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Chia-Yih Wang,1,2,* Shih-Wei Tsai,3,* Han-Hsiang Chien,1 Ting-Yu Chen,2,4 Shi-Yuan Sheu,5,6 Edmund Cheung So,7,8 Bu-Miin Huang1,9 1Department of Cell Biology and Anatomy, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 2Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 3Department of Obstetrics and Gynecology, An Nan Hospital, China Medical University, Tainan, Taiwan; 4Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 5School of Chinese Medicine for Post-Baccalaureate, I-Shou University, Kaohsiung, Taiwan; 6Department of Chinese Medicine, E-Da Cancer Hospital, Kaohsiung, Taiwan; 7Department of Anesthesia & Medical Research, An Nan Hospital, China Medical University, Tainan, Taiwan; 8Graduate Institute of Medical Sciences, Chang Jung Christian University Tainan, Tainan, Taiwan; 9Institute of Medical Research, China Medical University, Taichung, Taiwan*These authors contributed equally to this workCorrespondence: Edmund Cheung So; Bu-Miin Huang Tel +886-6-3553111 ext.1517; +886-6-2353535 ext. 5337Fax +886-6-3553111; +886-6-2093007Email edmundsotw@gmail.com; bumiin@mail.ncku.edu.twIntroduction: Human gestational choriocarcinoma, a type of gestational trophoblastic disease, occurs after miscarriage, abortion, ectopic pregnancy, or molar pregnancy. Despite recent advances in the mechanism of anticancer drugs that induce human gestational choriocarcinoma apoptosis or block its growth, new therapeutic approaches are needed to be established. Cordycepin is an active anti-cancer component extracted from Cordyceps sinensis. It prevents cell proliferation both in vitro and in vivo.Materials and Methods: Here, we examined cell growth by counting cell numbers, and performing a flow cytometry assay and EdU incorporation assay. Centrosome and cytoskeleton-related structures were observed by immunofluorescence assay. The DNA damage-related signaling was examined by Western blot assay.Results: Here, we showed that cordycepin inhibited human gestational choriocarcinoma cell proliferation and induced cell death. In addition, treatment with cordycepin activated DNA-PK and ERK, thus inducing centrosome amplification and aberrant mitosis. These amplified centrosomes also disrupted microtubule arrays and actin networks, thus leading to defective cell adhesion. Furthermore, cordycepin induced autophagy for triggering cell death.Conclusion: Thus, our study demonstrates that cordycepin inhibits cell proliferation and disrupts the cytoskeleton by triggering centrosome amplification.Keywords: human gestational choriocarcinoma, cordycepin, centrosome, cell growth, microtubule

Exostosis (also known as osteochondroma) is the most common benign bony lump of young people, usually arising at the distal femur. Vascular complications associated with exostoses are rare and include true aneurysm or pseudoaneurysm formation, deep vein thrombosis, arteriovenous fistula, and arterial insufficiency of the limbs. Few case reports describe pseudoaneurysms resulting from exostoses in mature adults, and no consensus has been reached regarding the optimal therapy. We report the case of a 51-year-old male patient complaining of persistent right thigh pain with a pulsatile mass and right calf swelling, without a history of trauma or hereditary multiple exostoses. The diagnosis was confirmed by computed tomography angiography, which showed a pseudoaneurysm of the popliteal artery resulting from an exostosis on the lateral aspect of the distal femur. A Doppler ultrasound examination confirmed popliteal vein thrombosis caused by the compression of the pseudoaneurysm. Surgical treatment consisted of removing the exostosis, excision of the pseudoaneurysm, and an end-to-end anastomosis. The deep vein thrombosis was treated with rivaroxaban for 3 months. The patient was discharged after 6 days and followed up for 6 months with satisfactory results.

ABSTRACT: Takayasu arteritis (TA) is a common autoimmune disease in the clinical setting. However, vertebral artery aneurysms caused by TA are rarely reported. We herein describe a 28-year-old man with multiple vertebral artery aneurysms and carotid artery aneurysms caused by TA, which showed typical wall thickening and lumen dilation with a “string of beads” appearance by Doppler ultrasound and radiology. Previous studies have shown that most TA-associated vertebral artery lesions are stenosis, occlusion, and dissection of the intracranial part of the artery. In this case, TA mainly affected the cervical segment of the vertebral artery (the intracranial segment was not obviously involved), and the main manifestations were aneurysms and occlusion. This case provides more information for further understanding of TA-associated vertebral artery lesions.

Objectives This study aimed to investigate the differentiation state and clinical significance of colorectal cancer cells, as well as to predict the immune response and prognosis of patients based on differentiation-related genes of colorectal cancer. Introduction Colorectal cancer cells exhibit different differentiation states under the influence of the tumor microenvironment, which determines the cell fates. Methods We combined single-cell sequencing (scRNA-seq) data from The Cancer Genome Atlas source with extensive transcriptome data from the Gene Expression Omnibus database. We obtained colorectal cancer differentiation-related genes using cell trajectory analysis and developed a colorectal cancer differentiation-related gene based molecular typing and prognostic model to predict the immune response and prognosis of patients with colorectal cancer. Results We identified 5 distinct cell differentiation subsets and 620 colorectal cancer differentiation-related genes. Colorectal cancer differentiation-related genes were significantly associated with metabolism, angiogenesis, and immunity. We separated patients into 3 subtypes based on colorectal cancer differentiation-related gene expression in the tumor and found differences among the different subtypes in immune infiltration status, immune checkpoint gene expression, clinicopathological features, and overall survival. Immunotherapeutic interventions involving a highly expressed immune checkpoint blockade may be selectively effective in the corresponding cancer subtypes. We built a risk score prediction model (5-year AUC: .729) consisting of the 4 most important predictors of survival (TIMP1, MMP1, LGALS4, and ITLN1). Finally, we generated and validated a nomogram consisting of the risk score and clinicopathological variables. Conclusion This study highlights the significance of genes involved in cell differentiation for clinical prognosis and immunotherapy in patients and provides prospective therapeutic targets for colorectal cancer.

Background/aim: Fibroblast growth factor 9 (FGF9) is a member of the human FGF family known for its pivotal roles in various biological processes, such as cell proliferation, tissue repair, and male sex determination including testis formation. Cordycepin, a bioactive compound found in Cordyceps sinensis, exhibits potent antitumor effects by triggering apoptosis and/or autophagy pathways. Our research has unveiled that FGF9 promotes proliferation and tumorigenesis in MA-10 mouse Leydig tumor cells, as the phenomena are effectively countered by cordycepin through apoptosis induction. Moreover, we have observed FGF9-mediated stimulation of proliferation and tumorigenesis in TM3 mouse Leydig progenitor cells, prompting an investigation into the potential inhibitory effect of cordycepin on TM3 cell proliferation under FGF9 treatment. Hence, we hypothesized that cordycepin induces cell death via apoptosis and/or autophagy in FGF9-treated TM3 cells., Materials and Methods: TM3 cells were treated with cordycepin and/or FGF9, and the flow cytometry, immunofluorescent plus western blotting assays were used to determine how cordycepin regulated Leydig cell death under FGF9 treatment., Results: Our findings reveal that cordycepin restricts cell viability and colony formation while inducing morphological alterations associated with cell death in FGF9-treated TM3 cells. Surprisingly, cordycepin fails to elicit the expression of key apoptotic markers, suggesting an alternate mechanism of action. Although the expression of certain autophagy-related proteins remains unaltered, a significant up-regulation of LC3-II, indicative of autophagy, is observed in cordycepin-treated TM3 cells under FGF9 influence. Moreover, the inhibition of autophagy by chloroquine reverses cordycepin-induced TM3 cell death, highlighting the crucial role of autophagy in this process., Conclusion: Our study demonstrates that cordycepin activates autophagy to induce cell death in TM3 cells under FGF9 treatment conditions., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

