Bluethner T, Niederhagen M, Caca K, Serr F, Witzigmann H, Moebius C, Mossner J, Wiedmann M. Inhibition of histone deacetylase for the treatment of biliary tract cancer: A new effective pharmacological approach. World J Gastroenterol 2007; 13(35): 4761-4770 [PMID: 17729398 DOI: 10.3748/wjg.v13.i35.4761]
Corresponding Author of This Article
Dr Marcus Wiedmann, Department of Internal Medicine II, University of Leipzig, Philipp-Rosenthal-Str. 27, 04103 Leipzig, Germany. wiedm@medizin.uni-leipzig.de
Article-Type of This Article
Basic Research
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Thilo Bluethner, Joachim Mossner, Marcus Wiedmann, Department of Internal Medicine II, University of Leipzig, Philipp-Rosenthal-Str. 27, Leipzig 04103, Germany
Frederik Serr, Helmut Witzigmann, Christian Moebius, Department of Surgery II, University of Leipzig, Liebigstrasse 20a, 04103 Leipzig, Germany
Manuel Niederhagen, Institute of Pathology, University of Leipzig, Liebigstr. 26, Leipzig 04103, Germany
Karel Caca, Department of Internal Medicine I, Klinikum Ludwigsburg, Posilipostr. 4, Ludwigsburg 71640, Germany
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Supported by the Deutsche Krebshilfe, No. 10-2106-Wi 1
Correspondence to: Dr Marcus Wiedmann, Department of Internal Medicine II, University of Leipzig, Philipp-Rosenthal-Str. 27, 04103 Leipzig, Germany. wiedm@medizin.uni-leipzig.de
Telephone: +49-341-9712230 Fax: +49-341-9712239
Received: May 12, 2007 Revised: June 1, 2007 Accepted: June 4, 2007 Published online: September 21, 2007
Abstract
AIM: To investigate in vitro and in vivo therapeutic effects of histone deacetylase inhibitors NVP-LAQ824 and NVP-LBH589 on biliary tract cancer.
METHODS: Cell growth inhibition by NVP-LAQ824 and NVP-LBH589 was studied in vitro in 7 human biliary tract cancer cell lines by MTT assay. In addition, the anti-tumoral effect of NVP-LBH589 was studied in a chimeric mouse model. Anti-tumoral drug mechanism was assessed by immunoblotting for acH4 and p21WAF-1/CIP-1, PARP assay, cell cycle analysis, TUNEL assay, and immunhistochemistry for MIB-1.
RESULTS: In vitro treatment with both compounds significantly suppressed the growth of all cancer cell lines [mean IC50 (3 d) 0.11 and 0.05 μmol/L, respectively], and was associated with hyperacetylation of nucleosomal histone H4, increased expression of p21WAF-1/CIP-1, induction of apoptosis (PARP cleavage), and cell cycle arrest at G2/M checkpoint. After 28 d, NVP-LBH589 significantly reduced tumor mass by 66% (bile duct cancer) and 87% (gallbladder cancer) in vivo in comparison to placebo, and potentiated the efficacy of gemcitabine. Further analysis of the tumor specimens revealed increased apoptosis by TUNEL assay and reduced cell proliferation (MIB-1).
CONCLUSION: Our findings suggest that NVP-LBH589 and NVP-LAQ824 are active against human biliary tract cancer in vitro. In addition, NVP-LBH589 demonstrated significant in vivo activity and potentiated the efficacy of gemcitabine. Therefore, further clinical evaluation of this new drug for the treatment of biliary tract cancer is recommended.
