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Background: Ovarian cancer (OvCa) is the leading cause of death of gynecological malignancies worldwide. Vascular endothelial growth factor A (VEGFA), the most potent angiogenic factor, is responsible for tumor growth and angiogenesis, but its role in OvCa chemotherapy resistance remains unclear. Methods: RT-PCR and Western blot were used to detect VEGFA expression in tumor cells and normal ovarian surface epithelial cells. Gene Ontology (GO) enrichment analysis was used to analyze GO terms correlated with VEGFA. In in vitro experiments, we knockdown VEGFA in tumor cells and detected the tumor cell viability and apoptosis after chemotherapy drug treatment by MTT assay and flow cytometry. Western blot was used to detect autophagy and apoptosis related proteins. Results: We proved that VEGFA was highly expressed in tumor cells comparted with normal ovarian surface epithelial cells, and enriched GO analysis of VEGFA showed that VEGFA was involved in anti-apoptotic process. Further in vitro experiments confirmed that expression of VEGFA was correlated with chemotherapy resistance and this effect was mediated by autophagy. Meanwhile tumor cells treated with chemotherapy drugs also promoted the expression of VEGFA. Knockdown VEGFA inhibited autophagy of tumor cells and thus potents the killing efficiency in DDP resistant tumor cells and this effect could be reversed by the addition of recombinant VEGFA. Conclusion: Taken together, our study demonstrates that VEGFA is involved in anti-apoptosis of tumor cells to chemotherapy, killing partly through autophagy, indicating that VEGFA may serve as a potential target to improve chemotherapy treatment. [ABSTRACT FROM AUTHOR]

Background: The dysregulation of the human papillomavirus 18 E6 and E7 oncogenes plays a critical role in the angiogenesis of cervical cancer (CC), including the proliferation, migration, and tube formation of vascular endothelial cells. Interfering E6/E7 increases the number of CC cell-derived microvesicles (CC-MVs). Additionally, microRNAs (miRNAs) can modulate CC angiogenesis and can be encapsulated in MVs. Objective: We aim to investigate whether E6/E7 affects CC angiogenesis via regulating miRNAs in CC-MVs. Methods: CC-MVs were isolated from a CC cell line (HeLa) which were transfected with small interfering RNAs (siRNAs) against E6/E7 or co-transfected with miR-377 mimics/inhibitors. The expression of several miRNAs in CC-MVs was detected using quantitative real-time PCR. After co-incubating CC-MVs with human umbilical vein endothelial cells (HUVECs), cell proliferation, migration, and tube formation of HUVECs were determined using cell counting kit-8, transwell, and tube formation assays, respectively. Results: MiR-377 was increased in E6/E7-interfering CC-MVs. Overexpressing miR-377 in CC-MVs suppressed HUVEC proliferation, migration, and tube formation. LPAR2, the cell surface G protein-coupled receptor, was the downstream target of miR-377 in HUVECs. The co-transfection of E6/E7 siRNAs and miR-377 inhibitors in CCs negated the effect of E6/E7 siRNAs on the elevation of miR-377 in CC-MVs. In HUVECs, the co-transfection of E6/E7 siRNAs and miR-377 inhibitors restored the LPAR2 expression which was reduced by the E6/E7 siRNA transfection. Meanwhile, miR-377 mimic reduced LPAR2 expression and inhibited HUVEC proliferation, migration, and tube formation, while such response was negated by LPAR2 overexpression. Conclusion: Interfering E6/E7 increased miR-377 in CC-MVs, and overexpressing miR-377 in CC-MVs inhibited angiogenesis of HUVECs via reducing LPAR2. [ABSTRACT FROM AUTHOR]

