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Modulation of angiogenesis-related protein synthesis by valproic acid
BBRC Biochemical and Biophysical Research Communications 316 (2004) 693–697 www.elsevier.com/locate/ybbrc
Modulation of angiogenesis-related protein synthesis by valproic acidq Dimitrios Zgouras,* Ute Becker, Stefan Loitsch, and J€ urgen Stein* Second Department of Medicine, Johann Wolfgang Goethe University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany Received 12 February 2004
Abstract Recent studies have attested to the antiangiogenic effects of HDAC inhibitors on solid human tumors. The HDAC inhibitor butyrate has been reported to impair tumor-cell-induced angiogenesis. However, due to its poor bioavailibility in vivo, the therapeutic use of butyrate is limited. On the other hand valproic acid, has inhibitory effects on carcinoma cells, is known to be well tolerated and has an excellent bioavailibility. We therefore set out to investigate whether the HDAC inhibitor valproic acid also impairs angiogenesis. Our findings indicate that valproic acid represses the relevant angiogenic factors VEGF and FGF in Caco-2 cells. Both, protein expression as well as mRNA levels of VEGF were reduced to a similar degree. Suppression of ubiquitin–proteasome activity could be a possible reason for valproic acid effects on regulatory angiogenesis proteins. These results suggest that the HDAC inhibitor valproic acid could become a valuable new addition in the attempt to develop alternative therapeutic approaches in the treatment of colon carcinomas. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Angiogenesis; Colon cancer; FGF; Histone deacetylase inhibitors; Valproic acid; VEGF
The inhibition of angiogenesis is considered to be one of the most promising strategies in the development of novel anti-neoplastic therapies [1]. Angiogenesis is defined as the de novo formation of blood vessels by sprouting and maturing from a pre-existing vasculature. This is essential in providing the tumor tissue with the oxygen and nutrients required for tumor growth, survival of solid neoplasms, and metastasis [2]. The vascular endothelial growth factor (VEGF) and the fibroblast growth factor (FGF) are important angiogenesis regulating factors. That FGF, one of the first angiogenic growth factors to be identified [3], plays a key role in tumor-induced angiogenesis has been well established [4]. VEGF, known to be one of the most potent angiogenic factors, stimulates endothelial cells to secrete proteases and plasminogen activators, which results in the degradation of the vessel basement mem-
q
Abbreviations: AMC, 7-amino-4-methylcoumarin; FCS, foetal calf serum; HAT, histone acetylase; HDAC, histone deacetylase; HRP, horse radish peroxidase; PBS, phosphate buffered saline; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; VPA, valproic acid. * Corresponding authors. Fax: +49-69-6301-83112. E-mail address: [email protected] (J. Stein). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.105
brane, in turn allowing cells to invade the surrounding matrix [5]. After subsequent migration and proliferation, the cells finally differentiate to form a new vessel. Enhanced expression of VEGF has been observed in many human cancers including colorectal, breast, nonsmall-cell lung, and ovarian cancers [6]. It has also been shown that HDAC inhibitors like butyrate reactivate gene expression and consequently repress the growth and survival of tumor cells [7]. The process of acetylation and deacetylation of histones plays a crucial role in gene expression or repression. Histone deacetylases (HDACs) are enzymes responsible for deacetylation whereas histone acetyltransferases (HAT) mediate the acetylation of histones. The inappropriate transcriptional repression mediated by HDACs [8,9] is a common molecular mechanism which leads to tumor formation. The effects of HDAC inhibitors like butyrate, namely their ability to reactivate gene expression and thus to affect cell growth in the colon, has been extensively studied and reviewed in detail [10,11]. Two recently published papers convincingly demonstrated the involvement of histone deacetylases in angiogenesis by negative regulation of tumour suppressor genes in vivo an in vitro [12,13]. We like others have
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recently conclusively documented butyrate’s angiogenesis-suppressing effects on colon carcinoma cells in vitro [14,15]. G€ ottlicher et al. [16] discovered that the well-tolerated drug valproic acid (VPA) is a class I HDAC inhibitor. This activity can be distinguished from its therapeutically exploited antiepileptic efficacy [17,18]. Since VPA has been used clinically for over two decades, the pharmacology and side effects of this drug have been studied in detail. As expected for HDAC inhibitory compounds, VPA induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute leukemia (AML) patients. The aim of the current study was to investigate the anti-angiogenic effects of the class I HDAC inhibitor valproic acid on colon carcinoma cells. In this study we show that valproic acid influences the angiogenesis-related factor VEGF. Further it leads to a significant reduction of VEGF secretion in Caco-2 cells. VPA inhibits protein expression as well as mRNA levels of VEGF. We hypothesize, that inhibition of the ubiquitin–poteasome proteolytic system activity by valproic acid, as observed in our experiments, could be a possible interpretation for HDAC-effects on angiogenesis regulating proteins.
