Analysis of apoptosome dysregulation in pancreatic cancer and of its role in chemoresistance

The apoptosome is a multiprotein complex mediating the mitochondrial pathway of cell death. Its importance during development has been clearly demonstrated by knocking out key genes in mouse. APAF1 is the core protein of the apoptosome and its dosage is also critical in various cancer types, i.e., melanoma, germ line tumor, gastrointestinal cancer and B-type chronic lymphocytic leukemia. This is generally due to inactivation of the APAF1 locus by epigenetic phenomena or by activity of promoter regulators. We investigated the putative roles of the apoptosome in pancreatic ductal adenocarcinoma (PDAC). We found that both APAF1 mRNA and protein are dysregulated in human PDAC samples. Similarly, several PDAC cell lines exhibited variable levels of both APAF1 protein and mRNA. The response to cell death induction and its biochemical features were assessed by treatment of each line with commonly used chemotherapeutic agents. We found that the apoptosome pathway was not functional in most cell lines upon cytochrome c release from mitochondria. In addition, we restored APAF1 and Caspase-9 dosage in Panc-1 cells, where the apoptosome is downregulated, by overexpressing the murine cDNA of the two molecules, and we improved the death response to chemotherapeutic agents.     

Ovarian cancer microenvironment: implications for cancer dissemination and chemoresistance acquisition

Ovarian adenocarcinoma is characterized by a late detection, dissemination of cancer cells into the whole peritoneum, and the frequent acquisition of chemoresistance. If these particularities can be explained in part by intrinsic properties of ovarian cancer cells, an increased number of studies show the importance of the tumor microenvironment in tumor progression. Ovarian cancer cells can regulate the composition of their stroma in promoting the formation of ascitic fluid, rich in cytokines and bioactive lipids, and in stimulating the differentiation of stromal cells into a pro-tumoral phenotype. In return, cancer-associated fibroblasts, cancer-associated mesenchymal stem cells, tumor-associated macrophages, or other peritoneal cells, such as adipocytes and mesothelial cells can regulate tumor growth, angiogenesis, dissemination, and chemoresistance. This review focuses on the current knowledge about the roles of stromal cells and the associated secreted factors on tumor progression. We also summarize the different studies showing that targeting the microenvironment represents a great potential for improving the prognosis of patients with ovarian adenocarcinoma.     

Galectin-3 induces ovarian cancer cell survival and chemoresistance via TLR4 signaling activation

Tumor Biology - Paclitaxel resistance becomes common in patients with aggressive ovarian cancer and results in recurrence after conventional therapy. Galectin-3 is a multifunctional lectin...     

凋亡抑制蛋白与卵巢癌化疗耐药

卵巢癌是严重威胁妇女健康的恶性肿瘤之一,其病死率在妇科肿瘤中占第一位.化疗耐药是导致其治疗失败、病死率居高不下的重要原因.已有的研究表明,抗凋亡机制在肿瘤细胞发生、发展及化疗耐药中起重要作用.凋亡抑制蛋白(IAPs)是重要的细胞凋亡调节因子,在细胞凋亡中起关键作用.本文主要综述IAPs与卵巢癌化疗耐药的相关进展.     

卵巢癌化疗耐药作用机制的基因研究进展

卵巢癌是妇科肿瘤中致死率最高的肿瘤,其主要原因之一是卵巢癌细胞易对化疗产生耐药而导致治疗失败。卵巢癌的发生与发展常常联系一系列基因的变化,从分子水平上了解卵巢癌细胞对化疗产生耐药机理,寻找更有效的化疗药物,是提高卵巢癌疗效的关键。本文将近年来此方面的研究进展作一概括。研究发现HER-2、XIAP和bcl-X(L)基因的高表达、Bcl-2和c-myc基因的低表达与卵巢癌患者对化疗药物产生耐药有关。一系列具抗耐药和与卵巢癌化疗药物有协同作用的基因SU5416、EpoB、Apo2L/TRAIL、PSC833、TP53、BCL-2、BAX和MEK1蛋白、MAPKs、JNK、p38和ERK的出现,为改善卵巢癌化疗效果,提高治愈率,带来了新的希望。     