Citation: Bluethner T, Niederhagen M, Caca K, Serr F, Witzigmann H, Moebius C, Mossner J, Wiedmann M. Inhibition of histone deacetylase for the treatment of biliary tract cancer: A new effective pharmacological approach. World J Gastroenterol 2007; 13(35): 4761-4770
Interactions between histones and DNA are regulated by the acetylation status of histones, which in eukyotic cells, plays a pivotal role in chromatin remodeling and in the regulation of gene expression: hyperacetylation determines transcription activation while hypoacetylation transcription repression. The balance between two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), can affect the acetylation status of histones. Altered HAT or HDAC activity has been identified in several cancers[1]. HDACs have been found to be associated with aberrant transcription factors, and can mediate the function of oncogenic translocation products in specific forms of leukemia and lymphoma. They are divided into three classes: classI(HDAC 1-3, 8, 11) is generally nuclear; class II (HDAC 4-7, 9, 10) is generally tissue-dependent for the expression, and can shuttle between the cytoplasm and the nucleus; and class III requires nicotinamide adenine dinucleotide (NAD) as cofactor with substrate; they were discovered recently and are poorly characterized[1]. To date, several structural unrelated classes of HDAC-inhibitors (HDACIs) demonstrating anti-tumor activity both in vitro and in vivo in animal models have been identified. These classes include carboxylic acids such as phenylbutyrate (PB), phenylacetate (PA), sodium butyrate (SB), AN-9 (Pivanex) and valproic acid; cyclic tetrapeptides such as trapoxin A; cyclic peptides such as depsipeptide or FK-228 and apicidine; benzamides such as MS27-275 and CI-994 (N-acetyldinaline); ketones such as trifluoromethyl ketone and α-ketomides; hydroxamic acids such as trichostatin A (TSA), suberoylanilide hydroxamic acid (vorinostat, SAHA), azelaic bis-hydroxamic acid (ABHA), scriptaid, oxamflatin, pyroxamide, m-carboxycinnamic acid bis-hydroxamide (CBHA), and the recently developed NVP-LAQ824, NVP-LBH589, and PXD101[2]. Multiple phaseI, II clinical trials are either completed or currently ongoing with several HDACIs, either as single agent or in combination with conventional chemotherapy, or biologicals. Clinical studies published so far have shown that HDACIs can be administered safely in humans and that treatment of some cancers with such agents seems to be beneficial[3]. NVP-LAQ824 and NVP-LBH589 are a new chemical entity belonging to a structurally novel class of cinnamic hydroxamic acid compounds[4-6] which are currently in phaseIclinical evaluation in advanced refractory solid tumors and hematologic malignancies[7-12]. However, little is known about their potential efficacy in biliary tract cancer, a rare tumor with a grim prognosis and up to now only limited treatment options. Therefore, the objectives of the current study were to investigate the efficacy of in vitro and in vivo treatment with the two novel pan-HDACIs NVP-LAQ824 and NVP-LBH589 and to evaluate the combination with gemcitabine.
MATERIALS AND METHODS
Materials
Seven biliary tract cancer cell lines - five extra-hepatic bile duct cancer cell lines (EGI-1, TFK-1, CC-SW-1, CC-LP-1, and SK-ChA-1)[13-17] and two gallbladder cancer cell lines (Mz-ChA-1, Mz-ChA-2)[16]-were examined. All cell lines were cultured with appropriate media and incubated at 37°C in a humidified atmosphere containing 50-100 mL/L CO2 in air, and the media were changed every three days. The HDACIs NVP-LAQ824 and NVP-LBH589 were provided by Novartis (Basel, Switzerland) and dissolved in dimethyl sulfoxide (DMSO) (10 mmol/L stock). Hoechst dye, sodium butyrate and monoclonal (mc) β-actin antibody were purchased from Sigma (Sigma-Aldrich Chemie GmbH Munich, Germany), mc p21WAF-1/Cip-1, and polyclonal (pc) cleaved-poly(ADP-ribose) polymerase (PARP) antibodies from Cell Signaling (Cell Signaling Technology, Beverly, USA), mc acH4 antibody from Upstate (Upstate Biotechnology, Lake Placid, USA), mc MIB-1 antibody from Dako (Glostrub, Denmark), and gemcitabine [diluted in 50 g/L dextrose in water (D5W) and 50 mL/L DMSO] and etoposide (dissolved in normal saline to 10 mmol/L stock) from our hospital pharmacy. Six to eight-week-old female athymic NMRI nude mice were supplied from Taconic (Taconic Europe, Ry, Denmark) and held under pathogen-free conditions. Humane care was administered, and study protocols complied with the Institutional Guidelines.
Inhibition of cell growth detected by MTT assay
Cytotoxic effects of both drugs were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldridge Chemie GmbH Munich, Germany) assay. About 1-5 × 103 cells were seeded in triplicate in 96-well plates (100 μL/well) and allowed to attach overnight. The medium was then replaced with medium (100 μL) containing the designated drug or vehicle control (50 mL/L DMSO in D5W), followed by an incubation for 3 or 6 d. For the 6-d experiment, medium was changed after 3 d. Three hours before the end of the incubation period, 10 μL of phosphate-buffered solution (PBS) containing 5 g/L MTT was added to each well. Following this, the medium was removed. The precipitate was then resuspended in 100 μL of lysis buffer (DMSO, 100 g/L SDS). The absorbance was measured on a plate reader at 590 nm and a reference wavelength of 630 nm. Each experiment was performed in triplicate.
