Flavopereirine Inhibits Autophagy via the AKT/p38 MAPK Signaling Pathway in MDA-MB-231 Cells

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.

Keywords: AKT; autophagy; breast cancer; flavopereirine; p38 MAPK.

Figure 1

Flavopereirine induced LC3-II accumulation in dose- and time-dependent manners. (A) MDA-MB-231 cells were treated with various concentrations of flavopereirine (5-15 μM) for 24 and 48 h. (B) MDA-MB-231 cells were incubated with 15 μM flavopereirine for 18, 24, 42, and 48 h. LC3-II accumulation and p62 expression were evaluated by Western blotting. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The data are representative of three independent experiments. The band signals were quantified and the ratio of LC3-II related to GAPDH is presented under the blots.

Figure 2

Flavopereirine inhibited autophagic flux in MDA-MB-231 cells. Representative fluorescent images of autophagosomes and autophagolysosomes in MDA-MB-231 cells were transduced with tandem RFP-GFP-LC3B and then independently treated with aloperine (100 μM), chloroquine (25 μM), flavopereirine, or a combination of the two drugs. In the merged images, autophagosomes are presented as yellow or orange puncta (RFP-GFP-LC3B), whereas red puncta (RFP-LC3B) indicate autophagolysosomes because acidification abolishes the green fluorescence. Images were obtained using a 63× oil immersion objective.

Figure 3

Flavopereirine inhibited autophagy through AKT/p38 MAPK signaling. (A) The expression level of p-AKT in MDA-MB-231 cells treated with increasing concentrations of flavopereirine was detected by Western blotting. (B, C) MDA-MB-231 cells transfected with plasmid constitutively expressing p-AKT (pAKT) or control plasmid (pBSSK) were treated with flavopereirine for 48 h and then analyzed for the expression of the indicated protein and their phosphorylation levels by Western blotting. (D) MDA-MB-231 cells transfected with p38 MAPK or negative control (NC) siRNAs were further treated with flavopereirine for 48 h. Western blotting was performed to detect p-p38 MAPK, p38 MAPK knockdown, and LC3 levels. The band signal was quantified and the ratio of LC3-II or p-p38 MAPK related to GAPDH is presented under the blots.

Figure 4

Cell viability of MDA-MB-231 cells treated with individual or a combination of flavopereirine with chloroquine. Cells (4 × 10⁵/well) were treated with different concentrations of flavopereirine, chloroquine (25 μM), or a combination of the two drugs, and cell viability was evaluated by a cell counting kit-8 (CCK-8) after treatment for 24 h (A) and 48 h (B). Histograms show the mean ± SD of three independent experiments performed in triplicate. * Indicates a significant difference from its respective untreated control as analyzed by one-way ANOVA followed by a Bonferroni test (p < 0.05).