Tetsuro Oishi

Combination chemotherapy of oxaliplatin and 5-fluorouracil may be an effective regimen for mucinous adenocarcinoma of the ovary: A potential treatment strategy

Resistance of ovarian mucinous adenocarcinoma to standard chemotherapy with paclitaxel and carboplatin is associated with poor prognosis, and an effective treatment is needed. The present study aimed to identify an effective chemotherapy for ovarian mucinous adenocarcinoma. Five human ovarian mucinous adenocarcinoma cell lines (MN‐1, OMC‐1, RMUG‐L, RMUG‐S, TU‐OM‐1) were used in this study. Sensitivity of the cells to the anticancer agents was determined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay, and we assessed drug sensitivity by calculating the assay area under the curve for each agent. Protein expression was confirmed by Western blot analysis. We also examined the efficacy of combination chemotherapy on survival in a xenograft model of nude mice. The IC50 to anticancer agents ranged widely. The assay area under the curve indicated that two of five cell lines (MN‐1, TU‐OM‐1) were sensitive to oxaliplatin, 5‐fluorouracil and etoposide, and only one (TU‐OM‐1) was sensitive to 7‐ethyl‐10‐hydroxycamptothecin, which is an active metabolite of camptothecin. All cell lines were resistant to cisplatin and paclitaxel. The combination of oxaliplatin and 5‐fluorouracil resulted in additive or synergistic effects on all cell lines. The combination of oxaliplatin and 5‐fluorouracil significantly prolonged survival in a ovarian mucinous adenocarcinoma xenograft model of nude mice. Protein expression levels of the excision repair cross‐complementation group 1 were lower in oxaliplatin sensitive cell lines. Exposure to 5‐fluorouracil down‐regulated cross‐complementation group 1 expression in ovarian mucinous adenocarcinoma cells. We conclude that combination chemotherapy consisting of oxaliplatin and 5‐fluorouracil was an effective treatment for ovarian mucinous adenocarcinoma and may be a pivotal candidate for a novel treatment strategy. (Cancer Sci 2009; 100: 546–551)

Expression of the c‐myc gene as a predictor of chemotherapy response and a prognostic factor in patients with ovarian cancer

The present study was conducted to determine whether and how expression of the c‐myc gene is related to the response to chemotherapy in patients with epithelial ovarian cancer. This study includes 101 consecutive patients with stage Ic to IV epithelial ovarian cancer who underwent primary surgery followed by platinum‐based chemotherapy. Immunohistochemical studies were performed to detect Ki‐67 and ARF proteins. Apoptotic cells were identified by the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate biotin nick‐end labeling method. Mutation of the p53 gene was screened by polymerase chain reaction (PCR)‐single strand conformation polymorphism analysis and confirmed by direct sequencing. mRNA expression of c‐myc was determined by means of reverse transcription‐PCR. Apoptotic index (AI) and ARF labeling index (LI) were significantly increased and Ki‐67 LI was decreased after chemotherapy in patients from whom specimens could be obtained before and after chemotherapy. AI, ARF LI, and Ki‐67 LI were not related to p53 gene status. A significant correlation between expression of c‐myc and ARF LI was observed. Of 38 patients with measurable lesion, 23 (60.5%) responded to chemotherapy and 15 (39.5%) did not. Tumors with the wild‐type p53 gene responded significantly better to chemotherapy than did tumors with the mutation. Responders showed a higher expression of c‐myc than nonresponders (468.76 vs. 187.68). The receiver operating characteristic (ROC) curve according to chemoresponse demonstrated that the cut‐off value of c‐myc expression was 200. Patients with c‐myc expression of more than 200 had a better 5‐year survival rate (69.8% vs. 43.5%; 101 patients). Multivariate analysis revealed that c‐myc expression was an independent prognostic factor. Our results suggest that the expression of c‐myc gene is related to chemoresponse and might be a useful prognostic factor in patients with epithelial ovarian cancer.

