Muscadine grape skin extract inhibits prostate cancer cells by inducing cell-cycle arrest, and decreasing migration through heat shock protein 40

Abstract

Previously we demonstrated that muscadine grape skin extract (MSKE), a natural product, significantly inhibited androgen-responsive prostate cancer cell growth by inducing apoptosis through the targeting of survival pathways. However, the therapeutic effect of MSKE on more aggressive androgen-independent prostate cancer remains unknown. This study examined the effects of MSKE treatment in metastatic prostate cancer using complementary PC-3 cells and xenograft model. MSKE significantly inhibited PC-3 human prostate cancer cell tumor growth in vitro and in vivo. The growth-inhibitory effect of MSKE appeared to be through the induction of cell-cycle arrest. This induction was accompanied by a reduction in the protein expression of Hsp40 and cell-cycle regulation proteins, cyclin D1 and NF-kBp65. In addition, MSKE induced p21 expression independent of wild-type p53 induced protein expression. Moreover, we demonstrate that MSKE significantly inhibited cell migration in PC-3 prostate cancer cells. Overall, these results demonstrate that MSKE inhibits prostate tumor growth and migration, and induces cell-cycle arrest by targeting Hsp40 and proteins involved in cell-cycle regulation and proliferation. This suggests that MSKE may also be explored either as a neo-adjuvant or therapeutic for castration resistant prostate cancer.

Keywords: Cancer research; Cell biology; Molecular biology.

Fig. 1

MSKE inhibits cell growth in PC-3 prostate cancer cells. (A) Cell cultures were treated with various concentrations of MSKE (2.5, 5, 10, 20, 40 μg/ml) during different time intervals (24, 72, 120 hours). The effect of MSKE on growth was determined by MTT proliferation assay in PC-3 cells. Sample absorption (indicative of formazan formation) was determined using an absorbance plate reader (OPTImax microplate reader, MTX Labsystems, Vienna, VA) at 490 nm. Results are expressed as mean absorbance plus or minus standard error of the mean (mean ± S.E. *). (B) Clonogenic assay confirms the inhibition of growth of the PC-3 prostate after treatment with various μg/ml concentrations of MSKE after 72 hours. (C) MSKE (20 and 40 μg/ml) did not significantly induce apoptosis in PC-3 cells after 24 hours using Annexin V-FITC. Columns mean absorbance; bars, ±SE. *, is the significant difference between MSKE treatment and ETOH and untreated control.

Fig. 2

MSKE arrested G0/G1 phase transition in PC-3 cells. (A) Describes the relative percentage of cells in the G0/G1 phase of cell cycle after 12 hours of MSKE treatment, (B) after 24 hours of MSKE treatment; Resveratrol treatment (25 μM) used as a control for G0/G1 arrest. The cells were fixed by adding 400 μl of ethanol and incubated on ice for 15 minutes. The cells were then centrifuged at 1500 rpm for 5 minutes and the pellet was re-suspended in 200 μl propidium iodide (PI) solution containing 50 μg/ml PI (Biotium), 0.1 mg/ml RNase A (Sigma-Aldrich), and 0.05% Triton X-100 (Sigma-Aldrich). The cellometer allows simultaneous acquisition of bright-field and fluorescence images for concentration, size, and viability measurement to measure cell cycle transition. The fluorescence intensity and size measurement were recorded for each cell contained within a data set, which was exported from the software into an Excel file, and imported into FlowJo (Tree Star, Oregon) for cytometric data analysis. Columns mean percentage of populated cells in G0/G1 phase of the cell cycle; bars, ±SE. * for three independent experiments, is the significant difference between treated and control.

Fig. 3

MSKE treatment decreases the expression of antibodies, Hsp40, NFKB/p65, and cyclin D1 (diluted 1:500) while increasing the mRNA expression of p21 independent of induced p53 in vitro. (A) Effects of MSKE treatment on protein expression of cell cycle markers by western blot in PC-3 cells. Proteins (50 μg) were separated using 10% or 16% pre-cast Tris-Glycine gels and dry-transferred for seven minutes using iBlot machine (Invitrogen, Gaithersburg, MD) onto PVDF membranes (Invitrogen, Gaithersburg, MD). (B) Confocal microscopy of PC-3 cells fixed and stained with Hsp40 antibody and Alexa Fluor-555 to detect protein expression, (C) Confocal microscopy of LNCaP cells fixed and stained with p53 antibody and Alexa Fluor-488 to detect protein expression of wild-type p53, (D) Fold-change of p21 mRNA in LNCaP and PC-3 cells determined by qRT-PCR analysis. (E) Effects of MSKE treatment on protein expression of p53ser15 and p21 in LNCaP cells. Columns represent mean of fold-change; bars, SE. *, is the significant difference between treatment and control.

