Monitoring tumor response after histone deacetylase inhibitor treatment using 3’-deoxy-3’-[18F]-fluorothymidine PET
Running Title: Imaging tumor response to ISAHA with 18F-FLT PET
*Pei-Chia Chan1,2, *Chun-Yi Wu3, Lin-Shan Chou1, Chung-Hsien Ho1, Chi-Wei Chang4, Shih-Hwa Chiou5, Wuu-Jyh Lin6, Fu-Du Chen7, C. Allen Chang1,2, Jeng-Jong
Hwang1, Ren-Shyan Liu3,8,9, and Hsin-Ell Wang1,2
1Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan
2Biophotonics & Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan
3Medical Cyclotron Center, Chung Shan Medical University Hospital, Taichung, Taiwan
4Department of Nuclear Medicine and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taipei, Taiwan
5Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan
6Institute of Nuclear Energy Research, Atomic Energy Council, Tao-Yuan County, Taiwan
7Graduate Institute of Biotechnology, Chinese Culture University, Taipei, Taiwan
8Department of Nuclear Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
9Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug
Testing Center, Taipei, Taiwan
* Pei-Chia Chan and Chun-Yi Wu contribute equally to this work.
Manuscript Category: Research Article
Corresponding authors: Hsin-Ell Wang, PhD, Department of Biomedical Imaging
and Radiological Sciences, National Yang-Ming University, 155, Sec. 2, Li-Nong Rd., Taipei, Taiwan 11221. FAX: +886-2-28201095. E-mail: [email protected]; Ren-Shyan Liu, MD, Department of Nuclear Medicine and National PET/Cyclotron Center, Taipei Veterans General Hospital, No.201, Sec. 2, Shi-Pai Rd., Beitou 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
District, Taipei, Taiwan 11217. E-mail: [email protected] 1
Abstract
Purpose: This study employed 3’-deoxy-3’-[18F]-fluorothymidine ([18F]FLT) microPET scanning to assess the treatment response of histone deacetylase inhibitors (HDACi), e.g., N1-hydroxy-N8-phenyloctanediamide (SAHA) and its iodinated derivative ISAHA, in a hepatoma mouse model.
Procedures: The in vitro cytotoxicity of HDACi in various hepatoma cell lines was
determined by MTT assay and flow cytometry. ISAHA and SAHA were used to treat HepG2 hepatoma xenograft-bearing mice. The treatment responses were characterized in terms of tumor burden, microPET imaging and immunohistochemical staining of tumor sections.
Results: ISAHA effectively inhibited HepG2 hepatoma cell survival and tumor
growth. A significantly reduced tumor uptake during HDACi treatment was noticed in [18F]FLT microPET imaging, which was consistent with the findings in immunohistochemical staining.
Conclusions: ISAHA can suppress tumor cell proliferation both in vitro and in vivo.
[18F]FLT PET is a promising modality for evaluating the in vivo therapeutic efficacy of HDACi at the early stage of treatment.
Key words: [18F]FLT, HDACi, treatment response, MicroPET, hepatocellular carcinoma 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Introduction
Hepatocellular carcinoma (HCC) is a malignant disease that is showing a continuously increasing incidence across the world, and is more prevalent in Taiwan than in either the United States of America or Western Europe. A history of chronic hepatitis B infection, a factor that increases by more than 100-fold risk of HCC, may account for the high prevalence of HCC in Taiwan . Despite advances in surgery, radiotherapy, and chemotherapy, HCC still remains difficult therapeutic challenge.
At present, it is believed that epigenetic alterations are crucial to the onset and progression of cancer and that these act in parallel with genetic defects such as gene mutation and gene deletion . In terms of epigenetic alterations, histone modifications, especially acetylation, are considered to be more labile than DNA methylation. The overall levels of histone acetylation are regulated by the balance between histone acetyltransferase (HAT) activity and histone deacetylases (HDAC) activity. Generally, any imbalance in these two enzymes will leads to various human diseases including cancers. Most cancers are characterized by overexpression of HDACs, which causes a global reduction in level of histone acetylation and eventually promotes tumor growth .
