Irene D. Román

Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography : Comparison with the radiochemical method

The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 &mgr;L packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl- l -methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures (y = 0.99 x + 0.33;x-axis, radiochemical assay;y-axis, HPLC-linked assay;r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.

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) 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.

RNAi‐mediated silencing of insulin receptor substrate‐4 enhances actinomycin D‐ and tumor necrosis factor‐α‐induced cell death in hepatocarcinoma cancer cell lines

Insulin receptor substrate‐4 (IRS‐4) transmits signals from the insulin‐like growth factor receptor (IGF‐IR) and the insulin receptor (IR) to the PI3K/AKT and the ERK1/2 pathways. IRS‐4 expression increases dramatically after partial hepatectomy and plays an important role in HepG2 hepatoblastoma cell line proliferation/differentiation. In human hepatocarcinoma, IRS‐4 overexpression has been associated with tumor development. Herein, we describe the mechanism whereby IRS‐4 depletion induced by RNA interference (siRNA) sensitizes HepG2 cells to treatment with actinomycin D (Act D) and combined treatment with Act D plus tumor necrosis factor‐α (TNF‐α). Similar results have been obtained in HuH 7 and Chang cell lines. Act D therapy drove the cells to a mitochondrial‐dependent apoptotic program involving cytochrome c release, caspase 3 activation, PARP fragmentation and DNA laddering. TNF‐α amplifies the effect of Act D on HepG2 cell apoptosis increasing c‐jun N‐terminal kinase (JNK) activity, IκB‐α proteolysis and glutathione depletion. IRS‐4 depleted cells that were treated with Act D showed an increase in cytochrome c release and procaspase 3 and PARP proteolysis with respect to control cells. The mechanism involved in IRS‐4 action is independent of Akt, IκB kinase and JNK. IRS‐4 down regulation, however, decreased γ‐glutamylcysteine synthetase content and cell glutathione level in the presence of Act D plus TNF‐α. These results suggest that IRS‐4 protects HepG2 cells from oxidative stress induced by drug treatment. J. Cell. Biochem. 108: 1292–1301, 2009.

Cyclosporin A induces apoptosis in rat hepatocytes in culture.

Abstract Cyclosporin A (CsA) at concentrations up to 1 μM induced apoptosis in a dose-dependent manner in cultured rat hepatocytes for 48 h in the presence of insulin and epidermal growth factor (EGF). The effect of CsA was evidenced by the DNA fragmentation pattern constituted by fragments of multiples of 180–200 base pairs, which is a characteristic of programmed cell death. The metabolic activity did not change significantly in the presence of 0.1 μM CsA and diminished to 49% of control in the presence of 1 μM CsA. Changes in the metabolic activity were correlated with a decrease in both [methyl-3H]thymidine uptake and DNA content, which reflects a decrease in the cell number. The treatment of cells with CsA (1 μM) decreased the metabolic activity/DNA content ratio by 24% with respect to dimethyl sulphoxide (DMSO) control, which also suggests, under these conditions, that the necrosis achieved is at most only 24%. In addition, the changes observed (apoptotic process, arrest of the cell cycle and apparition of a necrotic process) were correlated with an increase in the high-affinity guanosine triphosphatase (GTPase) enzymes.

Is the Autophagy Induced by Thiopurines Beneficial or Deleterious

Thiopurines (azathioprine, 6-mercaptopurine and 6-thioguanine), are drugs useful in the treatment of leukemia, autoimmune diseases, as well as in organ transplantation. After many years of use is still not well understood their mode of action. Recently, several groups have found that thiopurines can activate autophagy by different mechanisms. Autophagy is a process of auto-digestion. After an infection, radiation, injury, oxidative stress, or after drug treatment, the cellular organelles may be damaged. In those cases the damaged structures are recognized by the cell, isolated in a double-membrane vacuole and finally degraded in autolysosomes. The digestion gives rise to biosynthetic precursors needed to regenerate partially destroyed structures, so as to produce the energy essential in the anabolic process. During fasting, the protein aggregates, lipid droplets and glycogen deposits are degraded by this pathway for releasing nutrients to the blood. Therefore this process is of vital importance in the maintaining of cellular functions and in the systemic homeostasis of whole organism. The therapy with thiopurines leads to adverse effects such as myelosuppression and hepatotoxicity whose mechanism is not well understood today. Autophagy is also involved in liver degeneration induced by drugs, alcohol or viruses. Therefore, seems to be very attractive know whether the autophagy induced by thiopurines is the cause of the hepatotoxicity associated with these drugs, or rather, autophagy is a compensatory response that protects the liver against the deleterious effects of the thiopurines. Our results and previous data suggest that autophagy is beneficial for the liver because protects it against the deleterious effects of thiopurines.

Effect of flutamide-induced androgen-receptor blockade on adenylate cyclase activation through G-protein coupled receptors in rat prostate

The effect of the antiandrogen flutamide on the prostatic vasoactive intestinal peptide (VIP) receptor/effector system was studied in rats. Rats were s.c. injected with a daily dose of flutamide (15 mg/kg B.W.) or vehicle for 14 days. Drug treatment resulted in histological evidence of gland involution and increased plasma membrane fluidity as estimated by fluorescence spectroscopy. The number of VIP receptors and the stimulatory effect of VIP on adenylate cyclase activity in prostatic membranes decreased in flutamide-treated rats. However, the pattern of forskolin stimulation of the enzyme activity was not modified by this drug. Androgen-receptor blockade by flutamide also decreased the prostatic levels of alpha(s,) alpha(i1/2), and alpha(i3/0) G-protein subunits, as estimated by an immunological procedure. Whereas apoptotic DNA fragmentation was evidenced in prostate from 3-day castrated animals, a heterogeneous electrophoretic pattern was observed after flutamide treatment. Thus, androgen-receptor blockade by flutamide results in an important impairment of the components of the VIP receptor/effector system in rat prostate as well as in a modification of their coupling extent, which is presumably due to differences observed in plasma membrane fluidity. These results represent a crosstalk in the prostate between two mechanisms of signal transduction involved in cell proliferation.