Background: The ubiquitin-proteasome system and macroautophagy (hereafter referred to autophagy) are two complementary pathways for protein degradation. Emerging evidence suggests that proteasome inhibition might be a promising approach for tumor therapy. Accumulating data suggest that autophagy is activated as a compensatory mechanism upon proteasome activity is impaired. Method: Autophagy activation was measured using acridine orange staining and LC3 transition. Cell viability and apoptosis were measured using MTT assay and flow cytometry, respectively. Beclin 1 expression vectors or shRNA against Beclin 1 (shBeclin 1) were transfected to investigate the role of Beclin 1 in autophagy activation and cytotoxicity of ovarian cancer cells induced by proteasome inhibitors. Results: Proteasome inhibitors suppressed proliferation and induced autophagy in ovarian cancer cells. Neither phosphoinositide 3-kinase (PI3K) inhibitors nor shRNA against Beclin 1 could abolish the formation of acidic vacuoles and the processing of LC3 induced by proteasome inhibitors. Moreover, Beclin 1 overexpression enhanced anti-proliferative effects of proteasome inhibitors in ovarian cancer cells. Conclusions: For the first time, the current study demonstrated that proteasome inhibitors induced PI3K and Beclin 1-independent autophagy in ovarian cancer cells. In addition, this study revealed autophagy-independent tumor suppressive effects of Beclin 1 in ovarian cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: Background and Aim: miRNA is an important factor for tumorigenesis which could act as a potential molecular target for tumor diagnosis. The goal of this study was to explore a new method for visualizing the expression of let-7 in lung adenocarcinoma A549 cells by Cerenkov luminescence imaging (CLI) and gamma camera imaging. Methods: The human sodium/iodine symporter (hNIS) and 3′-UTR sequence of the ras gene (RU) that complementarily binds to let-7 were cloned with hNIS serving as the reporter gene. The expression of hNIS regulated by let-7 in the fusion gene hNIS-RU was constructed; the let-7 primer (pri-let-7), which could specifically bind to RU and the mir-143 primer (pri-mir143) not binding with RU, was cloned. A549 cells were transfected with hNIS or hNIS-RU, and additional cells were cotransfected with hNIS-RU and different concentrations of pri-let-7 or pri-mir143. The cells were incubated with 740kBq 131I-containing media for 1h, 24h after transfection. CLI, gamma camera imaging, and γ counting were subsequently conducted, and the correlation among CLI, gamma camera imaging, and γ counting was compared when cotransfected with pri-let-7. Results: CLI, gamma camera imaging, and radioactive counting showed that hNIS-transfected A549 cells had significantly higher uptake of 131I compared to non-transfected cells. The uptake of 131I in hNIS-RU transfected A549 cells decreased to approximately 70% compared to hNIS-transfected cells, since hNIS-RU expression was suppressed by intracellular let-7. After cotransfection with hNIS-RU and various concentrations of pri-let-7, 131I uptake gradually decreased with increasing pri-let-7, while 131I uptake remained roughly unchanged in the presence of hNIS-RU cotransfected with different amounts of pri-mir143. CLI was highly correlated with gamma camera imaging (r 2 =0.9893) and radioactivity counting (0.9779). Conclusions: Based upon miRNA-regulated reporter genes which mediate the uptake of the radionuclide, both CLI and gamma camera imaging can noninvasively detect miRNA expression in cells, which may provide a new way for the visualization of miRNA expression. [Copyright &y& Elsevier]

In early studies, it was shown that HINT2, which sensitizes cells to mitochondrial apoptosis, is down-regulated in hepatocellular carcinoma (HCC) cells (Martin et al., 2006). However, the molecular mechanism of this effect is unknown. Immunohistochemistry revealed that HINT2 expression is relatively low in pancreatic cancer tissues, compared to that in adjacent tissues (P < 0.05). Furthermore, its expression was related to pathological grade and lymph node metastasis (P = 0.0161 and 0.0108, respectively); in addition, down-regulation of HINT2 was found to be associated with relatively poor prognosis in pancreatic cancer patients. Up-regulation of HINT2 was shown to trigger pancreatic cancer cell apoptosis, decrease mitochondrial membrane potential (ΔΨm), promote intracellular reactive oxygen species (ROS) production, and elevate mitochondrial Ca2+ levels. However, co-treatment of HINT2 overexpressing BxPC-3 cells with ruthenium red partially inhibited HINT2-induced apoptosis, which was associated with a reduction in ΔΨm and an increase in intracellular ROS and mitochondrial Ca2+. According to our results, mitochondrial calcium uptake1 and 2 (MICU1 and MICU2) were down-regulated and the essential MCU regulator (EMRE) was up-regulated in cells transduced with Adv-HINT2. Therefore, we deduced that HINT2 triggers apoptosis in pancreatic cancer cells by regulating mitochondrial Ca2+ influx through the mitochondrial calcium uniporter (MCU). In addition, we found that HINT2 can sensitize BxPC-3 and L3.6pl cells to gemcitabine-induced apoptosis and that gemcitabine up-regulates HINT2 expression. This indicates that gemcitabine-induced apoptosis is related to HINT2 levels. [ABSTRACT FROM AUTHOR]

Alpha 1, 2-fucosyltransferase (FUT 1/2) is a rate-limiting enzyme that catalyzes the synthesis of Lewis y, a cell membrane-associated carbohydrate antigen. In human ovarian cancer, the upregulated expression of FUT1 and Lewis y is associated with advanced pathological stages and involved in cell proliferation, migration and invasion. However, the mechanism underlying the upregulation of FUT1 is largely unknown. Here, we identify an AP-1 binding site in FUT1 promoter in ovarian cancer cells. c-Jun promotes FUT1 expression, thereby enhancing Lewis y biosynthesis in various ovarian cancer cell lines. Moreover, EMSA, luciferase activity and ChIP assays demonstrate c-Jun directly interacts with FUT1 promoter. Furthermore, FUT1 mediates c-Jun-induced cell proliferation in ovarian cancer cells. In human ovarian cancer samples, c-Jun overexpression is linked to malignant degree and positively correlated to FUT1 and Lewis y expression. Taken together, c-Jun could transcriptionally modulate FUT1 expression in ovarian cancer, implicating the potential application of c-Jun inhibitors for human ovarian cancer therapy. [ABSTRACT FROM AUTHOR]