Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane

Abstract

Inhibition of metabolic re-programming represents an attractive approach for prevention of prostate cancer. Studies have implicated increased synthesis of fatty acids or glycolysis in pathogenesis of human prostate cancers. We have shown previously that prostate cancer prevention by sulforaphane (SFN) in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model is associated with inhibition of fatty acid metabolism. This study utilized human prostate cancer cell lines (LNCaP, 22Rv1 and PC-3), two different transgenic mouse models (TRAMP and Hi-Myc) and plasma specimens from a clinical study to explore the glycolysis inhibition potential of SFN. We found that SFN treatment: (i) decreased real-time extracellular acidification rate in LNCaP, but not in PC-3 cell line; (ii) significantly downregulated expression of hexokinase II (HKII), pyruvate kinase M2 and/or lactate dehydrogenase A (LDHA) in vitro in cells and in vivo in neoplastic lesions in the prostate of TRAMP and Hi-Myc mice; and (iii) significantly suppressed glycolysis in prostate of Hi-Myc mice as measured by ex vivo1H magnetic resonance spectroscopy. SFN treatment did not decrease glucose uptake or expression of glucose transporters in cells. Overexpression of c-Myc, but not constitutively active Akt, conferred protection against SFN-mediated downregulation of HKII and LDHA protein expression and suppression of lactate levels. Examination of plasma lactate levels in prostate cancer patients following administration of an SFN-rich broccoli sprout extract failed to show declines in its levels. Additional clinical trials are needed to determine whether SFN treatment can decrease lactate production in human prostate tumors.

Figure 1.

SFN treatment decreased ECAR and protein levels of HKII, PKM2 and LDHA in prostate cancer cells. Pharmacologic profiling of ECAR in LNCaP cells through real-time measurements using the Seahorse flux analyzer following 6- (A) or 9-h (B) treatment with DMSO or SFN (5 or 10 µM). After measurement of basal ECAR, the cells were treated with metabolic inhibitors, including 1 µM oligomycin (O), 300 nM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (F), 1 mM 2-deoxy glucose (2-DG) or 1 µM rotenone (R) at the indicated times. Basal ECAR was calculated using the difference between the mean of time points prior to injection of O (#1 to #4). Oligomycin-sensitive rate was calculated using the difference between the mean of time points prior to injection of F (#5 to #7). Quantitation of basal ECAR (C) and oligomycin-sensitive ECAR (D) in LNCaP cells following 9-h treatment with DMSO or SFN. Quantitative data for the 6-h time point are not shown as the difference was not significant. The experiment was repeated three times in triplicate, and combined data are expressed as mean ± SEM. Significantly different (*P < 0.05) compared with control by one-way ANOVA followed by Dunnett’s test. (E) Confocal images (×63 oil objective magnification) for HKII, PKM2 and LDHA (green fluorescence) in LNCaP and 22Rv1 cells following 24-h treatment with DMSO or 10 µM SFN. Nucleus was stained with DRAQ5 (blue fluorescence). (F) Quantitation of corrected total cell fluorescence (CTCF) using ImageJ software in LNCaP and 22Rv1 cells following treatment with DMSO or SFN for the specified time points. *Significant (P < 0.05) compared with DMSO-treated control by Student’s t-test. Results were consistent in two independent experiments.

Figure 2.

SFN treatment inhibited expression of glycolysis-related enzyme proteins in prostate cancer cells in vitro and in vivo. (A) Immunoblotting for HKII, PKM2 and LDHA proteins using lysates from LNCaP and 22Rv1 cells after 24-h treatment with DMSO or the indicated doses of SFN. Numbers above bands are fold changes in protein expression relative to respective DMSO-treated controls. (B) Immunohistochemical images for HKII, PKM2 and LDHA protein expression in representative prostate adenocarcinoma sections of TRAMP mice (×40 objective magnification, scale bar = 50 µm). (C) H-score for HKII, PKM2 and LDHA protein expression in TRAMP tumor section. The results shown are mean ± SD (n = 6). Statistical analysis was performed by Student’s t-test (*P < 0.05).

Figure 3.

SFN administration decreased lactate levels in the plasma and prostate adenocarcinoma of TRAMP mice in vivo. (A) Lactate levels in the plasma and prostate tumors of control and SFN-treated TRAMP mice. Results shown are mean ± SD (n = 18–19 for plasma and n = 13–20 for tumors). Plasma and tumor samples from different mice of each group were used for the assay. Statistical significance was determined by Student’s t-test (*P < 0.05). (B) Representative ex vivo¹H magnetic resonance spectrum of different metabolites in the prostate of a control Hi-Myc mouse. (C) Quantitation of the metabolite levels in the prostate of Hi-Myc mice. The results shown are mean ± SD (n = 6 for control group and n = 5 for SFN treatment group). Statistical significance was determined by Student’s t-test (*P < 0.05).

Figure 4.

SFN treatment downregulated expression of glycolysis-related proteins in PIN lesions of Hi-Myc mice. Immunohistochemical images for HKII, PKM2 and LDHA protein expression in representative prostate sections of control and SFN-treated Hi-Myc mice (×40 objective magnification, scale bar = 50 µm) are shown in the left panel. Quantitative data for H-score are shown are mean ± SD (n = 5) in the right panel. Statistical analysis was performed by Student’s t-test (*P < 0.05).

Figure 5.

Overexpression of Myc, but not constitutively active Akt (caAkt) partially attenuated SFN-mediated decrease in protein levels of HKII, PKM2, and LDHA in 22Rv1 cells. (A) Immunoblotting for Myc, total Akt, and phosphorylated s473 Akt using lysates from 22Rv1 cells stably transfected with empty vector, Myc or caAkt. (B) Immunoblotting for HKII, PKM2 and LDHA using lysates from 22Rv1 cells stably transfected with empty vector (EV) or Myc or caAkt plasmids and treated for 24 h with DMSO or the indicated doses of SFN. (C) Representative images of colonies resulting from 22Rv1 cells after 8 days of treatment with DMSO or the indicated doses of SFN. (D) Quantitation of colony formation. 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 or caAkt by one-way ANOVA followed by Bonferroni’s multiple comparisons test. Comparable results were obtained from replicate experiments. (E) Intracellular lactate levels in 22Rv1 cells stably transfected with EV or Myc or caAkt plasmids and treated for 24 h with DMSO or the indicated doses of SFN. 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 or caAkt by one-way ANOVA followed by Bonferroni’s multiple comparisons test. Each experiment was repeated at least two times.

Figure 6.

SFN-BSE administration did not alter plasma levels of lactate or pyruvate in a clinical study. Basal and post-SFN-BSE treatment plasma levels of lactate (A) and pyruvate (B). (C) A cartoon summarizing mechanistic effects of SFN in prostate cancer cells (22,23,25–27,41,42).