Azathioprine desensitizes liver cancer cells to insulin-like growth factor 1 and causes apoptosis when it is combined with bafilomycin A1.

Hepatoblastoma is a primary liver cancer that affects children, due to the sensitivity of this tumor to insulin-like growth factor 1 (IGF-1). In this paper we show that azathioprine (AZA) is capable of inhibiting IGF1-mediated signaling cascade in HepG2 cells. The efficiency of AZA on inhibition of proliferation differs in the evaluated cell lines as follows: HepG2 (an experimental model of hepatoblastoma)>Hep3B (derived from a hepatocellular carcinoma)>HuH6 (derived from a hepatoblastoma)>>HuH7 (derived from a hepatocellular carcinoma)=Chang Liver cells (a non-malignant cellular model). The effect of AZA in HepG2 cells has been proven to derive from activation of Ras/ERK/TSC2, leading to activation of mTOR/p70S6K in a sustained manner. p70S6K phosphorylates IRS-1 in serine 307 which leads to the uncoupling between IRS-1 and p85 (the regulatory subunit of PI3K) and therefore causing the lack of response of HepG2 to IGF-1. As a consequence, proliferation induced by IGF-1 is inhibited by AZA and autophagy increases leading to senescence of HepG2 cells. Our results suggest that AZA induces the autophagic process in HepG2 activating senescence, and driving to deceleration of cell cycle but not to apoptosis. However, when simultaneous to AZA treatment the autophagy was inhibited by bafilomycin A1 and the degradation of regulatory proteins of cell cycle (e.g. Rb, E2F, and cyclin D1) provoked apoptosis. In conclusion, AZA induces resistance in hepatoblastoma cells to IGF-1, which leads to autophagy activation, and causes apoptosis when it is combined with bafilomycin A1. We are presenting here a novel mechanism of action of azathioprine, which could be useful in treatment of IGF-1 dependent tumors, especially in its combination with other drugs.

Infliximab therapy reverses the increase of allograft inflammatory factor-1 in serum and colonic mucosa of rats with inflammatory bowel disease.

Abstract Objective: Our purpose was to study the molecular basis of infliximab (IFX) effect on colon mucosa in a colitis model and to identify new biomarkers of mucosal healing. Methods: Healthy rats and rats which were subjected to experimental colitis induced by dextran sulfate sodium, with or without IFX treatment (in the short- and long-term), were studied along with forty-seven IBD patients. Colon mucosal integrity by periodic acid Schiff (PAS) staining, intestinal damage by immunohistochemistry (proliferating cell nuclear antigen, β-catenin, E-cadherin, phosphotyrosine, p-p38, allograft inflammatory factor-1 (AIF-1) and colonic mucosal apoptosis by TUNEL staining were evaluated in rats while serum and colon AIF-1 levels were determined in IBD patients. Results: In rats with colitis, IFX reestablished the epithelial barrier integrity, recovered mucus production and decreased colon inflammation, as verified by reduced serum and colon AIF-1 levels; colon and serum AIF-1 levels were also lower in inactive IBD patients compare to active ones. P38 activation after IFX treatment tended to induce differentiation/proliferation of epithelial cells along the colonic crypt-villous axis. Conclusions: These findings support AIF-1 as a new biomarker of mucosal healing in experimental colitis and suggest that p38 activation is involved in the mucosal healing intracellular mechanism induced by IFX treatment.

Effect of Infliximab in oxidised serum albumin levels during experimental colitis

Abstract Infliximab (IFX) is widely used in ulcerative colitis and in Crohn’s disease treatment. Both diseases are characterised by increased oxidative stress, which may affect albumin oxidation. In order to test this hypothesis, the effect of IFX on colitis induced by dextran sulphate sodium (DSS) in rats was evaluated by measuring the Disease Activity Index, biochemical parameters, serum albumin oxidation and colonic mucosa oxidation. Rats with colitis showed an increase in oxidised serum albumin levels and in the oxidation of colon mucous cells. Both decreased after IFX treatment. This suggests that oxidised albumin could be a useful biomarker for monitoring inflammatory bowel disease.

Inhibitory Effect of Cyclosporin A Peptide on Rat Hepatocytes Proliferation Induced by Mitogens

Treatment of cultured rat hepatocytes with cyclosporin A (0.01-1 microM) for 24, 48, or 72 h in the presence of insulin and epidermal growth factor induced an inhibition on cell proliferation in a time- and concentration-dependent manner, with an IC50 = 0.05 microM CsA corresponding to 48-h treatment. The inhibitory effect of CsA at < or = 0.1 microM doses for 48 h on [3H]thymidine uptake was reversed after withdrawal of the drug and subsequent addition of insulin plus EGF or serum; however, at 1 microM CsA the effect was irreversible and numerous bright small vesicles were observed. The molecular mechanism involved in CsA action in hepatocytes seems to be independent on cAMP and pertussis-toxin sensitive G proteins.