A special micro LED whose light emitting area is laid out in a U-like shape is fabricated and integrated with colloidal quantum dots (CQDs). An inkjet-type machine directly dispenses the CQD layer to the central courtyard-like area of this U-shape micro LED. The blue photons emitted by the U-shape mesa with InGaN/GaN quantum wells can excite the CQDs at the central courtyard area and be converted into green or red ones. The U-shape micro LEDs are coated with Al 2 O 3 by an atomic layer deposition system and exhibit moderate external quantum efficiency (6.51% max.) and high surface recombination because of their long peripheries. Low-temperature measurement also confirms the recovery of the external quantum efficiency due to lower non-radiative recombination from the exposed surfaces. The color conversion efficiency brought by the CQD layer can be as high as 33.90%. A further continuous CQD aging test, which was evaluated by the strength of the CQD emission, under current densities of 100 A/cm 2 and 200 A/cm 2 injected into the micro LED, showed a lifetime extension of the unprotected CQD emission up to 1321 min in the U-shape device compared to a 39 min lifetime in the traditional case, where the same CQD layer was placed on the top surface of a squared LED.

XAV-939(XAV) is a chemical compound that inhibits the activity of tankyrase. However, the precise way in which XAV alters membrane ionic currents is not well understood. In this study,our goal was to examine the impact of XAV on the ionic currents in mouse MA-10 Leydig cells, specifically focusing on the magnitude, gating properties,and voltage-dependent hysteresis of erg-mediated K + currents(I K(erg) ). In our whole-cell current recordings we observed that the addition of XAV inhibited the density of I K(erg) in a concentration-dependent manner with an IC 50 of 3.1 μM. Furthermore we found that continued exposure to XAV, further addition of neither liraglutide nor insulin-like growth factor-1 counteracted XAV-mediated inhibition of I K(erg) . Additionally the presence of XAV suppressed the mean current versus voltage relationship of I K(erg) across the entire voltage-clamp step analyzed. This compound shifted the steady-state activation curve of I K(erg) to a less negative potential by approximately 12 mV. The presence of XAV increased the time constant of deactivating I K(erg) in MA-10 cells. The voltage-dependent clockwise hysteresis of I K(erg) responding to prolonged upright isosceles-triangular ramp voltage became diminished by adding XAV; moreover subsequent addition of NS3623 effectively reversed XAV-induced decrease of hysteretic area of I K(erg) . XAV also inhibited the proliferation of this cell line and the IC 50 value of XAV-induced inhibition of cell proliferation was 2.8M. Overall the suppression of I K(erg) by XAV may serve as a significant ionic mechanism that contribute to the functional properties of MA-10 cells. However, it is important to note that this effect cannot be attributed solely to the inhibition of tankyrase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Human adipose-derived stem cells (ASCs) have shown immense potential for regenerative medicine. Our previous work demonstrated that chitosan nano-deposited surfaces induce spheroid formation and differentiation of ASCs for treating sciatic nerve injuries. However, the underlying cell fate and differentiation mechanisms of ASC-derived spheroids remain unknown. Here, we investigate the epigenetic regulation and signaling coordination of these therapeutic spheroids. During spheroid formation, we observed significant increases in histone 3 trimethylation at lysine 4 (H3K4me3), lysine 9 (H3K9me3), and lysine 27 (H3K27me3), accompanied by increased histone deacetylase (HDAC) activities and decreased histone acetyltransferase activities. Additionally, HDAC5 translocated from the cytoplasm to the nucleus, along with increased nuclear HDAC5 activities. Utilizing single-cell RNA sequencing (scRNA-seq), we analyzed the chitosan-induced ASC spheroids and discovered distinct cluster subpopulations, cell fate trajectories, differentiation traits, and signaling networks using the 10x Genomics platform, R studio/language, and the Ingenuity Pathway Analysis (IPA) tool. Specific subpopulations were identified within the spheroids that corresponded to a transient reprogramming state (Cluster 6) and the endpoint cell state (Cluster 3). H3K4me3 and H3K9me3 were discovered as key epigenetic regulators by IPA to initiate stem cell differentiation in Cluster 6 cells, and confirmed by qPCR and their respective histone methyltransferase inhibitors: SNDX-5613 (a KMT2A inhibitor for H3K4me3) and SUVi (an SUV39H1 inhibitor for H3K9me3). Moreover, H3K9me3 and HDAC5 were involved in regulating downstream signaling and neuronal markers during differentiation in Cluster 3 cells. These findings emphasize the critical role of epigenetic regulation, particularly H3K4me3, H3K9me3, and HDAC5, in shaping stem cell fate and directing lineage-specific differentiation., (© The Author(s) 2023. Published by Oxford University Press.)

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID 50 ) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Background: Since most patients with oral cancer do not benefit from current treatments, new therapeutic strategies or drugs must be developed to improve patient prognosis. Qing Yan Li Ge Tang (QYLGT), a Chinese herbal medicine, is known for its anticancer activity. This study aimed to investigate whether QYLGT has anticancer effects on human OEC-M1 oral cancer cells., Methods: To evaluate whether QYLGT affects viability, morphology, and colony formation ability of the OEC-M1 cells, the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, morphology study, and colony formation assay were performed, respectively. Each assay was carried out in triplicate, and the whole set of experiments was performed three times independently. To investigate whether QYLGT induces apoptotic effects in OEC-M1 cells, the enzyme-linked immunosorbent (ELISA) was carried out to quantify cytokeratin 18 fragment (an apoptosis marker). Each assay was carried out in triplicate, and the whole set of experiments was performed three times independently. The immunoblotting assay was performed to detect the protein expression after QYLGT treatment. The whole set of experiments was performed two times independently., Results: The results from the MTT and colony formation assays indicate that QYLGT inhibited the cell viability and clonogenic growth capacity of OEC-M1 cells. The morphology study shows that QYLGT increased plasma membrane blebbing in OEC-M1 clles. The results of ELISA and an immunoblotting assay show that QYLGT increased cytokeratin 18 fragment release and poly ADP-ribose polymerase cleavage (another apoptosis marker) in OEC-M1 cells. In addition, the results from immunoblotting assay show that QYLGT also activated apoptotic executor proteins, including caspase-8, caspase-9, and caspase-3, and the results of ELISA indicate that treatment with the pan-caspase inhibitor, Z-VAD-FMK, inhibited QYLGT-induced cytokeratin 18 fragment release. These results indicate that QYLGT inhibited cell viability in OEC-M1 cells and induced OEC-M1 apoptosis through caspase activation. Additionally, QYLGT-activated c-Jun N-terminal kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and nuclear factor-kappa B (NF-κB), and the related inhibitors, including SP600125, PD184352, SB202190, and Bay11-7082, were used to confirm which signaling was involved in QYLGT-induced apoptosis. Moreover, only Bay11-7082, the NF-κB inhibitor, could suppress QYLGT-induced the release of cytokeratin 18 fragments from OEC-M1 cells., Conclusions: QYLGT induced apoptosis in OEC-M1 cells via the NF-κB pathway.

