Study On Anti-Hepatoma Mechanism Of MAP30 From Momordica Charantia

Background and objective:Liver cancer(Hepatocellular carcinoma(HCC)or malignant hepatoma),is one of the most lethal diseases worldwide,especially in China.Doxorubicin and Cisplatin are the common chemotherapeutical drugs in the treatment of liver cancer.However,with the prolongation of medicine,its anti-tumor effect becomes less obvious,and the expected therapeutic effect cannot be achieved.MAP30(Momordica anti-HIV-1 Protein of 30 k Da)is a kind of Ribosome Inactivating Proteins(RIPs)extracted from bitter melon seeds,which has anti-tumor,antivirus,antibacterial and other biological functions.In 1990,it was first discovered and reported by American scientist Sylvia Lee-Huang et al.that it has N-glycosidase activity,which can inhibit protein biosynthesis.Previous studies almost focused on the structural analysis and antiviral activities of MAP30,and just a few studies on its antitumor effects.At the same time,the method of original purification of MAP30 was cumbersome,so we plan to simplify its method steps and increased the yield,which laid a certain theoretical and experimental foundation for the subsequent development of anti-tumor drugs.Based on the previous studies,we combined several purification methods and chromatography techniques to successfully obtain the anti-tumor protein MAP30.And we explored on anti-liver cancer effects and the apoptosis and related apoptosis signaling pathways caused by MAP30.which can lay a certain theoretical and experimental basis for the protein as a potential antitumor drug.Materials and Methods:1.Preparation and identification of MAP30Fresh bitter melon seeds are used as materials,referring to the physicochemical and related literature reports of MAP30.We mainly use selective denaturation precipitation,ammonium sulfate fractionation,ion exchange chromatography,molecular sieve chromatography and hydrophobic interaction chromatography or SP-Sepharose ion exchange chromatography in combination with dialysis and ultrafiltration to separate and purify the target protein MAP30.MAP30 was characterized by PAGE,SDS-PAGE,IEF,RP-HPLC,protein amino acid sequencing and MALDI-TOF/MS.2.The anti-liver cancer activity in vitro(1)Hepatoma cell lines(Hep G2,MHCC-97 H,MHCC-97 L,HCC-LM3,SK-Hep-1)were treated with different concentrations of MAP30(0.012,0.06,0.3,1.5 mg/mL),and the CCK-8 kit and cell colony formation assay were used to assess the proliferation of hepatoma cells.(2)PI staining was used to detect the cell cycle of liver cancer cells after the treatment of MAP30.(3)The cell growth status,growth density,intercellular space,etc.were observed by an inverted microscope;The cells observed under electron microscopy for changes in organelles after MAP30.(4)Apoptosis rates detected by flow cytometry,and the activation of apoptosis marker proteins in cells was detected by western blotting.3.MAP30 induces apoptosis involved mitochondria in SK-Hep-1 cells(1)DCFH-DA probe detected intracellular ROS levels after the treatment of MAP30.(2)Mitochondrial membrane potential changes detected by JC-1 dye.(3)The expression of mitochondrial apoptosis-related proteins Bcl-2,Bax,cytochrome C was detected by western blotting.Results:1.MAP30 was purified by acidification,ammonium sulfate fractional precipitation,ion exchange chromatography and molecular sieve chromatography,63 mg of MAP30 was obtained from 100 g of bitter melon seeds.Under reduced or non-reducing conditions,the purified MAP30 SDS-PAGE showed a single protein colored band,corresponding to a molecular size of about 30 k Da,which was determined to be MAP30 protein by MALDI-TOF/MS and N-terminal amino acid sequencing.2.In vitro experimental studies have shown that MAP30 owned antitumor activity.A series of hepatoma cell lines(Hep G2,MHCC-97 H,MHCC-97 L,HCC-LM3,SK-Hep-1)was treated with different concentrations of MAP30(0.012,0.06,0.3,1.5 mg/mL)showed significant inhibition of cell growth,especially HCC-LM3 and SK-Hep-1 cell lines which showed obvious time-dose dependent manner.Hep G2,MHCC-97 H,HCCLM3,SK-Hep-1 can be blocked in G0/G1 phase.3.Through electron microscopy,MAP30 was found to cause apoptosis and autophagy in HCC-LM3 cells.The results of Annexin V-FITC/PI double staining and western blotting confirmed that MAP30 induced apoptosis in SK-Hep-1 and HCC-LM3 cells.And it also showed that the apoptosis rate,the apoptotic protein Caspase-3 and PARP1 activation increased and the cleaved Caspase-9 was upregulated when the dose of MAP30 was increased.4.After treatment of cells with MAP30,DCFH-DA probes detected an increase in intracellular ROS level.JC-1 staining observed the decrease of mitochondrial membrane potential.And bcl-2 protein was downregulated All of the results conclude that MAP30 can induce apoptosis involving the mitochondria in hepatoma cells.Conclusion:The purification method of MAP30 we modified simplifies steps,saves time,and obtains the purified MAP30.In vitro antitumor experiments have shown that it can inhibit the proliferation of Hep G2,MHCC-97 H,MHCC-97 L,HCC-LM3,SK-Hep-1 hepatoma cell lines,induce G0/G1 cycle arrest in Hep G2,MHCC-97 H,HCC-LM3,SK-Hep-1 cells,increase intracellular ROS levels,and promote apoptosis involving mitochondria.All results suggest that this natural MAP30 can be used as a novel potential drug against liver cancer in humans in clinical applications.