Immunoblotting
Cell culture monolayers were washed twice with ice-cold PBS, and lysed with RIPA-buffer containing Tris-HCl (50 mmol/L, pH 7.4), NP-40 (10 g/L), sodium-desoxycholate (2.5 g/L), NaCl (150 mmol/L), EDTA (1 mmol/L), sodium-orthovanadate (1 mmol/L), and one tablet of complete mini-EDTA-free protease inhibitor cocktail (Boehringer, Mannheim, Germany) (in 10 mL buffer). Histones for anti-acH4 immunoblotting were isolated by acid extraction [cells were lysed in ice-cold lysis buffer (HEPES 10 mmol/L; pH 7.9), MgCl2 (1.5 mmol/L), KCl (10 mmol/L), DTT (0.5 mmol/L), PMSF (1.5 mmol/L), and additional protease inhibitor]. One molar HCl was added to a final concentration of 0.2 mol/L, followed by incubation on ice for 30 min, and centrifugation at 13 000 g for 10 min. The supernatant was kept and dialysed against 200 mL of 0.2 mol/L acidic acid twice for 1 h and against 200 mL of H2O overnight. Proteins were quantified by Bradford protein assay (Bio-Rad, Munich, Germany) and stored at -80°C, and 50 μg of cell or tissue lysates were separated on SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Freiburg, Germany). Membranes were then incubated in blocking solution [50 g/L dry milk in 10 mmol/L Tris-HCl, 140 mmol/L NaCl, 1 g/L Tween-20 (TBS-T)], followed by incubation with the primary antibody at 4°C overnight (50 g/L BSA in TBS-T). The membranes were then washed in TBS-T, and incubated with horseradish peroxidase (HRPO)-conjugated secondary antibodies for 1 h at room temperature. Antibody detection was performed with an enhanced chemoluminescence reaction (SuperSignal West Dura, Pierce, Rockford, USA).
Cell cycle analysis
Cells (2 × 105) were seeded in T-25 flasks, treated with various concentrations of NVP-LAQ824 or NVP-LBH589 or vehicle control (50 mL/L DMSO in D5W) for 72 h, washed with PBS, trypsinized, centrifuged, and fixed in 750 mL/L ice-cold ethanol/phosphate-buffered saline containing 10 g/L EDTA. DNA was labelled with 100 mL/L propidium iodide. Cells were sorted by FACScan analysis, and cell cycle profiles were determined using ModFitLT V2.0 software (Becton Dickinson, San Diego, USA). Each experiment was performed in triplicate.
Animal studies
Tumors were induced by injecting 5 × 106 Mz-ChA-2 or EGI-1 cells in 200 μL of PBS subcutaneously into the flank region of NMRI nude mice. Treatment was started when an average tumor volume of 150 mm³ was reached (usually after 2 wk). The verum groups intraperitoneally received either NVP-LBH589 (40 mg/kg, 5 × weekly) or gemcitabine (5 mg/kg, 2 × weekly) or a combination of both (NVP-LBH589 at 20 mg/kg, 5 × weekly plus gemcitabine at 5 mg/kg, 2 × weekly), whereas the control group received placebo (carrier solution 50 mL/L DMSO in D5W) only. Treatment was continued for 28 consecutive days, tumors were daily measured with a Vernier caliper and tumor volumes were calculated using the formula: tumor volume = 0.5 ×L×W², where L represents the length and W the width of the tumor. When treatment was finished, animals were sacrificed and tumors were excised and weighed.
TUNEL POD test
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (In Situ Cell Death Detection Kit, POD) was used to detect apoptosis in paraffin sections from mouse tumor tissue. TUNEL test was carried out following the manufacturer's instructions (Roche, Penzberg, Germany) as previously described[18]. Apoptotic cells (red) were counted under a light microscope after fluorescence signal conversion using antibody with conjugated peroxidase and the substrate for peroxidase (DAB, Roche, Penzberg, Germany). The number of positive cells was counted by an experienced pathologist in a total of 8 high-power fields (HPFs) and expressed as mean percentage of total cells in these fields of the tumor. Necrotic tumor cells were excluded from cell count.
Immunohistochemical staining
For MIB-1 staining, we used paraffin sections following a protocol that has been described elsewhere[19]. The number of positive cells was counted in a total of 4 HPFs and expressed as mean percentage of total cells in these fields of the tumor.
Statistical analysis
Statistical calculations were performed using SPSS version 10.0 (SPSS Inc., Chicago, USA). Numeric data were presented as mean ± SD or SEM. Inter-group comparisons were performed using Student's t test. P < 0.05 was considered statistically significant.