Background/aim: Although clinical medicine has significantly progressed in treating nasopharyngeal carcinoma (NPC) in recent years, many patients still have poor prognoses due to distant metastasis. It is still relatively unclear why NPC has a highly metastatic ability. Especially whether the tumor microenvironment affects the invasion and metastasis of NPC still needs to be cleared. In this study, serum starvation was used to simulate nutrient deficiency in the tumor microenvironment to explore whether nutrient deficiency affects the malignancy of NPC cells., Materials and Methods: Semiquantitative reverse transcription-polymerase chain reaction, ELISA, immunoblotting assay, reporter gene assay, and Matrigel invasion assay were carried out., Results: Under serum starvation, NPC cells could induce the mRNA expression and protein secretion of matrix metalloproteinase 9 (MMP9). The ERK-AP1 pathway was activated under serum starvation in NPC cells, resulting in the expression of MMP9. In contrast, treatment with an MMP9 inhibitor or an MMP9 siRNA inhibited serum starvation-induced invasion., Conclusion: Serum starvation could up-regulate MMP9 expression in NPC cells, contributing to NPC invasion. Therefore, serum starvation may promote malignancy of NPC cells but also support MMP9 as a potential therapeutic target to prevent NPC cell invasion and metastasis., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Leydig cell tumor is the most frequent non-germ cell tumors of testis. The biggest challenge of using radiotherapy to treat testicular cancer is in effectively killing cancer cells and maintaining reproductive function after treatment. Our recently published article showed that cordycepin could enhance radiosensitivity to induce mouse Leydig tumor cell apoptosis by inducing cell cycle arrest, caspase pathway and endoplasmic reticulum (ER) stress. In the present study, the potency and mechanism of a previous combination treatment protocol on reactive oxygen species (ROS) induction and DNA damage were further investigated. Our results reveal that 25 μM cordycepin plus 4 Gy radiation leads to ROS accumulation accompanied by a decrease in heme oxygenase (HO)-1 protein expression in MA-10 mouse Leydig tumor cells. Subsequently, pronounced DNA damage with phosphorylated H2A histone family member X (γ-H2AX) increase and activation of DNA damage-related signaling pathways including double and single stranded break-induced ataxia telangiectasia mutated (ATM)/checkpoint kinase (Chk)2 and ataxia telangiectasia mutated and Rad3 related (ATR)/Chk1 signaling axes were identified. p53-dependent pathway was then initiated ultimately leading to cell death. Preincubated with free radical scavenger, N-acetylcysteine (NAC), down-regulated γ-H2AX expression in treated cells and partially reduced cell death, indicating that ROS overproduction is involved in combination treatment-induced DNA damage. Furthermore, the combination treatment effectively inhibited tumor growth as reflected in the reduction of tumor volume, size and weight, and high expression level of γ-H2AX in tumor tissue in vivo , suggesting that the combination treatment inhibited tumor growth via causing DNA damage in MA-10 cells. In summary, these results expound that the combination treatment of cordycepin and radiation induces MA-10 mouse Leydig tumor cell death through ROS accumulation and DNA damage. This finding can serve as a reference guideline for future clinical therapy of testicular cancer and provide potential targets for anti-cancer drug design., Competing Interests: None., (AJCR Copyright © 2023.)

Although arsenic is an environmental toxicant, arsenic trioxide (ATO) is used to treat acute promyelocytic leukemia (APL) with anticancer effects. Studies have demonstrated oral cancer is in the top 10 cancers in Taiwan. High rate of oral cancers is linked to various behaviors, such as excessive alcohol consumption and tobacco use. Similarly, betel chewing is a strong risk factor in oral cancer. In the present study, oral squamous carcinoma OC3 cells were investigated with the treatments of sodium arsenite (NaAsO2) and dimethylarsenic acid (DMA), respectively, to examine if arsenic compounds have anti‑cancer efforts. It was found that 1 µM NaAsO2 and 1 mM DMA for 24 h induced rounded contours with membrane blebbing phenomena in OC3 cells, revealing cell apoptotic characteristics. In addition, NaAsO2 (10‑100 µM) and DMA (1‑100 mM) significantly decreased OC3 cell survival. In cell cycle regulation detected by flow cytometry, NaAsO2 and DMA significantly augmented percentage of subG1 and G2/M phases in OC3 cells, respectively. Annexin V/PI double staining assay was further used to confirm NaAsO2 and DMA did induce OC3 cell apoptosis. In mechanism investigation, western blotting assay was applied and the results showed that NaAsO2 and DMA significantly induced phosphorylation of JNK, ERK1/2 and p38 and then the cleavages of caspase‑8, ‑9, ‑3 and poly ADP‑ribose polymerase (PARP) in OC3 cells, dynamically. In conclusion, NaAsO2 and DMA activated MAPK pathways and then apoptotic pathways to induce OC3 oral cancer cell apoptosis.

Arsenic compounds have been applied treating acute promyelocytic 1eukemia and solid tumors with brief mechanism investigations. In fact, we have demonstrated that sodium arsenite plus dimethylarsenic acid could activate apoptosis in MA-10 mouse Leydig tumor cells by inducing caspase pathways. However, detail underlying mechanisms how caspase cascade is regulated remains elusive. Therefore, the apoptotic mechanism of sodium arsenite plus dimethylarsenic acid were examined in MA-10 cells in this study. Our results reveal that Fas/FasL protein expressions were stimulated by sodium arsenite plus dimethylarsenic acid in MA-10 cells. In addition, reactive oxygen species (ROS) generation, cytochrome C release, Bid truncation, and Bax translocation were induced in MA-10 cells by arsenic compounds. Moreover, activation of p38, JNK and ERK1/2, MAPK pathways was stimulated while Akt phosphorylated levels and Akt expression were decreased by sodium arsenite plus dimethylarsenic in MA-10 cells. In conclusion, sodium arsenite and dimethylarsenic acid did activate MAPK pathway plus ROS generation, but suppress Akt pathway, to modulate caspase pathway and then induce MA-10 cell apoptosis., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Testes control the development of male reproductive system. The testicular interstitial Leydig cells (Leydig cells) synthesize testosterone for promoting spermatogenesis and secondary sexual characteristics. Type A platelet-derived growth factor (PDGF-AA) is one of the most important growth factors in regulating Leydig cell growth and function. Knockout of PDGF-AA or its congenital receptor PDGFR-α leads to poor testicular development caused by reducing Leydig cell numbers, supporting PDGF-AA/PDGFR-α signaling regulates Leydig cell development. Primary cilium is a cellular antenna that functions as an integrative platform to transduce extracellular signaling for proper development and differentiation. Several receptors including PDGFR-α are observed on primary cilia for initiating signaling cascades in distinct cell types. Here we showed that PDGF-AA/PDGFR-α signaling promoted Leydig cells growth, migration, and invasion via primary cilia. Upon PDGF-AA treatment, AKT and ERK signaling were activated to regulate these cellular events. Interestingly, active AKT and ERK were detected around the base of primary cilia. Depletion of ciliary genes (IFT88 and CEP164) alleviated PDGF-AA-activated AKT and ERK, thus reducing Leydig cell growth, migration, and invasion. Thus, our study not only reveals the function of PDGF-AA/PDGFR-α signaling in maintaining testicular physiology but also uncovers the role of primary cilium and downstream signaling in regulating Leydig cell development., (© 2022 Wiley Periodicals LLC.)

