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.
Prostate cancer continues to be a leading cause of cancer-related mortality among American men (1). Chemoprevention embodies a sensible alternative for reducing the mortality and morbidity from prostate cancer because many risk factors associated with this neoplasm are not easily modifiable (2–5). Chemoprevention of prostate cancer has been attempted clinically with 5α-reductase inhibitors finasteride (PCPT trial) and dutasteride (REDUCE trial) as well as with novel combinations such as vitamin E plus selenium in the SELECT trial (6–9). The PCPT and REDUCE trials showed a 23% to 25% decrease in the relative risk, but these agents were not approved by the FDA for prostate cancer chemoprevention because of the prevalence of high-grade tumors in the treatment arm (6, 7). High-grade prostate cancers were still more frequent in the finasteride arm when compared with controls in an 18-year follow-up of the PCPT trial (10). The SELECT trial did not show any preventative benefit of the vitamin E and selenium supplementation on prostate cancer (8, 9). Therefore, a safe and inexpensive intervention for chemoprevention of prostate cancer is still a clinically unmet need.
Inspired by the results of epidemiologic studies suggesting an inverse association between dietary intake of cruciferous vegetables and the risk of prostate cancer, isothiocyanate family of metabolic byproducts resulting from cutting or chewing of such vegetables has been studied rather extensively for the chemoprevention of prostate cancer (11–13). A study led by Cohen and colleagues (12) concluded that, after adjusting for covariates and total vegetable intake, the OR for prostate cancer risk for comparison of three or more servings/week of cruciferous vegetables with <1 serving/week was 0.59 with a two-sided Ptrend = 0.02. Phenethyl isothiocyanate (PEITC) is one such bioactive phytochemical in cruciferous vegetables (e.g., watercress) with strong in vitro and in vivo anticancer activity againt prostate cancer (11, 14–18). PEITC treatment also inhibited angiogenesis in vitro and ex vivo (19). The in vivo inhibitory effect of PEITC or its metabolite (N-acetylcysteine conjugate) on growth of human prostate cancer cells was reported in xenograft models (17, 20–22). For example, oral treatment with 12 μmol PEITC/day (5 times/week) significantly retarded growth of PC-3 human prostate cancer cells subcutaneously implanted in male athymic mice (17). In another study, dietary feeding of the N-acetylcysteine conjugate of PEITC (8 μmol/g diet) to PC-3 xenograft–bearing athymic mice resulted in a significant decrease in tumor size in 100% of the mice (20). Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model has been used to determine the efficacy of PEITC for chemoprevention of prostate cancer (23, 24). Dietary feeding of PEITC (3 μmol/g diet) decreased the incidence of poorly differentiated adenocarcinoma in the dorsolateral prostate of TRAMP mice by about 36% (P = 0.04) when compared with controls without any side effects (24).
Our prior in vitro cellular studies using the Seahorse flux analyzer revealed a statistically significant decrease in intracellular acidification rate, a measure of lactic acid production and glycolysis, upon PEITC treatment in human prostate cancer cells (18). However, the in vivo significance of these observations is still elusive. Because c-Myc is overexpressed in human prostate cancers and known to regulate glycolysis (25–27), the current study was undertaken to determine whether prostate cancer chemoprevention by PEITC was associated with suppression of c-Myc–regulated glycolysis. Human prostate cancer cell lines (LNCaP and 22Rv1), plasma from control and PEITC-fed TRAMP mice, and serum specimens from a clinical study were used to address this question. The results of the current study suggest that plasma lactate may be a useful biomarker of PEITC response in the TRAMP model.
Materials and Methods
Frozen plasma specimens from TRAMP mice fed basal AIN-76A diet or the same diet supplemented with 3 μmol PEITC/g diet for 19 weeks were used to measure lactate and pyruvate levels (24). This study was approved by the University of Pittsburgh Animal Care and Use Committee. A clinical study with PEITC is yet to be performed in subjects predisposed to or with prostate cancer, but serum specimens from a prior clinical study in smokers (28) were available and used to complement this study. This study was approved by the FDA under IND 74,037 (28). Male (n = 20 for the placebo arm and n = 25 for the PEITC arm) or female (n = 21 for the placebo arm and n = 20 for the PEITC arm) smokers were enrolled to the placebo group (n = 41) or PEITC group (n = 45; ref. 28). PEITC (10 mg) was dissolved in 1 mL of olive oil and administered through a plastic syringe 4 times per day (cumulative daily PEITC dose of 40 mg), once every 4 hours for 1 week (28). Placebo consisted of 1 mL of olive oil without PEITC that was similarly administered (28). Serum from the blood collected at baseline and posttreatment was used for this study.