RESULTS
Inhibition of cellular growth by NVP-LAQ824 and NVP-LBH589
After 3 d of incubation, all tested cell lines were sensitive to NVP-LAQ824 [mean IC50 (3 d) = 0.11 ± 0.06 μmol/L] and even more to NVP-LBH589 [mean IC50 (3 d) = 0.05 ± 0.05 μmol/L]. There was no significant difference in response between the group of extra-hepatic bile duct cancer cell lines [mean IC50/LAQ824 (3 d) = 0.10 ± 0.07 μmol/L and mean IC50/LBH589 (3 d) = 0.05 ± 0.06 μmol/L] and the group of the two gallbladder cancer cell lines [mean IC50/LAQ824 (3 d) = 0.12 ± 0.03 μmol/L and mean IC50/LBH589 (3 d) = 0.06 ± 0.05 μmol/L]. Inhibition of cell growth was more pronounced if incubation time was extended to 6 d, with a mean IC50 value of 0.05 + 0.02 μmol/L for NVP-LAQ824 and 0.02 + 0.01 μmol/L for NVP-LBH589. Once again, there was equal response between the group of extra-hepatic bile duct cancer cell lines [mean IC50/LAQ824 (6 d) = 0.05 ± 0.02 μmol/L and mean IC50/LBH589 (6 d) = 0.01 ± 0.01 μmol/L] and the group of the two gallbladder cancer cell lines [mean IC50/LAQ824 (6 d) = 0.06 ± 0.03 μmol/L and mean IC50/LBH589 (6 d) = 0.02 ± 0.02 μmol/L] (Figure 1 and Table 1). In addition, DMSO, the solvent of NVP-LAQ824 and NVP-LBH589, alone had no influence on cell growth (data not shown).
Table 1 Inhibition of cell growth by NVP-LAQ824 and NVP-LBH589.
Figure 1 In vitro treatment of biliary tract cancer cell lines with NVP-LAQ824 and NVP-LBH589.
A: 3-d incubation with NVP-LAQ824 (n = 3); B: 6-d incubation with NVP-LAQ824 (n = 3); C: 3-d incubation with NVP-LBH589 (n = 3); D: 6-d incubation with NVP-LBH589 (n = 3).
Mechanism of drug action
Treatment of cell lines EGI-1 and Mz-ChA-2 with 0.1 μmol/L NVP-LAQ824 or 0.1 μmol/L NVP-LBH589 for 24 h resulted in acetylation of histone H4 (Figure 2A). Protein extract from HELA cells that were treated with 5 mmol/L sodium butyrate served as positive control. The same treatment caused an induction of p21WAF-1/CIP-1 expression (Figure 2B). Cell lysate from HEK 293 cells served as positive control. A dose increase to 0.2 μmol/L NVP-LAQ824 or 0.2 μmol/L NVP-LBH589 corresponded with an increase of p21WAF-1/CIP-1 levels. Histone H4 acetylation was higher in treated Mz-ChA-2 than EGI-1 cells, whereas p21WAF-1/CIP-1 expression was higher in treated EGI-1 cells. Immunoblotting for cleaved-PARP was positive when cells were treated with 0.1 μmol/L NVP-LAQ824 or 0.1 μmol/L NVP-LBH589 for 24 h (Figure 2C, Lanes 2 and 5). PARP cleavage was more pronounced when the concentration of NVP-LAQ824 or NVP-LBH589 was increased to 0.2 μmol/L (Figure 2C, Lanes 3 and 6). Lysate from untreated NIH-3T3 cells (Figure 2C, Lane 7) and from cells that were treated with 25 μmol/L etoposide for 5 h [Figure 2C, Lane 8 (lane *)] served as negative control. Lysate from NIH-3T3 cells that were treated with 10 μmol/L etoposide for 24 h [Figure 2C, Lane 9 (lane **)] served as positive control. Positive and negative controls were selected according to the recommendations of the manufacturers of antibodies. Staining with β-actin antibody confirmed equal protein loading in all immunoblots.
Figure 2 Mechanism of drug action after in vitro treatment with NVP-LAQ824 and NVP-LBH589 for 24 h.
Lane *: NIH-3T3 cells treated with 25 μmol/L etoposide for 5 h (negative control); lane **: NIH-3T3 cells treated with 10 μmol/L etoposide for 24 h (positive control). A: acetylation of histone H4; B: p21WAF-1/CIP-1 expression; C: PARP cleavage.
