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Colorectal cancer (CRC) usually gives rise to transcoelomic spread and ultimately causes peritoneal carcinomatosis (PC). However, mechanism studies, especially the immunological basis of colorectal PC, are rarely revealed due to lack of a suitable PC model. Here we selected a mouse colorectal cancer cell line MC-38 for intraperitoneal inoculation in the C57BL/6 mice to mimic the development of colorectal PC. We demonstrated that the injected CRC cells preferentially and rapidly migrated and colonized in the visceral fat tissues, but not in other visceral organs. With flow cytometric analysis, we found the proportions of spleen T cells and B cells were not affected by PC progression, while the ratios of blood CD4+ and CD8+ T cells were largely influenced. Especially, the quantity or activity of CD4+ and CD8+ T cells in visceral fats were intimately regulated by PC development. Taken together, we successfully constructed a colorectal PC model in immune-competent mice and revealed the alteration of adaptive immunity in PC development. Our study might potentiate the research and therapy strategies of colorectal PC.

Autophagy is closely related to the growth and drug resistance of cancer cells, and autophagy related 4B (ATG4B) performs a crucial role in the process of autophagy. The long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed (CRNDE) promotes the progression of hepatocellular carcinoma (HCC), but it is unclear whether the tumor-promoting effect of CRNDE is associated with the regulation of ATG4B and autophagy. Herein, we for the first time demonstrated that CRNDE triggered autophagy via upregulating ATG4B in HCC cells. Mechanistically, CRNDE enhanced the stability of ATG4B mRNA by sequestrating miR-543, leading to the elevation of ATG4B and autophagy in HCC cells. Moreover, sorafenib induced CRNDE and ATG4B as well as autophagy in HCC cells. Knockdown of CRNDE sensitized HCC cells to sorafenib in vitro and in vivo. Collectively, these results reveal that CRNDE drives ATG4B-mediated autophagy, which attenuates the sensitivity of sorafenib in HCC cells, suggesting that the pathway CRNDE/ATG4B/autophagy may be a novel target to develop sensitizing measures of sorafenib in HCC treatment.

Liver cancer is one of the most common and high recurrence malignancies. Besides radiotherapy and surgery, chemotherapy also plays an essential role in the treatment of liver cancer. Sorafenib and sorafenib-based combination therapies have been proven efficacy against tumors. However, previous clinical studies have indicated that some patients with liver cancer are resistant to sorafenib treatment and the existing strategies are not satisfactory in the clinic. Therefore, it is urgent to investigate strategies to improve the effectiveness of sorafenib for liver cancer and to explore effective drug combinations. In the present study, we found that dichloroacetate (DCA) could significantly enhance the anti-tumor effect of sorafenib on liver cancer cells, including reduced viability and dramatically promoted apoptosis in liver cancer cells. Moreover, compared to sorafenib alone, the combination of DCA and sorafenib markedly increased the degradation of anti-apoptotic protein Mcl-1 by enhancing its phosphorylation. Overexpression of Mcl-1 could significantly attenuate the synergetic effect of DCA and sorafenib on apoptosis induction in liver cancer cells. Furthermore, we found that the ROS-JNK pathway was obviously activated in the DCA combined sorafenib group. The levels of ROS and p-JNK were dramatically up-regulated in the two drug combination groups. Antioxidant NAC could alleviate the synergetic effects of DCA and sorafenib on ROS generation, JNK activation, Mcl-1 degradation, and cell apoptosis. Moreover, DCA and sorafenib's effects on Mcl-1 degradation and apoptosis could also be inhibited by JNK inhibitor 'SP'600125. Finally, the synergetic effects of DCA and sorafenib on tumor growth suppression, Mcl-1 degradation and induction of apoptosis were also validated in liver cancer xenograft in vivo. These findings indicate that DCA enhances the anti-tumor effect of sorafenib via the ROS-JNK-Mcl-1 pathway in liver cancer cells. This study may provide new insights to improve the chemotherapeutic effect of sorafenib, which may be beneficial for further clinical application of sorafenib in liver cancer treatment.

MiR-124-3p has been identified as a novel tumor suppressor and a potential therapeutic target in hepatocellular carcinoma (HCC) through regulating its target genes. However, the upstream regulatory mechanisms of mir-124-3p in HCC has not been fully understood. The transcription factor liver X receptor (LXR) plays a critical role in suppressing the proliferation of HCC cells, but it is unclear whether LXR is involved in the regulation of mir-124-3p. In the present study, we demonstrated that the expression of mir-124-3p was positively correlated with that of LXR in HCC, and the cell growth of HCC was significantly inhibited by LXR agonists. Moreover, activation of LXR with the agonists up-regulated the expression of mir-124-3p, and in turn down-regulated cyclin D1 and cyclin-dependent kinase 6 (CDK6) expression, which are the target genes of mir-124-3p. Mechanistically, miR-124-3p mediates LXR induced inhibition of HCC cell growth and down-regulation of cyclin D1 and CDK6 expression. In vivo experiments also confirmed that LXR induced miR-124-3p expression inhibited the growth of HCC xenograft tumors, as well as cyclin D1 and CDK6 expression. Our findings revealed that miR-124-3p is a novel target gene of LXR, and regulation of the miR-124-3p-cyclin D1/CDK6 pathway by LXR plays a crucial role in the proliferation of HCC cells. LXR-miR-124-3p-cyclin D1/CDK6 pathway may be a novel potential therapeutic target for HCC treatment.

Metabolic reprogramming greatly contributes to the regulation of macrophage activation. However, the mechanism of lipid accumulation and the corresponding function in tumor-associated macrophages (TAMs) remain unclear. With primary investigation in colon cancer and confirmation in other cancer models, here we determine that deficiency of monoacylglycerol lipase (MGLL) results in lipid overload in TAMs. Functionally, macrophage MGLL inhibits CB2 cannabinoid receptor-dependent tumor progression in inoculated and genetic cancer models. Mechanistically, MGLL deficiency promotes CB2/TLR4-dependent macrophage activation, which further suppresses the function of tumor-associated CD8+ T cells. Treatment with CB2 antagonists delays tumor progression in inoculated and genetic cancer models. Finally, we verify that expression of macrophage MGLL is decreased in cancer tissues and positively correlated with the survival of cancer patients. Taken together, our findings identify MGLL as a switch for CB2/TLR4-dependent macrophage activation and provide potential targets for cancer therapy., Tumour associated macrophages (TAMs) have an altered lipid metabolism. Here the authors show that downregulation of monoacylglycerols lipase MGLL in TAMs induces lipid accumulation and tumor progression by polarizing TAMs toward tumor-promoting through activation of cannabinoid receptor CB2.

