Polygodial, a Sesquiterpene Dialdehyde, Activates Apoptotic Signaling in Castration-Resistant Prostate Cancer Cell Lines by Inducing Oxidative Stress

Abstract

Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. Surgery, radiation therapy, chemotherapy, and androgen deprivation therapy are currently the standard treatment options for PCa. These have poor outcomes and result in the development of castration-resistant prostate cancer (CRPC), which is the foremost underlying cause of mortality associated with PCa. Taxanes, diterpene compounds approved to treat hormonal refractory PCa, show poor outcomes in CRPC. Polygodial (PG) is a natural sesquiterpene isolated from water pepper (Persicaria hydropiper), Dorrigo pepper (Tasmannia stipitata), and mountain pepper (Tasmannia lanceolata). Previous reports show that PG has an anticancer effect. Our results show that PG robustly inhibits the cell viability, colony formation, and migration of taxane-resistant CRPC cell lines and induces cell cycle arrest at the G0 phase. A toxicity investigation shows that PG is not toxic to primary human hepatocytes, 3T3-J2 fibroblast co-cultures, and non-cancerous BPH-1 cells, implicating that PG is innocuous to healthy cells. In addition, PG induces oxidative stress and activates apoptosis in drug-resistant PCa cell lines. Our mechanistic evaluation by a proteome profiler-human apoptotic array in PC3-TXR cells shows that PG induces upregulation of cytochrome c and caspase-3 and downregulation of antiapoptotic markers. Western blot analysis reveals that PG activates apoptotic and DNA damage markers in PCa cells. Our results suggest that PG exhibits its anticancer effect by promoting reactive oxygen species generation and induction of apoptosis in CRPC cells.

Keywords: anoikis; castration-resistant prostate cancer; natural products; polygodial; prostate cancer; taxane-resistant CRPC.

Figure 1

Polygodial (PG) treatments are not toxic to hepatocytes and fibroblasts. MPCCs and 3T3-J2 fibroblast-only monocultures were cultured for 7 days. Cells were treated with various concentrations of PG every other day for 6 days (i.e., three total treatments), and cell viability was measured on the 9th, 11th, and 13th day of culture. The results show that the cell viability of MPCC (A) and fibroblast (B) is not significantly affected by PG treatment.

Figure 2

PG inhibits the cell viability of taxane-resistant PCa cell lines in a concentration-dependent manner. The taxane-resistant PCa cell lines (A) PC3-TXR and (B) DU145-TXR were treated with various concentrations of PG (treatment periods 24, 48, and 72 h). Viability was determined by MTT assay. (C) PC3-TXR and (D) DU145-TXR were treated with different inhibitors for 48 h. (E) depicts the effect of PG on the BPH-1 cell line. Data are represented as mean ± SD (n = 3) and describe three independent experiments performed in triplicate. * p < 0.05, *** p < 0.01, **** p< 0.001.

Figure 3

PG inhibits colony formation in taxane-resistant PCa cell lines. (A) PC3-TXR and (B) DU145-TXR represent images of colony formation after PG treatment. Compared to the control, a substantial decrease in the colonies’ number is observed with PG treatment. (C) and (D) display the quantified results of the average number of colonies plotted against varying concentrations of PG. Data are represented as mean ± SD (n = 3) and represent three trials performed in triplicate independently. *** p < 0.01, and **** p < 0.001.

Figure 4

PG inhibits cell migration in taxane-resistant PCa cell lines. (A) PC3-TXR and (B) DU145-TXR cell line images were taken at 0, 24, 48, and 72 h and analyzed using T-scratch software. (C) and (D) represent the % open area plotted against varying concentrations of PG for PC3-TXR and DU145-TXR. Images were taken at 10× magnification. Data are represented as mean ± SD (n = 3), and three independent trials were conducted in triplicate. * p < 0.05.

Figure 5

PG treatment elicits apoptosis in taxane-resistant PCa cell lines. (A) PC3-TXR and (C) DU145-TXR cell lines were treated with varying concentrations of PG for 48 h, and apoptosis was detected by annexin V FITC/PI staining. Images were taken via confocal microscopy and analyzed using ImageJ. In the taxane-resistant PCa cell lines, the relative annexin V FITC binding was higher in the treatment groups compared to that of controls. (B) and (D) show relative annexin V binding plotted against concentrations of PG for PC3-TXR and DU145-TXR, respectively. Images were taken at 60× magnification. Data are depicted as mean ± SD (n = 3) and represent three experiments performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

PG induces apoptosis in taxane-resistant PCa cell lines. (A) PC3-TXR and (B) DU145-TXR cell lines were treated with varying concentrations of PG, and apoptosis was detected by staining cells with annexin V FITC/PI followed by flow cytometry analysis. The results suggest that PG induces apoptosis in higher concentrations. (C) and (D) represent % apoptosis plotted against concentrations of PG for PC3-TXR and DU145-TXR. Data are shown as mean ± SD (n = 3) and denote three independent experiments in triplicate. * p < 0.05, ** p < 0.01, **** p < 0.001.