Expression of hypoxia-inducible factor 1α gene affects the outcome in patients with ovarian cancer

We conducted study to determine whether and how the expression of the hypoxia-inducible factor 1α (HIF-1α) gene relates to outcome in patients with epithelial ovarian cancer. A total of 66 patients with epithelial ovarian cancer, who underwent primary surgery followed by platinum-based chemotherapy, were entered into this study. We confirmed the expression of HIF-1α and the vascular endothelial growth factor (VEGF) by immunohistochemistry. To determine the quantity of HIF-1α and VEGF expression, messenger RNA of each gene was measured by real-time reverse transcription–polymerase chain reaction. The cutoff values were determined by the receiver-operating characteristic curve according to survival. The protein expressions of HIF-1α and VEGF were strongly observed in the cancer cells. The cutoff value of HIF-1α and VEGF gene expression was 6.0 and 3.0, respectively. The expression of HIF-1α did not relate to clinical stage, but tumor with low VEGF expression was observed more frequently in stage I patients. The response rate to chemotherapy did not differ between high and low expression of both genes. The overall survival for patients with high expression of HIF-1α was significantly lower, but disease-free survival did not differ between high and low expression of HIF-1α, whereas both overall and disease-free survival for patients with high expression of VEGF were significantly lower. Multivariate analysis revealed that FIGO stage and HIF-1α expression were independent prognostic factors but that VEGF was not. The present study suggested that the expression level of HIF-1α could be an independent prognostic factor in epithelial ovarian cancer

Genetic diagnosis for chemosensitivity with drug-resistance genes in epithelial ovarian cancer.

We conducted the present study to investigate whether and how chemosensitivity can be determined by means of genetic diagnosis using drug-resistance genes in patients with epithelial ovarian cancer. A total of 75 patients who had epithelial ovarian cancer with measurable lesions were entered into this study. Thirty-three patients received first-line chemotherapy, consisting of paclitaxel and carboplatin (TJ). Forty-two patients received second-line chemotherapy, 22 received EP therapy consisting of etoposide and cisplatin (CDDP), and 20 received irinotecan (CPT-11) and CDDP (CPT-11/CDDP) therapy. Tumor samples were obtained before chemotherapy. MessengerRNA expressions of the multidrug-resistance (MDR)-1 gene, MDR-associated protein-1 (MRP-1), topoisomerase (topo) I, and topo IIα were measured by real-time reverse transcription–polymerase chain reaction. The cutoff values of each gene were determined by the receiver operating characteristic curve. MDR-1 expression was significantly higher in patients who did not respond to TJ therapy. The expression of topo IIα was significantly higher in patients who did respond to EP therapy. The expression of topo I was significantly higher in patients who did respond to CPT-11/CDDP. MRP-1 expression did not differ between responders and nonresponders in all regimens. The cutoff value was 80 for MDR-1, 90 for topo IIα, and 200 for topo I. Next, to evaluate genetic diagnosis, 31 patients were newly added. The accuracy of this genetic diagnosis for chemosensitivity was 85.7% for TJ, 77.8% for EP, and 100.0% for CPT-11/CDDP therapy. The present study suggests that genetic diagnosis may be useful to determine chemosensitivity in patients with epithelial ovarian cancer

Galectin-3 may contribute to Cisplatin resistance in clear cell carcinoma of the ovary

Our previous findings suggested that lower cell proliferation of clear cell carcinoma (CCC) of the ovary may contribute to its resistance to chemotherapy. We conducted the present study to find the gene that regulates cell proliferation of CCC and to elucidate whether it contributes to cisplatin (CDDP) resistance. Complementary DNA microarray analysis revealed that the gene expression level of galectin-3 of CCC cell lines (KK, RMG-I, HAC-2) was over threefold higher than that of ovarian serous adenocarcinoma (SAC) cell lines (HRA, KF). S-phase fraction increased after knocking down galectin-3 using small interfering RNA in RMG-I, KK, and HAC-2 cells. The protein expression of p27 decreased after knocking down galectin-3. CDDP-induced apoptosis was increased after knocking down galectin-3, and this cytotoxic effect was canceled by roscovitine. Immunohistochemical staining showed that galectin-3 expression in tumors of 20 CCC was significantly more frequent than that of 20 SAC (70.0% vs 15.0%, P= 0.0004). The present study showed that the expression of galectin-3 in CCC might contribute to its lower cell proliferation and lead to CDDP resistance.

