Phenethyl isothiocyanate inhibits hypoxia-induced accumulation of HIF-1α and VEGF expression in human glioma cells

Food Chemistry 141 (2013) 1841–1846

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Phenethyl isothiocyanate inhibits hypoxia-induced accumulation of HIF-1a and VEGF expression in human glioma cells Brinda Gupta a, Linda Chiang b, KyungMin Chae c, Dae-Hee Lee d,e,⇑ a

Department of Chemistry, University of Virginia, USA Department of Computer Science and Neuroscience, University of Virginia, USA c Department of Biology, University of Virginia, USA d Department of Pharmacology and Surgery, University of Pittsburgh School of Medicine, USA e Department of Neurosurgery, University of Virginia School of Medicine, Charlottesville, VA 22908, USA b

a r t i c l e

i n f o

Article history: Received 10 January 2013 Received in revised form 16 April 2013 Accepted 2 May 2013 Available online 11 May 2013 Keywords: PEITC Hypoxia Hypoxia-inducible factor-1 VEGF Protein synthesis

a b s t r a c t Phenethyl isothiocyanate (PEITC), a natural dietary isothiocyanate, inhibits angiogenesis but the molecular mechanisms that underlie this effect are not known. In this study, under hypoxic conditions (1% O2), we examined the effect of PEITC on the intracellular level of the hypoxia inducible factor (HIF-1a) and extracellular level of the vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that PEITC suppressed the HIF-1a accumulation during hypoxia in human glioma U87, human prostate cancer DU145, colon cancer HCT116, liver cancer HepG2, and breast cancer SkBr3 cells. PEITC treatment also significantly reduced the hypoxia-induced secretion of VEGF. Suppression of HIF-1a accumulation during treatment with PEITC in hypoxia was related to PI3K and MAPK pathways. Taken together, these results suggest that PEITC inhibits the HIF-1a expression through inhibiting the PI3K and MAPK signalling pathway and provide a new insight into a potential mechanism of the anticancer properties of PEITC. Published by Elsevier Ltd.

1. Introduction Chemoprevention begins before tumour cells develop or become cancerous. There are three chemopreventive agents: inhibitors of carcinogen formation, blocking agents, and suppressing agents (Stoner, Morse, & Kelloff, 1997). This experiment focuses on a specific type of isothiocyanate, which is a blocking agent. Isothiocyanates are chemicals that contain a reactive thiocyanate group (–N@C@S) and are formed by hydrolysis of glucosinolates (Kassie & Knasmuller, 2000). According to Satyan et al. (2006), these chemicals are absorbed through the intestinal membranes via passive diffusion and bind to plasma protein thiols. Once isothiocyanates enter the cell, glutathione S-transferases convert the isothiocyanates into glutathione conjugates that ultimately lead to the depletion of intracellular glutathione. The

Abbreviations: PEITC, phenethyl isothiocyanate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulphate; HIF-1a, hypoxia-inducible factor-1; VEGF, vascular endothelial growth factor; uPA, urokinase-type plasminogen activator; MMP, matrix metallopeptidase; pVHL, von Hippel–Lindau protein; PHD, proline hydroxylase; CHX, cyclohexmide. ⇑ Corresponding author. Address: Department of Neurosurgery, University of Virginia, 135, Hospital Drive, Lane Road Loading Dock, Charlottesville, VA 22908, USA. Tel.: +1 434 924 5889; fax: +1 434 982 1416. E-mail address: [email protected] (D.-H. Lee). 0308-8146/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.foodchem.2013.05.006

