Empirical Study Of MeJA Antihepatocarcinoma Effect And Its Mechanism

[Background and objective]The HCC,briefly called hepatoma.is a kind of malignant tumor which is originated from hepatic cells or intrahepatic bile duct endothelial cells.In china which has the most issues of hepatoma in the world,the people who die from it annually could reach 110 thousand,accounting for 45%of the global totally.Today,Lots of Therapy method for HCC are under application,among which operation is thought the most effective for the early HCC.But the possibility of recurrence and diversion after operation is high and the prognosis is not.well.Neither radiotherapy nor generalized chemotherapy can perform well on HCC.Most of the routine chemotherapeutics don't function very well on hepatoma,no matter single or combined,for the effective power merely reach 10%to 20%.For the past few years,a kind of interventional therapy for HCC,the TACE which is noted for its precise targeting making it characterized by precise site of action,high topical centralization and prominent effect,has become the first choice for patients in advanced stage or with recurring after operation.However, the therapy has defects that the normal cellsare also greatly damaged as tumor cells are killed and wounded because of the use of traditional chemotherapeutics with comparatively large poisonous side effects and easy emerging of drug resistance, which confined the utilization of the therapy much.Therefore,to search newfasioned anticancer drugs characterized by high performance and less poisonous side effect is one of the significant subjects in research of medical domain at present.MeJA which is one of the main representatives of Jas hormone is a new type phylohormone,methyl derivate of jasmonic acid.Jasmonic acid generally reside in regnum vegetable distributing various kinds of the tissues and organs in the frond, especially in the bundles.It performs multiple effects of phylohormone and induce expression of many genes performing defense in frond,which makes it an important signaling molecule in harm reaction.Research report about MeJA in internal literature mostly restrict at field of botanic,very few at research in its anti-carcinoma function. It is found that MeJA can not only regulate plant stress reactions but also kill tumor cells of Mammalian as a kind of phylohormones in researches.Still no reports whether MeJA can inhibit HepG2 cells proliferation or cause apoptosis are published in literatures domestic or overseas.Therefore,this research aims to observe MeJA function that it serves to cause proliferation inhibition and apoptosis induction of HepG2 cells by empirical studies exterior and interior from the level of integer,cellular and molecular and investigate the possible mechanism and then to settle determinate theoretical and experimental roof for research in MeJA antihepatocarcinoma function.[Methods]1.Culture HepG2 cells in vitro,screen the effective working concentration of MeJA by MTT,determine its working concentration(1/2 IC₅₀),detect the mitochondrial respiratory changes in HepG2 cells;observe cell Morphologic changes in each group by inverted microscope and HE stain;detect the changes of AFP in HepG2 cells and peripheral blood of nude mice by radio-immunity;observe the cell ultrastructure in each group by TEM;detect the cell cycle and apoptosis by flow cytometry;detect cell apoptosis by AGE.2.UtilizeRT-PCR to detect the mRNA expression of Bcl-2,Bax,Caspase-3,hTERT,p53,p21 and p16 in cells and in tumors of nude mice;detect protein expression of Bcl-2, Bax,Cyclin D1,AFP,p53,Ki-67 and telomerase in cells and in tumors of nude mice by immunocytochemistry and immunohistochemistry.3.Detect mitochondrial transmembrane potential changes in HepG2 cells by confocal laster scanning microscopy;detect the expression of Cyt-C in mitochondria and cytosol in HepG2 cells by Western blotting.4.Settle the model of subcutaneously transplantation tumor from human hepatoma in nude mice.Observe and calculate the tumor development in nude mice; calculate the inhibition rate of tumor growth;observe the histomorphology changes of tumors in nude mice by light microscope with HE stain and observe the pathomorphism changes in heart,liver,lien,lung,kidney and pancreatic tissue.5.Subjects in the experiment can be grouped into four groups:(1)the control group;(2)MeJA group;(3)ATRA group;(4)MeJA+ATRA group.