Tannic Acid Induces Endoplasmic Reticulum Stress-Mediated Apoptosis in Prostate Cancer

Abstract

Endoplasmic reticulum (ER) stress is an intriguing target with significant clinical importance in chemotherapy. Interference with ER functions can lead to the accumulation of unfolded proteins, as detected by transmembrane sensors that instigate the unfolded protein response (UPR). Therefore, controlling induced UPR via ER stress with natural compounds could be a novel therapeutic strategy for the management of prostate cancer. Tannic acid (a naturally occurring polyphenol) was used to examine the ER stress mediated UPR pathway in prostate cancer cells. Tannic acid treatment inhibited the growth, clonogenic, invasive, and migratory potential of prostate cancer cells. Tannic acid demonstrated activation of ER stress response (Protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme 1 (IRE1)) and altered its regulatory proteins (ATF4, Bip, and PDI) expression. Tannic acid treatment affirmed upregulation of apoptosis-associated markers (Bak, Bim, cleaved caspase 3, and cleaved PARP), while downregulation of pro-survival proteins (Bcl-2 and Bcl-xL). Tannic acid exhibited elevated G₁ population, due to increase in p18^INK4C and p21^WAF1/CIP1 expression, while cyclin D1 expression was inhibited. Reduction of MMP2 and MMP9, and reinstated E-cadherin signifies the anti-metastatic potential of this compound. Altogether, these results demonstrate that tannic acid can promote apoptosis via the ER stress mediated UPR pathway, indicating a potential candidate for cancer treatment.

Keywords: ER stress; apoptosis; molecularly targeted therapeutics; tannic acid; unfolded protein response.

Figure 1

Tannic acid inhibited the growth of prostate cancer cells. (A) Effect of Tannic acid (TA) on cell proliferation of human prostatic epithelial cells (PWR1E) and prostate cancer (C4-2, DU 145 and PC-3) cells. Cells (5000) were seeded in each well of 96-well plate and allowed to grow overnight, the cells were then treated with the described concentrations for 48 and 72 h. The line graphs represent the percent proliferation compared with the vehicle-treated group cells. Data indicated TA is not toxic to PWR1E cells; (B) Effect of TA on clonogenic potential of prostate cancer cells. Representative colony images of control and TA treated C4-2, DU 145, and PC-3 cells; and, (C) Bar graphs indicating quantification of colony formation in C4-2, DU 145 and PC-3. Data represent the mean of triplicates ± SEM, ** p < 0.01, and *** p < 0.001.

Figure 2

Tannic acid induced ER stress in prostate cancer cells. (A) Western blot analysis of Protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring enzyme 1 alpha (IRE1α), and activating transcription factor 4 (ATF4) signaling in prostate cancer cells after dose-dependent treatment with TA. Briefly, cells were treated with indicated concentrations of TA, protein extracts were prepared and subjected for western blot analysis to detect the protein levels. β-Actin antibody served as an internal control; (B) Gene expression studies endoplasmic reticulum (ER) stress markers in TA treated cells for PERK, eukaryotic translation initiation factor 1 (EIF2S1), binding immunoglobulin protein (BiP), transcription factor C/EBP, homologous protein (CHOP), and ATF4 determined by qRT-PCR analysis. GAPDH was used as an internal control. Data represent the mean of triplicates ± SEM, ** p < 0.01; (C) Cells were grown and exposed to TA and Thapsigargin. Western blot analysis of regulatory protein expression of ER stress in TA and thapsigargin (TG) treated C4-2 and PC-3 cells.

Figure 3

Tannic acid induces G₁ phase arrest and apoptosis in prostate cancer cells. Prostate cancer cells were treated with TA for 24 h and cell cycle analysis was performed by flow cytometer. (A) Histogram plot of C4-2, DU 145 and PC-3 cells after treatment of 5, 10, and 20 µM TA. Untreated cells were used as control; (B) Directive role of TA on cell cycle regulatory proteins and its effect on G₁ phase arrest. Western blot analysis of G₁ phase cell cycle regulatory proteins in prostate cancer cells treated with TA. Tannic acid treated cell lysates were prepared and subjected for Western blot analysis. β-actin was probed for equal protein loading in each lane; and, (C) Cells treated with TA 10 and 20 µM for 24 h, cell lysates were collected and immunoblotted for apoptotic protein expressions.

Figure 4

Tannic acid inhibited migratory and invasive attributes of prostate cancer cells. (A) Wound healing assay. In cell migration assay, a uniform scratch was made in 80% confluent monolayer cultures of prostate cancer cells and the extent of closure was monitored in presence of TA (10 and 20 µM) under phase-contrast microscopy and imaged at 0 and 48 h at 20× magnification; (B) Percent wound closure with TA treatment in C4-2, DU 145 and PC-3 cells; (C) Boyden chamber assays of TA treated prostate cancer cells. Cells were imaged under phase-contrast microscopy at 20× magnification; (D) Bar graph displaying relative cell number of migrated cells (per unit area) in TA-treated groups; (E) Matrigel Invasion assays of TA treated prostate cancer cells; and, (F) Bar graph displaying relative cell number of cells invaded (per unit area) in TA-treated groups. Data represent the mean of triplicates ± SEM, ** p < 0.01 and *** p < 0.001.

Figure 5

Tannic acid altered the EMT regulatory protein markers in prostate cancer cells. (A) Western analysis of Epithelial to Mesenchymal Transition (EMT) markers in TA treated cells. Values shown above the blots are the densitometry analysis of each protein band normalized with respective β-actin value; (B) Gene expression studies of EMT markers, such as E-Cadherin, MMP2 and MMP9 in TA-treated cells. Data represent the mean of triplicates ± SEM, ** p < 0.01.

Figure 6

Molecular mechanism of TA in promoting unfolded protein response mediated ER stress and induced apoptosis in prostate cancer cells. (A) Heat map of differentially regulated genes in the ER Stress/EMT/Cell cycle regulatory/apoptosis signaling proteins in C4-2 and PC-3 prostate cancer cells; (B) Schematic illustration of TA-induced ER stress mediated apoptosis. .