Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Low Concentration of Quercetin Antagonizes the Cytotoxic Effects of Anti-Neoplastic Drugs in Ovarian Cancer

<div><p>Objective</p><p>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 <i>in vitro</i> and <i>in vivo</i>, along with the molecular mechanism of action.</p><p>Methods</p><p>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 <i>in vivo</i> studies. Immunohistochemical analysis was carried out to evaluate the ROS-induced injury and SOD1 activity of xenograft tumors.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>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.</p></div

The Role of BRCA Status on the Prognosis of Patients with Epithelial Ovarian Cancer: A Systematic Review of the Literature with a Meta-Analysis

<div><p>Objective</p><p>The role of BRCA dysfunction on the prognosis of patients with epithelial ovarian cancer (EOCs) remains controversial. This systematic review tried to assess the role of BRCA dysfunction, including BRCA1/2 germline, somatic mutations, low BRCA1 protein/mRNA expression or BRCA1 promoter methylation, as prognostic factor in EOCs.</p><p>Methods</p><p>Studies were selected for analysis if they provided an independent assessment of BRCA status and prognosis in EOC. To make it possible to aggregate survival results of the published studies, their methodology was assessed using a modified quality scale.</p><p>Results</p><p>Of 35 evaluable studies, 23 identified BRCA dysfucntion status as a favourable prognostic factor. No significant differences were detected in the global score of quality assessment. The aggregated hazard ratio (HR) of overall survival (OS) of 34 evaluable studies suggested that BRCA dysfunction status had a favourable impact on OS (HR = 0.69, 95% CI 0.61–0.79), and when these studies were categorised into BRCA1/2 mutation and low protein/mRNA expression of BRCA1 subgroups, all of them demonstrated positive results (HR = 0.67, 95% CI: 0.57–0.78; HR = 0.62, 95% CI: 0.51–0.75; and HR = 0.51, 95% CI: 0.33–0.78, respectively), except for the subgroup of BRCA1 promoter methylation (HR = 1.59, 95% CI: 0.72–3.50). The meta-analysis of progression-free survival (PFS), which included 18 evaluable studies, demonstrated that BRCA dysfunction status was associated with a longer PFS in EOC (HR = 0.69, 95% CI: 0.63–0.76).</p><p>Conclusions</p><p>Patients with BRCA dysfunction status tend to have a better outcome, but further prospective clinical studies comparing the different BRCA statuses in EOC is urgently needed to specifically define the most effective treatment for the separate patient groups.</p></div

Quercetin reduced ROS level of ovarian cancer cells in combination with Cisplatin treatment.

<p>Intracellular ROS levels of C13* cells of different treatment groups were detected by Reactive Oxygen Species Assay using flow cytometry. (A, B) The ROS levels of C13* cells treated by vehicle control or 20 µM Quercetin for 24 hours. (C, D) The ROS levels of C13* cells treated by 80 µM Cisplatin with or without 20 µM Quercetin for 24 hours. N = 3. *<i>P</i><0.001, #<i>P</i><0.001, Student t-test.</p

Characteristics of studies of patients of ovarian cancer with low BRCA1 mRNA expression or BRCA1 promoter methylation.

<p><b>Histology</b>: pathological histology of ovarian cancer, all = almost all of the epithelial ovarian cancer types, including serous, mucinous, clear cell cancer, etc.</p><p><b>Methods of methylation detecting</b>: methods of methylation detecting, MSP = methylation-specific polymerase chain reaction (PCR) analysis, MSRE = methylation-sensitive restriction enzyme digestion.</p><p><b>Treatment</b>: chemotherapy used, 2 = Platinum-based chemotherapy.</p

Characteristics of studies of patients with BRCA1/2 mutated ovarian cancer.

<p><b>Histology</b>: pathological histology of ovarian cancer, se = serous ovarian cancer, CCC = clear cell cancer of, Mu = mucinous ovarian cancer. all = almost all of the epithelial ovarian cancer types, including serous, mucinous, clear cell cancer, etc.</p><p><b>Laboratory methods</b>: laboratory methods used to detect BRCA1/2 mutation, PTT = Protein truncation test, SSCP = Single-Strand Conformation Polymorphism, seq = sequencing, DGGE = fluorescent multiplex denaturing gradient gel electrophoresis, MLPA = multiplex ligation-dependent probe amplification, DHPLC = Denaturing high performance liquid chromatography, RFLP = Restriction fragment length polymorphisms, F-CSGE = Fluorescence-based Conformation Sensitive Gel Electrophoresis.</p><p><b>Germline/somatic</b>: Germ = germline mutation, Mixed = BRCA1 germline/somatic mutation or BRCA1 promoter methylation.</p><p><b>Mutation types:</b> VUS = variants of unknown significance.</p><p><b>Treatment</b>: chemotherapy used, 1 = only Platinum was used, 2 = Platinum-based chemotherapy, 3 = other agents without Platinum, like Paclitaxel, etc.</p

Summary hazard ratios (HRs) and 95% confidence intervals (CIs) of ovarian cancer PFS for BRCA dysfunction status.

<p>Horizontal lines represent 95% CIs; diamonds represent summary estimates with corresponding 95% CIs. Test for heterogeneity: <i>P</i> = .118, <i>I</i>² = 29.3%. A fixed-effects model was used.</p

Subgroup meta-analysis of summary hazard ratios (HRs) and 95% confidence intervals (CIs) of ovarian cancer OS for different BRCA mutation statuses.

<p><b>A:</b> BRCA1 mutation. <b>B:</b> BRCA2 mutation. Horizontal lines represent 95% CIs; diamonds represent summary estimates with corresponding 95% CIs. Test for heterogeneity: <b>A: </b><i>P</i> = .251, <i>I </i>² = 20.2%, a fixed-effects model was used; <b>B: </b><i>P</i> = .023, <i>I</i>² = 55.1%, a random-effects model was used.</p

Begg’s funnel plots of the natural logarithm of the hazard ratios (HRs) and the SE of the natural logarithm of the HRs for all of the included studies reported with OS and PFS.

<p>A: Begg’s funnel plots for all of the included studies reported with OS, the dashed line represents 95% confidence intervals (CIs). Circles represent individual studies. Begg’s test: <i>P</i> = 0.221. B: Begg’s funnel plots for all of the included studies reported with PFS, the dashed line represents 95% confidence intervals (CIs). Circles represent individual studies. Begg’s test: <i>P</i> = 0.880.</p

Quercetin reduced oxidative injury of ovarian cancer cells caused by Cisplatin.

<p>(A), 8-OHdG and H2AX expression in C13* cells treated with Quercetin in combination with Cisplatin was detected by immunofluorescence assay. (B), Numbers of highly positive stained cells in five random high-power fields were counted by Image J and expressed as percentage of Cisplatin group values. Group of Cisplatin combined with Quercetin 20 µM treatment VS. Group of Cisplatin treatment, N = 3. *<i>P</i> = 0.004, #<i>P</i> = 0.001, Student t-test.</p