The Study Of The Effect And Mechanism Of Polyphyllin Ⅰ On Human Non-small Lung Cancer Cells In Vitro And In Vivo

Backgroud:Lung cancer is the leading malignant tumor and still prevalent,the incidence increases as much as 0.5%every year in the world.Non-small cell lung cancer (NSCLC)includes squamous cell carcinoma,adenocarcinoma,and large cell carcinoma.It accounts for 75-80%of all lung cancer patients and the overall five-year survival rate for NSCLC is only 8-15%.Surgery excision is the primary treatment of all therapies,but most patients have unrescctable disease.More than 75%patients need chemotherapy or combination of chemotherapy and radiotherapy in some stage of the disease.At present,the remission rate of the most effective chemotherapy regimen is only about 30%in advanced NSCLC.It is urgent to improve the therapeutic effect,to study and find a new effective drug in lung cancer therapy.Paris polyphylla,a famous folk medicine herb in south China,has been used for more than one thousand years in Chinese history,and is a common herb in anti-cancer prescription of traditional Chinese medicine.It is not only as an anti-cancer,anti-biotic,and anti-inflammatory drug but also to treat snake bite, chronic bronchitis and bone fracture,as well as haemostasis.The anti-cancer mechanism of extracts or monomers of Paris polyphylla is still unknown. Polyphyllin I,small molecular monomer extracted from Rhizoma of Paris polyphyllin,shows strong anti-cancer effect in our previous study.But the effect and mechanism of polyphyllin I(PP I)on NSCLC cels is still unclear.In this research,we investigated the mechanisms by which polyphyllin I elicits anti-cancer effects in NSCLS cells by means of various techniques,including cell culture system,MTT,flow cytometry,western blot,DNA microarray, quantitative reverse transefiption-polymerase(qRT-PCR),and semi-quantity RT-PCR.Material and Methods:1,Three NSCLC cell lines:human lung adenocareinoma A549 cells,human lung squamous cell carcinoma SK-MES-1 cells and human lung large cells carcinoma NCI-H460 cells,were cultured.The growth inhibition effects of different concentrations(0,0.625,1.25,2.5,10μg/ml)PP I on NSCLC cells was detected by MTT assay.The appropriate concentration and time were selected for further experiment.2,The apoptosis effect was detected by DNA ladder at 48h after 2.5μg/ml PP I treated NSCLC cells.The expression of protein proeaspase-3 and bel-2 were analyzed by Western blot at 24h after 2.5μg/ml PP I treated NSCLC cells.3,The effect of PP I on cell cycle of NSCLC cells was detected by flow cytometry (FCM).The apoptosis cells of A549 cells were detected by Annexin V-FITC and PI staining flow cytometry.4,Male,5 weeks old BALB/c nude mice were subcutaneous(s.c.)inoculated with 100μl of exponential growing A549 cells at the concentration of 5×10⁷/ml for formatting NSCLC xenograft.The mice were randomly divided into three groups:PP I group(n=11),cisplatin(DDP)group(n=11)and PBS group(n=11). The animals were treated with doses twice daily by intraperitoneal(i.p.) injection from day 2 to day 11.PP I at1.5mg/kg and DDP at 1mg/kg body weight were administrated to study the growth inhibition effect of PP I on NSCLC cells in vivo.Mice were euthanized on day 31.5,Exponential growing A549 cells were treated with PP I or PBS.After 6h or 12h,gene expression was analyzed with oligo cancer microarry of Super Array Company(Cat.Human OHS-802)for further discover the anti-cancer mechanism of PP I.6,Five selected differentially expressed genes were chosen to be validated by qRT- PCR using SYBR greenl as fluorescent dye.The values of glyceraldehyde 3-phosphate dehydrogenase(GAPDH)were used to normalize the expression data.7,The expressions of P53,GADD45α,GADD45γ,and GADD45βgenes were detected by RT-PCR at Oh,6h,12h or 24h after 2.5μg/ml PP I treated A549 cells.8,The phosphorylated and total AKT proteins were analyzed by Western blot at Oh, 6h,12h or 24h after 2.5μg/ml PP I treated A549 cells.9,Statistical Analysis:Results were expressed as the mean value±SD;the statistical significance of differences was determined by U's two-tailed test in rate of sample in two groups and one-way