Materials and methods Cell culture. Human colon cancer cells (Caco-2) were obtained from the American Type Culture Collection (ATCC) and grown under standard conditions. Caco-2 cells of passages 50–60 were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% foetal calf serum (FCS), 1% non-essential amino acids, and 1% penicillin/streptomycin. The medium was changed every second day and collected as described below. The cells were passaged at 80% (Caco-2) with Dulbecco’s PBS containing 0.25% trypsin and 1% EDTA. Cytotoxicity was assessed by a commercial kit measuring lactate dehydrogenase activity in the cell culture medium (LDH kit, Merck, Darmstadt, Germany). For the experiments, Caco-2 cells were grown until approx. 90% confluent and then switched to a serum-free medium containing either the solvent (PBS), valproic acid (1, 3 mM) (Merck–Schuchardt, Hohenbrunn, Germany), or butyrate (1, 3 mM) (Sigma Aldrich, Germany). After 12, 24, and 48 h, the cell culture medium was collected, centrifuged, and stored at )20 °C until use. Measurement of VEGF. In order to assess VEGF production in the supernatant of Caco-2 cells, the cells were incubated in serum-free medium or in serum-free medium containing valproic acid (1, 3 mM) or butyrate (1, 3 mM). After 12, 24, and 48 h, the media were collected, and VEGF was quantified by use of a colorimetric ELISA (CytELISA, CytImmune, Umkirch, Germany). Four VEGF splice variants have been identified until now. CytELISA detects the splice variant VEGF 165. SDS–polyacrylamide gel electrophoresis and immunoblot analysis. Caco-2 cells were incubated with valproic acid (1, 3 mM) or butyrate (1, 3 mM). After 24–72 h, the cells were harvested and the whole cell extract was quantified using the Bio-Rad protein colorimetric assay. VEGF and FGF were assessed by Western blot analysis using a primary antibody for VEGF (Transduction Laboratories) and a primary antibody for FGF (Santa Cruz Biotechnology). After washing, a sec-
ondary antibody (horseradish peroxidase-conjugated antibody, Vector Lab., Burlingame, USA) was applied. Afterwards the nitrocellulose mebranes were subjected to a chemoluminescence reaction (ECL, Amersham–Pharmacia Biotech, Buckinghamshire, UK) and the bands were visualized by exposure to Hyperfilm-MP (Amersham International plc, Buckinghamshire, UK). RT competitive multiplex PCR. Total cellular RNA was isolated by RNABee (Wak-Chemie, Bad Homburg, Germany), phenol/chloroform extracted, and then isopropanol precipitated. The RNA was reconstituted in diethylpyrocarbonate-treated water. RNA was reverse transcribed into cDNA by Superscript II reverse transcriptase (Invitrogen). Quantification by RT-competitive multiplex PCR was established as detailed elsewhere. Internal standards (IST) for reverse transcriptase competitive multiplex PCR were generated according to Celi et al. [26]. Primers and internal standards for GAPDH were described previously, whereas the primers and internal standards for VEGF (GenBank M32977) were kindly provided by Dr. Stefan Kippenberger (University of Frankfurt, Department of Dermatology). The primer sequences were: VEGF-A(67), 50 tgctgtcttgggtgcattgg-30 ; VEGF-B(289), 50 -acacaggatggcttgaagat-30 ; and VEGF-CB IST, 50 -acacaggatggcttgaagattctcgattggatggcagtag-30 . Primer combination VEGF-A/B amplifies a 222 bp product whereas the combination VEGF-A/CB generates an internal standard (competitor) of 178 bp. Aliquots of a master mixture containing KCl, Tris–HCl, MgCl2 , dNTPs, primers for the target genes and 0.5 ll of cDNA per 50 ll of total PCR volume were added to serial dilutions (1:3) of a mixture of the competitors. GAPDH was used as a control for initial RNA loading. Wildtype and competitors were amplified with Taq-Polymerase (Invitrogen). Each sample was electrophoresed in 2% agarose gels, stained with ethidium bromide and quantified with DocuGel IVSystem (MWG-Biotech, Ebersberg, Germany). The ratio of target to competitor was determined and, according to the method, a 1:1 ratio implies equivalent