Histone deacetylase inhibitors (HDACi), a new class of chemotherapeutics, are compounds that are able to inhibit the activity of HDACs. Hyperacetylation of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
histones can be achieved by employing HDACi to treat living cells and animals . Many preclinical studies using a broad range of cancer cells have demonstrated that HDACi have an anti-cancer effect that involves remodeling of the structure of chromatin and then the activation of multiple downstream signal pathways, namely cell cycle arrest, redifferentiation, apoptosis, mitotic disruption, enhanced accumulation of reactive oxygen species (ROS), and inhibition of angiogenesis . Moreover, these effects are restricted to cancer cells and do not seem to affect nonmalignant cells. To date, only two HDACi, vorinostat (SAHA) and romidepsin, have been approved by U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T cell lymphoma (CTCL) .
In fact, SAHA exhibits potent antitumor activity against solid tumors in addition to targeting hematologic malignancies as outlined earlier. For example, Emanuele et al. reported that SAHA is able to stimulate the extrinsic apoptotic pathway by increasing expression of both FasL and the FasL receptor; furthermore, it exerts a synergistic effect in HepG2 cells when combined with bortezomib, an inhibitor of the 26S proteasome . Bruzzese et al. indicated that SAHA enhances the anti-proliferative and apoptotic effects of gefitinib via regulation of the expression level of the ErbB receptor in H&N squamous cell carcinoma . At present, SAHA is being evaluated as a treatment for malignancies in phase II and III clinical trials as a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
monotherapy and as a combined therapy with other agents . Researchers around the world dedicated themselves to developing novel derivatives of SAHA. However, when studying the therapeutic efficiency of a new drug, it often is impractical to routinely perform a biopsy in order to determine the in vivo treatment response during treatment. Thus there is a need for a noninvasive approach that allows in vivo treatment responses to be assessed.
Uncontrolled cell proliferation is an important hallmark of cancer. The ultimate objective of therapeutics is always to impede the unlimited cell proliferation associated with tumors. Real-time and accurate information on the proliferating status of cancer would be helpful to physicians and allow them to optimize clinical management with minimal adverse effects and expense. Computed tomography (CT) and magnetic resonance imaging (MRI) are usually applied to determine changes in tumor size when evaluating treatment efficacy. However, tumor shrinkage is usually a delayed response during treatment and it has been recently recognized that these modalities are inappropriate and do not loyally reflect the pharmacological effect of cytostatic agents, particularly if the treatment only retards tumor growth and does not cause immediate cell death. Previous study using SAHA as a clinical treatment only showed retarded tumor progression . In contrast, nuclear medicine imaging, such as positron emission tomography (PET) and single photon emission computed 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
tomography (SPECT), with appropriate imaging probes that are specific for cell proliferation, are feasible ways to determine the treatment efficacy.
At present, monitoring DNA synthesis via the salvage pathway using radiolabeled nucleoside probes is considered to be the most specific approach available for assessing the cell proliferation rate of a tumor . [18F]FLT can be phosphorylated by cytosolic thymidine kinase 1 (TK1) and is then trapped in the cells as its phosphate form together with thymidine. Depending on the different tissue demands in terms of DNA precursors, the accumulation of [18F]FLT can be used as a reliable index for the quantification of the cell proliferation rate of a tumor; this has been confirmed by findings showing that there is a strong correlation between [18F]FLT uptake and the Ki67 staining index across various types of cancer . [18F]FLT PET has been employed to reveal the killing effects of various chemodrugs, such as cisplatin, 5-fluorouracil, and liposomal doxorubicin in tumor animal models or clinical patients . In addition, Leyton et al. and Na et al. revealed that [18F]FLT PET is able to detect the early therapeutic efficacy of HDACi, PAQ824 and PXD101 using a colon carcinoma xenograft mouse model .
In this study, we found beyond expectation that N1-hydroxy-N8-(4-iodophenyl)octanediamide (ISAHA), an analog of SAHA with a iodine substituent at the 4-position of benzene, showed higher potency than SAHA in retarding the growth 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
of HepG2 hepatoma cells, both in vitro and in vivo. [18F]FLT PET was applied to monitor the treatment response to ISAHA in a subcutaneous hepatoma-bearing mouse model.
