The Anti-tumor Ability Of Parthenolide To Cervical Cancer Hela Cells And The Study Of The Mechanism

Tumors were commonly diagnosed in clinical diseases in the decades.And majority of the malignant ones were incurable,which still was a big challenge for the doctors.Traditionally,the methods for tackling cancers were surgery,radiotherapy,as well as chemotherapy.Considering the toxic side effects of the chemistry drugs now clinically used for treatment,which cause great damages to patients' body,many researchers begin focusing on the traditional Chinese medicines with little side effects,aiming to improve the patient survivals through developing the novel anti-tumor drugs.Prthenolid(PTN)belongs to the half terpene lactones,as active ingredients of medicinal plant in Mexico and India.Prthenolid,as a commonly used herbal active ingredient,was mainly used in the treatment of migraine,inflammation,and tumor.With the deepening of the anti-tumor Chinese medicine research,more and more scholars begin to focusing on the study of prthenolid.This experiment accessed the antitumor effect of prthenolid on several kinds of tumor cell strains and the mechanism of inhibiting cervical cancer Hela cells,aiming to illustrate the antitumor mechanism of prthenolid.In the present study,we examined the effect of parthenolide on the proliferation inhibiting in a variety of cancer cells by MTT,including the human lung cancer A549 cell line,liver cancer HepG2 and Hep3B cell lines,cervical cancer Hela cell line,as well as the mouse melanoma B16 cell line.We detected the effects of parthenolide on the expression of Ki-67,Bcl-2,NF-KB,Caspase-9,Caspase-8 and Caspase-3 in Hela cells by immunohistochemistry(IHC).And we accessed the role of parthenolide on the morphology of Hela cells by HE staining,inverted microscope and transmission electron microscope.Through PI staining,the flow cytometry is used to test the apoptosis and cell cycle changes caused by parthenolide in human cervical cancer Hela cells.Additionally,The EMT-related protein Twist upon the treatment of parthenolide was examined by western blot.The effect of parthenolide on the migration of Hela cells was monitored by wound healing assay.Also,the levels of cytochrome C,which involves in the apoptosis pathway,P53,Bcl-2,and Bax were detected by western blot.The mRNA level of P53,Bcl-2,Bax,caspase-9,and caspase-3 was detected by PCR analysis.Also,the protein levels of caspase-9 and caspase-3 were determined by ELISA method.At the same time the SOD and CAT activity tested.In this study,we sought to elucidated the mechanism of parthenolide in the suppression of cervical cancer and 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 all detected cell lines,such as lung cancer A549 cell line,liver cancer HepG2 and Hep3B cell lines,cervical cancer Hela cell line,as well as the mouse melanoma B16 cell line.2.Morphological observations We 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.Also,the number of apeptotic and dead cells was increased,with visible apoptotic bodies,in parthenolide treatment group observed by transmission electron microscopy(SEM).3.The Flow cytometry detection resultsWe observed altered cell cycle distribution caused by parthenolide when the Hela cells were treated by PI staining.In control group,the cells in G0/G1 phase at 24 h accounted for 42.39%,while the G2/M phase and S phase cells for 15.68%and 31.05%,respectively.The GO/G1 phase cells in parthenolide group increased obviously,accounting for 61.85%,whereas the percentage of G2/M phase or S phase cells is less than the control group,with 11.52%or 24.67%,respectively.At 48 h,the percentage of GO/G1 phase cells in control group was 47.06%,G2/M phase cells 14.81%,and S phase cells 32.38%,while the parthenolide treating group composed of 67.35%of GO/G1 phase cells,9.28%of G2/M phase cells,and 26.51%of S phase cells;Compared with control group,the experimental group had statistical significance(P<0.01),however,the results obtained at 24 h and 48 h displayed no significant difference.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.4.The result of RT-PCR The mRNA levels of P53,bax,caspase9 and caspase3 in the control groups showed a low level.However,after treated by parthenolide for 24 h or 48 h,the expression of those proteins,excepting caspase 9,was greatly increased in a time-dependent manner,with a significant difference(P<0.01),compared with the control group.The level of aspase 9 between 24 h and 48 h had no statistic significance(P>0.05).The mRNA of Bcl-2 had a high level at 0 h,and displayed a decreased expression upon parthenolide treatment for 24 h or 48 h,with a statistic significance(P<0.05,P<0.01),relative to the Oh group.5.The effect of parthenolide on the level of cytochrome C in Hela cells A specific band was seen when the cell lysate was detected by the monoclone antibody against cytochrome C.And the band was greatly strengthened and had a significant difference after treating the cells with parthenolide for 8h,relative to the Oh.The cytochrome C level increased to a peak at 24h,and then exerted a declining trend.6.Parthenolide on HeLa activated caspase 9 and caspase 3 expression effect ELISA results showed that the activity of intracellular caspase-9 or caspase-3 was obviously enhanced when the cells were treated with parthenolide for 24 h or 48 h,and had a significant difference(P<0.01),compared with Oh.But,the data between 24 h and 48 h group had no statistic significance(P>0.05).7.Cell monitoring by immunohistochemistry(IHC)We observed dramatically changed expression levels of Bcl-2,Bax,NF-KB,Caspase-9,Caspase-8,and Caspase-3 in Hela 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,NF-KB,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,bcl-2,bax,respectively.As expected,we observed the significantly deceased level of bcl-2,and enhanced levels of the suppressor p53 and bax,upon the parthenolide treating,with significant difference(P<0.01).between the experimental and control group9.Detection of the cell migration From the wound healing analysis,we observed that the Hela cells displayed a slowly accumulating rate upon the parthenolide treating,with an enlarged intercellular space and a round shape of the periphery cells,compared to the control cells which continued to migrate toward and,sooner or later,heal the wound region(P<0.01).10.The effect parthenolide on SOD and CAT activity in HeLa cells.The SOD and CAT in the cells displayed alliterating activities and the dynamic curve had a rising trend upon the parthenolide treating for different times.SOD or CAT activity was significantly increased after treating for 24h or 48h,and had a significant difference(P<0.01),relative to the control group.Conclusion1.The parthenolide has a broad-spectrum anti-tumor effect.2.The parthenolide can inhibit the proliferation of human cervical cancer Hela cells.3.The parthenolide can induce the apoptosis of Hela cells,perhaps via the mitochondrial pathway.Parthenolide can promote the outflow of mitochondrial cytochrome C and cause apoptosis.4.The apoptotic induction of parthenolide in Hela cells may be related to its ability of arresting cells in the G1/S phase.5.Parthenolide can influence the level of active oxygen in Hela cells,leading to apoptosis.6.The parthenolide is able to decrease the cell migration,which may be related to its inhibition of twist expression.7.The parthenolide has a promising potential to be a novel anti-tumor drug.