| Summary: | 碩士 === 國立臺灣師範大學 === 生命科學研究所 === 102 === 一.Previously, we have screened more than five hundred compounds with small molecular weight through MTT assay. The findings have identified that lower concentrations of a small molecular compound A containing functional groups indole and quinoline was effective to inhibit the growth rate of wild-type p53 NSCLC cells and suppress tumor growth, but the drug is less sensitive to p53-null H1299 cells. All western blot, MTT, and flow cytometry experiments showed that the induced apoptosis in NSCLC cells involved wild-type tumor suppressor genes p53. The cells without p53 were less sensitive to compound A.The experiments keep on exploring the mechanism on how compound A regulates the growth and association with p53 status in cells. We have further tested the effect of the compound in p53-null H1299 cells with stable expression of mutated (H1299/p53R267P) and wild-type p53 (H1299/p53), respectively. Through MTT assay, after treated 10 µM compound A 48 hours, the data was shown more drug effective in H1299/p53 cells than H1299/p53R267P cells. The results proved that the efficacy is related to p53 genotype. Using flow cytometry and western blot experiments, we found that compound A can reduce proliferation rate through apoptotic cell death in the H1299/p53 cells, and the apoptosis percent of H1299/p53 cells is 39.2％ higher than H1299/p53R267P cells. We have also shown that the p53-dependent apoptotic cell death pathway began with activating p53, attenuating Bcl-2, activating caspase family proteins, and finally cleaving poly (ADP-ribose) polymerase. These results demonstrate that the cells with wild-type p53 were more sensitive to compound A. In the future, we will continue to explore the mechanisms on how compound A inhibits the growth and metastasis in lung cancer stem-like models.
二.In recent years, the Chinese herb medicine therapy is taken seriously because this therapy combine with Radiotherapy can reduce side effect and improve constitution in cancer patients. The experiment based on the lung cancer stem-like cells model, which may be higher metastasis and resistance. To evaluate Chinese herb medicine Sophora flavescens , Brucea javanica , Prunella vulgaris and triep teroxirone can potentially eradicate lung cancer stem-like cells, we used human lung cancer stem-like cells as established from the parental cells. First, we used fluorescence microscopy to confirm that H460, A549 and H12PP/p53 stem-like cells changed in size and in numbers after the drugs treated. Finally, we used western blot experiment to confirm that teroxirone reduced the H460 stem-like cells numbers resulted from apoptotic cell death, and the apoptosis pathway is associated with p53and apoptosis proteins. Previously, we showed that the Chinese herb medicine Prunella vulgaris inhibited the metastasis capacity in liver cancer cells. Besides, Sophora flavescens and Brucea javanica inhibited A549 tumors growth.We also showed that teroxirone inhibited NSCLC cell growth that was caused by apoptotic cell death. In this work, we continued to use stem-like models to evaluate whether these drugs inhibit cells growth and metastatic property of the stem-like cells.
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