Toshiya Nakatani

The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti‐angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)‐chelating agent for patients with Wilsons disease of penicillamine intolerance. In our study, we examined the effect of Cu‐chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu‐deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu‐chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

Inhibition of renin–angiotensin system attenuates liver enzyme-altered preneoplastic lesions and fibrosis development in rats

BACKGROUND/AIMS It is suggested that the renin-angiotensin system (RAS) is involved in tumor development and fibrogenesis. The aim of the present study was to examine the effect of RAS inhibition on the liver enzyme-altered preneoplastic lesions and fibrosis development. METHODS The effects of the clinically used angiotensin-I converting enzyme inhibitor (ACE-I), perindopril (PE), on two different rat model of liver carcinogenesis models induced separately by diethylnitrosamine (DEN) and a choline-deficient L-amino acid-defined (CDAA) diet were studied. This CDAA model was also used to elucidate the effect of PE on liver fibrosis development. RESULTS The immunohistochemical evaluation revealed that the glutathione S-transferase placental form (GST-P), and gamma-glutamyltransferase (GGT)-positive preneoplastic foci significantly decreased in the livers of the PE-treated groups. In CDAA-induced liver fibrosis model, PE revealed a marked inhibitory effect of liver fibrosis development. The hepatic hydroxyproline, serum fibrosis markers, alpha-smooth muscle actin (alpha-SMA) immunopositive cells in number, and alpha-(III) pro-collagen mRNA expression were significantly suppressed by PE treatment. These inhibitory effects of PE were achieved even at a clinically comparable dose (2 mg/kg per day). CONCLUSIONS These results suggested that the RAS is involved in liver carcinogenesis and fibrosis development.

Particle-mediated gene transfer into murine livers using a newly developed gene gun

Although particle-mediated gene transfer using gene gun technology has been applied for gene transfer into epidermis, applications of this technology to visceral tissues have not been well investigated. Although all helium gas-driven gene gun instruments have used macrocarriers to discharge DNA-coated microprojectiles so far, we used a newly developed gene gun instrument, in which a hammering bullet is used to discharge microprojectiles. With the gene gun, gold particles coated with lacZ expression plasmid were discharged to murine livers. LacZ expression was induced much more profoundly in the liver by particle-mediated gene transfer than by simple plasmid injection and electroporation-mediated gene transfer. LacZ expression was broadly and randomly distributed throughout the bombarded livers, indicating that particle-mediated gene transfer can induce transgene expression even at relatively distant areas from the surface of the bombarded tissue. Furthermore, although transgene expression was at its peak on day 2 after the bombardment, it was still detectable even on day 28. These results indicate that particle-mediated gene transfer with a newly developed gene gun may provide a new approach to gene therapy for human diseases.

Expression of a retrovirally transduced gene under control of an internal housekeeping gene promoter does not persist due to methylation and is restored partially by 5-azacytidine treatment

Although expression of transgenes under the control of a retroviral long terminal repeat (LTR) promoter has been shown not to persist due to methylation, it has been observed that internal promoter may be active even if expression from the LTR promoter is silent. We constructed a retroviral vector carrying the herpes simplex virus thymidine kinase (HSVtk) gene under the control of the albumin gene promoter and transduced the HSVtk gene into hepatocellular carcinoma cells. Three of 14 mice, however, could not eradicate HSVtk-transduced grafts completely despite ganciclovir (GCV) treatment. These GCV-refractory cell lines exhibited resistance to GCV after recultivation. Subsequent Southern blot analysis revealed that the HSVtk gene was not deleted but extensively or completely methylated in GCV-refractory lines. Treatment with 5-azacytidine, a demethylating agent, partially restored the sensitivity of GCV-refractory lines to GCV. These results indicate that expression of retrovirally transduced gene may not persist in vivo due to methylation even when the gene is directed by an internal housekeeping gene promoter. These observations may also have important implications for future clinical applications of retrovirus-mediated gene therapy.

Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter

We have recently isolated carcinoembryonic antigen (CEA) promoter regions consisting of 419 bp and 204 bp from CEA-producing human colorectal carcinoma (CRC). We constructed CEA419/CD and CEA204/CD retroviruses carrying the bacterial cytosine deaminase (CD) gene directed by the CEA promoter regions. pCD2 retroviruses carrying the CD gene directed by the retrovirus long terminal repeat promoter were also used. CEA419/CD or CEA204/CD retrovirus-infected CRC cells were found to be susceptible to 5-fluorocytosine (5-FC), while non-CRC cells infected with the same retroviruses were not. CD-transduced CRC xenografts in nude mice were sensitive to 5-FC treatment, resulting in arrest of tumor growth. When mice with intraperitoneally disseminated CRCs were given intraperitoneal injections of CEA419/CD retrovirus-producing cells followed by 5-FC treatment, significantly prolonged survival rates were observed compared with animals injected with pCD2 retrovirus-producing cells followed by 5-FC treatment. Importantly, bone marrow suppression was not observed in animals injected with CEA419/CD retrovirus-producing cells and 5-FC, while profound bone marrow suppression was observed in those injected with pCD2 retrovirus-producing cells and 5-FC. These results indicate that effective and safe in vivo gene therapy for advanced CRC may be feasible by transferring the CD gene controlled by the CEA promoter followed by 5-FC treatment.

Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice

Murine hepatocellular carcinoma cells were retrovirally transduced with the bacterial cytosine deaminase (CD) gene. CD‐transduced cells exhibited more than 120‐fold higher sensitivity to 5‐fluorocytosine (5‐FC) compared with parental cells. When syngeneic immunocompetent mice were inoculated s.c. with parental hepatocellular carcinoma cells containing as little as 5% CD‐transduced cells, significant inhibition of tumor formation was induced by 5‐FC treatment. Furthermore, established solid tumors in immunocompetent mice containing only 5% CD‐transduced cells were infiltrated markedly with CD4+ and CD8+ T lymphocytes and macrophages by 5‐FC treatment, such that significant reduction or even complete regression of tumors was observed. These tumor‐free mice resisted subsequent rechallenge with wild‐type tumor. Conversely, when athymic nude mice were inoculated with a cell mixture containing CD‐transduced cells and parental cells at a ratio of 40:60, all developed tumors despite 5‐FC treatment. Our results indicate that gene therapy using the CD/5‐FC system can induce efficient anti‐tumor effects and protective immunity in immunocompetent mice but not in athymic immunodeficient mice, suggesting that the hosts immunocompetence may be a critical factor for achieving successful gene therapy against cancer. Int. J. Cancer 81:592–597, 1999.

Assessment of efficiency and safety of adenovirus mediated gene transfer into normal and damaged murine livers

BACKGROUND When recombinant adenoviruses are infused directly into the circulation, transgene expression is almost completely restricted to the liver. AIMS Efficiency and safety of adenovirus mediated gene transfer into damaged livers were examined in mice with liver cirrhosis or fulminant hepatitis. METHODS Liver cirrhosis and fulminant hepatitis were induced by intraperitoneal administration of thioacetamide and d-galactosamine followed by lipopolysaccharide, respectively. Mice were infused with adenoviruses carrying the Escherichia coliβ-galactosidase gene, lacZ gene, into the tail vein. Transduction efficiency of thelacZ gene was estimated histochemically by X-gal staining and quantitatively using a chemiluminescent assay. Activation of adenovirus specific T cells and development of neutralising antibodies against adenovirus were also examined. RESULTS Histochemical evaluation revealed that approximately 40%, 80%, and 40% of cells in normal, cirrhotic, and fulminant hepatitis livers, respectively, were stained blue using X-gal staining. Quantitative analyses revealed that levels of lacZ expression in cirrhotic livers were approximately 2.5-fold and sixfold greater than those in normal and fulminant hepatitis livers, respectively. Although transgene expression in fulminant hepatitis livers was significantly lower than that in normal livers, marked levels of transgene expression were achieved even in fulminant hepatitis livers. Significant adverse effects of adenoviruses were not observed in damaged livers. There were no significant differences in cellular or humoral immune responses to adenoviruses among animals with normal, cirrhotic, and fulminant hepatitis livers. CONCLUSIONS Our results suggest that gene therapy with adenoviruses may be used efficiently and safely, even in patients with severe liver disease.