isothiocyanates, that were not converted into glutathione conjugates, react with cellular proteins by thiocarbamoylation, and when coupled with depletion of intracellular glutathione, signal transduction pathways and apoptosis are activated (Satyan et al., 2006). Isothiocyanates are considered blocking agents because they hinder the effects of carcinogens, by inhibiting the enzymes that are needed for carcinogen bioactivation, carcinogen excretion, and detoxification (Kassie & Knasmuller, 2000). One particular isothicyanate of interest is phenethyl isothiocyanate (PEITC), which can be found in vegetables such as watercress. According to Hwang and Lee (2010), the glucosinolate, gluconasturtiin, is hydrolysed to produce PEITC. When the aromatic compound is absorbed and metabolised within the body, it undergoes glutathionine conjugation followed by conversion to produce a conjugate of N-acetylcysteine. It is believed that PEITIC inhibits carcinogenesis induced by carcinogens such as, 12-dimethylbenz[a]anthracene, benzo[a]pyrene, diethylnitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and methylbenzylnitrosamine. According to Satyan et al., this is done by inducing the NADPH quinine reductase glutathione-S-transferase, a phase II detoxification enzyme, and by inhibiting the enzymes involved in the metabolic activation of the carcinogens. PEITC also prolongs the activation of JNK and the stress-activated protein kinase pathway, which sensitises the cell to undergo apoptosis.

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Such PEITC-induced apoptosis involves p53, activation of JNK, and induction of caspase-3-like activity (Chen, Han, Kori, Kong, & Tan, 2002). When there is poor blood supply for tumour cells, the transcriptional activator hypoxia-inducible factor-1 (HIF-1), a heterodimer consisting of a and b subunits, increases to promote angiogenesis and anaerobic metabolism (Maxwell et al., 1997). Normally the a subunit of HIF-1 (HIF-1a), forms a complex with the von Hippel– Lindau (VHL) protein that requires oxygen and iron to be present for proline residue hydroxylation in the HIF-1a protein (Semenza, 2007). The HIF-1a protein is then degraded by ubiquitination. However, in hypoxic conditions, proline residue hydroxylation is blocked (Jaakkola et al., 2001). This prevents the formation of the HIF-1a/VHL protein complex, and the subsequent ubiquitination and degradation of the complex and results in an accumulation of HIF-1a. HIF-1a plays an important role in angiogenesis by regulating the transcription of the vascular endothelial growth factor (VEGF) (Forsythe et al., 1996). Recent studies show that PEITC inhibits the accumulation of HIF-1a, through suppression of the mTOR pathway that plays a major role in breast cancer progression (Wang, Cavell, Syed Alwi, & Packham, 2009). As a consequence, the report that PEITC inhibits tumour angiogenesis suggests that PEITC would be an ideal agent to prevent tumour growth. Consistent with our previous experiments, our results show that PEITC suppresses accumulation of HIF-1a during hypoxic conditions, in human glioma cells, and suggest that PEITC is a viable agent in the treatment of tumours in addition to chemoprevention. Thus, in this study, we examined whether PEITC can affect HIF-1a accumulation and VEGF expression differently under hypoxic conditions. We observed that PEITC inhibits HIF-1a accumulation under hypoxic conditions in human brain cancer U87, colon cancer HCT116, prostate cancer DU145, and breast cancer SkBr3 cell lines. This study provides important information for the understanding of the mechanism of action of angiogenesis and resistance to this class of compounds. Angiogenesis plays a critical role early in tumour development, and its inhibition may play a major role in the chemopreventive/anti-cancer effects of PEITC. Since HIF plays a central role in angiogenesis, we have investigated the effects of PEITC on HIF activity. 2. Materials and methods 2.1. Reagents and antibodies PEITC (>99% pure), CoCl2, and DFX were obtained from Sigma Chemical Co. (St. Louis, MO, USA). MAPK inhibitor PD98059 and PI3K inhibitor LY294002 were obtained from Calbiochem (San Diego, CA, USA). Monoclonal antibodies were purchased from the following companies: anti-HIF-1a from BD Biosciences (San Jose, CA, USA), and anti-actin antibody from ICN (Costa Mesa, CA, USA). Anti-phospho-ERK, Anti-ERK, Anti-MMP-2, Anti-MMP-9, Anti-uPA, Anti-phospho-Ser473-Akt, and anti-Akt antibodies were from Cell Signalling (Beverly, MA, USA). 2.2. Cell culture Human glioma cancer U87, prostate cancer DU145, colon cancer HCT116, liver cancer HepG2, and breast cancer SKBR-3 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). U87 and SKBr-3 cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA), DU145 cells were cultured in DMEM medium (Gibco BRL, Gaithersburg, MD, USA), and HCT116 cells were cultured in McCoy’s 5A medium (Invitrogen, Carlsbad, CA, USA) respectively, with 10% foetal bovine serum