[Results]Results from the first section of the methods:1.It can be concluded that the MeJA inhibition effect on the HepG2 cell growth offers time-effect dependence and dose-effect dependence when put MeJA with a concentration of 0.01μmol/L,0.1μmol/L,1μmol/L,10μmol/L,100μmol/L in turn with HepG2 for 24h,36h,48h,72h separately.2.Half inhibition concentration IC₅₀MeJA functioning at different time is 8.60μmol/L.Make the half of IC₅₀at 48h(4.3μmol/L)as the MeJA working concentration for the experiment later.3.The result from morphology observing MeJA function on cell growth inhibiting shows:The cells in control group grow well in the state of adherence and HE stain of cells is uneven because of the comparatively deep stain of the cell in stacking-pile arrangement.In the MeJA experimental group,it is the fact that part of the cells ablate,adherent cells decrease obviously,and the HE stain of cells is relatively even.The fact of the ATRA group is that part of the cells ablate as well,adherent cells decease,the cell nucleus is in anachromasis by HE stain and some cells become small and round.ATRA+MeJA group:adherent cells decrease obviously and HE stain of cells is in uniformity.4.Result from ultramicrostructure observing MeJA function on HepG2 cells by TEM in ultramicrostructure:The facts that HepG2 cells are in low differentiation is morphologic character of the typical malignant tumor cells.In MeJA group, cell junction and microvillus have disappeared and apoptotic body has appeared in part of the cells.Changes of caryocinesia image and cell apoptosis appears in part of the cells in group ATRA.The fact in MeJA+ATRA group is that some mitochondria swell,microvillus disappear and apoptotic body appears. 5.Result from observing by flow cytometry MeJA function on cell cycle of HepG2 human hepatocellular carcinoma cell:The fact in control group is that ratio ot cells at stage G1 is 40.8%,24.6%at stage S and the ratio is 34.6%at the stage G2.The fact of MeJA group is that the cell cycle has obvious depression at stage G1,the ratio of cells at stage G1 rises from 40.8%to 62.1%while the ratio declines from 24.6%to 1.7.3%at stage S;the ratio at stage G2 declines from 34.6%to 20.6%,the celi cycle has been blocked to stage G1;apoptosis may happen to some cells and peak of diploid may appear.The fact in ATRA:the ratio of cells at stage G1 has increased to some extent reaching to 55.9%;the ratio at stage S has declined somewhat to 17.2%,the ratio at stage G2 is low as well,reaching 26.9%;the cell cycle has been obviously blocked to stage G1.The fact in MeJA+ATRA grohp:cell cycle has obvious depression at stage G1,the ratio of cells at stage G1 reaches up to 61.2%,ratio at stage S obviously declines,only reaching 7.1%,.the ratio at stage G2 increases somewhat,reaching 31.7%;most cells have been blocked to stage G1,that is to say blocking effect on cells mainly takes place at stage G1 with certain blocking effect happening at stage G2/M at the same;the blocking effect at stage G0/G1 still plays the vital role and the blocking effect of MeJA+ATRA is stronger than the effect of single MeJA or ATRA,typical peak of apoptosis diploid appears.6.Result from observing by DNA AGE MeJA function on HepG2 cells apoptosis induction:DNA breaking ladder straps don't appear in the control group. Obviously typical ladder straps with interval between 180bp and 200bp or its integer times are all true in HcpG2 cells in the group MeJA,ATRA, ATRA+MeJA after 48h.Result from the second section of methods:1.The effect of MeJA on the mRNA expression of Bcl-2,Bax,Caspase-3,hTERT, p53,p21 and p16 in HepG2 cells.(1)Result from observing mRNA expression of Bcl-2 and Bax by mRNA AGE:The HepG2 cells in the control group have certain mRNA expression of Bcl-2 and Bax;with the function of the three experimental groups for 48h,the mRNA expression of Bcl-2 has significantly declined(P＜0.05)while the mRNA expression of Bax obviously surpasses the expression of the control groups(p＜0.05).