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Materials and Methods
Ethics statement in relation to animal studies
The animal studies were performed in strict accordance with the recommendations presented in the Guide for the Care and Use of Laboratory Animals of the National Laboratory Animal Center. The animal experiment protocol was approved by the Institutional Animal Care and Use Committee of the National Yang-Ming University, Taipei, Taiwan (Permits Number: 971214). The imaging studies were performed under 1% to 3% isoflurane anesthesia. All animals were sacrificed by carbon dioxide narcosis and all efforts were made to minimize suffering. All applicable institutional and national guidelines for the care and use of animals were followed
Synthesis of HDAC inhibitors
The preparation of SAHA and ISAHA was carried out using a slightly modified method based on the one reported by Stowell et al. (supplementary method) The overall chemical yields of SAHA and ISAHA were 17% and 25%, respectively. Preparation of [18F]FLT
This followed the method reported by Kim et al. whereby [18F]FLT was prepared with a radiochemical yield of 12±3% (decay corrected from end of bombardment) and with a radiochemical purity of >98 % after HPLC separation. Cell culture 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
HepG2 human hepatocellular carcinoma cell line was provided by Dr. Liu (Taiwan Mouse Clinic, Taipei, Taiwan). The Mahlavu, PLC, and Hep3B human hepatocellular carcinoma cell lines were kindly provided by Dr. Chiou (Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan). The HepG2 cell line was cultured in Eagle’s Minimum Essential Medium, while the Mahlavu, PLC, and Hep3B cell lines were cultured in Dulbecco's modified Eagle's
medium in a humidified atmosphere with 5% CO2 at 37˚C, both supplemented with
10% fetal bovine serum (Thermo, MA, USA) and 1% PEN-STREP-AMPHO solution (PSA, Biological Industries, Israel).
Cell viability assay
Cell viability was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, MO, USA) assay using five replicates. A cell suspension (200 μL) containing an adequate number of cells (3.5 × 103 for Mahlavu, PLC, and Hep3B cell lines and 4.0 × 103 for HepG2 cell line) were added to a 96-well plate and incubated at 37°C in humidified air with 5% CO2 for 24 hours. Then, 200 μL of culture medium containing serial dilutions of either ISAHA or SAHA (0–500 μM) was added to these cell cultures. After 48 hours, the medium were removed and an aliquot of 150 μL of MTT solution (diluted by culture medium, 0.1mg/mL) was added to each well and the plate was incubated for 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
another 2 h. The supernatant was aspirated before the insoluble formazan crystals were dissolved in 100 μL of dimethyl sulfoxide (DMSO). Cell viability was determined by comparing the absorbance of treated wells with that of blank wells (cells incubated with culture medium containing vehicle).
Cell cycle analysis
The effect of ISAHA and SAHA on cell cycle arrest was determined by flow cytometry. HepG2 cells (3 × 106/each) were seeded into 10-cm diameter Petri dishes, and exposed to increasing concentration of ISAHA and SAHA (1, 2, 5, and 10 μM) or vehicle only (0.05% DMSO). Three independent experiments were conducted. After 24 h of treatment, cells were harvested and pelleted by centrifugation. The cells were then fixed in ice-cold 70% ethanol for 2 h at -20°C. Next, the fixed cells were incubated with propidium iodide solution (20 μg/mL propidium iodide, 0.2 mg/mL RNase A, and 0.1% Triton X-100) at ambient temperature for 30 min. Finally, the cells were analyzed by flow cytometry using a Cytomic FC 500 flow cytometry system (Beckman Coulter, CA, USA) and cell cycle profiles determined using the CXP software (Beckman Coulter, CA, USA).
Western blot analysis
Western blot analysis of HepG2 cells was performed. The cells were first exposed to increasing concentration of ISAHA and SAHA (1, 2, 5, 10 μM) or vehicle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
only (0.05% DMSO) for 24 h. The cells were harvested and lysed on ice using CytoBuster protein extraction reagent (Novagen, EMD Millipore, MA, USA) with Complete Protease Inhibitor Cocktail Tablets (Roche, Germany). An aliquot of 50 μg of protein was then separated by 12% SDS-PAGE and transferred onto a polyscreen PVDF membrane (PerkinElmer, MA, USA). Proteins were detected by incubation with specific primary antibodies diluted in blocking buffer at 4°C overnight. The primary antibodies used were acetyl Histone-H3, acetyl Histone-H4, anti-Cdk1, anti--actin, and anti-caspase 3 (EMD Millipore, MA, USA). The membranes were washed and then incubated with IgG horseradish peroxidase conjugated secondary antibody. Bands were visualized using chemiluminescence reagent (EMD Millipore, MA, USA).
