The Experimental Study On Apoptosis Of Lung Cancer A549 Cells And The Growth Of Xenografts By Parthenolide Combined With Gefitinib

The non-small-cell lung carcinoma(NSCLC)is the most common pathologic type of lung cancer.The surgery,radiotherapy,chemotherapy,targeting therapy and differe nt combinations are the most common treatments.Chemotherapy and targeting drugs are the main treatments of advanced NSCLC patients,and chemotherapy is not a perf ect therapy.Targeting drugs areused extensively because of the superiority with target ing and less side effects.Gefitinib is an epidermal growth factor receptoe-tyrosine kin ase inhibitor(EGFR-TKIs),as a first line therapy with EGFR mutant advance NSCLC patients.According to clinical research,not selected recurrence or chemo-failure NSC LC patients could be treated with Gefitinib,as it has the same therapeutic effect with second chemotherapy,and patients find it easy to tolerate.It's remind that may be try EGFR-TKIs to EGFR unknown advanced NSCLC patients after chemo-failure.How to let these patients benefit from Gefitnib is the key point that enhances thetherapeu tic effect of Gefitinib.Autophagy is a cell survival mechanism to reply stress.Autoph agy hasshown to have double the effect of cell survival:one is protect cell survival,another is promote cell apoptosis.Mitophagy is the one of the special types in autopha gy which eliminates damaged mitochondria by autophagy electively.The research has shown that defectiveor over autophagy could affect therapeutic effect of EGFR-TKIs.Parthenolide is the principal sesquiterpene lactone isolated from the herb feverfew an treated with fever,migraine,rheumatoid arthritis.Many studies have shown thatparth enolide hasan anti-cancer effect and induce autophagy in multiple tumors,andenhan ce effect of chemo and radiotherapy in the tumor cells.So far,there are few reports ab out parthenolidecombined with gefitnib in lung cancer.In this study,to observe the ef fect of parthenolide combined with gefitinib on proliferation,apoptosis,mitochondrial damage,autophagy and mitophagy in lung cancer A549 cells in vitro,and investigatin g its mechanism.Then,in vivo,we will establish xenografts of lung caner cells A549 in nude mice,and observe inhibitory effects of tumor growth,apoptosis-relating prote in,autophagy-relating protein and STAT3 activation in xenografts treatment with Gefi tinib and Parthenilide,and investigating its mechanism.These findings in vitro and vivo may provide a reliable theoretical basis forthechemo-failure and EGFR unkno wn advanced NSCLC patients treatment with targeting drug and a novelcombination.Part1The effect of mitophagy on gefitinib-inducing apoptosis of lung cancer A549 cells enhanced by parthenolideObjective:To investigate the effect of mitophagy on gefitinib-inducing apoptosis of human lung cancer A549 cells enhanced by parthenolide.Methods:First,the cell survival rate were detected by MTT assay with different concentration of parthenolide and gefitinib in A549 cells and then the harf-inhibitory concentration(IC50)was calculated.The A549 cells were divided in to 4 groups:Control group(C,no drug),Parthenolide group(P,5?mol/L),Gefitinib group(G,10?mol/L),Parthenolide and Gefitinib group(PG,Parthenolide5?mol/L+Gefitinibl0?mol/L).The cell survival was detected by MTT assay;The cell proliferationwas detected by wound healing assay;TUNEL staining was used to detect the formation of apoptotic cells;The cell apoptosis was detected by Annexin V-FITC/PI assay;The mitochondrial membrane potential(MMP)was detected by JC-1 fluorescence probe labeling method;The level of reactive oxygen species(ROS)was detected by DCFH-DA fluorescence probe labeling method;The expression of cell apoptosis-relating protein Bcl-2,Bax,caspase-3,caspase-9 and autophagy related protein LC3-?/?,Beclin-1,PINK1 was detected by Western Blotting analysis;The autophagosome was analyzed by transmission electron microscopy(TEM);Using Annexin V-FITC/PI assay to detect cell apoptosis after added 3-MA which is autophagy inhibitor with parthenolide and gefitinib;Using western blotting detect expression of protein Beclin-1,caspase-3,Bax,Bcl-2.Results:1.The IC50 of parthenolide and gefitinib on A549 cell is(35.77±2.05)?mol/L,(15.38±1.13)?mol/L,respectively.The A549 cells were treated with 5?mol/LParthenolide and 10?mol/LGefitinib,the cell survival rate of group G and P lower than C group(all P<0.05),and the group PG lower than group P and G(all P<0.05).2.Observation from microscopy,when Oh,the distance of cell wound healing was the same in all groups,and no migration cells in the healing.When 48h with drugs,the distance of wound healing with C,G,P groups shrank obviously and have migration cells in the healing space.The wound healing rate of group PG was weaker than the other group(all P<0.05).3.Observation from fluoroscope,the apoptotic cells volume narrowed,the connection between cells disappeared,separated from the near cells,the density was increased,but the membrane structure still was completed.The apoptotic cells of G and P group were more than C group(all P<0.05).The more apoptotic cells were observed from group PG than other group(all P<0.05).4.The cell apoptosis rate of G and P group were higher than C group(all P<0.05).The cell apoptosis rate of PG was higher than G and P group(all P<0.05).5.The expression of Bcl-2/Bax ratio,caspase-3,caspase-9 in G group were no different with C group(all P>0.05).The Bcl-2/Bax