concentrations of competitor and input target mRNA. Proteasome activity assay. Untreated, valproic acid-treated (1, 3 mM) and butyrate-treated (1, 3 mM) Caco-2 cells were harvested after 24 h exposure. Proteasome activity was assessed in cell lysate with 20S proteasome activity assay kit (Chemicon). The assay is based on detection of the fluorophore 7-amino-4-methylcoumarin (AMC) after its cleavage from the labeled Subtrate LLVY-AMC by chymotrypsinlike activity of the proteasome. Free AMC is detected by fluorimetric quantification (380/460 nm). Statistical analysis. Data were expressed as means SD. Differences between two values were tested for statistical significance using the Student’s unpaired t test (SigmaPlot, SPSS Chicago, IL). A p value <0.05 considered to indicate a significant difference.
Results To elucidate whether valproic acid induces angiosuppressive effects on colon tumor cells, direct inhibition of angiogenic growth factors was quantified. We measured VEGF165 concentration, a splice variant of VEGF, in the conditioned medium over time. VEGF is known to be one of the most important angiogenic factors. As shown in Fig. 1, VEGF165 secretion to the cell culture medium was inhibited by VPA in Caco-2 cells. The highest inhibition of VEGF production occurred after 48 h (23.2%). Valproic acid showed a similar repression of VEGF165 production as after butyrate application. Decreased VEGF165 secretion was also observed after 24 h.
D. Zgouras et al. / Biochemical and Biophysical Research Communications 316 (2004) 693–697
Fig. 1. VEGF165 amounts in pre-conditioned medium obtained from Caco-2 cells either basally, or after treatment with valproic acid (3 mM) or butyrate (3 mM) were observed by ELISA. Means SD, n ¼ 6, *p < 0:05 and **p < 0:01.
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Fig. 3. Western blot analysis for detection of FGF expression in total cell lysate of Caco-2 cells. The cells were treated with valproic acid or butyrate (1 or 3 mM) for 48 h. Protein was separated by SDS–PAGE followed by immunoblotting and detection with the polyclonal antiFGF IgG antibody. Results are representative of three independent experiments. The graphs show the quantification by densitometric analysis normalized by the internal control (b-actin). Means SD, n ¼ 3, **p < 0:01, ***p < 0:001.
Fig. 2. Western blot analysis of VEGF expression in total cell lysate of Caco-2 cells. The cells were treated with valproic acid or butyrate (1 or 3 mM) for 72 h. Protein was separated by SDS–PAGE followed by immunoblotting and detection with the polyclonal anti-VEGF IgG antibody. Essentially identical results were obtained in three independent experiments. The graphs show the quantification by densitometric analysis normalized by the internal control (b-actin). Means SD, n ¼ 3, *p < 0:05, **p < 0:01.
Furthermore, intracellular VEGF- and FGF-isoforms were detected by Western blot in Caco-2 cell lysate. VEGF and FGF were highly expressed in untreated Caco-2 cells. Repression of VEGF protein was induced by VPA in a dose-dependent manner after 72 h (Fig. 2). The highest inhibition rate was reached after treatment with 3 mM valproic acid for 72 h. In Western blot analysis of FGF protein valproic acid decreased FGF levels after 48 h treatment (Fig. 3). Stronger effects in FGF protein expression were yet recognized in cells treated by butyrate. We further determined the expression and modulation of VEGF genes by valproic acid in Caco-2 cells (Fig. 4). RT-PCR experiments showed a decrease in VEGF mRNA levels of
Fig. 4. (A) RT competitive multiplex PCR of VEGF together with GAPDH and respective competitors 24 h after incubation without (controls) or with butyrate (3 mM) or valproic acid (3 mM). The lanes in each sample correspond to 1:3 serial dilutions of the competitors. (B) Relative decrease of VEGF mRNA expression in Caco-2 cells 24 h after incubation without (controls) or with valproic acid (3 mM) or butyrate (3 mM), related to GAPDH mRNA expression levels. Means SD, n ¼ 3, *p < 0:05, **p < 0:01.
