Original Article
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World J Gastroenterol. Sep 14, 2011; 17(34): 3899-3911
Published online Sep 14, 2011. doi: 10.3748/wjg.v17.i34.3899
Preclinical evaluation of azathioprine plus buthionine sulfoximine in the treatment of human hepatocarcinoma and colon carcinoma
Borja Hernández-Breijo, Jorge Monserrat, Sara Ramírez-Rubio, Eva P Cuevas, Diana Vara, Inés Díaz-Laviada, M Dolores Fernández-Moreno, Irene D Román, Javier P Gisbert, Luis G Guijarro
Borja Hernández-Breijo, Sara Ramírez-Rubio, Eva P Cuevas, M Dolores Fernández-Moreno, Irene D Román, Luis G Guijarro, Molecular Hepatic Toxicology Unit, Biochemistry and Molecular Biology Department, University of Alcalá, Networked Biomedical Research Center of Hepatic and Digestive Diseases (CIBEREHD), Alcalá de Henares 28871, Spain
Jorge Monserrat, Medicine Department, University of Alcalá, Alcalá de Henares 28871, Spain
Diana Vara, Inés Díaz-Laviada, Neuroendocrinology of Cannabinoid/Vanilloid System Unit, Biochemistry and Molecular Biology Department, University of Alcalá, Alcalá de Henares 28871, Spain
Javier P Gisbert, Gastroenterology Unit, Hospital de la Princesa, Institute of Health Research Princesa, Networked Biomedical Research Center of Hepatic and Digestive Diseases (CIBEREHD), Madrid 28006, Spain
Author contributions: Hernández-Breijo B performed the majority of the in vitro and in vivo experiments; Monserrat J performed cytometry; Ramírez-Rubio S and Cuevas EP performed in vitro experiments; Vara D performed the in vivo experiments; Fernández-Moreno MD, Román ID performed analysis of glutathione levels and reviewed the manuscript; Díaz-Laviada I and Gisbert JP reviewed the manuscript; Guijarro LG designed the study and wrote the manuscript.
Supported by Grants from SAF2008-05355 and CCG07-UAH/BIO-2085
Correspondence to: Dr. Luis González Guijarro, Professor, Biochemistry and Molecular Biology Department, University of Alcalá, Alcalá de Henares 28871, Spain. luis_guijarro@telefonica.net
Telephone: +34-91-885-4865 Fax: +34-91-885-4585
Received: January 10, 2011
Revised: March 26, 2011
Accepted: April 2, 2011
Published online: September 14, 2011
Abstract

AIM: To evaluate the efficacy and the safety of azathioprine (AZA) and buthionine sulfoximine (BSO) by localized application into HepG2 tumor in vivo.

METHODS: Different hepatoma and colon carcinoma cell lines (HepG2, HuH7, Chang liver, LoVo, RKO, SW-48, SW-480) were grown in minimal essencial medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic solution and maintained in a humidified 37 °C incubator with 5% CO2. These cells were pretreated with BSO for 24 h and then with AZA for different times. We examined the effects of this combination on some proteins and on cellular death. We also studied the efficacy and the safety of AZA (6 mg/kg per day) and BSO (90 mg/kg per day) in HepG2 tumor growth in vivo using athymic mice. We measured safety by serological markers such as aminotransferases and creatine kinase.

RESULTS: The in vitro studies revealed a new mechanism of action for the AZA plus BSO combination in the cancer cells compared with other thiopurines (6-mercaptopurine, 6-methylmercaptopurine, 6-thioguanine and 6-methylthioguanine) in combination with BSO. The cytotoxic effect of AZA plus BSO in HepG2 cells resulted from necroptosis induction in a mitochondrial-dependent manner. From kinetic studies we suggest that glutathione (GSH) depletion stimulates c-Jun amino-terminal kinase and Bax translocation in HepG2 cells with subsequent deregulation of mitochondria (cytochrome c release, loss of membrane potential), and proteolysis activation leading to loss of membrane integrity, release of lactate dehydrogenase and DNA degradation. Some of this biochemical and cellular changes could be reversed by N-acetylcysteine (a GSH replenisher). In vivo studies showed that HepG2 tumor growth was inhibited when AZA was combined with BSO.

CONCLUSION: Our studies suggest that a combination of AZA plus BSO could be useful for localized treatment of hepatocellular carcinoma as in the currently used transarterial chemoembolization method.

Keywords: Azathioprine; Buthionine sulfoximine; Transarterial chemoembolization; Glutathione; Apoptosis; Necrosis