| Lung cancer is a disease with the highest incidence and mortality rate in all malignant tumors and is the leading cause of cancer-related death worldwide. In China, lung cancer also has a rather high incidence rate, which is ranked No.1 among all other kinds of cancers, especially in the urban population. Therefore, lung cancer has posed a serious threat to people’s lives and health. Lung cancer is categorized into small cell lung cancer(SCLC) and non-small cell lung cancer(NSCLC). The most common types of non-small cell carcinomas take up about 80-85% of all lung cancers. Chemotherapy is very important for the treatment of lung cancer. However,the traditional methods of chemical treatment, due to the low specificity, can inhibit a large number of normal tissue cells, resulting in the inhibition of bone marrow hematopoietic function, mucocutaneous reaction and have many other side effects.Drug resistance is also quite common in the clinical practice, which compromises the efficacy and severely restrict the application of chemotherapy drugs in the treatment of non-small cell lung cancer. Therefore, it is important to recognize the mechanism of lung cancer tumorigenesis and progression at the molecular level, and it is urgently needed that new chemotherapeutic drugs are developed.Esophageal cancer is also one of the most common malignant tumors in the world, and is the seventh leading cause of cancer deaths, esophageal squamous cell carcinoma(SCC)accounted for most of the total esophageal cancer. Although in the last few decades, surgery and adjuvant chemoradiotherapy have resulted in relieving symptoms and improving the life quality of patients suffering from esophageal cancer, a 5-year survival Rate is still less than 30%. Similarly, in the traditional chemical treatment process, there are problems like inhibition of a large number of normal tissues and cells, the emergence of bone marrow suppression, gastrointestinalreaction and many others. Consequently, exploring more effective therapeutic agents is necessary for more effective esophageal cancer treatment.In recent years, a series of studies on the traditional Chinese medicine as a new type of anti-tumor drugs have been highly concerned. These drugs provide a new method for tumor prevention and treatment by controlling the molecular targets of cell cycle and apoptosis signaling pathway. Related studies have shown that a large number of signaling pathways are activated in the formation of malignant tumors,resulting in tumor cell proliferation, invasion, metastasis, and ultimately to the death of patients, the molecular mechanism of these biological events can potentialy be used for the prevention and treatment of malignant tumors.Costunolide(CT) is a sesquiterpene lactone compound, one of the main chemical components of Chinese herb Saussurea lappa. CT has a wide range of pharmacological activities like anti-ulcer, anti-inflammatory, anti-fungal and anti-viral effects. Its anti-tumor effect is the main pharmacological activity, and showed a good inhibitory effect on many kinds of tumor cells. Cepharanthine(CEP)is a biscoclaurine alkaloid, extracted from the roots of Stephania cepharantha Hayata,and is known to have anti-inflammatory and immunomodulatory activities. During the last several decades, many reports reavealed that CEP has anti-tumor activity Like tumor invasion inhibitory and pro-apoptotic effects in many cancer cells.However, there is no report about the effect and mechanism of CT and CEP on lung cancer and esophageal cancer. Therefore, the aim of our study was to explore the inhibitory effects of CT and CEP on human NSCLC cells and human esophageal squamous cell carcinoma.In the present study, we uesd three NSCLC cell lines, human lung squamous cell carcinoma SK-MES-1 cells, human lung adenocarcinoma cell line A549, human non-small cell lung cancer cell line H1299 and human esophageal squamous cell carcinoma cell line Eca-109 cells for in vitro study. NSCLC cells were cultured in vitro. CEP cells were inhibited by MTT. CT can inhibit the proliferation of Eca-109 cells. And the effect of this growth inhibition was significantly related to the concentration of the drug. In order to explore the mechanism, we carried out thefollowing research.Experiment one, we mostly use human lung squamous cancer cell SK-MES-1cells to study the anti-tumor mechanism of CT. Costunolide induced morphological changes on the structure of SK-MES-1 cells, and inhibited the proliferation of cancer cells. Flow cytometry analysis showed that the costunolide induce SK-MES-1 cells to apoptosis, and cell cycle arrest in G1 / S phase. Protein analysis by Western blot showed CT can up-regulation of expression of p53, p21 and Bax, down-regulated Bcl-2, activated caspase-3, cleavage of substrate poly ADP-ribose polymerase(PARP). The result also found that mitochondrial membrane potential(MMP) was destroy. These conclusions clearly proved mitochondria mediated caspase activation pathway exists in costunolide induced apoptosis of SK. These findings suggest that novel compounds costunolide in the future is expected to become resistant to treatment of lung cancer.Experiment two, we use human Non-small cell lung cancer A549, H1299 cells as the experimental model to study the anticancer mechanism of small molecule drug CEP. It was found that cepharanthine inhibited the growth of H1299 and A549 cells in a dose-dependent manner which was associated with the generation of reactive oxygen species(ROS) and the dissipation of mitochondrial membrane potential(Δψm). These effects were markedly abrogated when cells were pretreated with N-acetylcysteine(NAC), a specific ROS inhibitor, indicating that the apoptosis-inducing effect of cepharanthine in lung cancer cells was mediated by ROS. In addition, cepharanthine triggered apoptosis in non-small lung cancer cells via the upregulation of Bax, downregulation of Bcl-2 and significant activation of caspase-3 and PARP. These results provide the rationale for further research and preclinical investigation of cepharanthine’s anti-tumor effect against human non-small-cell lung cancer.Experiment three, we use human esophageal squamous cell carcinoma Eca-109 cells as the experimental model to study the mechanism of CT. The cell cycle distribution was detected by flow cytometry. The results showed that the Eca-109 cell cycle was blocked in G1 / G0 phase by CT. CT can significantly induceapoptosis of human esophageal squamous cell carcinoma cell line Eca-109 by flow cytometry, and it can also increase the level of ROS and destroy the mitochondrial membrane potential. The expression of apoptosis pathway was observed by Western blot. The results showed increased p53, p21 and pro-apoptotic protein Bax expression, decreased anti-apoptotic protein Bcl-2 expression and activation of caspase-3 with the cleavage of PARP. The above phenomena can be inhibited by NAC. These data suggest that CT inhibits the growth of human esophageal squamous cell carcinoma, and plays an important role in the increase of ROS level and the activation of mitochondrial pathway mediated by p53.In this study, we used human non small cell lung cancer cell line and human esophageal cancer cell line as the research object, screening of natural small molecular compounds with antitumor activity and their mechanism of action. These provided a new idea for the treatment of lung cancer and esophageal cancer. This study shows that both CT and CEP are potential anticancer drug candidates, and further provides a theoretical basis for the clinical treatment. |
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