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Background: To reveal roles of reactive oxygen species (ROS) status in chemotherapy resistance and to develop a ROS scoring system for prognosis prediction in ovarian cancer., Methods: We tested the sensitizing effects of ROS elevating drugs to cisplatin (cDDP) in ovarian cancer both in vitro and in vivo. A ROS scoring system was developed using The Cancer Genome Atlas (TCGA) database of ovarian cancer. The associations between ROS scores and overall survival (OS) were analyzed in TCGA, Tothill dataset, and our in-house dataset (TJ dataset)., Results: ROS-inducing drugs increased cisplatin-induced ovarian cancer cell injury in vitro and in vivo. ROS scoring system was established using 25 ROS-related genes. Patients were divided into low (scores 0-12) and high (scores 13-25) score groups. Improved patient survival was associated with higher scores (TCGA dataset hazard ratio (HR) = 0.43, P < 0.001; Tothill dataset HR = 0.65, P = 0.022; TJ dataset HR = 0.40, P = 0.003). The score was also significantly associated with OS in multiple datasets (TCGA dataset r 2  = 0.574, P = 0.032; Thothill dataset r 2  = 0.266, P = 0.049; TJ dataset r 2  = 0.632, P = 0.001) and with cisplatin sensitivity in ovarian cancer cell lines (r 2  = 0.799, P = 0.016) when used as a continuous variable. The scoring system showed better prognostic performance than other clinical factors by receiver operating characteristic (ROC) curves (TCGA dataset area under the curve (AUC) = 0.71 v.s. 0.65, Tothill dataset AUC = 0.73 v.s. 0.67, TJ dataset AUC = 0.74 v.s. 0.66)., Conclusions: ROS status is associated with chemotherapy resistance. ROS score system might be a prognostic biomarker in predicting the survival benefit from ovarian cancer patients.

Currently, acquired resistance to cisplatin (DDP) is a substantial obstacle to reducing the morbidity and mortality due to ovarian malignant tumors. Nevertheless, cisplatin plays a vital role in killing the tumor cells while it may also be a 'primer' involved in chemotherapy resistance. We found that the cisplatin-induced chemokine (C-C motif) ligand 5 (CCL5) secretion derived from cancer-associated fibroblasts (CAFs) promoted ovarian cancer cell resistance to cisplatin. Via a cytokine chip assay, we identified a spectrum of secreted proteins that were derived from the CAFs through cisplatin-induced treatment. Among these, CCL5 significantly attenuated the cytotoxic effect of cisplatin chemotherapy in vitro and in vivo. Additionally, CCL5 expression was also detected in 62 serous ovarian cancer patient tissue specimens using IHC, and the results demonstrated that chemotherapy resistant patients displayed higher expression of CCL5 than the chemo-sensitive patients (P<0.05). Mechanistically, we found that CCL5 notably increased STAT3 and Akt phosphorylation levels in ovarian cancer cells. These results indicated that cisplatin- induced CCL5 secretion derived from the CAFs may promote cisplatin resistance, which was mediated by regulation of the STAT3 and PI3K/Akt signal pathways.

Objective: To explore the effect of ataxia telangiectasia mutated and RAD3 related protein (ATR) expression and ATR kinase activity on the sensitivity to cisplatin in ovarian cancer SKOV3 cells., Methods: SiRNA targeting ATR was transfected into SKOV3 cells for 48 h to reduce the ATR protein level, and ATR kinase inhibitor VE-821 was used for 12 h to inhibit the ATR pathway activity. The alteration of cell viability was examined by CCk-8 assay. Expression levels of ATR, p-ATR and γ-H2AX proteins were detected by Western blot. The DNA double strand breaks (DSB) marker γ-H2AX and homologous recombination repair key protein RAD51 and their co-localization in the cells were examined under the confocal microscope. The status of DNA double strand breaks (DSB) in single cells was visualized by alkaline comet assay. Finally, the cell cycle distribution was assessed using flow cytometry., Results: DDP caused evident DNA double strands breaks and activated ATR kinase pathway. ATR-siRNA notably reduced ATR protein level, the 48 h IC(50) value of DDP was 72.12 µmol/L and 41.25 µmol/L, respectively, in the NC-siRNA and ATR-siRNA groups (P < 0.05). Confocal microscopic assay presented decreased recruitment of RAD51 at the DSB loci and comet assay showed enhanced DSB in the cells after ATR knocking down. After the inhibition of ATR kinase by VE-821, the 48 h IC(50) value of DDP was 75.32 µmol/L and 45.64 µmol/L, respectively, in the DMSO and VE-821 groups (P < 0.05 for both), confocal microscopic assay demonstrated reduced RAD51 recruitment, and comet assay showed increased DSB in cells after ATR kinase inhibition. Flow cytometry showed that percentage of cells distributed in G(0)/G(1), S and G(2)/M phases was 71.2%, 13.4% and 15.4%, repectively, after 40 µmol/L DDP treatment for 24 h. Compared with that of control group (G(0)/G(1): 54.2%, S: 21.3% and G(2)/M: 24.4%), DDP induced G(0)/G(1) phase arrest. DDP intervention resulted in the cell cycle status (G(0)/G(1): 43.2%, S: 20.4%, G(2)/M: 36.4%) in the ATR-siRNA group and (G(0)/G(1): 40.2%, S: 22.5%, G(2)/M: 37.3%) in the VE-821 group, indicating that the inhibition of ATR or ATR kinase could abrogate the effect of G(0)/G(1) phase arrest induced by DDP., Conclusions: Suppression of ATR can affect the homologous recombination repair in ovarian cancer cells, leading to accumulation of DNA double strand breaks in the cell nuclei as well as reduction of DDP-caused G(0)/G(1) phase arrest, finally enhances the sensitivity to cisplatin in the ovarian cancer SKOV3 cells.