TGFBI promoter hypermethylation correlating with paclitaxel chemoresistance in ovarian cancer

The purpose of this study is to determine the methylation status of Transforming growth factor-beta-inducible gene-h3 (TGFBI) and its correlation with paclitaxel chemoresistance in ovarian cancer. The methylation status of TGFBI was examined in ovarian cancer and control groups by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP). The TGFBI expression and cell viability were compared by Quantitative Real-Time PCR, Western Blotting and MTT assay before and after demethylating agent 5-aza-2''-deoxycytidine (5-aza-dc) treatment in 6 cell lines (SKOV3, SKOV3/TR, SKOV3/DDP, A2780, 2780/TR, OVCAR8). In our results, TGFBI methylation was detected in 29/40 (72.5%) of ovarian cancer and 1/10 (10%) of benign ovarian tumors. No methylation was detected in normal ovarian tissues (P < 0.001). No statistical correlation between RUNX3 methylation and clinicopathological characteristics was observed. A significant correlation between TGFBI methylation and loss of TGFBI mRNA expression was found (P < 0.001). The methylation level of TGFBI was significantly higher in paclitaxel resistant cell lines (SKOV3/TR and 2780/TR) than that in the sensitive pairs (P < 0.001). After 5-aza-dc treatment, the relative expression of TGFBI mRNA and protein increased significantly in SKOV3/TR and A2780/TR cells. However, no statistical differences of relative TGFBI mRNA expression and protein were found in other cells (all P > 0.05), which showed that re-expression of TGFBI could reverse paclitaxel chemoresistance. Our results show that TGFBI is frequently methylated and associated with paclitaxel-resistance in ovarian cancer. TGFBI might be a potential therapeutic target for the enhancement of responses to chemotherapy in ovarian cancer patients.     

Intracellular clusterin negatively regulates ovarian chemoresistance: compromised expression sensitizes ovarian cancer cells to paclitaxel

Understanding the molecular events that lead to paclitaxel (TX) resistance is necessary to identify effective means to prevent chemoresistance. Previously, results from our lab revealed that secretory clusterin (CLU) form positively mediates TX response in ovarian cancer cells. Thus, we had interest to study the role of another non-secreted form (intracellular clusterin (i-CLU)) in chemo-response. Here, we provide evidences that i-CLU form localizes mainly in the nucleus and differentially expressed in the TX-responsive KF cells, versus TX-resistant, KF-TX, ovarian cancer cells and negatively regulate cellular chemo-response. I-CLU was cloned, by deleting the secretion-leading signaling peptide from full-length CLU cDNA, and transiently over-expressed in OVK-18 cells. Forced expression of truncated i-CLU was mainly detectable in the nuclei and significantly reduced cellular growth, accumulating cells in G1 phase which finally died through apoptosis. Importantly, compromised expression of i-CLU under an inducible promoter was tolerated and did not induce apoptosis but sensitized ovarian cancer cells to TX. We then demonstrated that this sensitization mechanism was cell cycle independent and relied on i-CLU/Ku70 binding probably due to controlling the free amount of Ku70 available for DNA repair in the nucleus. Results from CLU immunehistochemistry in ovarian tumor tissues verified the retardation of nuclear CLU staining in the recurrent tumor even though their primary counterparts showed nuclear CLU staining. Thus, the controversial data on CLU function in chemo-response/resistance may be explained by a shift in the pattern of CLU expression and intracellular localization as well when tumor acquires chemoresistance.     

Exosomal transfer of miR-429 confers chemoresistance in epithelial ovarian cancer

The development of multidrug resistance during chemotherapy is the main obstacle for epithelial ovarian cancer (EOC) treatment. Exosomal transfer of carcinogenic microRNAs (miRNAs) might strengthen chemoresistance in recipient cells. Here, we identified through microarray analysis higher miR-429 expression in multidrug-resistant SKOV3 cells and their secreted exosomes (SKOV3-EXO) than in sensitive A2780 cells and their secreted exosomes. SKOV3-derived exosomes were internalized by A2780 cells, which permitted the transfer of miR-429. Exosomal miR-429 enhanced the proliferation and drug resistance of A2780 cells by targeting calcium-sensing receptor (CASR)/STAT3 pathway in vitro and in vivo. In addition, NF-κB-p65 was predicted to bind to the miR-429 promoter region, and the inhibition of NF-κB reduced the expression of miR-429 and led to the sensitivity of EOC cells. Consistently, A2780 cells co-incubated with SKOV3 pretreated with an NF-κB inhibitor or miR-429 antagomir showed sensitivity to cisplatin and exhibited attenuated cell proliferation. Based on our data, exosomal miR-429 functions as a primary regulator of the chemoresistance and malignant phenotypes of EOC by targeting CASR through a mechanism promoted by NF-κB and might be a therapeutic target for EOC.     