The BH3 mimetic (-)-gossypol (-)-G has shown promising efficacy to kill several kinds of cancer cells or potentiate current chemotherapeutics. But it induces limited apoptosis in cancer cells with high level of Bcl-2. The nuclear receptor PPARγ and its agonist rosiglitazone can suppress various malignancies. More importantly, rosiglitazone is able to enhance the anti-tumor effects of chemotherapy drugs such as carboplatin and tyrosine kinase inhibitors. In this study, we for the first time demonstrated that rosiglitazone could sensitize (-)-G to induce apoptosis in cancer cells with high level of Bcl-2. Furthermore, we found that (-)-G increased the mRNA level and protein stability of Mcl-1, which weakened the pro-apoptotic effect of (-)-G. Rosiglitazone attenuated the (-)-G-induced Mcl-1 stability through decreasing JNK phosphorylation. Additionally, rosiglitazone upregulated dual-specificity phosphatase 16 (DUSP16), leading to a reduction of (-)-G-triggered JNK phosphorylation. Animal experiments showed that rosiglitazone could sensitize (-)-G to repress the growth of cancer cells with high level of Bcl-2 in vivo. Taken together, our results suggest that the PPARγ agonists may enhance the therapeutic effect of BH3 mimetics in cancers with high level of Bcl-2 through regulating the DUSP16/JNK/Mcl-1 singling pathway. This study may provide novel insights into the cancer therapeutics based on the combination of PPARγ agonists and BH3 mimetics.

// Fan Yang 1, 2, * , Qingjian Wu 3, * , Yan Zhang 1 , Haojun Xiong 1 , Xinzhe Li 1 , Bo Li 1 , Wei Xie 2 , Le Zhang 2 , Min Xu 4 , Kebin Zhang 2 and Fengtian He 1 1 Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China 2 Central Laboratory, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China 3 Department of Urology, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China 4 Center for Disease Control and Prevention, Chengdu Military Region, Chengdu 610021, China * These authors have contributed equally to this work Correspondence to: Fengtian He, email: hefengtian66@163.com Kebin Zhang, email: zhangkebin12@163.com Min Xu, email: 603537726@qq.com Keywords: LOC653786; FOXM1; long noncoding RNA; renal cell carcinoma; cell growth Received: August 02, 2017 Accepted: January 02, 2018 Published: January 08, 2018 ABSTRACT Renal cell carcinoma (RCC) is the most common kidney malignancy with poor prognosis. Recently, long noncoding RNAs (lncRNAs) have been demonstrated as important regulators in multiple cancers including RCC. LOC653786 is a lncRNA, but its role in cancer remains unclear. In this study, we for the first time found that LOC653786 was upregulated in RCC tissues and cell lines, and this lncRNA promoted growth and cell cycle progression of RCC cells. Moreover, we showed that LOC653786 elevated the expression of forkhead box M1 (FOXM1) and its downstream target genes cyclin D1 and cyclin B1 in RCC cells. Reporter assay revealed that LOC653786 enhanced the transcriptional activity of FOXM1 gene promoter. Additionally, knockdown of FOXM1 attenuated the LOC653786-enhanced growth and cell cycle progression of RCC cells. Meanwhile, silencing of LOC653786 suppressed RCC cell growth and cell cycle progression, which was alleviated by overexpression of FOXM1. The in vivo experiments in nude mice showed knockdown of LOC653786 repressed xenograft tumor growth and FOXM1 expression. In conclusion, our results demonstrate that LOC653786 accelerates growth and cell cycle progression of RCC cells via upregulating FOXM1, suggesting that the ‘LOC653786/FOXM1’ pathway may serve as a novel target for RCC treatment.

p53 deficiency, a frequent event in multiple kinds of malignancies, decreases the sensitivity of diverse targeted chemotherapeutics including the BCL-XL/BCL-2 inhibitor ABT-263. Loss of p53 function can activate mTOR complex 1 (mTORC1), which may make it a vulnerable target. Metformin has shown anti-neoplastic efficiency partially through suppressing mTORC1. However, it remains unknown whether mTORC1 activation confers ABT-263 resistance and whether metformin can overcome it in the p53-defective contexts. In this study, we for the first time demonstrated that metformin and ABT-263 synergistically elicited remarkable apoptosis through orchestrating the proapoptotic machineries in various p53-defective cancer cells. Mechanistic studies revealed that metformin sensitized ABT-263 via attenuating mTORC1-mediated cap-dependent translation of MCL-1 and survivin and weakening internal ribosome entry site (IRES)-dependent translation of XIAP. Meanwhile, ABT-263 sensitized metformin through disrupting the BCL-XL/BIM complex. However, metformin and ABT-263 had no synergistic killing effect in p53 wild-type (p53-WT) cancer cells because the cotreatment dramatically induced the senescence-associated secretory phenotype (SASP) in the presence of wild type p53, and SASP could aberrantly activate the AKT/ERK–mTORC1–4EBP1–MCL-1/survivin signaling axis. Blocking the axis using corresponding kinase inhibitors or neutralizing antibodies against different SASP components sensitized the cotreatment effect of metformin and ABT-263 in p53-WT cancer cells. The in vivo experiments showed that metformin and ABT-263 synergistically inhibited the growth of p53-defective (but not p53-WT) cancer cells in tumor xenograft nude mice. These results suggest that the combination of metformin and ABT-263 may be a novel targeted therapeutic strategy for p53-defective cancers. Mol Cancer Ther; 16(9); 1806–18. ©2017 AACR.