Fibroblast growth factor 9 (FGF9) modulates cell proliferation, differentiation and motility for development and tissue repair in normal cells. Growing evidence shows that abnormal activation of FGF9 signaling is associated with tumor malignancy. We have previously reported that FGF9 increases MA-10 mouse Leydig tumor cell proliferation, in vitro , and tumor growth, in vivo . Also, FGF9 promotes the tumor growth and liver metastasis of mouse Lewis lung cancer cells, in vivo . However, the effects of FGF9 in the early stage of tumorigenesis remains elusive. In this study, TM3 mouse Leydig progenitor cells, that are not tumorigenic in immunocompromised mice, were used as a model cell line to investigate the role of FGF9 in tumorigenesis. The results demonstrated that FGF9 significantly induced cell proliferation and activated the MAPK, PI3K and PLCγ signaling pathways in TM3 cells. The percentage of the cell number in G1 phase was reduced and that in S and G2/M phases was increased after FGF9 stimulation in TM3 cells. Cyclin D1, cyclin A1, CDK2, CDK1, and p21 expressions and the phosphorylation level of Rb were all induced in FGF9-treated TM3 cells. In addition, FGF9 increased the expression of FGF receptor 1-4 in TM3 cells, suggesting the positive feedback loop between FGF9 and FGFRs. Furthermore, in the allograft mouse model, FGF9 promoted the tumorigenesis of TM3 cells characterized by higher expression of tumor markers, such as tumor necrosis factor alpha (TNFα) and α-fetoprotein (AFP), in the subcutaneously inoculated TM3 cell tissue. Conclusively, FGF9 induced cell cycle to increase cell proliferation of TM3 cells through FAK, MAPK, PI3K/Akt and PLCγ signaling pathways, in vitro , and promoted the tumorigenesis of TM3 cell allograft tissue, in vivo , which is a potential marker for tumor as well as a target for cancer therapeutic strategies., Competing Interests: None., (AJCR Copyright © 2022.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of its late diagnosis and chemoresistance. Primary cilia, the cellular antennae, are observed in most human cells to maintain development and differentiation. Primary cilia are gradually lost during the progression of pancreatic cancer and are eventually absent in PDAC. Here, we showed that cisplatin-resistant PDAC regrew primary cilia. Additionally, genetic or pharmacological disruption of primary cilia sensitized PDAC to cisplatin treatment. Mechanistically, ataxia telangiectasia mutated (ATM) and ATM and RAD3-related (ATR), tumor suppressors that initiate DNA damage responses, promoted the excessive formation of centriolar satellites (EFoCS) and autophagy activation. Disruption of EFoCS and autophagy inhibited primary ciliogenesis, sensitizing PDAC cells to cisplatin treatment. Collectively, our findings revealed an unexpected interplay among the DNA damage response, primary cilia, and chemoresistance in PDAC and deciphered the molecular mechanism by which ATM/ATR-mediated EFoCS and autophagy cooperatively regulate primary ciliogenesis., (© 2022 Wiley Periodicals LLC.)

Cordycepin, a bioactive compound extracted from Cordyceps sinensis , can induce apoptosis in human OEC-M1 oral cancer cells. However, the exact mechanism is still unclear. The present study aimed to investigate the underlying mechanism of cordycepin-induced apoptosis in OEC-M1 cells. Following treatment with cordycepin, apoptosis was examined and quantified using a DNA laddering assay and a cytokeratin 18 fragment enzyme-linked immunosorbent assay, respectively. Expressions of mitogen-activated protein kinases (MAPKs) and apoptosis-related proteins were detected by the western blot analysis. Our results show that a pan-caspase inhibitor, Z-VAD-FMK, could significantly inhibit cordycepin-induced apoptosis in OEC-M1 cells. In addition, treatment with cordycepin not only activated caspase-8, caspase-9, and caspase-3 but also induced Bid and poly ADP-ribose polymerase cleavages. Furthermore, cordycepin also induced the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase, and p38 MAPKs. Among MAPKs, activation of JNK solely contributed to cordycepin-induced apoptosis with the activation of caspase-8, caspase-9, and caspase-3 and cleavage of PARP. Taken together, the present study demonstrated that cordycepin activated JNK and caspase pathways to induce apoptosis in OEC-M1 cells., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Kuo-Lung Tung et al.)

Water yield is an important ecosystem service. It is the key to maintain ecological security and sustainable development to accurately grasp the spatial heterogeneity characteristics and identify the key driving factors of water yield in different regions. Taking Yunnan Province with significant geospatial heterogeneity as the research area, we used InVEST model to simulate the temporal and spatial variations of water yield in Yunnan Province from 1992 to 2019. The spatial characteristics of driving factors, such as climate, vegetation, soil, terrain, land use, on water yield service were analyzed through Geodetector. The results showed that water yield of Yunnan Province showed a fluctuating trend of increasing at first and decreasing later from 1992 to 2019, with a similar spatial distribution pattern in each year, and an overall trend of gradually decreasing from northwest, west and southwest to central and east. Climatic factors (precipitation and actual evapotranspiration) were the main driving factors leading to spatial differentiation of water yield. In different watershed divisions, the impact of each driving factor on water yield had significant spatial heterogeneity: the watersheds dominated by precipitation were mainly distributed in the west of Yunling-Yuanjiang line, the Irrawaddy River, the upper reaches of Nujiang River, the lower reaches of Nujiang River, the upper reaches of Lancang River, the upper reaches of Jinsha River, Yuanjiang River and Lixian River areas. As for the east of Yunling-Yuanjiang line, in karst landform areas (Nanguang River, Chishui River, Wujiang River, Youjiang River and Panlong River) and the lower reaches of Jinsha River with high population density and shortage of cultivated land, actual evapotranspiration and land use type were the leading factors controlling the spatial pattern of water yield. The results could provide a reference for water resources management policy and ecological civilization construction in Yunnan Province, with a certain guiding significance to promote the optimization and sustainable development of ecosystem services in complex plateau-mountain area.

Radiotherapy is a localized treatment commonly used in various types of cancer. However, major limitation of radiotherapy is the development of resistance of tumor cells to radiosensitivity. Cordycepin, a predominant functional component of the Cordyceps sinensis , is considered to use in treating tumor cells. In the present study, we investigated the anticancer effect of the combination of radiation and cordycepin in the treatment of Leydig tumor cells. Results showed that the combination treatment has a synergistic effect significantly suppress cell viability and enhance the radiosensitivity in MA-10 mouse Leydig tumor cells. The combination treatment induced MA-10 cell apoptosis through increasing levels of cleaved caspase-3/-8/-9, poly ADP-ribose polymerase (PARP), and cytochrome c and decreasing levels of B-cell lymphoma 2 (Bcl-2). In addition, prolonged sub-G1 and G2/M arrest accompany with cell cycle-related protein regulation was observed in cells that received the combination treatment. The endoplasmic reticulum (ER) stress-related protein expressions were regulated after MA-10 cells treating with a combination of 100 μM cordycepin and 4 Gy radiation. Furthermore, the combination treatment also decreased the Leydig tumor mass by increasing cell apoptosis in tumor-bearing mice. In conclusion, cordycepin enhances radiosensitivity to induce mouse Leydig tumor cells toward apoptosis in vitro and in vivo . This study will provide a scientific basis for the development of therapeutic regimen of testicular cancer., Competing Interests: None., (AJCR Copyright © 2022.)

