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Shi Y, Wang X, Zhu Q, Chen G. The Ribosomal Protein L28 Gene Induces Sorafenib Resistance in Hepatocellular Carcinoma. Front Oncol 2021; 11:685694. [PMID: 34307151 PMCID: PMC8299949 DOI: 10.3389/fonc.2021.685694] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2021] [Accepted: 06/15/2021] [Indexed: 11/17/2022] Open
Abstract
Background Sorafenib is the first molecular-targeted drug for the treatment of advanced hepatocellular carcinoma (HCC). However, its treatment efficiency decreases after a short period of time because of the development of drug resistance. This study investigates the role of key genes in regulating sorafenib-resistance and elucidates the mechanism of drug resistance in hepatocellular carcinoma. Methods The HCC HepG2 cells were used to generate a sorafenib-resistant cell model by culturing the cells in gradually increasing concentration of sorafenib. RNA microarray was applied to profile gene expression and screen key genes associated with sorafenib resistance. Specific targets were knockdown in sorafenib-resistant HepG2 cells for functional studies. The HCC model was established in ACI rats using Morris hepatoma3924A cells to validate selected genes associated with sorafenib resistance in vivo. Results The HepG2 sorafenib-resistant cell model was successfully established. The IC50 of sorafenib was 9.988μM in HepG2 sorafenib-resistant cells. A total of 35 up-regulated genes were detected by expression profile chip. High-content screening technology was used and a potential drug-resistance related gene RPL28 was filtered out. After knocking down RPL28 in HepG2 sorafenib-resistant cells, the results of cell proliferation and apoptosis illustrated that RPL28 is the key gene involving in drug resistance. Furthermore, it was found that both RNA and protein expression of RPL28 increased in HepG2 sorafenib-resistant specimens of Morris Hepatoma rats. In addition, the expression of proliferative protein Ki-67 increased in sorafenib-resistant cells. Conclusion Our study suggested that RPL28 is a key gene inducing sorafenib resistance in HCC and could be a potential target for the treatment of drug-resistant HCC.
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Affiliation(s)
- Yi Shi
- Departments of Molecular Pathology, Fujian Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou, China.,The School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
| | - Xiaojiang Wang
- Departments of Molecular Pathology, Fujian Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou, China.,The School of Basic Medical Sciences, Fujian Medical University, Fuzhou, China
| | - Qiong Zhu
- Departments of Molecular Pathology, Fujian Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou, China
| | - Gang Chen
- Departments of Molecular Pathology, Fujian Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou, China.,Departments of Pathology, Fujian Cancer Hospital, Fujian Medical University Cancer Hospital, Fuzhou, China
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Choi JW, Cho HR, Lee K, Jung JK, Kim HC. Modified Rat Hepatocellular Carcinoma Models Overexpressing Vascular Endothelial Growth Factor. J Vasc Interv Radiol 2018; 29:1604-1612. [PMID: 30293733 DOI: 10.1016/j.jvir.2018.07.005] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2017] [Revised: 06/21/2018] [Accepted: 07/05/2018] [Indexed: 10/28/2022] Open
Abstract
PURPOSE To compare tumor vascularity in 4 types of rat hepatocellular carcinoma (HCC) models: N1S1, vascular endothelial growth factor (VEGF)-transfected N1S1 (VEGF-N1S1), McA-RH7777, and VEGF-transfected McA-RH7777 (VEGF-McA-RH777) tumors. MATERIALS AND METHODS The N1S1 and McA-RH7777 cell lines were transfected with expression vectors containing cDNA for rat VEGF. Eighty-eight male Sprague-Dawley rats (weight range, 400-450 g) were randomly divided into 4 groups (ie, 22 rats per model), and 4 types of tumor models were created by using the N1S1, VEGF-N1S1, McA-RH7777, and VEGF-McA-RH777 cell lines. Tumor vascularity was evaluated by perfusion computed tomography (CT), enzyme-linked immunosorbent assay of VEGF, CD34 staining, angiography, and Lipiodol transarterial embolization. Intergroup discrepancies were evaluated by Kruskal-Wallis test. RESULTS Arterial perfusion (P < .001), portal perfusion (P = .015), total perfusion (P < .001), tumor VEGF level (P = .002), and microvessel density (MVD; P = .007) were significantly different among groups. VEGF-McA-RH7777 tumors showed the greatest arterial perfusion (46.7 mL/min/100 mL ± 15.5), total perfusion (60.7 mL/min/100 mL ± 21.8), tumor VEGF level (3,376.7 pg/mL ± 145.8), and MVD (34.5‰ ± 7.5). Whereas most tumors in the N1S1, VEGF-N1S1, and McA-RH7777 groups showed hypovascular staining on angiography and minimal Lipiodol uptake after embolization, 5 of 6 VEGF-McA-RH7777 tumors (83.3%) presented hypervascular tumor staining and moderate to compact Lipiodol uptake. CONCLUSIONS McA-RH7777 tumors were more hypervascular than N1S1 tumors, and tumor vascularity was enhanced further by VEGF transfection. Therefore, the VEGF-McA-RH7777 tumor is recommended to mimic hypervascular human HCC in rats.