Highly upregulated in liver cancer (HULC), a lncRNA overexpressed in hepatocellular carcinoma (HCC), has been demonstrated to be involved in the carcinogenesis and progression of HCC. However, the mechanisms of HULC promoting the abnormal growth of HCC cells are still not well elucidated. In the present study, we for the first time demonstrated that HULC promoted the growth of HCC cells through elevating COX-2 protein. Moreover, the study of the corresponding mechanism by which HULC upregulated COX-2 showed that HULC enhanced the level of ubiquitin-specific peptidase 22 (USP22), which decreased ubiquitin-mediated degradation of COX-2 protein by removing the conjugated polyubiquitin chains from COX-2 and finally stabilized COX2 protein. In addition, knockdown of USP22 or COX-2 attenuated HULC-mediated abnormal growth of HCC cells. In conclusion, our results demonstrated that "USP22/COX-2" axis played an important role in HULC promoting growth of HCC cells. The identification of this novel pathway may pave a road for developing new potential anti-HCC strategies.

// Haojun Xiong 1 , Yan Zhang 1 , Shan Chen 1 , Zhenhong Ni 1 , Jintao He 2 , Xinzhe Li 1 , Bo Li 1 , Kai Zhao 1 , Fan Yang 1 , Yijun Zeng 1 , Bingbo Chen 3 and Fengtian He 1 1 Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China 2 Battalion 17 of Students, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China 3 Laboratory Animal Center, Third Military Medical University, Chongqing 400038, China Correspondence to: Fengtian He, email: hefengtian06@aliyun.com Bingbo Chen, email: chenbb81@126.com Keywords: HCC, LXR, SOCS3 Received: April 28, 2017 Accepted: June 10, 2017 Published: July 18, 2017 ABSTRACT Liver X receptor (LXR), a member of nuclear receptor superfamily, is involved in the regulation of glucose, lipid and cholesterol metabolism. Recently, it has been reported that LXR suppress different kinds of cancers including hepatocellular carcinoma (HCC). However, the corresponding mechanism is still not well elucidated. In the present study, we found that activation of LXR downregulated cyclin D1 while upregulated p21 and p27 by elevating the level of suppressor of cytokine signaling 3 (SOCS3), leading to the cell cycle arrest at G1/S phase and growth inhibition of HCC cells. Moreover, we demonstrated that LXRα (not LXRβ) mediated the induction of SOCS3 in HCC cells. Subsequently, we showed that LXR activation enhanced the mRNA stability of SOCS3, but had no significant influence on the transcriptional activity of SOCS3 gene promoter. The experiments in nude mice revealed that LXR agonist inhibited the growth of xenograft tumors and enhanced SOCS3 expression in vivo . These results indicate that “LXRα-SOCS3-cyclin D1/p21/p27” is a novel pathway by which LXR exerts its anti-HCC effects, suggesting that the pathway may be a new potential therapeutic target for HCC treatment.

// Bo Li 1, * , Xinzhe Li 1, * , Haojun Xiong 1 , Peng Zhou 1 , Zhenhong Ni 1 , Teng Yang 1 , Yan Zhang 1 , Yijun Zeng 1 , Jintao He 2 , Fan Yang 1 , Nan Zhang 1 , Yuting Wang 1 , Yingru Zheng 3 and Fengtian He 1 1 Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China 2 Battalion 17 of Students, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China 3 Department of Obstetrics and Gynecology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China * These authors have contributed equally to this work Correspondence to: Yingru Zheng, email: zyrdaping@aliyun.com Fengtian He, email: hefengtian06@aliyun.com Keywords: dichloroacetate, COX2, celecoxib, QKI, cervical cancer Received: February 07, 2017 Accepted: May 06, 2017 Published: June 16, 2017 ABSTRACT Dichloroacetate (DCA), a traditional mitochondria-targeting agent, has shown promising prospect as a sensitizer in fighting against malignancies including cervical cancer. But it is unclear about the effect of DCA alone on cervical tumor. Moreover, previous reports have demonstrated that the increased cyclooxygenase-2 (COX2) expression is associated with chemoresistance and poor prognosis of cervical cancer. However, it is still unknown whether COX2 can affect the sensitivity of DCA in cervical cancer cells. In this study, we found that cervical cancer cells were insensitive to DCA. Furthermore, we for the first time revealed that DCA could upregulate COX2 which impeded the chemosensitivity of DCA in cervical cancer cells. Mechanistic study showed that DCA reduced the level of RNA binding protein quaking (QKI), leading to the decay suppression of COX2 mRNA and the subsequent elevation of COX2 protein. Inhibition of COX2 using celecoxib could sensitize DCA in repressing the growth of cervical cancer cells both in vitro and in vivo . These results indicate that COX2 is a novel resistance factor of DCA, and combination of celecoxib with DCA may be beneficial to the treatment of cervical cancer.

Sorafenib is the first‑line drug used in the treatment of liver cancer; however, drug resistance seriously limits the clinical response to sorafenib. The present study investigated the molecular mechanisms of sorafenib resistance in liver cancer cells. The data indicated that forkhead box M1 (FoxM1) was significantly overexpressed in sorafenib‑resistant cells, at the mRNA and protein levels. Knockdown of FoxM1 rendered drug‑tolerant cells sensitive to sorafenib. Furthermore, FoxM1 was upregulated at the transcriptional level. Overexpression of c‑jun was associated with the upregulation of FoxM1. The results of a reporter gene assay, electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that there is an activator protein‑1 (AP1) binding site in the promoter of FoxM1, located at ‑608 to ‑618. Knockdown of c‑jun significantly decreased the levels of FoxM1, accompanied by enhanced cell sensitivity to sorafenib. Furthermore, the activation of AKT contributed to the upregulation of c‑jun and FoxM1. Inhibition of AKT using BEZ‑235 markedly suppressed the upregulation of c‑jun and FoxM1, and increased the sensitivity of drug‑resistant cells to sorafenib in vitro and in vivo. The data indicated that the activation of the AKT/AP1/FoxM1 signaling axis is an important determinant of sorafenib tolerance.

