The Role Of Taurine On Apoptosis Of Tumor Cells And The Initial Mechanism

The tumor is one of the most serious diseases for human health. In our country,the malignant tumor has become first cause of death in the spectrum of death inrecent years, it serious harm for people’s health and life. Therefore, prevention andcontrol of cancer have become the most important one of the health work. Researchshows that human about40%to60%of the cancer is caused by chemical factors，thatis main related to the diet. Therefore, healthy diet is a positive cancer preventionstrategy. Now, the anticancer material of diet has become a chemical preventionresearch for tumor development. Taurine(Tau) is a simple chemical structure of sulfuramino acids. There is much Tau in human body, accounts for about0.1%of bodyweight, almost with the free form existing in all organs. A large number of domesticand foreign research and clinical application show that Tau has a wide range ofphysiological functions, are endogenous substances against injury, but, there are lessof the research for Tau antitumor, action mechanism is not clear yet.Objective:Observed the effective of Tau on tumor cell apoptosis, and explored its possiblemechanism.Methods:Detection the effect of tumor cells growth after Tau dealing with tumor cells.The role of Tau detection after the influence of the tumor cells growth. Experimentwith four kinds of cancer cells and one kind of normal cells, respectively is humanbreast cancer cells MCF-7(p53wild type, p53+/+) and MDA-MB-231(p53mutationstype, human colon cancer cells LoVo (p53+/+) and HT-29(p53-/-) and normal embryorenal cells of293T. With different Tau concentration (0mM,10mM,20mM,40mM,80mM and160mM) dealt with four kinds of tumor cells and embryos normal renalcells for different hours, tested the cells active change with MTT methods. Flowcytometer to detect tumor cell apoptosis rate change. Observation morphology changeby Hoechst33342nuclear staining method; RT-PCR and Western blot methodsdetected the expression changes of mRNA and proteins of PUMA, BAX and Bcl-2;and then we used spectrophotometric detection of MDA-MB-231cells ofCaspase-3activity change.Transfected PUMA specificity siRNA (PUMA-siRNA, si-PUMA) to humanLoVo colon cancer cells by liposome. Detected apoptosis rate induced by Tau afterPUMA silence. Different concentrations synthetic siRNA segment of fluorescentmarker FAM-siRNA (si-FAM) packaged with lipofectamine2000(lipo2000) aretransfected LoVo cells, tested the transfection efficiency. In screening high si-PUMAfragments transfection efficiency, this experiment was divided into6groups:(1)Control group,(2) Ⅰ siRNA clips,(3) Ⅱ siRNA clips,(4) Ⅲ siRNA clips,(5)Positive Control group (Positive Control, PC),(6) Negative Control group(Negative Control, NC). After48hours, the detection of each group of PUMAexpression by RT-PCR and Western blot. To test the role of the PUMA, transfectedPUMA specificity siRNA instantaneously to LoVo cells, trials are divided into fourgroups:(1)Control group,(2)NC group,(3)si-PUMA group,(4)thesi-PUMA+Tau80mM group. After48h, detected LoVo cells apoptosis rate by flowcytometer; detected PUMA, BAX and Bcl-2proteins expression changes by Westernblot methods.Results:With the Tau concentration and the processed time increases, the activity of4kinds of tumor cells appeared to reduce trend, the inhibition rate appeared graduallyincreasing trend. Calculation of IC50after dealing with Tau for48h，and then foundthat human breast cancer cells MCF-7IC50(133.61mM) is1.05times ofMDA-MB-231IC50(127.21mM), human LoVo colon cancer cells IC50(297.81mM)is4.76times of HT-29IC50(45.66mM). It showed that Tau inhibited p53-/-cancercells more apparently. But in embryo293T cells after dealing with24and48h, onlyafter72h, slight inhibition of293T cells on a higher concentration group. Thisprompted that the Tau is not obvious for the proliferation of normal cells. Flowcytometer found that, with the increase of the concentration of Tau, the apoptosis rateof two groups tumor cells increased gradually, and the cell apoptosis rate ofp53-/-cells increase more apparently than p53+/+cells. It was similarly with MTTresults. Morphology detection of tumor cells after dealing with80mM Tau by Hoechst33342nuclear staining showed that tumor cells nuclear were in differentdegree of dense hyper chromatic or pieces of density of typical hyper chromatic cellapoptosis form. RT-PCR image analysis showed: with the increasing of theconcentration of Tau, two groups of tumor cells to promote PUMA, BAX mRNAexpression level are raised as Tau concentration rising, but Bcl-2mRNA expressionlevel is down. Western Blot resulted show that with the increase of the concentrationof Tau, PUMA and BAX level of two groups of cells apparently rise, Bcl-2levelsclearly down.Caspase-3activity was detected, the MDA-MB-231cells was dealt withTau48h, and then we found that Caspase-3activity was significantly elevated, theTau40mM group was (1.33±0.13) times of Control group, the Tau80mM was(3.25±0.16) times of Control group, the Tau160mM group was (4.39±0.24) ofControl group.Different concentrations of si-FAM transfected LoVo cells, showed the differenttransfection efficiency. When si-FAM concentration is100nM that showed hightransfection efficiency, was61.34%. RT-PCR and Western blot results found thatthree siRNA clips rut PUMA mRNA and PUMA protein expression in degree. butsiRNA clips Ⅱ cut PUMA mRNA and PUMA protein expression most significantly.After siRNA specific interference PUMA gene expression on LoVo cells, earlyapoptosis rate (5.16±0.19)%and late apoptosis rate (4.29±0.35)%of si-PUMAgroup were significantly lower than early apoptosis rate (9.39±0.32)%and lateapoptosis rate (7.50±0.34)%of Control group, the difference was statisticallysignificant. The early apoptosis rate (9.52±0.35)%and late apoptosis rate (9.28±0.11)%of si-PUMA and Tau group appeared to riser compared si-PUMA group, thedifference was statistically significant. the early apoptosis rate of si-PUMA and Tauand Control group was no obvious difference, but the late apoptosis rate(7.50±0.34)%of si-PUMA and Tau group was more likely high than Control group.Western blot results showed that the specific interference PUMA of LoVo cells, after48h, PUMA and BAX protein expression of si-PUMA group were down, Bcl-2protein was increased, it has significant difference compared with Control group.Continue dealt with the80mM Tau, compared with the si-PUMA group, PUMA and BAX proteins expression of si-PUMA and Tau group appeared slightly raised, Bcl-2protein expression appears slightly down, it has statistically significant difference.Conclusion:1. Tau suppression of breast cancer and colon cancer cells proliferation and canpromote apoptosis occurs, so as to achieve the effect of the prevention and treatmentof tumors, and Tau is more pronounced inhibition of p53-/-tumor cells than p53+/+.2. Tau pro-apoptotic effect may be through the raised PUMA expression,thereby causing BAX up-regulation and Bcl-2down-regulation, ultimately leading tothe increase in Caspase-3activity mediated.3. Tau raised PUMA protein expression is no relationship with tumor cells p53state.4. Pro-apoptosis protein PUMA is a key molecule of Tau induced apoptosis oftumor cells.