| BackgroundGlobal Cancer Statistics in 2012 indicates that,lung cancer has become the most common malignant tumor in the word.Its morbidity and mortality rank first among all malignant tumors,which is a serious danger to the mankind.Lung cancer belongs to primary malignant tumor,which could be divided to small cell lung cancer and non-small cell lung cancer by clinical pathology.Non-small cell lung cancer accounts for 80-85%of lung cancer.Nearly 70%of patients with a diagnosis of non-small cell lung cancer present with higher malignant-grade in medium or advanced-stage cancer.Most patients with lung cancer are generally diagnosed at stages that are too late for surgical intervention.With the increasing use of chemotherapy as an adjunct to surgical management,systemic chemotherapy will ultimately be used to treat the majority of patients with non-small cell lung cancer.However,resistance to chemotherapeutic agents leading to a failure of the chemotherapy is a significant issue in the management of patients with non-small cell lung cancer.Therefore,it is urgent to explore a new and effective drug,for the purpose of improving the therapeutic efficacy of clinical treatment of non-small cell lung cancer in our country and even around the world.At present,the induction of cell cycle arrest and apoptosis is an important strategy to treat cancer in the development of antitumor drug.Apoptosis is a physiological process with multiple factors,involving in gene regulation and signal transduction.Apoptosis occurs via activation of two different signaling pathways,the pathway mediated by receptor,triggered by the activation of the cell-surface death receptors and the pathway mediated by non-receptor,activated by mitochondrial function.Apoptosis is a natural barrier in the development of tumor.Apoptosis evasion of cancer cells is the leading cause of human cancer,and also one of the reasons for drug resistance or treatment failure.Recently,it has been demonstrated that Chinese herbal medicine and their main components possesses antitumor activities through inducing cell apoptosis.Thus,it is an effective method to screen for Chinese herbal medicine via inducing cell apoptosis.Rhizoma Paridis is commonly used to the treatment of cancer for thousands of years.The active components of Rhizoma Paridis are steroidal saponins,which have a wide range of pharmacological effects,including antitumor,anti-inflammatory,antibacterial,hemostatic,and immunity-enhancing.Polyphyllin VI(PVI)and polyphyllin VII(PVII)are monomers extracted from the Chinese medical herb Phizoma Paridis.It is reported that these two saponins have cytotoxic activities against cancer cells.PVII was shown to inhibit the growth of HepG2,MCF-7 and PC-3 cancer cells and the growth of the HepG2 xenograft tumors in vitro.However,the in vivo and in vitro antitumor activities against non-small cell lung cancer of PVI and PVII remain poorly understood.In previous experiments,we found that PVI and PVII can inhibit the proliferation of non-small cell lung cancer A549 and NCI-H1299 cells in vitro.However,which proteins or signaling pathways can be regulated by PVI and P? in the growth and proliferation of human non-small cell lung cancer A549 and NCI-H1299 cells?Whether A549 xenograft tumors will be inhibited by PVI and PVII in vivo?Hence,we conducted a series of experiments to elucidate the antitumor activities against non-small cell lung cancer of PVI and PVII.ObjectivesIn this study,our aim was to explore the antitumor activities of PVI and PVII in human non-small cell lung cancer A549 and NCI-H1299 cells.In addition,we proposed to construct the A549 xenograft model in nude mice,further evaluating the antitumor activities and the related mechanism of P? and P? in human non-small cell lung cancer.Findings of this study will help us better understand the mechanisms of P? and P? against lung cancer and provide deeper theoretical basis in the development of drug and clinical application of lung cancer.MethodsTo investigate the antitumor effects and mechanisms of P? and P? in lung cancer in vitro and in vivo,various commonly used methods are listed and discussed below,such as MTT assay,flow