Anti-tumor activities of Panax quinquefolius saponins and potential biomarkers in prostate cancer

Abstract

Background: Prostate carcinoma is the second most common cancer among men worldwide. Developing new therapeutic approaches and diagnostic biomarkers for prostate cancer (PC) is a significant need. The Chinese herbal medicine Panax quinquefolius saponins (PQS) have been reported to show anti-tumor effects. We hypothesized that PQS exhibits anti-cancer activity in human PC cells and we aimed to search for novel biomarkers allowing early diagnosis of PC.

Methods: We used the human PC cell line DU145 and the prostate epithelial cell line PNT2 to perform cell viability assays, flow cytometric analysis of the cell cycle, and FACS-based apoptosis assays. Microarray-based gene expression analysis was used to display specific gene expression patterns and to search for novel biomarkers. Western blot and quantitative real-time PCR were performed to demonstrate the expression levels of multiple cancer-related genes.

Results: Our data showed that PQS inhibited the viability of DU145 cells and induced cell cycle arrest at the G1 phase. A significant decrease in DU145 cell invasion and migration were observed after 24 h treatment by PQS. PQS up-regulated the expression levels of p21, p53, TMEM79, ACOXL, ETV5, and SPINT1 while it down-regulated the expression levels of bcl2, STAT3, FANCD2, DRD2, and TMPRSS2.

Conclusion: PQS promoted cells apoptosis and inhibited the proliferation of DU145 cells, which suggests that PQS may be effective for treating PC. TMEM79 and ACOXL were expressed significantly higher in PNT2 than in DU145 cells and could be novel biomarker candidates for PC diagnosis.

Keywords: ACOXL, Acyl-CoA oxidase-like protein; Chinese medicinal herbs; DRD2, dopamine receptor D2; ETV5, ETS variant 5; FACS, fluorescence-activated cell sorting; FANCD2, fanconi anemia group D2; PC, prostate cancer; PQS, Panax quinquefolius saponins; Panax quinquefolius; Potential biomarkers; Prostate cancer cells; SPINT1, serine peptidase inhibitor Kunitz type 1; STAT3, signal transducer and activator of transcription 3; TCM, Traditional Chinese Medicine; TMEM79, transmembrane protein 79; TMPRSS2, transmembrane protease serine 2; bcl2, B-cell lymphoma 2; p21, cyclin-dependent kinase inhibitor p21; p53, tumor suppressor p53; qRT-PCR, quantitative real-time PCR; saponins.

Fig. 1

Quality control of six batches of purified PQS by UHPLC-QTOF-MS/MS. The fingerprints of ginsenosides in the methanol fraction include Re, Rg1, Rc, Rb1, Rb2, Rb3, Rd, and Rg3. The characteristic peaks were consistent among all six batches repeated within one month.

Fig. 2

PQS inhibited the viability of DU145 cells in a dose-dependent manner. Human PC DU145 cells and prostate epithelial PNT2 cells were treated without (0 μg/mL, vehicle control) or with increasing concentrations of PQS (5, 15, 50, 100, and 150 μg/mL) for 48 h. Cells were photographed using a microscope (A) and cell viability (% of control, B) was measured using a Cell Titer-Blue Cell Viability assay kit. Error bars represent standard deviations (SD) from n ≥ 3 independent experiments in triplicate. ∗ and ∗∗∗∗ represent p < 0.05 and p < 0.0001, as obtained by one-way ANOVA.

Fig. 3

PQS induced the apoptosis of DU145 cells. DU145 cells were treated with PBS as a negative control, 100% ethanol as a positive control, and PQS (150 μg/mL) for 24 and 48 h and measured by FACS using a FITC Annexin V Apoptosis Detection Kit with PI. Cells treated with PQS exhibited a significantly increased apoptosis in a time-dependent manner. Total apoptosis of cells increased from 78.6% at the time point of 24 h to 96.1% total apoptosis after 48 h of PQS treatment. The viable cell percentage was significantly reduced from 11.9% after 24 h to 2.7% after 48 h of treatment.