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Affiliation(s)
- Jin Woo Choi
- Department of Radiology, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea
| | - Hye Rim Cho
- Department of Radiology, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea
| | - Kyoungbun Lee
- Department of Pathology, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea
| | - Jae Kyung Jung
- Department of Radiology, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea
| | - Hyo-Cheol Kim
- Department of Radiology, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea.
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Vogl TJ, Qian J, Tran A, Oppermann E, Naguib NN, Korkusuz H, Nour Eldin NEA, Bechstein WO. Study on the effect of chemoembolization combined with microwave ablation for the treatment of hepatocellular carcinoma in rats. Diagn Interv Radiol 2017; 23:150-155. [PMID: 28185998 DOI: 10.5152/dir.2016.16617] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
PURPOSE We aimed to evaluate the combining effects of transarterial chemoembolization (TACE) and open local thermal microwave ablation in a hepatocellular carcinoma animal model. METHODS Tumor cubes were implanted into the liver of 30 male inbred ACI rats. Groups of 10 animals were treated at 13 days (TACE or microwave ablation) and 16 days (microwave ablation) postimplantation with combined therapy of TACE (0.1 mg mitomycin C; 0.1 mg iodized oil; 5.0 mg degradable starch microspheres) and microwave ablation (2450 Mhz; 45 s; 35 W) (study group A), TACE alone (control group B), or microwave ablation alone (control group C). At day 12 and day 25 tumor size was measured via magnetic resonance imaging and the relative growth ratio was calculated. Hepatic specimens were immunohistochemically examined for the expression of vascular endothelial growth factor (VEGF). RESULTS Mean growth rates were 1.34±0.19 in group A, 3.19±0.13 in group B, and 4.18±0.19 in group C. Compared with control groups B and C, tumor growth rate in group A was significantly inhibited (P < 0.01). The VEGF-antibody reaction in peritumoral tissue (staining intensity at portal triad, percent antibody reaction and staining intensity at central vein) was significantly lower in group A compared with group B (P < 0.01). No significant difference between group A and group C could be observed. CONCLUSION This investigation shows improved results of TACE followed by microwave ablation as treatment of hepatocellular carcinoma in a rat model, compared with single therapy regimen regarding the inhibition of growth rate and reduction of VEGF-level in peritumoral tissue.
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Affiliation(s)
- Thomas Josef Vogl
- Institute for Diagnostic and Interventional Radiology, Frankfurt University Hospital, Frankfurt/Main, Germany.
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Hwang GL, van den Bosch MA, Kim YI, Katzenberg R, Willmann JK, Paulmurugan R, Gambhir SS, Hofmann L. Development of a High-Throughput Molecular Imaging-Based Orthotopic Hepatocellular Carcinoma Model. Cureus 2015; 7:e281. [PMID: 26180705 PMCID: PMC4494575 DOI: 10.7759/cureus.281] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2014] [Accepted: 06/17/2015] [Indexed: 02/06/2023] Open
Abstract
We have developed a novel orthotopic rat hepatocellular (HCC) model and have assessed the ability to use bioluminescence imaging (BLI), positron emission tomography (PET), and ultrasound for early tumor detection and monitoring of disease progression. Briefly, rat HCC cells were stably transfected with click beetle red as a reporter gene for BLI. Tumor cells were injected under direct visualization into the left or middle lobe of the liver in 37 rats. In six animals, serial PET, BLI, and ultrasound imaging were performed at 10-time points in 28 days. The remainder of the animals underwent PET imaging at 14 days. Tumor implantation was successful in 34 of 37 animals (91.9%). In the six animals that underwent serial imaging, tumor formation was first detected with BLI on Day 4 with continued increase through Day 21, and hypermetabolic activity on PET was first noted on Days 14-15 with continued increase through Day 28. PET activity was seen on Day 14 in the 28 other animals that demonstrated tumor development. Anatomic tumor formation was detected with ultrasound at Days 10-12 with continued growth through Day 28. The first metastases were detected by PET after Day 24. We have successfully developed and validated a novel orthotopic HCC small animal model that permits longitudinal assessment of change in tumor size using molecular imaging techniques. BLI is the most sensitive imaging method for detection of early tumor formation and growth. This model permits high-throughput in vivo evaluation of image-guided therapies.