Figure 7

PG promotes anoikis in taxane-resistant PCa cell lines. (A) PC3-TXR and (B) DU145-TXR cell lines were treated with varying concentrations of PG for 48 h and stained with calcein AM and EthD-1 to detect anoikis. We observed that with the increase in the concentration of PG, the number of viable cells stained with calcein-AM (green fluorescence) decreased, and the number of dead cells stained with EthD-1 (red fluorescence) increased. (C) and (D) % cell viability plotted against various concentrations of PG for PC3-TXR and DU145-TXR, respectively. Images were taken at 10× magnification. Data are represented as mean ± SD (n = 3), and a total of three experiments were performed independently in triplicate. *** p < 0.05, **** p < 0.01.

Figure 8

PG promotes anoikis in taxane-resistant PCa cell lines via the activation of PTEN. (A) PC3-TXR and (B) DU145-TXR cell lines. Our data illustrate that PG induces upregulation in the expression of PTEN. Data are represented as mean ± SD (n = 3), and a total of three experiments were performed independently in triplicate. * p < 0.05, ** p < 0.01.

Figure 9

PG induces G0 phase cell cycle arrest in taxane-resistant PCa cell lines. (A) PC3-TXR and (B) DU145-TXR cell lines were treated with varying concentrations of PG for 48 h and stained with PI, and flow cytometry analysis was performed. We observed that PG 50 µM significantly blocks the cell cycle at the G0 phase in taxane-resistant PCa cell lines. (C) and (D) represent the quantified results of % of cells plotted against varying PG treatment of PC3-TXR and DU145-TXR. Data are represented as mean ± SD (n = 3), and three experiments were performed in triplicate. * p < 0.05.

Figure 10

PG treatment induces ROS generation, leading to oxidative stress in taxane-resistant PCa cell lines. The taxane-resistant PCa cell lines were treated with varying concentrations of PG for 6 h and stained with DCFH2-DA. The images were taken and analyzed using ImageJ software. (A) PC3-TXR and (B) DU145-TXR show an increase in green fluorescence with an increase in PG treatment up to PG 20 µM compared to the control, but PG 50 µM shows slight green fluorescence. (C),D) represent the quantified results of relative ROS levels plotted against concentrations of PG of PC3-TXR and DU145-TXR. Images were taken at 40× magnification. Data are represented as mean ± SD (n = 3), and three experiments were carried out in triplicate. * p < 0.05, ** p < 0.01, **** p < 0.001.

Figure 11

PG induces ROS production, resulting in oxidative stress in CRPC cells. PC3-TXR and DU145-TXR cells were stained with DCFH2-DA and then treated with different concentrations of PG for 4 h, followed by flow cytometry analysis. (A) PC3-TXR and (B) DU145-TXR show an increase in the amount of ROS generated with PG treatment compared to the control. (C) and (D) represent the quantified ROS levels’ results plotted against PG concentrations of PC3-TXR and DU145-TXR compared to the untreated control.

Figure 12

PG modulates the expression of various apoptotic markers in PC3-TXR. Figure (A) represents PC3-TXR control and (B) represents PC3-TXR treated with PG 20µM, in which the cells were seeded and treated with PG 20 µM for 48 h. Expression of the various apoptotic markers was found using a proteome profiler-human apoptosis array. PG promotes downregulation in the antiapoptotic markers Bcl-2, Bcl-xL, and IAP family, in addition to an upregulation in cytochrome c and cleaved-caspase-3. Numbers used to depict the expression of selected markers compared to reference spots: 1 = reference spot, 2 = Bcl family, 3a = pro-caspase 3, 3b = cleaved caspase 3, 4,5,8 = IAP family, 6 = cytochrome c, 7 = survivin. (C) represents the quantified results of the relative band intensities calculated using ImageJ, and relative expression levels were plotted against PG treatment. Data are represented as mean ± SD. * p < 0.05.

Figure 13

PG differentially modulates the expression of key apoptotic markers and DNA damage markers in taxane-resistant PCa cell lines. (A) PC3-TXR and (B) DU145-TXR cells were treated with varying concentrations of PG, and an immunoblot assay was performed. PG induces downregulation of various apoptotic markers, such as pro-caspase-3, XIAP, and cIAP-2. We also observed that the DNA damage marker pH2AX is upregulated, and PARP-1 is downregulated. (C) and (D) represent the quantified results of relative band intensities calculated using ImageJ, and relative expression levels were plotted against PG treatment. (E) and (F) depict the expression of cleaved PARP-1 in PC3-TXR and DU145-TXR, respectively, confirming that PG induces taxane-resistant PCa cell lines to commit apoptosis. (G) and (H) are graphical representations of the relative band intensities calculated using ImageJ, and fold change in expression levels were plotted against various PG treatments.

Figure 14

Proposed mechanism of PG action in taxane-resistant CRPC cell lines. We identified that ROS generation by PG induces DNA damage and apoptosis by the intrinsic signaling apoptosis pathway, indicating that PG has a potential therapeutic effect on taxane-resistant PCa.