Preclinical evaluation of azathioprine plus buthionine sulfoximine in the treatment of human hepatocarcinoma and colon carcinoma.
OBJECTIVE
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.


CONCLUSIONS
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.
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.
Borja
B
Hernández-Breijo
Jorge
J
Monserrat
Sara
S
Ramírez-Rubio
Eva P
EP
Cuevas
Diana
D
Vara
Inés
I
Díaz-Laviada
M Dolores
MD
Fernández-Moreno
Irene D
ID
Román
Javier P
JP
Gisbert
Luis G
LG
Guijarro
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D001379:Azathioprine / Q000494:pharmacology / Q000627:therapeutic use; D019328:Buthionine Sulfoximine / Q000494:pharmacology / Q000627:therapeutic use; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy*; D002417:Cattle; D045744:Cell Line, Tumor / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy*; D004359:Drug Therapy, Combination; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation
D000964:Antimetabolites, Antineoplastic; D019328:Buthionine Sulfoximine; D001379:Azathioprine