The omega-3 polyunsaturated fatty acid DHA induces simultaneous apoptosis and autophagy via mitochondrial ROS-mediated Akt-mTOR signaling in prostate cancer cells expressing mutant p53

Abstract

Docosahexaenoic acid (DHA) induces autophagy-associated apoptotic cell death in wild-type p53 cancer cells via regulation of p53. The present study investigated the effects of DHA on PC3 and DU145 prostate cancer cell lines harboring mutant p53. Results show that, in addition to apoptosis, DHA increased the expression levels of lipidated form LC3B and potently stimulated the autophagic flux, suggesting that DHA induces both autophagy and apoptosis in cancer cells expressing mutant p53. DHA led to the generation of mitochondrial reactive oxygen species (ROS), as shown by the mitochondrial ROS-specific probe mitoSOX. Similarly, pretreatment with the antioxidant N-acetyl-cysteine (NAC) markedly inhibited both the autophagy and the apoptosis triggered by DHA, indicating that mitochondrial ROS mediate the cytotoxicity of DHA in mutant p53 cells. Further, DHA reduced the levels of phospho-Akt and phospho-mTOR in a concentration-dependent manner, while NAC almost completely blocked that effect. Collectively, these findings present a novel mechanism of ROS-regulated apoptosis and autophagy that involves Akt-mTOR signaling in prostate cancer cells with mutant p53 exposed to DHA.

Figure 1

DHA induces cytotoxicity in PC3 and DU145 prostate cancer cells. (a) Reduced cell viability in response to DHA. PC3 and DU145 cells were exposed to 0−50 μM DHA for 24 h and cell viability was measured. (b) Representative images of PC3 (bottom) and DU145 (top) cells exposed to DHA for 24 h (scale bar, 200 μm). (c), (d) Different effects of ω3- and ω6-PUFAs on the viability of PC3 and DU145 cells. PC3 cells (c) and DU145 cells (d) were exposed to 50 μM DHA, EPA, and AA for 24 h and cell viability was measured by MTT. Data are displayed as means ± SD (n > 5; *P < 0.05).

Figure 2

DHA induces apoptosis and autophagic activation in PC3 and DU145 prostate cancer cells. (a) Cell cycle analysis of PC3 cells exposed to DHA. PC3 cells were incubated with 0−50 μM DHA for 24 h and then subjected to flow cytometry after staining with PI. (b) Increased number of TUNEL-positive cells in DHA-treated cells. PC3 (left) and DU145 (right) cells were incubated with 50 μM DHA for 24 h before staining with TUNEL reagents (scale bar, 200 μm). (c) Levels of cleaved PARP and expression levels of LC3-II in response to DHA. PC3 (left) and DU145 (right) cells were incubated with 0−50 μM DHA for 24 h, and PARP cleavage and the expression levels of LC3-II were monitored by Western blot analysis. (d) Representative images of PC3 (top) and DU145 (bottom) cells transiently expressing GFP-LC3B upon exposure to 50 μM DHA for 2 h (magnification, 600x). (e) Effect of DHA on autophagic flux. PC3 (left) and DU145 (right) cells were incubated with 50 μM DHA and/or 2 μM CQ for 24 h and were then subjected to Western blotting for the detection of LC3-II. Black triangle indicates the lipidated form LC3B (LC3-II), and asterisk indicates nonlipidated form of LC3B (LC3-I).

Figure 3

Mitochondrial ROS overproduction mediates DHA-induced apoptosis and autophagy. (a), (b) Increased levels of intracellular ROS induced by DHA in PC3 and DU145 cells. DHE-loaded PC3 and DU145 cells were examined for intracellular ROS accumulation by microscopy (a) and flow cytometry (b) after incubating with 50 μM DHA in the absence or presence of 5 mM NAC for 2 h (scale bar, 200 μm). (c), (d) Effect of the ROS scavenger NAC on the reduction in cell viability induced by DHA. PC3 (c) and DU145 (d) cells were exposed to 50 μM DHA and/or 5 mM NAC for 12 h and cell viability was measured by MTT. Data are displayed as means ± SD. (n > 5; *P < 0.05). (e) Effect of NAC on the levels of cleaved PARP and on the expression levels of LC3-II in DHA-treated cells. PC3 (left) and DU145 (right) cells were exposed to 50 μM DHA and/or 5 mM NAC for 12 h, and PARP cleavage and the expression levels of LC3-II were examined by Western blot analysis. Black triangle indicates the lipidated form LC3B (LC3-II), and asterisk indicates nonlipidated form of LC3B (LC3-I). (f) Increased levels of mitochondrial ROS induced by DHA. MitoSOX-loaded PC3 (top) and DU145 (bottom) cells were examined for mitochondrial ROS levels by flow cytometry after incubating with 50 μM DHA in the absence or presence of 5 mM NAC for 2 h.

Figure 4

Inhibition of Akt-mTOR signaling contributes to the ROS-mediated apoptosis and autophagy. (a) Inhibition of Akt-mTOR signaling induced by DHA. PC3 (left) and DU145 (right) cells were incubated with 0−50 μM DHA for 24 h, and then the expression levels of Akt-mTOR pathway molecules were detected by Western blot analysis. (b) Effect of NAC on the expression levels of Akt-mTOR pathway molecules in DHA-treated cells. PC3 (left) and DU145 (right) cells were exposed to 50 μM DHA and/or 5 mM NAC for 12 h, and the expression levels of Akt-mTOR pathway molecules were examined by Western blot analysis.

Figure 5

Proposed mechanism of DHA-induced apoptosis and autophagy in prostate cancer cells with altered p53 status.