Shunji Kamazawa

Sensitivity to anticancer agents and resistance mechanisms in clear cell carcinoma of the ovary.

We conducted the present study to determine the chemoresistance mechanisms in clear cell carcinoma of the ovary (CCC). Five human CCC cell lines (HAC‐2, RMG‐I, RMG‐II, KK, and KOC‐7c) were used in this study. The sensitivity of the cells to the anticancer agents was determined by 3–(4,5–dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and we assessed drug sensitivity by calculating assay area under the curve (AUC) for each agent. The expression of multi‐drug resistance genes (MDR‐1, MRP‐1, MRP‐2) was detected by reverse transcription‐polymerase chain reaction (RT‐PCR). Glutathione (GSH) concentration was measured by an enzymatic assay. Topoisomerase (topo) I activity was assayed in terms of relaxation of supercoiled plasmid substrate DNA. The IC50 to anticancer agents ranged widely. The assay AUC indicated that 3 of 5 cell lines (RMG‐I, RMG‐II, and KK) were sensitive to paclitaxel (PTX), 3 (HAC‐2, RMG‐I, and RMG‐II) were sensitive to 7–ethyl‐10‐hydroxycamptothecin (SN‐38), which is an active metabolite of camptothecin (CPT‐11), and only one (HAC‐2) was sensitive to cisplatin (CDDP). All cell lines were resistant to mitomycin‐C (MMC) and etoposide (VP‐16). The MRP‐1 gene was detected in all cell lines. Only one cell line showed both MRP‐2 and MDR‐1 gene expression. Except for HAC‐2 cells, expression of MRP genes was related to CDDP resistance, and MDR‐1 gene expression was associated with PTX resistance. GSH concentrations increased after exposure to CDDP or MMC in all cell lines. There was a significant correlation between topo‐I enzymatic activity and the response to SN‐38. The present study revealed several resistance mechanisms in CCC and the results suggested that PTX and CPT‐11 might be effective agents to treat CCC.

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.

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.

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.

Telomerase activity and p53-dependent apoptosis in ovarian cancer cells.

We conducted the present study to determine the relationship between p53-dependent apoptosis and telomerase activity in ovarian cancer cells. A human ovarian adenocarcinoma cell line, SK-OV-3 that had homozygous deletion of the p53 gene was used in this study. Wild-type p53 genes were transducted to SK-OV-3 cells with a recombinant adenovirus that contained a wild-type p53 gene (AxCAp53). IC50 to cisplatin (CDDP) was 12.9 μM for SK-OV-3 cells and 9.2 μM for p53 gene-transducted SK-OV-3 cells. The apoptotic index for cells with p53 gene transduction was significantly higher than cells without transduction. Additionally, p53 gene transduction significantly enhanced CDDP-induced apoptosis. Bax protein in SK-OV-3 cells did not differ before and after exposure to CDDP. In SK-OV-3 cells with transduction of the p53 gene, the expression of p53 and Bax proteins increased after exposure to CDDP. Expression of Bcl-xL decreased after exposure to CDDP in SK-OV-3 cells with and without transduction. The telomerase activity in SK-OV-3 cells with the p53 gene was significantly lower compared with the cells without the p53 gene. CDDP exposure did not affect telomerase activity and human telomerase reverse transcriptase (hTERT) expression in both cell lines. We suggest that the p53 gene may relate to telomerase activity, but that p53-dependent apoptosis does not affect the activity.

Alteration of telomerase activity in ovarian cancer after chemotherapy

To elucidate the relationship between telomerase activity and chemosensitivity in epithelial ovarian cancer, telomerase activity and telomere length (TRF) were examined before and after chemotherapy. Of 21 patients, 9 patients responded to chemotherapy and 12 did not. The positivity of telomerase activity did not significantly differ between responders and nonresponders. There were no differences in the mean length and the distribution of TRF between the two groups. Those distributions became narrow after chemotherapy in both groups. Seven nonresponders (58.3%) exhibited an increase in telomerase activity after chemotherapy but none of the responders showed an increase in activity. Telomerase activity may relate to chemosensitivity in epithelial ovarian cancer.

