Human prostate cancer PC-3 cells were purchased from Procell Life Science & Technology Co., Ltd. The cells were cultured in Ham's F-12K media (Wuhan Pricella Biotechnology Co., Ltd.; Cat. CM0185) supplemented with 10% fetal bovine serum (FBS) (Gibco. Cat. 26140-079), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. PSP was purchased from Shanghai Yuanye Biotechnology Co., Ltd. with a purity of 70%. The compounds were dissolved in PBS at a concentration of 20 mg/ml to prepare stock solution, which was stored at −20°C for in vitro studies. Before each assay, the stock solution was diluted with the medium.

The effect of PSP on cell proliferation was assessed using the CCK-8 assay. Cells were seeded into 96-well tissue culture plates at a density of 1×10⁴ cells/well and incubated at 37°C with PSP for 24, 48, 72, 96 or 120 h. Untreated cells served as the control. Following the treatment period, 10 µl CCK-8 reagent (Fluorescence, cat. no. DCM7122) was added to each well and then incubated for an additional 4 h at 37°C. The absorbance at 450 nm was measured using a microplate reader. Cell viability was determined by the following formula: Cell viability (%)=[(A_s-A_b)/(A_c-A_b)] ×100%, where A_s is the absorbance of PSP-treated groups, A_c is the absorbance of 0 µg/ml PSP-treated groups, and A_b is the absorbance of the blank group.

Based on the results of the CCK-8 assay, PC-3 cells were plated into 6-well plates at a density of 2×10⁵ cells/well and treated with several concentrations of PSP solution (0, 250, 500 µg/ml, 1, 2 and 4 mg/ml) at 37°C for 72 h. The apoptosis rate of PC-3 cells was evaluated using the Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer's instructions (Beijing 4A Biotech Co., Ltd. Cat. FXP018). Cultured cells were collected, washed with cold PBS and resuspended in a binding buffer. To this buffer, 5 µl Annexin V-FITC and 5 µl PI were added, followed by incubation at room temperature for 15 min in the dark. Annexin V-FITC binds to phosphatidylserine on the outer membrane of apoptotic cells, whilst PI penetrates and stains cells with compromised membrane integrity, binding to and labeling DNA. Data collection was performed using a flow cytometer (BeamCyte-1026; Bidaiko Biotechnology (Suzhou) Co.,Ltd, and the data were analyzed using FlowJo software 10.8.1 (BD Biosciences).

For the cell cycle assay, PC-3 cells were trypsinized, washed with cold PBS and fixed with 70% ethanol at 4°C overnight. The fixed cells were collected, resuspended in cold PBS and stained with PI. After staining, the cells were incubated for 30 min at 37°C in the dark. Data were collected using a flow cytometer (BeamCyte-1026; Bidaiko Biotechnology (Suzhou) Co., Ltd) and the data were analyzed with FlowJo software 10.8.1 (BD Biosciences).

A Transwell assay was used to analyze cell invasion and/or migration. 2×10⁵ cells treated with PSP (0, 500 µg/ml, 1, 2 and 4 mg/ml) were suspended in an FBS-free medium and 4×10⁴ cells seeded into the upper chamber of 24-well plates, which were either coated with Matrigel at 37°C for 24 h (for invasion assays) or uncoated (for migration assays). The lower chamber was filled with a medium containing 10% FBS as a chemoattractant. After a 24-h incubation at 37°C, non-invaded or non-migrated cells on the upper surface of the membranes were carefully removed using cotton swabs, whilst cells that had transverse the membrane were fixed with 75% carbinol for 10 min at room temperature and stained using 0.1% Giemsa (Beyotime, C0133) for 10 min at room temperature. The stained cells were then counted under an invert light microscope (Leica DM3000).

PC-3 cells were seeded in 12-well microplates at a density of 1.5×10⁵ cells/well and incubated at 37°C for 24 h (confluence reached 90–100%). Confluent monolayers were scratched with 10 µl tips. After washing with PBS, the cells were cultured at 37°C in low serum medium (0.5% FBS) with different concentrations of PSP (0, 500 µg/ml, 1, 2 and 4 mg/ml). Images of scratched areas were captured by fluorescence microscope at 0, 24, 48 and 72 h of incubation in the dark. Image J v1.8.0 software (National Institutes of Health) was used to analyze the cell migration distance. The migration inhibition rate was expressed as the % scratch closure change according to the following formula: Scratch closure change (%)=[(A_t0-A_tc)/A_t0] ×100%, where A_t0 is the scratch area at time 0 and A_tc is the corresponding scratch area at 24, 48 and 72 h.