Alteration of Telomerase Activity Associated With Development and Extension of Epithelial Ovarian Cancer

Objective To assess telomerase activity associated with the development and extension of epithelial ovarian cancer and to investigate the relationship between p53 gene status and telomerase activity. Methods A total of 53 samples (41 epithelial ovarian cancers, five borderline epithelial tumors, four benign adenomas, and three surface epithelia) were examined for telomerase activity by the polymerase chain reaction (PCR) -based telomeric repeat amplification protocol assay. Mutations in the p53 gene were determined by PCR-single strand conformation polymorphism analysis. Results Telomerase activity was detected in 33 of 41 epithelial ovarian cancers and in three of five borderline malignancies but was not detectable in either benign tumors or normal surface epithelium. The mean (± standard deviation [SD]) intensity of telomerase activity in cancers was significantly higher than that in borderline malignancies (10.6 ± 8.2 versus 3.6 ± 1.3). The positivity of telomerase activity did not correlate with any clinical findings, but the intensity (±SD) of telomerase activity was significantly higher in tumors with lymph node involvement (12.2 ± 8.3 versus 3.8 ± 1.1). Mutations of the p53 gene were observed in 44% of ovarian cancers; p53 gene status did not relate to telomerase activity. Multivariate analysis showed that the intensity of telomerase activity was not an independent factor for prognosis of patients with ovarian cancer. Conclusion Telomerase activity may be associated with development and extension of epithelial ovarian cancer.

A newly developed adenovirus-mediated transfer of a wild-type p53 gene increases sensitivity to cis-diamminedichloroplatinum (II) in p53-deleted ovarian cancer cells

A new recombinant adenovirus carrying a wild-type p53 gene (AxCAp53) was developed and the combination effect of p53 gene transfer and cis-diamminedichloroplatinum (II) (CDDP) was examined in an ovarian cancer cell line, SK-OV-3, with deletion of the p53 gene. AxCAp53 showed a high efficiency of gene transduction and increased sensitivity to CDDP in the SK-OV-3 cells. It was found that the sensitivity of the cells to CDDP correlated with the amount of infectious units of virus per cell of AxCAp53 which correlated with p53 protein expression. The results suggest that the combination of CDDP and AxCAp53 may be a potential strategy for the therapy of CDDP-resistant ovarian cancer.

Checkpoint kinase inhibitor AZD7762 overcomes cisplatin resistance in clear cell carcinoma of the ovary.

Objective Checkpoint kinase (Chk) inhibitors are thought to increase the cytotoxic effects of DNA-damaging agents and are undergoing clinical trials. The present study was aimed to assess the potential to use the Chk1 and Chk2 inhibitor, AZD7762, with other anticancer agents in chemotherapy to treat ovarian clear cell carcinoma. Methods Four ovarian clear cell carcinoma cell lines were used in this study. We treated the cells with AZD7762 and anticancer agents, then assessed cell viability, cell cycle distribution, apoptosis, and the expression of protein in apoptotic pathways and molecules downstream of the Chk signaling pathways. We also investigated the effects of these drug combinations on tumor growth in a nude mouse xenograft model. Results Synergistic effects from the combination of AZD7762 and cisplatin were observed in all 4 cell lines. However, we observed additive effects when AZD7762 was combined with paclitaxel on all cell lines tested. AZD7762 effectively suppressed the Chk signaling pathways activated by cisplatin, dramatically enhanced expression of phosphorylated H2A.X, cleaved caspase 9 and PARP, decreased the proportion of cells in the gap 0/ gap 1 phase and the synthesis-phase fraction, and increased apoptotic cells. Combinations of small interfering RNA against Chk 1 and small interfering RNA against Chk2 enhanced the cytotoxic effect of cisplatin in both RMG-I and KK cells. Finally, treating mice-bearing RMG-I with AZD7762 and cisplatin significantly suppressed growth of tumors in a xenograft model. Conclusions The present study indicates that chemotherapy with AZD7762 and cisplatin should be explored as a treatment modality for women with ovarian clear cell carcinoma.