cytometry,cell cycle arrest detection,western blotting and nude mice xenograft model etc.1.Determination of cell viability by MTT assayCells were seeded at a density of 3000/well in 96-well plates and grown overnight.Then,the medium was replaced with a fresh medium containing polyphyllin V,polyphyllin H,gracillin,CL-8,P? and P? at 0,0.78,1.56,3.12,6.25,12.5 ?M for 48 h in A549 cells.And A549 and NCI-H1299 cells were treated with P? and P? at 0,0.78,1.56,3.12,6.25,12.5?M for 24,48,72 h.After treatment,MTT solution in the phosphate buffer solution(PBS)was added to each well and the plates were incubated for another 4 h.The supernatants were discarded,and 150?l of dimethyl sulfoxide was added to each well.After shaking the plates at room temperature,the stained formazan product was determined at 570 nm with a multimode plate reader.2.Determination of cell cycle distribution by flow cytometryA549 and NCI-H1299 cells were seeded at a density of 2×105/well in six-well plates and grown overnight,and then the medium was replaced with a fresh medium containing PVI at 0,1,2,4?M and PVII at 0,0.5,1,2 for 24 h.After treatment,cells were collected and washed twice with PBS,then fixed with ice-cold 70%ethanol in PBS at 4? overnight.The cells were collected and incubated with 500 ?M PBS containing 25 ?l of 1mg/ml propidium iodide(PI)and 0.5 ?l of 10 mg/ml RNase A at 37? for 0.5 h in the dark.The cells were resuspended and then the distribution of cell cycle was analyzed by flow cytometry within 1 h.3.Determination of cell apoptosis by flow cytometryA549 and NCI-H1299 cells were seeded at a density of 2 x 105/well in six-well plates and grown overnight,and then the medium was replaced with a fresh medium containing P? at 0,1,2,4 ?M and PVII at 0,0.5,1,2 ?M for 48 h.After treatment,cells were collected and washed twice with PBS after centrifugation.The cells were resuspended with 100 ?l/Tube 1 × Binding Buffer from FITC Annexin V Apoptosis Detection Kit.And then each tube was added and incubated with 5 ?l of Annexin V-FITC and PI at room temperature for 15 min in the dark.The cells were resuspended with 400 ?l/Tube of 1 × Binding Buffer.The ratio of apoptosis was analyzed by Flow cytometer within 1 h.4.Determination of apoptotic proteins and G2/M correlative proteins by western blottingA549 and NCI-H1299 cells were treated with PVI at 0,1,2,4 ?M and PVII at 0,0.5,1,2 ?M for 48 h.After treatment,the cells were collected and added with RIPA lysis buffer.The total proteins were extracted and quantified.The expression levels of Fas,DR3,DR5,DcR3,PARP,Cleaved PARP and Cleaved Caspase-3 proteins and G2/M checkpoint correlative proteins Cyclin B1 expressions were determined by western blotting.5.Evaluation of P? and P? on the antitumor effects in vivo in nude mice xenograft tumor model.A method to establish nude mice xenograft tumor model in human lung cancer cells is as followings.4 to 6 weeks of female BALB/c nu/nu mice were used in this study.A549 cells were resuspended and subcutaneously inoculated with 2 x 106/mouse in 0.2 ml of PBS into the right flank of the nude mice.Body weight was measured and tumor volume(TV)was measured using micrometer calipers twice or three times a week.The formula TV(mm3)=1/2 ×(L x W2)was used to calculate TV,where L is the long diameter and W is the short diameter.Growth curve of tumor was plotted between the groups.When an average of tumor volume reached approximately 100 mm3,the mice were divided randomly into eight groups(n=8),including control group(0.9%NaCl),P?-treated groups(4,3,2 mg/kg),P?-treated groups(3,2,1 mg/kg),positive group(cyclophosphamide,25 mg/kg).The treatments were intraperitoneally administered five times a week for 4 weeks.After treatment,the nude mice were sacrificed,and the tumors were segregated and measured.A fraction of tumor tissue was lysed with RIPA buffer,and then the extractive proteins were quantified.The expressions of DR3,DR5 and DcR3 proteins and G2/M checkpoint correlative proteins Cyclin B1 expressions were determined by western blotting.6.Statistical analysisStatistical significance of the data was determined by one-way ANOVA using SPSS 13.0 software.Data were plotted using GraphPad Prism 