Fig. 4

MSKE decreased migration and invasiveness of PC-3 cells. (A) Representative photographs of the wounded PC-3 cell monolayer. Typical result from two independent experiments conducted in triplicates (Magnification power [di1] 200X, Opelco Olympus 1 × 71^® inverted microscope (Optical Elements Cooperation) and DP70 BSW Olympus software Opelco Olympus 1 × 71^® inverted microscope (Optical Elements Cooperation) and DP70 BSW Olympus software), (B) Quantitative assessment of relative cell migration inhibition rate of PC-3 prostate cancer cells. Error bar indicates the standard error of the mean of two independent experiments. Bars, SE*.

Fig. 5

Hsp40 knockout attenuates growth and migration. (A) The transfected PC-3 cells of Hsp40 knockout CRISP/Cas9 plasmid were prepared for evaluating protein expression of Hsp40, Hsp70, Hsp90, and NF-κBp65 in a western analysis. (B)The transfected cells were than trypsanized and the percentage of live and dead cells were counted using trypan blue exclusion assay for cell viability. (C) Moreover, transfected cells were prepared for examining migration using Basement Membrane, Colorimetric Format (Cell Biolabs, INC., San Diego, CA) following the manufacturer's instructions. Columns mean relative percentage of cells that migrate; bars, ±SE. *, is the significant difference between treatment and control.

Fig. 6

Dietary administration of MSKE decreased growth of PC-3 prostate cancer cell in vivo. (A) PC-3 cells were injected two week after the acclimation period. Animals remained on their respective diets for seven weeks after cell injection, until tumors reached 2–3 cm³ in volume, and were then sacrificed. Athymic nude mice were administered by gavage either control or MSKE diet (50 mg/kg/week) after injection of PC-3 human prostate cancer cells at week 1. This produced palpable tumors within 5 days. Cancer preventive efficacy of the treatments was assessed once a week by measuring tumor volume (cm³) = 0.523 × [length × width² (cm²)]. Mice were killed after seven additional weeks on their respective diets. (B) Mean weekly food consumption of control and MSKE treated mice was measured weekly. (C) Tumor volume of PC-3 xenografts after dietary administration was measured weekly. Values are means ± SE. *, is the significant difference between MSKE treatment and control at p < 0.05. (D) MSKE decreased protein expression of Hsp40, Hsp60, and NF-κB under in vivo conditions. Equal loading of protein was confirmed by probing blots for beta actin.

Fig. 7

(A) MSKE inhibits cell growth in E006AA prostate cancer cells. Cell cultures were treated with various concentrations of MSKE (2.5, 5, 10, 20, 40 μg/ml) during different time intervals (24, 48, and 72 hours). At the end of each experiment MTT assays were performed as described in the methods section to estimate cell numbers. (B) MSKE treatment decreases protein expression of Hsp27, Hsp40, Hsp60, hsp70, hsp90, NF-κB, and cyclin D1 in E006AA after 24 hours. (C) Protein array used to examine the effect of MSKE (10, 20 μg/ml) on proteins involved in cell cycle and apoptosis compared to an ethanol control after 24 hours. Results presented as columns represent mean fold-change over ethanol, bars ± SE, *. (D) IHC analysis of Hsp40 was performed in paraffin-embedded tumor samples from control and MSKE-treated mice. Representation of Hsp40 from three IHC samples from tumor specimen. For statistical analyses, an average of three fields of view per sample was used for counting and the average percent positive cells were counted and graphed. Statistical analyses were performed using the Students' t-test with p ≤ 0.05 establishing significance. Values are means ± SE. *, is the significant difference between MSKE treatment and control.

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