Objective: To establish an intramedullary transplantation model of primary megakaryocytes to evaluate the platelet-producing capacity of megakaryocytes and explore the underlying regulatory mechanisms. Methods: Donor megakaryocytes from GFP-transgenic mice bone marrow were enriched by magnetic beads. The platelet-producing model was established by intramedullary injection to recipient mice that underwent half-lethal dose irradiation 1 week in advance. Donor-derived megakaryocytes and platelets were detected by immunofluorescence staining and flow cytometry. Results: The proportion of megakaryocytes in the enriched sample for transplantation was 40 to 50 times higher than that in conventional bone marrow. After intramedullary transplantation, donor-derived megakaryocytes successfully implanted in the medullary cavity of the recipient and produce platelets, which showed similar expression of surface markers and morphology to recipient-derived platelets. Conclusion: We successfully established an in vivo platelet-producing model of primary megakaryocytes using magnetic-bead enrichment and intramedullary injection, which objectively reflects the platelet-producing capacity of megakaryocytes in the bone marrow.

For a number of years, oral cancer has remained in the top ten most common types of cancer, with an incidence rate that is steadily increasing. In total, ~75% oral cancer cases are associated with lifestyle factors, including uncontrolled alcohol consumption, betel and tobacco chewing, and the excessive use of tobacco. Notably, betel chewing is highly associated with oral cancer in Southeast Asia. Arsenic is a key environmental toxicant; however, arsenic trioxide has been used as a medicine for the treatment of acute promyelocytic leukemia, highlighting its anticancer properties. The present study aimed to investigate the role of arsenic compounds in the treatment of cancer, using FaDu oral squamous carcinoma cells treated with sodium arsenite (NaAsO 2 ) and dimethyl arsenic acid (DMA). The results demonstrated that FaDu cells exhibited membrane blebbing phenomena and high levels of apoptosis following treatment with 10 µM NaAsO 2 and 1 mM DMA for 24 h. The results of cell viability assay demonstrated that the rate of FaDu cell survival was markedly reduced as the concentration of arsenic compounds increased from 10 to 100 µM NaAsO 2 , and 1 to 100 mM DMA. Moreover, flow cytometry was carried out to further examine the effects of arsenic compounds on FaDu cell cycle regulation; the results revealed that treatment with NaAsO 2 and DMA led to a significant increase in the percentage of FaDu cells in the sub‑G1 and G2/M phases of the cell cycle. An Annexin V/PI double staining assay was subsequently performed to verify the levels of FaDu cell apoptosis following treatment with arsenic compounds. Furthermore, the results of the western blot analyses revealed that the expression levels of caspase‑8, ‑9 and ‑3, and poly ADP‑ribose polymerase, as well the levels of phosphorylated JNK and ERK1/2 were increased following treatment with NaAsO 2 and DMA in the FaDu cells. On the whole, the results of the present study revealed that treatment with NaAsO 2 and DMA promoted the apoptosis of FaDu oral cancer cells, by activating MAPK pathways, as well as the extrinsic and intrinsic apoptotic pathways.

Clerodane diterpene, a class of bicyclic diterpenoids, is found in hundreds of plant species. 16-hydroxycleroda-3,13-dien-15,16-olide (CD) can be isolated from the plant Polyalthia longifol ia and has been applied against oral cancer and glioma by xenograft model. In this study, we aim to explore its antitumour action by examining its histone deacetylase (HDAC) activity and integrin-associated intracellular signalling pathway on T24 human bladder cancer (BC) cells. Our results revealed that CD-inhibited colony formation, HDAC activity, HDAC (1, 2 and 3) mRNA and cell spreading on fibronectin-coated surfaces in a concentration-dependent manner. Furthermore, decreased cFLIP and increased caspase-8 cleavage accompanied CD-induced cell death. At non-toxic concentrations, CD blocked the migration and invasion of T24 cells. CD hindered migration and invasion by the downregulation of fibronectin, integrin α 5 β 1, β -catenin, FAK, vinculin and Rho A, as well as by reduction of phosphorylated glycogen synthase kinase 3 β (pGSK3 β ), pSrc, pstat3 and pNF κ B. We observed that the MMP9 gene was closely linked with prognosis of patients with bladder cancer. MMP9 protein levels and activity were largely attenuated by CD in a concentration-dependent manner. In conclusion, CD-induced caspase-8-dependent apoptosis and suppressed migration and invasion by blocking several intracellular signalling pathways, including downregulation of HDAC activity and integrin-FAK and MMP9 pathways.

Despite improvements in cancer treatments resulting in higher survival rates, the proliferation and metastasis of tumors still raise new questions in cancer therapy. Therefore, new drugs and strategies are still needed. Midazolam (MDZ) is a common sedative drug acting through the γ-aminobutyric acid receptor in the central nervous system and also binds to the peripheral benzodiazepine receptor (PBR) in peripheral tissues. Previous studies have shown that MDZ inhibits cancer cell proliferation but increases cancer cell apoptosis through different mechanisms. In this study, we investigated the possible anticancer mechanisms of MDZ on different cancer cell types. MDZ inhibited transforming growth factor β (TGF-β)-induced cancer cell proliferation of both A549 and MCF-7 cells. MDZ also inhibited TGF-β-induced cell migration, invasion, epithelial-mesenchymal-transition, and Smad phosphorylation in both cancer cell lines. Inhibition of PBR by PK11195 rescued the MDZ-inhibited cell proliferation, suggesting that MDZ worked through PBR to inhibit TGF-β pathway. Furthermore, MDZ inhibited proliferation, migration, invasion and levels of mesenchymal proteins in MDA-MD-231 triple-negative breast cancer cells. Together, MDZ inhibits cancer cell proliferation both in epithelial and mesenchymal types and EMT, indicating an important role for MDZ as a candidate to treat lung and breast cancers.

Since a portion of patients with nasopharyngeal carcinoma (NPC) do not benefit much from current standard treatments, it is still needed to discover new therapeutic drugs to improve the prognosis of the patients. Considering that Chinese traditional medicine plays a role in inhibiting tumor progression, in this study, we aimed to investigate whether a Chinese herbal formula, Qing Yan Li Ge Tang (QYLGT), has the anticancer activity in NPC cells and explore the underlying mechanism as well. MTT assay, colony formation assay, immunoblotting assay, and DNA laddering assay were performed to assess cell viability, cell colony formation, protein expression, and DNA fragmentation, respectively. Results show that QYLGT was able to inhibit the cell viability and decrease colony formation ability in NPC cells. QYLGT could also increase the formation of intracellular vacuoles and induce the autophagy-related protein expressions, including Atg3, Atg6, and Atg12-Atg5 conjugate in NPC cells. Treatment with an autophagy inhibitor, 3-methyladenine, could significantly recover QYLGT-inhibited cell viability of NPC cells. In addition, QYLGT did not significantly induce apoptosis in NPC cells. We also found that QYLGT had the ability to activate phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway. Treatment with PI3K inhibitors, LY294002 and wortmannin, or mTOR inhibitors, rapamycin and Torin 1, could not only recover QYLGT-inhibited cell viability of NPC cells but also inhibit Atg3 expression. Taken together, our results demonstrated that QYLGT could induce autophagic cell death in NPC cells through the PI3K/Akt/mTOR pathway., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2021 Ching-Huey Yang et al.)

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis., (Copyright © 2021. Published by Elsevier Inc.)