5.0 and expressed as the Mean± SD.Statistical significance was considered at P<0.05.Results1.The growth inhibition of six Paris saponins on lung cancer cells.The growth inhibition ratios of six Paris saponins on lung cancer cells were determined by MTT assay.Results were shown that P? and P? have the strongest inhibitory effects on A549 cells.In addition,the treatment of PVI and P? resulted in dose dependent inhibiting effects on the viability of A549 and NCI-H1299 cells.The IC50 values of PVI in A549 cells after 24,48,and 72 h of treatment were 4.63 ± 0.58?M,4.53± 0.56 ?M,and 4.24 ±0.59 ?M,respectively,and those of P? in the same cells were 1.80 ± 0.14 pM,1.59± 0.12 ?M,and 1.52± 0.20 ?M,respectively.Then,the IC50 values of PVI in NCI-H1299 cells after 24,48,and 72 h of treatment were 5.64± 0.65 ?M,5.46± 0.45 ?M,and 5.54±0.91 ?M,respectively,and those of P?in the same cells were 2.08±0.05 ?M,1.87± 0.09 ?M,and 1.71 ±0.25 ?M,respectively.These findings were shown that P? and P? displayed strongly inhibitory effects on A549 and NCI-H1299 cells.2.P? and P? arrested the cells in the G2/M phase.Cell cycle arrest was analyzed by flow cytometry.Compared with control group,the ratios of the G2/M phase cells were significantly in P?-and P?-treated groups in a concentration-dependent manner.These findings were shown that A549 and NCI-H1299 cells were arrested in the G2/M phase by P? and P?.In addition,the expression of Cyclin B1 was significantly increased in the P?-and P?-treated A549 cells,determined by western blotting,especially at high concentration.3.P? and P? induced apoptosis in A549 and NCI-H1299 cells.Apoptotic cells were detected by flow cytometry.Compared with control group,the ratios of Annexin V-FITC-positive cells were significantly increased in P?-and P?-treated groups,in a concentration-dependent manner.These results suggested that P? and P? reduced cell viability by inducing apoptosis.In addition,P? and P? significantly up-regulated the expression levels of Fas,DR3,DR5,PARP,Cleaved PARP and Cleaved Caspase-3 but reduced the expression levels of DcR3 in A549 and NCI-H1299 cells by western blotting.4.P? and P? inhibited the growth of A549 nude mice xenograft.A549 nude mice xenograft model revealed 100%tumor formation of A549 cell transplanted tumor.P? and P? can significantly inhibit the growth of A549 nude mice xenograft in vivo.Compared with control group,the inhibition of A549 nude mice xenograft has significantly difference in P?-and P?-treated groups.P?significantly inhibited the growth of 25.74%,34.62%,and 40.43%A549 cells and P? inhibited the growth of 25.63%,41.71%,and 40.41%A549 cells,respectively,at high dose,medium dose and low dose group,after 4 weeks of treatment.In addition,these treatments had no significant influence on the body weight of the nude mice.Furthermore,P? and P? treatments in A549 xenograft tumor significantly increased the expression levels of DR3,DR5 but downregulated the expression levels of Cyclin B1 proteins,as determined by western blotting.ConclusionsOur findings revealed that P? and P? can inhibit the proliferation of non-small cell lung cancer cells in vitro and in vivo,induce cell apoptosis and arrest cell cycle.1.Six Paris saponins showed inhibitory effects on A549 cells,and the inhibitory effects of P? and P? were the strongest.What's more,P? and P? can significantly reduce cell viability of A549 and NCI-H1299 cells.2.P? and P? arrested A549 and NCI-H1299 cells in the G2/M phase.P?and P? significantly reduced the expression level of Cyclin B1 in A549 and NCI-H1299 cells.3.P? and P? induced apoptosis in A549 and NCI-H1299 cells.The rates of apoptosis were increased in a dose-dependent manner.P? and P? significantly upregulated the expression levels of Fas,DR3,DR5,Cleaved PARP and Cleaved Caspase-3,but reduced the expression levels of DcR3 in A549 and NCI-H1299 cells.4.P? and P? can significantly inhibit the growth of A549 nude mice xenograft and have antitumor activities in vivo.Results were showed that P? and P? can inhibit the proliferation of A549 cells by arresting cell cycle in the G2/M phase and triggering cell apoptosis. |
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