INVESTIGATION OF MECHANISMS OF CDDP-INDUCED APOPTOSIS IN THE CDDP-RESISTANT A431 CELL LINE

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Cisplatin, cis-diamminedichloroplatinum (II) (CDDP) is one of the most important anticancer agents, producing initial good responses in various tumors. However, the resistance to this drug often develops in various tumors, and the additional administration declines its chemotherapeutic efficacy in the treatment of the cancer patient. Despite of this clinically important issue, the precise mechanisms of acquisition of resistance to this drug is still uncertain. We established the CDDP-resistant subline A431/CDDP2 from human epidermoid carcinoma cell line A431. These resistant sublines were constituted by mutagenic induction with mutagen (A431/CDDP2). A431/CDDP2 have developed 2.7 times more resistance to CDDP than the original A431 cell in terms of ICィイD250ィエD2. The CDDP-resistant subline showed a cross-resistance to CDDP analogue, Carboplatin (CBDCA) , but no cross-resistance to other chemotherapeutic drugs such as Adriamycin (ADR) and 5-Fluorouracil (5-FU)> This CDDP-resistant subline … More were transplanted into nude mice to demonstrate the resistance to CDDP treatment in vivo. The established CDDP-resistant sublines may be used in further trials to improve the understanding of the mechanisms of resistance to CDDP.Recent reports have demonstrated that chemotherapy can induce apoptosis in some cancer cells indicating that apoptosis may play a very important role in cancer therapy. Therefore, to investigate whether modulation of apoptosis influences CDDP resistance. The DNA gel electrophoresis, and ELISA of CDDP-resistant cell showed a reduced apoptosis when compared with the A431 cells treated with CDDP. We determined the p53, Bcl-2, Bax and CPP32 protein levels by western blotting. This analysis demonstrated a marked increase in Bcl-2 protein levels and reduction of CPP32 protein levels in CDDP-resistant cells. Our results indicate that reduction of apoptosis was one of the CDDP resistant mechanisms and that reduced apoptosis in CDDP-resistant cells was influenced by Bcl-2 and CPP32 protein.Recent studies have revealed that apoptosis induced by CDDP could be mediated by the activation of CPP32, one of ICE/CED-3 family protease. The parent A431 cells (A431/P) and the A431/CDDP2 were exposed to CDDP with or without ICE/CED-3 family protease inhibitors (Z-Asp-CHィイD22ィエD2-DCB). In the A431/P, the cytotoxity of CDDP was clearly reduced by Z-Asp-CHィイD22ィエD2-DCB compared with the A431/CDDP2. Furthermore, quantitative analysis of DNA fragmentation revealed that Z-Asp-CHィイD22ィエD2-DCB surely inhibited the DNA fragmentation induced by CDDP in A431/Pcells, but A431/CDDP2 were not inhibited the DNA fragmentation. We determined the CPP32 protein level by Western Blotting. This analysis demonstrated a marked reduction of CPP32 protein levels in A431/P treated with Z-Asp-CHィイD22ィエD2-DCB. In the A431/CDDP2, this protein levels were no change with or without Z-Asp-CHィイD22ィエD2-DCB. These findings suggest that CPP32 may mediate apoptosis induced by CDDP, and its induction could represent a novel approach for the effective treatment of malignant tumors. Less