B cell activating factor (BAFF), a member of the tumor necrosis factor (TNF) family, plays a critical role in the pathogenesis and progression of rheumatoid arthritis (RA). Chlorogenic acid (CGA) is a phenolic compound and exerts antiarthritic activities in arthritis. However, it is not clear whether the anti-inflammatory property of CGA is associated with the regulation of BAFF expression. In this study, we found that treatment of the collagen-induced arthritis (CIA) mice with CGA significantly attenuated arthritis progression and markedly inhibited BAFF production in serum as well as the production of serum TNF-α. Furthermore, CGA inhibits TNF-α-induced BAFF expression in a dose-dependent manner and apoptosis in MH7A cells. Mechanistically, we found the DNA-binding site for the transcription factor NF-κB in the BAFF promoter region is required for this regulation. Moreover, CGA reduces the DNA-binding activity of NF-κB to the BAFF promoter region and suppresses BAFF expression through the NF-κB pathway in TNF-α-stimulated MH7A cells. These results suggest that CGA may serve as a novel therapeutic agent for the treatment of RA by targeting BAFF.

// Bo Li 1, * , Xinzhe Li 1, * , Zhenhong Ni 1 , Yan Zhang 1 , Yijun Zeng 1 , Xiaohuan Yan 2 , Yan Huang 3 , Jintao He 4 , Xilin Lyu 1 , Yaran Wu 1 , Yuting Wang 1 , Yingru Zheng 2 , Fengtian He 1 1 Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing 400038, China 2 Department of Obstetrics and Gynecology, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China 3 Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing 400042, China 4 Battalion 17 of Students, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China * These authors have contributed equally to this work Correspondence to: Yingru Zheng, email: zyrdaping@aliyun.com Fengtian He, email: hefengtian06@aliyun.com Keywords: dichloroacetate, metformin, Mcl-1, cancer metabolism, ovarian cancer Received: January 24, 2016 Accepted: July 09, 2016 Published: July 19, 2016 ABSTRACT Both dichloroacetate (DCA) and metformin (Met) have shown promising antitumor efficacy by regulating cancer cell metabolism. However, the DCA-mediated protective autophagy and Met-induced lactate accumulation limit their tumor-killing potential respectively. So overcoming the corresponding shortages will improve their therapeutic effects. In the present study, we found that DCA and Met synergistically inhibited the growth and enhanced the apoptosis of ovarian cancer cells. Interestingly, we for the first time revealed that Met sensitized DCA via dramatically attenuating DCA-induced Mcl-1 protein and protective autophagy, while DCA sensitized Met through markedly alleviating Met-induced excessive lactate accumulation and glucose consumption. The in vivo experiments in nude mice also showed that DCA and Met synergistically suppressed the growth of xenograft ovarian tumors. These results may pave a way for developing novel strategies for the treatment of ovarian cancer based on the combined use of DCA and Met.

Dexamethasone (Dex) has been commonly used in lymphoma and leukemia treatment, but the detailed mechanisms are not fully understood. Suppressor of AP-1 regulated by interferon (SARI) has tumor-selective growth inhibitory effect. However, it's unclear whether SARI is involved in the Dex-mediated lymphoma growth suppression. In this study, we found that Dex-treated B lymphoma tissues had a higher level of SARI. Dex repressed the growth of B lymphoma cells and upregulated SARI expression by activating glucocorticoid receptor (GR) in vitro and in vivo. Silencing of SARI attenuated the Dex-mediated growth suppression of B lymphoma cells and inhibition of AP-1 activity. Reporter assays revealed that activation of GR enhanced the transcriptional activity of SARI promoter. EMSA and ChIP assays showed that GR directly bound to the ER9 element in SARI promoter region. These results for the first time demonstrated that SARI is a novel target gene of GR, and the upregulation of SARI plays an important role in Dex's killing effect on B lymphoma cells, suggesting that SARI may serve as a novel target and a potential indicator of Dex sensitivity in B lymphoma treatment.

// Xiaoqing Wu 1, 2, 3 , Wenhua Tang 1, 2 , Rebecca T. Marquez 1 , Ke Li 1 , Chad A. Highfill 1 , Fengtian He 1, 2, 4 , Jiqin Lian 2, 4 , Jiayuh Lin 5 , James R. Fuchs 6 , Min Ji 3 , Ling Li 2, 7 , Liang Xu 1, 2 1 Departments of Molecular Biosciences and Radiation Oncology, University of Kansas, Lawrence, KS, USA 2 Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, MI, USA 3 School of Chemistry and Chemical Engineering, Southeast University, Nanjing, Jiangsu, China 4 Department of Biochemistry and Molecular Biology, Third Military Medical University, Chongqing, China 5 Department of Pediatrics, College of Medicine, Ohio State University, Columbus, OH, USA 6 Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Ohio State University, Columbus, OH, USA 7 Department of Cell Biology and Cell Engineering Research Centre, State Key Laboratory of Cancer Biology, Fourth Military Medical University, Xi’an, Shanxi, China Correspondence to: Liang Xu, e-mail: xul@ku.edu Ling Li, e-mail: liling25@fmmu.edu.cn Keywords: chemo/radio-resistance, pSTAT3, pancreatic cancer, lip-FLLL32, CSCs Received: September 22, 2015 Accepted: January 23, 2016 Published: February 12, 2016 ABSTRACT Chemo/radio-therapy resistance to the deadly pancreatic cancer is mainly due to the failure to kill pancreatic cancer stem cells (CSCs). Signal transducer and activator of transcription 3 (STAT3) is activated in pancreatic CSCs and, therefore, may be a valid target for overcoming therapeutic resistance. Here we investigated the potential of STAT3 inhibition in sensitizing pancreatic cancer to chemo/radio-therapy. We found that the levels of nuclear pSTAT3 in pancreatic cancer correlated with advanced tumor grade and poor patient outcome. Liposomal delivery of a STAT3 inhibitor FLLL32 (Lip-FLLL32) inhibited STAT3 phosphorylation and STAT3 target genes in pancreatic cancer cells and tumors. Consequently, Lip-FLLL32 suppressed pancreatic cancer cell growth, and exhibited synergetic effects with gemcitabine and radiation treatment in vitro and in vivo . Furthermore, Lip-FLLL32 reduced ALDH1-positive CSC population and modulated several potential stem cell markers. These results demonstrate that Lip-FLLL32 suppresses pancreatic tumor growth and sensitizes pancreatic cancer cells to radiotherapy through inhibition of CSCs in a STAT3-dependent manner. By targeting pancreatic CSCs, Lip-FLLL32 provides a novel strategy for pancreatic cancer therapy via overcoming radioresistance.

