Effect And Mechanism Research Of Alantolactone On The Proliferation Of Non Small Cell Lung Cancer

Lung cancer is the most prevalent type of malignant tumor and the leading cause of cancer-related death worldwide. Lung cancer can be divided into non small cell lung cancer and small cell lung cancer, and the former is the most common subtype accounting for about 85% of lung cancer. Non small cell lung cancer(NSCLC) includes adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Despite advances in surgical techniques and chemo-radiotherapy, the prognosis of the most patients diagnosed with NSCLC remains poor due to its chemotherapy resistance. Currently, goals in lung cancer treatment are focused on finding new therapeutic agents that inhibit the proliferation of lung squamous carcinoma cells. Recently, it is well accepted that chemotherapy using naturally occurring substances is an attractive option in cancer therapy.Sesquiterpene lactones are plant-derived compounds, having been used for the inflammation treatment. Over the past few years, pharmacological studies have provided convincing evidence of the anti-cancer property of sesquiterpene lactones against various human cancers. Alantolactone, derived from Inula helenium L.root, is one of the major sesquiterpene lactone compounds.Recent studies have shown that alantolactone has diverse pharmacological effects, such as anti-inflammation, anti-bacteria and antifungi effects. Recently, the anti-proliferative property of alantolactone against various human malignancies was widely reported. For example, it has been shown that alantolactone could trigger apoptosis in human colorectal cancer, hepatocellular carcinoma, and leukemia cells. In addition, alantolactone also induces cell cycle arrest in human liver cancer cell. Apoptosis occurs mainly via two signal pathways: the intrinsic pathway(also called mitochondrial pathway) and the extrinsic pathway. In the extrinsic pathway, caspase-8 is activated, while caspase-9 is involved in the intrinsic pathway. The changes in the expression of proapoptotic protein(such as Bax) versus antiapoptotic proteins(such as Bcl-2) activate the intrinsic apoptotic pathway. It has reported that alantolactone could trigger mitochondrial apoptotic pathway via Bcl-2 down-regulation and caspase-9 activation in liver cancer cell. However, it is unclear whether alantolactone could suppress the growth of NSCLC. In this regard, firstly we examined the effect of alantolactone on NSCLC. To further clarify the molecular events underlying the antitumor effects of alantolactone, we determined apoptotic cells and cell cycle distribution by flow cytometry. Moreover, we also investigated the expression of apoptosis factors including caspase-8, 9, 3, PARP, Bax and Bcl-2. We also invested the levels of MMP and ROS. In addition, we examined G1/G0 phase transition modulators, such as cyclin D1, cyclin D3, CDK4, CDK6, p21 and p27 systemically.In the present study, alantolactone exhibited inhibitory effects on lung cancer cell proliferation, and alantolactone-mediated inhibition of cell growth might be through triggering apoptosis and arresting cells at G1/G0 phase in SK-MES-1 cells. Furthermore, induction of apoptosis by alantolactone in SK-MES-1 seems regulators including cyclin D1, cyclin D3, CDK4, CDK6, p21 and p27 play critical roles in alantolactone-induced cell cycle arrest in SK-MES-1 cells. Alantolactone inhibited the proliferation of A549 cells by triggering apoptosis. Furthermore, induction of apoptosis by alantolactone in A549 seems to be mediated via modulating the protein expression of Bax and Bcl-2, decreasing MMP and increasing ROS.In our study alantolactone significantly down-regulated Bcl-2 expression, and upregulated expression of Bax in NSCLC cells, suggesting the involvement of mitochondrial apoptotic pathway in alantolactone-mediated apoptosis. Furthermore, the decreased level of MMP was also detected which was well-known indicators of intrinsic apoptosis. Our findings suggested that Bcl-2 protein family could be an important molecular mechanism through which alantolactone induced mitochondrial apoptosis in NSCLC cells. In addition, the activation of caspase-9, caspase-3 and PARP by alantolactone were also confirmed the involvement of mitochondrial pathway in SK-MES-1 cell. Further exploration showed that treatment of alantolactone activated caspase-8 which cleavage represents the hallmark of extrinsic apoptosis, suggesting that the extrinsic pathway may be also involved in alantolactone-induced apoptosis in SK-MES-1 cell. It suggested that the intrinsic and extrinsic pathways were both involved in alantolactone-induced apoptosis in SK-MES-1 cell.Cell cycle arrest is an essential mechanism involved in the induction of cell growth inhibition. Our findings showed that the treatment with alantolactone resulted in G1/G0 cell cycle arrest in SK-MES-1 cells in a dose-dependent manner by flow cytometric analysis. Similarly other studies also have shown that alantolactone is an inhibitor of cell-cycle progression in human erythroleukemia and liver cancer cells. Lots of studies were shown that cell cycle progression is dependent on the activation of cyclin dependent kinases(CDKs) complexed with their activatory cyclins and in G1 and G1-S transition cyclin D-CDK4/CDK6 complexes act predominantly. Three types of cyclin D(D1, D2 and D3) which are associated with CDK4 or CDK6 in mammals have been identified. During the G1 phase, the cell accumulates cyclin D, which forms a complex with CDK4 or CDK6. The complex phosphorylates the retinoblastoma protein(p Rb), releasing transcription factors to promote the transcription of the genes required for cell cycle progression. In present study alantolactone-treated cells showed low expression of cyclin D1, cyclin D3 and their partner CDK4 and CDK6, it was suggested that the depletion of cyclin D1, cyclin D3, CDK4 and CDK6 proteins may cause the cell cycle arrest at G1/G0 phase in alantolactone-treated cells. In addition, cell cycle is also negatively regulated by CDK inhibitory protein which is responsible for slowing or arresting the progression of cell cycle. P21 and p27 are two important CDK inhibitors. In our study the expression of p21 and p27 were up-regulated in SK-MES-1 cells treated with alantolactone.Reactive Oxygen Species(ROS) is an important regulator in the cell. The excessive ROS can induce oxidative stress, inducing apoptosis via the intrinsic pathway by affecting the expression of the Bcl-2 family proteins. ROS is involved in the process of lipid peroxidation which leading the opening of permeability transition pore(PTP). It was also reported that ROS could induce apoptosis in other studies. In order to further explore the mechanism of alantolactone mediated inhibition in NSCLC, intracellular ROS level was detected. Results showed that ROS was increased in NSCLS cells treated with alantolactone. It indicated that the intrinsic apoptosis may be mediated by ROS. ROS also influence the process of cell cycle. It was reported that ROS could mediate cell cycle arrest in G1 phase in other studies. During these researches, the expression of cyclin D was low while p21 was up-regulated which was the same with our results. The cell cycle arrest at G1/G0 phase in SK-MES-1 cells may related to the increased level of ROS.Alantolactone inhibited the growth of human squamous lung cancer cells by induction of apoptosis and G1/G0 cell cycle arrest. Alantolactone induced apoptosis via increasing level of ROS, down-regulation of Bcl-2 and up-regulation of Bax followed by decreasing of MMP, activation of caspase-9, 8, 3 and PARP. Alantolactone-mediated cell cycle arrest was associated with a marked decrease in protein expression of cyclin D1, cyclin D3, CDK4 and CDK6. Alantolactone also inhibited the growth of human lung adenocarcinoma cells. Alantolactone induced apoptosis via increasing level of ROS, down-regulation of Bcl-2 and up-regulation of Bax followed by decreasing of MMP in A549 cells. Therefore, these findings may be an important implication in the development of targeted therapeutics for NSCLC.