The Effects And Mechanisms Of Baicalin Against CA46 Cells And Its Xenografts In Nude Mice

【Objective】(1)To investigate the effects of baicalin on proliferation inhibition and apoptosis induction in human Burkitt lymphoma cell line CA46 cells;the effects of baicalin combined with adriamycin(ADR),daunorubicin(DNR), homoharringtonine(HH) and etoposide(VP16) on CA46 cells respectively.(2)To investigate the effects of baicalin on proliferation and apoptosis related genes and proteins expression in CA46 cells and explore its mechanisms.(3)To investigate the role of PI3K/Akt and MAPK(ERK1/2) signal pathways in the apoptosis of CA46 cells induced by baicalin.(4)To investigate the effects of baicalin on CA46 cell xenografts in nude mice.【Methods】(1)CA46 cells were exposed to various dosages of Baicalin. Proliferation inhibition was detected by both MTT assay and clone formation assay. The effects of baicalin combinating with ADR,DNR,HH and VP16 on CA46 cells were evaluated by Jin formula respectively.The ability of baicalin to induce apoptosis of CA46 cells was examined by Annexin V FITC/PI double staining analysis,DNA fragamentation,TUNEL labeling method and acridine orange/ethidium bromide(AO-EB) double fluorescence staining respectively.The effects of baicalin on cell cycle of CA46 cells were examined by DNA ploid analysis. (2)The expressions of c-myc,bcl-2,bax,hTERT,pim-2,P21,Mcl-1 and TGFβ1 mRNA were detected by RT-PCR.The expressions of c-myc,bcl-2,procaspase-9,procaspase-3,PARP,Akt, pAkt, IκB-α,pIκB-α,NF-κB,mTOR,pmTOR,GSK-3β,pGSK-3β,MAPK(44/42) and pMAPK(44/ 42) proteins were detected by Western-blot.(3)The nude mice with CA46 cell xenografts were divided into six groups:negative control group,15mg/kg baicalin group,30mg/kg baicalin group,60mg/kg baicalin group,4mg/kg VP16 positive control group,and combination group(30mg/kg baicalin+2mg/kg VP16). Drugs were administered via intraperitoneal injection for 12 days. The changes of CA46 cell xenografts'weight and histology were used to evaluate the effects of baicalin on xenografts growth in nude mice.The apoptosis of xenograft cells was tested by tranmission electron microscope assay,and the Akt, pAkt, mTOR and pmTOR proteins extracted from xenografts were detected by western blot.The blood was gained to detected the hematocyte count,liver and renal function.The important organs including liver,spleen,renal,heart and lung were detected by histology assay to evaluate the toxic action of baicalin to nude mice.The other nude mice with CA46 cell xenografts in all groups were fed until died (from the time receiving intraperitoneal injection) in order to evaluate the effects of balcalin on survival time of nude mice with CA46 cell xenografts.【Results】(1)The results of MTT assay and clone formation assay showed that baicalin could remarkably inhibit the CA46 cell proliferation, and the IC50 value at 48h of treatment was about 10μM.The Q value and synergistic grade revealed that baicalin combinated with ADR,DNR and VP16 had the synergistic effects on inhibiting the CA46 cell proliferation,especially in a low dose baicalin.The results of Annexin V FITC/PI double staining analysis,DNA fragamentation,TUNEL labeling method and AO-EB double fluorescence staining revealed that baicalin induced the apoptosis of CA46 cells in dose-dependent manner.The DNA ploid analysis revealed that the cell cycle of CA46 cells treated by baicalin was blocked in G0/G1 phase. (2)The results of RT-PCR and/or western blot showed the expressions of c-myc,bcl-2,hTERT, pim-2,Mcl-1, procaspase-9,procaspase-3 and PARP(116KD) decreased,while bax,P21,TGFβ1 and PARP(85KD) increased after baicalin treatment.(3)Baicalin could downregulate the expressions of pAkt, pIκB, NF-κB,mTOR,pmTOR,pGSK-3βand pMAPK(44/42) proteins in CA46 cells,but the expressions of Akt,IκB,GSK-3βand MAPK(44/42) were not changed remarkably.(4)In vivo baicalin could inhibit the growth of CA46 cell xenografts in a dose-dependent manner with the tumor weight of 1.71±0.17g(P>0.05),1.35±0.35g(P<0.05) and 0.97±0.38g(P<0.01)in the group where baicalin was injected to nude mice with a dose of 15mg/kg,30mg/kg and 60mg/kg for 12 days respectively.Baicalin combined with VP16 had the synergistic effect on inhibiting the growth of CA46 cell xenografts in nude mice with the inhibition rate of 60.4% in combination group(30mg/kg baicalin+2mg/kg VP16),which was higher than 51.1% in 4mg/kg VP16 positive control group and 46.7% in 60mg/kg baicalin group.There were more necrotic and apoptotic cells in baicalin treatment groups and combination group than that in negative control group by histology assay and tranmission electron microscope assay.The expressions of pAkt,mTOR and pmTOR protein in high-dose baicalin group and combination group were downregulated remarkably,which revealed that baicalin could inhibit the proliferation of CA46 cells in vivo by downregulating the PI3K/Akt/mTOR signal pathway.The median survival time in 60mg/kg baicalin group,30mg/kg baicalin group and 15mg/kg baicalin group were 48,58 and 72 days respectively,which revealed that baicalin could raise the survival time of nude mice with CA46 cell xenografts in a dose-dependent manner.The median survival time in combination group(30mg/kg baicalin+2mg/kg VP16) was raised to 88 days,which was higher than that in negative control group significantly(P<0.05). 【Conclusions】(1)Baicalin could efficiently inhibit cell proliferation,block cell cycle in G0/G1 phase,and induce chondriosome pathway-apoptosis in CA46 cells,which could be correlated with the down-regulation expressions of c-myc, bcl-2,hTERT,pim-2 and Mcl-1,and the up-regulation expressions of bax,P21 and TGFβ1.(2)Baicalin combined with ADR,DNR and VP16 could have the synergistic effect on inhibiting the CA46 cell proliferation. (3)PI3K/Akt and MAPK(ERK1/2) signal pathways could be involved in proliferation inhibition and apoptosis induction of CA46 cells after treatment with baicalin. (4)Baicalin could inhibit growth and induce cell apoptosis of CA46 cell xenografts in nude mice,and raise the survival time of nude mice with CA46 cell xenografts significantly if combined with VP16,which mechanism could be correlated with the downregulation of PI3K/Akt/mTOR signal pathway. Baicalin combined with VP16 could have the synergistic effect on inhibiting growth of CA46 cell xenografts in nude mice.