B-lymphocyte stimulator (BLyS) plays a critical role in the pathogenesis and progression of rheumatoid arthritis (RA). Liver X receptor (LXR), a nuclear receptor, has an important anti-inflammatory effect. However, it is unclear whether the BLyS expression is regulated by LXR. In this study, we found that treatment with LXR agonist in collagen-induced arthritis (CIA) mice significantly attenuated arthritis progression, and markedly decreased BLyS production in serum and splenocytes as well as the production of serum IFNγ and TGFβ. Activation of LXR in B lymphocytes dramatically suppressed the basal and IFNγ/TGFβ-induced BLyS expression. Moreover, LXR agonist prominently suppressed the binding of NF-κB to BLyS promoter region, and decreased the promoter's transcriptional activity. Additionally, activation of LXR obviously repressed IFNγ-induced STAT1 activation and TGFβ-induced SMAD3 activation. These results indicated that downregulation of BLyS may be a novel mechanism by which LXR ameliorates RA, and LXR/BLyS pathway may serve as a novel target for the treatment of RA.

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease that results in synovitis, cartilage destruction, and even loss of joint function. The frequent and long-term administration of anti-rheumatic drugs often leads to obvious adverse effects and patient non-compliance. Therefore, to specifically deliver dexamethasone (Dex) to inflamed joints and reduce the administration frequency of Dex, we developed Dex-loaded reactive oxygen species (ROS)-responsive nanoparticles (Dex/Oxi-αCD NPs) and folic acid (FA) modified Dex/Oxi-αCD NPs (Dex/FA-Oxi-αCD NPs) and validated their anti-inflammatory effect in vitro and in vivo. In vitro study demonstrated that these NPs can be effectively internalized by activated macrophages and the released Dex from NPs significantly downregulated the expression of iRhom2, TNF-α, and BAFF in activated Raw264.7. In vivo experiments revealed that Dex/Oxi-αCD NPs, especially Dex/FA-Oxi-αCD NPs significantly accumulated at inflamed joints in collagen-induced arthritis (CIA) mice and alleviated the joint swelling and cartilage destruction. Importantly, the expression of iRhom2, TNF-α, and BAFF in the joint was inhibited by intravenous injection of Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs. Collectively, our data revealed that Dex-loaded ROS-responsive NPs can target inflamed joints and attenuate arthritis, and the 'iRhom2-TNF-α-BAFF' pathway plays an important role in the treatment of RA with the NPs, suggesting that this pathway may be a novel target for RA therapy.

Background and aims Human telomerase reverse transcriptase (hTERT) plays an important role in cancer invasion, but the relevant mechanism is not well known. This study aims to investigate the role and mechanism of hTERT in gastric cancer metastasis. Design Proteomics analysis, qPCR and western blotting were used to screen for hTERT-regulated candidate molecules in gastric cancer invasion. Chromatin immunoprecipitation (ChIP) qPCR was performed to identify the binding sites of hTERT at the regulatory region of the integrin β1 ( ITGB1 ) gene. ChIP assays were further applied to elucidate the transcription factors that bound to the regulatory region. The interactions between hTERT and the transcription factors were tested by co-immunoprecipitation (Co-IP) and glutathione S-transferase (GST) pull-down experiments. Moreover, the revealed pathway was verified in tumour-bearing nude mice and human gastric cancer tissues. Results ITGB1 was identified as a downstream gene of hTERT, and there were two hTERT-binding regions within this gene. hTERT alleviated the binding of forkhead box O3 (FOXO3a) to FOXO3a binding element (+9972∼+9978), but it enhanced the binding of forkhead box M1 (FOXM1) to FOXM1 binding element (−1104∼−1109) in ITGB1 gene. Importantly, FOXO3a played a major role in hTERT-induced ITGB1 expression, and the hTERT/murine double minute 2 (MDM2) complex promoted the ubiquitin-mediated degradation of FOXO3a. Moreover, hTERT increased ITGB1 expression in xenograft gastric cancer, and the level of hTERT was positively correlated with that of ITGB1 in human gastric cancer tissues. Conclusions The hTERT/MDM2–FOXO3a–ITGB1 pathway markedly contributes to hTERT-promoted gastric cancer invasion, suggesting that this pathway might be a novel target for the prevention and treatment of gastric cancer metastasis.

Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that belongs to the Bcl-2 family. The aberrant expression of Mcl-1 is important for sensitivity to chemotherapy drugs in gastric cancer. However, the regulatory mechanism of Mcl-1 in gastric cancer cells remains unclear. In this study, we first found that Forkhead box M1 (FOXM1) and Mcl-1 expression levels were positively correlated in human gastric cancer specimens and that both are associated with poor prognosis of patients treated with oxaliplatin. Second, we demonstrated that the expression level of Mcl-1 was correlated with FOXM1 expression in gastric cancer cells. Third, reporter assays showed that FOXM1 upregulated the promoter activity of the Mcl-1 gene. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays further demonstrated that FOXM1 could bind to a particular site (-635acaaacaa-628) in the promoter region of the Mcl-1 gene. Moreover, CCK-8 assays and analyses of apoptosis revealed that the suppression of the FOXM1/Mcl-1 pathway induced apoptosis and thus increased sensitivity to oxaliplatin in gastric cancer cells, whereas the enhancement of the FOXM1/Mcl-1 pathway inhibited apoptosis and decreased sensitivity to oxaliplatin in gastric cancer cells. Taken together, this study is the first to not only show that Mcl-1 is a novel target gene of FOXM1 but also suggest that targeting FOXM1/Mcl-1 may be a novel strategy to enhance sensitivity to oxaliplatin in gastric cancer.

ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence "GTFG," and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as "F-D value," can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (R(2) = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.