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Affiliation(s)
| | | | - Young I Kim
- Radiology, Seoul National University College of Medicine
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Variability of apoptosis and response in N1-S1 rodent hepatomas to benzamide riboside and correlation to early changes in water apparent diffusion coefficient and sodium MR imaging. J Vasc Interv Radiol 2013; 24:894-900. [PMID: 23566523 DOI: 10.1016/j.jvir.2013.02.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2012] [Revised: 01/18/2013] [Accepted: 02/14/2013] [Indexed: 11/20/2022] Open
Abstract
PURPOSE This pilot trial assesses variability of apoptosis and response 1 day after hepatic intraarterial (IA) benzamide riboside (BR) in rodent hepatomas and its correlation to water apparent diffusion coefficient (ADC) and single-quantum (SQ) and triple-quantum-filtered (TQF) sodium-23 ((23)Na) magnetic resonance (MR) imaging. MATERIALS AND METHODS Sprague-Dawley rats (n = 8) were inoculated with 10(6) N1-S1 cells. IA BR (20 mg/kg) was infused after 14 days. Animals were killed 1 day (n = 4) or 21 days (n = 4) after therapy. Imaging was performed 1 day before and after treatment. Volume was assessed over 2 weeks. Percentage apoptosis was counted from terminal deoxynucleotidyl transferase dUTP nick-end labeling-stained slides at 400×magnification. Kruskal-Wallis tests were used to compare apoptosis, and Wilcoxon signed-rank tests were used to compare MR signal intensity (SI). RESULTS Apoptosis was marginally greater in tumor than in nontumor (6.7% vs 1.3%; P = .08), varying from 2% to 10%. Before treatment, MR SI was greater in tumor than in nontumor (ADC, 1.18 vs 0.76 [P = .0078]; SQ, 1.20 vs 1.04 [P = .03]; TQF, 0.55 vs 0.34 [P = .03]). After treatment, tumors increased in volume (0.62 vs 0.33; P = .016) variably over 2 weeks. MR SI remained greater in tumor than in nontumor (ADC, 1.20 vs 0.77 [P = .0078]; SQ, 1.76 vs 1.15 [P = .016]; TQF, 0.84 vs 0.49 [P = .03]). SQ and TQF SI increased by 47% (P = .016) and 53% (P = .016) in tumors, whereas ADC did not change. CONCLUSIONS Apoptosis was marginal and varied from 2% to 10%. Water ADC, SQ, and TQF MR imaging distinguished tumor from nontumor. Changes in water ADC and sodium MR imaging correlated to apoptosis and volume in select cases, but additional animals are needed to validate this trend against tumor growth.
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Babsky AM, Ju S, Bennett S, George B, McLennan G, Bansal N. Effect of implantation site and growth of hepatocellular carcinoma on apparent diffusion coefficient of water and sodium MRI. NMR IN BIOMEDICINE 2012; 25:312-321. [PMID: 21823182 DOI: 10.1002/nbm.1752] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/14/2010] [Revised: 04/15/2011] [Accepted: 04/18/2011] [Indexed: 05/31/2023]
Abstract
Hepatocellular carcinoma (HCC) and liver metastases are an increasing problem worldwide. Non-invasive methods for the early detection of HCC and understanding of the tumor growth mechanisms are highly desirable. Both the diffusion-weighted (1)H (DWI) and (23)Na MRI reflect alterations in tissue compartment volumes in tumors, as well as physiological and metabolic transformation in cells. Effects of untreated growth on apparent diffusion coefficient of water (ADC), single quantum (SQ) and triple quantum-filtered (TQF) (23)Na MRI were compared in intrahepatically and subcutaneously implanted HCCs in rats. Animals were examined weekly for 4 weeks after injection of N1S1 cells. ADC of intrahepatic HCC was 1.5-times higher compared to the nearby liver tissue, and with growth, the ADC did not increase. ADC of subcutaneous HCC was lower compared to intrahepatic HCC and it increased with growth. Untreated growth of both intrahepatic and subcutaneous HCCs was associated with an increase in SQ and TQF (23)Na signal intensity suggesting an increase in tissue Na(+) and intracellular Na(+) (Na(+)(i)), respectively, most likely due to an increase in relative extracellular space and Na(+)(i) concentration as a result of changes in tissue structure and cellular metabolism. Thus, SQ and TQF (23)Na MRI may be complementary to diffusion imaging in areas susceptible to motion for characterizing hepatic tumors and for other applications, such as, predicting and monitoring therapy response.
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Affiliation(s)
- Andriy M Babsky
- Department of Radiology and Imaging Sciences, Indiana University, Indianapolis, IN 46202-5181, USA.
