In vitro Anticancer Effects of JI017 on Two Prostate Cancer Cell Lines Involve Endoplasmic Reticulum Stress Mediated by Elevated Levels of Reactive Oxygen Species

Abstract

Prostate cancer is the second most commonly diagnosed cancer, and prostate cancer is the second most common cause of cancer death in United States men after lung cancer. Many therapies are used to treat prostate cancer, and chemotherapy is one of the most relevant treatments. However, chemotherapy has many side effects, and repeated administration of chemotherapeutic agents leads to acquired resistance. Thus, new drugs with few side effects are needed. We investigated the molecular mechanism of action of JI017 in human prostate cancer cells. We identified an endoplasmic reticulum (ER) stress pathway that depended on the reactive oxygen species (ROS) pathway and played a crucial role in JI017-induced apoptosis. We measured cell viability by the MTS assay to determine the effect of JI017. Analysis of apoptosis, mitochondrial dysfunction, and cell cycle features was performed by flow cytometry. We used western blot and RT-PCR to measure the levels of the proteins of the unfolded protein response (UPR) pathway and apoptosis markers. Immunoprecipitation assay and transfection were used to determine the expression levels of proteins interacting with the pathways influenced by JI017 in prostate cancer cells. The anticancer effects induced by JI017 were evaluated. JI017 induced cell death that regulated apoptotic molecules and caused cell cycle arrest that inhibited the proliferation of cancer cells. Moreover, JI017 generated ROS. Accumulation of ROS caused ER stress through the PERK-eIF2α-CHOP and IRE1α-CHOP pathways. Furthermore, persistent activation of the UPR pathway induced by JI017 treatment triggered mitochondrial dysfunction, including dissipation of mitochondrial membrane potential, which activated intrinsic apoptotic pathway in human prostate cancer cells. The data indicated that N-acetyl-L-cysteine diminished apoptosis. We demonstrated that JI017 induced ER stress and cell death. Anticancer properties of JI017 in prostate cancer cells and in a human prostate cancer model involved ROS-mediated ER stress. Thus, JI017 treatment provides a new strategy for chemotherapy of prostate cancer.

Keywords: CHOP; JI017; ROS; cancer; er stress; mitochondria cell death.

FIGURE 1

HPLC profile of JI017. Identification of the components in JI017; nodakenin, aconitine, 6-gingerol, and decursin were detected at 17, 33, 36, and 46 min, respectively.

FIGURE 2

The anticancer effect of JI017 treatment induced the G2/M cell cycle arrest and apoptosis of DU145 and PC3 cells. DU145 and PC3 cells were treated with JI017 (40, 80, 120, and 200 μg/ml) for 24 h (A). Cell viability was measured by MTS assay. DU145 and PC3 cells were exposed to JI017 (40, 80, and 120 μg/ml) for 14 days (B). Cell cycle distribution was analyzed using flow cytometry (C). The expression of p21, p27, and cyclin B mRNA was measured by RT-PCR (D). The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to untreated cells.

FIGURE 3

DU145 and PC3 cells were treated by indicated drugs for 24 h and used in the Annexin V/7-AAD assay (A). Whole cell lysates were analyzed by western blotting with anti-PARP, anti-cleaved caspase-3 and -9, anti-Bax, anti-Bcl-2, and anti-GAPDH antibodies (B). Pretreatment with Z-VAD-FMK (20 μM) for 1 h followed by treatment with various concentrations of JI017. Cell viability was then measured by MTS assay (C). The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to untreated cells.

FIGURE 4

JI017 treatment induced ROS accumulation in prostate cancer cells. Intracellular ROS levels in DU145 and PC3 cells treated with JI017 (40, 80, 120, and 200 μg/ml) for 24 h were determined by flow cytometry. Cells were labeled with DCFH-DA (10 μM) for 0.5 h (A). Pretreatment with NAC (10 mM) for 1 h was followed by treatment with various concentrations of JI017. Cell viability was measured using the MTS assay (B). The levels of ROS were measured by flow cytometry (C). The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to untreated cells.

FIGURE 5

JI017 treatment activated UPR signaling in DU145 and PC3 cells. DU145 and PC3 cells were treated with JI017 (40, 80, and 120 μg/ml) for 24 h. Whole cell lysates were analyzed by western blotting with anti-GRP78/BIP, anti-p-PERK, anti-p-ELF2α, anti-p-IRE1 α, anti-p-JNK, anti-CHOP and anti-GAPDH antibodies (A). The expression of CHOP mRNA was measured by RT-PCR (B). Pretreatment with 4-PBA (10 mM) for 1 h was followed by JI017 treatment. Cell viability was measured by the MTS assay (C). DU145 and PC3 cells were treated with JI017 (120 μg/ml) for 24 h. BIP was immunoprecipitated in DU145 and PC3 cells. Then, the immunoprecipitated proteins were detected by western blotting with anti-PERK and anti-IRE1α antibodies (D). Pretreatment with NAC (10 mM) for 1 h was followed by JI017 treatment. Whole cell lysates were analyzed by western blotting with an anti-CHOP antibody (E). The expression of CHOP mRNA was measured by RT-PCR (F). The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to untreated cells.

FIGURE 6

Induction of the mitochondrial dysfunction response by UPR signaling. DU145 and PC3 cells were treated with JI017 for 24 h and subjected to the rhodamine 123 assay (A). DU145 and PC3 cells were treated with JI017 for 24 h and stained with MitoTracker (300 nM) (B). The expression of BAK, NOXA, and PUMA mRNAs was measured by RT-qPCR (C). Mitochondrial, and cytosolic extracts of cultured cells were prepared and analyzed by western blotting to detect the levels of cytochrome c and Smac/Diablo (D). Effect of an ER stress inhibitor (4-PBA) on JI017-induced mitochondrial apoptosis pathway. Pretreatment with 4-PBA (10 mM) for 1 h was followed by JI017 treatment. Whole cell lysates were analyzed by western blotting with anti-CHOP, anti-cleaved caspase-9, and anti-GAPDH antibodies (E). The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to untreated cells.

FIGURE 7

Knockdown of CHOP signaling blocks apoptosis induced by JI017 treatment of prostate cell lines. DU145 and PC3 cells were transfected with CHOP siRNA and treated with JI017. Cell viability was measured by the MTS assay (A). Whole cell lysates were analyzed by western blotting with anti-CHOP, anti-cleaved caspase-3, anti-cleaved caspase-7, anti-cleaved caspase-9, anti-CHOP, and anti-GAPDH antibodies (B). Schematic representation of JI017 treatment of prostate cancer cells (C). The data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to untreated cells.