Caco-2 cells being treated by valproic acid (3 mM) (see Fig. 4). It has been shown that the Ub/26S proteasome system, being responsible for protein degradation of regulatory proteins, plays a crucial role in regulation of important angiogenic factors like VEGF and HIF1-a [19,20]. The 20S proteasome, catalytic core of the
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Fig. 5. Proteasome activity in cell lysate from Caco-2 cells after treatment with valproic acid or butyrate (3 mM) over 24 h was determined by a 20S proteasome activity assay kit. Means SD, n ¼ 6, *p < 0:05.
proteasomecomplex, is responsible for the breakdown of key proteins involved with apoptosis, cell cycle control, and angiogenesis [21]. Effects of valproic acid and butyrate on proteasome activity were studied by a proteasome activity assay (Chemicon) in Caco-2 cell lysate (Fig. 5). Our data indicate valproic acid significantly suppresses proteasome activity (19% vs. control) in Caco-2 cells after 24 h treatment.
Discussion Formation of new blood vessels in malignant tumors is a complex multi-step process including degradation of the basement membrane, migration and proliferation of endothelial cells leading to the formation of solid endothelial cell sprouts. This process called angiogenesis is essential for the continuous growth and survival of solid neoplasms and their metastasis, providing oxygen, and nutrients to the tumor tissue. Regulation of tumorinduced angiogenesis is controlled by the tumor tissue itself producing and secreting several growth and regulating factors. Especially VEGF and FGF play a crucial role in the angiogenesis process consequently becoming also potential targets in new anticancer strategies. It has long been known that the short-chain fatty acid butyrate, an HDAC inhibitor, induces apoptosis, and differentiation of colon cancer cells [22], but most recently research has demonstrated that it also serves as a potent anti-angiogenic agent [23,24]. Current research has suggested that this effect is the result of the regulation and inhibition of VEGF [14]. We have been able to prove, confirming previous studies, that butyrate impairs the secretion of VEGF in colon cancer cells (HT29, Caco-2) in a dose-dependent manner [15]. We in turn hypothesized that the HDAC inhibitor valproic acid might similarly have this effect on Caco-2-induced angiogenesis. Its better bioavailibility since it is only slowly
metabolized, in contrast to butyrate, which is degraded rapidly, could be an additional benefit and prove of enormous significance for new anti-cancer applications. In the present study we have investigated whether colon cancer cell-induced angiogenesis is affected by valproic acid a class I HDAC inhibitor known to induce differentiation, apoptosis, and impairing cell proliferation in carcinoma cells. We could show that valproic acid influences angiogenesis by down-regulating either VEGF secretion or the intracellular protein expression in the Caco-2 cell line. We also demonstrated that the HDAC inhibitor reduces the protein expression of FGF in Caco-2 cell lysate. Translation of VEGF was further examined by RT-PCR. Both HDAC inhibitors showed repressive effects on VEGF mRNA level in Caco-2 cells. As shown in our experiments treatment with valproic acid significantly decreased proteasome activity in Caco2 cell lysate. Our findings suggest that alterations in the cellular proteasome activity by HDAC inhibitors may influence angiogenesis regulatory proteins like VEGF and HIF-1a. In the final analysis, our results of valproic acid effects on colon carcinoma cell-induced angiogenesis correspond to the results of Kwon et al. [25]. They verified that HDACs regulate the expression of crucial angiogenesis-related genes and observed that HDAC inhibitors suppress human hepatoblastoma cells [1]. The anti-angiogenic activities of valproic acid observed in this study suggest that the HDAC inhibitor valproic acid could become a valuable new addition in the attempt to develop alternative therapeutic approaches in the treatment of human colorectal malignancies. Its better bioavailibility compared to butyrate could be an additional and significant benefit. Acknowledgment This study was supported by the Else Kr€ oner-Fresenius-Foundation, Bad Homburg, Germany.
