Masakuni Takahashi

Chemosensitivity and p53-Dependent Apoptosis in Epithelial Ovarian Carcinoma

Although p53 gene mutation frequently is observed in ovarian carcinoma, the function of the p53 gene in chemosensitivity has not been defined conclusively. The objective of the current study was to elucidate the relation between chemotherapy‐induced apoptosis through the p53 pathway and chemosensitivity to ovarian carcinoma.

Mechanisms of Cisplatin Resistance in Clear Cell Carcinoma of the Ovary

Resistance of clear cell carcinoma (CCC) of the ovary to platinum-based chemotherapy is associated with a poor prognosis. However, the mechanism underlying the resistance of CCC to platinum has not yet been understood. We conducted the present study to clarify the mechanism of cisplatin (CDDP) resistance in CCC cells. Eleven CCC and 5 serous adenocarcinoma (SAC) cell lines were used in this study. The IC50 to CDDP ranged from 1.3 to 18.0 µM for CCC cells and from 2.2 to 13.0 µM for SAC cells. There was no correlation between multidrug resistance-associated protein expression and the sensitivity to CDDP in CCC cells. In contrast, the doubling time for CCC cells was significantly longer than that for SAC cells (61.4 vs. 29.8 h). A significant reverse correlation between the S-phase fraction and the response to CDDP was observed (r = 0.647, p < 0.05). The present study suggests that the resistance of CCC to CDDP may be caused by low cell proliferation.

Cisplatin-resistant HeLa cells are resistant to apoptosis via p53-dependent and -independent pathways.

Since HeLa cells possess very little functional p53 activity, they could be originally resistant to genotoxic stress‐induced apoptosis. Therefore, it is likely that the drug‐resistant cells derived from HeLa cells are more resistant to apoptosis. The aim of this study was to determine whether cisplatin‐resistant cells derived from HeLa cells have an apoptosis‐resistant phenotype. A cisplatin‐resistant cell subline, HeLa/CDDP cells, showed a 19‐fold resistance to cisplatin compared with the parent cells. The subline showed a collateral sensitivity to paclitaxel. An equitoxic dose (IC50) of cisplatin produced DNA fragmentation in HeLa cells but not in HeLa/CDDP cells. Transfection of wild‐type p53gene enhanced the cytotoxicity of cisplatin and cisplatin‐induced apoptosis in HeLa cells but not in HeLa/CDDP cells, although it caused p53 overexpression in both cell lines. The expression of caspase 1 (interleukin‐1β‐converting enzyme, ICE) mRNA and the overexpression of bax protein were observed only in HeLa cells. Paclitaxel‐induced DNA fragmentation appeared less in HeLa/CDDP cells than in HeLa cells. p53gene transfection did not affect the extent of DNA fragmentation in either cell line, suggesting that paclitaxel may induce p53‐independent apoptosis. These findings suggest that HeLa/CDDP cells may have an acquired phenotype that is resistant to p53‐dependent and ‐independent apoptosis.

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.

Significance of cytoreductive surgery including bowel resection for patients with advanced ovarian cancer.

The aim of this study was to determine the significance of bowel resection in advanced ovarian cancer. A total of 64 women with stage IIIc or IV epithelial ovarian cancer, who consecutively received primary treatment between 1991 and 1995, were entered in this prospective study. The outcome of the patients undergoing bowel resection was evaluated. Thirty-nine patients underwent cytoreductive surgery at initial surgery. Of them, 16 patients could undergo optimal operation without bowel resection. Twenty-three patients received bowel resection at initial surgery. Of these 23 patients, 16 underwent optimal operation and 7 did not. Among 25 patients judged as inoperable cases at initial surgery, 21 responded to chemotherapy and underwent second surgery. Of 21 patients receiving second surgery, 15 underwent optimal operation (7 without bowel resection and 8 with bowel resection). The 3-year survival rate for 24 patients undergoing optimal operation with bowel resection (46.8%) was not significantly different from that for 23 patients without bowel resection (59.1%). Postoperative complications were seen in 8 patients (21.6%) of the patients receiving bowel resection and 3 (13.0%) of those without bowel resection. Cytoreductive surgery including bowel resection is effective for an improvement of the survival in patients with advanced ovarian cancer, if an optimal operation can be performed.

