviously improve the motor function in CCI rats, which may be associated with its function in down-regulating the expression of p-p38 and p-p53 proteins in L4-L5 DRGs, restraining p38 MAPK signaling. The therapeutic effect of DEA is evidently better than that of SEA.

Lymphoplasmacytic lymphoma (LPL) is a marrow-based lymphoma, rarely involving extramedullary sites, particularly the pleural cavities. The distinction of lymphomatous pleural effusion (PE) in LPL patients from benign effusion is challenging. We conducted this study to examine whether MYD88 L265P mutation analysis is useful in distinguishing benign from lymphomatous PE in four patients with LPL, in which the initial marrow specimens were all positive for MYD88 mutation. In one case each with plasma cell- or lymphocyte-predominant PE, MYD88 mutation was positive, confirming lymphomatous effusion. The other lymphocyte-predominant PE was negative for MYD88 mutation, but was clonally related to a previous nodal biopsy and this PE was also considered to have LPL involvement. The fourth case developed large B-cell lymphoma in the PE 30 months later. The PE specimen was negative for MYD88 mutation but was clonally related to the diagnostic marrow tissue, indicating large cell transformation. Four cases of small lymphocyte-predominant benign PE from patients without history of lymphoma were examined and were all negative for MYD88 L265P mutation. In conclusion, in this small case series we showed that MYD88 L265P mutation analysis could serve as a useful adjunct in distinguishing benign from lymphomatous PE in patients with LPL., (© 2019 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Objective: To observe the effect of different frequencies (2 Hz, 100 Hz) of electroacupuncture (EA) on limb locomotion and the expression of inflammatory factors IL-1β, IL-6, TNF-α in sciatic nerve, and nuclear factor kappa B (NF-κB) in lumber(L)4-L5of spinal cord in rats with sciatic nerve injury (SNI), so as to reveal its mechanisms underlying improvement of SNI., Methods: A total of 48 SD rats (half male and half female) were equally divided into blank control, model, low frequency (2 Hz) EA and high frequency (100 Hz) EA groups. The SNI model was established by clamping the spinal nerve. EA intervention (2 Hz, 100 Hz, 1 mA), starting on the 8 th day after modeling, was applied to "Huantiao" (GB30) on the injured side for 15 min, once daily for 14 consecutive days. The sciatic function index (SFI) was calculated to assess the injured hindlimb recovery with reference to BAIN's and colleagues' methods. Histopathological changes of the sciatic nerve were displayed by H.E. staining. The expressions of IL-1β, IL-6 and TNF-α in the sciatic nerve tissue were detected by immunohistochemistry, and the expression of NF-κB in the spinal cord was detected by using Western blot., Results: After modeling, the SFI level on day 8 was significantly decreased in the model group ( P <0.01), and no significant differences were found among the model, low frequency EA and high frequency EA groups before the EA intervention ( P >0.05). Following the treatment (at the 22 nd day), the SFI values of both low frequency EA and high frequency EA groups were significantly increased ( P <0.01), suggesting an improvement of the limb motor function, and the SFI of the low frequency EA group was notably higher than that of the high frequency EA group ( P <0.01). In comparison with the blank control group, the expression levels of IL-1β, IL-6, TNF-α in the sciatic nerve and NF-κB protein in the spinal cord were significantly up-regulated ( P <0.05). Following EA intervention, the increased expression levels of IL-1β, IL-6, TNF-α and NF-κB proteins were significantly down-regulated in both low frequency EA and high frequency EA groups ( P <0.05), and the therapeutic effect of low frequency EA was markedly superior to that of high frequency EA in down-regulating the expression levels of IL-1β, IL-6, TNF-α and NF-κB protein ( P <0.05). H.E. staining showed increase of Schwann cells in number, cellular swelling, and disintegration of the axons and myelin sheath, and appearance of vacuolar degeneration in the model group, which was relatively milder in both low frequency EA and high frequency EA groups, particularly in the low frequency EA group., Conclusion: EA of GB30 at 2 Hz and 100 Hz can promote the recovery of hindlimb motor function in SNI rats, which is probably related to its function in inhibiting the inflammatory response, and facilitating the repair of the damaged sciatic nerve. 2 Hz EA is better than 100 Hz EA in the therapeutic effect.