PEITC (purity ≥ 98%) was purchased from LKT laboratories. Stock solution of PEITC (65 mmol/L) was prepared in DMSO. Cell culture reagents were purchased from Life Technologies-Thermo Fisher Scientific or Corning. The antibodies against c-Myc, hexokinase II (HKII), pyruvate kinase M2 (PKM2), and lactate dehydrogenase A (LDHA) were from Cell Signaling Technology, an antibody against GAPDH was from GeneTex, the anti–β-actin antibody was from Sigma-Aldrich, the mouse monoclonal anti-LDHA antibody was from Santa Cruz Biotechnology, and the Alexa Fluor 488- or 568-conjugated goat anti-rabbit antibody and Alexa Fluor 488-conjugated anti-mouse antibody were from Life Technologies. Kits for colorimetric measurements of lactate and pyruvate were purchased from BioVision.
The LNCaP, 22Rv1, and PC-3 human prostate cancer cell lines were purchased from the ATCC and cultured by following the supplier's recommendations. These cell lines were last authenticated by us in March 2017 and found to be of human origin. PC-3 cells stably transfected with empty pcDNA3 vector or pcDNA3-Myc plasmid were maintained as described by us previously (29). The 22Rv1 cells were stably transfected with empty pcDNA3 vector or the same vector encoding Myc and selected in the presence of 400 μg/mL of G418. A normal human prostate stromal cell line (PrSC) was purchased from Lonza and cultured as instructed by the supplier.
LNCaP (2 × 104) or 22Rv1 (3 × 104) cells were plated on coverslips in 24-well plates. After overnight incubation, cells were treated with DMSO or the indicated dose of PEITC for 8, 16, and/or 24 hours. The cells were then fixed and permeabilized with 2% paraformaldehyde and 0.5% Triton X-100, respectively. After blocking with PBS containing 0.5% BSA and 0.15% glycine, cells were treated overnight at 4°C with the desired primary antibody followed by Alexa Fluor 488- or 568-conjugated goat anti-rabbit antibody or Alexa 488–conjugated goat anti-mouse antibody for 1 hour and then counterstained with DRAQ5 (nuclear stain) for 5 minutes at room temperature in the dark. Corrected total cell fluorescence was quantitated using ImageJ software.
LNCaP or 22Rv1 cells were cotransfected with 2 μg pBV-Luc wt MBS1-4 plasmid and 0.2 μg of pCMV-RL using FuGENE6. Twenty-four hours after cotransfection, cells were treated with DMSO or the indicated doses of PEITC for 24 hours. Luciferase activity was determined using Dual-Luciferase Reporter Assay kit from Promega, and by following the manufacturer's instructions.
Colony formation assay
Empty vector–transfected control and c-Myc–overexpressing PC-3 (500 cells) or 22Rv1 (1,000 cells) were seeded in 6-well plates in triplicate and allowed to attach by overnight incubation. Cells were treated with DMSO or desired doses of PEITC. The medium containing DMSO or PEITC was replaced every third day. After 8 days of treatment, cells were fixed with 100% methanol for 5 minutes at room temperature and stained with 0.5% crystal violet solution in 20% methanol for 30 minutes at room temperature. The colonies from control and PEITC treatment groups were counted under an inverted microscope.
RNA-Seq expression profile in The Cancer Genome Atlas
Association of Myc expression with that of glycolysis-related genes in prostate cancer was determined from the RNA sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) using University of California Santa Cruz Xena Browser (http://xena.ucsc.edu/public-hubs/).
Determination of metabolites
Lactate or pyruvate levels in lysates of 22Rv1 cell, the plasma of TRAMP mice, and the serum of human subjects were measured using commercially available kits according to the supplier's instructions.
Statistical significance of difference for cellular in vitro parameters and mouse specimens were determined by Student t test or one-way ANOVA, followed by Dunnett adjustment or Bonferroni multiple comparisons test using GraphPad Prism (Version 7.02). Statistical significance of difference in human serum lactate levels between placebo and PEITC groups was determined by mixed effects ANOVA using R (Version 3.4.2).
PEITC treatment inhibited expression and transcriptional activity of c-Myc in prostate cancer cells
The level of c-Myc protein was decreased dose dependently upon PEITC exposure in both LNCaP and 22Rv1 cells as evidenced by immunoblotting (Fig. 1A). PEITC-mediated downregulation of c-Myc protein was confirmed by immunocytochemistry (Fig. 1B). The c-Myc protein staining was predominant in the nucleus of each cell line, which was decreased significantly upon 24-hour treatment with 2.5 μmol/L PEITC (Fig. 1C). PEITC treatment resulted in a dose-dependent and statistically significant decrease in transcriptional activity of Myc as evidenced by luciferase reporter assay (Fig. 1D). These results indicated suppression of protein level as well as transcriptional activity of Myc in androgen-responsive (LNCaP) and castration-resistant (22Rv1) human prostate cancer cell lines following exposure to plasma achievable levels of PEITC (24).