It has been reported that paclitaxel activates cell cycle arrest and increases caspase protein expression to induce apoptosis in head and neck squamous cell carcinoma (HNSCC) cell lines. However, the potential signaling pathway regulating this apoptotic phenomenon remains unclear. The present study used OEC-M1 cells to investigate the underlying molecular mechanism of paclitaxel-induced apoptosis. Following treatment with paclitaxel, cell viability was assessed via the MTT assay. Necrosis, apoptosis, cell cycle and mitochondrial membrane potential (∆Ψm) were analyzed via flow cytometric analyses, respectively. Western blot analysis was performed to detect the expression levels of proteins associated with the MAPK and caspase signaling pathways. The results demonstrated that low-dose paclitaxel (50 nM) induced apoptosis but not necrosis in HNSCC cells. In addition, paclitaxel activated the c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38 mitogen-activated protein kinase. The paclitaxel-activated JNK contributed to paclitaxel-induced apoptosis, activation of caspase-3, -6, -7, -8 and -9, and reduction of ∆Ψm. In addition, caspase-8 and -9 inhibitors, respectively, significantly decreased paclitaxel-induced apoptosis. Notably, Bid was truncated following treatment with paclitaxel. Taken together, the results of the present study suggest that paclitaxel-activated JNK is required for caspase activation and loss of ∆Ψm, which results in apoptosis of HNSCC cells. These results may provide mechanistic basis for designing more effective paclitaxel-combining regimens to treat HNSCC., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Lan et al.)

Natural products have long been considered as a kind of complementary medicine. In this study, we investigate the apoptotic effect of essential oils of Toona sinensis roots (TSR) on human clear cell renal cell carcinomas (ccRCC). The sesquiterpene content of TSR essential oil was determined via GC/MS analysis. TSR decreased ccRCC cell viabilities, inducing ROS generation and reduction of the mitochondrial membrane potential. Moreover, TSR inhibited Bcl-2 and Hsp90 expression but increased PARP-1 cleavage and cytochrome c release. Akt, mTOR and NF-κB phosphorylation and HIF-α expression were all inhibited, which likely contributed to the anti-proliferative and anti-adhesive effects of TSR.

Polyalthia belong to the Annonaceae family and are a type of evergreen tree distributed across many tropical and subtropical regions. Polyalthia species have been used long term as indigenous medicine to treat certain diseases, including fever, diabetes, infection, digestive disease, etc. Recent studies have demonstrated that not only crude extracts but also the isolated pure compounds exhibit various pharmacological activities, such as anti-oxidant, anti-microbial, anti-tumor, anti-cancer, etc. It is known that the initiation of cancer usually takes several years and is related to unhealthy lifestyle, as well as dietary and environmental factors, such as stress, toxins and smoking. In fact, natural or synthetic substances have been used as cancer chemoprevention to delay, impede, or even stop cancer growing. This review is an attempt to collect current available phytochemicals from Polyalthia species , which exhibit anti-cancer potentials for chemoprevention purposes, providing directions for further research on the interesting agents and possible clinical applications.

Cordycepin is an adenosine derivative isolated from Cordyceps sinensis , which has been used as an herbal complementary and alternative medicine with various biological activities. The general anti-cancer mechanisms of cordycepin are regulated by the adenosine A3 receptor, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), and glycogen synthase kinase (GSK)-3β, leading to cell cycle arrest or apoptosis. Notably, cordycepin also induces autophagy to trigger cell death, inhibits tumor metastasis, and modulates the immune system. Since the dysregulation of autophagy is associated with cancers and neuron, immune, and kidney diseases, cordycepin is considered an alternative treatment because of the involvement of cordycepin in autophagic signaling. However, the profound mechanism of autophagy induction by cordycepin has never been reviewed in detail. Therefore, in this article, we reviewed the anti-cancer and health-promoting effects of cordycepin in the neurons, kidneys, and the immune system through diverse mechanisms, including autophagy induction. We also suggest that formulation changes for cordycepin could enhance its bioactivity and bioavailability and lower its toxicity for future applications. A comprehensive understanding of the autophagy mechanism would provide novel mechanistic insight into the anti-cancer and health-promoting effects of cordycepin.

Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell's K + currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K + current ( I K(DR) ) in concentration-, time-, and state-dependent manners. The IC 50 for MDZ-mediated reduction of I K(DR) density was 5.87 μM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on I K(DR) density was estimated with a dissociation constant of 5.14 μM. In addition, the inactivation curve of I K(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of I K(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca 2+ -activated K + (IK Ca ) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both I K(DR) and IK Ca -channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of I K(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.

The DNA-PK maintains cell survival when DNA damage occurs. In addition, aberrant activation of the DNA-PK induces centrosome amplification, suggesting additional roles for this kinase. Here, we showed that the DNA-PK-p53 cascade induced primary cilia formation (ciliogenesis), thus maintaining the DNA damage response under genotoxic stress. Treatment with genotoxic drugs (etoposide, neocarzinostatin, hydroxyurea, or cisplatin) led to ciliogenesis in human retina (RPE1), trophoblast (HTR8), lung (A459), and mouse Leydig progenitor (TM3) cell lines. Upon genotoxic stress, several DNA damage signaling were activated, but only the DNA-PK-p53 cascade contributed to ciliogenesis, as pharmacological inhibition or genetic depletion of this pathway decreased genotoxic stress-induced ciliogenesis. Interestingly, in addition to localizing to the nucleus, activated DNA-PK localized to the base of the primary cilium (mother centriole) and daughter centriole. Genotoxic stress also induced autophagy. Inhibition of autophagy initiation or lysosomal degradation or depletion of ATG7 decreased genotoxic stress-induced ciliogenesis. Besides, inhibition of ciliogenesis by depletion of IFT88 or CEP164 attenuated the genotoxic stress-induced DNA damage response. Thus, our study uncovered the interplay among genotoxic stress, the primary cilium, and the DNA damage response.

Proper development of neuronal cells is important for brain functions, and impairment of neuronal development may lead to neuronal disorders, implying that improvement in neuronal development may be a therapeutic direction for these diseases. Huntington's disease (HD) is a neurodegenerative disease characterized by impairment of neuronal structures, ultimately leading to neuronal death and dysfunctions of the central nervous system. Based on previous studies, fibroblast growth factor 9 (FGF9) may provide neuroprotective functions in HD, and FGFs may enhance neuronal development and neurite outgrowth. However, whether FGF9 can provide neuronal protective functions through improvement of neuronal morphology in HD is still unclear. Here, we study the effects of FGF9 on neuronal length in HD and attempt to understand the related working mechanisms. Taking advantage of striatal cell lines from HD knock-in mice, we found that FGF9 increases total neuronal length and upregulates several structural and synaptic proteins under HD conditions. In addition, activation of nuclear factor kappa B (NF-kB) signaling by FGF9 was observed to be significant in HD cells, and blockage of NF-kB leads to suppression of these structural and synaptic proteins induced by FGF9, suggesting the involvement of NF-kB signaling in these effects of FGF9. Taken these results together, FGF9 may enhance total neuronal length through upregulation of NF-kB signaling, and this mechanism could serve as an important mechanism for neuroprotective functions of FGF9 in HD.

