魚藤酮引起MCF-7乳癌細胞凋亡及兒茶素抑制CL1-5肺癌細胞轉移之機制探討

Abstract

Cancer is still a serious problem in the world. Therefore, it is important to find ways to prevent or cure cancer. Because cancer cells possess abilities to prolong cell growth and invade to other tissues, it is still thorny to control cancer disease. Therefore, how to suppress cell growth in cancer cells and prevent cancer cells invade to other tissues is of great urgency. Phytochemicals are a group of compounds from natural plants. Recent reports show that they are bioactive components in natural plant to execute chemoprevention or other beneficial effects such as prevent diabetes, protect neuron cells and prevent obesity. Rotenone is a phytochemical from the roots of the Derris and Lonchorcarpus species. Rotenone is an inhibitor of electron transfer chain complex Ι (NADH dehydrogenase complex) and it can promote production of reactive oxygen species (ROS). It has been used as a botanical insecticide for at least 150 years to control crop pests and used even longer as a fish poison by native tribes in South America and East Africa. Recent reports show that rotenone can induce apoptosis in human melanoma cells and leukemia cells. Because anti-apoptosis plays an important role in anti-cancer, we want to determine whether rotenone can induce apoptosis in MCF-7 breast cancer cells which threaten women worldwide. In this study, we found that rotenone can induce apoptosis in MCF-7 human breast cancer cells by using MTT assay, trypan blue exclusion assay, alamar blue assay, flow cytometry and Hoechst 33258 staining and they are dose- and time-dependent effects. To further confirm these results, we used western blot analysis to investigate apoptosis-related proteins, PARP, Bcl-2 and Bax. We showed that rotenone can promote PARP cleavage, downregulate anti-apoptotic Bcl-2 and upregulate apoptotic Bax. In addition, rotenone can reduce mitochondria membrane potential. Because rotenone is attributed to irreversible binding and inactivation of mitochondrial electron transport chain complex Ι, we investigated whether rotenone indeed promote production of ROS in MCF-7 cells. Rotenone can result in an increase of the reactive oxygen species (ROS). We treated MCF-7 cells with rotenone and indeed found that ROS was increased. Then, we want to determine the signaling pathways under treatment of EGCG in MCF-7 cells. Using western blotting analysis, we found that rotenone induced JNK and p38 activation, whereas it attenuated ERK1/2 activation. Then, apoptosis was induced. In addition, metastasis is a fundamental property of high malignant cancer cells with poor clinical outcome. Therefore, to investigate how to suppress metastasis is an important urgency. Cleavage of extracellular matrix to allow cells invade into blood and transfer to other tissues is an important step in metastasis and MMPs play important roles in this step. Because lung cancer has the highest rate of cancer mortality worldwide and the highest rate of lung cancer mortality is due to their high potential of metastasis, we used highly invasive CL1-5 human lung cancer cells to investigate possible mechanisms of suppression tumor invasion in cancer cells. Green tea contains anti-cancer effects, so we want to determine whether EGCG, the major bioactive compound in green tea, can attenuate metastasis in highly invasive CL1-5 human lung cancer cells. First, we found that EGCG suppress cell growth at concentration higher than 20 μM by using MTT assay. Further, we want to investigate whether EGCG induce apoptosis or cell cycle arrest at concentrations higher than 20 μM. We used flow cytometry analysis and found that EGCG induced G2/M arrest in CL1-5 cells at concentrations of 30, 40 and 50 μM. Next, we want to investigate whether EGCG can attenuate invasion ability of CL1-5 cells. Using transwell invasion assay, EGCG could suppress invasion of CL1-5 cells. Because MMP-2 plays important roles to degrade extracellular matrix and is closely related to invasion in patients of lung cancer, we want to investigate whether EGCG can affect expression of MMP-2 in CL1-5 cells. Using gelatin zymography, western blot analysis and PCR, we found that EGCG can repress MMP-2 expression at the transcriptional level. Furthermore, by western blot analysis we showed that EGCG could suppress activation of JNK and attenuate translocation of MMP-2 transcription factor, NF-κB and Sp1, from the cytosol into the nucleus. In addition, docetaxel, a clinical drug used in the treatment of lung cancer, performs serious side effect, we want to clarify synergistic effects of EGCG and docetaxel. Combine EGCG and docetaxel can repress MMP-2 expression at low dosages of docetaxel. Taken together, rotenone and EGCG may be potential phytochemicals to chemoprevent breast cancer and lung cancer respectively and they may play synergistic effects with clinical drug.