Jun Naniwa

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.

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

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.

The PI3K/mTOR dual inhibitor NVP-BEZ235 reduces the growth of ovarian clear cell carcinoma

Patients with clear cell carcinoma of the ovary (OCCC) have poor survival due to resistance to standard chemotherapy. OCCC has frequent activating mutations of the PIK3CA gene. The present study was conducted to clarify the efficacy of the inhibition of the PI3K-AKT-mTOR pathway in OCCC. We used 8 OCCC cell lines and 5 ovarian serous adenocarcinoma (OSAC) cell lines. The mutation status of the PIK3CA and KRAS genes was examined by direct sequencing. The IC50 values of NVP-BEZ235 (BEZ235) and temsirolimus were determined by WST-8 assay. Protein expression levels of PI3K-AKT-mTOR pathway molecules were examined by western blotting. Cell cycle distribution was analyzed by flow cytometry. Annexin V staining was used for detecting apoptosis. We also investigated the effects of BEZ235 on OCCC tumor growth in a nude mouse xenograft model. Four of the 8 OCCC cell lines showed a PIK3CA mutation while none of the 5 OSAC cell lines showed a mutation. The IC50 values of BEZ235 for the OCCC cell lines were lower than these values for the OSAC cell lines. The IC50 value of temsirolimus was higher than BEZ235 in the OCCC cell lines. The PIK3CA mutation was more frequently noted in OCCC than OSAC cells, but the sensitivity of these cell lines to BEZ235 or temsirolimus was not related to the mutation status. pHER3 and pAkt proteins were expressed more frequently in OCCC compared with OSAC. However, protein expression levels were distributed widely, and were not related to the sensitivity. Treatment with BEZ235 suppressed expression of pAkt, although treatment with temsirolimus did not. OCCC cells exhibited G1 phase arrest after treatment with BEZ235 and apoptosis with a higher concentration of the agent. BEZ235 significantly inhibited tumor growth in mice bearing OVISE and TU-OC-1 cell tumors. The present study indicated that the PI3K-AKT-mTOR pathway is a potential target for OCCC, and that BEZ235 warrants investigation as a therapeutic agent.

Preoperative and intraoperative assessments of depth of myometrial invasion in endometrial cancer.

Objective: Preoperative and intraoperative assessments of myometrial invasion (MI) are commonly used for planning surgical procedures such as dissection of the para-aortic node; however, the assessments often differ from the final diagnosis determined by pathological examination. The present study evaluated the accuracy of preoperative and intraoperative assessments of MI. Methods: A total of 191 patients with endometrial cancer, who underwent hysterectomy from 1995 to 2007 in Tottori University Hospital, were included in this study. One hundred seventy-four patients underwent endometrial curettage or Pipelle biopsy preoperatively. Histological grade was compared between preoperation and postoperation. Magnetic resonance imaging (MRI) was performed before surgery, and the depth of MI was assessed as 3 levels (no MI, <50%, and >50%). During surgery, the uterine wall was incised at the most invasive part, and then, intraoperative gross assessment was evaluated as less than or greater than 50%. Results: Histological evaluation revealed that 34 patients had no invasion, 97 had less than 50% MI, and 60 had greater than 50% MI. On MRI assessment, 135 patients had correct diagnoses, and the accuracy was 70.7%. Regarding the diagnosis of greater than 50% MI depth, the accuracy, the sensitivity, and the specificity of the MRI assessment were 83.2%, 75.0%, and 85.7%, respectively. Seventeen patients were overestimated, and 15 patients were underestimated by the MRI assessment. On intraoperative gross assessment, 162 patients had correct diagnoses, 8 patients were overestimated, and the remaining 21 patients were underestimated. The accuracy of the gross assessment was 84.8%, the sensitivity was 65.0%, and the specificity was 93.9%. The preoperative grading accuracy was 71.8% (125/174). A discrepancy between preoperative and postoperative grades was more frequent in a low-grade tumor. The incidence of underdiagnosis was significantly higher in patients with a grade 3 (G3) tumor than in those with a G1 or G2 tumor in both assessments. Conclusions: The present study suggests that gross assessment may be useful to determine MI of less than 50%, although patients with a G3 tumor were more frequently underestimated.

PTEN is involved in the signal transduction pathway of contact inhibition in endometrial cells

PTEN is involved in the regulation of normal cellular functions in addition to its well–known role as a tumor suppressor. In the present study, we have shown that stable transfection of the PTEN gene into PTEN–mutated endometrial carcinoma cells leads to contact inhibition accompanied by a decreased level of phosphorylated–Akt (p–Akt) expression, an increase in p27Kip1, and a decrease in β–catenin. PTEN–induced cells with contact inhibition exhibit G0–G1 cell-cycle arrest, and the Ki–67 labeling index is reduced. These changes are canceled by transfection of a double–stranded short–interfering RNA against the PTEN gene. Normal endometrial stromal cells increase their PTEN expression when reaching confluence; this is followed by changes in the expression of Akt–related proteins in the same way as in tumor cells. These results indicate that PTEN, p–Akt, p27, and β–catenin are involved in the signal transduction of contact inhibition and suggest that PTEN may, in part, control the proliferation of endometrial carcinoma cells through the induction of contact inhibition.

Activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway overcomes cisplatin resistance in ovarian carcinoma cells.