(Hyclone, Logan, Utah) and 26 lM sodium bicarbonate for monolayer cell culture. Cells were cultured at 37 °C humidified atmosphere and 5% CO2 in air. 2.3. Hypoxia and drug treatments Before the experiments, cells were grown to approximately 80% confluence in 60 or 100 mm tissue culture dishes. For hypoxia treatment, Petri dishes containing cells were incubated in a hypoxic chamber (Forma Scientific, Marietta, OH, USA) with a 94:5:1 mixture of N2/CO2/O2. Deoxygenated mediums were prepared prior to each experiment, by equilibrating the medium with a hypoxic gas mixture containing 5% CO2, 94% N2, and 1% O2 at 37 °C. Exponentially growing cells (70–80% confluence) in complete medium were co-treated with different concentrations of PEITC, followed by continual incubation in normal culturing conditions or exposure to hypoxia (1% O2) for indicated time intervals according to the purpose of the experiment. 2.4. Enzyme-linked immunosorbent assay for detection of VEGF U87 cells were plated in a 60 mm plate at a density of 1  105 cells/ml in RPMI medium and incubated overnight before the cells were subjected to treatment. After treatment, the cell culture media was removed for storage at 80 °C. Levels of VEGF protein in the medium were determined by ELISA using a commercial kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions and our previous reports (Lee & Lee, 2009). 2.5. Immunoblot analysis For Western blot analysis, we followed the protocol described in Lee and Lee (2009). Proteins were separated by SDS–PAGE and electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose membrane was blocked with 5% nonfat dry milk in PBS–Tween-20 (0.1%, v/v) for 1 h. The membrane was incubated with primary antibody (diluted according to the manufacturer’s instructions) at 4 °C overnight. Horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG was used as the secondary antibody. Immunoreactive protein was visualised by the chemiluminescence protocol (ECL, Amersham, Arlington Heights, IL, USA). For the immunoblotting experiments, the assays were performed repeatedly at least three times. Densitometric analysis was performed with a computer using a gel image analysis program. 2.6. Invasion assay Transwell invasion experiments were performed using 24-well BD BioCoat Growth Factor Reduced Matrigel Invasion Chambers (BD Biosciences, Bedford, MA, USA). The invasion chambers consist of a BD Falcon cell culture insert with a 8 lm pore size PET membrane coated with BD Matrigel Matrix, which serves as a reconstituted basement membrane in vitro. Tumour cells under normoxia or hypoxia treatment were prepared for cell suspension in serum free culture medium and at a density of 1  105 cells/ml U87 cells with or without PEITC. Membranes with cells attached to the lower membrane surface (invaded cells) were fixed with methanol and stained with haematoxylin. Membranes were permanently mounted and all invaded cells were counted under a microscope at a 100 magnification. All experiments were performed in triplicate. Results are expressed as the average cell number per filter. 2.7. Statistical analysis Statistical significance was examined using Student’s t test. The two-sample t test was used for two-group comparisons. Values

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were reported as the mean ± SD. P < 0.05 were considered significant and indicated by asterisks in the figures. Bands of Western blotting were calculated by average densitometric analysis using ImageJ software (NIH).

the presence of both CoCl2 and DFX. To examine whether our observations could be generalised, we tested with four different cell lines, HepG2, Sk-Br3, HCT116 and DU145. Fig. 2 shows that HIF-1a accumulation was suppressed by treatment with PEITC in hypoxic conditions in all four cell lines.

