Inhibition of Glycolysis in Prostate Cancer Chemoprevention by Phenethyl Isothiocyanate

Abstract

We have shown previously that dietary administration of phenethyl isothiocyanate (PEITC), a small molecule from edible cruciferous vegetables, significantly decreases the incidence of poorly differentiated prostate cancer in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice without any side effects. In this study, we investigated the role of c-Myc-regulated glycolysis in prostate cancer chemoprevention by PEITC. Exposure of LNCaP (androgen-responsive) and 22Rv1 (castration-resistant) human prostate cancer cells to PEITC resulted in suppression of expression as well as transcriptional activity of c-Myc. Prostate cancer cell growth inhibition by PEITC was significantly attenuated by stable overexpression of c-Myc. Analysis of the RNA-Seq data from The Cancer Genome Atlas indicated a significant positive association between Myc expression and gene expression of many glycolysis-related genes, including hexokinase II and lactate dehydrogenase A Expression of these enzyme proteins and lactate levels were decreased upon PEITC treatment in prostate cancer cells, and these effects were significantly attenuated by ectopic expression of c-Myc. A normal prostate stromal cell line (PrSC) was resistant to lactic acid suppression by PEITC treatment. Prostate cancer chemoprevention by PEITC in TRAMP mice was associated with a significant decrease in plasma lactate and pyruvate levels. However, a 1-week intervention with 10 mg PEITC (orally, 4 times/day) was not sufficient to decrease lactate levels in the serum of human subjects. These results indicated that although prostate cancer prevention by PEITC in TRAMP mice was associated with suppression of glycolysis, longer than 1-week intervention might be necessary to observe such an effect in human subjects. Cancer Prev Res; 11(6); 337-46. ©2018 AACR.

Figure 1

PEITC treatment inhibited c-Myc protein expression in prostate cancer cells. (A) Immunoblotting for c-Myc protein using lysates from LNCaP and 22Rv1 following 8, 16, and 24 h treatment with DMSO or specified concentrations of PEITC. Numbers above bands are fold changes in c-Myc protein expression relative to respective DMSO-treated control. (B) Confocal images (63× oil objective magnification) depicting effect of 24 h PEITC treatment on c-Myc expression (red fluorescence) in LNCaP and 22Rv1 cells. DRAQ5 (blue fluorescence) was used for nuclear staining. (C) Quantitation of corrected total cell fluorescence (CTCF) for c-Myc expression using ImageJ software. Results shown are mean ± SD (n=20). *Statistically significant (P < 0.05) compared with DMSO-treated control by Student’s t-test. (D) c-Myc-associated luciferase activity in LNCaP and 22Rv1 cells after 24 h treatment with DMSO or the indicated doses of PEITC. Results shown are mean ± SD (n = 3). *Statistically significant (P < 0.05) compared with control by one-way ANOVA with Dunnett’s adjustment. Each experiment was repeated at least twice and the results were consistent.

Figure 2

Overexpression of c-Myc partially attenuated PEITC-mediated decrease in colony formation in 22Rv1 and PC-3 cells. (A) Immunoblotting for c-Myc and GAPDH using lysates from 22Rv1 and PC-3 cells stably transfected with empty vector (EV) or the same vector encoding c-Myc (Myc). (B) Representative images of colonies from 22Rv1 and PC-3 cells after 8 days of treatment with DMSO or the indicated doses of PEITC. (C) Quantitation of colony formation. Combined results from two independent experiments are shown as mean ± SD (n = 6). Statistically significant (P < 0.05) compared with the *corresponding DMSO-treated control or ^#between cells transfected with EV and Myc by one-way ANOVA followed by Bonferroni’s multiple comparisons test. (D) Correlation of Myc expression with that of key glycolysis enzymes in prostate tumors from TCGA. Pearson test was used to determine correlation.

Figure 3

PEITC treatment decreased protein levels of HKII, PKM2, and LDHA in prostate cancer cells. (A) Immunocytochemistry for HKII, PKM2, and LDHA (green fluorescence) in LNCaP and 22Rv1 cells following 24 h treatment with DMSO or 5 μmol/L PEITC. Nucleus was stained with DRAQ5 (blue fluorescence). Results were consistent in two independent experiments. (B) Quantitation of corrected total cell fluorescence (CTCF) for HKII, PKM2, and LDHA expression using ImageJ software. Results shown are mean ± SD (n=20). *Statistically significant (P < 0.05) compared with DMSO-treated control by Student’s t-test.

Figure 4

PEITC-mediated decrease in protein levels of HKII and LDHA were attenuated by c-Myc overexpression. (A) Immunoblotting for HKII, PKM2, and LDHA using lysates from LNCaP and 22Rv1 cells after 24 h treatment with DMSO or specified concentrations of PEITC. Numbers above bands represent fold changes in protein expression relative to corresponding DMSO-treated control. (B) Immunoblotting for HKII, PKM2, and LDHA using lysates from 22Rv1 and PC-3 cells stably transfected with EV or Myc plasmid and treated for 24 h with DMSO or the indicated doses of PEITC. Increased expression of HKII, PKM2, and LDHA protein was observed in both cell lines upon stable overexpression of c-Myc protein. (C) Intracellular lactate levels in 22Rv1 cells stably transfected with EV or Myc plasmid and treated for 24 h with DMSO or indicated doses of PEITC. Results shown are mean ± SD (n = 3). Statistically significant (P < 0.05) compared with the *corresponding DMSO-treated control or ^#between cells transfected with EV and Myc by one-way ANOVA followed by Bonferroni’smultiple comparisons test.

Figure 5

PEITC administration decreased lactate and pyruvate levels in the plasma of TRAMP mice. Levels of lactate (A) and pyruvate (B) in the plasma of TRAMP mice fed basal AIN-76A diet (n = 17 for lactate and n = 11 for pyruvate) or the same diet supplemented with PEITC (n = 16 for lactate and n = 11 for pyruvate). Results shown are mean ± SD. Statistical significance was determined by Student’s t-test. (C) Baseline and posttreatment lactate levels in the serum of smokers after 1-week intervention with placebo (n = 41) or PEITC (n = 45, 10 mg PEITC, orally in olive oil, 4 times/day).

Figure 6

Schematic summary of the mechanism underlying glycolysis inhibition by PEITC. PEITC = Phenethyl Isothiocyanates; HKII = Hexokinase II; GPI = Glucose-6-phosphate isomerase; PFK1= Phosphofructokinase-1; ALDOA = Fructose-bisphosphate aldolase A; GAPDH = Glyceraldehyde-3-phosphate dehydrogenase; PGK1 = Phosphoglycerate kinase 1; PGAM1 = Phosphoglycerate mutase 1; ENO = Enolase; PKM2 = Pyruvate kinase M2; LDHA = Lactate dehydrogenase A.