Resistomycin Suppresses Prostate Cancer Cell Growth by Instigating Oxidative Stress, Mitochondrial Apoptosis, and Cell Cycle Arrest

Abstract

Globally, prostate cancer is among the most threatening and leading causes of death in men. This study, therefore, aimed to search for an ideal antitumor strategy with high efficacy, low drug resistance, and no or few adverse effects. Resistomycin is a natural antibiotic derived from marine actinomycetes, and it possesses various biological activities. Prostate cancer cells (PC3) were treated with resistomycin (IC₁₂.₅: 0.65 or IC₂₅: 1.3 µg/mL) or 5-fluorouracil (5-FU; IC₂₅: 7 µg/mL) for 24 h. MTT assay and flow cytometry were utilized to assess cell viability and apoptosis. Oxidative stress, apoptotic-related markers, and cell cycle were also assessed. The results revealed that the IC₅₀ of resistomycin and 5-FU on PC3 cells were 2.63 µg/mL and 14.44 µg/mL, respectively. Furthermore, treated cells with the high dose of resistomycin showed an increased number of apoptotic cells compared to those treated with the lower dose. Remarkable induction of reactive oxygen species generation and lactate dehydrogenase (LDH) leakage with high malondialdehyde (MDA), carbonyl protein (CP), and 8-hydroxyguanosine (8-OHdG) contents were observed in resistomycin-treated cells. In addition, marked declines in glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in PC3 cells subjected to resistomycin therapy were observed. Resistomycin triggered observable cell apoptosis by increasing Bax, caspase-3, and cytosolic cytochrome c levels and decreasing Bcl-2 levels. In addition, notable downregulation of proliferating cell nuclear antigen (PCNA) and cyclin D1 was observed in resistomycin-treated cancerous cells. According to this evaluation, the antitumor potential of resistomycin, in a concentration-dependent manner, in prostate cancer cells was achieved by triggering oxidative stress, mitochondrial apoptosis, and cell cycle arrest in cancer cells. In conclusion, our investigation suggests that resistomycin can be considered a starting point for developing new chemotherapeutic agents for human prostate cancer.

Keywords: 5-FU; apoptosis; oxidative stress; prostate cancer; resistomycin.

Figure 1

Structural formula of resistomycin (1).

Figure 2

Effects of different concentrations of resistomycin or 5-FU treatment (24 h) on PC3, DU-145, MCF-7, and Caco-2 cell lines. The cell viability rate was indicated as a % of control cells without the test sample. Values represent the mean of three experiments ± SD. ∆: significant with respect to the control (p ˂ 0.05).

Figure 3

Effects of resistomycin (0.65 or 1.3 µg/mL) or 5-FU treatment (24 h) on LDH leakage in PC3 cells. Values represent the mean of three experiments ± SD. ∆∆: significant with respect to the control (p ˂ 0.01). ∆∆∆: significant with respect to the control (p ˂ 0.001). ΨΨ: significant with respect to the 5-FU (p ˂ 0.01).

Figure 4

Effects of resistomycin (0.65 or 1.3 µg/mL) or 5-FU treatment (24 h) on the antioxidant enzymatic activities of SOD, CAT, and GPx in PC3 cells. Values represent the mean of three experiments ± SD. ∆: significant with respect to the control (p ˂ 0.05). ∆∆: significant with respect to the control (p ˂ 0.01). ∆∆∆: significant with respect to the control (p ˂ 0.001). Ψ: significant with respect to the 5-FU (p ˂ 0.05). ΨΨ: significant with respect to the 5-FU (p ˂ 0.01). ΨΨΨ: significant with respect to the 5-FU (p ˂ 0.001).

Figure 5

Effects of resistomycin (0.65 or 1.3 µg/mL) or 5-FU treatment (24 h) on ROS in PC3 cells. Values represent the mean of three experiments ± SD. ∆∆: significant with respect to the control (p ˂ 0.01). ∆∆∆: significant with respect to the control (p ˂ 0.001). ΨΨ: significant with respect to the 5-FU (p ˂ 0.01).

Figure 6

Effects of resistomycin (0.65 or 1.3 µg/mL) or 5-FU treatment (24 h) on the non-enzymatic oxidative stress markers (GSH, MDA, CP, and 8-OHdG) in PC3 cells. Values represent the mean of three experiments ± SD. ∆∆: significant with respect to the control (p ˂ 0.01). ∆∆∆: significant with respect to the control (p ˂ 0.001). Ψ: significant with respect to the 5-FU (p ˂ 0.05). ΨΨΨ: significant with respect to the 5-FU (p ˂ 0.001).

Figure 7

Effects of resistomycin (0.65 or 1.3 µg/mL) or 5-FU treatment (24 h) on the levels of apoptotic markers (Bcl-2, Bax, Cas-3, and cytochrome c) in PC3 cells. Values represent the mean of three experiments ± SD. ∆: significant with respect to the control (p ˂ 0.05). ∆∆: significant with respect to the control (p ˂ 0.01). ∆∆∆: significant with respect to the control (p ˂ 0.001). ΨΨ: significant with respect to the 5-FU (p ˂ 0.01).

Figure 8

Apoptosis-induced cell death after treatment (24 h) with resistomycin (0.65 or 1.3 µg/mL) or 5-FU in PC3 cell line. Apoptosis-induced cell death was determined through annexin V and PI staining. Percentages of viable, early, late, and necrotic cells are shown when possible. Experiments were performed in triplicate. Values represent the mean of three experiments ± SD. ∆: significant with respect to the control (p ˂ 0.05). Ψ: significant with respect to the 5-FU (p ˂ 0.05). (A): Control, (B): Resistomycin, (C): 5-FU, (D): Histograms showing the different percentages of cell types. Black dots in (A–C) are 12,000 events acquired and Red dots are the positive FITC and/or PI cells.

Figure 9

Effects of resistomycin (0.65 or 1.3 µg/mL) or 5-FU treatment (24 h) on the cell-cycle-related markers (PCNA and cyclin D1) in PC3 cells. Values represent the mean of three experiments ± SD. ∆∆: significant with respect to the control (p ˂ 0.01). ∆∆∆: significant with respect to the control (p ˂ 0.001). Ψ: significant with respect to the 5-FU (p ˂ 0.05). ΨΨ: significant with respect to the 5-FU (p ˂ 0.01).