A total of 2×10⁵ Cells treated with PSP for 72 h were harvested and lysed using RIPA buffer (Solarbio life sciences, Cat. R0010), and protein concentrations were measured using a BCA quantification kit. The lysates including 50 µg total proteins were then separated using 10% SDS-PAGE and transferred onto PVDF membranes (cat. no. ISEQ00010; MilliporeSigma; Merck KGaA), then blocked with 5% BSA (Solarbio life sciences, Cat. SW3015) at room temperature for 1 h. The membranes were incubated overnight at 4°C with primary antibodies (Affinity Biosciences, Ltd.) diluted at 1:1,000, including those targeting Akt (cat. AF6261), p-Akt (Cat. AF0016), PI3K (Cat. AF6241), p-PI3K (Cat. AF3241), caspase-3 (Cat. no. AF6311), P65 (Cat. AF5006), p-P65 (Cat. AF2006) and β-actin (Cat. AF7018). After being washed thrice with 1×PBS-T (1% Tween-20), the membranes were incubated with HRP-conjugated sheep anti-rabbit secondary antibodies (1:3,000, Medical Discovery Leader, MD912565) at room temperature for 1 h. Finally, the protein bands were visualized using enhanced chemiluminescence. The intensity of the bands was semi-quantified by the ChemiDoc MP Chemiluminescence Imaging System with Image Lab software 6.0.1 (Bio-Rad Laboratories, Inc.). β-actin was used as internal control.

2×10⁵ PC-3 cells were treated with different concentrations of PSP (0, 500 µg/ml, 1, 2, 4 mg/ml) at 37°C for 72 h, after which total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA concentration was determined using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). A total of 1 µg RNA was then reverse transcribed into cDNA using the SuperRT cDNA Synthesis Kit (Jiangsu CoWin Biotech Co., Ltd. cat. CW0741m), with β-actin serving as the internal control, the condition for reverse transcription are incubated at 42°C for 30–50 min and at 85 ° C for 5 min. The expression levels of multidrug resistance-1 (MDR1) mRNA were assessed using the UltraSYBR Mixture (Low ROX; Jiangsu CoWin Biotech Co., Ltd.; cat. CW2601M). The data were normalized with β-actin using the following formula: Relative mRNA expression=2^−ΔΔCq (22), where Cq is the cycle threshold. The primer sequences used were as follows: MDR1 (forward), 5′-TGCTCAAGTTAAAGGGGCTA-3′ and (reverse), 5′-CAGTGTTAGTTGCCAACCAT-3′; and β-actin (forward), 5′-TCCTCCTGAGCGCAAGTACTCC-3′ and (reverse), 5′-CATACTCCTGCTTGCTGATCCAC-3′. The thermocycling conditions were as follows: Pre-denatured at 95°C for 5 min, 40 cycles at 95°C for 10 sec, 58°C for 20 sec and 72°C for 20 sec.

PC-3 cells were seeded into 6-well plates with sterile slides at a density of 2×10⁵ cells/well, treated with several concentrations of PSP solution (0 and 500 µg/ml and 1, 2, 4 mg/ml) at 37°C for 72 h, fixed with pre-cooled 4% paraformaldehyde at room temperature for 20 min, and then blocked with 10% goat serum (Beyotime, C0265) for 30 min at 37°C. The slides were subsequently stained with MDR1/P-glycoprotein (P-gp) primary antibodies (1:1,000; Affinity Biosciences, Ltd.; Cat. AF5185) overnight at 4°C. Following this, the slides were incubated with HRP-conjugated secondary anti-rabbit IgG (1:5,000, MDL Biotech; cat. no. MD912565) at 37°C for 40 min. Slides were visualized using DAB, counterstained with hematoxylin at room temperature for 5 min and observed under a light microscope (Leica DM2000 LED).