Treatment of cell lines EGI-1 and Mz-ChA-2 with 25 nmol/L NVP-LAQ824 or 25 nmol/L NVP-LBH589 for 72 h resulted in G2/M phase arrest. This arrest was more pronounced if the dose of NVP-LAQ824 or NVP-LBH589 was increased to 50 nmol/L. For both concentrations, the effect of NVP-LBH589 was stronger than the effect of NVP-LAQ824 (Table 2). Further increase of the NVP-LAQ824 and NVP-LBH589 concentration to 100 nmol/L and 200 nmol/L led to no further significant changes in G2/M phase arrest (data not shown). Sub-G1-peak percentage was not analyzed.
Inhibition of tumor cell growth by NVP-LBH589 ± gemcitabine in nude mice
Tumors were induced in nude mice by subcutaneous injection of Mz-ChA-2 and EGI-1 cell lines. These cell lines were selected because they had shown the best growth capability in nude mice in our previous studies[20,21]. Since NVP-LBH589 seemed to have a higher in vitro activity than NVP-LAQ824 in these two cell lines (Table 1), only NVP-LBH589 was investigated in vivo in order to save costs. Treatment of mice consisted of intraperitoneal injections with NVP-LBH589, gemcitabine, NVP-LBH589 plus gemcitabine (COMBO) or placebo (50 mL/L DMSO in D5W). Starting at d 19, respectively at d 26, of the experiment, EGI-1 cell tumors showed a significantly reduced volume in comparison to control after treatment with NVP-LBH589 or COMBO (n = 7 for each group; P < 0.05). The statistically significant difference was maintained until the end of the experiment. In contrast to that, treatment of mice with gemcitabine alone did not result in any significant reduction of tumor growth compared to control (Figure 3A). Treatment of Mz-ChA-2 tumors with NVP-LBH589, COMBO, or gemcitabine resulted in a significantly reduced volume in comparison to control at d 5, 5, and 8 (P < 0.01), respectively (n = 7 for each group; Figure 3B). The statistically significant difference was maintained until the end of the experiment. Regarding tumor latency (representing the time for the tumor to increase to 150% of the initial volume when treatment was initiated), the extrapolated data were as follows for EGI-1: placebo d 6, LBH d 26, COMBO d 12, and gemcitabine d 4. The data for Mz-ChA-2 were: placebo d 3, LBH d 14, COMBO "not reached", and gemcitabine d 7. At the end of the experiment at d 29, tumor mass in EGI-1 cells-bearing mice was significantly diminished as compared to placebo after treatment with NVP-LBH589 (-66%) or COMBO (63%) (P < 0.05; Figure 3C). In contrast to that, treatment of mice with gemcitabine alone (-6%) did not result in any significant reduction of tumor mass as compared to control (Figure 3C). For Mz-ChA-2 cells-bearing mice, tumor mass was significantly diminished after treatment with either NVP-LBH589 (-87%), COMBO (96%), or gemcitabine (-74%), respectively (P < 0.01; Figure 3C). For both cell lines, EGI-1 and Mz-ChA-2, the combination of NVP-LBH589 and gemcitabine was more effective in tumor mass reduction in comparison to gemcitabine alone (P < 0.05 and P < 0.01, respectively). Moreover, for tumor cell line Mz-ChA-2, COMBO reduced tumor mass at a higher degree than NVP-LBH589 alone (P < 0.05), although NVP-LBH589 was administered at half dose in the COMBO group. Concerning side effects of the different drugs used in our experiments with EGI-1 and Mz-ChA-2 cells tumor-bearing mice, NVP-LBH589 alone (-17% and -19%, respectively) and COMBO (-7% and -10%, respectively) induced weight loss in the animals at d 29 of therapy as compared to the initial body weight, whereas gemcitabine alone did not. One animal died at d 12 (EGI-1 experiment) in the COMBO group, and 2 animals died at d 22 and d 26 (Mz-ChA-2 experiment) in the NVP-LBH589 group from significant weight loss. These three mice were replaced by back-up mice in order to guarantee equal numbers in the different groups (n = 7) for the comparisons of tumor volume and tumor mass at the end of the experiment. Finally, NVP-LBH589 alone at a dose of 40 mg/kg (5 × weekly) caused diarrhea and exsiccation in the animals starting at d 16 of the experiment. We performed no further microscopic evaluation for organ damage in these animals. However, there was no macroscopic tissue damage.
Figure 3 In vivo treatment with NVP-LBH589 ± gemcitabine in chimeric mice.
A: Effect on tumor volume of EGI-1 cells (aP < 0.05, NVP-LBH589 vs control; cP < 0.05, COMBO vs control); B: Effect on tumor volume of Mz-ChA-2 cells (aP < 0.05, bP < 0.01, NVP-LBH589 vs control; cP < 0.05, dP < 0.01, COMBO vs control; fP < 0.01, gemcitabine vs control); C: Effect on tumor mass (aP < 0.05, NVP-LBH589 or COMBO vs control; bP < 0.01, NVP-LBH589 or COMBO or gemcitabine vs control).