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Aprahamian M, Bour G, Akladios CY, Fylaktakidou K, Greferath R, Soler L, Marescaux J, Egly JM, Lehn JM, Nicolau C. Myo-InositolTrisPyroPhosphate treatment leads to HIF-1α suppression and eradication of early hepatoma tumors in rats. Chembiochem 2011; 12:777-783. [PMID: 21370375 DOI: 10.1002/cbic.201000619] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2010] [Indexed: 01/20/2023]
Abstract
Myo-inositol trispyrophosphate (ITPP), a synthetic allosteric effector of hemoglobin, increases the regulated oxygen-releasing capacity of red blood cells (RBCs), leading to suppression of hypoxia-inducible factor 1α (HIF-1α) and to down-regulation of hypoxia-inducible genes such as vascular endothelial growth factor (VEGF). As a consequence, tumor growth is markedly affected. The effect of weekly intravenous injection of ITPP on an orthotopic, syngenic rat hepatocellular carcinoma (HCC) model was compared to that for untreated animals and animals subjected to conventional Doxorubicin chemotherapy. The longitudinal examination of HCC was performed by microCT imaging, and the cellular and molecular changes were evaluated by histology and Western blotting analysis of HIF-1α, VEGF, and caspase-3 gene expression in the tumor and in the surrounding liver. Hematologic impact was evaluated by blood cell-count measurement and determination of P50 (oxygen partial pressure for a 50 % oxygen saturation of hemoglobin). The HCC evaluation by microCT revealed a high potency of ITPP for tumor growth inhibition, thus allowing long-term survival and even cure of almost all the treated animals. The P50 value of hemoglobin in RBCs underwent a shift of 30 % following ITPP injection. Under these conditions, HIF-1α activity was strongly decreased, VEGF expression was down-regulated, and apoptosis was induced in HCC and surrounding liver cells, as indicated by Caspase-3 expression. ITPP did not affect hematologic parameters during treatment. The observations of in vivo tumor eradication suggest a significant clinical potential for ITPP in cancer therapy.
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Affiliation(s)
- Marc Aprahamian
- Institut National pour Santé et Recherche Médicale, Unit 701, IRCAD Strasbourg, France.
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Akladios CY, Bour G, Balboni G, Mutter D, Marescaux J, Aprahamian M. [Contribution of microCT structural imaging to preclinical evaluation of hepatocellular carcinoma chemotherapeutics on orthotopic graft in ACI rats]. Bull Cancer 2011; 98:120-132. [PMID: 21382793 DOI: 10.1684/bdc.2011.1303] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Animal experimentation is a prerequisite for preclinical evaluation of treatments such as chemotherapy. It's strictly regulated with the purpose of reducing the number of experimental animal as well as their pain. Small animal imaging should provide a painless longitudinal follow up of tumor progression on a single animal. The aim of the study is to validate small animal imaging by microscanner (μscan) in longitudinal follow up of a hepatocellular carcinoma (HCC) and to demonstrate its interest for in vivo evaluation of tumor response to different therapeutics. An HCC model achieved by orthotopic graft of the MH3924A cell line in ACI rats was followed using a Imtek/Siemens microscanner (μscan) with contrast agents (Fenestra(®) LC/VC). The procedures giving the optimal enhancement of the liver as well as a reliable determination of tumor volumes by μscan were validated. Three protocols for therapeutic assessment through μscan longitudinal follow up were performed. Each consisted in three groups testing a chemotherapy (gemcitabine, gemcitabine-oxaliplatine or sorafenib) versus two control groups (placebo and doxorubicine). Comparison was done on tumor volumes, median and actual survivals. There was a significant correlation between tumor volumes measured by μscan and autopsy. Treatment by sorafenib, at the contrary of gemcitabine alone or with oxaliplatine, resulted in a significant reduction in tumor volumes and prolongation of actuarial survival. These results are consistent with available clinical data for these diverse therapeutics. In conclusion, small animal imaging with μscan is a non-invasive, reliable, and reproducible method for preclinical evaluation of antitumor agents.
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Noninvasive magnetic resonance imaging of the development of individual colon cancer tumors in rat liver. Biotechniques 2008; 44:529-35. [PMID: 18476817 DOI: 10.2144/000112695] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Monitoring tumor development is essential for the understanding of mechanisms involved in tumor progression and to determine efficacy of therapy. One of the evolving approaches is longitudinal noninvasive magnetic resonance imaging (MRI) of tumors in experimental models. We applied high-resolution MRI at 7 Tesla to study the development of colon cancer tumors in rat liver. MRI acquisition was triggered to the respiratory cycle to minimize motion artifacts. A special radio frequency (RF) coil was designed to acquire detailed T1-weighted and T2-weighted images of the liver. T2-weighted images identified hyperintense lesions representing tumors with a minimum diameter of 2 mm, enabling the determination of growth rates and morphological aspects of individual tumors. It is concluded that high-resolution MRI using a dedicated RF coil and triggering to the respiratory cycle is an excellent tool for quantitative and morphological analysis of individual diffusely distributed tumors throughout the liver. However, at present, MRI requires expensive equipment and expertise and is a time-consuming methodology. Therefore, it should preferably be used for dedicated applications rather than for high-throughput assessment of total tumor load in animals.
