Methanolic Extracts of Bitter Melon Inhibit Colon Cancer Stem Cells by Affecting Energy Homeostasis and Autophagy
Figure 4
Bitter melon extracts induce autophagy in SW480 and HT-29 cells. (a) Electron micrograph of bitter melon treated SW480 and HT-29 cells. Investigation of ultrastructural morphology and autophagic vacuoles 12 h after treatment with 100 μg/mL of bitter melon whole fruit extract. Rapamycin 25 μM treatment for 12 hours was used as positive control. Insets in the TEM images show magnified autophagic vacuoles. Both SW480 and HT-29 show presence of autophagic vacuoles following treatments with BMW and rapamycin. (b) Effect of bitter melon extracts on LC3B cleavage. Immunocytochemistry analysis of SW480 and HT-29 cells treated with 100 μg/mL of bitter melon whole fruit extract for 24 h shows enhanced accumulation of LC3B in cytoplasm. Immunostaining was performed using rabbit anti-LC3B antibody followed by Cyanine Dye (Cy3) labeled anti-rabbit IgG. This method only picks up cleaved LC3B. (c) Western blot analysis of SW480 and HT-29 cells treated with increasing concentration of bitter melon whole fruit extract showed increased levels of cleaved LC3B. (d) Monodansylcadaverine accumulation measured by fluorometric analysis showed increased accumulation of the dye within the cells treated with increasing concentration of bitter melon whole fruit extract for 48 h. For a positive control, the cells were serum starved for the same amount of time. (e) Western blot analysis for autophagy markers Bcl-2, Beclin-1, Atg 7 and 12. Both the cells lines treated with increasing concentration of bitter melon whole fruit extract showed decreased expression of Bcl-2 and increased expression of all the other markers in a concentration dependent manner.