In order to assess the anti-tumoral drug mechanism, paraffin sections of mouse tumors were stained with hematoxylin-eosin (HE), MIB-1 (proliferation marker) and TUNEL (apoptosis marker) (Figure 4). Treatment with NVP-LBH589, gemcitabine, and COMBO reduced proliferation (reduced MIB-1 staining) and induced apoptosis (increased TUNEL staining) (Figure 4, Table 3).
Table 3 MIB-1- and TUNEL-staining of mouse tumor specimens (mean, %).
Figure 4 Hematoxilin-eosin (HE), MIB-1 (proliferation marker) and TUNEL (apoptosis marker) staining of mouse tumors (SABC, × 40).
A: cell line Mz-ChA-2; B: cell line EGI-1.
DISCUSSION
Cholangiocarcinoma (CC) is the second most common primary hepatic tumor, with increasing incidence and a high mortality[22-24]. Tumors may occur anywhere along the biliary ductal system, and are arbitrarily defined anatomically as intra-hepatic or extra-hepatic CC as well as adenocarcinoma of the gallbladder[25,26]. Unfortunately, the vast majority of patients with CC typically seek treatment with advanced disease, and often these patients are deemed poor candidates for curative surgery. Moreover, conventional chemotherapy and radiation therapy have not been shown to be effective in prolonging long-term survival, and although photodynamic therapy combined with stenting has been reported to be effective as palliative treatment, it is not curative[27-30]. Thus, there is a need to develop novel therapeutic strategies for CC based on exploiting selected molecular targets that would have an impact on clinical outcome[31,32].
One possible approach may be the use of HDACIs, perhaps in combination with conventional chemotherapy or other so called biologicals. Therefore, in our current study, we investigated the two novel cinnamic hydroxamic acid compounds NVP-LAQ824 and NVP-LBH589 for in vitro and in vivo treatment of CC. Cell-growth inhibition by NVP-LAQ824 and NVP-LBH589 was studied in vitro in 7 human biliary tract cancer cell lines by MTT assay. Treatment with the both compounds significantly suppressed the growth of all cancer cell lines after 3 and 6 d with a mean IC50 (3d) of 0.11 and 0.05 μmol/L, and with a mean IC50 (6d) of 0.05 and 0.01 μmol/L, respectively. In previous in vitro studies, NVP-LAQ824 exhibiteda potent anti-proliferative activity against the HCT116 colon carcinoma cell line (IC50 = 0.01 μmol/L), as well as against H1299 (IC50 = 0.15 μmol/L) and A549 non-small cell lung carcinoma cells (IC50 = 0.15 μmol/L), DU145 (IC50 = 0.018 μmol/L), PC3 prostate cancer cell lines (IC50 = 0.023 μmol/L), Cal27 (IC50 = 0.04 μmol/L), SCC25 (IC50 = 0.095 μmol/L), SCC9 (IC50 = 0.245 μmol/L), FaDu (IC50 = 0.340 μmol/L) head and neck squamous carcinoma cells, and MDA-MB-435 (IC50 = 0.039 μmol/L) and BT-474 (0.03 μmol/L) human breast adenocarcinoma cells after 72 h of exposure[6,33,34]. The in vitro effects of NVP-LAQ824 on hematologic malignancies have been examined in several human cell lines, including AML (HL60, K562), lymphoma (Namalwa, DHL10), and multiple myeloma (IM9, 8226). Death of more than 90% of all cells was seen in all cell lines following 48 h of drug incubation, with exposures as low as 0.1 μmol/L[35-37]. NVP-LBH589, the second investigated compound, was even more effective in vitro for the treatment of human chronic myeloid leukemia blast crisis K562 and LAMA-84, multiple myeloma, and acute leukemia MV4-11 cells[5,38-40].
