| Lung cancer is a kind of malignant tumor with increasing risk of the morbidity and mortality, which is severely threatening human health. In many big cities in China, lung cancer is becoming the main cause for the death and morbidity of patients with malignant tumor in male, and the second cause in female. Although operation, combined chemotherapy and radiotherapy are playing an active role in the treatment of lung cancer, 70-80% patients have lost the chance of early radical resection due to the lack of effective methods for early diagnosis. The recurrence rate after the operation, radiotherapy and chemotherapy is high with resistance so that the 5-year survival rate of the patients has stayed at 20-30% for a long time.The traditional Chinese medicine has unique theories on the treatment of tumors. The relevant reports about the treatment of lung cancer with traditional Chinese medicines indicated that the traditional Chinese medicine could prolong the life span of the patients with lung cancer as well as increase their quality of life. The curative effect of combination of traditional Chinese medicine with radiotherapy and chemotherapy is better than that of singly application of chemotherapy or radiotherapy. The traditional Chinese medicine could increase the sensitivity to chemotherapy and radiotherapy, and reduce the adverse effects of chemotherapy and radiotherapy, so that it has the functions of inhibiting tumors and synergia. The natural Chinese medicine has raised broad interests of worldwide researchers due to its extensive and certain curative effect and low toxicity.In this study, we selected many extracts or monomer of the traditional Chinese medicines to screen the one(s) that can inhibit the growth or proliferation of lung cancer cells at low concentration (the level ofμg/mL) through in vitor experiments. Based on these results, we choose the extract or monomer of the traditional Chinese medicine with the most significant efficiency and studied the relation between its effect of inhibiting the growth or proliferation of lung cancer cells and the relevant signal pathways as well as its effect on the sensitivity of lung cancer cells to chemotherapy. These studies will provide the possibility of finding some new ways in the development of new drugs for the treatment of lung cancer and provide experimental bases for the further application of traditional Chinese medicines in the treatment of tumors. Based on the above mentioned contents, we carried out the study from the following four aspects:1. The screening of Chinese herb with anti-lung cancer effect: we preliminarily screened 25 kinds of monomer or crude extracts of the traditional Chinese medicines which can effectively inhibit the proliferation of lung cancer cells as shown under the light microscope and found that 12 kinds of them had the effect of inhibiting the tumor growth (there were obvious morphological changes of the cell, such as cell shrinkage, disruption, flotation, etc), which include curcumine, silymarin, emodin, tea polyphenols, pine polyphenols, soybean isoflavone, pueraria isoflavone, genistein, ginger extract, podophyllotoxin, forsythia extract and cyathea spinulosa wall extract. Based on these results, we choose 4 kinds of drugs, including curcumine, emodin, silymarin and soybean isoflavone, with high purity (>80%) and significant inhibitory effect on the growth of tumor cells to further confirm their inhibitory effects on the lung cancer cell lines through the MTT test, our results showed that silymarin had strong inhibitory effect on all tested cell lines; emodin had inhibited the growth of Anip973 and A549, and it had only weak effects on NCI-H292 and PLA-801; curcumine had effects on A549 and NCI-H292, and it only had weak effects on Anip973 and PLA-801; soybean isoflavone affected on Anip973 and PLA-801, and it had only weak effects on A549 and NCI-H292. These results suggested that the drugs had different effects on various cell lines and the mechanism leading to this phenomenon needed to be further studied.2. The study of the mechanism of cell apoptosis of the lung cancer cell line with high-metastatic phenotype (Anip973) induced by silymarin: In this experiment, we took lung cancer cell line Anip973 to study the apoptosis of the lung cancer cells at different time points treated with silymarin of different concentrations, and we found that silymarin could induce apoptosis of Anip973 cells through the morphological observations and flow cytometer analysis the apoptosis is time-dependent and concentration-dependent. The caspase family and Bcl-2 family play important roles in the process of apoptosis. Moreover, the results of Western blot indicated that, when Anip973 cells with high-metastatic phenotype were treated by silymarin, the expression of the anti-apoptosis protein Bcl-2 decreased significantly, in the meanwhile the expression of the apoptosis protein Bax increases significantly, which suggested that the promotion of apoptosis induced by silymarin was relevant to the decreased ratio of Bcl-2/Bax.3. Silymarin reversed the drug resistance of A549/DDP cells to cisplatin through the MAPK signal pathway: We found that silymarin could reverse the drug resistance of A549/DDP cells through the MTT test and the effect was time-dependent. The analysis results of flow cytometer indicated that the combination of silymarin and cisplatin could induce the apoptosis or death of A549/DDP cells and the effect was time-dependent. To further study the mechanism of the apoptosis of A549/DDP induced by silymarin, we detected the changes of the expression levels and the degrees of activation of MAPK family members including JNK, ERK and p38 through the Western blot with the application of specific antibodies of the phosphorylated and non-phosphorylated forms of those above mentioned kinases. There was no significant change on the expression levels of these kinases when the cells were treated by silymarin of different concentrations for 24h, however, their phosphorylation degrees were significantly changed compared with the negative control group treated by DMSO. The phosphorylation of ERK was significantly inhibited, but the phosphorylation levels of JNK and p38MAPK increased.4. The effects of silymarin on the tumorigenicity in nude mice, proliferation and apoptosis of the lung cancer cell line Anip973 cells and angiogene: In this study, the Anip973 cells were subcutaneously inoculated on the back of the nude mice to establish the subcutaneous xenotransplanted tumor Model of human lung cancer in nude mice. The results indicated that, in the established models treated by intraperitoneal injection of silymarin, the tumor formation was delayed, the tumor growth was slower and the volume and weight of tumors were significantly decreased compared with the nude mice without treatment. The HE staining of the tumor specimens of each group indicated that the heteromorphism of the cancer cells were obvious, the sizes and shapes of cells are different and the cancer cells grow densely. Cell apoptosis was scarcely observed in the untreated tumor specimens, however, cell apoptosis increased in the tumor specimens treated by silymarin, and the apoptosis cells presented cell rounding, pyknosis, cell membrane blebbing, chromatin condensation and deep staining of nucleolus.In conclusion, it is suggested that silymarin had the function of inhibiting the proliferation of lung cancer cells and promoting apoptosis by regulating the caspase family and Bcl-2 family. Meanwhile silymarin could reverse the drug resistance of lung cancer cell line A549/DDP cells by regulating MAPK signal pathway and increase the sensitivity of the cells to the chemotherapy with cisplatin. This study will give new light for the development of new drugs for the treatment of lung cancer and provide experimental bases for the further application of traditional Chinese medicines in the treatment of tumors.
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