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Okubo H, Takei Y, Serizawa N, Enomoto N, Ikejima K, Sato N. Orthotopic hepatocellular carcinoma model with a controlled and reproducible tumorigenicity. J Gastroenterol Hepatol 2007; 22:423-8. [PMID: 17295777 DOI: 10.1111/j.1440-1746.2006.04520.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
BACKGROUND To explore therapeutic strategies for hepatocellular carcinoma (HCC) there is a need for a suitable and reproducible animal model. However, most models produced thus far have drawbacks such as high rates of artificial tumor dissemination and complexity of the implantation technique. To circumvent these issues, we selected an appropriate HCC cell line coupled with a simple modality to prevent unintended tumor cell dissemination. METHOD KDH-8 cells were inoculated into the rat liver. To prevent tumor cell leakage, a human fibrinogen/thrombin-coated collagen patch was attached on the site after withdrawal of the needle. In some animals, ligation of the hepatic artery was performed. RESULTS Early after injection, all rats (n = 60) developed a solitary nodule. Successful inoculation was observed in all animals with a leakage (dissemination) rate of 0%. Computed tomography (CT) demonstrated a well-demarcated low density mass accompanied with a central necrosis that mimicked a typical CT image of human HCC. Doppler ultrasonography demonstrated a vascularized property of the tumor. Tumor volumes increased with time, reaching 3299.5 +/- 290.0 mm3 at day 28 after inoculation. Thus the formed KDH-8 hepatoma was pathologically classified into a poorly differentiated HCC demarcated with surrounding connective tissue. After hepatic arterial ligation, tumor growth was impeded with an inhibition of 59.1 and 70.4% (day 21 and 28, respectively). CONCLUSIONS The current orthotopic hepatoma model enables a controlled and reproducible tumorigenicity and displays properties and histopathology resembling human HCC. It may be a useful tool in the investigation of antiangiogenic and anticancer therapeutics.
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Affiliation(s)
- Hironao Okubo
- Department of Gastroenterology, Juntendo University School of Medicine, Tokyo, Japan
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Graepler F, Lemken ML, Wybranietz WA, Schmidt U, Smirnow I, Gross CD, Spiegel M, Schenk A, Graf H, Lauer UA, Vonthein R, Gregor M, Armeanu S, Bitzer M, Lauer UM. Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model. World J Gastroenterol 2006; 11:6910-9. [PMID: 16437592 PMCID: PMC4717030 DOI: 10.3748/wjg.v11.i44.6910] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model. METHODS To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC), we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase (YCD) and the yeast uracil phosphoribosyltransferase (YUPRT) gene. RESULTS In vitro stably transduced Morris rat hepatoma cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH cells stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin (MH BCD), respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions (P<0.01) under both high dose as well as low dose systemic 5-FC application, whereas MH tumors without transgene expression (MH naive) showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness (SuperCD>> YCD>>BCD>>>negative control) was defined as a result of a direct in vivo comparison of all three suicide genes. CONCLUSION Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model, thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.
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Affiliation(s)
- Florian Graepler
- Department of Internal Medicine I, Medical University Clinic Tübingen, Germany.
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Graepler F, Verbeek B, Graeter T, Smirnow I, Kong HL, Schuppan D, Bauer M, Vonthein R, Gregor M, Lauer UM. Combined endostatin/sFlt-1 antiangiogenic gene therapy is highly effective in a rat model of HCC. Hepatology 2005; 41:879-86. [PMID: 15739185 DOI: 10.1002/hep.20613] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Hepatocellular carcinoma (HCC) is regarded as a suitable target for antiangiogenic strategies. However, antiangiogenic agents aimed at single targets can be neutralized by upregulation of other proangiogenic factors. Therefore, combined approaches addressing at least two angiogenic targets should be more effective. Employing an appropriate rat hepatoma model, we examined the effects of sFlt-1 (soluble vascular endothelial growth factor [VEGF] receptor 1 as an indirect inhibitor of angiogenesis) and endostatin (a direct inhibitor of angiogenesis) in both single-agent as well as combined approaches under in vitro and in vivo conditions. Similar to human HCC, rat Morris hepatoma (MH) cells secreted high levels of VEGF, but no endogenous sFlt-1. Parental MH or MHES(r) cells, stably expressing rat endostatin, were adenovirally transduced either with AdsFlt-1 (encoding sFlt-1) or control vector Adnull (containing no transgene), followed by subcutaneous inoculation into syngeneic ACI rats. Compared with MH/Adnull cells, expressing no antiangiogenic factors at all, tumor weights were reduced fourfold in the MHES(r)/Adnull group, 19-fold in the MH/AdsFlt-1-group, and 77-fold in the MHES(r)/AdsFlt-1 combination therapy group. Analysis of variance did not show a significant interaction between the effects of the two factors ES(r) and sFlt-1; their effects multiplied. In conclusion, combined expression of sFlt-1 and endostatin effectively suppresses HCC growth under in vivo conditions. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
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Affiliation(s)
- Florian Graepler
- Department of Internal Medicine, University Clinic Tübingen, D-72076 Tübingen, Germany.