3. Results 3.1. PEITC inhibits the accumulation of HIF-1a in hypoxic conditions

3.2. Inhibition of HIF-1 accumulation by PEITC during hypoxia is AKT and ERK signalling pathway-dependent

In the present study, we investigated whether PEITC inhibits hypoxia-induced accumulation of HIF-1a in U87 cells. We first examined whether PEITC induces cytotoxicity under hypoxic conditions and whether PEITC-induced cytotoxicity is responsible for the suppression of HIF-1a accumulation. The effects of PEITC on cell viability and drug-induced PARP-1 cleavage, the hallmark feature of apoptosis (data not shown), were determined by the trypan blue exclusion dye assay and western blotting, respectively (Fig. 1A). When U87 cells were treated with various concentrations of PEITC (1–50 lM) for 8 h in hypoxia, no significant concentration-dependent reduction of the viability was observed (Fig. 1A). We checked that HIF-1a accumulation was time-dependent during hypoxia (Fig. 1B). We also showed that exposure to hypoxia, led to transient activation of both ERK1/2 and Akt in U87 cells (Fig. 1B). Surprisingly, we observed that reduction of HIF-1a accumulation, occurred during treatment with PEITC in hypoxic condition (Fig. 1C). The reduction of HIF-1a accumulation was dependent upon PEITC concentrations during hypoxia. We also studied the effect of two other known HIF-1 inducers, CoCl2 and DFX, with PEITC treatment on U87 cells. As shown in the Fig. 1D, HIF-1a protein levels were markedly decreased after treatment with PEITC, in

Recent studies indicate that expression of HIF-1a can be regulated by PI3K/AKT and two kinases of the MAPK signalling pathway, as ERK1/2 has been implicated in the regulation of HIF-1a expression (Blancher, Moore, Robertson, & Harris, 2001; Fukuda et al., 2002; Richard, Berra, Gothie, Roux, & Pouyssegur, 1999). To examine whether the inhibition of HIF-1a accumulation by treatment with PEITC during hypoxia occurs by inhibiting the PI3K and MAPK signalling pathway, cells were treated with the PI3K inhibitor LY294002 or MAP Kinase inhibitor PD98059 prior to hypoxia alone, PEITC treatment alone, or combined hypoxia and PEITC treatment. Fig. 3A and B show that LY294002 and PD98059 caused the dephosphorylation (inactivation) of Akt and ERK regardless of oxygen tensions. Interestingly, LY294002 and PD98059 inhibited accumulation of HIF-1a during hypoxia (Fig. 3A and B). Next, to check whether PEITC inhibits expression of HIF-1a through these signalling pathways, we determined the effects of PEITC on the activation of AKT and ERK by immunoblotting. We found that PEITC inhibited Akt and ERK phosphorylations in U87 cells (Figs. 1C and 3C). These results suggest that PEITCinduced inhibition of HIF-1a accumulation during hypoxia, is due to dephosphorylation of Akt and ERK.

Cell Viability (%)

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Akt Actin Actin Fig. 1. Effect of hypoxia and PEITC on the intracellular level of HIF-1a in U87 cells. (A) Cells were treated with various concentrations (1–50 lM) of PEITC for 24 h. The cytotoxic effect of PEITC on U87 cells was determined using the trypan blue dye exclusion assay as described in Section 2. Error bars represent standard error of the mean (SEM) from three separate experiments. (B) Kinetics of HIF-1a accumulation in hypoxic conditions. (C) Kinetics of HIF-1a accumulation during treatment with PEITC in hypoxic conditions. (D) Cells were exposed to hypoxia, cobalt chloride, and DFX in combination with PEITC for 8 h, then harvested. Cells lysates containing equal amounts of protein (20 lg) were separated by SDS–PAGE and immunoblotted with anti-HIF-1a antibody. Actin was used as a loading control. Symbols denote a response that was significantly different from the control (⁄p < 0.05, ⁄⁄p < 0.01).

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Fig. 2. Effect of PEITC on HIF-1a accumulation during hypoxia in various cancer cell lines. HepG2, HCT116, SkBr3, and DU145 cells were exposed to 1% O2 alone or were treated with 5 lM PEITC in combination with 1% O2 for 8 h. Cell lysates containing equal amounts of protein (20 lg) were separated by SDS–PAGE and immunoblotted with anti-HIF-1a antibody. Actin was used as a loading control.