Animal tumor model and drug treatment
Six- to eight-week-old male mice were obtained from National Laboratory Animal Center (Taipei, Taiwan). For the subcutaneous hepatocellular carcinoma-bearing mouse model, 1 × 107 HepG2 human hepatoma cells were inoculated subcutaneously into the right hind flank of the BALB/c nu/nu mice to produce a tumor. Tumor dimension was measured using a caliper and tumor volume was calculated by the following equation: volume = Length × Width2 / 2. After the tumor volume had reached 100±50 mm3 (day 0), the mice were randomized into four groups. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Mice in each group were intraperitoneally (i.p.) administered with ISAHA (25 and 100 mg/kg), SAHA (100 mg/kg) or vehicle (1 mL/kg of DMSO) five days a week for three weeks. The average tumor growth inhibition (TGI) was expressed as 100 %×(Vt-Vc)/Vc, where Vc and Vt are the tumor volume of the control and the treated mice, respectively, on day 22. Mice with a tumor volume of ≥ 2000 mm3, or a body weight of ≤ 15 g, were euthanized.
3’-Deoxy-3’-[18F]-fluorothymidine ([18F]FLT) microPET imaging
[18F]FLT PET study was carried out on a microPET R4 scanner (Concorde Microsystems, Inc.) with a resolution of 1.8 mm at full width at half maximum. The features of this instrument have been described previously . Static [18F]FLT microPET imaging was conducted for 10 min at 60 min post-injection of 3.7 MBq of [18F]FLT on days 0 and 8. During the examination, the mice were placed in the prone position with the long axis parallel to the table of the scanner, and anesthetized with isoflurane. Regions of interest (ROIs) were drawn around the tumors and the contralateral muscles. The pixels within the ROIs were corrected by subtracting the background levels of radioactivity, which had been measured in remote areas away from the animal body. The radioactivity concentrations in the tumors were normalized against that of muscle and expressed as the tumor-to-muscle ratio (T/M), which was also regarded as the specific tumor uptake.
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Immunohistological examination
Immunohistological examination of tumor sections was performed in order to evaluate the changes in tumor cell proliferation after 1 week of treatment. One day after [18F]FLT PET scanning, Itreated (25 mg/kg or 100 mg/kg) mice, SAHA-treated (100 mg/kg) mice and the controls were sacrificed and their tumors were excised. Dehydration, paraffin embedding, and sectioning were conducted by routine procedures. Mouse monoclonal Ki67 antibody (Millipore, MA, USA) was used to evaluate the tumor cell proliferation rate . The number of Ki67 positive cells and the total number of cells were counted across five randomly selected fields of view per section. The relative staining index (rSI) was calculated using the equation rSI = (Ki67-positive cells/total number of cells) × 100 %.
Statistical analysis
The unpaired t-test was used for group comparisons. Differences were considered statistically significant when p < 0.05. Linear regression was used to analyze the correlation between the T/M derived from the microPET images and the expression of Ki67 (proliferation marker) obtained by immunohistological staining. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Results
ISAHA inhibits cell proliferation in vitro
The inhibition of cell growth by ISAHA and SAHA were assessed using various hepatocellular carcinoma cell lines. Both ISAHA and SAHA inhibited cell proliferation significantly in a dose dependent manner, but with ISAHA being more potent than SAHA (Fig. 1). The IC50 values for ISAHA were: HepG2, 1.66±0.03 μM; Mahlavu, 5.08±0.89 μM; Hep3B, 19.78±4.49 μM and PLC, 21.16±2.90 μM, which are all about 2-fold lower than those for SAHA at HepG2, 3.52±0.18 μM, Mahlavu, 7.50±0.90 μM, Hep3B, 31.59±1.17 μM, and PLC, 46.07±1.73 μM, after 48 h of incubation.
