Abstract
Autophagy is a potential target for the treatment of triple negative breast cancer (TNBC). Because of a lack of targeted therapies for TNBC, it is vital to find optimal agents that avoid chemoresistance and metastasis. Flavopereirine has anti-proliferation ability in cancer cells, but whether it regulates autophagy in breast cancer cells remains unclear. A Premo™ Tandem Autophagy Sensor Kit was used to image the stage at which flavopereirine affects autophagy by confocal microscopy. A plasmid that constitutively expresses p-AKT and siRNA targeting p38 mitogen-activated protein kinase (MAPK) was used to confirm the related signaling pathways by Western blot. We found that flavopereirine induced microtubule-associated protein 1 light chain 3 (LC3)-II accumulation in a dose- and time-dependent manner in MDA-MB-231 cells. Confocal florescent images showed that flavopereirine blocked autophagosome fusion with lysosomes. Western blotting showed that flavopereirine directly suppressed p-AKT levels and mammalian target of rapamycin (mTOR) translation. Recovery of AKT phosphorylation decreased the level of p-p38 MAPK and LC3-II, but not mTOR. Moreover, flavopereirine-induced LC3-II accumulation was partially reduced in MDA-MB-231 cells that were transfected with p38 MAPK siRNA. Overall, flavopereirine blocked autophagy via LC3-II accumulation in autophagosomes, which was mediated by the AKT/p38 MAPK signaling pathway.
Highlights
Autophagy is a cellular self-digestion pathway that removes unnecessary or dysfunctional cellular components via sequestration in double membrane vesicles, which subsequently fuse with lysosomes for acidic degradation
Autophagic flux is often examined through the turnover of the autophagy marker light chain 3 (LC3) (LC3-II) by Western blotting
We tested the significance of flavopereirine in autophagy in MDA-MB-231 cells and found that flavopereirine induced the accumulation of LC3-II in a dose- and time-dependent manner (Figure 1)
Summary
Autophagy is a cellular self-digestion pathway that removes unnecessary or dysfunctional cellular components via sequestration in double membrane vesicles, which subsequently fuse with lysosomes for acidic degradation. Autophagy plays a critical role in the survival of breast cancer cells and is considered a novel therapeutic target [2,3,4]. Inducing autophagy was demonstrated to be a potential therapeutic strategy for TNBC treatment [9]. It is still unclear whether autophagy in TNBC has a protective or cytotoxic role [4]. We demonstrated that flavopereirine induced TNBC (MDA-MB-231) cell cycle arrest and apoptosis through the AKT/p38 mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK)1/2 signaling pathway [13]. Because autophagy has become a potential therapeutic target for breast cancer, we further investigated whether flavopereirine has a role in autophagy regulation. We used flavopereirine-treated MDA-MB-231 cells to examine the mechanism by which flavopereirine is involved in autophagy
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