The Anti-tumor Ability Of Parthenolide To Melanoma B16 Cells And The Study Of The Mechanism

Be the most difficult disease to cure,the tumor disturb the clinic for a long time.The tumor,whatever we do after we discover the dead rate of the tumor is still very high,and a lot of cancers are not can be cure now,how to do with the tumor is a problem of the medical world.The tumors are threatening our world.The method of the cure is operation,indurstry medicine and radiotherapy,so made lots damage to the patients bady,the survivel rate is very little,recidivity is very commen,many surgeons adviced the patients to go to see the traditional Chinese medicine doctors.But the traditional Chinese medicine doctors's cure way is lack of the molecular basis,and can not accepted by the medical world.We use the parthenolide to remedy the tumor,and investigate the molecular mechanism,in order to state the anti tumor function of the parthenolide,and expect to discover a new medicine to the tumor.The parthenolide originate in Tongxiang of Zhejiang Province,is a relish of tea,the commen component of medicine is it's lactone.It is often used in headache?inflamation and sevrel kinds of tomors.Our experiment is in order to illustrate the mechanism of how to anti the melanoma B16 cell line.In the present study,we examined the effect of parthenolide on the proliferation inhibiting of B16 cells by MTT.We tested the effects of parthenolide on the expression of Bcl-2,fas,Caspase-9,Caspase-8 and Caspase-3 in B16 cells by immunohistochemistry(IHC).And we accessed the role of parthenolide on the morphology of B16 cells by HE staining,AO/EB staining,hochest staining and transmission electron microscope.Through PI staining,the flow cytometry is used to test the apoptosis and cell cycle changes caused by parthenolide in B16 cells.P53,P65,cyclinD1and Ki-67 were detected by western blot.The mRNA level of P65,cyclinD1and Ki-67 was detected by PCR analysis.In order to provide the theoretical evidence for the development of parthenolide as a novel anti-tumor drug.Result:1.Cell apoptosis detecting by MTT assay.The results from MTT assay indicated that parthenolide has a significant inhibition on the proliferation of B16 cell lines.2.He stainingWe found the unfirmed attachment of the cultured cells which exhibited a round shape and inhibited growth when treating the media with parthenolide,while the control cells displayed a robust adherent growth.3.AO/EB stainingThe result showed that the survival cells chromatin was stained to be green,but the structure were normal,the viable apoptosis cells chromatin were stained to be green but the margine of the cells wore irregular,the later period apoptosis cells chromatin were stained to be jacinth,and the cell-nucleus were shrinked.And the treatment group was much more than the control group.4.Hochest stainingThe treatment group cells showed obviously apoptosis state,Uv stimulated emission clear blue fluorescence,The nucleus edge can appear fold or crease appearance.Some cells chromatin condensation marginalized status.5.The Flow cytometry detection resultsWe observed altered cell cycle distribution caused by parthenolide when the Hela cells were treated by PI staining.The data demonstrated that parthenolide can disturb the cell cycle through prevent the cells entering the S/G2 phase and retaining them in G1 phase.Also,the cell apoptosis index in Parthenolide treating group is greatly higher than the control group,exhibiting a significant difference.6.The result of RT-PCRAfter treated by parthenolide for 24h,the expression of those proteins,was greatly increased in a time-dependent manner,with a significant difference(P<0.05),The mRNA levels of P65?cyclinD1?Ki-67 in the control groups showed a high level.However,after treated by parthenolide for 24 h,the expression of those proteins,with a significant difference(P<0.05).7.Cell monitoring by immunohistochemistry(IHC)We observed dramatically changed expression levels of Bcl-2,fas,Caspase-9,Caspase-8,and Caspase-3 in B16 cells upon parthenolide treatment by IHC analysis.The positive ratio of the proteins was calculated and the difference was statistically significant between the cells with or without the addition of parthenolide(P<0.05).The result showed that parthenolide was able to decrease the expression of Bcl-2,and elevate the expression of Bax,Caspase-9,Caspase-8,and Caspase-3.8.Western blotWe monitored the effect of parthenolide on the expression of P53?Bax?P65?cyclinD1?Ki-67 respectively.As expected,we observed the significantly inhanced the P53?Bax,reduced the level of P65?cyclinD1?Ki-67,upon the parthenolide treating,with significant difference(P<0.01).between the experimental and control groupConclusion1.The parthenolide has a significant anti-tumor effect to B16 cell line.2.The parthenolide can induce the apoptosis of B16 cells,perhaps via the mitochondrial pathway through affect the expression of Bcl-2?Bax?Caspase-9 and Caspase-3.3.The apoptotic induced apoptosis of mouse melanoma B16 cells function can also be done through death receptor pathway through affect the expression of Caspase-8 and Caspase-3.4.The parthenolide effect on inducing apoptosis of B16 cells may be involved in the G1/S phase block the cell cycle5.The parthenolide has a promising potential to be a novel anti-tumor drug.