Anthricin Isolated from Anthriscus sylvestris (L.) Hoffm. Inhibits the Growth of Breast Cancer Cells by Inhibiting Akt/mTOR Signaling, and Its Apoptotic Effects Are Enhanced by Autophagy Inhibition

Figure 2

Anthricin induces autophagy. (a) p62 levels in anthricin-treated breast cancer cells. Cells were treated with varying concentrations of anthricin for 8 h. Protein levels were analyzed by immunoblotting. (b) Immunoblot analysis of the LC3 protein. The levels of endogenous LC3-II increased in breast cancer cells treated with anthricin. The cells were cotreated with anthricin and chloroquine (CQ) for 8 h. (c) Immunostaining of LC3 in MDA-MB-231 cells. The cells were treated with CQ for 8 h to induce autophagosome accumulation. After fixation and permeabilization, autophagosomes were stained with an anti-LC3B rabbit polyclonal antibody and visualized with Alexa Fluor 488 goat anti-rabbit IgG. (d) MCF-7 cells were transduced with GFP-LC3. Two days later, cells were cotreated with anthricin and CQ for 8 h. (e) Quantitative analysis of the formation of GFP-LC3 puncta.