Fig. 4

PQS treatment induced caspase-3/7 activity (A), expression levels of cleaved caspase-3 (B) and cytotoxicity (C). DU145 cells were treated with PQS (0, 15, 50, 100, and 150 μg/mL) for 48 h for the ApoTox-Glo Triplex assay and western blots, and for 24 h and 48 h for LDH cytotoxicity assay. Caspase-3/7 activity was determined by luminescence measurement and is shown as fold increase of treated cells versus untreated cells (vehicle) (A). Western blot analysis of cleaved caspase-3 (B) showed that PQS treatment raised the levels of the cleaved caspase-3 in a dose-dependent manner, which is in agreement with the data of caspase-3/7 activity assay. Cytotoxicity was determined using a LDH release assay by fluorescence measurement at 485Ex/520Em (C). PQS caused a significant, time- and dose-dependent increase of LDH release at concentrations of 15 to 150 μg/mL in comparison with the control group. Error bars represent standard deviations (SD) from n ≥ 3 independent experiments in triplicate. ∗, ∗∗, and ∗∗∗∗ represent p < 0.05, p < 0.01, and p < 0.0001, as obtained by one-way ANOVA (A).

Fig. 5

PQS treatment inhibited the migration of DU145 cells as shown by a wound healing assay. DU145 and PNT2 cells were treated with the indicated concentration of PQS and were tested using the CytoSelect Wound Healing Assay kit. Wound closures were photographed at 0 h and 24 h after wounding (A). The wound closure of DU145 cells was significantly smaller than that of PNT2 cells at concentrations of 50 and 150 μg/mL (B). Error bars represent standard deviations (SD) from n ≥ 3 independent experiments in triplicate. ∗∗∗∗ represents p < 0.0001 as obtained by one-way ANOVA.

Fig. 6

PQS treatment reduced the invasion of DU145 cells. Cells were treated with different concentrations of PQS (50 and 150 μg/mL) for 24 h. Invasion assays were performed using the CytoSelect Invasion Assay kit. Both concentrations of PQS inhibited DU145 cell invasion compared to untreated cells. Only 43.28% of DU145 cells were able to invade through the polycarbonate membrane after treatment with 150 μg/mL PQS. Error bars represent standard deviations (SD) from n ≥ 3 independent experiments in triplicate. ∗∗ and ∗∗∗∗ represent p < 0.01 and p < 0.0001 as obtained by two-way ANOVA.

Fig. 7

PQS treatment induced the cell cycle arrest at the G1 phase in DU145 cells. DU145 cells were treated with the indicated concentrations of PQS for 48 h. Propidium iodide was used for FACS analysis of DNA content in fixed cells. PQS increased the number of cells in the G1 phase from 46.58% to 58.73% and decreased the number of cells in the G2 phase from 31.39% to 26.32% and in the S phase from 22.04% to 14.95% in a dose-dependent manner.

Fig. 8

PQS treatment regulated the expression levels of multiple cancer-related genes and pathways. An oligonucleotide microarray was used to analyze the miRNA expression profiles in DU145 and PNT2 cells after PQS treatment (A, B). Hierarchical clustering analysis was employed to evaluate differential miRNA expression between treated and untreated groups. p21, p53, ETV5, SPINT1, ACOXL, and TMEM79 were up-regulated, whereas bcl2, DRD2, TMPRSS2, FANCD2, and STAT3 were down-regulated in the treatment groups. The enrichment scores of ten significant up- and down-regulated pathways are shown (C, D).

Fig. 9

PQS treatment regulated the expression of multiple cancer-related genes at the mRNA level. DU145 cells were treated with indicated concentrations of PQS for 48 h. mRNA of cells was extracted for qRT-PCR. The expression levels of p21, p53, TMEM79, ACOXL, SPINT1, and ETV5 were up-regulated after PQS treatment (A–F). The expression levels of bcl2, STAT3, FANCD2, TMPRSS2, and DRD2 were down-regulated after PQS treatment (G–K). The qRT-PCR was performed using a SYBR Green PCR Master Mix kit. Error bars represent standard deviations (SD) from n ≥ 3 independent experiments in triplicate. ∗, ∗∗, ∗∗∗, and ∗∗∗∗ represent p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 as obtained by one-way ANOVA.

Fig. 10

PQS treatment regulated the expression of multiple cancer-related genes at the protein level. DU145 cells were collected after PQS treatment for 48 h. Cell lysates were prepared for SDS-PAGE and western blotting. β-Actin served as the control protein. The expression levels of p21, p53, TMEM79, and ACOXL were up-regulated and the expression levels of bcl2, FANCD2, TMPRSS2, and DRD2 were down-regulated after PQS treatment in a dose-dependent manner. Proteins were detected using BCIP/NBT color development substrate.