Timing of G-CSF administration based on the circadian rhythm in patients with ovarian cancer.

The aim of this study was to determine the relationship between the timing of granulocyte colony-stimulating factor (G-CSF) administration and its efficacy in patients with chemotherapy-induced granulocytopenia. Twenty patients in whom chemotherapy-induced leukopenia developed after the first course were enrolled in this prospective study. Subjects were randomly divided in two groups according to G-CSF injection time as follows: at 7:00 am and 7:00 pm. Before the G-CSF injection, the plasma G-CSF level for all patients was significantly lower at 7:00 am than that at 7:00 pm. After the injection, plasma G-CSF level did not differ between the two groups. The nadir of the leukocyte was 2,554 ± 379/mm3 (granulocyte 1,530 ± 689) for the group injected at 7:00 am, and 2,300 ± 426/mm3 (granulocyte 1,203 ± 848) for the group injected at 7:00 pm. The duration of leukocytes less than 2,000/mm3 and granulocytes less than 1,000/mm3 were 2.8 ± 1.8 days and 3.2 ± 1.8 days, respectively. Those differences were not significant. The present study showed the circadian rhythm of G-CSF levels in patients with ovarian cancer with chemotherapy-induced granulocytopenia, but there was no remarkable difference depending on administration time.

Mechanism of the combination effect of wild-type TP53 gene transfection and cisplatin treatment for ovarian cancer xenografts

To clarify the effect of a combination treatment consisting of a recombinant adenovirus carrying a wild-type TP53 gene (AxCATP53) and cisplatin (CDDP), we examined p53-dependent apoptosis in ovarian cancer xenografts with and without the wild-type TP53 gene. Severe combined immunodeficiency (SCID) mice were implanted with ovarian cancer cell lines consisting of SK-OV-3 cells without the TP53 gene and KF cells with the TP53 gene. In SK-OV-3 and KF tumours, the inhibitory effect of the combination treatment on tumour growth was significant, compared with a single treatment with CDDP alone or AxCATP53 alone. The apoptotic index increased significantly after combination treatment in the SK-OV-3 tumours. The expression of Bax protein in SK-OV-3 tumours was weak, but strengthened after TP53 gene transfection. In contrast, AxCATP53 transfection did not affect CDDP-induced apoptosis in the KF tumours. Therefore, combination treatment of AxCATP53 and CDDP may be a new strategy for treating ovarian cancer with or without the TP53 gene.

Alteration of a p53 gene status affects outcome of patients with recurrent ovarian cancer.

The aim of this longitudinal study was to examine whether and how the p53 gene is altered in patients with recurrent ovarian cancer and to determine the significance of p53 mutation in recurrent tumors. The primary and recurrent tumors were examined in 15 patients who had recurrent epithelial ovarian cancer, and whose primary tumor contained a wild-type p53 gene. The interval between cytoreductive surgery and the appearance of recurrence ranged from 5.2 to 63.6 months (mean 23.4 months). Mutations in the p53 gene were screened by polymerase chain reaction single strand conformation polymorphism analysis and determined by cycle sequencing. Mutation of the p53 gene in the recurrent tumor was found in 7 of the 15 patients (46.7%). Estimated 3- and 5-year survival rates were 57.1 and 0%, respectively, for patients with p53 gene mutation detected in the recurrence tumor, and 75.0% and 37.5% for patients without the mutation (p = 0.0155). The interval between cytoreductive surgery and the appearance of recurrence did not differ between those groups (549.7 ± 102.2 vs. 832.9 ± 283.8 days). Mean survival time after recurrence was significantly better in the patients without mutation (438.6 ± 56.4 vs. 873.0 ± 157.5 days, p = 0.0125). The present study suggests that p53 gene mutation frequently occurs in recurrent ovarian cancer and that alteration of p53 gene status affects salvage chemotherapy. This phenomenon affects the prognosis of recurrent disease and may predict outcome.