Cellular Efflux Pump and Interaction between Cisplatin and Paclitaxel in Ovarian Cancer Cells

Objective: The aim of this study was to evaluate the combination effect of paclitaxel (PTX) and cisplatin (CDDP) and to determine the mechanisms of interaction between these agents. Methods and Results: We used human ovarian adenocarcinoma cell lines, namely a parent cell line (KF), a CDDP-resistant cell line (KFr) and a PTX-resistant cell line (KFTx).The combination effect of PTX and CDDP was synergistic on KF and KFTx and additive on KFr. The incidence of anaphase or telophase, evaluated by immunofluorescence microscopy, decreased with PTX and significantly decreased with PTX and CDDP in KF and KFTx. The concentration of PTX, which was measured by high-performance liquid chromatography, was higher in KF and KFTx cells treated with a combination of PTX and CDDP than those treated with PTX alone. Multidrug resistance gene mRNA appeared in KFTx and its expression decreased after exposure to PTX and CDDP. After exposure to CDDP, the expression of multidrug resistance-associated protein (MRP) and the concentration of glutathione increased in KF, but not in KFr or KFTx. MRP expression slightly increased in KF and KFTx after exposure to PTX. In contrast, its expression decreased in KFr. Conclusion: The present study suggests that CDDP enhances PTX accumulation and that the interaction of these agents is synergistic in CDDP-sensitive cells.

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.

Sensitivity to paclitaxel is not related to p53-dependent apoptosis in ovarian cancer cells

We conducted this study to determine whether the sensitivity of ovarian cancer cells to paclitaxel (PTX) relates to cells undergoing p53-dependent apoptosis. Human ovarian adenocarcinoma cell lines (SK-OV-3, KF and KP cells) were used in this study. In SK-OV-3 and KP cells, which have a homozygous deletion of the TP53 gene, wild-type TP53 gene-transduction markedly enhanced the sensitivity to cisplatin (CDDP), but did not enhance the sensitivity to PTX. In all cells, the apoptotic index was increased by CDDP or PTX. After exposure to CDDP, p53 and Bax protein expression increased and Bcl-xL expression decreased in the KF cells and TP53 gene-transducted SK-OV-3 cells. However, these proteins did not change in KP cells. Therefore, the role of p53 in CDDP-induced apoptosis depends upon the cell type. In contrast, TP53 gene status did not correlate with PTX-induced cytotoxicity in any of the cell lines with differing apoptotic pathways. In conclusion, the sensitivity to PTX may not be related to p53-dependent apoptosis in ovarian cancer cells.

Topoisomerase‐I activity and response to second‐line chemotherapy consisting of camptothecin‐11 and cisplatin in patients with ovarian cancer

The aim of the present study was to clarify the relationship between topoisomerase‐I (topo‐I) activity and sensitivity to second‐line chemotherapy consisting of cisplatin and camptothecin‐11 (CPT‐11) in patients with ovarian cancer. Thirty Japanese women with relapsed epithelial ovarian cancer who received treatment at Tottori University Hospital or Kurume University Hospital between 1992 and 1997 were included in this study. All patients had initially undergone chemotherapy consisting of cisplatin, doxorubicin and cyclophosphamide (CAP). All subjects exhibited measurable lesions and received second‐line chemotherapy consisting of 50 to 60 mg/m2 CPT‐11 on days 1, 8 and 15 and 60 mg/m2 cisplatin on day 1. Tumor samples were obtained in the period between initial and second‐line chemotherapy. Topo‐I activity was assayed by relaxation of supercoiled plasmid substrate DNA. Of the 30 patients, 18 responded to second‐line chemotherapy and 12 did not. We found no significant difference in patient characteristics in responders and non‐responders. The interval from the end of the initial course of chemotherapy to the beginning of the second‐line chemotherapy did not significantly differ in the 2 groups. The minimum amount of extraction showing complete DNA relaxation in non‐responders was significantly greater than that in responders (201.7 ± 92.5 vs. 124.1 ± 59.4 ng; p = 0.0164). In 8 cases whose samples could be obtained before and after CAP, the amount of protein significantly decreased after CAP therapy (286.4 ± 142.1 vs. 138.5 ± 97.8 ng; p = 0.0294). Topo‐I activity, which is enhanced by CAP therapy, can play an important role in sensitivity to CPT‐11. Int. J. Cancer (Pred. Oncol.) 84:521–524, 1999.