(2)Result from observing mRNA expression of p53,Caspase-3å’ŒhTERT by mRNAAGE:The HepG2 cells in the control group have certain mRNA expression of p53,Caspase-3å’ŒhTERT; with the effect of the three experimental groups on HepG2 cells for 48h,the mRNA expression of p53 in the three groups is obviously lower than the expression of control group(P＜0.01);group comparison among the three experimental groups has difference(P＜0.05).With the effect of the three experimental groups on HepG2 cells for 48h,the mRNA expression of Caspase-3 in each group has increased to different degree and expression in MeJA+ATRA group is higher than expression in control group and ATRA group(p＜0.05). With the effect of the three experimental groups on HepG2 cells for 48h,the mRNA expression of hTERT in the three groups is obviously lower than the expression in control group(P＜0.05);the expression in group MeJA and MeJA+ATRA is lower than expression in ATRA group(P＜0.05).(3)Result from observing the mRNA expression of p21,p16 by mRNA AGE:with the effect of the three experimental groups on HepG2 cells for 48h,the mRNA expression of p16 surpasses the expression in control group(P＜0.05);the mRNA expression of p16 in group MeJA and MeJA+ATRA is higher than expression in group ATRA (P＜0.05).The mRNA expression of p21 surpasses the expression in control group(P＜0.05);the mRNA expression of p21 in group MeJA and MeJA+ATRA is higher than expression in group ATRA(P＜0.05)2.The MeJA effect on protein expression of Bcl-2,Bax,CyclinD₁,p53,Ki-67 and telomerase in HepG2 cells.(1)Result from observing protein expression of Bcl-2 and Bax by immunocytochemistry:With the effect of the three experimental groups on HepG2 cells for 48h,the protein expression of Bcl-2 in endochylema of the three groups is obviously lower than the expression in control group (P＜0.01);the difference in protein expression of Bcl-2 in group ATRA is significant compared with group MeJA and group MeJA+ATRA(P＜0.01).With the effect of the three experimental groups,the protein expression of Bax of the three groups obviously increases compared with control group(P＜0.01);the protein expression of Bax in group MeJA is higher than the expression in group ATRA,so is the expression in group MeJA+ATRA(P＜0.01).(2)Result from observing protein expression of cyclinD1 and p53 by immunocytochemistry: With the effect of the three experimental groups on HepG2 cells for 48h,the protein expression of cyclinD1 in the three groups is obviously lower than the expression in control group(P＜0.01);compared with group ATRA,the protein expression of cyclinD1 in group MeJA and MeJA+ATRA is lower(P＜0.05)。With the effect of the three experimental groups on HepG2 cells for 48h,the protein expression of p53 in the three groups is obviously lower than the expression in control group(P＜0.01);compared with group MeJA+ATRA,the protein expression in group MeJA and ATRA is higher(P＜0.01);the expression in group MeJA is lower than the expression in group ATRA (P＜0.05).(3).Result from observing the protein expression of Ki-67 and telomerase by immunocytochemistry:With the effect of the three experimental groups on HepG2 cells for 48h,the protein expression of Ki-67 in the three groups is obviously lower than the expression in control group(P＜0.01); compared with group ATRA,the protein expression of Ki-67 in group MeJA and MeJA+ATRA is lower(P＜0.05).The difference in expression is significant between group ATRA and group MeJA(P＜0.01).With the effect of group MeJA and MeJA+ATRA on HepG2 cells for 48h,the protein expression of telomerase in the two groups is lower than the expression in control group(P＜0.01).The expression in group ATRA is lower than the expression in control group (P＜0.05);compared with group ATRA,the expression in group MeJA and group MeJA+ATRA is lower(P＜0.01);the expression in group MeJA+ATRA is lower than group MeJA(P＜0.01).Result from the third section of methods:1.Result from observing MeJA effect on mitochondrial respiratory function of HepG2 hepatoma carcinoma cell by MTT experiment:Compared with the control group,the mitochondrial respiratory function of HepG2 cells has decreased obviously in the three experimental groups after their effect on HepG2 cell for 48h(P＜0.05).2.Result from observing MeJA effect on apoptosis of HepG2 hepatoma carcinoma cells by flow cytometry:The ratio of apoptosis occurrence in control group is 13.1, 36.3%in group ATRA,41.5%in group MeJA and 43.9%in group MeJA+ATRA, among which the ratio in group MeJA+ATRA is highest.3.Result from observing MeJA effect on mitochondrial transmembrane potential of HepG2 hepatoma carcinoma cells by confocal laster scanning microscopy:With the effect of the three experimental groups on HepG2 cells for 48h,the mitochondrial transmembrane potential in the three groups has obviously declined compared with control group(P＜0.01);the difference in expression by