Complete cure of established murine hepatocellular carcinoma is achievable by repeated injections of retroviruses carrying the herpes simplex virus thymidine kinase gene

Although xenotransplantation of retrovirus-producing cells into a tumor has been shown to be effective for the treatment of cancer, injections of recombinant retroviruses are much more feasible for clinical applications. We established a clone producing retroviruses carrying the herpes simplex virus thymidine kinase (HSVtk) gene with titers of up to 4 × 107 colony-forming units/ml, and examined the effectiveness of in vivo gene therapy against cancer. Syngeneic mice were inoculated subcutaneously with murine hepatocellular carcinoma (HCC) cells, BNL1ME A.7R.1, and the treatment was initiated after tumors were established. When mice were given an intratumoral injection of HSVtk-carrying retroviruses or their producing cells followed by ganciclovir (GCV) treatment, significantly pro- longed survival periods were observed. When mice were treated with repeated intratumoral injections of HSVtk-carrying retrovirus-producing cells, significant antitumor responses and some cures were induced by GCV treatment. Furthermore, repeated intratumoral injections of HSVtk-carrying retroviruses and GCV treatment resulted in complete regression of established HCC tumors in all animals used in the experiment. Mice that completely eradicated tumors exhibited protective immunity against wild-type HCC tumors. These results suggest that repeated injections of HSVtk-carrying retroviruses followed by GCV treatment is a potent modality for the treatment of solid tumors.

Comparison of Gene Therapy with the Herpes Simplex Virus Thymidine Kinase Gene and the Bacterial Cytosine Deaminase Gene for the Treatment of Hepatocellular Carcinoma

BACKGROUND Bystander effects induced by suicide gene/prodrug systems play an essential role in achieving successful antitumor effects. Although it has been shown in several in vitro studies that the bacterial cytosine deaminase (CD) gene/5-fluorocytosine (5-FC) system is superior to the herpes simplex virus thymidine kinase (HSV-TK) gene/ganciclovir (GCV) system, we examined here which suicide gene system was more promising in vivo for the treatment of hepatocellular carcinoma (HCC). METHODS BNL1ME A.7R.1 murine HCC cells were retrovirally transduced with the HSV-TK or CD gene, and bystander effects caused by the appropriate prodrug treatment were examined not only in vitro but also in vivo. RESULTS The CD/5-FC system was superior to the HSV-TK/GCV system in HCC cell elimination in vitro. The bystander effect of the HSV-TK/GCV was shown to be substantially dependent on cell-to-cell contact, whereas that of the CD/5-FC was not. However, antitumor effects on HCC and tumor immunity to parental HCC induced by the HSV-TK/GCV system were not inferior and even superior to those induced by the CD/5-FC system. Bystander effects induced by the suicide gene/prodrug systems in immunocompetent syngeneic mice were much more profound than those induced in vitro. However, significant bystander effects were not observed in athymic nude mice. CONCLUSIONS These results suggest that both HSV-TK/GCV and CD/5-FC systems are useful for the treatment of HCC. The results also suggest that T-cell-mediated immune responses elicited by the suicide gene/prodrug systems play a substantial role in antitumor effects in vivo.

Tissue-specific expression of HSV-tk gene can induce efficient antitumor effect and protective immunity to wild-type hepatocellular carcinoma.

The efficacy of expression of the herpes simplex virus thymidine kinase (HSV‐tk) gene under the transcriptional control of the liver‐specific albumin gene promoter, followed by ganciclovir treatment, was investigated both in vitro and in vivo. Murine and rat hepatocellular carcinoma (HCC) cells infected with retroviruses carrying the HSV‐tk gene under the control of the murine albumin gene promoter were selectively killed by ganciclovir treatment in vitro, whereas non‐HCC cells, such as murine mammary tumor cells and fibroblast cells, which were infected with the same retroviruses, were not. Susceptibility of the retroviral‐infected HCC cells to ganciclovir was more than 100‐fold higher than that of the retroviral‐infected non‐HCC cells. When mice bearing a bulky HCC mass consisting of the retroviral‐infected HCC cells were treated with systemic ganciclovir administration, complete regression of the tumors was observed without any signs of overt toxicity. Profound antitumor effects on preestablished murine HCCs were observed when wild‐type HCC cells were implanted into animals with a small percentage of the retroviral‐infected counterparts. When only 5% of the cells were infected with retroviruses carrying the HSV‐tk gene, significant inhibition of tumor development was observed with systemic ganciclovir treatment. Importantly, animals that were treated with implantation of mixtures of the retroviral‐infected and parental HCC cells, followed by ganciclovir administration, did not exhibit tumor formation and resisted subsequent rechallenge with wild‐type HCC cells. Our results indicate the feasibility of combination therapy with the HSV‐tk gene and ganciclovir for the treatment of HCC. Int. J. Cancer 71:470‐475, 1997.