Isoalantolactone Increases the Sensitivity of Prostate Cancer Cells to Cisplatin Treatment by Inducing Oxidative Stress

Abstract

Prostate cancer is the most common malignancy among men worldwide. Platinum (II)-based chemotherapy has been used to treat a number of malignancies including prostate cancer. However, the potential of cisplatin for treating prostate cancer is restricted owing to its limited efficacy and toxic side effects. Combination therapies have been proposed to increase the efficacy and reduce the toxic side effects. In the present study, we investigated how isoalantolactone (IATL), a sesquiterpene lactone extracted from the medicinal plant Inula helenium L., acts synergistically with cisplatin on human prostate cancer cells. We show that IATL significantly increased cisplatin-induced growth suppression and apoptosis in human prostate cancer cells. Mechanistically, the combined treatment resulted in an excessive accumulation of intracellular reactive oxygen species (ROS), which leads to the activation of endoplasmic reticulum (ER) stress and the JNK signaling pathway in human prostate cancer cells. Pretreatment of cells with the ROS scavenger N-acetylcysteine (NAC) significantly abrogated the combined treatment-induced ROS accumulation and cell apoptosis. In addition, the activation of ER stress and the JNK signaling pathway prompted by IATL and cisplatin was also reversed by NAC pretreatment. In vivo, we found that IATL combined with cisplatin showed the strongest antitumor effects compared with single agents. These results support the notion that IATL and cisplatin combinational treatment may be more effective for treating prostate cancer than cisplatin alone.

Keywords: cisplatin; endoplasmic reticulum stress; isoalantolactone; oxidative stress; prostate cancer.

FIGURE 1

IATL and cisplatin synergistically inhibit cells growth and induce apoptosis. (A,B) DU145 or PC-3 cells were treated with IATL or cisplatin alone or their combination at the indicated concentration for 24 h; cell viability was determined by an MTT assay. (C,D) Combination index (CI) values were calculated by the Calcusyn software, and a CI value of less than 1 indicates synergism. (E–H) DU145 or PC-3 cells were treated with IATL or cisplatin alone or their combination at the indicated concentration for 24 h; cell apoptosis rate was analyzed by flow cytometry. (I,J) DU145 or PC-3 cells were treated with IATL or cisplatin alone or their combination at the indicated concentration for 20 h; caspase-3 and caspase-9 activity were measured by an assay kit. *p < 0.05, **p < 0.01.

FIGURE 2

IATL and cisplatin combination triggers ROS-dependent apoptosis in prostate cancer cells. (A,B) DU145 or PC-3 cells were treated with IATL or cisplatin alone or their combination at the indicated concentration for 2 h; cellular ROS levels were detected by using the DCFH-DA probe. (C,D) DU145 or PC-3 cells were preincubated with NAC (5 mM) for 2 h before exposure to the combination of IATL and cisplatin for 2 h; cellular ROS levels were detected by using the DCFH-DA probe. (E,F) DU145 or PC-3 cells were preincubated with NAC (5 mM) for 2 h; cell viability was detected by an MTT assay after being treated with the combination of IATL and cisplatin for 24 h. (G,H) DU145 or PC-3 cells were preincubated with catalase (2,000 U/mL) for 2 h; cell viability was detected by an MTT assay after being treated with the combination of IATL and cisplatin for 24 h. (I–L) DU145 or PC-3 cells were preincubated with NAC (5 mM) for 2 h; the cell apoptosis rate was detected by flow cytometry after being treated with the combination of IATL and cisplatin for 24 h. (M,N) DU145 or PC-3 cells were preincubated with NAC (5 mM) for 2 h; caspase-3 and caspase-9 activity was measured by an assay kit after being treated with the combination of IATL and cisplatin for 20 h. *p < 0.05, **p < 0.01.

FIGURE 3

IATL and cisplatin co-operate to activate ER stress in prostate cancer cells. (A,B) DU145 cells were treated with IATL (15 μM) and cisplatin (20 μM) combination for the indicated time periods; the expression of p-eIF2α and ATF4 was measured by Western blot. (C,D) DU145 cells were treated with IATL (15 μM) or cisplatin (20 μM) alone or their combination (15 μM IATL and 20 μM cisplatin); the expression of p-eIF2α and ATF4 was measured by Western blot. (E) DU145 cells were treated with IATL (15 μM) or cisplatin (20 μM) alone or their combination (15 μM IATL and 20 μM cisplatin) for 6 h; the mRNA level of CHOP was determined by qRT-PCR. (F) DU145 cells were treated with IATL (15 μM) or cisplatin (20 μM) alone or their combination (15 μM IATL and 20 μM cisplatin) for 12 h; the expression of CHOP was measured by Western blot. (G) DU145 cells were infected with CHOP siRNA; the mRNA level of CHOP was determined by qRT-PCR after being treated with the combination of IATL (15 μM) and cisplatin (20 μM) for 6 h. (H,I) DU145 cells transfected with CHOP siRNA were treated with the combination of IATL (15 μM) and cisplatin (20 μM) for 20 h; caspase-3 and caspase-9 activity was measured by an assay kit. (J) DU145 cells transfected with CHOP siRNA were treated with the combination of IATL (15 μM) and cisplatin (20 μM) for 24 h; cell viability was measured by an MTT assay. *p < 0.05, **p < 0.01.

FIGURE 4

IATL and cisplatin co-operate to activate ROS-dependent ER stress. (A,B) DU145 cells were preincubated with NAC (5 mM) for 2 h before exposure to the combination of IATL (15 μM) and cisplatin (20 μM); the expression of p-eIF2α and ATF4 was measured by Western blot. (C) DU145 cells were preincubated with NAC (5 mM) for 2 h; the mRNA level of CHOP was determined by qRT-PCR after being treated with IATL (15 μM) and cisplatin (20 μM) combination for 6 h. (D,E) DU145 cells were preincubated with NAC (5 mM) for 2 h before exposure to the combination of IATL (15 μM) and cisplatin (20 μM) for 12 h; the expression of CHOP was measured by Western blot. **p < 0.01.

FIGURE 5

IATL and cisplatin co-operate to induce JNK phosphorylation in prostate cancer cells. (A,B) DU145 cells were treated with the combination of IATL (15 μM) and cisplatin (20 μM) for the indicated time periods; the expression of p-JNK and JNK was measured by Western blot. (C,D) DU145 cells were treated with IATL (15 μM) or cisplatin (20 μM) alone or their combination (15 μM IATL and 20 μM cisplatin); the expression of p-JNK and JNK was measured by Western blot. (E) DU145 cells were preincubated with SP600125 for 2 h; cell viability was detected by an MTT assay after being treated with the combination of IATL (15 μM) and cisplatin (20 μM) for 24 h. (F,G) DU145 cells were preincubated with SP600125 for 2 h before being treated with the combination of IATL (15 μM) and cisplatin (20 μM) for 20 h; caspase-3 and caspase-9 activity was measured by an assay kit. (H) DU145 cells were preincubated with NAC (5 mM) for 2 h before being treated with the combination of IATL (15 μM) and cisplatin (20 μM); the expression of p-JNK and JNK was measured by Western blot. *p < 0.05, **p < 0.01.

FIGURE 6

IATL and cisplatin synergistically inhibit tumor growth in nude mice. (A,B) IATL in combination with cisplatin markedly suppressed tumor volume (A) and tumor weight (B). (C) The body weight of nude mice. (D,E) The levels of blood urea nitrogen (BUN) and creatinine (Cr). (F) Caspase-3 activity in the tumor tissues. (G) MDA levels in the tumor tissues. *p < 0.05, **p < 0.01.