Cisplatin-resistant Bladder Cancer Cells Research Articles

Cisplatin sensitivity of testis tumour cells is due to deficiency in interstrand-crosslink repair and low ERCC1-XPF expression

BackgroundCisplatin based chemotherapy cures over 80% of metastatic testicular germ cell tumours (TGCT). In contrast, almost all other solid cancers in adults are incurable once they have spread beyond the primary site. Cell lines derived from TGCTs are hypersensitive to cisplatin reflecting the clinical response. Earlier findings suggested that a reduced repair capacity might contribute to the cisplatin hypersensitivity of testis tumour cells (TTC), but the critical DNA damage has not been defined. This study was aimed at investigating the formation and repair of intrastrand and interstrand crosslinks (ICLs) induced by cisplatin in TTC and their contribution to TTC hypersensitivity.ResultsWe observed that repair of intrastrand crosslinks is similar in cisplatin sensitive TTC and resistant bladder cancer cells, whereas repair of ICLs was significantly reduced in TTC. γH2AX formation, which serves as a marker of DNA breaks formed in response to ICLs, persisted in cisplatin-treated TTC and correlated with sustained phosphorylation of Chk2 and enhanced PARP-1 cleavage. Expression of the nucleotide excision repair factor ERCC1-XPF, which is implicated in the processing of ICLs, is reduced in TTC. To analyse the causal role of ERCC1-XPF for ICL repair and cisplatin sensitivity, we over-expressed ERCC1-XPF in TTC by transient transfection. Over-expression increased ICL repair and rendered TTC more resistant to cisplatin, which suggests that ERCC1-XPF is rate-limiting for repair of ICLs resulting in the observed cisplatin hypersensitivity of TTC.ConclusionOur data indicate for the first time that the exceptional sensitivity of TTC and, therefore, very likely the curability of TGCT rests on their limited ICL repair due to low level of expression of ERCC1-XPF.

Enhanced S100 calcium‐binding protein P expression sensitizes human bladder cancer cells to cisplatin

• To investigate the role of S100 calcium-binding protein P (S100P) in the gain of cis-diamminedichloroplatinum (II) (cisplatin) resistance in bladder cancer, having previously found, with cDNA microarrays using two pairs of parental (T24, KK47) and their cisplatin-resistant bladder cancer cell lines (T24/DDP10, KK47/DDP20), that S100P mRNA expression was significantly reduced in cisplatin-resistant cells. • S100P mRNA and protein expression levels were investigated by northern and western blot analyses, respectively. • Intracellular S100P localization was examined by immunocytochemistry and immunohistochemistry. • S100P over-expression, obtained by transfection with S100P expression plasmid, was used to investigate whether or not S100P affected cellular resistance to cisplatin. • S100P mRNA showed increased expression by cisplatin stimulation in parental cell lines. • On the other hand, S100P mRNA and protein expression levels were markedly reduced in cisplatin-resistant cells. • The over-expression of S100P in resistant cells resulted in an increased sensitivity to cisplatin. • In bladder cancer cells, S100P was expressed and localized mainly in the nucleus. • S100P expression was also involved in cisplatin sensitivity. • S100P might thus represent a molecular marker predicting cisplatin sensitivity and a molecular therapeutic target for cisplatin-based chemotherapy.

Augmentation of cisplatin sensitivity in cisplatin‐resistant human bladder cancer cells by modulating glutathione concentrations and glutathione‐related enzyme activities

To investigate the roles of glutathione and glutathione-S-transferase (GST) in cisplatin-resistance mechanisms in human bladder cancer, by using glutathione-depleting or GST-blocking agents. Cisplatin-resistant human bladder cancer cell lines were established by continuous exposure of T24 cells to increasing concentrations of cisplatin. Buthionine sulphoximine (BSO), ethacrynic acid and indomethacin were used to deplete glutathione or block GST. Intracellular glutathione content, GST activity and cisplatin cytotoxicity were determined after exposing parental and drug-resistant cell lines to these agents. Intracellular glutathione content and GST activity were significantly decreased, and cisplatin cytotoxicity significantly enhanced, in both parental and resistant cell lines by glutathione-depleting or GST-blocking agents. However, the resistance of cisplatin-resistant cell lines did not fully recover to that of the parental cells with combined BSO and indomethacin. Both increased glutathione content and GST activity are significant in the cisplatin resistance of human bladder tumour cells. Because BSO, ethacrynic acid and indomethacin caused a partial recovery of resistance in the cisplatin-resistant cell line, further studies are needed to investigate their efficacy for treating patients with metastatic bladder carcinoma resistant to cisplatin.

Inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1) modulates the acquisition of cisplatin resistance in bladder cancer cell lines

To investigate the molecules that regulate the acquisition of cis-diamminedichloroplatinum (II) (cisplatin) resistance, we performed cDNA microarrays using two pairs of parental and cisplatin-resistant bladder cancer cell lines. We found a markedly reduced expression of inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1), endoplasmic reticulum membrane protein, in cisplatin-resistant cells. The suppression of IP3R1 expression using small interfering RNA in parental cells prevented apoptosis and resulted in decreased sensitivity to cisplatin. Contrarily, overexpression of IP3R1 in resistant cells induced apoptosis and increased sensitivity to cisplatin. These results suggest that cisplatin-induced downregulation of IP3R1 expression was closely associated with the acquisition of cisplatin resistance in bladder cancer cells.

200 Human BCL-2 related BFL-1/A1 gene is up regulated in cisplatin-resistant human bladder cancer cells

200 Human BCL-2 related BFL-1/A1 gene is up regulated in cisplatin-resistant human bladder cancer cells

Ultrastructural change due to acquired cisplatin resistance in human bladder cancer cells

Cis-diaminedichloroplatinum (cisplatin) has therapeutic efficacy against advanced bladder cancer. However, the response is often limited due to appearance of drug-resistant tumor cells. Recently, we established a cisplatin-resistant human bladder cancer cell line, T24/R2, from T24 human bladder cancer cell line, which shows 18-fold resistance to cisplatin by the stepwise exposure of increasing concentrations of cisplatin. A light microscopic (LM) and transmission electron microscopic (EM) examination was performed to investigate the morphological and ultrastructural changes during induction of drug resistance. In LM, the cytoplasm of T24R2 cells showed plumper pattern than that of T24 parent cells. In EM, the chromatin pattern of T24R2 cells was finely dispersed compared to T24 cells, which were coarse and aggregated. The mitochondrial volume, rough endoplasmic reticulum, polyribosomes and ribosomes were moderately increased in T24R2 cells. The cell membrane showed ruffled border and great amount of double membrane vesicles and pinocytic vesicles were observed in the cell surface of T24R2 cells, which were seldom observed in T24 cells. With these findings, we concluded that human bladder cancer cells underwent morphological and ultrastructural changes during acquiring of resistance to cisplatin. We could suggest that these changes might be involved in the drug resistance mechanism in human bladder cancer cells.

Antisense Bcl2 oligonucleotide in cisplatin-resistant bladder cancer cell lines.

To determine the change of expression of Bcl2 in cisplatin-resistant bladder cancer cell lines and the reversibility of chemoresistance to cisplatin with antisense oligonucleotide against Bcl2, as higher expression of Bcl2 is associated with drug resistance in many different cancer cell lines. In the cisplatin-resistant bladder tumour cell lines T24R1 and T24R2, the expression of Bcl2 was determined by reverse transcription-polymerase chain reaction and Western blot assay, and antisense oligonucleotide targeting of the Bcl2 coding sequence was administered with lipofectin. The expression of Bcl2 mRNA and protein was greater in T24R1 and T24R2 cells than in the parent T24 cells. Short-term exposure to cisplatin up-regulated Bcl2 mRNA and protein expression in parent T24 cells. Treatment with antisense oligonucleotide down-regulated Bcl2 protein expression and significantly enhanced the cytotoxicity of cisplatin. Up-regulation of Bcl2 protein expression might be one of the mechanisms of cisplatin resistance in bladder cancer cells, and antisense Bcl2 oligonucleotide may be helpful in chemotherapy for bladder cancer by reversing cisplatin resistance.

Loss of p73 induction in a cisplatin-resistant bladder cancer cell line.

Cisplatin (CDDP) plays an important role in the treatment of transitional-cell carcinoma (TCC), but resistance develops. The mechanism is not entirely understood. To assess acquired resistance to CDDP, we established a CDDP-resistant subclone, CL8-2, of T24, which is a bladder cancer cell line. We examined the changes in the various pathways leading to apoptosis in the parent line and CL8-2. The drug caused apoptosis of T24 cells but not CL8-2 cells. The CL8-2 cells were 6.4 times more resistant to CDDP than was T24. In both cell lines, the mismatch repair genes hMLH-1 and hMSH-2 were expressed at high levels. The p53 protein was not detected in either cell line but p73 protein was induced by CDDP treatment in T24 cells, which was followed by activation of caspases 3, 8, and 9. This phenomenon was not observed in CL8-2 cells. These results suggest that loss of p73 induction may lead to CDDP resistance of TCC.