Interdependence of DNA mismatch repair proteins MLH1 and MSH2 in apoptosis in human colorectal carcinoma cell lines.

The mammalian DNA mismatch repair (MMR) system consists of a number of proteins that play important roles in repair of base pair mismatch mutations and in maintenance of genomic integrity. A defect in this system can cause genetic instability, which can lead to carcinogenesis. For instance, a germline mutation in one of the mismatch repair proteins, especially MLH1 or MSH2, is responsible for hereditary non-polyposis colorectal cancer. These MMR proteins also play an important role in the induction of apoptosis. Accordingly, altered expression of or a defect in MLH1 or MSH2 may confer resistance to anti-cancer drugs used in chemotherapy. We hypothesized that the ability of these two MMR proteins to regulate apoptosis are interdependent. Moreover, a defect in either one may confer resistance to chemotherapy by an inability to trigger apoptosis. To this end, we studied three cell lines-SW480, LoVo, and HTC116. These cell lines were selected based on their differential expression of MLH1 and MSH2 proteins. SW480 expresses both MLH1 and MSH2; LoVo expresses only MLH1 but not MSH2; HCT116 expresses only MSH2 but not MLH1 protein. MTT assays, a measure of cytotoxicity, showed that there were different cytotoxic effects of an anti-cancer drug, etoposide, on these cell lines, effects that were correlated with the MMR status of the cells. Cells that are deficient in MLH1 protein (HCT116 cells) were resistant to the drug. Cells that express both MLH1 and MSH2 proteins (SW480 cells) showed caspase-3 cleavage, an indicator of apoptosis. Cells that lack MLH1 (HCT116 cells) did not show any caspase-3 cleavage. Expression of full-length MLH1 protein was decreased in MMR proficient (SW480) cells during apoptosis; it remained unchanged in cells that lack MSH2 (LoVo cells). The expression of MSH2 protein remained unchanged during apoptosis both in MMR proficient (SW480) and deficient (HCT116) cells. Studies on translocation of MLH1 protein from nucleus to cytosolic fraction, an indicator of apoptosis, showed that MLH1 translocation only occurred in MMR proficient (SW480) cells upon induction of apoptosis further suggested a MSH2 dependent role of MLH1 in apoptosis. These data suggest a role of MLH1 in mediation of apoptosis in a MSH2-dependent manner. Taken together, our data supported an interdependence of mismatch repair proteins, particularly MLH1 and MSH2, in the mediation of apoptosis in human colorectal carcinoma cell lines.

Metabolic reprogramming by Dichloroacetic acid potentiates photodynamic therapy of human breast adenocarcinoma MCF-7 cells.

Aberrant glycolytic metabolism is one of the hallmarks of carcinogenesis and therefore reversal of metabolic transformation is a promising drug target in cancer treatment strategies. Dichloroacetic acid (DCA) is known to target the glycolytic pathway in cancer cells and facilitates reversal of metabolic transformation from aerobic cytosolic accumulation of pyruvic acid, "the Warburg effect", to mitochondrial oxidative phosphorylation. Recently, combination therapy particularly involving photodynamic therapy (PDT) has received considerable attention in oncology. We hypothesized that if DCA and PDT are combined, they might potentiate mitochondrial dysfunction and induce apoptosis by a reactive oxygen species (ROS) dependent pathway. We used MCF-7 cells as our in vitro model and 5-aminolevulinic acid (5-ALA) dependent PDT therapy to test our hypothesis. We found that combinatorial treatment of MCF-7 cells with PDT and DCA not only increased cell growth inhibition, but also affected mitochondrial membrane integrity perhaps via production of ROS, and enhanced apoptosis. Further, our results on ATP release during the combined treatment demonstrate that immunogenic cell death (ICD) is likely to be a potential mechanism by which PDT and DCA induce cancer cell death. Taken together, our study suggests a novel way of sensitizing MCF-7 cells for accelerated induction of apoptosis and ICD in these cells. The findings included in this study might have direct relevance in breast cancer treatment strategies.