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

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

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

<p>(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). * <i>p</i><0.05 vs DMSO control; # <i>p</i><0.05 vs curcumin-treated group.</p

A schematic diagram of curcumin-induced Akt degradation.

<p>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.</p

Effect of curcumin on the ubiquitin proteasome system.

<p>(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). * <i>p</i><0.05 vs DMSO control.</p

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

<p>(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). ** <i>p</i><0.01 vs DMSO control. ## <i>p</i><0.01 vs curcumin-treated group.</p

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

<p>(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).*<i>p</i><0.05 vs indicated group.</p

Curcumin regulated Akt ubiquitination and aggregation.

<p>(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).*<i>p</i><0.05 vs control group. § <i>p</i><0.05 vs 3 h-treated group. # <i>p</i><0.05 vs curcumin-treated group.</p