group comparison among the three experimental groups has no statistical significance(P＞0.05).4.Result from observing MeJA effect on protein expression of Cyt-C in mitochondria and endochylema of HepG2 hepatoma carcinoma cells by Western-Blot:It is showed by Western blot that the Cyt-C enjoys a high expression in the HepG2 cell mitochondria in the control group after 48h while the expression in the three experimental groups(MeJA group,ATRA group,MeJA+ATRA group) was low;The Cyt-C expression is relatively low in the HepG2 cell cytosol in control group while the expression has significantly increased in the three experimental groups(MeJA group,ATRA group,MeJA+ATRA group).Result from the fourth section of methods:1.result from observing the MeJA effect on growth inhibiting of subcutaneously transplantation tumor in nude mice from HepG2 cells:All the absorbent gland in the body surface and celiac lymph nodes have no divertion in all the nude mice of each group and no abdominal membrane infiltration or abdominal dropsy appears; after the function of the three experimental groups,the volume and the weight of the transplantation tumor from HepG2 cell share a obviously lower level in the three groups than the control group and the difference is significant(P＜0.01). The group MeJA+ATRA makes the highest tumor inhibiting rate,group MeJA the next in order and group ATRA makes the lowest.but difference by group comparison among the three groups has no statistical significance(P＞0.05).2.Result from observing by pathologic histomorphology with HE stain:Cells in tumor tissues share the same character with primary cell line.In the three experimental groups the tumor tissues have the same changes that the tumor cell density in the tumor tissue has decreased,fibrosis occurs and some of the cancer nest perform lamellar necrosis differently.No metastasis appear in heart,liver,lien,lung,kidney and pancreatic tissues in nude mice of each groups.3.Result from detecting AFP in peripheral blood of nude mice bearing cancer by radio-immunity:Compared with the control group,AFP value has obviously declined in the three experimental groups(P＜0.01).4.MeJA effect on mRNA expression of I Bcl-2,Bax,Caspase-3,hTERT,p53,p21and p16 in tumor in nude mice:(1).Compared with the control group,the mRNA expression of Bcl-2 has decreased obviously in the tumor issues of each group(P＜0.05)while the mRNA expression of Bax has increased greatly(P＜0.05).(2).Compared with the control group,the mRNA expression of Caspase-3 has increased obviously in group MeJA and the group MeJA+ATRA(P＜0.05).Compared with group ATRA,group MeJA and MeJA+ATRA enjoy a higher expression(P＜0.05).Compared with the control group,the mRNA expression of hTERT has obviously declined among the three experimental groups(P＜0.05).Compared with group ATRA,group MeJA and MeJA+ATRA enjoy a higher expression(P＜0.05).(3).Compared with the control group,the mRNA expression of p53 has obviously declined among the three experimental groups(P＜0.05).Compared with group ATRA,group MeJA and MeJA+ATRA enjoy a lower expression(P＜0.05).Compared with the control group,the mRNA expression of p21 has obviously increased in the group MeJA and group ATRA(P＜0.05).The expression in group MeJA+ATRA is higher than group ATRA(P＜0.05).Compared with the control group,the mRNA expression of p16 has obviously increased among the three experimental groups(P＜0.05). 5.MeJA effect on protein expression of Bcl-2,Bax,Cyclin D₁,AFP,p53,Ki-67 and telomerase in tumors of nude mice:(1)Result from detecting protein expression of Bcl-2 and Bax by immunohistochemistry:The expression of Bcl-2 protein has obviously declined among the three experimental groups compared with the control group(P＜0.05)and the difference by group comparison among the three groups has no statistical significance(P＞0.0.5).The expression of Bax protein has obviously increased in the three experimental groups(P＜0.05)compared with the control group the difference by group comparison among them has no statistical significance(P＞0.05).(2)Result from detecting protein expression of Cyclin D1 and AFP by immunohistochemistry:The protein expression of Cyclin D₁ among the three experimental groups has obviously declined(P＜0.01).The expression in group MeJA is lower than expression in group MeJA(P＜0.05)and the difference is obvious between group MeJA+ATRA and group ATRA.