A chemotherapy response classifier based on support vector machines for high-grade serous ovarian carcinoma.

Long-term outcome of high-grade serous epithelial ovarian carcinoma (HGSOC) remains poor as a result of recurrence and the emergence of drug resistance. Almost all the patients were given the same platinum-based chemotherapy after debulking surgery even though some of them are naturally resistant to the first-line chemotherapy. No method could verify this part of patients right after the surgery currently. In this study, we used 156 paraffin-embedded high-grade HGSOC specimens for immunohistochemical analysis with 37 immunology markers, and association between the expression levels of these markers and the chemoresponse were evaluated. A support vector machine (SVM)-based HGSOC prognostic classifier was then established, and was validated by a 95-patient independent cohort. The classifier was strongly predictive of chemotherapy resistance, and divided patients into low- and high-risk groups with significant differences progression-free survival (PFS) and overall survival (OS). This classifier may provide a potential way to predict the chemotherapy resistance of HGSOC right after the surgery, and then allow clinicians to make optimal clinical decision for those potentially chemoresistant patients. The potential clinical application of this classifier will benefit those patients with primary drug resistance.

[Proteasome inhibitors sensitize ovarian cancer cells to cisplatin].

OBJECTIVE: To explore the sensitivity and the molecular mechanism of cisplatin-resistance ovarian cancer cell line C13 to proteasome inhibitors and the combination with cisplatin. METHODS: After different treatments, methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability, annexin-V/propidium iodide (PI) apoptosis detection kit was used to determine the apoptosis rate of different groups, western blot assay was introduced to evaluate the expression levels of Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein (cFLIPs), and the activity of caspase-8 was examined. RESULTS: MTT assay shown that the cell viability ratios of combination group at serial time points from 12, 24, 36, 48, 60, 72 hours were (56.0 ± 8.4) %, (44.7 ± 7.3) %, (33.7 ± 11.2) %, (27.6 ± 8.0) %, (27.6 ± 7.6) % and (28.1 ± 2.4) %, which were much lower than those of cisplatin group (P < 0.05). After treated for 24 hours, apoptosis rates of cisplatin group, bortezomib group and combination group were (16.7 ± 1.7) %, (23.4 ± 2.1) % and (26.9 ± 1.6) %, respectively. The rate of combination group was much higher than that of non-treated group and that of cisplatin group or bortezomib group (P < 0.05). Western blot assay showed the changes of expression levels of cFLIPs, which were down-regulated seriously after cisplatin, bortezomib or combination treatment [(43.2 ± 2.3) % vs (75.7 ± 3.0) % vs (67.9 ± 2.1) %, P < 0.05]. The caspase-8 activity of combination group was (5.6 ± 1.6) folds than that of non-treated group, which was higher than those of other two groups [(2.3 ± 1.0) and (4.2 ± 0.9) folds, P < 0.05]. CONCLUSIONS: The tumor cell lethal effect of cisplatin could be increase significantly by the combination application of proteasome inhibitors, bortezomib. And the cFLIPs/caspase-8 signaling pathway may be play an important role in the molecular mechanism of the combination treatment.

[Proteasome inhibitors sensitize ovarian cancer cells to paclitaxel induced apoptosis].

OBJECTIVE: To explore the sensitivity of ovarian cancer cell line SKOV3 to paclitaxel, proteasome inhibitors, bortezomib, and their combination. METHODS: The methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability after treatment. The annexin V-propidium iodide apoptosis detection kit was used to determine the apoptosis rate of different groups. Western blot assay was used to evaluate the expression levels of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3 beta (GSK-3beta). RESULTS: In MTT assay, the cell viability ratios of the combination group at serial time points from 12, 24, 36, 48 and 72 hours were (65.2 +/- 5.8)%, (58.3 +/- 14.4)%, (35.3 +/- 5.0)%, (19.2 +/- 1.5)%, and (11.4 +/- 2.5)%, which were significantly lower than those of the paclitaxel group (P < 0.05). After drug treatments, apoptosis rates of paclitaxel group, bortezomib group and the combination group were (14.7 +/- 0.5)%, (15.1 +/- 0.8)% and (20.5 +/- 0.7)% respectively. The rate of the combination group was significantly higher than that of non-treated group and paclitaxel group (P < 0.05). Western blot assay showed the changes in expression levels of phosphorylated AKT and GSK-3beta, which were decreased significantly after paclitaxel and bortezomib combination treatment [(3.2 +/- 0.8)%, (19.3 +/- 0.4)%; P < 0.05]. CONCLUSIONS: The lethal effect of paclitaxel on tumor cells could be increased significantly by its combination with proteasome inhibitors, bortezomib. The AKT/GSK-3beta signaling pathway plays an important role in the molecular mechanism of the combination treatment.

