Effects Of Baicalin On Resistant HL-60/ADR Cells And HL-60 Cell Xenografts In Nude Mice

Objective(1)To study the effects of baicalin on proliferation inhibition and apoptosis induction on adriamycin-resistant human myeloid leukemia cell line HL-60/ADR cells and on expressions of related apoptotic proteins and mRNA,and to study the change of PI3K/Akt/mTOR,MAPK signal pathway in baicalin-induced HL-60/ADR cells.(2)To investigate the effects of baicalin on the expression of drug-resistance related genes in HL-60/ADR cells and explore its reversal mechanism (.3)To observe the effects of baicalin on HL-60 cell xenografts in nude mice.Methods(1)HL-60/ADR cells were in vitro cultured and exposed to baicalin at different dosages.Its proliferation inhibition and colony formation were detected by MTT assay and colony formation assay respectively.The cell apoptosis was tested by Annexin V FITC/PI double staining analysis, cell cycle analysis, DNA fragmentation and TUNEL labeling method.RT-PCR was used to analyze the c-myc, bcl-2, hTERT, pim-2, mcl-1 and p21 mRNA expression.The expressions of apoptosis related proteins and signaling molecular PI3K/AKT proteins were examined by western blot.(2)The effect of Adriamycin (ADR),Daunorubicin (DNR) ,Cytarabine (Ara-C) and Etoposide (VP16) on the proliferation of HL-60 and HL-60/ADR cells were observed by MTT assay.The inhibitory effects of all these chemotherapeutic agents used alone or incomebination with baicalin on the proliferation of HL-60 and HL-60/ADR cells were evaluated by MTT assay. The reversal fold of drug resistance was calculated. The expressions of mrp-1, lrp,GSTπ,TopoⅠ,TopoⅡαand TopoⅡβmRNA were measured by RT-PCR.The proteins level of mrp-1 and GSTπwere detected by western blot.The positive rate of ADR fluorescence was analyzed by flow cytometry.(3)Xenograft tumor model of HL-60 cell in nude mice was established,which was divided randomly into six groups:negative control group (5%NaHCO3 group),25mg/ kg baicalin group,50mg/kg baicalin group,100mg/kg baicalin group,combination group (50mg/kg baicalin+2mg/kg VP16),VP16 positive control group(4mg/kg). Nude mice growth state was observed, average weigh and inhibition rate of transplanted tumor were calculated, and the ultramicrostructure change of xenografts cells were tested by transmission electron microscope .Histopathology were used to observed the change of chief organs in nude mice.The expression of Akt, p-Akt, mTOR and p-mTOR proteins extracted from xenografts were detected by western blot. The effects of balcalin on survival rate and median survival time in nude mice with HL-60 cell xenografts were evaluated.Result(s1)Baicalin effectively inhibited HL-60/ADR cells, proliferation, with an IC50 value of 28μmol/L.Cell colony formation was obviously inhibited by baicalin. Apoptosis occurred with dose dependent manners(20μmol/L, 40μmol/L,80μmol/L), and its earlier and later stages were both detected by Annexin V FITC/PI double staining analysis, DNA fragmentation and TUNEL labeling method. G0/G1 phase cell population increased while S phase cells decreased in 40μmol/L and 80μmol/L groups compared with control group(P<0.01).The typical hypo-diploid peak (apoptotic peak) appeared in each dose group.The mRNA expressions of c-myc, bcl-2, hTERT,pim-2 and mcl-1 in baicalin treated cells decreased in a time-dependent manner(12h,24h, 48h), while the p21 mRNA expression increased. Meanwhile, protein expressions of c-myc, bcl-2, caspase-3(35kD)and PARP(116kD)were down-regulated in a time- dependent manner, and the expressions of caspase-3(17kD,19kD),PARP(85kD) and bad were up-regulated.Moreover,western blot showed the expressions of p-Akt, NF-κB, p-NF-κB, p-IκB-α, mTOR, p-mTOR, p-GSK-3βand p-MAPK protein decreased in HL-60/ ADR cells after baicalin treatment , but the expression of Akt , IκB-α, GSK-3βand MAPK showed no difference compared to control group(.2)The multidrug resistance (MDR) in HL-60/ADR cells can be partially reversed by baicalin. Proliferation inhibition of ADR incombination with baicalin on the HL-60/ADR cells were significantly lower than that of ADR used alone, there was no difference of proliferation inhibition on HL-60 cells between ADR used alone and ADR incombination with baicalin.Baicalin could down-regulate the expression level of mrp-1, lrp,GSTπmRNA and mrp-1, GSTπprotein, and up-regulate TopoⅡαand TopoⅡβmRNA(p<0.05), but TopoⅠdid not change obviously. Furthermore,it was found that baicalin could elevated positive rate of ADR fluorescence in HL-60/ADR cells.(3)Baicalin could inhibit the growth of transplanted tumors in dose dependent manner.The transplanted tumor weight in 100 mg/kg baicalin group and combination group(50mg/kg baicalin+2mg/kg VP16) were significantly lower than that in negative control group(p<0.01) .There were more necrotic and apoptotic cells in baicalin treatment groups and combination group than that in negative control group by histopathology and transmission electron microscope methods.Baicalin could inhibit the proliferation of HL-60 cells in vivo by down-regulating the PI3K/Akt/mTOR signal pathway, where the expressions of p-Akt, mTOR and p-mTOR proteins decreased compared to negative control group, and Akt did not change significantly. Baicalin could prolong the median survival time of nude mice.Compared with negative control group, the median survival time in combination group was significantly different(p<0.05), which illustrated that baicalin combinated with VP16 have the synergistic effect on inhibiting growth of HL-60 cell xenografts in nude mice. The baicalin administration was well tolerated by the nude mice, and no significantly adverse effect was shown.Conclusion(1)Baicalin could efficiently inhibit proliferation and induce apoptosis in HL-60/ADR cells, which could be correlated with the down-regulated expressions of c-myc,bcl-2,hTERT, mcl-1, and up-regulated expressions of p21and bad. PI3K/Akt /mTOR and MAPK signal pathways may have involved in the process of proliferation inhibition and apoptosis in HL-60/ADR cells induced by baicalin.(2)Baicalin could partially reverse the multidrug resistance of HL-60/ADR cell, and the reversal effect may be related with the reduction of the expression of mrp-1, lrp, GSTπmRNA or protein, increase of TopoⅡαand TopoⅡβmRNA expression level, and elevation of drug accumulation in HL-60/ADR cells.(3)Baicalin could inhibit growth and induce apoptosis of HL-60 cell xenografts in nude mice, and prolong its median survival time. The possible mechanisms may be related to inhibition of Akt activity and down- regulation ation of the PI3K/Akt /mTOR signal pathway. The combination of baicalin and VP16 shows a synergistic effect on inhibiting growth in HL-60 cell xenografts in nude mice.