| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 101 === Autophagy is a homeostatic cellular recycling mechanism that mediates removal of old or dysfunctional proteins and organelles. Cells could activate cytoprotective autophagy when in response to metabolic stress for cell survival, such as hypoxia, starvation, chemotherapy. There exists a high degree of correlation between cellular tumorigenicity and metastatic potential in the nude mouse and anchorage-independent growth in vitro. It has been reported that cells with the anchorage-independent growth ability significantly increase autophagy level. According to previous studies, we assume that tumor cells could activate autophagy for adapting to the unfavorable metabolic environment in the condition of anchorage-independent growth of tumorspheres. Therefore, we use the anchorage-independent growth of cance cells as a platform for anticancer drug screening.
First, we compared the autophagy expression of the anchorage-independent growth for tumorspheres culture and the conventional monolayer culture. We found that Beclin 1 mRNA expression is upregulated in 7-day CT26 tumorspheses compared with the conventional monolayer culture, suggesting that autophagy would be activated in the condition of the anchorage-independent growth. We treat mouse colon carcinoma cell line CT26 and mouse Lewis lung carcinoma LL/2 with autophagy inhibitor monensin. Monensin (0.1 μg/ml) inhibits anchorage-independent growth of CT26 cells and LL/2 cells in a dose-dependent manner. Autophagy inhibitor monensin (1 μg/ml) in combination with 5-FU (0.5 uM) could significantly suppress the anchorage-independent growth of CT26 cells, indicating that autophagy may serve as a survival mechanism against 5-FU. We used soft agar colony formation assay as platform for screening anticancer herbs. We found that herbal extracts AgPi, CoTe, LoCa, PhWi, PtMu, SeDo, SoNi at low dose (20 μg/ml) or medium dose (100 μg/ml) could inhibit the anchorage independent growth of LL/2 cells while herbal extracts AgPi, CiLa, CoTe, CuCh, HeDi, HoCo, LoCa, PrVu, PtMu, RhPa, SaOf, SoLy and SoNi could inhibit the anchorage independent growth of CT26 cells. To develop potential autophagy inhibitors from herbal extracts, we used the anticancer drug 5-FU-induced autophagy model to evaluate whether herbal extracts could inhibit 5-FU-induced autophagy. We found that herbal extract CoTe (20 μg/ml), HoCo (100 μg/ml), SoLy (100 μg/ml), SaOf (100 μg/ml) and PhWi (20 μg/ml) could inhibit 5-FU-induced autophagy. We also found that berberine could decrease the LC3 conversion (LC3-I conversion to LC3-II) and the accumulation of autophagic substrate p62 protein, indicating berberine may possess autophagy inhibition activity. herbal extract CoTe (20 μg/ml), HoCo (50 μg/ml), SoLy (50 μg/ml) in combination with 5-FU could significantly potentiate the anticancer effect of 5-FU on anchorage-independent growth of CT26 cells.
Our data show that autophagy may serve as a cytoprotective mechanism for cell survival in the condition of anchorage-independent growth. We could use autophagy inhibitor to overcome cancer relapse and metastasis or use herbal extracts in in combination with anticancer drugs to enhance the anticancer activity of the anticancer drugs and decrease the possibility of cancer recurrence.
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