Adenovirus type 5 E1A gene therapy for ovarian clear cell carcinoma: a potential treatment strategy

Resistance of ovarian clear cell carcinoma (CCC) to platinum-based chemotherapy is associated with poor prognosis, and an effective treatment for advanced disease is urgently needed. HER2/neu is up-regulated more often in CCC than in other histologic types of epithelial ovarian cancer. The purpose of this study was to assess possible treatment for ovarian CCC with the anti-HER2 antibody trastuzumab or human adenovirus type 5 E1A. We treated 10 CCC cell lines with trastuzumab or E1A and assessed cell viability, proliferation, and colony formation and the expression of HER2 and wild-type p53 proteins and molecules downstream of those signaling pathways. HER2 protein was detected at various levels in all 10 cell lines by Western blotting and in 5 CCC cell lines by immunohistochemical staining; HER2 gene amplification was detected (by fluorescence in situ hybridization) in only one cell line (RMG-I). Trastuzumab did not inhibit proliferation in any of the four CCC cell lines tested (RMG-I, SKOV-2, OVTOKO, and OVSAYO). However, transfection with E1A (as compared with control vectors) reduced colony formation in all 10 CCC cell lines regardless of HER2 expression level. Infection of RMG-I and SMOV-2 cells with an adenoviral vector encoding E1A led to significant (P < 0.05) suppression of proliferation and enhancement of cell death; this effect required stabilization of p53 (but not p73) protein and was associated with the up-regulation of Bax and the cleavage of caspase-9. Other mechanisms, such as p53-independent apoptosis, may also be involved in E1A-mediated cell death in CCC. Finally, treatment with E1A prolonged survival in a CCC xenograft model (P < 0.001). E1A gene therapy, because of its ability to stabilize wild-type p53, is worth exploring as a treatment modality for women with ovarian CCC, which typically expresses wild-type p53. [Mol Cancer Ther 2007;6(1):227–35]

Significance of histologic pattern of carcinoma and sarcoma components on survival outcomes of uterine carcinosarcoma

BACKGROUND To examine the effect of the histology of carcinoma and sarcoma components on survival outcome of uterine carcinosarcoma. PATIENTS AND METHODS A multicenter retrospective study was conducted to examine uterine carcinosarcoma cases that underwent primary surgical staging. Archived slides were examined and histologic patterns were grouped based on carcinoma (low-grade versus high-grade) and sarcoma (homologous versus heterologous) components, correlating to clinico-pathological demographics and outcomes. RESULTS Among 1192 cases identified, 906 cases were evaluated for histologic patterns (carcinoma/sarcoma) with high-grade/homologous (40.8%) being the most common type followed by high-grade/heterologous (30.9%), low-grade/homologous (18.0%), and low-grade/heterologous (10.3%). On multivariate analysis, high-grade/heterologous (5-year rate, 34.0%, P = 0.024) and high-grade/homologous (45.8%, P = 0.017) but not low-grade/heterologous (50.6%, P = 0.089) were independently associated with decreased progression-free survival (PFS) compared with low-grade/homologous (60.3%). In addition, older age, residual disease at surgery, large tumor, sarcoma dominance, deep myometrial invasion, lymphovascular space invasion, and advanced-stage disease were independently associated with decreased PFS (all, P < 0.01). Both postoperative chemotherapy (5-year rates, 48.6% versus 39.0%, P < 0.001) and radiotherapy (50.1% versus 44.1%, P = 0.007) were significantly associated with improved PFS in univariate analysis. However, on multivariate analysis, only postoperative chemotherapy remained an independent predictor for improved PFS [hazard ratio (HR) 0.34, 95% confidence interval (CI) 0.27-0.43, P < 0.001]. On univariate analysis, significant treatment benefits for PFS were seen with ifosfamide for low-grade carcinoma (82.0% versus 49.8%, P = 0.001), platinum for high-grade carcinoma (46.9% versus 32.4%, P = 0.034) and homologous sarcoma (53.1% versus 38.2%, P = 0.017), and anthracycline for heterologous sarcoma (66.2% versus 39.3%, P = 0.005). Conversely, platinum, taxane, and anthracycline for low-grade carcinoma, and anthracycline for homologous sarcoma had no effect on PFS compared with non-chemotherapy group (all, P > 0.05). On multivariate analysis, ifosfamide for low-grade/homologous (HR 0.21, 95% CI 0.07-0.63, P = 0.005), platinum for high-grade/homologous (HR 0.36, 95% CI 0.22-0.60, P < 0.001), and anthracycline for high-grade/heterologous (HR 0.30, 95% CI 0.14-0.62, P = 0.001) remained independent predictors for improved PFS. Analyses of 1096 metastatic sites showed that carcinoma components tended to spread lymphatically, while sarcoma components tended to spread loco-regionally (P < 0.001). CONCLUSION Characterization of histologic pattern provides valuable information in the management of uterine carcinosarcoma.