Targeting multiple pro-apoptotic signaling pathways with curcumin in prostate cancer cells

Abstract

Curcumin, an extract from the turmeric rhizome (Curcuma longa), is known to exhibit anti-inflammatory, antioxidant, chemopreventive and antitumoral activities against aggressive and recurrent cancers. Accumulative data indicate that curcumin may induce cancer cell death. However, the detailed mechanism underlying its pro-apoptotic and anti-cancer effects remains to be elucidated. In the present study, we examined the signaling pathways triggered by curcumin, specifically, the exact molecular mechanisms of curcumin-induced apoptosis in highly metastatic human prostate cancer cells. The effect of curcumin was evaluated using for the first time in prostate cancer, a gel-free shotgun quantitative proteomic analysis coupled with Tandem Mass Tag isobaric labeling-based-signaling networks. Results were confirmed at the gene expression level by qRT-PCR and at the protein expression level by western blot and flow cytometry. Our findings revealed that curcumin induced an Endoplasmic Reticulum stress-mediated apoptosis in PC3. The mechanisms by which curcumin promoted cell death in these cells were associated with cell cycle arrest, increased reactive oxygen species, autophagy and the Unfolded Protein Response. Furthermore, the upregulation of ER stress was measured using key indicators of ER stress: Glucose-Regulated Protein 78, Inositol-Requiring Enzyme 1 alpha, Protein Disulfide isomerase and Calreticulin. Chronic ER stress induction was concomitant with the upregulation of pro-apoptotic markers (caspases 3,9,12) and Poly (ADP-ribose) polymerase. The downregulated proteins include anti-apoptotic and anti-tumor markers, supporting their curcumin-induced pro-apoptotic role in prostate cancer cells. Taken together, these data suggest that curcumin may serve as a promising anticancer agent by inducing a chronic ER stress mediated cell death and activation of cell cycle arrest, UPR, autophagy and oxidative stress responses.

Fig 1. Curcumin inhibits cell proliferation and promotes cell death.

(A) Optical micrograph of PC3 confluency after treatment with either Curcumin or DMSO. (B) Percentage of death cells stained with 7AAD, analyzed by flow cytometry and compared by unpaired t-test, p≤0.05. (C) Cell cycle analysis by flow cytometry; statistical analysis was determined by Two-way ANOVA, *p≤0.05, **p≤0.01.

Fig 2. Curcumin induces caspase-mediated apoptosis.

(A) Cleaved caspase 3 protein expression determined by flow cytometry. (B) Validation of cleaved caspase 3 protein expression by PathScan Sandwich ELISA. (C) Uncleaved caspase 3 gene expression analyzed by qRT-PCR. (D-F) Caspase 9, PARP and Caspase 12 protein expressions assessed by western blotting. Statistical significance between Curcumin and DMSO was determined by unpaired t-test, * p≤0.05, ** p≤0.01.

Fig 3. Curcumin triggers ER stress.

(A-C) Protein expression of ER stress markers: GRP78, IRE-1α and Calreticulin analyzed by western blotting. (D) Phosphorylated expression of eIF2α protein assessed by flow cytometry. Statistical significance between Curcumin and DMSO was determined by unpaired t-test, * p≤0.05, ** p≤0.01, ***p≤0.001.

Fig 4. Curcumin promotes oxidative stress and autophagy.

(A) Increased production of ROS was measured using the H₂DCFDA technique by flow cytometry. (B) Acridine orange fluorescence micrographs in curcumin and DMSO treated PC3 cells. (C) LC3B II protein expression analyzed by western blot. D. Total protein expression by india ink staining. Statistical significance between Curcumin and DMSO was determined by unpaired t-test, ** p≤0.01, **** p≤0.0001.

Fig 5. Proposed molecular mechanism of Curcumin’s cytotoxic effects in metastatic prostate cancer cells (PC3).

Curcumin causes a chronic ER and oxidative stress inducing autophagy, cell cycle arrest, ERAD, cytoskeletal changes and caspase mediated apoptosis. Upward (↑) and downward (↓) arrows indicate upregulation or downregulation of proteins respectively.