Bufalin Exerts Antitumor Effects By Triggering Apoptosis And Enhancing Chemosensitivity To Cisplatin In NSCLC Cells

The incidence of lung cancer ranked the first in the human malignancies, non-small-cell lung cancer (NSCLC), which accounts for more than 80% of all lung cancer, is the leading cause of cancer mortality. The biological characteristics of NSCLC is prone to local invasion and distant metastasis, and the 5-year survival rate of patients suffering from NSCLC is merely 15%, which largely due to the fact that most of them are diagnosed at advanced and later stages. For patients with locally advanced or metastatic NSCLC, cisplatin-based chemotherapy is the main mode of treatment. However, the development of resistance of NSCLC against cisplatin often reduce treatment effect. Therefore, it is tremendously significant to exploit novel chemicals to prevent and treat NSCLC.Previous research and clinical studies have demonstrated that many natural products derived from traditional Chinese medicine(TCM) could be effective antitumor compounds, indicating that TCM is a promising source for developing new anti-cancer drugs. As major component of Chan-Su extracts, bufalin has a relative molecular mass of 386.5. There have been researches demonstrating that bufalin could effectively inhibit the proliferation of many cancers including pancreatic cancer, gastric cancer and breast cancer. In Other reports have proved that bufalin could also inhibit the proliferation of lung cancer yet its mechanism is not clear. Besides, researches also indicated that bufalin could increase the sensitivity to regular chemotherapeutics, even reverse drug resistance of serval kinds of tumor cells. However, the underlying molecular mechanisms of the antitumor effect and whether it could enhance chemosensitivity to cisplatin especially have not been well understood yet in NSCLC until now.In this study, we tested the effects of bufalin on NSCLC cells and the underlying mechanisims, meanwhile we further explored the enhancement effects of bufalin on chemosensitivity to cisplatin and its potential mechanisms. Our work is presented in three parts as follows.Part oneThe inhibitory effects of bufalin on the proliferation of A549 and H1975 cellsObjective:To investigate the influence of bufalin on the proliferation and migration ability of NSCLC cells.Methods:A549 and H1975 cells were treated with different concentrations of bufalin for 24h,48h and 72h, CCK-8 assay was used to test the proliferation of cells, wound healing assay were used to investigate the influence on migration ability.Results:After exposure to various concentrations of bufalin(2.5nM,5nM, 10nM,20nM,40nM,80nM, 100nM,200nM), The result showed that after treated with relatively lower amount (2.5,5 nmol/L) of bufalin, the survival rate of A549 and H1975 cells were about 98%, no obvious proliferation inhibition effects observed; after cells were treated with higher amount (10-200 nM) of bufalin, the proliferation of A549 and H1975 cells dramatically inhibited in a time-and dose-dependent manner. Morever, the migration ability of cells after bufalin treatment was dramatically inhibited, as manifested by wound healing assay.Conclusion:Bufalin could inhibited the proliferation and migration ability of A549 and H1975 cells in a time-and dose-dependent manner.Part twoThe effects of bufalin on the apoptotic rates and the underlying mechanismsObjective:To investigate the potential molecular mechanisms by which bufalin exerts anti-tumor effects.Methods:After A549 and H1975 cells were being treated with different concentrations of bufalin(0-200nM) for the indicated time(48h), c the fraction of apoptotic cells was analyzed by flow cytometry; western blotting analysis was used to detect the expression level of apoptosis-related proteins and the downstream modulating molecules, we also examined the transcripts change of oct-4.Results:The results of flow cytometry indicated that the apoptotic rates of A549 and H1975 cells increased significantly in a dose-dependent manner, which means that bufalin could actually induce apoptosis in NSCLC cells. Meanwhile, we used western blotting analysis to further investigate the potential mechanisms of bufalin-induced apoptosis in NSCLC cells. Our results showed us that after treatment with bufalin, the expression level of β-catenin and its downstream molecule c-myc and cyclinD1 was down-regulated in a dose-dependent manner and the protein level of oct-4 was also decreased. In addition, we found that PARP is degraded after treatment with bufalin, and the expression level of cleaved-PARP was up-regulated in A549 and H1975 cells.Conclusion:Bufalin might target Wnt/(3-catenin and CSCs subsets, and thus regulate the key apoptosis-related proteins to eventually trigger apoptosis in NSCLC cells.Part threeBufalin affected the anti-cancer activity of cisplatin and resistance-associated protein in NSCLCObjective:To explore the potential mechanisms of bufalin enhance NSCLC chemosensitivity to cisplatin.Methods:A549 and H1975 cells were treated with bufalin or cisplatin alone or their combinations for 48h, cells treated with 0.1% DMSO served as control. After treatment, the results were analyzed with CCK-8 assay. Western blotting analysis was used to detect the expression level of NF-κB and the downstream modulating molecules to explore the potential mechanisms of bufalin enhance NSCLC chemosensitivity to cisplatin.Results:Our data showed that the combination treatment could suppress the proliferation of these two NSCLC cell lines more than either bufalin or cisplatin alone. This result suggested that low concentration of bufalin could enhance the chemosensitivity of A549 and H1975 cells. In addition, Western blotting results showed that compared with cisplatin monotherapy group, the expression of NF-κB and its downstream molecule c-myc was down-regulated in combined therapy group.Conclusion:Bufalin might enhance the chemosensitivity to cisplatin in NSCLC by inhibiting the activation of NF-κB signaling pathway.