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Modulation of angiogenesis-related protein synthesis by valproic acidq Dimitrios Zgouras,* Ute Becker, Stefan Loitsch, and J€ urgen Stein* Second Department of Medicine, Johann Wolfgang Goethe University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany Received 12 February 2004
Abstract Recent studies have attested to the antiangiogenic effects of HDAC inhibitors on solid human tumors. The HDAC inhibitor butyrate has been reported to impair tumor-cell-induced angiogenesis. However, due to its poor bioavailibility in vivo, the therapeutic use of butyrate is limited. On the other hand valproic acid, has inhibitory effects on carcinoma cells, is known to be well tolerated and has an excellent bioavailibility. We therefore set out to investigate whether the HDAC inhibitor valproic acid also impairs angiogenesis. Our findings indicate that valproic acid represses the relevant angiogenic factors VEGF and FGF in Caco-2 cells. Both, protein expression as well as mRNA levels of VEGF were reduced to a similar degree. Suppression of ubiquitin–proteasome activity could be a possible reason for valproic acid effects on regulatory angiogenesis proteins. These results suggest that the HDAC inhibitor valproic acid could become a valuable new addition in the attempt to develop alternative therapeutic approaches in the treatment of colon carcinomas. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Angiogenesis; Colon cancer; FGF; Histone deacetylase inhibitors; Valproic acid; VEGF
The inhibition of angiogenesis is considered to be one of the most promising strategies in the development of novel anti-neoplastic therapies [1]. Angiogenesis is defined as the de novo formation of blood vessels by sprouting and maturing from a pre-existing vasculature. This is essential in providing the tumor tissue with the oxygen and nutrients required for tumor growth, survival of solid neoplasms, and metastasis [2]. The vascular endothelial growth factor (VEGF) and the fibroblast growth factor (FGF) are important angiogenesis regulating factors. That FGF, one of the first angiogenic growth factors to be identified [3], plays a key role in tumor-induced angiogenesis has been well established [4]. VEGF, known to be one of the most potent angiogenic factors, stimulates endothelial cells to secrete proteases and plasminogen activators, which results in the degradation of the vessel basement mem-
q
Abbreviations: AMC, 7-amino-4-methylcoumarin; FCS, foetal calf serum; HAT, histone acetylase; HDAC, histone deacetylase; HRP, horse radish peroxidase; PBS, phosphate buffered saline; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; VPA, valproic acid. * Corresponding authors. Fax: +49-69-6301-83112. E-mail address: [email protected] (J. Stein). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.02.105
brane, in turn allowing cells to invade the surrounding matrix [5]. After subsequent migration and proliferation, the cells finally differentiate to form a new vessel. Enhanced expression of VEGF has been observed in many human cancers including colorectal, breast, nonsmall-cell lung, and ovarian cancers [6]. It has also been shown that HDAC inhibitors like butyrate reactivate gene expression and consequently repress the growth and survival of tumor cells [7]. The process of acetylation and deacetylation of histones plays a crucial role in gene expression or repression. Histone deacetylases (HDACs) are enzymes responsible for deacetylation whereas histone acetyltransferases (HAT) mediate the acetylation of histones. The inappropriate transcriptional repression mediated by HDACs [8,9] is a common molecular mechanism which leads to tumor formation. The effects of HDAC inhibitors like butyrate, namely their ability to reactivate gene expression and thus to affect cell growth in the colon, has been extensively studied and reviewed in detail [10,11]. Two recently published papers convincingly demonstrated the involvement of histone deacetylases in angiogenesis by negative regulation of tumour suppressor genes in vivo an in vitro [12,13]. We like others have
694
D. Zgouras et al. / Biochemical and Biophysical Research Communications 316 (2004) 693–697
recently conclusively documented butyrate’s angiogenesis-suppressing effects on colon carcinoma cells in vitro [14,15]. G€ ottlicher et al. [16] discovered that the well-tolerated drug valproic acid (VPA) is a class I HDAC inhibitor. This activity can be distinguished from its therapeutically exploited antiepileptic efficacy [17,18]. Since VPA has been used clinically for over two decades, the pharmacology and side effects of this drug have been studied in detail. As expected for HDAC inhibitory compounds, VPA induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute leukemia (AML) patients. The aim of the current study was to investigate the anti-angiogenic effects of the class I HDAC inhibitor valproic acid on colon carcinoma cells. In this study we show that valproic acid influences the angiogenesis-related factor VEGF. Further it leads to a significant reduction of VEGF secretion in Caco-2 cells. VPA inhibits protein expression as well as mRNA levels of VEGF. We hypothesize, that inhibition of the ubiquitin–poteasome proteolytic system activity by valproic acid, as observed in our experiments, could be a possible interpretation for HDAC-effects on angiogenesis regulating proteins.