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Fig. 3. Role of ERK and Akt in the accumulation of HIF-1a in U87 cells. Cells were treated with 10 lM PEITC in combination with 1% O2 in the presence or absence of 10 lM LY294002 for 8 h. Cells were pretreated with PD98059 (10 lM) and LY294002 (10 lM) for 30 min, followed by treatment with PEITC and/or hypoxia for 8 h in the presence of PD98059 and LY294002. Cell lysates containing equal amounts of protein (20 lg) were separated by SDS–PAGE and immunoblotted with anti-HIF-1a, anti-phospho-Akt (S473), or anti-Akt antibody. Actin was used as a loading control.

3.3. PEITC inhibited hypoxia-induced VEGF expression and cell invasion VEGF is an immediate downstream target gene of HIF-1a and plays a critical role in tumour angiogenesis (Hicklin & Ellis,

2005). To determine whether PEITC could inhibit the hypoxiainduced VEGF expression, we examined PEITC VEGF expression by Western blot. Our results showed that treatment of U87 cells with PIETC resulted in a dose-dependent decrease of hypoxiainduced VEGF expression at protein levels (data not shown). We

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Fig. 4. PEITC inhibits production of VEGF and invasion during hypoxia. (A) U87 cells were exposed to 1% O2 for various times (2–24 h). (B) U87 cells were treated with various concentrations of PEITC (1, 2.5, and 5 lM) for 16 h during hypoxia. The concentration of VEGF protein in the culture media was determined by ELISA. The assays were performed with triplicate experimental samples. The results represent the mean values of VEGF concentrations and error bars represent standard error of the mean from triplicate samples. (C) Invasion indicators are expressed as the mean ± SD of three independent experiments. The data was evaluated for statistical significance by Student’s ttest. The means noted with an asterisk were statistically different from the matched normoxic control. (D) Western blot analysis shows that PEITC inhibits invasion related proteins in U87 cells during an hypoxic condition. Symbols denote a response that was significantly different from the control (⁄p < 0.05, ⁄⁄p < 0.01).

also examined whether reduction of HIF-1a accumulation during treatment with PEITC in hypoxic conditions, results in a decrease in the extracellular level of VEGF. Fig. 4A shows an increase in VEGF in a time dependent manner (up to 24 h) during hypoxia in U87 cells. However, VEGF induction was decreased by treatment with PEITC in hypoxic conditions (Fig. 4B). We next examined whether hypoxic conditions enhance the invasiveness of U87 cells and whether PEITC can suppress tumour migration. Recent studies have shown that hypoxia has stimulatory effects on cancer cell invasion and migration (Zhang et al., 2005). To investigate whether PEITC suppresses hypoxia-induced cancer cell invasion, an in vitro cell invasion assay was done. As shown in Fig. 4C, under normoxic conditions, an increase in the baseline invasiveness of U87 cells, was observed under culture conditions of 1% hypoxia as compared with 21% normoxia (Fig. 4C). On the other hand, pretreatment with 5 lM of PEITC suppressed the 1% hypoxia-stimulated invasiveness of U87 cells (Fig. 4C). Also, we confirmed that PEITC inhibits invasion related proteins, MMP-2, MMP-9, uPA, and p-ERK, in hypoxic conditions (Fig. 4D). These results indicate that PEITC suppresses hypoxia-stimulated invasiveness of U87 cells.

4. Discussion In this study, we observed that PEITC inhibits hypoxia-induced HIF-1a accumulation in a dose-dependent manner, in several human cancer cell lines. Inhibition of HIF-1a accumulation by PEITC is mediated through the inhibition of protein synthesis, under hypoxic conditions. Angiogenesis, a characteristic hallmark of cancer, sustains a tumour’s invasive nature. As brain cancers are known to express low

