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

AIM: To evaluate the efficacy and the safety of azathioprine (AZA) and buthionine sulfoximine (BSO) bylocalized 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 ℃ 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 eff icacy 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 localizedtreatment of hepatocellular carcinoma as in the currently used transarterial chemoembolization method.

Study on effect of BSO on esophageal cancer cell line TE-1

Objective: The aim of the study was to investigate the sensitizing effect of buthionine sulfoximine (BSO) and radiation on esophageal cancer cell line TE-1. Methods: Methyl thiazolyl tetrazolium (MTT) assay was used to observe the inhibition of BSO and radiation on cell proliferation, and to investigate the sensitizing effect of BSO on esophageal cancer cell line TE-1. Flow cytometry (FCM) was used to observe the effect of BSO and radiation on cell apoptosis and cycle. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to observe the effect of BSO on manganese superoxide dismutase (MnSOD) mRNA and protein expression. Results: BSO could inhibit the proliferation of TE-1 esophageal cancer cells, and had significant dose- and time-dependent radiosensitizing effects on TE-1 esophageal cancer cells. After the combined effects of BSO and radiation on TE-1 cells, the rate of apoptosis and G2/M phase proportion increased significantly, and MnSOD mRNA and protein expression decreased. Conclusion: BSO may reduce MnSOD mRNA and protein expression by affecting TE-1 cell cycle, thus inhibiting and inducing the apoptosis of esophageal cancer cells and enhancing the killing effect of the radiation on esophageal cancer cells.

Arsenic trioxide induces multiple myeloma cell apoptosis via disruption of mitochondrial transmembrane potentials and activation of caspase-3

OBJECTIVE: To investigate the response of multiple myeloma (MM) cells to arsenic trioxide (As2O3) and their possible mechanisms. METHODS: Two MM-derived cell lines RPMI8226 and U266 cells were used as in vitro models. Cell apoptosis was assessed by morphology, flow cytometry, and DNA gel electrophoresis. Mitochondrial transmembrane potentials (delta psi m) were evaluated by measuring cellular Rhodamine 123 staining intensity. Protein expression was analyzed using Western blot. RESULTS: Zero point one to 0.5 mumol/L As2O3 inhibited cell proliferation and 2.0 mumol/L As2O3 induced cell apoptosis, while 1.0 mumol/L As2O3 inhibited proliferation with a weak degree of apoptosis induction in RPMI8226 and U266 cell lines. As2O3-induced apoptosis was accompanied by mitochondrial transmembrane potentials (delta psi m) collapse and caspase-3 activation in the presence of intact membrane. Glutathione depleter buthionine sulfoximine enhanced, while disulfide bond-reducing agent dithiothreitol partially antagonized As2O3-induced delta psi m collapse and apoptosis in MM cells. All-trans retinoic acid (ATRA) could also induce apoptosis in RPMI8226 cells, but it did not show any cooperative effects with As2O3. CONCLUSION: As2O3 exerts apoptosis-inducing and growth-inhibiting effects on MM cells, and mitochondrium is a pivotal and common target of As2O3 for apoptosis induction.