Objective This study was aimed to elucidate the roles of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′-kinase (PI3K)/Akt pathways in regulating cytotoxicity induced by cisplatin (CDDP) in ovarian carcinoma cells. Methods We treated 7 ovarian cancer cell lines with CDDP alone or with CDDP and either a PI3K inhibitor (LY294002), a MEK inhibitor (PD98059), or a MEK/ERK activator (phorbol 12-myristate 13-acetate [PMA]) and assessed cell viability, expression of MEK/ERK and PI3K/Akt, cell cycle distribution, and apoptosis. We also investigated the effect of combination treatment on survival in a xenograft model. Results The cell lines showed half-maximal inhibitory concentrations (IC50) of CDDP from 2.4 to 26.9 µmol/L. KFr, a CDDP-resistant cell line developed from KF cells, showed an IC50 of CDDP of 9.6 µmol/L. Five of the cell lines with IC50 values of 9.6 µmol/L or greater were defined as CDDP-resistant. Cisplatin and LY294002 had an additive effect on inhibiting cell growth, and CDDP and PD98059 had and antagonistic effect on cell growth in all cell lines. In CDDP-resistant cells, CDDP and PMA dramatically suppressed the cell growth, up-regulated the expression of phosphorylated ERK and cleaved caspase-9, down-regulated the expression of checkpoint kinases, and increased the proportion of cells in the synthesis-phase fraction and apoptotic cells. The treatment of nude mice with CDDP and PMA prolonged survival in an ovarian cancer xenograft model. Conclusions The present study indicates that further study is warranted to determine the effectiveness of combination treatment with CDDP and PMA for platinum-resistant ovarian carcinoma.

Activity of docetaxel in paclitaxel-resistant ovarian cancer cells

PurposeThe aim of this study was to determine the behavior of docetaxel (DTX) in ovarian cancer cells resistant to paclitaxel (PTX).MethodsWe used human ovarian adenocarcinoma cell lines KF, KFTx (PTX-resistant KF), SK-OV-3, and HAC-2. The sensitivity of the cells to PTX or DTX was determined by the MTT assay. Cellular accumulation of PTX and DTX was measured by high-performance liquid chromatography. mRNA of MDR-1 was detected by RT-PCR. Cell cycle distribution was determined by flow cytometry after exposure to the IC50 of each drug. Bcl-2 phosphorylation was determined by Western blot analysis. Activity for tubulin polymerization of each drug was examined by a β-tubulin polymerization assay.ResultsKFTx cells had a 5.5-fold greater resistance to PTX and a 7.3-fold greater resistance to DTX than KF cells, indicating that KFTx cells had acquired cross-resistance to DTX. SK-OV-3 cells were sensitive and HAC-2 cells were resistant to both PTX and DTX. The gene expression of MDR-1 increased after exposure to DTX in KF and KFTx cells. Residual cellular accumulation of PTX and DTX was significantly lower in KFTx cells than in KF cells. In contrast, MDR-1 expression was not detected in SK-OV-3 and HAC-2 cells. Flow cytometric analysis indicated no differences in alterations of cell cycle distribution following exposure to the two drugs. Bcl-2 phosphorylation occurred after exposure to DTX at a concentration equivalent to the clinical dose, but did not occur after exposure to PTX in KFTx cells. In HAC-2 cells, Bcl-2 phosphorylation was not detected after exposure to DTX or PTX at concentrations equivalent to the clinical doses. DTX showed greater tubulin polymerization activity than PTX in KFTx cells. β-tubulin polymerization did not correlate with the concentration of PTX or DTX, suggesting that alteration in the tubulin reaction might contribute to the resistance in HAC-2 cells.ConclusionsThe present study suggests that the mechanisms involved in cytotoxicity of and resistance to PTX and DTX do not differ, but DTX has a greater cytotoxic potential in PTX-resistant cells with an efflux system.

Leptomycin B enhances CDDP-sensitivity via nuclear accumulation of p53 protein in HPV-positive cells.

Cervical cancer, which commonly contains a wild‐type p53 gene, is highly correlated with human papilloma virus (HPV) infection. Because the oncoprotein E6, derived from HPV, inhibits the function of p53 protein, the inhibition of apoptosis via the p53 pathway by HPV may be related to cisplatin (CDDP)‐sensitivity in cervical cancer. We conducted the present study to determine whether and how HPV is related to CDDP‐sensitivity in HPV‐positive cervical cancer cells. We used cervical carcinoma cell lines HeLa with integrated HPV 18 and SiHa with integrated HPV 16. An HPV‐negative cell line, Yumoto, with wild‐type p53 gene was used as a control. Leptomycin B (LMB) enhanced sensitivity to CDDP and CDDP‐induced apoptosis in HeLa and SiHa cells, but not in Yumoto cells. After exposure to LMB or CDDP alone, we observed weak p53 staining in HeLa, SiHa and Yumoto cells. Nuclear p53 staining was significantly increased by combined treatment with CDDP and LMB in HeLa and SiHa cells, but not in Yumoto cells. The expression of p53 and Bax protein increased with exposure to CDDP and was enhanced by LMB in HeLa and SiHa cells. The present study demonstrated that LMB enhanced CDDP‐sensitivity via nuclear accumulation of p53 protein in HPV‐positive cells.