levels of oxygen, debilitating the growth of new blood vessels, through the use of dietary chemotherapeutic agents, may provide an effective strategy to delay the cancer’s progression. In this study, it was observed that PEITC diminishes the extent of hypoxia, through the decreased accumulation of HIF-1a, as a result of the inhibition of phosphorylated ERK and Akt. In response, the decreased expression of VEGF shows a marked reduction in the levels of MMP-2, MMP-9, and uPA, all proteins associated with vascularisation. Our previous studies, indicated that inactivation of the PI3K/Akt and ERK 1/2 signalling pathways reduces the hypoxia-driven growth of tumour cells (Soeda et al., 2009). Because the phosphorylation of Akt and ERK was significantly attenuated, PEITC modulated the hypoxic response through control of these signalling pathways. Since the phosphorylation events are downstream of the activation of EGFR, it is probable that PEITC exerts its inhibitory effect on EGFR. Recent studies on the role isothiocyanates play in the control of prostate cancer, as well as ovarian cancer, attribute the antiproliferative effects of PEITC to the inhibition of EGFR (Kim et al., 2006; Loganathan, Kandala, Gupta, & Srivastava, 2012). The significance of EGFR targeting in controlling GBM is evidenced by the finding that EGFR is overexpressed in approximately 50–60% of glioblastoma tumours (Heimberger et al., 2005). Since inhibition of the ERK1/2 signalling pathway was found to be linked to a decrease in VEGF production, the use of phosphorylated ERK as a marker of cell invasiveness was verified. The concentration of VEGF, heightened in hypoxic conditions, decreased along with the diminishing levels of MMP-2, MMP-9, and uPA in the presence of PEITC (Fig. 4). The overexpression of MMPs, endopeptidases necessary for basement membrane degradation, has been observed in several types of cancers and is associated with tumour

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angiogenesis, metastasis and growth factor release from the extracellular matrix (ECM) (Mandal, Rao, Tran, & Pendurthi, 2005). The proteolytic cleavage and destruction of the ECM to which tumour cells adhere, leads to cancer cell metastasis. In concordance with the published literature, it was found that PEITC inhibits MMP-2, MMP-9, and uPA. The aberrations in protein levels were manifested in a decrease in the percentage of invasive cells under hypoxic conditions. The anti-angiogenic response elicited by PEITC might, in part, be related to the interference of reactive-oxygen species (ROS) metabolism. Previous studies report that elevated levels of ROS may activate HIF-1a. VEGF expression through EGF induced activation of the PI3K/Akt and ERK pathways, has been shown to have been modulated by ROS (Liu et al., 2006). In this study, PEITC was able to further diminish levels of the phosphorylated proteins in combination with LY294002, an Akt inhibitor, or with PD98059, an inhibitor of ERK. Therefore, the use of PEITC in concert with currently used chemotherapeutic drugs may clinically strengthen the anti-cancer physiological response. Elucidation of the molecular basis by which PEITC is able to inhibit angiogenesis, through the suppression of phosphorylated Akt and ERK, HIF-1a and ultimately VEGF may be useful for the development of a reasonable brain cancer therapy. References Blancher, C., Moore, J. W., Robertson, N., & Harris, A. L. (2001). Effects of ras and von Hippel–Lindau (VHL) gene mutations on hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, and vascular endothelial growth factor expression and their regulation by the phosphatidylinositol 3-kinase/Akt signaling pathway. Cancer Research, 61, 7349–7355. Chen, Y. R., Han, J., Kori, R., Kong, A. N., & Tan, T. H. (2002). Phenylethyl isothiocyanate induces apoptotic signaling via suppressing phosphatase activity against c-Jun N-terminal kinase. Journal of Biological Chemistry, 277, 39334–39342. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., et al. (1996). Activation of vascular endothelial growth factor gene transcription by hypoxiainducible factor 1. Molecular and Cellular Biology, 16, 4604–4613. Fukuda, R., Hirota, K., Fan, F., Jung, Y. D., Ellis, L. M., & Semenza, G. L. (2002). Insulinlike growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells. Journal of Biological Chemistry, 277, 38205–38211. Hicklin, D. J., & Ellis, L. M. (2005). Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. Journal of Clinical Oncology, 23, 1011–1027. Heimberger, A. B., Hlatky, R., Suki, D., Yang, D., Weinberg, J., Gilbert, M., et al. (2005). Prognostic effect of epidermal growth factor receptor and EGFRvIII in glioblastoma multiforme patients. Clinical Cancer Research, 11, 1462–1466.

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