ANOVA in multiple groups.P values <0.05(marked with a single asterisk)were considered statistically significant. All data were analyzed with the SPSS13.0 statistical software. Results:1,The inhibition of PP I on the proliferation of three NSCLC cell lines:The inhibiting effect was detected by MTT assay in A549,NCI-H460,SK-MES-1 cells at 24h,48h,72h after treated with different concentration(0,0.625,1.25,2.5,5,10μg/ml)PP I.The obviously inhibiting effects were found in the three NSCLC cell lines at 24h,48h and 72h after treated with 2.5μg/ml PP I.The inhibiting rates of A549 cells ware 51.9±7.1%,76.9±2.8%,84.7±4.8% respectively according with time.In NCI-H460 cells,the rates were 60.3±10.6%,49.1±7.5%and 52.1±2.2%respectively.And in SK-MES-1 cells, the figures were 67.8±8.9%,60.2±2.7%and 71.9±2.9%respectively.While the concentration PP I was 10μg/ml,the rates reached about 90%in three lines.The 50%inhibiting concentration(IC₅₀)values in A549,NCI-H460 and SK-MES-1 cells were 1.24,2.40 and 2.33μg/ml at 48h.2,The apoptosis of NSCLC cells was induced by PP I in vitro:PP I elicited typical apoptosis morphologic changes and the distinct DNA ladder was clearly observed in three NSCLC cell lines.After treated with 2.5μg/ml PP I in three cell lines,western blot analysis showed that the expression of bcl-2 was strongly inhibited as well as procaspase-3.Through flow cytometry assay,the apoptotic peak was detected in A549 cells induced by 2.5μg/ml PP I,and increased with time-dependent.In contrast,the percentage of G0/G1,S and G2/M phase cells decreased markedly.The rate of apoptotic cells detected by Annexin V-FITC assay is 39.68%at 24h after A549 cells treated with 2.5μg/ml PP I.3,The inhibition of PP I on the growth of A549 xenograft tumors in vivo: Mice with PP I,DDP or PBS administrated intraperitoneal for ten days were well and did not show any drug-related deaths.The PP I group and DDP group revealed significantly reduced gross tumor volume and weight of xenograft tumors,compared with PBS group.PP I was more effective than DDP.The tumor static rate in PP I group>DDP group>PBS group(P<0.05).4,Results of DNA microarray(SuperArray Human OHS-802):There were 104 and 34 genes up-regulated(≥2 folds)respectively,5 and 72 genes down-regulated(≤2 folds)respectively at 6h and 12h after treatment of 2.5μg/ml PP I on A549 cells.We found overexpression of many genes related to apoptosis,but BCl-2 and BAX genes changed slightly at 6h and 12h.Some overexpressed genes related to anti-cancer effect of PP I on tumor signaling pathways.5,qRT-PCR verification of DNA mieroarray data:Five genes(PIK3CB,PIK3CG, PTEN,JUN,PCTK3)associated with tumor signaling pathways were chosen to verify the DNA microarray experiments.The changes,observed with this technique confirmed results obtained by the DNA microarrays,although the variations in magnitude of the changes between the two technologies were observed.6,Expression of P53 and GADD45 genes detected by RT-PCR:The expression level of GADD45αin PP I group was higher than that in blank control group at 6h after A549 cells induced by 2.5μg/ml PP I,it decreased slightly after 12h although was higher than control.The expression levels of GADD45βand GADD45γdid not change.The overexpression of p53 gene was observed at 6h, 12h and 24h after treatment of PP I on A549 cells.7,The phosphorylation of AKT analyzed by Western blot:After A549 cells treated with 2.5μg/ml PP I,the expression level of total AKT did not changed at Oh,6h,12h and 24h,but that of phosphorylated AKT decreased distinctly. The data suggests that PP I induces A549 cells apoptosis may be via the deactivation of AKT. Conclusion:1,Polyphyllin I can inhibit growth of human NSCLC cells in vitro and in vivo.2,Polyphyllin I can induce apoptosis of human NSCLC cells in vitro.3,Polyphyllin I exhibits pleiotropic effects,simultaneously up-regulating or down-regulating cytotoxic and cytoprotective genes that function cooperatively in determining cell fate.4,Polyphyllin I may induce apoptosis of A549 cells via P53/GADD45/JUN signal pathway,further more the GADD45αpathway.5,PP I induces A549 cells apoptosis may be via the deactivation of AKT.