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Maataoui A, Qian J, Vossoughi D, Khan MF, Oppermann E, Bechstein WO, Vogl TJ. Transarterial chemoembolization alone and in combination with other therapies: a comparative study in an animal HCC model. Eur Radiol 2004; 15:127-33. [PMID: 15580507 DOI: 10.1007/s00330-004-2517-8] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2004] [Revised: 08/27/2004] [Accepted: 09/02/2004] [Indexed: 12/14/2022]
Abstract
The purpose of this study is to compare transarterial chemoembolization (TACE) alone and in combination with other therapies in an animal model. Subcapsular implantation of a solid Morris hepatoma 3924A in the liver was carried out in 50 male ACI rats (day 0). Tumor volume (V1) was measured by MRI (day 13). After laparotomy and retrograde placement of a catheter into the gastroduodenal artery (day 14), the following protocols of the interventional procedure were applied: TACE (mitomycin C + lipiodol) + immunotherapy (group A: TNFalpha + IL-2, group B: OK-432 + IL-2); TACE + antiangiogenesis therapy (group C: TNP-470, group D: endostatin); TACE alone in group E (control group). Tumor volume (V2) was assessed by MRI and the mean ratio of x (V2/V1) was calculated. Data were analyzed using Dunnett's t test (comparing therapeutic groups with the control group) and the Student-Newman-Keuls test (comparing significant therapeutic groups). Multivariate analysis showed a significant reduction in the tumor growth rate (P<0.05) in groups B (x=6.53) and C (x=4.01) compared to the mean ratio of the control group E (x=9.14). Significant results were observed in group C (P<0.05) in comparison with the other therapeutic groups. TACE combined with immunotherapy (OK-432) and antiangiogenesis therapy (TNP-470) retards tumor growth compared with TACE alone in an HCC animal model.
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Affiliation(s)
- A Maataoui
- Institute for Diagnostic and Interventional Radiology, Johann Wolfgang Goethe University, Frankfurt/Main, Germany.
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Qian J, Vossoughi D, Woitaschek D, Oppermann E, Bechstein WO, Li WY, Feng GS, Vogl T. Combined transarterial chemoembolization and arterial administration of Bletilla striata in treatment of liver tumor in rats. World J Gastroenterol 2003; 9:2676-80. [PMID: 14669311 PMCID: PMC4612030 DOI: 10.3748/wjg.v9.i12.2676] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate and compare the effect of combined transarterial chemoembolization (TACE) and arterial administration of Bletilla striata (a Chinese traditional medicine against liver tumor) versus TACE alone for the treatment of hepatocellular carcinoma (HCC) in ACI rats.
METHODS: Subcapsular implantation of a solid Morris hepatoma 3924A (2 mm3) in the liver was carried out in 30 male ACI rats. Tumor volume (V1) was measured by magnetic resonance imaging (MRI) on day 13 after implantation. The following different agents of interventional treatment were injected after retrograde catheterization via gastroduodenal artery (on day 14), namely, (A) TACE (0.1 mg mitomycin + 0.1 ml Lipiodol) + Bletilla striata (1.0 mg) (n = 10); (B) TACE + Bletilla striata (1.0 mg) + ligation of hepatic artery (n = 10), (C) TACE alone (control group, n = 10). Tumor volume (V2) was assessed by MRI (on day 13 after treatment) and the tumor growth ratio (V2/V1) was calculated.
RESULTS: The mean tumor volume before (V1) and after (V2) treatment was 0.0355 cm3 and 0.2248 cm3 in group A, 0.0374 cm3 and 0.0573 cm3 in group B, 0.0380 cm3 and 0.3674 cm3 in group C, respectively. The mean ratio (V2/V1) was 6.2791 in group A, 1.5324 in group B and 9.1382 in group C. Compared with the control group (group C), group B showed significant inhibition of tumor growth (P < 0.01), while group A did not (P > 0.05). None of the animals died during implantation or in the postoperative period.
CONCLUSION: Combination of TACE and arterial administration of Bletilla striata plus ligation of hepatic artery is more effective than TACE alone in the treatment of HCC in rats.
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Affiliation(s)
- Jun Qian
- Department of Radiology, Xiehe Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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Ling CQ, Li Q, Liu XH, Chen QH, Peng YH, Luo RY, Huang XQ. Infusion of Melittin-poly lactic-co-glycolic acid microspheres via hepatic artery for hepatocarcinoma in rats. Shijie Huaren Xiaohua Zazhi 2003; 11:900-903. [DOI: 10.11569/wcjd.v11.i7.900] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To observe the therapeutic effects of poly lactic-co-glycolic acid (PLGA) microspheres containing Melittin (M-MS) infused via artery on hepatocarcinoma in rats.