Materials and methods Cell culture. Human colon cancer cells (Caco-2) were obtained from the American Type Culture Collection (ATCC) and grown under standard conditions. Caco-2 cells of passages 50–60 were grown in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% foetal calf serum (FCS), 1% non-essential amino acids, and 1% penicillin/streptomycin. The medium was changed every second day and collected as described below. The cells were passaged at 80% (Caco-2) with Dulbecco’s PBS containing 0.25% trypsin and 1% EDTA. Cytotoxicity was assessed by a commercial kit measuring lactate dehydrogenase activity in the cell culture medium (LDH kit, Merck, Darmstadt, Germany). For the experiments, Caco-2 cells were grown until approx. 90% confluent and then switched to a serum-free medium containing either the solvent (PBS), valproic acid (1, 3 mM) (Merck–Schuchardt, Hohenbrunn, Germany), or butyrate (1, 3 mM) (Sigma Aldrich, Germany). After 12, 24, and 48 h, the cell culture medium was collected, centrifuged, and stored at )20 °C until use. Measurement of VEGF. In order to assess VEGF production in the supernatant of Caco-2 cells, the cells were incubated in serum-free medium or in serum-free medium containing valproic acid (1, 3 mM) or butyrate (1, 3 mM). After 12, 24, and 48 h, the media were collected, and VEGF was quantified by use of a colorimetric ELISA (CytELISA, CytImmune, Umkirch, Germany). Four VEGF splice variants have been identified until now. CytELISA detects the splice variant VEGF 165. SDS–polyacrylamide gel electrophoresis and immunoblot analysis. Caco-2 cells were incubated with valproic acid (1, 3 mM) or butyrate (1, 3 mM). After 24–72 h, the cells were harvested and the whole cell extract was quantified using the Bio-Rad protein colorimetric assay. VEGF and FGF were assessed by Western blot analysis using a primary antibody for VEGF (Transduction Laboratories) and a primary antibody for FGF (Santa Cruz Biotechnology). After washing, a sec-
ondary antibody (horseradish peroxidase-conjugated antibody, Vector Lab., Burlingame, USA) was applied. Afterwards the nitrocellulose mebranes were subjected to a chemoluminescence reaction (ECL, Amersham–Pharmacia Biotech, Buckinghamshire, UK) and the bands were visualized by exposure to Hyperfilm-MP (Amersham International plc, Buckinghamshire, UK). RT competitive multiplex PCR. Total cellular RNA was isolated by RNABee (Wak-Chemie, Bad Homburg, Germany), phenol/chloroform extracted, and then isopropanol precipitated. The RNA was reconstituted in diethylpyrocarbonate-treated water. RNA was reverse transcribed into cDNA by Superscript II reverse transcriptase (Invitrogen). Quantification by RT-competitive multiplex PCR was established as detailed elsewhere. Internal standards (IST) for reverse transcriptase competitive multiplex PCR were generated according to Celi et al. [26]. Primers and internal standards for GAPDH were described previously, whereas the primers and internal standards for VEGF (GenBank M32977) were kindly provided by Dr. Stefan Kippenberger (University of Frankfurt, Department of Dermatology). The primer sequences were: VEGF-A(67), 50 tgctgtcttgggtgcattgg-30 ; VEGF-B(289), 50 -acacaggatggcttgaagat-30 ; and VEGF-CB IST, 50 -acacaggatggcttgaagattctcgattggatggcagtag-30 . Primer combination VEGF-A/B amplifies a 222 bp product whereas the combination VEGF-A/CB generates an internal standard (competitor) of 178 bp. Aliquots of a master mixture containing KCl, Tris–HCl, MgCl2 , dNTPs, primers for the target genes and 0.5 ll of cDNA per 50 ll of total PCR volume were added to serial dilutions (1:3) of a mixture of the competitors. GAPDH was used as a control for initial RNA loading. Wildtype and competitors were amplified with Taq-Polymerase (Invitrogen). Each sample was electrophoresed in 2% agarose gels, stained with ethidium bromide and quantified with DocuGel IVSystem (MWG-Biotech, Ebersberg, Germany). The ratio of target to competitor was determined and, according to the method, a 1:1 ratio