Both brain native inflammatory cells and infiltrated peripheral white blood cells (WBCs) are primary participants in the brain inflammatory damage post-TBI. Metabotropic glutamate receptor 5 (mGluR5) has been reported to regulate microglias and astrocytes to affect inflammation after TBI, but its effect on modulating infiltrated peripheral WBCs remains unclear. In a mouse moderate TBI model, we found that mGluR5 knockout (KO) significantly reduced neutrophil infiltration and inflammatory cytokine expression in the brain at 24 hours post TBI, which was accompanied by improved neurological dysfunction. Further investigation indicated that mGluR5 KO reduced the permeability of blood-brain barrier (BBB), the entrance for neutrophils to enter brain, and markedly decreased the mRNA levels of neutrophil-associated chemokines in brain tissue, including CXCL1, CXCL2, CCL2, CCL4 and CCL5. Using brain microvascular endothelial cells (BMECs), neutrophils and a BBB model in vitro, we confirmed the inhibitory effect of mGluR5 deficiency on neutrophil infiltration and demonstrated that blockade of protein kinase C (PKC) signaling was involved in it. These results provide insight into the role of mGluR5 in the regulation of inflammation in the acute phase of TBI, which may provide novel clues for TBI therapy.

Background— Long noncoding RNAs (lncRNAs) have recently been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results— We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically downregulated in atherosclerotic plaques of ApoE −/− mice, an animal model for atherosclerosis. Through loss- and gain-of-function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and to bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in patients with coronary artery disease. Conclusions— Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders.

The aberrant activation of telomerase is critical for the initiation and development of human cervical cancer, which is dependent on the activation of human telomerase reverse transcriptase (hTERT). Recently, Pin2/TRF1-interacting protein X1 (PinX1) has been identified as a suppressor of hTERT. It has been found that the telomerase is activated while the level of PinX1 is decreased in cervical cancer. However, the regulatory mechanism of PinX1 in cervical cancer cells remains unclear. In the present study, we demonstrated that the level of PinX1 is regulated by p53, and p53 functions as a transcriptional factor to directly activate the expression of PinX1 in cervical cancer cells. Moreover, we found that HPV16 E6 suppresses the expression of PinX1 via inhibiting p53 transcriptional activity, resulting in the enhancement of telomerase activity. This study not only for the first time shows that PinX1 is a novel target gene of p53 but also suggests that suppression of p53/PinX1 pathway may be a novel mechanism by which HPV16 E6 enhances the telomerase activity in cervical cancer cells.

Renal fibrosis is the common pathway of most chronic kidney disease progression to end-stage renal failure. The nuclear receptor FXR (farnesoid X receptor), a multiple functional transcription factor, plays an important role in protecting against fibrosis. The TGFβ-Smad signaling has a central role in kidney fibrosis. However, it remains unclear whether FXR plays direct anti-fibrotic effect in renal fibrosis via regulating TGFβ-Smad pathway. In this study, we found that the level of FXR was negatively correlated with that of Smad3 and fibronectin (a marker of fibrosis) in human fibrotic kidneys. Activation of FXR suppressed kidney fibrosis and downregulated Smad3 expression, which was markedly attenuated by FXR antagonist. Moreover, the FXR-mediated repression of fibrosis was significantly alleviated by ectopic expression of Smad3. Luciferase reporter assay revealed that FXR activation inhibited the transcriptional activity of Smad3 gene promoter. The in vivo experiments showed that FXR agonist protected against renal fibrosis and downregulated Smad3 expression in UUO mice. These results suggested that FXR may serve as an important negative regulator for manipulating Smad3 expression, and the FXR/Smad3 pathway may be a novel target for the treatment of renal fibrosis.

Increased expression of BAFF (B cell-activating factor belonging to the TNF family) and its receptors has been identified in numerous B-cell malignancies. A soluble human BAFF mutant (mBAFF), binding to BAFF receptors but failing to activate B-lymphocyte proliferation, may function as a competitive inhibitor of BAFF and may serve as a novel ligand for targeted therapy of BAFF receptor-positive malignancies. Pin2/TRF1-interacting protein X1 (PinX1), a nucleolar protein, potently inhibits telomerase activity and affects tumorigenicity. In this study, we generated novel recombinant proteins containing mBAFF, a polyarginine tract 9R and PinX1 (or its C/N terminal), to target lymphoma cells. The fusion proteins PinX1/C-G(4)S-9R-G(4)S-mBAFF and PinX1/C-9R-mBAFF specifically bind and internalize into BAFF receptor-positive cells, and subsequently induce growth inhibition and apoptosis. The selective cytotoxicity of the fusion proteins is a BAFF receptor-mediated process and depends on mBAFF, PinX1/C and 9R. Moreover, the fusion proteins specifically kill BAFF receptor-expressing Burkitt's lymphoma (BL) cells by inhibiting telomerase activity and the consequent shortening of telomeres. Therapeutic experiments using PinX1C-G(4)S-9R-G(4)S-mBAFF in severe combined immunodeficient (SCID) mice implanted with Raji cells showed significantly prolonged survival times, indicating the in vivo antitumor activity of the fusion protein. These results suggest the potential of PinX1/C-G(4)S-9R-G(4)S-mBAFF in targeted therapy of BL.

A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (-)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy through blocking Bcl-2-Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (-)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (-)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (-)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.

In this study, we examined the long-term influence of a high-fat (HF) diet compared with a standard chow (SC) diet on glucose metabolism in genetically normal Sprague-Dawley rats. As the diet was given for a period of 14 months it represents almost a life-long exposure of the rats to the diet. Results showed that (1) after exposure to 14 months of SC or HF feeding, the fasting glucose levels were below 5.1 mmol/l in both genders; (2) the 2 h plasma glucose concentration (2 h PG) of male rats after an intraperitoneal glucose tolerance test for the SC groups and for the HF groups was significantly higher than for females; (3) the liver triglycerides content was significantly enhanced by HF feeding as compared with SC-feeding, and histological staining showed that both genders of the HF group developed severe liver steatosis; (4) the fasting insulin concentration in blood of HF-fed males was significantly higher than that of females and homeostasis model assessment of insulin resistance was also significantly higher; (5) the insulin secretion capabilities in response to glucose stimulation were significantly higher in HF-fed female rats than that of males. Taken together, these data indicate that glucose intolerance produced by a HF diet in rats shows a gender difference, and females are protected against development of impaired glucose tolerance under HF condition.