Effect of ISAHA on the cell cycle profile, acetylation and apoptosis-related protein levels in HepG2 cells
The effect of ISAHA and SAHA on the cell cycle profiles of HepG2 cells was determined by flow cytometry analysis. Cell cycle arrest occurred in the G2/M phase after ISAHA and SAHA treatment. ISAHA is more cytotoxic than SAHA. A concentration of 2 μM of ISAHA showed a visible cytotoxicity to HepG2 cells (Fig. 2a and 2b). HDACi like Panobinostat has been shown to induce an increase in acetylation of histone proteins and apoptosis-related proteins in cancer cells . A dose-related increase in acetylated histone levels (acetyl-H3 and acetyl-H4) was also found by Western blotting of ISAHA/SAHA-treated HepG2 cells (Fig. 2c). ISAHA showed 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
higher potency than SAHA. In addition, a decrease in the level of the cell cycle regulation protein Cdk1 was also noticed in the ISAHA-treated cells. The expression of caspase 3 (an indicator of apoptosis) was found to be upregulated after ISAHA treatment in a dose-dependent manner. These findings demonstrate that ISAHA is a promising HDAC inhibitor, is able to induce apoptosis and cell cycle arrest in G2/M phase at an appropriate concentration.
ISAHA retards HepG2 tumor growth
ISAHA effectively retards the growth of subcutaneous HepG2 tumor xenografts in vivo. A comparison of tumor growth with the control group after treatment of the mice with SAHA (100 mg/kg) and ISAHA (25 and 100 mg/kg) from day 0 to day 22 is shown in Figure 3a. At the early stage of treatment, namely before day 10, there is no significant difference in tumor volume between the various groups of mice that underwent the different treatment protocols. At the late stage (on day 22), the ISAHA (25 and 100 mg/kg)-treated mice had remarkably lower tumor burdens (520.6±222.76 and 308.53±125.46 mm3, respectively) than those treated with SAHA (888.5±386.6 mm3) and the controls (1228.0±453.0 mm3; p<0.01). The mice treated with a dose of 25 mg/kg of ISAHA all lived until the end of experiment period (22 days) with an average body weight loss of 14%. However, the mice treated with a higher dose (100 mg/kg) of ISAHA, though showed more remarkable tumor growth inhibition (TGI = 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
75% at day 22), also showed a significantly larger average body weight loss (23% on day 22, Fig. 3b) together with a lower survival rate (42%, data not shown).
[18F]FLT PET is able to monitor tumor response during treatment
[18F]FLT microPET imaging was conducted before and one week after the initiation of treatment of hepatoma-bearing mice to monitor the tumor response non-invasively (Fig. 4). The mice treated with HDACi all showed significantly reduced tumor uptake after treatment (p < 0.05), while that of the control remained unchanged.
The T/M derived from [18F]FLT microPET images was about 7.71 on day 0 (before
treatment, n=12) and decreased to 5.70±0.73 (100 mg/kg of SAHA, n=3), 3.38±0.06 (100 mg/kg of ISAHA, n=3) and 4.80±0.54 (25 mg/kg of ISAHA, n=3) on day 8; while the T/M of the control was 8.10±1.48 (n=3) on day 8. Quantitative analysis of FLT PET showed no significant difference in T/M between 25 mg/kg of ISAHA-treated and 100 mg/kg of SAHA-ISAHA-treated mice on day 8, indicated a more potency of ISAHA than SAHA in inhibition of tumor cell proliferation. In addition, statistical difference was noticed in T/M between 25 mg/kg and 100 mg/kg of ISAHA-treated mice. [18F]FLT PET demonstrated a promising approach that allows, at an early stage of tumor treatment, the noninvasive evaluation of the therapeutic efficacy of HDACi. ISAHA decreases Ki67 expression in tumors
In order to assess the change in tumor cell proliferation rate during treatment, immunohistological staining of tumor sections was conducted. The Ki67 expression in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
tumors (expressed as the relative staining index, rSI) decreased after one week of SAHA (100 mg/kg) and ISAHA (25 mg/kg, 100 mg/kg) treatment (Fig. 5). The rSI of the tumor was 0.52±0.06 (initial value) on day 0, and was 0.41±0.08 (100 mg/kg of SAHA), 0.24±0.04 (100 mg/kg of ISAHA) and 0.37±0.06 (25 mg/kg of ISAHA) on day 8; while remain steady (0.54±0.03) in the control group. The significantly reduced rSI clearly indicates that proliferation of the HepG2 tumor cells was being impeded in a dose dependent manner by ISAHA treatment. The T/M derived from [18F]FLT microPET imaging showed good correlation with the rSI obtained from the immunohistochemical staining (r2 = 0.98, p < 0.05). 1 2 3 4 5 6 7 8 9
Discussion
SAHA has been demonstrated to inhibit remarkably the proliferation of HepG2 cells as well as being able to induce apoptosis in HepG2 cells . To noninvasively determine the in vivo distribution of SAHA, we synthesized a radioiodinated derivative, [131I]ISAHA, as a radioactive surrogate of SAHA. In vitro studies showed that the uptake of [131I]ISAHA by HepG2 cells increased with time and reached a plateau after 4 h of incubation (Fig. S1). The authentic compound ISAHA, in which the iodine substituent had been introduced at the 4-position of benzene, was found to exhibit superior antitumor activities than SAHA, both in vitro and in vivo. Compared with SAHA, ISAHA showed a better anti-proliferative activity to various hepatocellular carcinoma cell lines in vitro (Fig. 1). The potency of ISAHA in terms of induction of histone hyperacetylation and apoptosis was confirmed by the Western blot analysis and the cell cycle profile analysis (Fig. 2). In addition, a remarkable level of retardation of established HepG2 xenograft tumor growth was also detected (Fig. 3). These findings suggested that ISAHA is a potent HDAC inhibitor, and is more efficacious compared to SAHA in cancer treatment.