[Application of RNAi library to oncology investigation].

RNA interference (RNAi) library was pioneered in C. elegans in a broad range of organism-based screens. During the past few years, RNAi has become a powerful tool to silence the expression of genes and analyze their loss-of-function phenotype in mammalian cells. There are two types of RNAi library, synthetic oligonucleotide library and vector library, and two screening strategies, high throughput and selective screenings. Short hairpin RNA (shRNA), which is processed intracellularly into short duplex RNAs and has siRNA-like properties, can mediate persistent gene silencing after stable integration of the vector into the host-cell genome. In combination with the shRNA vector library and suitable screening strategies, much greater depth will be added to the functional understanding of therapeutic applications of potential targets in oncology.

[Reversal of drug resistance in human ovarian cancer cells by wild-type PTEN gene and its mechanisms].

OBJECTIVE: To examine expression of PTEN gene in ovarian cancer cisplatin-sensitive cell line OV2008 cells and cisplatin-resistant cell line C13K cells, and evaluate the effect of wild-type PTEN gene on reversing cisplatin-resistance of C13K cells and underlying mechanisms. METHODS: The expression of PTEN mRNA and protein in OV2008 and C13K cells were detected by semi-quantitative RT-PCR and western blot. Recombinant eukaryotic expression plasmid containing human wild-type PTEN gene was transfected into C13K cells by lipofectamine 2000. The expression of PTEN mRNA was monitored by RT-PCR and the expression of PTEN, protein kinase B (AKT), phospho-AKT (p-AKT) protein were analyzed by western blot in PTEN transfected and untransfected C13K cells. Proliferation and chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium (MTT), and cell apoptosis was detected by flow cytometry after treatment with cisplatin. RESULTS: (1) The expression of PTEN mRNA and protein (1.02 +/- 0.05, 1.02 +/- 0.07) in OV2008 cells were significantly higher than those in C13K cells, which were 0.45 +/- 0.03 and 0.55 +/- 0.03 respectively (P < 0.05). (2) After transfected with PTEN gene for 48 hours, the expression of PTEN mRNA and protein in C13K cells were 2.04 +/- 0.10, 0.94 +/- 0.04 respectively. Compared with C13K cells transfected with empty vector (1.04 +/- 0.04, 0.36 +/- 0.03) and untransfected C13K cells (1.03 +/- 0.05, 0.37 +/- 0.03), the difference was significant respectively (P < 0.01). The expression of p-AKT protein (0.94 +/- 0.07) was lower than those in control groups (1.66 +/- 0.10, 1.68 +/- 0.14; P < 0.05). (3) The 50% inhibition concentration (IC(50)) to cisplatin of C13K cells transfected with PTEN [(7.2 +/- 0.3) micromol/L] was obviously lower than those of empty-vector transfected cells and untransfected cells [(12.7 +/- 0.4), (13.0 +/- 0.3) micromol/L; P < 0.05]. (4) The apoptosis ratio of C13K cells with wild-type PTEN transfection, empty vector transfection and untransfected were (41.7 +/- 0.9)%, (18.6 +/- 0.7)% and (15.3 +/- 0.8)% respectively (P < 0.01). CONCLUSIONS: 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 multidrug-resistant human ovarian cancer cell line C13K by decreasing the expression of p-AKT.