Malignant hematologic diseases are highly malignant and refractory to conventional therapies. Ligand-mediated targeting of liposomal anticancer drugs to surface receptors expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. BAFF plays an important role in the maintenance of normal B-cell development and homeostasis and the expression of its receptors is significantly increased in numerous B-cell malignancies. mBAFF (a soluble BAFF mutant with amino acid 217-224 being replaced by two glycine residues) may be used as a competitive inhibitor for BAFF to treat relevant malignant hematologic diseases. It may also hold promise as a novel ligand for targeted anticancer therapy. In this study, we show that liposomes that are sterically stabilized by PEG and surface decorated with mBAFF exhibited strong affinity and specificity to cultured human Raji B lymphoma cells. Vincristine formulated in the targeted liposomes showed significantly higher levels of cytotoxicity towards Raji cells than the nontargeted liposomal drug. Therapeutic experiments in SCID mice implanted with Raji cells showed significantly prolonged survival time with targeted liposomal vincristine compared to either free VCR or vincristine formulated in nontargeted liposomes. These studies suggest the potential of the mBAFF-modified liposomal drugs in targeted therapy of B-cell malignancies.

Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation and are associated with the development of cancers. To investigate the important role and mechanism of lncRNAs in the progression of gastric cancer, we screened lncRNAs in gastric cancer tissues and corresponding adjacent tissues, and assessed the effects on gastric cancer. Here, we report that BC032469, a novel lncRNA, expressed highly in gastric cancer tissues, and the upregulation was clinically associated with larger tumor size, poor differentiation and shorter survival of gastric cancer patients. Downregulation of BC032469 resulted in a significant inhibition of proliferation in vitro and in vivo. Mechanistically, BC032469 could directly bind to miR-1207-5p and effectively functioned as a sponge for miR-1207-5p to modulate the derepression of hTERT. Thus, BC032469 may function as a ceRNA to impair miR-1207-5p-dependent hTERT downregulation, suggesting that it may be clinically valuable as a poor prognostic biomarker of gastric cancer.

The aim of the present study was to evaluate whether the anti-Rheumatoid arthritis (RA) effect of curcumin is associated with the regulation of B cell-activating factor belonging to the TNF family (BAFF) production.Collagen-induced arthritis (CIA) was induced in DBA/1 J mice by immunization with bovine type II collagen. To investigate the anti-arthritic effect of curcumin in the CIA model, mice were injected intraperitoneally with curcumin (50 mg/kg) on every other day either from day 1 or from day 28 after the first immunization. The clinical severity of arthritis was monitored. BAFF, interleukin-6 (IL-6) and interferon-γ (IFNγ) production in serum were measured. Furthermore, the effect of curcumin on IFNγ-induced BAFF expression and transcriptional activation in B lymphocytes was determined by qPCR, Western Blot, and luciferase assay. Finally, IFNγ related signal transducers and activators of transcription 1 (STAT1) signaling in B lymphocytes were studied using Western Blot.Curcumin dramatically attenuated the progression and severity of CIA in DBA/1 J mice, accompanied with decrease of BAFF production in serum and spleen cells as well as decrease of serum IFNγ and IL-6. Treatment of B lymphocytes with curcumin suppressed IFNγ-induced BAFF expression, STAT1 phosphorylation and nuclear translocation, suggesting that curcumin may repress IFNγ-induced BAFF expression via negatively interfering with STAT1 signaling.The results of the present study suggest that suppression of BAFF production may be a novel mechanism by which curcumin improves RA.

Octamer-binding transcription factor 4 (Oct4), an important embryonic transcriptional factor, is highly expressed in several tumors and is considered as a hallmark of cancer stem cells. Knowledge about the expression and regulatory mechanisms of Oct4 can contribute to the treatment of cancers. As for cervical cancer, however, details remain obscure about Oct4 expression and its regulatory mechanism. In this study, we found that the level of Oct4 in human papillomavirus 16 (HPV16)- positive cervical cancer cells (CaSki cells) was higher than that in HPV-negative cervical cancer cells (C-33A cells), whereas both the level of histone deacetylase 1 (HDAC1) and DNA methyltransferase 3A (DNMT3A) were lower in CaSki cells than those in C-33A cells. Treatment with valproic acid, an HDAC inhibitor, could significantly increase the expression of Oct4 in C-33A cells, but only slightly increased Oct4 in CaSki cells. Co-immunoprecipitation assays showed that HDAC1 and DNMT3A existed in a common complex. The co-immunoprecipitated DNMT3A or HDAC1 was dose-dependently decreased with valproic acid treatment. These results indicated that HDAC1/DNMT3A-containing complex is associated with the suppression of Oct4 in cervical cancer cells, and the activity of HDAC1 is required in the repression of Oct4.

The colony-forming ability of cervical cancer is affected by many factors. Oct4, an important transcription factor, is highly expressed in several tumors and promotes the colony-forming ability of cancer cells. Thus, it is considered a potential target for the treatment of cancer. However, we know little about the expression level of Oct4 and its epigenetic regulatory mechanism in cervical cancer cells. In this study, we are the first to observe that human papillomavirus (HPV)-positive cervical cancer cell lines (HeLa, Caski) have a stronger colony-forming ability than HPV-negative cervical cancer cell lines (C-33A). Moreover, the expression level of Oct4 in both HeLa and Caski cells was also higher than that in C-33A cells. We then confirmed that there was a negative correlation between the expression of Oct4 and DNMT3A in these three types of cervical cancer cells, whereas DNA methyltransferase 1 and 3B had no differences among the cell lines. However, after DNA methylation in both key regulatory regions of the Oct4 gene and the genomic levels were analyzed, we found that DNA methyltransferase 3A could neither regulate the expression of Oct4 nor affect the whole level of genomic DNA methylation. These results suggest three points: (1) Oct4 might be treated as a new target for the treatment of cervical cancer, (2) we could not inhibit the expression of Oct4 by DNA demethylation, and (3) HPV virus might initiate cervical carcinogenesis by upregulation of Oct4 expression.

Overexpression of B lymphocyte stimulator (BLyS) is closely involved in the pathogenesis and progression of some autoimmune diseases. Curcumin, a pharmacologically safe agent, has been shown to possess potent anti-inflammatory properties. However, it is not clear whether curcumin affects the expression of BLyS. In this study, we report that curcumin inhibits the expression of BLyS and that a DNA-binding site for the transcriptional factor NF-κB in the BLyS promoter region is required for this regulation. Moreover, we find that curcumin reduces the DNA-binding activity of NF-κB to the BLyS promoter region and suppresses nuclear translocation of p65, suggesting that curcumin may suppress BLyS expression via negatively interfering with NF-κB signaling. These results suggest that curcumin may serve as a novel therapeutic agent in the treatment of autoimmune diseases by targeting BLyS.