All experiments were performed in triplicate. Data were analyzed using SPSS 19.0 statistical software (IBM Corp.) and are expressed as the mean ± standard deviation. One-way analysis of variance was used to compare data among groups when they had a normal distribution and homogeneous variance, and Tukey's was used for the post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The CCK-8 assay was used to determine whether PSP inhibits the proliferation of PC-3 cells at varying concentrations. PC-3 cells were treated with different concentrations of PSP (0, 125, 250, 500 µg/ml, 1, 2 and 4 mg/ml) for 24, 48, 72, 96 and 120 h. The results demonstrated there was no significant inhibition observed across the concentrations at 24 h. However, at 48 h, inhibition increased with higher concentrations of PSP, particularly at 4 mg/ml. By 72 and 96 h, significant inhibition was observed at concentrations of 2 and 4 mg/ml (Fig. 1). These results indicate that PSP effectively inhibits the proliferation of PC-3 cells.

The PC-3 cells were treated with different concentrations of PSP for 72 h, after which the apoptosis rate was analyzed using flow cytometry. The results demonstrated that the apoptosis rate in the PSP-treated groups was significantly higher than in the blank control group, and this increase was concentration-dependent (P=0.001; Fig. 2A and B).

As activated caspase-3 is a specific marker of apoptosis (14), the present study evaluated whether its activation contributed to PSP-induced apoptosis. Caspase-3 activity was assessed using western blot analysis following the treatment of PC-3 cells with PSP for 72 h. PSP concentrations of 0, 500 µg/ml, 1, 2 and 4 mg/ml were selected based on the results of the apoptosis assay. The results indicated a concentration-dependent increase in caspase-3 activity (Fig. 2C and D), suggesting the involvement of the intrinsic apoptotic pathway in PSP-induced apoptosis in PC-3 cells.

Cell cycle analysis revealed a marked increase in the % cells in the S phase after 72 h of PSP treatment compared with the blank control. Specifically, at a concentration of 4 mg/ml, PSP significantly increased the portion of PC-3 cells in the S phase from 19.7 to 49.0% (Fig. 3).

These results indicate that PSP significantly promotes apoptosis in PC-3 cells and arrests the cell cycle in the S phase. Furthermore, PSP appears to induce apoptosis of PC-3 cells in vitro through the activation of the caspase-3 pathway.

To assess the impact of PSP on the invasion and migration of PC-3 cells, the cells were treated with several concentrations of PSP for 72 h, and invasion and migration were measured using a Transwell assay. The results revealed a significant reduction in both invasion and migration cell numbers with increasing PSP concentrations, particularly at 2 and 4 mg/ml compared to concentration of 0 µg/ml (Figs. 4 and 5). Wound healing assays also demonstrated that the wound healing of PC-3 cells was inhibited by PSP in a dose-dependent manner (Fig. 6A). Furthermore, the scratch closure change indicated that PSP could significantly prevent wound healing, especially at concentrations of 2 and 4 mg/ml compared to 0 µg/ml, at 24, 48 and 72 h (Fig. 6B). These results indicate that PSP could inhibit the invasion and migration of PC-3 cells.

Elevated expression of MDR-1 is known to be associated with hormone-independent prostate cancer, making it crucial to identify effective drugs to reverse multidrug resistance (MDR) and enhance the efficacy of prostate cancer chemotherapy (23,24). As PC-3 cells are androgen-independent, the present study used PC-3 cells as an MDR model to assess the effects of PSP on the MDR-1 gene and P-gp protein expression. This was evaluated using reverse transcription-quantitative PCR and immunocytochemistry. The results indicated that MDR-1 gene expression decreased gradually with increasing PSP concentrations, with the most significant reduction observed at 4 mg/ml, where MDR-1 gene expression was significantly lower compared with at other concentrations (Fig. 7A). Similarly, the expression of P-gp also decreased gradually with higher PSP concentrations, particularly at 4 mg/ml (Fig. 7B). These findings suggest that PSP may have the potential to reverse MDR in PC-3 cells.

To assess the potential mechanisms through which PSP affects the biological behavior of prostate cancer cells, western blot analysis was performed on proteins involved in the PI3K-AKT and NF-kB signaling pathways after PC-3 cells were treated with several concentrations of PSP for 72 h. The results revealed no significant differences in the levels of total AKT, PI3K and P65 among the groups. However, the phosphorylated forms, p-P65, p-PI3K and p-AKT, demonstrated a concentration-dependent decrease, with the most pronounced reduction observed at 4 mg/ml. At this concentration, the levels of p-P65, p-PI3K and p-AKT were significantly lower compared with at other concentrations (Fig. 8).