The in vitro anti-tumoral drug mechanism in our study was assessed by immunoblotting for acH4 and p21WAF-1/CIP-1, PARP assay, and cell cycle analysis. Treatment with both compounds was associated with hyperacetylation of nucleosomal histone H4, increased expression of p21WAF-1/CIP-1, induction of PARP cleavage, and cell cycle arrest at G2/M checkpoint. Hyperacetylation of nucleosomal histones H3 and H4 has been previously reported as the primary mechanism of action of NVP-LAQ824 and NVP-LBH589 for the treatment of HCT116, A549, MDA-MB-231, BT-474, MCF-7 (human breast adenocarcinoma), HL-60, K562, MV4-11, and multiple myeloma cells[5,6,34,35,41]. Inactivation of tumor suppressor p14/mdm-2/p53/p21WAF-1/CIP-1 signaling pathway is a phenomenon that occurs frequently in CC, mostly due to p53 mutations or by up-regulation of mdm-2, an inhibitor of p53[42]. P21WAF-1/CIP-1 binds to the cell division kinase (CDK) 4:cyclin D complex and prevents it from phosphorylating Rb. Therefore, the release of the bound E2F transcription factor that regulates genes encoding for proteins critical to entrance into the S-phase of the cell cycle is prevented[25]. Up-regulation of p21WAF-1/CIP-1 induced by NVP-LAQ824 and NVP-LBH589 has been described for BT-474, MCF-7, MDA-MB-468 (human breast adenocarcinoma), human umbilical vein endothelial cells (HUVEC), LNCaP (prostate cancer), K562, LAMA-84, MV4-11 and multiple myeloma cells[5,34-36,38,43,44]. P21WAF-1/CIP-1 is likely to be transcriptionally activated by a p53-independent mechanism, since it has been shown for NVP-LAQ824 to activate the p21WAF-1/CIP-1 promoter[6]. Other commonly induced tumor suppressor genes include p53, p57 and the CDK inhibitor p27kip1[34,36,38,40].
Another mechanism of in vitro drug action found in our study was induction of apoptosis as shown by the increase of PARP cleavage, a process mediated by active effector caspase-3. This phenomenon, induced by NVP-LAQ824 and NVP-LBH589, has been described previously for several other cell lines[5,34-36,39,43]. Furthermore, transcriptional up-regulation of pro-apoptotic genes such as FAS, TRAIL, BID or BAX and down-regulation of anti-apoptotic genes such as BCL-XL or FLIP, although not examined in our study, may explain apoptotic cell death induced by HDACIs, besides priming of malignant cells for innate immune effector mechanisms[36,45-49]. Whereas a number of different mechanisms were described for HDACI-induced cell death, including the death receptor-activated caspase cascade, usually the intrinsic apoptotic pathway, mediated by mitochondrial membrane disruption, is commonly activated, which has also been shown for NVP-LAQ824 and NVP-LBH589[38,50]. Among several genes repressed by HDACIs are cyclin D1 and thymidylate synthase. The result is a G1 or G2-M cell cycle arrest and differentiation, common mechanisms of the anti-proliferative effect of HDACIs. Further potential growth inhibitory mechanisms include induction of other cell cycle regulatory genes such as GADD45α and β and up-regulation of transforming growth factor β (TGF-β) receptor signaling, leading to repression of c-myc and cell cycle arrest. Several studies, including ours, investigating NVP-LAQ824 and NVP-LBH589, could demonstrate G2-M cell cycle arrest, induced by those compounds[6,34,44], others detected G1-arrest[5,35,36].
Encouraged by our in vitro results, we decided to test the most effective drug NVP-LBH589 in vivo in comparison to placebo using the chimeric mouse model. At d 29 of the experiment, NVP-LBH589 significantly reduced tumor mass in comparison to placebo and potentiated the efficacy of gemcitabine. The NVP-LBH589 dose of 40 mg/kg (5 d/wk) was selected according to a study testing different intravenous doses of NVP-LAQ824 between 5 and 100 mg/kg (5 d/wk) in a similar chimeric mouse model using the human colon cancer cell line HCT116[6]. In vivo data for NVP-LBH589 were only available after completion of our study, using human prostate carcinoma cell PC-3 xenografts, being able to show tumor reduction at a dose of 10 mg/kg per day[44]. Surprisingly, weight loss of the animals, as observed in our study, was not previously reported for NVP-LAQ824[6]. Therefore, further studies are required to examine animals microscopically for organ damage. Unfortunately, this was not done in our experiments. Since we already experienced this phenomenon in a small pilot study with 3 animals (data not shown), we decided to have the dose of NVP-LBH589 for the combination therapy with gemcitabine. The latter drug was selected since it is a commonly used drug for CC monotherapy in the palliative setting[30]. Even with this dose reduction of NVP-LBH589, our results do strongly support an additive effect which, however, requires further investigation. In human breast cancer cell lines SKBR-3 and BT-474, NVP-LAQ824 also enhanced taxotere-, epithilone B-, and gemcitabine-induced apoptosis in vitro[34]. For head and neck squamous carcinoma cells, the combination of NVP-LAQ824 with gemcitabine was more effective in vitro than a combination with docetaxel, paclitaxel, or cisplatin, especially when used in the sequence of the cytotoxic agent first for 24 h, followed by 48 h of NVP-LAQ824[33]. There might be also a synergistic in vitro effect of a combination of NVP-LAQ824 and VEGFR-inhibitor PTK787/ZK222584 as demonstrated for PC3 and MDA-MB-435 cells[41]. Similar results were observed in vitro for a combination of NVP-LBH589 and proteasome inhibitor bortezomib[39], NVP-LBH589 and tyrosine kinase inhibitor AMN107[40], and NVP-LBH589 and heat-shock protein 90 (hsp90) inhibitor 17-allyl-amino-demethoxy geldanamycin (17-AAG)[5].
