Mir-30b-3p affects the migration and invasion function of ovarian cancer cells by targeting the CTHRC1 gene

BACKGROUND: The aim of this study was to investigate the effect role and mechanism of miR-30b-3p on ovarian cancer cells biological function. METHODS: The expression of miR-30b-3p was detected in ovarian cancer cell lines and normal ovarian epithelial cell line by qRT-PCR. Mir-30b-3p mimic was transfected into OVCAR3 cells. Cell-counting kit-8 (CCK-8) assay was conducted to explore the effect of mir-30b-3p on the OVCAR3 cells' proliferation. Cell cycle and apoptosis were detected by Flow cytometry. Cell invasion ability was detected by Transwell test. The regulation of putative target of miR-30b-3p was verified by double luciferase reporter assays and Western blot. RESULT: We found that miR-30b-3p was downregulated in OVCAR3 cells. Overexpression of miR-30b-3p suppressed proliferation, promoted apoptosis, slowed cell cycle and inhibited migration and invasion of OVCAR3 cells. Bioinformatics analysis identified 3'-untranslated region (3'UTR) of Collagen triple helix repeat-containing 1 (CTHRC1) as the presumed binding site for miR-30b-3p. Detection of double luciferase reporter and Western-Blot result confirmed that CTHRC1 was the target gene of miR-30b-3p. Furthermore, E-cadherin, ß-cadherin and Vimentin protein expression level were changed after transfection of miR-30b-3p. CONCLUSION: miR-30b-3p function as an anti-cancer gene. Overexpression of miR-30b-3p can inhibit the biological function of ovarian cancer cells. MiR-30b-3p targets CTHRC1 gene plays an important role in epithelial-mesenchymal transformation (EMT), and supports miR-30b-3p as a potential biological indicator for ovarian cancer in the future.

Effect of HE4 and PAX8 gene knockdown on malignant biological behaviors of epithelial ovarian cancer cells treated with TC regimen / 中国肿瘤生物治疗杂志

@#Objective: To investigate the effect of human epididymal protein 4 (HE4) and paired box gene 8 (PAX8) gene knockdown on proliferation, migration, invasion and apoptosis of human epithelial ovarian cancer OVCAR3 cells treated with TC regimen (paclitaxel+carboplatin). Methods: Sequences of single-target siRNA (HE4-siRNA or PAX8-siRNA) and double-target siRNA (HE4+PAX8siRNA) as well as negative siRNAwere respectively designed and synthesized, and then linked with plasmid vector pGCsi-H1 to obtain the recombinant plasmids. The obtained recombinant plasmids were then transfected into human epithelial ovarian cancer OVCAR3 cells, namely HE4-siRNA group, PAX8-siRNA group, HE4+PAX8-siRNA group and siRNA-NC group, respectively. The blank control group was also set up (without any treatment). The cells in above five groups were treated with TC regimen (paclitaxel 3.13 g/ml+carboplatin 2.82 µg/ml), and the changes in proliferation, migration, invasion and apoptosis of the cells were detected by MTT, wound-healing assay, Transwell chamber assay, and flow cytometry, respectively. Results: After knocking down the HE4 and PAX8 genes, compared with siRNA-NC group and blank control group, the proliferation, migration and invasion abilities of OVCAR3 cells in HE4-siRNA group, PAX8-siRNA group and HE4+PAX8-siRNA group significantly decreased (all P<0.01), and the apoptosis rate significantly increased (P<0.01), especially in HE4+PAX8-siRNA group. Conclusion: Knockout of either HE4 or PAX8 can enhance the effect of TC regimen on inhibiting proliferation, migration and invasion as well as promoting apoptosis of epithelial ovarian cancer cells, and the effect of simultaneous down-regulation of HE4 together with PAX8 is better.

Detection of chemosensitivity using K18-Asp(396) (M30) antibody in HeLa and OVCAR-3 cell lines treated with anticancer agents / 대한산부인과학회지

OBJECTIVE: The aim of this study was to detect the levels of M30-antigens as a biomarker of apoptosis in cells and their culture media after treatments with anticancer drugs as a preclinical study. METHODS: After HeLa and OVCAR-3 cells were treated respectively with paclitaxel, cisplatin, and camptothecin, the harvested cells were stained sequentially with M30 monoclonal antibodies and propidium iodide (PI). Afterwards, they were analyzed using a FACScan flow cytometer and observed under an immunofluorescence microscope for M30-FITC immunofluorescences. Levels of M30 antigens were also detected in their culture media using M30-Apoptosense ELISA kit. RESULTS: The levels of M30-FITC immunofluorescences were elevated in both cell lines after each drug treatments compared with those of control cells. The levels of M30 antigens detected by ELISA in media culturing each cell line treated with each of drugs were elevated compared with those of control cells. CONCLUSION: This study suggests that M30-antigens representing chemotherapy induced apoptosis may be a useful biomarker for predicting and monitoring the response of neoadjuvant chemotherapy in patients with gynecologic cancers.