Objective: The role of Quercetin in ovarian cancer treatment remains controversial, and the mechanism is unknown. The aim of this study was to investigate the therapeutic effects of Quercetin in combination with Cisplatin and other anti-neoplastic drugs in ovarian cancer cells both in vitro and in vivo, along with the molecular mechanism of action., Methods: Quercetin treatment at various concentrations was examined in combination with Cisplatin, taxol, Pirarubicin and 5-Fu in human epithelial ovarian cancer C13* and SKOV3 cells. CCK8 assay and Annexin V assay were for cell viability and apoptosis analysis, immunofluorescence assay, DCFDA staining and realtime PCR were used for reactive oxygen species (ROS)-induced injury detection and endogenous antioxidant enzymes expression. Athymic BALB/c-nu nude mice were injected with C13*cells to obtain a xenograft model for in vivo studies. Immunohistochemical analysis was carried out to evaluate the ROS-induced injury and SOD1 activity of xenograft tumors., Results: Contrary to the pro-apoptotic effect of high concentration (40 µM-100 µM) of Quercetin, low concentrations (5 µM-30 µM) of Quercetin resulted in varying degrees of attenuation of cytotoxicity of Cisplatin treatment when combined with Cisplatin. Similar anti-apoptotic effects were observed when Quercetin was combined with other anti-neoplastic agents: Taxol, Pirarubicin and 5-Fluorouracil (5-Fu). Low concentrations of Quercetin were observed to suppress ROS-induced injury, reduce intracellular ROS level and increase the expression of endogenous antioxidant enzymes, suggesting a ROS-mediated mechanism of attenuating anti-neoplastic drugs. In xenogeneic model, Quercetin led to a substantial reduction of therapeutic efficacy of Cisplatin along with enhancing the endogenous antioxidant enzyme expression and reducing ROS-induced damage in xenograft tumor tissue., Conclusion: Taken together, these data suggest that Quercetin at low concentrations attenuate the therapeutic effects of Cisplatin and other anti-neoplastic drugs in ovarian cancer cells by reducing ROS damage. Quercetin supplementation during ovarian cancer treatment may detrimentally affect therapeutic response.

Background: Expression of BRCA1 is commonly decreased in sporadic ovarian cancer, and this is associated with platinum sensitivity and favorable prognosis. However, multiple mechanisms underlying low BRCA1 expression are not fully understood., Methods: A bioinformatics-driven microRNA (miR) library screening was used to identify miRs that regulate BRCA1 expression. The effects of miR-9 on cisplatin (cDDP) and PARP inhibitor sensitivity were measured in ovarian cancer cells and C13* xenograft mice (n = 6 per group). The roles of miR-9 on prognosis were assessed in a cohort of ovarian cancer patients (n = 113) with Kaplan-Meier and Cox proportional hazards analyses. All statistical tests were two-sided., Results: Reverse miR library screening revealed that miR-9 reduced the normalized luciferase activity to 60.3% (95% confidence interval [CI] = 52.0% to 68.5%; P < .001). miR-9 bound directly to the 3'-UTR of BRCA1 and downregulated BRCA1 expression in ovarian cancer cells. Treatment with miR-9 agomiR sensitized BRCA1-proficient C13* xenograft tumors to cisplatin and AG014699. In serous ovarian cancer, higher levels of miR-9 were inversely correlated with BRCA1 expression (Spearman rank correlation: R (2) = 0.379; P = .003). Patients with higher levels of miR-9 had better chemotherapy response, platinum sensitivity, and longer progression-free survival (PFS) (high vs low miR-9 expression: median PFS = 26.4 months, 95% CI = 13.8 to 39.0 months vs median PFS = 15.4 months, 95% CI = 6.8 to 23.9 months, P = .01)., Conclusions: miR-9 mediates the downregulation of BRCA1 and impedes DNA damage repair in ovarian cancer. miR-9 may improve chemotherapeutic efficacy by increasing the sensitivity of cancer cells to DNA damage and may impact ovarian cancer therapy.

Background: P21(WAF1/Cip1) binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer., Methods: RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry., Results: p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment., Conclusions: Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors that are refractory to conventional treatment.

