P2Y1 agonist HIC in combination with androgen receptor inhibitor abiraterone acetate impairs cell growth of prostate cancer

Abstract

P2Y receptors belong to the large superfamily of G-protein-coupled receptors and play a crucial role in cell death and survival. P2Y1 receptor has been identified as a marker for prostate cancer (PCa). A previously unveiled selective P2Y1 receptor agonist, the indoline-derived HIC (1-(1-((2-hydroxy-5-nitrophenyl)(4-hydroxyphenyl)methyl)indoline-4-carbonitrile), induces a series of molecular and biological responses in PCa cells PC3 and DU145, but minimal toxicity to normal cells. Here, we evaluated the combinatorial effect of HIC with abiraterone acetate (AA) targeted on androgen receptor (AR) on the inhibition of PCa cells. Here, the presence of HIC and AA significantly inhibited cell proliferation of PC3 and DU145 cells with time-dependent manner as a synerfistic combination. Moreover, it was also shown that the anticancer and antimetastasis effects of the combinratorial drugs were noticed through a decrease in colony-forming ability, cell migration, and cell invasion. In addition, the HIC + AA induced apoptotic population of PCa cells as well as cell cycle arrest in G1 progression phase. In summary, these studies show that the combination of P2Y1 receptor agonist, HIC and AR inhibitor, AA, effectively improved the antitumor activity of each drug. Thus, the combinatorial model of HIC and AA should be a novel and promising therapeutic strategy for treating prostate cancer.

Keywords: Abiraterone acetate; Apoptosis; Indoline; Proliferation; Prostate cancer.

Fig. 1

Sensitivity of PCa and noncancer cells to HIC and AA. Structure of HIC and AA (A). Percentage of cell viability with HIC treatment (B) and AA treatment (C) were determined using MTT assay. PC3, DU145, HEK293, and MEF cells were treated with increasing doses of HIC and AA for 48 h. Values are presented as the means ± SD of three biological experiments. *p < 0.05 relatives to DMSO-treated group

Fig. 2

HIC and AA co-treatment reduces cell growth in PCa cells. Percentage of PC3 (A) and DU145 (B) cells viability in the presence of HIC and AA, alone or in combination with indicated concentrations. Percentage of cell viability for HEK293 and MEF cells with co-treated HIC and AA (C). Representative images of PC3 (D) and DU145 (E) cells treated with HIC, AA, or a combination of both in Clonogenic survival assay. The bar graph presented the colony formulation of PCa cells with HIC and AA treatment based on the normalization with DMSO groups (F). Data were analyzed by one-way ANOVA; *p < 0.05 relatives to DMSO-treated group

Fig. 3

Combination of HIC and AA inhibits the proliferation of PCa cells in a time-dependent manner. Percentage of cell viability in A PC3 and B DU145 cells treated with HIC, AA, and HIC + AA combination for 24, 48, and 72 h. The percentage of cell survival was normalized to DMSO groups. The experiment was performed with n = 6. Statistical significance was considered with *p < 0.05

Fig. 4

Combinatorial effect of HIC and AA induces apoptosis, caspase 3/7 activation, and ROS production. Representative images of PC3 (A) and DU145 (B) cells stained with DAPI/Annexin-V/PI in DMSO, HIC, AA, and cotreated drugs condition. Percentage of apoptotic and necrotic cells were presented in the corresponding conditions. C Fold changes of caspases 3/7 activity in PC3 and DU145 cells compared to DMSO groups. D Fold change of ROS production in PC3 and DU145 cells were calculated using fluorescence intensities based normalized against DMSO control. Biological and technical replicates were performed to analyze the results, with mean ± SD, *p < 0.05, n = 6

Fig. 5

Combination of HIC and AA suppresses the G1 phase of the PCa cell cycle. PC3 and DU145 cells were treated with HIC and AA alone or in combination for 48 h. Cells were fixed and stained with PI. Microscopic images of PC3- (A) and DU145- (B) treated cells were captured and analyzed to detect cell-cycle distribution. The percentage of cells in each phase was calculated and presented. Data is shown as mean ± SD, *p < 0.05, n = 6

Fig. 6

Inhibition of metastatic properties of PCa cells. A The wounds of PC3 and DU145 cells were created using a scratcher, and then cells were incubated with HIC, AA, alone or a combination of both. Representative images were captured at 0 and 24 h. A mark in these images was placed to locate the same area on the scratch. Percentage of relative wound closure was shown as a bar graph. B Invaded cells were captured under the microscope. The bar graph presented is the percentage of cell invasion based on DMSO groups. Experiments were repeated three times with technical repeats, n = 6, *p < 0.05, ANOVA test