Measurement of apoptosis using M30 in culture media of cell lines treated with anti-cancer agents / 대한산부인과학회지

OBJECTIVE: We investigated a possible use of the induced apoptosis as a biomarker in the cells and their media treated with commonly used anti-cancer agents in gynecologic malignancies. METHODS: After treatments with low and high concentrations of paclitaxel, cisplatin, and camptothecin in HeLa and OVCAR-3 cells, the levels of M30 antigen were detected in the cells and their media by immunofluorescence staining and ELISA methods, respectively. RESULTS: The percentages of M30-fluoresein isothiocyanate (FITC) positive cells in HeLa and OVCAR-3 cells treated with paclitaxel, cisplatin, and camptothecin were 4.3% vs 18.1% vs 34.87% and 4.07% vs 18.6% vs 32.63%, 4.3% vs 17.87% vs 32.38% and 4.07% vs 16.83% vs 32%, and 4.3% vs 16.75% vs 31.3% and 4.07% vs 15.18% vs 29.9% in control, low dose, and hight dose groups, respectively (P<0.001). M30 antigen levels (U/L) measured in culture media of HeLa and OVCAR-3 cells treated with paclitaxel, cisplatin, and camptothecin were 53.03 vs 101.53 vs 355.59 and 86 vs 114.41 vs 412.04, 53.03 vs 79.84 vs 327.64 and 86 vs 125.44 vs 385.09, and 53.03 vs 88.41 vs 295.005 and 86 vs 108.42 vs 263.1 in control, low dose, and hight dose groups, respectively (P<0.001). CONCLUSION: Our results obtained in this preclinical study suggests that measurement of the levels of M30 antigen may help to predict the clinical responses and to select the effective anti-cancer agents in clinical settings, rapidly and quantitatively.

Effect of Thalidomide on Angiogenesis in Implanted Human Tumors of Nude Mice / 中国药房

OBJECTIVE:To observe the effects of thalidomide in the growth of the human ovarian cancer cells (OVCAR-3) and HCT-8 as well as the angiogenesis in tumor tissue through building a model of heterograft of nude mice with human tumors. METHODS: After implanting human tumors in nude mice for modeling, the inoculated nude mice were intragastrically administered different dose of thalidomide, with tumor growth condition observed and compared with control group. The diversity of microvessel density(MVD) in tumor tissue was detected by immunohistochemistry. RESULTS: Middle and high dose thalidomide could significantly inhibit the growth of OVCAR-3 human tumor in nude mice(P

Antiproliferative and Apoptotic Effects of Zinc-Citrate Compound (CIZAR(R)) on Human Epithelial Ovarian Cancer Cell (OVCAR3) / 대한산부인과학회지

OBJECTIVE: Human seminal plasma has diverse biological activities including cytotoxic effect. It contains high concentrations of zinc and citric acid. Zinc inhibits several carcinoma cell growths through induction of cell cycle arrest and apoptosis. We tried to investigate the effects of zinc-citrate compound (CIZAR(R)) on normal human ovarian epithelial (NOSE) cells and human epithelial ovarian cancer cells, OVCAR-3. METHODS: To investigate the potential effect of CIZAR(R) on cell growth and survival, cells were treated with different dose and exposed to different time. Mitochondrial(m)-aconitase activity was determined in cell extracts using aconitase assay. The flow cytometric assay, DNA laddering, telomerase activity and morphological analysis were done to investigate apoptosis of OVCAR-3 cells. Molecular mechanism of apoptosis was investigated by p53, Bcl-XL, Bcl-2, Bax protein, and caspase activity. RESULTS: Treatment of OVCAR-3 cells with CIZAR(R) resulted in a time- and dose-dependent decrease in cell number in comparison with NOSE cells. M-aconitase activity was significantly decreased in OVCAR-3 cells but relatively constant in NOSE cells. The flow cytometric assay, DNA laddering and morphological analysis indicated apoptosis of OVCAR-3 cells. CIZAR(R) did not affect p53 but increased the expression of p21waf1 upon the indicated times and induced reduction of telomerase activity. CIZAR(R) reduced expression of Bcl-2 and Bcl-xL proteins but induced expression of Bax protein. CIZAR(R) induced apoptosis of OVCAR-3 cells by activation of caspase-3 pathway. CONCLUSION: These results show that CIZAR(R) prevent the proliferation of OVCAR-3 cells by inactivation of m-aconitase activity and induce apoptosis by induction of apoptotic genes and repression of antiapoptotic genes without adverse effect on normal ovarian epithelial cells. These results will offer new window in prevention and treatment of epithelial ovarian cancer.