implies equivalent concentrations of competitor and input target mRNA. Proteasome activity assay. Untreated, valproic acid-treated (1, 3 mM) and butyrate-treated (1, 3 mM) Caco-2 cells were harvested after 24 h exposure. Proteasome activity was assessed in cell lysate with 20S proteasome activity assay kit (Chemicon). The assay is based on detection of the fluorophore 7-amino-4-methylcoumarin (AMC) after its cleavage from the labeled Subtrate LLVY-AMC by chymotrypsinlike activity of the proteasome. Free AMC is detected by fluorimetric quantification (380/460 nm). Statistical analysis. Data were expressed as means SD. Differences between two values were tested for statistical significance using the Student’s unpaired t test (SigmaPlot, SPSS Chicago, IL). A p value <0.05 considered to indicate a significant difference.
Results To elucidate whether valproic acid induces angiosuppressive effects on colon tumor cells, direct inhibition of angiogenic growth factors was quantified. We measured VEGF165 concentration, a splice variant of VEGF, in the conditioned medium over time. VEGF is known to be one of the most important angiogenic factors. As shown in Fig. 1, VEGF165 secretion to the cell culture medium was inhibited by VPA in Caco-2 cells. The highest inhibition of VEGF production occurred after 48 h (23.2%). Valproic acid showed a similar repression of VEGF165 production as after butyrate application. Decreased VEGF165 secretion was also observed after 24 h.
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Fig. 1. VEGF165 amounts in pre-conditioned medium obtained from Caco-2 cells either basally, or after treatment with valproic acid (3 mM) or butyrate (3 mM) were observed by ELISA. Means SD, n ¼ 6, *p < 0:05 and **p < 0:01.
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Fig. 3. Western blot analysis for detection of FGF expression in total cell lysate of Caco-2 cells. The cells were treated with valproic acid or butyrate (1 or 3 mM) for 48 h. Protein was separated by SDS–PAGE followed by immunoblotting and detection with the polyclonal antiFGF IgG antibody. Results are representative of three independent experiments. The graphs show the quantification by densitometric analysis normalized by the internal control (b-actin). Means SD, n ¼ 3, **p < 0:01, ***p < 0:001.
Fig. 2. Western blot analysis of VEGF expression in total cell lysate of Caco-2 cells. The cells were treated with valproic acid or butyrate (1 or 3 mM) for 72 h. Protein was separated by SDS–PAGE followed by immunoblotting and detection with the polyclonal anti-VEGF IgG antibody. Essentially identical results were obtained in three independent experiments. The graphs show the quantification by densitometric analysis normalized by the internal control (b-actin). Means SD, n ¼ 3, *p < 0:05, **p < 0:01.
Furthermore, intracellular VEGF- and FGF-isoforms were detected by Western blot in Caco-2 cell lysate. VEGF and FGF were highly expressed in untreated Caco-2 cells. Repression of VEGF protein was induced by VPA in a dose-dependent manner after 72 h (Fig. 2). The highest inhibition rate was reached after treatment with 3 mM valproic acid for 72 h. In Western blot analysis of FGF protein valproic acid decreased FGF levels after 48 h treatment (Fig. 3). Stronger effects in FGF protein expression were yet recognized in cells treated by butyrate. We further determined the expression and modulation of VEGF genes by valproic acid in Caco-2 cells (Fig. 4). RT-PCR experiments showed a decrease in VEGF mRNA levels of
Fig. 4. (A) RT competitive multiplex PCR of VEGF together with GAPDH and respective competitors 24 h after incubation without (controls) or with butyrate (3 mM) or valproic acid (3 mM). The lanes in each sample correspond to 1:3 serial dilutions of the competitors. (B) Relative decrease of VEGF mRNA expression in Caco-2 cells 24 h after incubation without (controls) or with valproic acid (3 mM) or butyrate (3 mM), related to GAPDH mRNA expression levels. Means SD, n ¼ 3, *p < 0:05, **p < 0:01.