Background/purpose: To explore the association of patient injury patterns and entrapped locations inside damaged buildings in the 2016 Taiwan earthquake., Methods: A retrospective analysis was conducted using the Tainan incident registry system. Residents inside nine conjunctive, 16-story (49.3 m in height) reinforced concrete buildings were categorized as non-injured, injured, and dead. Residents were classified into different groups according to their entrapped locations in height and the severity of building damage. The field triage acuity and trauma severity among groups were compared. Statistical significance was set at the level of 0.05., Results: There were 309 enrollees with 76 (24.6%) non-injured, 118 (38.2%) injured, and 115 (37.2%) dead. Residents either in the high floors (odds ratio [OR] = 2.9, 95% CI: 1.5-5.8, p = 0.003) or in the collapsed buildings (OR = 18.2, 95% CI: 7.6-43.6, p < 0.001) were more likely to be dead. Injured patients who were located in the high floors were more likely to have severe field triage acuities (adjusted OR = 14.7, 95% CI: 1.8-118.0, p = 0.012); intracranial hemorrhage (12.5%), intrathoracic injury (18.8%), or intra-abdominal damage (12.5%) (All p < 0.05); the need for emergency surgical intervention (31.3%, p = 0.035); and major trauma (18.8%, p = 0.001). Residents in the collapsed buildings were more likely to have a crush injury (80.0%, p < 0.001) or crush syndrome (80.0%, p < 0.001)., Conclusion: People entrapped at different heights of floors or in differently damaged buildings could have a distinct pattern of injury. Our findings may facilitate strategic approaches of patients entrapped in damaged buildings and may contribute to future training for field searches and rescues after earthquakes., (Copyright © 2018. Published by Elsevier B.V.)

Endometrial carcinoma is a cancer derived from oncogenesis of the regenerating uterine cavity, in which cytokine stimulation shapes cell differentiation and tissue remodeling. Expression of the stem cell factors SOX2, OCT4, NANOG, and MYC has been linked to tumor malignancy in several cancers. However, how these stem cell factors crosstalk with cytokine signaling to promote malignancy in endometrial carcinoma is still elusive. Here we report that the expression of SOX2 and MYC, but not that of OCT4 and NANOG, correlate with poor histological differentiation and prognosis, while SOX2 expression is negatively associated with MYC level. We found that SOX2-high endometrial carcinoma cells possessed a higher colony-forming ability than their SOX2-low counterparts, and knockdown of SOX2 attenuated the colony-forming ability. We observed that SOX2 regulated EGFR expression in a SOX2-EGFR positive feedback loop. EGF stimulation induced SOX2 expression and promoted migration of endometrial carcinoma cells, whereas TGF-β stimulation inhibited SOX2 expression and attenuated the colony-forming ability. Immunohistochemistry analysis revealed that SOX2 expression correlated with lymph node infiltration of endometrial carcinoma. Our findings support that cytokine-induced stem cell factor SOX2 possesses oncogenic properties, with the potential to serve as a prognostic biomarker in endometrial carcinoma.