Aims: Huntington's disease (HD) is a neurodegenerative disease that causes deficits in neurite outgrowth, which suggests that enhancement of neurite outgrowth is a potential direction by which to improve HD. Our previous publications showed that fibroblast growth factor 9 (FGF9) provides anti-apoptosis and anti-oxidative functions in striatal cell models of HD through the extracellular signal-regulated kinases (ERK) pathway, and FGF9 also stimulates cytoskeletons to enhance neurite outgrowth via nuclear factor kappa B (NF-kB) signaling. In this study, we further demonstrate the importance of the ERK pathway for the neurite outgrowth induced by FGF9 in HD striatal models., Materials and Methods: FGF9 was treated with ERK (U0126) or NF-kB (BAY11-7082) inhibitors in STHdh Q7/Q7 and STHdh Q111/Q111 striatal knock-in cell lines to examine neurite outgrowth, cytoskeletal markers, and synaptic proteins via immunofluorescence staining and Western blotting. NF-kB activity was analyzed by NF-kB promoter reporter assay., Key Findings: Here, we show that suppression of ERK signaling significantly inhibits FGF9-induced neurite outgrowth, cytoskeletal markers, and synaptic proteins in HD striatal cells. In addition, we also show suppression of ERK signaling significantly decreases FGF9-induced NF-kB activation, whereas suppression of NF-kB does not decrease FGF9-induced ERK signaling. These results suggest that FGF9 activates ERK signaling first, stimulates NF-kB upregulation, and then enhances neurite outgrowth in HD striatal cells., Significance: We elucidate the more detailed mechanisms of neurite outgrowth enhanced by FGF9 in these HD striatal cells. This study may provide insights into targeting neurite outgrowth for HD therapy., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Arsenic is a well‑documented environmental toxicant that can induce neurotoxicity and peripheral vascular diseases. In fact, arsenic trioxide has been used to treat various cancer types. Oral cancer has been in the top ten common cancers for decades in Taiwan, and the incidence rate is continuously increasing. The majority of oral cancers are associated with excessive tobacco, alcohol consumption and betel chewing. To the best of our knowledge, no study has revealed the effect of arsenic compounds on oral cancers. Thus, the present study used OEC‑M1 oral squamous carcinoma cells treated with sodium arsenite (NaAsO2) and dimethylarsenic acid (DMA) to determine whether both arsenic compounds could exert anticancer effects on oral cancer. The results demonstrated that NaAsO2 and DMA induced rounding up and membrane blebbing in OEC‑M1 cells, which are morphological characteristics of apoptosis. Annexin V/PI double staining analysis further confirmed that both arsenic compounds induced apoptosis of OEC‑M1 cells. In addition, NaAsO2 and DMA significantly decreased the survival rate and increased the percentage of OEC‑M1 cells in the subG1 and G2/M phases (P<0.05). Furthermore, both arsenic compounds significantly activated the cleavage of caspase‑8, ‑9, ‑3 and PARP, and the phosphorylation of JNK, ERK1/2 and p38 in OEC‑M1 cells (P<0.05). Collectively, the findings of the present study indicated that NaAsO2 and DMA stimulate extrinsic and intrinsic apoptotic pathways through the activation of the MAPK pathways to induce apoptosis of OEC‑M1 cells, suggesting that NaAsO2 and DMA may be used as novel anticancer drugs for oral cancers.

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organ developments and is involved in diverse physiological events. Loss of function of FGF9 exhibits male-to-female sex reversal in the transgenic mouse model and gain of FGF9 copy number was found in human 46, XX sex reversal patient with disorders of sex development. These results suggested that FGF9 plays a vital role in male sex development. Nevertheless, how FGF9/Fgf9 expression is regulated during testis determination remains unclear. In this study, we demonstrated that human and mouse SRY bind to -833 to -821 of human FGF9 and -1010 to -998 of mouse Fgf9, respectively, and control FGF9/Fgf9 mRNA expression. Interestingly, we showed that mouse SRY cooperates with SF1 to regulate Fgf9 expression, whereas human SRY-mediated FGF9 expression is SF1 independent. Furthermore, using an ex vivo gonadal culture system, we showed that FGF9 expression is sufficient to switch cell fate from female to male sex development in 12-16 tail somite XX mouse gonads. Taken together, our findings provide evidence to support the SRY-dependent, fate-determining role of FGF9 in male sex development., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Cordycepin, a bioactive constituent from the fungus Cordyceps sinensis , could inhibit cancer cell proliferation and promote cell death via induction of cell cycle arrest, apoptosis and autophagy. Our novel finding from microarray analysis of cordycepin-treated MA-10 mouse Leydig tumor cells is that cordycepin down-regulated the mRNA levels of FGF9, FGF18, FGFR2 and FGFR3 genes in MA-10 cells. Meanwhile, the IPA-MAP pathway prediction result showed that cordycepin inhibited MA-10 cell proliferation by suppressing FGFs/FGFRs pathways. The in vitro study further revealed that cordycepin decreased FGF9-induced MA-10 cell proliferation by inhibiting the expressions of p-ERK1/2, p-Rb and E2F1, and subsequently reducing the expressions of cyclins and CDKs. In addition, a mouse allograft model was performed by intratumoral injection of FGF9 and/or intraperitoneal injection of cordycepin to MA-10-tumor bearing C57BL/6J mice. Results showed that FGF9-induced tumor growth in cordycepin-treated mice was significantly smaller than that in a PBS-treated control group. Furthermore, cordycepin decreased FGF9-induced FGFR1-4 protein expressions in vitro and in vivo. In summary, cordycepin inhibited FGF9-induced testicular tumor growth by suppressing the ERK1/2, Rb/E2F1, cell cycle pathways, and the expressions of FGFR1-4 proteins, suggesting that cordycepin can be used as a novel anticancer drug for testicular cancers.

A clerodane diterpene compound 16-hydroxycleroda-3,13-dien-15,16-olide (CD) is considered a therapeutic agent with pharmacological activities. The present study investigated the mechanisms of CD-induced apoptosis in T24 human bladder cancer cells. CD inhibited cell proliferation in a concentration and time-dependent manner. CD-induced overproduction of reactive oxygen species and reduced mitochondrial membrane potential, associated with reduced expression of Bcl-2 and increased levels of cytosolic cytochrome c, cleaved PARP-1 and caspase-3. In addition, CD treatment led to cell cycle arrest at the G0/G1 phase and inhibited expression of cyclin D1 and cyclin-dependent kinases 2 and 4 and led to increased levels of p21, p27Kip1 and p53. All of these events were accompanied with a reduction of pEGFR, pMEK1/2, pERK1/2, pAkt, pmTOR, pP70S6K1, HIF-1α, c-Myc and VEGF. RNAseq-based analysis revealed that CD-induced cell death was characterised by an increased expression of stress and apoptotic-related genes as well as inhibition of the cell cycle-related genes. In summary, CD induces apoptosis in T24 bladder cancer cells through targeting multiple intracellular signaling pathways as a result of oxidative stress and cell cycle arrest.

Background: Excessive microglial activation is implicated in the pathogenesis of various age-related neurodegenerative diseases. In addition to neurons, brain-derived neurotrophic factor (BDNF) and its receptor TrkB are also expressed in microglia. However, the direct effect of BDNF on age-related microglial activation has rarely been investigated., Methods: We began to address this question by examining the effect of age on microglial activation and the BDNF-TrkB pathway in mice. By using pharmacological and genetic approaches, the roles of BDNF and downstream signaling pathways in microglial activation and related neurotoxicity were examined in microglial cell line and primary microglial cells., Results: We showed that microglial activation was evident in the brains of aged mice. The levels of BDNF and TrkB in microglia decreased with age and negatively correlated with their activation statuses in mice during aging. Interestingly, aging-related microglial activation could be reversed by chronic, subcutaneous perfusion of BDNF. Peripheral lipopolysaccharide (LPS) injection-induced microglial activation could be reduced by local supplement of BDNF, while shTrkB induced local microglial activation in naïve mice. In cultured microglial cell line and primary microglial cells, BDNF inhibited LPS-induced microglial activation, including morphological changes, activations of p38, JNK, and NF-кB, and productions of proinflammatory cytokines. These effects were blocked by shTrkB. BDNF induced activations of ErK and CREB which then competed with LPS-induced activation of NF-кB for binding to a common coactivator, CREB-binding protein., Conclusions: Decreasing BDNF-TrkB signaling during aging favors microglial activation, while upregulation BDNF signaling inhibits microglial activation via the TrkB-Erk-CREB pathway.