TLR-4 signaling promotes tumor growth and paclitaxel chemoresistance in ovarian cancer

Evidence suggests that an inflammatory profile of cytokines and chemokines persisting at a particular site would lead to the development of a chronic disease. Recent studies implicate bacterial infection as one possible link between inflammation and carcinogenesis; however, the crucial molecular pathways involved remain unknown. We hypothesized that one possible upstream signaling pathway leading to inflammation in carcinogenesis may be mediated by Toll-like receptors (TLR). We describe for the first time an adaptive mechanism acquired by ovarian cancer cells that allows them to promote a proinflammatory environment and develop chemoresistance. We propose that the TLR-4-MyD88 signaling pathway may be a risk factor for developing cancer and may represent a novel target for the development of biomodulators. Our work explains how bacterial products, such as lipopolysaccharide, can promote, directly from the tumor, the production of proinflammatory cytokines and the enhancement of tumor survival. In addition, we provide new evidence that links TLR-4 signaling, inflammation, and chemoresistance in ovarian cancer cells.     

Aurora-A induces cell survival and chemoresistance by activation of Akt through a p53-dependent manner in ovarian cancer cells

Aurora-A is frequently altered in epithelial malignancies. Overexpressing Aurora-A induces centrosome amplification and G2/M cell cycle progression. We have previously shown elevated level of Aurora-A in ovarian cancer and activation of telomerase by Aurora-A in human mammary and ovarian epithelia. Here we report that Aurora-A protects ovarian cancer cells from apoptosis induced by chemotherapeutic agent and activates Akt pathway in a p53-dependent manner. Ectopic expression of Aurora-A renders cells resistant to cisplatin (CDDP), etoposide and paclitaxel-induced apoptosis and stimulates Akt1 and Akt2 activity in wild-type p53 but not p53-null ovarian cancer cells. Aurora-A inhibits cytochrome C release and Bax conformational change induced by CDDP. Knockdown of Aurora-A by RNAi sensitizes cells to CDDP-induced apoptosis and decreases phospho-Akt level in wild-type p53 cells. Reintroduction of p53 decreases Akt1 and Akt2 activation and restores CDDP sensitivity in p53-null but not p53-null-Aurora-A cells. Inhibition of Akt by small molecule inhibitor, API-2, overcomes the effects of Aurora-A-on cell survival and Bax mitochondrial translocation. Taken collectively, these data indicate that Aurora-A activates Akt and induces chemoresistance in a p53-dependent manner and that inhibition of Akt may be an effective means of overcoming Aurora-A-associated chemoresistance in ovarian cancer cells expressing wild-type p53.     

Contribution of c-erbB-2 and topoisomerase IIalpha to chemoresistance in ovarian cancer.