Caco-2 cells being treated by valproic acid (3 mM) (see Fig. 4). It has been shown that the Ub/26S proteasome system, being responsible for protein degradation of regulatory proteins, plays a crucial role in regulation of important angiogenic factors like VEGF and HIF1-a [19,20]. The 20S proteasome, catalytic core of the
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Fig. 5. Proteasome activity in cell lysate from Caco-2 cells after treatment with valproic acid or butyrate (3 mM) over 24 h was determined by a 20S proteasome activity assay kit. Means SD, n ¼ 6, *p < 0:05.
proteasomecomplex, is responsible for the breakdown of key proteins involved with apoptosis, cell cycle control, and angiogenesis [21]. Effects of valproic acid and butyrate on proteasome activity were studied by a proteasome activity assay (Chemicon) in Caco-2 cell lysate (Fig. 5). Our data indicate valproic acid significantly suppresses proteasome activity (19% vs. control) in Caco-2 cells after 24 h treatment.
Discussion Formation of new blood vessels in malignant tumors is a complex multi-step process including degradation of the basement membrane, migration and proliferation of endothelial cells leading to the formation of solid endothelial cell sprouts. This process called angiogenesis is essential for the continuous growth and survival of solid neoplasms and their metastasis, providing oxygen, and nutrients to the tumor tissue. Regulation of tumorinduced angiogenesis is controlled by the tumor tissue itself producing and secreting several growth and regulating factors. Especially VEGF and FGF play a crucial role in the angiogenesis process consequently becoming also potential targets in new anticancer strategies. It has long been known that the short-chain fatty acid butyrate, an HDAC inhibitor, induces apoptosis, and differentiation of colon cancer cells [22], but most recently research has demonstrated that it also serves as a potent anti-angiogenic agent [23,24]. Current research has suggested that this effect is the result of the regulation and inhibition of VEGF [14]. We have been able to prove, confirming previous studies, that butyrate impairs the secretion of VEGF in colon cancer cells (HT29, Caco-2) in a dose-dependent manner [15]. We in turn hypothesized that the HDAC inhibitor valproic acid might similarly have this effect on Caco-2-induced angiogenesis. Its better bioavailibility since it is only slowly
metabolized, in contrast to butyrate, which is degraded rapidly, could be an additional benefit and prove of enormous significance for new anti-cancer applications. In the present study we have investigated whether colon cancer cell-induced angiogenesis is affected by valproic acid a class I HDAC inhibitor known to induce differentiation, apoptosis, and impairing cell proliferation in carcinoma cells. We could show that valproic acid influences angiogenesis by down-regulating either VEGF secretion or the intracellular protein expression in the Caco-2 cell line. We also demonstrated that the HDAC inhibitor reduces the protein expression of FGF in Caco-2 cell lysate. Translation of VEGF was further examined by RT-PCR. Both HDAC inhibitors showed repressive effects on VEGF mRNA level in Caco-2 cells. As shown in our experiments treatment with valproic acid significantly decreased proteasome activity in Caco2 cell lysate. Our findings suggest that alterations in the cellular proteasome activity by HDAC inhibitors may influence angiogenesis regulatory proteins like VEGF and HIF-1a. In the final analysis, our results of valproic acid effects on colon carcinoma cell-induced angiogenesis correspond to the results of Kwon et al. [25]. They verified that HDACs regulate the expression of crucial angiogenesis-related genes and observed that HDAC inhibitors suppress human hepatoblastoma cells [1]. The anti-angiogenic activities of valproic acid observed in this study suggest that the HDAC inhibitor valproic acid could become a valuable new addition in the attempt to develop alternative therapeutic approaches in the treatment of human colorectal malignancies. Its better bioavailibility compared to butyrate could be an additional and significant benefit. Acknowledgment This study was supported by the Else Kr€ oner-Fresenius-Foundation, Bad Homburg, Germany.
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