(P＜0.01)The protein expression of AFP has obviously declined among the three experimental groups compared with control group(P＜0.01).Then make the group comparison among them.The difference in expression is obvious between group MeJA and group ATRA,so is the difference between group MeJA+ARTA and group ATRA(P＜0.01).(3)Result from detecting protein expression of p53,ki-67and Telomerase by immunohistochemistry:The protein expression of p53,ki-67and Telomerase has obviously declined among the three experimental groups compared with the control group(P＜0.01).The protein expression of p53 in group ATRA and MeJA+ATRA is higher than the expression in group MeJA(P＜0.05) The protein expression of ki-67 in the three experimental groups has declined(P＜0.01),so has the protein expression of Telomerase in the three experimental groups(P＜0.01).[conclusions]1.MeJA function on obvious proliferation inhibiting of HepG2 cells with different acting concentration at different time takes on time-effect dependence and doze-effect dependence. 2.MeJA effect on proliferation inhibiting is that it can reduce the number of survival HepG2 cells,it inhibits HepG2 cells proliferation,it has HepG2 cells blocked at stage G0/G1,it obviously reduce the cells at stage S and lower the AFP level of HepG2 cells.3.The MeJA effect on cell apoptosis inducing is that it can alter the ultramicrostructure of HepG2 cells and cause typical apoptosis morphology changes.4.MeJA performs better than ATRA in aspect of HepG2 cells apoptosis inhibition.5.The function of MeJA and ATRA on proliferation inhibition and apoptosis induction can be combined with a certain role.6.The mechanism of MeJA antihepatocarcinoma effect may work at the level of transcription and translation.7.The fact that MeJA declines expression of cell circle regulating-related genes and protein CyclinD₁,Ki-67,mtp53 and raise the mRNA expression ofp16 and p21 causes blockage in tumor cell circle and then inhibit the proliferation of hepatoma carcinoma cell.8.MeJA can trigger apoptosis cascade reaction by the mechanism that it declines the protein and mRNA expression of apoptosis -depressing gene Bcl-2,raises the mRNA and protein expression of apoptosis-promoting gene Bax and activate Caspase-3.The mechanism by which MeJA induces tumor cells apoptosis is that it inhibits mRNA expression of hTERT and depresses the activity of telomerase.9.MeJA can cause HepG2 human hepatocellular carcinoma cell mitochondrial respiratory dysfunction,decrease mitochondrial transmembrane potential,make intramitochondrial cytochrome C release to cytolymph,trigger the cascade activation of Caspase and induce apoptosis.10.The fact that MeJA can change mitochondrial function of HepG2 cells may be one significant mechanism of its antihepatocarcinoma effect.11.MeJA can make the transplantation from HepG2 cells deflates and become light and it has relatively strong growth inhibiting effect on subcutaneously transplantation from HepG2 cells in nude mice. 12.MeJA can cause decreasing of the cell density in transplantation tumors in nude mice and different lamellar necrosis of some cancer nests and inhibit the growth of transplantation tumor from HepG2 cells.13.MeJA has no obvious influence on morphosis of heart,liver,lien,lung,kidney and pancreatic tissues in nude mice bearing cancer.14.MeJA can reduce the content of AFP in peripheral blood and depress AFP depression in nude mice bearing cancer,which can cause malignant phenotype of hepatoma carcinoma cell takes reverse.15.The mechanism by which MeJA inhibits tumor growth of nude mice bearing cancer is that MeJA declines expression of cell circle regulating-related genes and protein CyclinD₁,Ki-67,mtp53 and raise the mRNA expression of p16 and p21 causes blockage in tumor cell circle and then inhibit the proliferation of hepatoma carcinoma cell;it declines the protein and mRNA expression of apoptosis -depressing gene Bcl-2,raises the mRNA-and protein expression of apoptosis-promoting gene Bax,activate Caspase-3 and trigger apoptosis cascade reaction;it inhibits mRNA expression of hTERT,depresses the activity of telomerase and induce apoptosis in tumor cells.16.MeJA performs better than ATRA in aspect of HepG2 cells apoptosis inhibition and the function of MeJA and ATRA on proliferation inhibition and apoptosis induction can be combined with a certain role.17.MeJA has promising prospect to be a new kind phylohormone anticancer drug with character of less poison and high performance.