Preliminary Investigation On The Inhibition And Mechanism Of Cancer Cell By Alantolactone

Inula helenium L.is a kind genus inula flower of the compositae herbaceous,which roots has been used as a medicine,it can strengthen the stomach,diminish inflammation,dewarm and other effects.Inula helenium grows mianly in Asia and Europe.The reseach of Inula helenium L.began in 1970 s,and the currently report showed that it contains more than 40 different components,and the main compositions are inulin and volatile terpenoids,and it also contains a bit of alkaloids,flavonoids,amino acids and so on.Alantolactone is a sesquiterpene lactones extraction from Inula helenium L.with strong biological activity,and the studies are focusing on its anti-inflammatory,antibacterial and antitumor activity,especially the effect of antitumor is particularly prominent.Many reports have pointed out that alantolactone can against breast cancer,colon cancer,liver cancer,prostate cancer,cervical cancer and leukemia cell,but there are few reports about the activity and mechanism of inhibition of gastric cancer and lung cancer.Therefore,in this study,two kinds of gastric carcinoma cell line BGC823 and SGC7901,NCI-H1299 and Anip973 of lung cancer cell lines have been choosen to explore the inhibition of alantolactone on gastric cancer cells and lung cancer cell and its possible mechanism.In this experiment,gastric carcinoma cell line BGC823 and SGC7901 and lung cancer cell lines NCI-H1299 and Anip973 have been choosen as the research object.Examining the cell morphology after incubating by alantolactone(0,5,10,20 and 30μM)under the microscope.The cells were treated by alantolactone(0,5,10,20 and 30μM)in different time points(24h,48 h and 72h),and cell viability was detected by MTT assay.Using Hoechst33258 and Annexin V/PI staining to detect the inhibition of tumor cell growth and apoptotic rate.Using wound healing assay,transwell method and colony assay to measure the invasion,migration and colony forming abilities of the caner cells in vitro after incubating with different concentration alantolactone.Real-time quantitative PCR is using to detect the influence of the transcription expression of mRNA of apoptosis and metastasis related genes.The expressin level of apoptosis related proteins Bcl-2,Bax,p53,and metastasis associated protein MMP9,MMP7,MMP2 were measured by Western blot,and it also detected the expression of p38 and NF-κB.The results: the microscopic observation showed that alantolactone could change the shape of cancer cells,the cell morphology turned from diamond or spindle to circle and the number of the cells decresed significantly with the concentration increased,these indicated that alantolactone can transform the cell morphology and inhibit cell growth as well.MTT assay showed that alantolactone inhibited cancer cells viability remarkably with dose-dependent and time-dependent.The results of Hoechst33258 and flow cytometry indicated that alantolactone can induce the apoptosis of cancer cells with a dose-dependent.The experimental results of wound healing assay showed alantolactone can reduce the migration of cancer cells compared with the control group,and transwell results showed alantolantne can prevent the cross of cancer cells,and the number of the cells were decresed with the concentration increased.The experimental results of colony assay revealed alantolactone can inhibit colony ability of cancer cells.After incubating by alantolactone for 12 h and 24 h,the expression of apoptosis inhibiting gene in Bcl-2 was significantly reduced,and the pro-apoptotic gene Bax and p53 expression level of mRNA were increased,metastasis related genes MMP-9,MMP-7 and MMP-2 mRNA expression levels were apparent down-regulated.Alantolactone may inhibit the proliferation,invasion and migration by regulating the apoptosis related genes and MMPs genes.Western blot results showed that alantolactone can activate p38 MAPK and induce the phosphorylation,indued the apoptosis of tumor cells by inceasing pro-apoptotic proteins and down regulate the expression of apoptosis protein,alantolactone can also prevent the nuclear transfer of p65,which inhibited NF-κB(p65)activation and down-regulation the expression of NF-κB(p65)induced the down-regulation expression of matrix metalloproteinases.In conclusion,alantolactone can inhibit the proliferation and metastasis of gastric cancer cells BGC823 and SGC7901 and NCI-H1299 and Anip973 in lung cancer cells,and induce apoptosis.In the process of induction,alantolactone activation of p38 MAPK pathway by regulating the expression of Bcl-2 proteins and p53 protein.At the same time,the inhibition of NF-κB signaling pathway prevented matrix metalloproteinase family proteins expression,which can inhibit proliferation and metastasis,and inducing apoptosis ultimately.The above results showed that alantolactone has the potential to be an antitumor drug,and it can provide some experimental basis for the clinical application of tumor therapy.