Prostate cancer cell growth inhibition by PEITC was attenuated by c-Myc overexpression
We stably overexpressed c-Myc protein in 22Rv1 cells (hereafter empty vector–transfected control cells and c-Myc–overexpressing cells abbreviated as EV and Myc, respectively) to determine the functional significance of c-Myc suppression by PEITC. The c-Myc–overexpressing PC-3 cells generated for another project (29) were also included in these experiments. Immunoblotting confirmed overexpression of c-Myc protein in stably transfected 22Rv1 and PC-3 cells in comparison with corresponding EV-transfected controls (Fig. 2A). Overexpression of c-Myc significantly increased colony formation efficiency in both cell lines when compared with corresponding EV cells (Fig. 2B). Colony formation was dose dependently and significantly inhibited by PEITC treatment in EV and Myc cells (Fig. 2C). Moreover, c-Myc overexpression conferred significant protection against PEITC-mediated inhibition of colony formation in both 22Rv1 and PC-3 cells (Fig. 2C). Collectively, these results provided evidence for functional significance of c-Myc downregulation by PEITC.
PEITC treatment inhibited c-Myc–regulated glycolysis in prostate cancer cells
Analysis of the RNA-Seq data in prostate cancer from TCGA revealed a statistically significant positive association between Myc expression and that of HKII and LDHA, but not PKM2 (Fig. 2D) or LDH-B (data not shown). We also examined the association of Myc expression with that of other genes of the glycolytic pathway. These analyses revealed a significant positive association of Myc expression with that of phosphofructokinases and phosphoglycerate kinase (data not shown). No such association was observed for glucose-6-phosphate isomerase, aldolase A, GAPDH, or enolase (data not shown). On the other hand, a negative association was observed for expression of Myc and that of phosphoglycerate mutase 1 and pyruvate kinase isozymes M1 (data not shown).
Representative microscopic images for protein levels of key glycolysis enzymes, including HKII (which phosphorylates six-carbon sugars including glucose), PKM2 (which catalyzes the last step of glycolysis of conversion of phosphoenolpyruvate to pyruvate), and LDHA (which is responsible for conversion of pyruvate to lactate) in LNCaP and 22Rv1 cells following 24-hour treatment with DMSO or PEITC are shown in Fig. 3A. Quantitation of the immunocytochemistry data indicated a statistically significant decrease in protein levels of HKII, PKM2, and LDHA in PEITC-treated LNCaP and 22Rv1 cells especially at 16- and 24-hour time points compared with corresponding controls (Fig. 3B).
Western blotting confirmed suppression of HKII, PKM2, and LDHA protein expression following 24-hour treatment with 2.5 and 5 μmol/L PEITC in both LNCaP and 22Rv1 cells when compared with vehicle-treated control cells (Fig. 4A). These results indicated downregulation of key glycolysis enzymes upon PEITC exposure in prostate cancer cells. Next, we determined the effect of c-Myc overexpression on PEITC-mediated downregulation of glycolysis enzyme proteins. Overexpression of c-Myc resulted in upregulation of HKII, PKM2, and LDHA in both 22Rv1 and PC-3 cells (Fig. 4B). Similar to untransfected cells (Fig. 4A), the protein levels of HKII, PKM2, and LDHA were decreased following PEITC treatment in 22Rv1-EV and PC-3-EV cells. PEITC-mediated decrease in HKII and LDHA protein levels, and to a lesser extent PKM2, was markedly attenuated by c-Myc overexpression in 22Rv1 and PC-3 cells. For example, HKII protein level was decreased by about 80% upon 24-hour exposure of 22Rv1-EV cells to 5 μmol/L PEITC. A similar PEITC treatment resulted in a decrease of only about 10% in HKII protein level in c-Myc–overexpressing 22Rv1 cells (Fig. 4B). These results indicated that PEITC-mediated downregulation of glycolysis enzyme proteins was due to suppression of c-Myc activity.
PEITC treatment decreased lactate levels in prostate cancer cells in vitro
The in vitro effect of PEITC treatment on intracellular lactate level was determined using 22Rv1 cells stably transfected with EV or c-Myc plasmid. Intracellular lactate level was decreased by 27% and 66% upon 24-hour treatment of 22Rv1-EV cells with 2.5 and 5 μmol/L PEITC, respectively, when compared with vehicle-treated control (Fig. 4C). The PEITC-mediated decline in intracellular lactate level was attenuated by c-Myc overexpression (Fig. 4C). On the other hand, a normal human prostate stromal cell line (PrSC) was resistant to lactic acid inhibition by PEITC treatment (24-hour treatment with 2.5 and 5 μmol/L; data not shown).