Adhesion molecule CD44 is a cell surface glycoprotein, involved in adhesion of extracellular matrix, angiogenesis, cell proliferation, differentiation and migration. Elevated CD44 expression was correlated with poor prognosis in many malignancies, including pancreatic cancer. Recent studies suggest that CD44 is a marker for cancer initiating cells or cancer stem cells. The effects of anti-CD44 monoclonal antibody (MAb) on pancreatic cancer, especially combination therapy with radiation remain largely unknown. In the current study, we evaluated the efficacy of anti-CD44s MAb treatment for human pancreatic cancer both in vitro and in vivo in xenograft models. The anti-CD44s MAb, H4C4, was Protein-A/G affinity purified from ascites and its specific reactivity with cell surface CD44 was confirmed using cellular ELISA. Two human pancreatic cancer cell lines were tested, including MiaPaCa-2 and Panc-1. Anti-CD44s MAb H4C4 significantly inhibited cell growth in MiaPaCa-2 and Panc-1 cells in vitro as compared with control normal mouse IgG (NIgG). Since as a cell adhesion molecule, CD44 plays an important role in anchorage-independent cell growth, we carried out tumorsphere culture assay by culturing cancer cells on ultra-low attachment plates in serum-free condition with either H4C4 or NIgG. The results show that H4C4 dramatically decreased the tumorsphere formation and sphere size in the CD44+ cells. In tumor formation assays, 1 hr pre-incubation of MiaPaCa-2 cells with H4C4 totally blocked tumorigenesis after s.q. implantation in nude mice, but not with NIgG. To evaluate the efficacy of anti-CD44s MAb treatment in vivo, we employed the pancreatic cancer MiaPaCa-2 and Panc-1 orthotopic models in nude mice. The mice received H4C4 or NIgG 4 mg/kg, i.v., three times per week for 5 weeks. H4C4 treatment significantly inhibited tumor growth and metastasis (p Taken together, our data demonstrate that anti-CD44s MAb strongly inhibits the CD44+ tumor growth, metastasis and recurrence in pancreatic cancer models. Mechanism studies indicate that H4C4 blocked CD44-STAT3 signaling pathway, accompanied by reduction of pancreatic cancer stem cells. Our data demonstrate that CD44 may be a promising molecular target for inhibiting metastasis and post-radiotherapy recurrence of human pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 565. doi:10.1158/1538-7445.AM2011-565

Radioresistance markedly impair the efficacy of cancer therapy. Anti-apoptotic Bcl-2 family proteins such as Bcl-xL, Bcl-2 and Mcl-1 are overexpressed in castration-resistant prostate cancer (CRPC) and contribute to prostate tumor initiation, progression and resistance to radiotherapy. A natural BH3-mimetic, small molecule inhibitor of Bcl-2, (−)-gossypol, shows promise in ongoing Phase II clinical trials for human prostate cancer. We have recently shown that (−)-gossypol preferentially induces autophagy in CRPC cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent cells with low Bcl-2 and sensitive to apoptosis. Our results demonstrate for the first time that (−)-gossypol can interrupt the interaction between Beclin1 and Bcl-2/Bcl-xL at the endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. (−)-Gossypol-induced autophagy is Beclin1- and Atg5-dependent, together with Bcl-2 downregulation and Beclin1 upregulation. (−)-Gossypol increased autophagic cell death induced by X-ray radiation in the CRPC cells. Orally administered (−)-gossypol achieved a much greater efficacy with long-term tumor regression when used in combination with ionizing radiation. We have also explored a series of gossypol derivatives and identified two novel BH3-mimetic lead compounds that are more potent than the parental compound in inducing autophagic cell death in CRPC. Taken together, (−)-gossypol significantly enhances the anti-tumor activity of radiotherapy in vitro and in vivo, and represents a promising new regime for treatment of castration-resistant human prostate cancer with overexpression of Bcl-2. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which would facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5085. doi:10.1158/1538-7445.AM2011-5085

Radioresistance markedly impair the efficacy of cancer therapy and involve anti-apoptotic signal transduction pathways that prevent the treatment-induced cell death. Anti-apoptotic Bcl-2 family proteins such as Bcl-xL, Bcl-2 and Mcl-1 are overexpressed in prostate cancer and contribute to prostate tumor initiation, progression and resistance to radiotherapy. (−)-Gossypol, a natural product from cottonseed, has recently been identified as a potent small molecule inhibitor of Bcl-2/Bcl-xL/Mcl-1 and is now in advanced clinical trials. Since Bcl-2 also inhibits autophagy and radiation induces autophagy, we hypothesize that (−)-gossypol may improve prostate cancer's response to radiotherapy by inducing autophagy in apoptosis-resistant prostate cancer cells with high Bcl-2. (−)-Gossypol inhibits tumor cell growth and preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and resistant to apoptosis, both in vitro and in vivo, but not in cells with low Bcl-2. (−)-Gossypol radiosensitized AI-prostate cancer PC-3 and CL-1 cells in clonogenic assay. (−)-Gossypol increased autophagy induced by X-ray radiation in these cells. Orally administered (−)-gossypol achieved a much greater efficacy with long-term tumor regression when used in combination with ionizing radiation. Immunoprecipitation /pull-down assay showed that (−)-gossypol induced autophagy via blocking Bcl-2-Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1 and activating autophagic pathway signaling. Knockdown of the Beclin1 or atg5 expression blocked the autophagy induced by (−)-gossypol or radiation. Taken together, our data demonstrate that (−)-Gossypol exerts its radiosensitizing activity through promoting radiation-induced autophagy, via modulating Bcl-2-Beclin1 interaction and atg5. (−)-Gossypol significantly enhances the anti-tumor activity of radiotherapy in vitro and in vivo, and represents a promising new regime for treatment of hormone-refractory human prostate cancer with overexpression of Bcl-2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4851.