Selective Protection of Normal Proliferationg Cells Against Anti-Neoplastic Chemotherapy by Stausporine Pretreatment / 대한산부인과학회잡지

OBJECTIVE: A major limiting factor in human cancer chemotherapy is toxicity in normal cells and tissues. Our goal was to determine whether normal proliferating cells could be protected from chemotherapeutic agents by taking advantage of the differential drug sensitivity of cell cycle G1 checkpoint in normal and cancer cells. METHODS: Normal peripheral blood mononuclear cells (PBMC) and ovarian cancer cell lines (OVCAR- 3 and SKOV-3) were initially treated with 10 nM of staurosporine for 48 hours. After removal of staurosporine contained media, both PBMC and ovarian cancer cells were treated with 20 nM of paclitaxel for 24 hours. Cells were then allowed to recover in drug-free medium for 4 days. The DNA contents and cell cycle changes were detected by FACScan flow cytometer in the cells harvested whenever the medium was changed. RESULTS: After pretreatment of ovarian cancer cell lines (OVCAR-3 and SKOV-3) with 10 nM of staurosporine followed by treatment with 20 nM of paclitaxel, both OVCAR-3 and SKOV-3 cells were selectively arrested in G2M phase of cell cycle by paclitaxel and they resumed their proliferative cycle to some extents after the drugs were removed and cultured with fresh media. However. pretreatment with 10 nM of staurosporine protected normal circulating PBMC that had been induced to proliferate in vitro with phytohemagglutinin from paclitaxel. Staurosporine-induced arrest of PBMC in G0/G1 phase was reversible, and arrested cells tolerated 10 nM of paclitaxel in culture. CONCLUSION: OVCAR-3 and SKOV-3 cancer cells can be targeted specifically with paclitaxel, following staurosporine-mediated, selective and reversible G0/G1 arrest in PBMC.

Flow Cytometric Detection of Apoptosis and p53 Protein in OVCAR-3 and SKOV-3 Ovarian Cancer Cell Lines / 대한산부인과학회잡지

OBJECTIVE: Taxol (paclitaxel)-induced apoptosis was studied to understand their biological mechanism correlated with the expression of p53 in the SKOV-3 and OVCAR-3 ovarian cancer cell lines. MATERIALS AND METHODS: The SKOV-3 and OVCAR-3 cell lines were cultured in RPMI 1640 medium without taxol (control group) and with taxol for 24 h and 48 h (experimental group). After harvest, the cells were stained with annexin V-FITC (fluorescein isothiocyanate) and anti-cytokeratin antibodies (clone CAM5.2 and clone MNF116). They were washed and stained with p53 antibody. After then the secondary antibodies, i.e., FITC- or phycoerythrin (PE)-conjugated goat anti-mouse (GAM) immunoglobulin G (GAM IgG-FITC or GAM IgG-PE) were added in the cells and they were incubated in the dark. DNA of these cells were stained sequentially with propidium iodide (PI). Standard FACScan equipped with a 488 nm single laser was used for the analysis of these cells. RESULTS: Both of SKOV-3 and OVCAR-3 cell lines were arrested in the G2M phase after treatment of taxol, suggesting that these cells would eventually enter into the stage of cell death. Fractions of negative cytokeratin and positive annexin V and amount of sub-G0G1 fraction indicative of apototic fractions were lower in the SKOV-3 cell line compared with that in OVCAR-3 cell line, probably as a result of lower sensitivity of SKOV-3 cell line to the taxol. p53 expression were not detected in SKOV-3 cell line. On the basis of observed findings in SKOV-3 cell line and findings of high expressions of p53 regardless of taxol treatment, no increases in their expressions according to culturing time, and gradual increases in sub-G0G1 fractions and in fractions of negative cytokeratin and positive annexin V indicative of apoptosis in OVCAR-3 cell line, we concluded that the expression of p53 would not be associated with cell cycle changes and the arrest in the G2M pahse but associated with the appearance of apotosis. CONCLUSION: Our results suggest that flow cytometric detection of the apoptotic fractions would be an effective, fast, and accurate method for the chemosensitivity test in tumor cells before the administration of anti-cancer drugs in gynecologic cancer patients.