Chemotherapy is the preferred therapeutic approach for advanced ovarian cancer, but a successful long-term treatment is prevented by the development of drug resistance. Recent works have underlined the involvement of non-coding RNAs, microRNAs (miRNAs) in cancer development, with several conjectures regarding their possible involvement in the evolution of drug resistance. This study is to investigate the promoting effects and mechanism of miR-125b involved in the development of chemoresistance in ovarian cancer. The different expression of miR-125b in cisplatin-sensitive ovarian cancer cell line (OV2008) and its resistant variant (C13*) was identified by real-time PCR. An in vitro cytotoxicity assay and apoptosis assay using CCK-8 assay and flow cytometry, were carried out to detect the effect of miR-125b and Bak1 on cisplatin resistance of cells. Real-time PCR, Western blotting and luciferase reporter assay were used to detect whether Bak1 is a target of miR-125b. As compared with OV2008 cells, the expression levels of miR-125b in C13* cells were increased. It was found that the up-regulation of microRNA-125b caused a marked inhibition of cisplatin-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to cisplatin in OV2008 and C13* cells. Moreover, Bak1 was a direct target of miR-125b, and down-regulation of Bak1 suppressed cisplatin-induced apoptosis and led to an increased resistance to cisplatin. Our study indicates that miR-125b has a significantly promoting effect on chemoresistance of C13* cells and up-regulation of miR-125b expression contributes to cisplatin resistance through suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming cisplatin resistance in ovarian cancer.

Purpose: The aim of this study was to explore role of PTEN gene in chemosensitivity to cisplatin in human ovarian cancer cells and related mechanisms., Method: A PTEN-targeted short hairpin RNA (shRNA) expression vector and a wild-type sense PTEN plasmid were constructed, human ovarian cisplatin-sensitive cancer cell line OV2008 and its resistant variant C13 * cells were transfected with PTEN shRNA or wild-type PTEN plasmid, respectively, and cells were then treated with cisplatin. Next, AKT activity was regulated with co-transfection of antisense or sense AKT plasmid in OV2008 /PTENshRNA cells or C13 */p-PTEN cells, respectively. Effects of transfection of above vectors on cell growth, apoptosis and expression of PTEN and AKT were evaluated., Results: Expression of PTEN in OV2008 cells was significantly higher than that in C13 * cells. Transfection of PTEN shRNA into OV2008 cells remarkably down-regulated expression of PTEN and up-regulated expression of phospho-AKT protein, with transfected cells being resistant to cisplatin. Overexpression of PTEN by transfection with sense PTEN obviously enhanced cisplatin-induced apoptosis of C13 * cells. Furthermore, decreased AKT activity could increase cisplatin-induced apoptosis in OV2008/PTENshRNA cells; while, transfection of pcDNA3.1-AKT plasmid into C13 */p-PTEN cells resulted in increased activity of AKT, with cisplatin-induced apoptosis being inhibited significantly., Conclusions: PTEN might reverse chemoresistance to cisplatin in human ovarian cancer cells through inactivation of the PI3K/AKT cell survival pathway and may serve as a potential molecular target for the treatment of chemoresistant ovarian cancer.

The reversing effect of wild-type PTEN gene on resistance of C13K cells to cisplatin and its inhibitory effect on the phosphorylation of protein kinase B (AKT) were studied. The expression of PTEN mRNA and protein in OV2008 cells and C13K cells were semi-quantitatively detected by using RT-PCR and Western blotting. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, Akt, p-Akt protein were analyzed by Western blotting in PTEN-transfected and non-transfected C13K cells. Proliferation and chemosensitivity of cells to DDP were measured by MTT, and cell apoptosis was detected by flow cytometry after treatment with cisplatin. The expression of PTEN mRNA and protein in OV2008 cells were significantly higher than those in C13K cells. After transfection with PTEN gene for 48 h, the expression of PTEN mRNA and protein in C13K cells were 2.04 +/- 0.10, 0.94 +/- 0.04 respectively and the expression of p-Akt protein (0.94 +/- 0.07) was lower than those in control groups (1.68 +/- 0.14, 1.66 +/- 0.10) (P < 0.05). The IC(50) of DDP to C13K cells transfected with PTEN (7.2 +/- 0.3 micromol/L) was obviously lower than those of empty-vector transfected cells and non-transfected cells (12.7 +/- 0.4 micromol/l, 13.0 +/- 0.3 micromol/L) (P<0.05). The apopototis ratio of wild-type PTEN-transfected, empty vector transfected and non-transfected C13K cells were (41.65 +/- 0.87)%, (18.61 +/- 0.70)% and (15.28 +/- 0.80)% respectively, and the difference was statistically significant (P<0.05). PTEN gene plays an important role in ovarian cancer multidrug resistance. Transfection of PTEN could increase the expression of PTEN and restore drug sensitivity to cisplatin in human ovarian cancer cell line C13K with multidrug-resistance by decreasing the expression of p-Akt.