PEITC treatment decreased lactate levels in the plasma of TRAMP mice in vivo
Archived plasma specimens from our previously published chemoprevention study in TRAMP mice (24) were used to determine the in vivo relevance of these cellular observations. As can be seen in Fig. 5A, lactate level was significantly lower in the plasma of PEITC-fed TRAMP mice when compared with control mice. The pyruvate level was similarly decreased in the plasma of PEITC-fed TRAMP mice in comparison with control (Fig. 5B).
Finally, we used serum specimens collected from male and female smokers after a 1-week PEITC intervention to determine levels of lactate. On average, lactate levels were not significantly different between serum samples at baseline and post PEITC treatment when the results were analyzed cumulatively before or after adjustment for age and sex (Fig. 5C). These results indicated that although prostate cancer chemoprevention by PEITC in TRAMP mice was associated with a significant decrease in plasma lactate levels, oral supplementation of 40 mg PEITC/day for one week did not render significant changes in serum lactate levels at least in active smokers.
Myc is a valid target for chemoprevention of prostate cancer based on the following observations: (i) Myc plays an important role in cancer cell metabolism and proliferation (26); (ii) Myc overexpression was shown to induce PIN in association with loss of a candidate tumor suppressor (Nkx3.1) in mouse luminal epithelial cells (31); (iii) c-Myc is frequently amplified in metastatic prostate cancer, and its nuclear overexpression is observed in early prostate cancer (25, 27); and Myc-driven murine prostate cancer (Hi-Myc mice) exhibits molecular overlap with human prostate cancers (32). The current study indicates that PEITC suppresses c-Myc expression and activity at plasma achievable concentrations, and this effect is not a cell line–specific phenomenon. Downregulation of c-Myc is functionally important as cell proliferation inhibition by PEITC in vitro is partially attenuated by c-Myc overexpression.
This study shows repression of c-Myc transcriptional activity upon PEITC treatment in cultured prostate cancer cells. Previous studies have indicated a critical role for AP-1 and E2F1 in regulation of c-Myc expression (33, 34). Published data suggest that AP-1 may not be engaged in c-Myc downregulation by PEITC. One study showed activation of MAPK-mediated activation of AP-1 in PEITC-treated PC-3 cells (35). In another study using bladder cancer cells, suppression of AP-1 activation failed to alter anticancer effects of PEITC (36). Hence, further work is needed to elucidate the mechanism underlying c-Myc downregulation by PEITC.
Identification of mechanistic biomarkers is essential for clinical development of potential chemopreventive agents as primary cancer incidence is too rigorous of an endpoint for malignancies with long latency, including prostate cancer. The current study is the first to demonstrate that prostate cancer chemoprevention by PEITC in the TRAMP mice is associated with a significant decrease in plasma lactate as well as pyruvate levels as schematically summarized in Fig. 6. In addition to glycolysis, β-oxidation of fatty acids is another source of energy especially in prostate cancer (37). In this context, we have shown previously that PEITC administration to TRAMP mice also results in a significant decrease in total free fatty acid level in the plasma in comparison with control mice (38). On the basis of these findings, we propose that plasma levels of lactate/pyruvate and free fatty acids represent novel noninvasive biomarkers of PEITC for future clinical investigations in human subjects at increased risk of prostate cancer. We also suspect that PEITC intervention longer than 1 week may be required to observe a decrease in plasma/serum lactate and free fatty acids based on analysis of human serum samples in the current study.
In conclusion, this study shows c-Myc inhibition by PEITC treatment in cultured androgen-responsive and androgen-independent human prostate cancer cells. Chemoprevention of prostate cancer by PEITC in TRAMP model is also associated with suppression of plasma lactate levels.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: S.V. Singh
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.-M. Yuan
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K.B. Singh, E.-R. Hahm, L.H. Rigatti, D.P. Normolle, S.V. Singh
Writing, review, and/or revision of the manuscript: K.B. Singh, E.-R. Hahm, D.P. Normolle, J.-M. Yuan, S.V. Singh
Study supervision: S.V. Singh
This work was supported by the grant RO1 CA101753 awarded by the NCI (S.V. Singh). This research used the Animal Facility, the Biostatistics Facility, and the Tissue and Research Pathology Facility supported in part by Cancer Center Support Grant from the NCI (P30 CA047904; Robert L. Ferris- principal investigator).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.