In this study, the mice treated with a higher dose (100 mg/kg) of ISAHA showed remarkable tumor growth inhibition (Fig. 3a), however, the adverse effects showed a larger average body weight loss (Fig. 3b) together with a lower survival
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rate. The most frequently encountered side effects observed in clinical trials with histone deacetylase inhibitors were gastrointestinal related diarrhea (52%), nausea (41%), dysguesia (28%), thrombocytopenia (26%), and body weight loss (21%) . We have observed the diarrhea and loss of appetite in the mice received high dosage of ISAHA (100 mg/kg), with results of significant body weight loss and lower survival rate. Another possible influence factor is the route of drug administration. Owing to the low solubility of ISAHA in water, the HDACi was administered via intraperitoneal injection in this study, while that used in clinical application is via oral administration. When decreasing the dosage of ISAHA to 25 mg/kg, no significant adverse effects can be observed, and the effective tumor growth inhibition was still noticed.
SAHA has been indicated for the treatment of patients with cutaneous T-cell lymphoma; however, the drug is not ideal due to its low in vivo stability and limited membrane permeability . The half-lives of SAHA and its glucuronide metabolites are both around 2 h, while that of 4-anilino-4-oxobutanoic acid is around 6~9 h . The hydroxamate group of SAHA was believed to be the factor that is associated with the low in vivo stability of the drug . The newly developed [123/131I]ISAHA and the authentic ISAHA are stable in normal saline, decomposed slowly in culture medium, and are rapidly metabolized in vivo as that of SAHA (Fig. S2). The intact [131I]ISAHA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
accounted for less than 2% and free [131I]iodide for more than 60% of the radioactivity in blood and urine at 1 h post intravenous injection in mice. The rapid appearance of [131I]iodide originated from in vivo deiodination suggests that the main metabolite of ISAHA is SAHA, which remains an effective HDAC inhibitor. The extended metabolic pathway of ISAHA may partly account for the findings that ISAHA displays a higher toxicity profile than SAHA both in vitro and in vivo (Fig. 1 and 4). Further investigations are needed to determine the oral bioavailability and in vivo permeability of ISAHA.
Over the past decades, various HDAC inhibitors have been identified as being able to inhibit the growth of tumor cells both in vitro and in vivo. However, instead of tumor shrinkage, tumor stabilization was observed in patients with advanced cancer after HDACi treatment . The employment of a functional imaging modality to obtain the information of tumor responses to HDACi during a treatment course may be an alternative to biopsy. [18F]FLT PET has been shown to be a reliable tool for quantifying proliferation rate of tissues . The application of [18F]FLT PET for monitoring the killing effects of chemodrugs has also been demonstrated in various other studies . Thymidine kinase 1 (TK1) is responsible for [18F]FLT accumulation in cells . A study by Leyton et al. showed that the level of TK1 in HCT116 colon
carcinoma is down-regulated in a dose dependent manner after HDACi (LAQ824)
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treatment, which indicates the appropriateness of [18F]FLT PET to the monitoring of the biological effects of HDACi . Na et al. also applied [18F]FLT PET to monitor the tumor responses after HDACi (PXD101)/topoisomerase I inhibitor (Irinotecan) treatment in colon carcinoma-bearing mice . In the present study, [18F]FLT PET was applied to monitor the tumor responses after treatment with ISAHA, a novel HDACi. A remarkable decrease in the T/M of ISAHA-treated HepG2 tumor xenograft mice was noticed at day 8, while no significant difference in tumor burden could be observed till day 10. The T/M derived from [18F]FLT PET royally reflects the inhibition of proliferation rate after various HDACi treatments. [18F]FLT PET was demonstrated a promising strategy to evaluate, or even to predict, the efficacy in the early stage of treatment.