Propofol is an anesthetic agent moderating GABA receptors in the nervous system. A number of studies have demonstrated that propofol exerts a negative effect on neural stem cell development in the neonatal mouse hippocampus. However, to the best of our knowledge, there is no study available to date illustrating whether neonatal exposure to propofol affects Leydig stem/progenitor cell development for normal male reproductive development and functions, and the regulatory mechanism remains elusive. In the present study, TM3 cells, a mouse Leydig stem/progenitor cell line, was treated with propofol. The data illustrated that propofol significantly reduced TM3 cell viability. TM3 subG1 phase cell numbers were significantly increased by propofol assayed by flow cytometric analysis. Annexin V/PI double staining assay of the TM3 Leydig cells also demonstrated that propofol increased TM3 cell apoptosis. In addition, cleaved caspase‑8, ‑9 and ‑3 and/or poly(ADP‑ribose) polymerase (PARP) were significantly activated by propofol in the TM3 cells. Furthermore, the expression levels of phospho‑JNK, phospho‑ERK1/2 and phospho‑p38 were activated by propofol in the TM3 cells, indicating that propofol induced apoptosis through the mitogen‑activated protein kinase (MAPK) pathway. Additionally, propofol diminished the phosphorylation of Akt to increase the apoptosis of TM3 cells. On the whole, the findings of the present study demonstrate that propofol induces TM3 cell apoptosis by activating caspases and MAPK pathways, as well as by inhibiting the Akt pathway in TM3 cells. These findings illustrate that propofol affects the viability of Leydig stem/progenitor cells possibly related to the development of the male reproductive system.

Objective: To evaluate the efficacy and safety of lurasidone compared with quetiapine for treatment of delirium in critically ill patients., Design: Prospective, observational cohort study., Setting: Single-center community teaching hospital., Patients: Forty adult intensive care unit (ICU) patients with delirium (Confusion Assessment Method in the ICU positive), tolerating enteral nutrition, and without active alcohol withdrawal or prior use of atypical antipsychotics., Interventions: Patients were treated at the discretion of the prescriber with either lurasidone or quetiapine for delirium. Dose escalation and/or discontinuation were determined at the discretion of individual providers., Results: Baseline characteristics differed with a higher severity of illness in patients in the quetiapine group (n = 20) and a higher baseline QTc interval in the lurasidone group (n = 20). No significant difference was seen in the time to delirium resolution (3.2 vs 3.4 days), average daily haloperidol requirements (5.7 vs 6.9 mg), hospital length of stay (LOS; 23.6 vs 27.9 days), or ICU LOS (12.1 vs 14.2 days). Lurasidone was associated with fewer ventilator support days (4.0 [interquartile range, IQR: 2.3-6.8] days vs 7 [IQR: 4.0-9.8; P = .0295] days) but also a fewer number of delirium-free days (0 [IQR: 0-1.0] days vs 2 [IQR: 0-3.0; P = .0231] days). Additionally, no difference was seen for ICU mortality (20% vs 20%), percentage of time oversedated (2.8% vs 2.7%), or incidence of QTc prolongation (10.0% vs 10.0%)., Conclusions: Lurasidone for the treatment of delirium in critically ill patients did not differ in the time to delirium resolution when compared to quetiapine. Additionally, the incidence of QTc prolongation between agents does not appear to be different. Future randomized trials should evaluate dose escalation schemes and a larger proportion of patients to evaluate differences in mortality, efficacy, and life-threatening arrhythmias associated with atypical antipsychotic use.

Rationale : The formation of adipose-derived stem cells (ASCs) into spheres on a chitosan-coated microenvironment promoted ASCs differentiation into a mixed population of neural lineage-like cells (NLCs), but the underline mechanism is still unknown. Since the fibroblast growth factor 9 (FGF9) and fibroblast growth factor receptors (FGFRs) play as key regulators of neural cell fate during embryo development and stem cell differentiation, the current study aims to reveal the interplay of FGF9 and FGFRs for promoting peripheral nerve regeneration. Methods : Different concentration of FGF9 peptide (10, 25, 50, 100 ng/mL) were added during NLCs induction (FGF9-NLCs). The FGFR expressions and potential signaling were studied by gene and protein expressions as well as knocking down by specific FGFR siRNA or commercial inhibitors. FGF9-NLCs were fluorescent labeled and applied into a nerve conduit upon the injured sciatic nerves of experimental rats. Results : The FGFR2 and FGFR4 were significantly increased during NLCs induction. The FGF9 treated FGF9-NLCs spheres became smaller and changed into Schwann cells (SCs) which expressed S100β and GFAP. The specific silencing of FGFR2 diminished FGF9-induced Akt phosphorylation and inhibited the differentiation of SCs. Transplanted FGF9-NLCs participated in myelin sheath formation, enhanced axonal regrowth and promoted innervated muscle regeneration. The knockdown of FGFR2 in FGF9-NLCs led to the abolishment of nerve regeneration. Conclusions : Our data therefore demonstrate the importance of FGF9 in the determination of SC fate via the FGF9-FGFR2-Akt pathway and reveal the therapeutic benefit of FGF9-NLCs., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide and accounts for over 90% of malignant neoplasms of the oral cavity, with a 5-year survival rate of less than 50%. The long-term survival rate of OSCC patients has not markedly improved in recent decades due to its heterogeneous etiology and treatment outcomes. We investigated the anticancer effect of the combination of irradiation (IR) and cordycepin in the treatment of human OSCC cells in vitro. The type of cell death, especially autophagy and apoptosis, and the underlying mechanisms were examined. We found synergistic effects of cordycepin and IR on the viability of human oral cancer cells. The combination of cordycepin and IR treatment induced apoptosis, cell cycle arrest, and autophagic cell death. Furthermore, cordycepin induced S-phase arrest and prolonged G2/M arrest in the cells that received the combination treatment compared with those that received irradiation alone. Combined treatment induced the upregulation of ATG5 and p21 in an autophagy cascade-dependent manner, arrested the cell cycle in the G2/M phase, and repressed cell proliferation. Thus, we conclude that the combination of cordycepin and IR treatment could be a potential therapeutic strategy for OSCC., Competing Interests: The authors declare no conflict of interest.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease found worldwide. The present study aimed to evaluate the mechanisms of inhibiting lipid accumulation in free fatty acid (FFA)-treated HepG2 cells caused by bark and fruit extracts of Toona sinensis (TSB and TSF). FFA induced lipid and triglyceride (TG) accumulation, which was attenuated by TSB and TSF. TSB and/or TSF promoted phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-coA carboxylase and peroxisome proliferator-activated receptor alpha upregulation. Furthermore, TSB and TSF suppressed FFA-induced liver X receptor, sterol regulatory element-binding transcription protein 1, fatty acid synthase, and stearoyl-CoA desaturase 1 protein expression. Moreover, TSB and/or TSF induced phosphorylation of Unc-51 like autophagy-activating kinase and microtubule-associated protein 1A/1B-light chain 3 expressions. Therefore, TSB and TSF relieve lipid accumulation by attenuating lipogenic protein expression, activating the AMPK pathway, and upregulating the autophagic flux to enhance lipid metabolism. Moreover, TSB and TSF reduced TG contents, implying the therapeutic use of TSB and TSF in NAFLD.