Cisplatin resistance in an ovarian carcinoma is associated with a defect in programmed cell death control through XIAP regulation

Abstract

Chemoresistance is a major impediment to the successful treatment of cancer. It involves various mechanisms, including defects in the apoptosis program that is induced by anticancer drugs. To further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin (CDDP) treatment, we compared the effect of CDDP on expression of X-linked inhibitor of apoptosis protein (XIAP), a direct inhibitor of caspase-3, -7, and -9, Fas, Fas-ligand (Fas-L), and pro- and antiapoptotic proteins in a CDDP-sensitive human ovarian carcinoma cell line (2008) and its CDDP-resistant subclone (2008C13). In this article, we show that cisplatin treatment led to a differential expression of distinct apoptotic targets in the CDDP-sensitive cell line (2008) and its CDDP-resistant subclone (2008C13). The acquisition of cisplatin resistance was associated with the ability of the treated cells to enhanced expression of XIAP, whereas the death inducer Fas-L was abrogated in 2008C13 following treatment with CDDP. However, the CDDP-sensitive cells failed to activate XIAP but increased Fas-L expression, indicating that distinct regulatory mechanisms are operative. These findings suggest that the expression of XIAP and downregulation of Fas-L are linked to chemoresistance in ovarian carcinoma cells and may represent one of the potential antiapoptotic mechanisms involved during this process.

Figure 1

CDDP-induced apoptosis in ovarian carcinoma cells. (a) Dose response of CDDP-induced apoptosis. The 2008 and 2008C13 cells were incubated for 1 h in 96-well plates (2 × 10³ cells/well) with CDDP at the concentrations indicated, then cultured in a fresh CDDP-free medium for the remaining time. The percentage of live cells was measured 4 days later by using a fluorescence-based nucleic acid method for cell viability (CyQUANT cell proliferation assay kit). (b) Analysis of CDDP-induced DNA fragmentation pattern. The 2008 ovarian carcinoma cells were treated with 20 μM of CDDP for 1 h, and cells were collected at the indicated time for DNA fragmentation analysis.

Figure 2

Expression of pro- and antiapoptotic proteins in CDDP-treated ovarian carcinoma 2008 and 2008Cl3 cells. CDDP-sensitive and CDDP-resistant cells were treated for 1 h with 20 μM CDDP. At the indicated time, whole cell lysates were prepared and proteins were separated (70 μg) and analyzed by Western blotting with the indicated antibodies.

Figure 3

Expression of XIAP and Fas/Fas-L following CDDP treatment. (a) Fas-L mRNA was enhanced in 2008 cells and abrogated in 2008C13 cells after stimulation with CDDP. Cells were incubated with medium or with 20 μM CDDP for 1 h. At time points indicated, total RNA was purified, and the expression of Fas-L and β-actin mRNAs was examined by RT-PCR using specific primers. (b) Same experiment as in (a) with XIAP and GAPDH as the specific primer pairs.