METHODS M-MS was prepared with biodegradable poly lactic-co-glycolic acid by multiple emulsions in liquid evaporation process. Rats bearing transplanted hepatoma were established and were randomly divided into control group, melittin group, blank microsphere (B-MS) and M-MS group with 16 rats in each group, in each respective group, normal saline (NS, 1.5 mL/kg), melittin (0.35 mg/kg), blank microspheres (10 mg/kg), and M-MS(10 mg/kg) were infused via gastro duodenal artery into hepatic artery. The tumor growth, severity of necrosis and survival of rats were documented.
RESULTS Compared with control group, the tumor growth in melittin and B-MS groups were significantly inhibited (12.4±7.1, 10.1±8.2 vs 28.3±13.6, P<0.01) and the tumor necrosis degree were dominantly in low- and moderate-grade, but the survival were not prolonged obviously (15.8±2.0 d, 16.5±3.0 d vs 13.7±2.2, P>0.05). Meanwhile, the tumor growth in M-MS group (1.11.1) was much slower than that of other 3 groups and tumor necrosis degree were mainly in severe and the survival (31.03.9 d) of rats was also significantly prolonged (P<0.01).
CONCLUSION The anti-tumor effect of M-MS administered via hepatic artery is much higher than that of melittin and B-MS.
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Affiliation(s)
- Chang-Quan Ling
- Department of Traditional Chinese Medicine, Changhai Hospital Affiliated Second Military Medcical University, 174 Changhai Road, Shanghai 200433, China
| | - Qi Li
- Department of Traditional Chinese Medicine, Changhai Hospital Affiliated Second Military Medcical University, 174 Changhai Road, Shanghai 200433, China
| | - Xiao-Hua Liu
- Shanghai Institute of pharmaceutical Industry, Shanghai 200437, China
| | - Qing-Hua Chen
- Shanghai Institute of pharmaceutical Industry, Shanghai 200437, China
| | - Yong-Hai Peng
- Department of Traditional Chinese Medicine, Changhai Hospital Affiliated Second Military Medcical University, 174 Changhai Road, Shanghai 200433, China
| | - Ruo-Yin Luo
- Department of Traditional Chinese Medicine, Changhai Hospital Affiliated Second Military Medcical University, 174 Changhai Road, Shanghai 200433, China
| | - Xue-Qiang Huang
- Department of Traditional Chinese Medicine, Changhai Hospital Affiliated Second Military Medcical University, 174 Changhai Road, Shanghai 200433, China
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Hehr T, Budach W, Lamprecht U, Belka C, Classen J, Trübenbach J, Wehrmann M, Dietz K, Bamberg M. Experimental thermoradiotherapy in malignant hepatocellular carcinoma. Int J Radiat Oncol Biol Phys 2003; 55:1374-80. [PMID: 12654450 DOI: 10.1016/s0360-3016(02)04615-1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
PURPOSE The human liver is known to be a relatively radiosensitive organ that develops clinically relevant late radiation hepatitis subsequent to whole liver treatment with total doses above 30 Gy in conventional fractionation. Experimental data, as well as clinical series, have demonstrated that hyperthermia of solid tumors in addition to radiotherapy enhances tumor growth inhibition and tumor control probability. We therefore developed an experimental model for combined radiotherapy and hyperthermia of the liver in transplantable rat Morris hepatoma 3924A. METHODS AND MATERIALS A cube of approximately 8 mm(3) was implanted subcapsularly into the middle liver lobe of 59 male syngenic ACI rats weighing approximately 180-200 g. On Day 16 after tumor implantation, irradiation of the tumor-bearing liver with either 0 Gy/25 Gy/35 Gy/45 Gy total dose in 10 fractions +/- hyperthermia (target temperature 40-42 degrees C) twice a week was initiated. Energy deposition was monitored by temperature probes in the liver and esophagus of the rats. Determination of tumor volume with magnetic resonance imaging was performed 2 to 5 weeks after the end of therapy. The tumor growth rates could be estimated for 44 rats. If the growth rate was positive (37 rats), the inverse of the growth rate was interpreted as the time to 10-fold tumor volume. Otherwise the maximum observation time was considered as a censored value in a parametric survival analysis. RESULTS Intrahepatic temperature probes showed a temperature plateau of greater than 40 degrees C after 5 to 8 min subsequent to initiation of hyperthermia. The target temperatures could be maintained for at least 22 min > or =40 degrees C and 10 min > or =41 degrees C, respectively. Median plateau temperature in liver, esophagus, and epicutaneously was 41.2 degrees C (standard deviation [SD] 0.7 degrees C; range 38.2 to 43.3 degrees C), 40.4 degrees C (SD 1.08 degrees C; range 38.9 to 41.8 degrees C), and 40.8 degrees C (SD 0.8 degrees C; range 38.2 to 42.7 degrees C), respectively. Elevation of the temperature in the esophagus correlated with intrahepatic temperatures in the range of 39-42 degrees C, r = 0.957. The increase in time to 10-fold tumor volume for each step of irradiation dosage was by 34% (95% confidence interval [CI] 20% to 49%) without hyperthermia and by 60% (95% CI 47% to 80%) with hyperthermia (p < 0.0001). CONCLUSION Treatment outcome after experimental percutaneous thermoradiotherapy in intrahepatically implanted Morris hepatoma 3924A was related to total dose of irradiation and concurrently administered regional hyperthermia. An increased radiosensitivity due to hyperthermia (<42 degrees C) has to be assumed.
