Study Of EAAL Cells Combined With All Trans Retinoic Acid On Human Lung Adenocarcinoma Cell Lines A549 In Vitro And In Vivo Effect

Objective: To evaluate the effect of killing ability by using expandedactivated autologouse lymphocytes(EAAL) with all-trans retinoic acid(ATRA)on lung cancer A549 cell lines and nude mice which orthotopic transplantedwith human lung adenocarcinoma cell lines. We try to discuss the mechanismof the combination effects of EAAL cells and ATRA to provide some labevidences for the further combination therapy of human lung adenocarcinoma.Methods: 1 experiment of lung cancer A549 cell lines in vitroWe set up 4 groups of Control group,EAAL group,ATRA group andEAAL+ATRA combination group.(1) Observing each group’s morphological change by optical microscope(2) SRB assay : To test each group’s growth and proliferation changes.(3) The wound scrape assay was used to detect the migration ability ofeach group’s.(4) Using flow cytometry analysis to test each group’s distribution of cellapoptosis and cell cycle arrest condition.(5) RT-PCR was performed to observe the different transcription expres-sion of apoptotic related-genes(Bcl-2,Bax and Survivin).(6) To observe the different protein expression of apoptotic related- genes(Bcl-2,Bax and Survivin) by immunohistochemical staining and western blot.2 experiment of orthotopic transplanted tumor of human lungadenocarcinoma in nude mice36 nude mice which orthotopic transplanted with lung cancer cell linesA549 were randomly divided into 6 groups, those were control group(physiological saline group), positive control group(2mg/kg, DDP), ATRAgroup(15mg/kg), EAAL cells group(1×107/mice), EAAL with ATRA groupand EAAL after ATRA group.(1) All the nude mice were put to death after medication.We strippedtumors completely, measured tumor size, calculated the tumor inhibition rateand observed morphological changes by HE staining.(2) Expressions of protein about apoptotic related-genes Bcl-2, Bax andSurvivin in nude mice transplanted tumor were detected byimmunohistochemical method.Results:1 The A549 cells begin to show some morphological changes such asbeing shrink, turnning round, shedding, becoming smaller and the lowerquantities.In addition to apoptotic bodies appear in the cells, with theconcentration of ATRA and effect target ratio of EAAL cells increasing.Whereas the cells of control group are in well growth condition of uniformsize.Compared with the control group, the killing effect of two separatetreatment groups morphology on A549 cells is remarkable, and the killingeffect of combination group is obviously enhanced compared with the abovetwo separate treatment groups.2 The SRB result showed that, ATRA and EAAL cells has significantinhibitory effect on the proliferation of to A549 cells, and the presence ofconcentration(effect target ratio)-dependent and time-dependent effect. Theoptimal concentration of ATRA is 10-5mol/L and the optimal effect target ratioof EAAL cell is 50:1. In addition, the cytotoxic activity of the EAAL andATRA combination group was significantly increased compared with thecontrol group and two separate treatment groups(P<0.05).3 After the treatment of ATRA(10-5mol/L) and EAAL cells(50:1) aloneor in combination, the migration speed of A549 cells in each experimentalgroup has slowed down compared with the control group, the migration speedof combination group has slowed more significantly(P<0.05).4 Flow cytometry analysis showed, the cell cycle of each experimentalgroup was delayed compared with the control group, which G1 phase wassignificantly increased, S, G2/M phase was decreased. The cell cycle ofcombination group was delayed significantly compared with and two separatetreatment groups.5 RT-PCR result showed that, compared with the control group, ATRA(10-5mol/L) and EAAL cells(50:1) treatment alone or in combination, theexpression level of apoptosis inhibition related-genes(Bcl-2 and Survivin)m RNA were decreased, and the expression level of combination groupdecreased more significantly compared with two separate treatment groups;the expression level of pro apoptotic protein Bax m RNA increased in eachexperimental group,and the expression level of combination group increasedmore significantly compared with two separate treatment groups(P<0.05).6 Immunohistochemistry of cell climbing piece and Western blot resultshow that, ATRA(10-5mol/L) and EAAL cells(50:1) treatment alone or incombination, the protein expression level of apoptosis inhibition related-genes(Bcl-2 and Survivin) were decreased,and the expression level of combinationgroup decreased more significantly compared with two separate treatmentgroups; the expression level of pro-apoptotic protein Bax increased in eachexperimental group, and the expression level of combination group increasedmore significantly compared with two separate treatment groups(P<0.05).7 Injected A549 cells which in the logarithmic growth phase to rightaxilla subcutaneously in nude mice, 36 nude mice all growed tumor, tumorformation rate were 100%. No obvious abnormality of the nude mice diet andmental state on each groups(control group, DDP group, ATRA group, EAALgroup, EAAL with ATRA group, EAAL after ATRA group) during the courseof medication, The differences of each groups nude mice body weight were nostatistically significant before treatment(P>0.05), and the differences were nostatistically significant in each groups nude mice body weight on the first dayafter treatment(P>0.05). The tumor size were still increasing during thetreatment, the tumor size of each groups(control group, DDP group, ATRAgroup, EAAL group, EAAL with ATRA group, EAAL after ATRA group).Tumor inhibition rate were 74.2%, 30.8%, 44.2%, 73.2%, 73.6% respectivelyin each treatment groups, significant differences were found in each groups(P<0.05), in addition to the tumor inhibition rate of the two combinationtreatment groups have obvious increasing differences to the two individualtreatment groups(P<0.05).8 Immunohistochemical results showed:(1)The protein expression levelof EAAL and ATRA individual treatment groups of Bcl-2 gradually decreased(P<0.05), significant differences were not found in the two individualtreatment groups(P>0.05). In addition, compared with the control group andtwo individual treatment groups,the protein expression level of the twocombination groups decreased more significantly(P<0.05),but the twocombination groups have no significant differences(P>0.05); the proteinexpression level of the two combination groups of Bax increased moresignificantly according to the immune reaction integral(P<0.05).(2) Theprotein expression level of each experimental group of survivin graduallydecreased,and two combination groups decreased more significantly(P<0.05).9 ELISA result showed:The level of IL-2 and IFN-γin the blood serumof each group nude mice increased more significantly than the control and twoindividual treatment groups(P<0.01).Conclusions:1 The combination treatment of EAAL and ATRA on the lungadenocarcinoma A549 cell lines in the proliferation inhibition, cell cycle arrestand apoptosis aspect has obvious synergistic effect.2 Synergistic antitumor effects of combined application of EAAL cellsand ATRA may be closely related to cell apoptosis. Expression level of thecombined application of Bcl-2 and Survivin can be down regulated andup-regulated the expression of Bax,and up-regulated the level of IL-2 andIFN-γin the blood serum. The result point out the combination effect is likelyto play a role by affecting the expression of apoptosis related gene to inducethe apoptosis of tumor cells and increase the level of IL-2 and IFN-γ, therebyinhibiting the growth of transplanted tumor in nude mice.