Anti-tumoral drug mechanism in our in vivo model was assessed by TUNEL assay, and immunohistochemistry for MIB-1, which revealed increased apoptosis (TUNEL) and reduced cell proliferation (MIB-1), confirming our in vitro data. Surprisingly, the calculated numbers were much smaller than expected from the in vitro experiments.
Finally, it should be mentioned that other mechanisms of anti-tumor activity of HDACIs include anti-angiogenic properties through alteration of VEGF-, HIF-1α-, Ang-2/Tie-2-, and survivin-signaling, down-regulation of endothelial nitric oxide synthase (eNOS), suppression of tumor invasion through negative regulation of matrix metalloproteinases expression, and depletion of several oncoproteins that are normally stabilized by binding to the hsp90 multi-chaperone complexes, including bcr-abl, ErbB2, Raf-1, or AKT (HDACIs may induce acetylation of hsp90 via inhibition of HDAC 6, thus inhibiting chaperone activity, resulting in polyubiquination and proteasomal degradation of the hsp90 client proteins), which was not further investigated in our study[44,51].
In conclusion, our findings suggest that NVP-LBH589 and NVP-LAQ824 are active against human biliary tract cancer in vitro. In addition, NVP-LBH589 demonstrated significant in vivo activity and potentiated the efficacy of gemcitabine. Therefore, further clinical evaluation of NVP-LBH589 alone or its combination with gemcitabine for the treatment of biliary tract cancer is recommended.
ACKNOWLEDGMENTS
We thank Annett Kluge, Katrin Schoepp, Karen Rother, and Ines Sommerer for technical assistance and Novartis Pharma for the provision of NVP-LAQ824 and NVP-LBH589.
COMMENTS
Background
Carcinoma of the biliary tree are rare tumors of the gastrointestinal tract with worldwide rising incidence for intrahepatic cholangiocarcinoma during the last several years. At present, complete resection is the only potentially curative therapy.But, most of the patients present with already advanced disease. In the palliative setting, systemic chemotherapy (chemoradiation) has not been clearly proven to prolong survival significantly.
Research frontiers
Receptor tyrosine kinase (TKs), cyclooxygenase-2 (COX-2), mammalian target of rapamycin (m-TOR), and histondeacetylase (HDAC) inhibitors are currently under preclinical and clinical evaluation as new treatment options.
Innovations and breakthroughs
In 2006, the results of a two-stage phaseIand a phase II study suggested a therapeutic benefit for EGFR blockade with erlotinib and combined erbB-1/erbB-2 signaling inhibition with GW572016 (Lapatinib) in patients with biliary cancer. In 2007, a phase II study was presented examining the combination of gemcitabine, oxaliplatin and angiogenesis inhibitor bevazicumab (GEMOX-B) in unresectable or metastatic biliary tract cancer/gallbladder cancer. Early evidence of antitumor activity was seen.
Applications
The aim of our study was to investigate the in vitro and in vivo treatment with histone deacetylase inhibitors NVP-LAQ824 and NVP-LBH589 in biliary tract cancer. Our findings suggested that NVP-LBH589 > NVP-LAQ824 are active against human biliary tract cancer in vitro. In addition, NVP-LBH589 demonstrated significant in vivo activity and potentiated the efficacy of gemcitabine.
Terminology
Histones (positively charged proteins) are the major components of chromatin. Histone acetylation and deacetylation modulate chromosome structure and regulate gene transcription. Two families of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), activate and repress gene expression, respectively. Aberrant HAT or HDAC activity is associated with various epithelial and hematologic cancers. HDACs may play an important role in human oncogenesis through HDAC-mediated gene silencing and interaction of HDACs with proteins involved in tumorigenesis. HDAC inhibition could potentially restore normal processes in transformed cells without affecting normal cells.
Peer review
This manuscript is original, interesting and well-written. It deserves to be published.
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