Overexpression of the c-erbB-2 (HER-2/neu) oncogene, which encodes a transmembrane receptor tyrosine kinase, has been shown to be associated with poor prognosis in ovarian and breast cancer. Recent studies indicate that c-erbB-2 may also be involved in determining the chemosensitivity of human cancers. In the present study, we examined the role of c-erbB-2 for chemoresistance in ovarian cancer. Overexpression of c-erbB-2 mRNA in tumor tissue was associated with a shorter survival of patients with primary ovarian cancer (P = 0.0001; n = 77) and was an independent prognostic factor in the proportional-hazard model adjusted for International Federation of Gynecologists and Obstetricians stage, residual disease, chemotherapy, and age (P = 0.035). A significant association between expression of c-erbB-2 mRNA and survival was obtained for the subgroup of patients who received a standard chemotherapy with carboplatin or cisplatin and cyclophosphamide (P = 0.0003), whereas only a nonsignificant trend was observed for patients who did not receive a standard chemotherapy (P = 0.124). In addition, the application of a standard chemotherapy improved the survival of patients with relatively low c-erbB-2 expression (P = 0.013) but not of patients with overexpression of c-erbB-2 (P = 0.359). Expression of c-erbB-2 mRNA correlated with expression of topoisomerase IIalpha mRNA determined by a reverse semiquantitative PCR technique (P = 0.009), whereas expression of c-erbB-2 and topoisomerase IIbeta mRNA did not correlate (P = 0.221). To examine the hypothesis that coamplified and/or coregulated topoisomerase IIalpha contributes to the resistance of c-erbB-2-overexpressing carcinomas, we established a chemosensitivity assay using primary cells from an ovarian carcinoma that overexpressed both c-erbB-2 and topoisomerase IIalpha. The combination of carboplatin with nontoxic concentrations of the topoisomerase II inhibitors etoposide or novobiocin enhanced the toxicity of carboplatin. In contrast, the tyrosine kinase inhibitor emodin exhibited no chemosensitizing effect in cells of this individual carcinoma. In conclusion, overexpression of c-erbB-2 was associated with poor prognosis and poor response to chemotherapy. The data suggest that topoisomerase IIlalpha, which correlates with c-erbB-2 expression, contributes to the resistance of c-erbB-2-overexpressing carcinomas.     

卵巢癌化疗耐药及逆转耐药基因水平研究进展

卵巢癌细胞对化疗产生耐药的机制主要有肿瘤细胞内有效药物浓度降低、DNA损伤修复功能异常和细胞凋亡调控异常3个方面.逆转卵巢癌细胞耐药主要包括反义基因治疗、RNA干预、化疗药物的联合应用等.     

Decreased ARID1A expression is correlated with chemoresistance in epithelial ovarian cancer

Objective

Loss of ARID1A is related to oncogenic transformation of ovarian clear cell adenocarcinoma. The present study was conducted in epithelial ovarian cancer of all tissue types to investigate whether an increased or decreased expression level of ARID1A can be a prognostic factor for ovarian cancer or can influence the sensitivity to anticancer drugs.

Methods

The expression level of ARID1A was investigated in 111 patients with epithelial ovarian cancer who received initial treatment at the Hirosaki University Hospital between 2006 and 2011. The expression level of ARID1A was immunohistochemically graded using staining scores, which were calculated by multiplying the staining intensity of the nuclei by the stain-positive area.

Results

The level of ARID1A was significantly lower in clear cell adenocarcinoma than in other histologic types. Among the patients with stage III, IV cancer (n=46), the level of ARID1A was significantly lower (p=0.026) in patients who did not achieve complete response (CR; n=12) than in patients who achieved CR (n=34). The level of ARID1A was relatively lower (p=0.07) in patients who relapsed after achieving CR (n=21) than in patients who did not relapse (n=13). When the staining score of 0 was defined as ARID1A-negative and other staining scores were defined as ARID1A-positive, there was significant difference in progression-free survival between ARID1A-negative (n=11) and ARID1A-positive (n=35) patients in stage III, IV disease.

Conclusion

The result suggests that decreased ARID1A expression is correlated with chemoresistance and may be a predictive factor for the risk of relapse of advanced cancer after achieving CR.     

Hsp70 promotes chemoresistance by blocking Bax mitochondrial translocation in ovarian cancer cells

Cisplatin can induce apoptosis in ovarian cancer cells through the mitochondrial death pathway, and dysregulation of this pathway contributes to cisplatin resistance in ovarian cancer cells. Here we show that cisplatin induces mitochondrial proteins such as Smac/DIABLO, Cytochrome c, and HrtA2/Omi release to the cytosol and apoptosis in cisplatin-sensitive, but not -resistant ovarian cancer cells. Bax translocation to mitochondria is required for mitochondrial protein release and cisplatin-induced apoptosis in human ovarian cancer cells. Hsp70 is highly expressed in cisplatin-resistant cells. Hsp70 promotes chemoresistance, in part, by blocking Bax translocation to the mitochondria and mitochondrial protein release to cytosol in human ovarian cancer cells.