The Mechanism Research Of Microcystin-LR Induced Apoptosis In Colorectal Cancer Cells

Background and porposeMicr?ystin-LR(MC-LR)is one of the most toxic cyclic 7-peptide molecules produced by cyanobacteria,which can cause serious damage to multiple organs such as liver,kidney and reproduction in animals and humans.MC-LR enters cells through a membrane transport receptor family of OATP.In cells,MC-LR covalently binds withand inhibits the activity of protein phosphatase 2A(PP2A),resulting in a high degree of phosphorylation of intracellular protein signal molecules.High phosphorylation enhances the ROS production in cells and damages cellular cytoskeletal,DNA and protein structure leading to endoplasmic reticulum stress.MC-LR induced ROS can also disrupt the function of mit?hondrial.Recently,it has been d?that MC-LR induces iNOS expression by activating NF-kappaB for induce of apoptosis in iNS-1 cells.Moreover,iNOS and NF-kappaB is critical for MC-LR induced apoptosis.iNOS produces a high level of NO,which regulates cellular function through post-translational modification of targeting protein by covalently modifies the sulfhydryl groups to form a nitrosothiol covalent bond,called S-nitrosylation(SNO).SNO regulates multiple signal pathways for the regulation of cell functions.High concentration of NO causes intracellular nitrogen pressure(RNS)and is an important factor for apoptosis regulation.It has been reported that glyceraldehyde-3-phosphate dehydrogenase(GAPDH),a key glycolysis enzyme,works as a sensor for NO stress through modification by S-nitrosylation(SNO-GAPDH).SNO-GAPDH decreases its glycolysis activity but promotes its binding to SiaH1 for nuclear transl ? ation,where SNO-GAPDH activates the nuclear apoptotic pr?esses through translation of its nitrosylation group.Thus,SNO-GAPDH-SiaHl is a unique mechanism for NO related apoptosis.It has been proved that MC-LR increases cellular NO.However,the role of NO on MC-LR-induced cytotoxicity remains unclear.The purposes of this study are to clarify the mechanism of NO/SNO-GAPDH-SiaHl involved in MC-LR related cellular toxicity and explores the significance of MC-LR in the treatment of colon cancer cells.Moreover,GAPDH is an important glycolytic enzyme;we also analized the effect of MC-LR on the reglation of glycol-metabolism in SW480 cells through 13C-label-glucose tracer combinding with gas chromatography-mass spectrometry(GC-MS).Methods1.MTT assay was used to detect the effect of MC-LR on cell proliferation of five cell lines,including human colorectal cancer cell line SW480,CaCo2,mouse colonic cancer cell line CT26,hepatoma cell HepG2,and hepatic normal cell line L02;Annexin-V FITC/PI double staining and flow cytometry were used for analysis of MC-LR-induced cell death.2.NO2-/N03-fluorescence method used for the detection of N02-/N03-concentration released by culture cells and NOS inhibitors(L-NAME/N-PLA/1400W)used for the explore of source of NO in cells stimulated with MC-LR;Biotech-switch and western blot were used to detect modification of S-nitrosylation on targeting protein induced by MC-LR.3.The nuclear transfer of GAPDH induced by MC-LR was analyzed by nuclear plasma protein separation technique with western-bloting and immunofluorescence methods;4.siRNA method was used to analyzed the involvement of GAPDH and Siahl in MC-LR induced apoptosis.5.13C-glucose tracer combined with gas chromatography-mass spectrometry(GC-MS)technique Was used to analyze the regulatory effect of MC-LR on the glycometabolism of colon cancer SW480 cells.Results1.The effect of MC-LR on cellular proliferation and viability is complicated detected by MTT assay.Low dose of MC-LR(?luM)promotes cellular proliferation while high dose of MC-LR(?luM)inhibited the cellular viability.The sensitivity of MC-LR toxicity of five cell lines(SW480,CaCo2,HepG2,LO2 and CT26)was different.Flow cytometry comformed that MC-LR increased the apoptosis population(PI+ and Annexin-V+cells);and Western blot showed that MC-LR increased the levels of apoptosis-related protein Bax and decreased anti-apoptosis protein Bcl-2 in SW480 cells.2.The expression of NOS1 and NOS2 in SW480 were induced by MC-LR detected by Western blot;and N02-/N03-fluorescence detection indicated the increase of endogenous NO in cells after treatment with MC-LR.MC-LR induces NO was synthesis by NOS1 and NOS2 since Specific inhibitors of NOS1 and NOS2(N-PLA and 1400W)could reversed the level of NO.3.S-nitrosylation of GAPDH was increased by MC-LR treatment which triggers its nuclear transl?ation.Moreover,the NOS inhibitors could decreased the level of S-nitrosylated GAPDH and reverses its nuclear transfer indicated that MC-LR induces GAPDH nuclear transfer through intracellular NOS dirived NO for GAPDH-SNO.4.MC-LR-induced apoptosis of SW480 decreased by siRNA-Siahl or siRNA-GAPDH or NOS inhibitor(L-NAME)suggests SNO-GAPDH-Siahl mechanism is involved in MC-LR-induced apoptosis and cytotoxicity.5.13C full-label glucose-gas chromatography-mass spectrometry technique showed that glycolysis was increased while TCA-cycle was decreased by MC-LR treatment,indicating that induction of mit?hondrial damage by MC-LR might contributes to the inhibiton of TCA cycle.However,the increase of glycolysis was due to the active role of MC-LR on glycolysis emzyms or gluco-metabolism transfer drived by mit?ondrial damage need to be further study.Conclusion1.Consistent with published reports,Low dose of MC-LR(?1uM)promotes cellular proliferation while high dose of MC-LR(>luM)inhibited the cellular viability.MC-LR mainly produces cytotoxic effects by inducing apoptosis.2.MC-LR induced NO synthesis leads to S-nitrosylation of GAPDH and cellular apoptosis through the SNO-GAPDH-siahl mechanism.3.The increase of glycolysis while decrease of TCA-cycle by MC-LR exposure consistent with effect of MC-LR on mit?hondrial damage.