Several literatures reported that a combination of HDACi and anti-cancer drug might be synergistic in terms of tumor treatment. Cheriyath et al. reported that HDACi in combination with doxorubicin significantly decrease the survival of myeloma cells by inducing cytoplasmic cathepsin B mediated apoptosis . In our another study, treatment of orthotopic SASVO3 human tongue squamous carcinoma xenograft-bearing mice with ISAHA followed by liposomal doxorubicin (PLD) achieved a better in vivo outcome compared with those treated with either ISAHA or PLD alone (Fig. S3). [18F]FLT PET also demonstrated a reduced T/M in ISAHA/PLD-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
treated group compared with the control (Fig. S4). Further experiments are required to elucidate the exact mechanism that leads to a better outcome after the treatment of this combinational regimen.
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Conclusion
The application of non-invasive imaging to the monitoring of real time tumor responses during treatment may provide valuable information and help the clinical physician to optimize treatment protocols. This study demonstrated that [18F]FLT PET is a promising modality to evaluate the therapeutic efficacy of the HDACi at an early stage of treatment. [18F]FLT PET showed reduced T/M in ISAHA-treated HepG2-bearing mice, indicated that ISAHA is a promising HDACi which is able to impede tumor cell proliferation.
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Acknowledgements
The authors thank the financial support from National Science Council, Taiwan (NSC 101-2623-E-010-006-NU, NSC100-2623-E-010-003-NU and NSC99-NU-E-010-003). The authors also appreciate the technical support provided by the Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, Taipei, Taiwan.
Conflict of interest
The authors declare that they have no conflict of interest. 1 2 3 4 5 6 7 8
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Figure Legends
Figure 1. The cytotoxicity of ISAHA and SAHA on hepatocellular carcinoma cell
lines. The PLC, Hep3B, Mahlavu, and HepG2 cells were treated with 0.5 to 500 μM of ISAHA or SAHA for 48 h. Cell viability was assessed by MTT assay. The dose-dependent effect of ISAHA (left) and SAHA (right) was observed.
Figure 2. The effect of ISAHA on the cell cycle and the levels of various protein
expression. a Cell cycle profiles of vehicle-treated, ISAHA-treated and SAHA-treated HepG2 cells were assessed by flow cytometry. b Summary of cell cycle distribution that was derived from the cell cycle profiles using the CXP software. * p < 0.05. c Western blotting was used to detect acetyl-H3 (Ac-H3), acetyl-H4 (Ac-H4), cell cycle regulation protein cdk1 and apoptosis-related protein caspase 3 of the HepG2 cells that were treated with increasing concentrations of ISAHA and SAHA for 24 h.
Figure 3. a The effect of ISAHA on HepG2 tumor growth. HepG2 tumor-bearing
mice were intraperitoneally administered with ISAHA (25 and 100 mg/kg), SAHA (100 mg/kg) or vehicle (1 mL/kg of DMSO) for five days a week over three weeks. Seven mice per group were used. b The relative body weight of the drug-treated mice and the control is expressed as 100 %×(W-W0)/W0, where W and W0 are the body weight of mice on the day of measurement and on day 0, respectively
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Figure 4. [18F]FLT microPET images of HDACi-treated HepG2 tumor-bearing mice and the control. HepG2 tumor-bearing mice were intraperitoneally administered with ISAHA (25 and 100 mg/kg), SAHA (100 mg/kg) or vehicle (1 mL/kg of DMSO) for five days a week. MicroPET imaging was conducted at 1 h post the intravenous injection of approximately 3.7 MBq of [18F]FLT on day 0 and day 8. Arrows indicate tumor lesions. The tumor-to-muscle ratios (T/Ms) were derived from the regions of interest (ROIs), which were drawn around the tumor and the contralateral muscle
Figure 5. Effect of ISAHA on tumor cell proliferation of HepG2 tumor-bearing mice.
Ki67-staining was performed on the tumor sections obtained before and one week after HDACi treatment.
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