ratio of group P was lower than C group(allP<0.05).PG group less than G and P group(all P<0.05).The expression of caspase-3,caspase-9 of group P was higher than C group(all P<0.05).PG group more than G and P group(all P<0.05).6.There was red fluorescence and no green in C group.In the G group,there was slight decreased red and slight green fluorescence.In the P group,there was decreased red and little strong green fluorescence.In the PG group,there was strong decreased red and strong green fluorescence.Compared the MMP:There is no different between G and C group(P>0.05).The P group was decreased than C group(P<0.05).PG group was decreased than G and P group(all P<0.05).7.The ROS level of group C,G,P,PG were 0.94±0.15,1.53±0.06,2.03±0.12,3.14±0.16,respectively.The generation of ROS level of P group higher than C group(P<0.05).There is no different between G and C group(P>0.05).The ROS level in group PG higher than G and P group(all P<0.05).8.Observed from TEM:There was no obvious injury of organelles were completed and no autophagosome in group C.There is no autophagosomes in group G.The small amount of autophagosomes were founded in group P.The amounts of autophagosomes were founded in group PG.9.The expression of LC3-?/? ratio,Beclin-1,PINK1 in G group were no different with C group(all P>0.05).The expression of LC3-?/? ratio,Beclin-1,PINK1(mito)in group P werehigher than C group(all P<0.05).The PG group were more than G and P group(all P<0.05).10.The PG group made cells apoptosis about(16.52±1.48)%and 3-MA+PG group decreased cells apotosis for(9.64±0.73)%.The expression of Beclin-1 and casepase-3 protein in 3-MA+PG group lower than PG group(P<0.05).The expression of Beclin-1,caspase-3 protein in 3-MA+PG group lower than PG group(all P<0.05).The Bcl-2/Bax ratio of 3-MA+PG group higher than PG group(P<0.05).Conclusions:1.Parthenolide as a autophagy-inducing agent enhanced effect of inhibiting proliferation and inducing apoptosis by gefitinib in lung cancer A549 cells;2.Parthenolide enhanced apoptosis inducing by gefitinib in lung cancer A549 cells through mitoptosis pathway;3.The activated autophagy may promote cells apotosis by parthenolide combined with gefitinib;4.Mitophagy might invoved in gefitinib-inducing apoptosis enhanced by parthenolide in A549 lung cancer cells.Part2A study of the growth and mechanism of parthnolide combined with gefitinib on the xenografts of A549 cellsObjective:To investigate the study of the growth and mechanism of parthnolide combined with gefitinib on the xenografts of A549 cells in nude mice.Methods:Established subcutaneous xenografts model of human lung cancer A549 ce lls in nude mice.10 days later,the nude mice were randomly divided into 4 groups:Control group(C),Gefitinib group(G),Parthenolide group(P),Parthenolide+Gefitinib group(PG).Each group will be administration by Gefitinib 25mg/kg/d,Parthenolide 25mg/kg/d,Parthenolide 25mg/kg/d+Gefitinib 25mg/kg/d,and Control group only re ceived same volume of saline.Administration 3 times in a week,tumor volume was detected every 2 days.After 3 weeks kill the nude mice,then pick off the tumor tissue.Final tumor volume and tumor inhibitory rate was calculated after 3 weeks interventi on.Observate tissue lung cancer cells morphologic change with HE staining.Theexpr ession of apoptosisi-ralating protein Bax,Bcl-2,caspase-3 and autophagy-ralating pro tein Beclin-1,LC3-? and p-SATA3,STAT3 protein were deteted by Western Blot.Results:1.After intervention with 3 weeks,the tumor volume(TV)of xenografts in group C,G,P,PG were(732.21±36.34)mm3,(627.01±76.04)mm3,(459.63±29.76)mm3,(311.27±33.67)mm3,respectively.The tumor volume of group G is no different with C group(P>0.05).The tumor volume of group P,GP smaller than group C(allP<0.05).The tumor volume of group PG smaller than group P,G(all P<0.05).2.The tumor inhibitory rate of C,G,P,PG were 0%,14.36%,37.22%,57.48%,respec tively.The tumor inhibitory rate of G is no different with C group(P>0.05).The tumor inhibitory rate of P groups were higher than group C(all P<0.05).The tumorinhibito ry rate of group PG smaller than P,Ggroup(all P<0.05).3.Observation from microscope with HE staining,the active proliferation of tumor cells were detected in Cgroup,and the little loosen intercellular space was detected in G group,and the little necrosis area and loosen intercellular space was detected in P group,and a plenty of necrosis area and loosen intercellular space was detected in PG group.4.The expression of caspase-3 protein in G,P group higher than C group,significantly(all P<0.05).The expression of caspase-3 protein in PG group higher than G and P group(all P<0.05).The Bcl-2/Bax radio of G,Pgroup lower than C group(all P<0.05).The Bcl-2/Bax radio of PG group lower than G and P group(all P<0.05).5.The expression of Beclin-1,LC3-? protein in G group were no different withC group(all P>0.05).The expression of Beclin-1,LC3-? protein in P group higher than C group(all P<0.05),and the expression of Beclin-1 protein in PG group higher than P and G group(all P<0.05).The expression of p-STAT3 protein in G,Pgroup lower than C group(all P<0.05).The expression of p-STAT3 proterin in PG group lower than G and P group,especially(all P<0.05).Conclusion:1.Gefitinib can not inhibits tumor growth on xenografts of A549 cells;2.Parthenolide enhanced effect of inhibits tumor growth on xenograft,the mechanism isinducing apopotosis and inhibiting activation of STAT3 and activating autophagy.