Background: Primary cutaneous peripheral T-cell lymphomas (PC-PTCLs) are classified into mycosis fungoides (MF) and other rare specific types; and those do not fit into any specific entities are designated as PTCL, not otherwise specified (NOS), an aggressive neoplasm. Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is an aggressive primary intestinal T-cell lymphoma with enteropathy in the non-neoplastic mucosa. We report a rare case of PC-PTCL-NOS with a late relapse solely in the ileum after complete remission. We discuss the importance of evaluating enteropathy, megakaryocyte-associated tyrosine kinase (MATK) immunostaining, and the implication of clonal relationship of metachronous lymphomas., Case Report: We reviewed the histopathology and immunohistochemistry of the skin tumor from a 68-year-old female and the relapsed intestinal T-cell lymphoma. The tumor cells "trans-regressed" from large and pleomorphic in the skin to small/medium-sized cells with clear cytoplasm in the ileum; and furthermore, there was immunophenotypic alteration. However, there was no enteropathy in the non-tumoral ileal mucosa adjacent to the tumor proper and both the cutaneous and ileal tumors were negative for MATK. Clonality study showed clonal TRG and TRB rearrangement with identical band sizes of the amplicons, confirming primary cutaneous tumor with a late relapse in the ileum., Conclusions: Although PC-PTCL-NOS is an aggressive neoplasm, rare cases such as this might have a long-term survival. Furthermore, the late relapse mimicking MEITL is intriguing and exceptional, in spite the fact that MEITL is a primary intestinal T-cell lymphoma with a typical histopathology and immunophenotype. Detailed clinicopathological and molecular studies are mandatory to elucidate the clonal relationship of metachronous lymphomas, as this has important clinical implication for treatment. Evaluation of the non-tumoral intestinal mucosa for enteropathy and immunostaining for MATK might help to differentiate a mimicker from a true MEITL., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Objectives: High respiratory hazards among search and rescue workers (SRWs) emerged after the World Trade Center attacks on 11 September 2001. There have been limited studies on respiratory symptoms among earthquake SRWs. We investigated the respiratory symptoms and the use of respiratory protective equipment among the SRWs who responded to the 2016 Taiwan earthquake., Methods: On 6 February 2016, a 6.4-magnitude earthquake struck southern Taiwan and caused 513 injuries and 117 deaths. During the 9-day field operation, 519 firefighters affiliated with the Tainan City Government Fire Bureau participated in the search and rescue response. A standardised, self-completed questionnaire was used to collect data on demographics, dust exposures, personal protective measures and health outcomes 3 weeks after the earthquake. Descriptive and multivariate analyses adjusting for demographics and exposure variables were performed for new or worsened outcomes., Results: Of the 519 SRWs, 414 (80%) responded to the questionnaire. Of these SRWs, 153 (37%) reported new or worsened respiratory symptoms, with cough (23%) as the leading symptom, followed by rhinorrhoea or nasal congestion (22%) and chest tightness (6%). More than 90% of the symptoms persisted to the third week after the earthquake. The prevalence of new or worsened respiratory symptoms was significantly higher among SRWs with a higher level of exposure to dust. Prior training in response to respiratory pollutants was only 5%., Conclusions: There were significant respiratory hazards among earthquake SRWs. The persistent symptoms and low coverage of training warrant further regular examination and occupational health programmes., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. No commercial re-use. See rights and permissions. Published by BMJ.)

Novel biomarkers for pancreatic adenocarcinoma are urgently needed because of its poor prognosis. Here, by using The Cancer Genome Atlas (TCGA) RNA-seq data, we evaluated the prognostic values of the differentially expressed miRNAs and constructed a five-miRNA signature that could effectively predict patient overall survival (OS). The Kaplan-Meier overall survival curves of two groups based on the five miRNAs were notably different, showing overall survival in 10.2% and 47.8% at five years for patients in high-risk and low-risk groups, respectively. The ROC curve analysis achieved AUC of 0.775, showing good sensitivity and specificity of the five-miRNA signature model in predicting pancreatic adenocarcinoma patient survival risk. The functional enrichment analysis suggested that the target genes of the miRNA signature may be involved in various pathways related to cancer, including PI3K-Akt, TGF-β, and pluripotent stem cell signaling pathways. Finally, we analyzed expression of the five specific miRNAs in the miRNA signature, and validated the reliability of the results in 20 newly diagnosed pancreatic adenocarcinoma patients using qRT-PCR. The expression results of qRT-PCR were consistent with the TCGA results. Taken together, these findings suggested that the five-miRNA signature (hsa-miR-203, hsa-miR-424, hsa-miR-1266 hsa-miR-1293, and hsa-miR-4772) could be used as a prognostic marker for pancreatic adenocarcinoma.