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Affiliation(s)
- Thomas Hehr
- Department of Radiation Oncology, Eberhard-Karls University, Tübingen, Germany.
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Qian J, Truebenbach J, Graepler F, Pereira P, Huppert P, Eul T, Wiemann G, Claussen C. Application of poly-lactide-co-glycolide-microspheres in the transarterial chemoembolization in an animal model of hepatocellular carcinoma. World J Gastroenterol 2003; 9:94-8. [PMID: 12508359 PMCID: PMC4728258 DOI: 10.3748/wjg.v9.i1.94] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To introduce an animal model of hepatocellular carcinoma (HCC) in ACI-rats, and to evaluate the therapeutic effects of Poly-lactide-co-glycolide(Plcg)-microspheres in the transarterial chemoembolization (TACE) in this model, as well the value of this model in the experiments of interventional therapy.
METHODS: Subcapsular implantation of a solid Morris Hepatoma 3924A (1 mm3) in the livers was carried out in 11 male ACI-rats. The tumor volume (V1) was measured by magnetic resonance imaging (MRI) (13 days after implantation). After laparotomy and retrograde placement of catheter into the gastroduodenal artery (14 days after implantation), the following protocols of interventional treatment were performed: (A) mitomycin C+Poly-lactide-co-glycolide(Plcg)-microspheres (n = 4); (B) 0.9% NaCl (control group, n = 7). 13 days after these therapies the change of the tumor volume (V2) was determined by MRI again.
RESULTS: The success rate of tumor implantation reached to 100%. The mean tumor volume before TACE (V1) were 0.082 cm3 in group A and 0.096 cm3 in group B respectively. The mean tumor volume after TACE (V2) were 0.230 cm3 in group A and 1.347 cm3 in group B respectively. The mean V2/V1 were 2.860 in group A and 27.120 in group B respectively. Compared to the control group (group B), groups A showed a significant reduction of tumor growth (P = 0.004) in the period of observation.
CONCLUSION: The growth of liver tumor could be obviously prevented by utilizing Plcg-mitomycin-microspheres in TACE in animal model. This rat model of HCC is suitable for the experimental studies of interventional therapy.
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Affiliation(s)
- Jun Qian
- Department of Diagnostic Radiology, Eberhard-Karls-University Tubingen, Germany.
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Wybranietz WA, Gross CD, Phelan A, O'Hare P, Spiegel M, Graepler F, Bitzer M, Stähler P, Gregor M, Lauer UM. Enhanced suicide gene effect by adenoviral transduction of a VP22-cytosine deaminase (CD) fusion gene. Gene Ther 2001; 8:1654-64. [PMID: 11895004 DOI: 10.1038/sj.gt.3301564] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The low transduction efficiency of viral and nonviral vectors is a major limitation in tumour gene therapy. The HSV-1 tegument protein VP22 has been shown to exhibit a novel intercellular transport property. VP22 wild-type as well as VP22 fusion proteins efficiently spread from the original expressing cell to numerous neighbouring cells, so that protein transport by VP22 chimaeric polypeptides into the surrounding cells offers a possible compensation for the inadequate gene transfer efficiencies. To improve the therapeutic efficacy of the E. coli cytosine deaminase (CD) suicide gene we made use of the VP22 transport property in CD transducing adenoviral (Ad) vectors. C- and N-terminal fusions of CD linked in-frame with VP22 were generated and cloned into recombinant adenoviral vectors. Following in vitro transduction immunofluorescence analysis of Ad-transduced producer cells coplated with naive cells confirmed that the characteristic foci pattern of central producer and adjoining neighbour cells displaying nuclear staining was retained. After transduction of rat hepatoma cells with adenoviral vectors and subsequent incubation with the prodrug 5-FC, we observed enhanced cell cytotoxicity when comparing the CD-VP22 fusion (Ad-CD-VP22) with Ad-vectors expressing the CD gene only (Ad-CD). Thereby employment of Ad-vectors encoding VP22 fusion proteins opens up new possibilities to potentiate the efficiency of suicide gene therapy for the treatment of solid tumours.
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Affiliation(s)
- W A Wybranietz
- Internal Medicine I, Medical University Clinic Tübingen, Germany
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