Curcumin Suppresses Proliferation and Migration of MDA-MB-231 Breast Cancer Cells through Autophagy-Dependent Akt Degradation

Abstract

Previous studies have evidenced that the anticancer potential of curcumin (diferuloylmethane), a main yellow bioactive compound from plant turmeric was mediated by interfering with PI3K/Akt signaling. However, the underlying molecular mechanism is still poorly understood. This study experimentally revealed that curcumin treatment reduced Akt protein expression in a dose- and time-dependent manner in MDA-MB-231 breast cancer cells, along with an activation of autophagy and suppression of ubiquitin-proteasome system (UPS) function. The curcumin-reduced Akt expression, cell proliferation, and migration were prevented by genetic and pharmacological inhibition of autophagy but not by UPS inhibition. Additionally, inactivation of AMPK by its specific inhibitor compound C or by target shRNA-mediated silencing attenuated curcumin-activated autophagy. Thus, these results indicate that curcumin-stimulated AMPK activity induces activation of the autophagy-lysosomal protein degradation pathway leading to Akt degradation and the subsequent suppression of proliferation and migration in breast cancer cell.

Fig 1. Effect of curcumin (Cur) on proliferation and migration of MDA-MB-231 cells.

(A and B) Proliferation determined by MTT assay. (C and D) Cell apoptosis determined by TUNEL assay. (E) A representative western blot image of cleaved caspase-3. (F and G) Quantitative analysis of wound closure. (H and I) Mean values for the number of migrated cells counted in the closed areas. Data are means ± SEM. (n = 3).*p<0.05, ** p<0.01 vs DMSO control.

Fig 2. Effect of curcumin on Akt expression in MDA-MB-231 cells.

(A and B) Dose- and time-responses of curcumin (Cur) on Akt protein levels. Left panels are representative western blot bands of Akt. Right panels are quantitative analysis of Akt levels. (C) Stable mRNA expression of three AKT isoforms in curcumin-treated cells. (D) Cycloheximide (CHX) chase assay. (E) Akt protein level in (D) was analyzed quantitatively in a line graph. Data are means ± SEM. (n = 3).* p<0.05, ** p<0.01 vs DMSO control.

Fig 3. Effect of curcumin on autophagy in MDA-MB-231 cells.

(A and B) Protein expressions of autophagy marker p62 and LC3. Left panel are the representative images of western blot. Right panels are LC3-II/LC3-I ratio analyzed quantitatively in a bar graph. (C) GFP-LC3 puncta formation. Left panel is representative images of GFP-LC3 puncta. Right panel is a quantitative analysis of dot number in cells. (D) Autophagic flux. Left panel is a representative western blot band of LC3. Right panel is a quantitative analysis of autophagy flux. Autophagic flux was determined by calculating the difference in LC3-II turnover between the experimental groups. Data are means ± SEM. (n = 4).* p<0.05, ** p<0.01 vs DMSO control.

Fig 4. Effect of curcumin on the ubiquitin proteasome system.

(A) GFPu expression. (B) GFPu/RFP ratio in (A) was analyzed quantitatively in a bar graph. (C) Proteasomal chymotrypsin-like peptidase activity. (D) Total protein ubiquitination. Data are means ± SEM. (n = 4). * p<0.05 vs DMSO control.

Fig 5. Effect of autophagy inhibition and UPS inhibition on curcumin-reduced Akt expression in MDA-MB-231 cells.

(A) A representative western blot band of Akt in cells treated with proteasome inhibitor MG132 and autophagy inhibitor chloroquine (CQ). (B) Quantitative analysis of Akt levels in (A). (C) A representative western blot band of Akt in Atg5/7 knockdown cells. (D) Quantitative analysis of Akt levels in (C). Data are means ± SEM. (n = 4). ** p<0.01 vs DMSO control. ## p<0.01 vs curcumin-treated group.

Fig 6. Effect of autophagy inhibition on proliferation and migration of curcumin-treated MDA-MB-231 cells.

(A) Proliferation determined by MTT assay. (B) Quantitative analysis of Tunel positive cells. (C) A representative western blot image of cleaved caspase-3. (D) Quantitative analysis of wound closure. (E) Mean values for the number of migrated cells. Data are means ± SEM. (n = 3).*p<0.05 vs indicated group.

Fig 7. Curcumin regulated Akt ubiquitination and aggregation.

(A) Akt ubiquitination. (B) Akt expression in insoluble fractionation of curcumin-treated cell. (C) Akt expression in insoluble fractionation of Atg5 knockdown cell. Data are means ± SEM. (n = 3).*p<0.05 vs control group. § p<0.05 vs 3 h-treated group. # p<0.05 vs curcumin-treated group.

Fig 8. AMPK mediated curcumin action on autophagy in MDA-MB-231 cells.

(A) A representative western blot band of autophagosome markers in cells treated with AMPK specific inhibitor compound C (Comp-C). (B) Quantitative analysis of LC3-II/LC3-I ratio in (A). (C) A representative western blot band of autophagosome markers in AMPK1α knockdown cells. (D) Quantitative analysis of LC3-II/LC3-I ratio in (C). Data are means ± SEM. (n = 4). * p<0.05 vs DMSO control; # p<0.05 vs curcumin-treated group.

Fig 9. A schematic diagram of curcumin-induced Akt degradation.

Autophagy is upregulated in curcumin-treated MDA-MB-231 breast cancer cells via an AMPK-mediated mechanism. The precise mechanism that regulates Akt ubiquitination and aggregation remains unclear; however, the presence of activated autophagy will result in degradation of the aggregated Akt leading to a reduction in Akt levels.