A variety of cancers carry mutations in genes encoding DNA mismatch repair (MMR) proteins (e.g. MLH1, MSH2). MMR is a post-replicative process that recognizes and corrects DNA biosynthetic errors and elicits apoptosis in tumor cells exposed to some types of DNA modifying chemicals. Defect in MMR hence fails to detect modified DNA and to initiate cell cycle attenuation and apoptosis. MMR defect is thus associated with an anticancer drug resistance phenotype (e.g. cisplatin). Lipoplatin is a novel liposomal cisplatin formulation that is highly effective against cancers and shows reduced systemic toxicity and adverse reactions frequently observed for cisplatin. Although lipoplatin is anticipated to exert its cytotoxic effect by mechanisms similar to those of naked cisplatin, the possibility remains that additional processes owing to the different mechanism of uptake of lipoplatin (endocytosis) might contribute to its cytotoxic effect, which eventually may overrule the effects triggered by MMR. We report that MLH1-deficient human colorectal adenocarcinoma cells were 1.6-fold resistant to a 1h exposure (IC50: 556 ± 56 uM vs. 359 ± 39 uM, p=0.01) and 2.3-fold resistant to a 24h exposure (IC50: 70 ± 11 uM vs. 38 ± 8 uM, p=0.002) to lipoplatin as compared to MLH1-proficient cells (clonogenic assay). These data indicate that MLH1 is involved in lipoplatin-mediated cytotoxicity. The extent of DNA fragmentation (i.e. fraction of TUNEL-positive cells) was substantially lower in MLH1-deficient than in -proficient cells (500 uM lipoplatin for 24h; 7% vs. 37%). Proteolytic cleavage of PARP-1, caspase-3, and caspase-7 was only minor under these conditions and was comparable in MLH1-deficient and MLH1-proficient cells. This indicates that lipoplatin may not grossly induce apoptosis, suggesting that the higher lipoplatin sensitivity observed in the clonogenic assay as well as the higher fraction of DNA fragmentation in MMR-proficient cells eventually may owe to other cell death processes (e.g. necrosis). Trypan Blue-inclusion data showed that MLH1-deficient cells were 2-fold less susceptible to membrane disruption (450 uM lipoplatin, 30 ± 16% vs. 63 ± 5%, p=0.017), indicating that MLH1 may regulate necrosis. Lipoplatin treatment also induced a substantial and sustained arrest at the G2/M transition of the cell cycle 24h post lipoplatin treatment in MLH1-deficient as well as in MLH1-proficient cells, indicating that MLH1-deficient cells retain the ability to efficiently attenuate cell cycle progression at this checkpoint. These results suggest that the cytotoxic effect of lipoplatin is dependent on MMR, that this MMR-dependent effect may be paralleled by necrosis rather than by apoptosis, and that lipoplatin resistance in MMR-deficient cells is not due to misregulation of cell cycle control. Lipoplatin may thus not be recommended for treatment of cancers mutated in MMR genes.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]