Small Cell Lung Cancer Expressing Glutamate Decarboxylase with Latent Autoimmune Diabetes in Adults.

Small cell lung cancer (SCLC) causes paraneoplastic syndromes, such as diabetes mellitus, by eliciting the expression of various antibodies including anti-glutamate decarboxylase (GAD) antibody. A 62-year-old woman presented to our hospital with a 1-week history of progressive dyspnea and difficulty in walking. Computed tomography showed a tumor obstructing the left bronchus and obstructive lung abscesses with pleural effusions. A biopsy during bronchoscopy revealed SCLC, and the clinical stage was ultimately determined to be IIIB. SCLC was complicated by diabetes mellitus with high titers of serum anti-GAD antibody. An immunohistochemical analysis showed GAD expression in the cancer cells, which is a novel finding.

The Bcl-xL inhibitor, ABT-737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib.

UNLABELLED: Tumor cells are characterized by uncontrolled proliferation, often driven by activation of oncogenes, and apoptosis resistance. The oncogenic kinase inhibitor sorafenib can significantly prolong median survival of patients with advanced hepatocellular carcinoma (HCC), although the response is disease-stabilizing and cytostatic rather than one of tumor regression. Bcl-xL (B cell lymphoma extra large), an antiapoptotic member of the B cell lymphoma-2 (Bcl-2) family, is frequently overexpressed in HCC. Here, we present in vivo evidence that Bcl-xL overexpression is directly linked to the rapid growth of solid tumors. We also examined whether ABT-737, a small molecule that specifically inhibits Bcl-xL but not myeloid cell leukemia-1 (Mcl-1), could control HCC progression, especially when used with sorafenib. Administration of ABT-737, even at an in vivo effective dose, failed to suppress Huh7 xenograft tumors in mice. ABT-737 caused the levels of Mcl-1 expression to rapidly increase by protein stabilization. This appeared to be related to resistance to ABT-737, because decreasing Mcl-1 expression levels to the baseline by a small interfering RNA-mediated strategy made hepatoma cells sensitive to this agent. Importantly, administration of ABT-737 to Mcl-1 knockout mice induced severe liver apoptosis, suggesting that tumor-specific inhibition of Mcl-1 is required for therapeutic purposes. Sorafenib transcriptionally down-regulated Mcl-1 expression specifically in tumor cells and abolished Mcl-1 up-regulation induced by ABT-737. Sorafenib, not alone but in combination with ABT-737, efficiently induced apoptosis in hepatoma cells. This combination also led to stronger suppression of xenograft tumors than sorafenib alone. CONCLUSION: Bcl-xL inactivation by ABT-737 in combination with sorafenib was found to be safe and effective for anti-HCC therapy in preclinical models. Direct activation of the apoptosis machinery seems to unlock the antitumor potential of oncogenic kinase inhibitors and may produce durable clinical responses against HCC.

FOXP3/Scurfin gene polymorphism is associated with adult onset type 1 diabetes in Japanese, especially in women and slowly progressive-type patients.

PURPOSE: Some reports have shown that the cause of type 1 diabetes is associated with dysfunction of regulatory T cells (Tregs). The FOXP3/Scurfin gene is known to be a master gene of Tregs. Therefore, we tried to analyze the relation between the gene polymorphism and adult onset type 1 diabetes and its subtype in the Japanese population. METHODS: In this study, we recruited 316 Japanese patients with type 1 diabetes (155 male and 161 female, mean onset age 35.4 years) and 432 healthy Japanese controls (263 male and 169 female, mean age 44.4 years). Then we subdivided the patients by onset type, sex, and islet-associated autoantibody positivity. RESULTS: The genotype frequency of (GT)(16)/(GT)(16) in female patients with overall type 1 diabetes was especially lower than that in controls (19.9% vs. 38.5%, p = 0.0002). Moreover, the genotype frequency of (GT)(16)/(GT)(16) in female patients with slowly progressive type 1 diabetes was significantly lower than that in controls (15.4% vs. 38.5%, p = 0.002). CONCLUSION: Our data showed that the (GT)n microsatelloite polymorphism in the FOXP3/Scrufin gene was associated with Japanese adult onset type 1 diabetes, especially in females, and slowly progressive type 1 diabetes.

Cremophor EL, a non-ionic surfactant, promotes Ca(2+)-dependent process of cell death in rat thymocytes.

Cremophor EL, a surfactant for pharmaceutical products, augments the cytotoxicity of hydrogen peroxide in rat thymocytes [Iwase, K., Oyama, Y., Tatsuishi, T., Yamaguchi1, J., Nishimura1, Y., Kanada, A., Kobayashi, M., Maemura, Y., Ishida, S., Okano, Y., 2004. Cremophor EL augments the cytotoxicity of hydrogen peroxide in lymphocytes dissociated from rat thymus glands. Toxicol. Lett. 154, 143-148]. The effect of cremophor EL on Ca(2+)-dependent process of cell death has been examined using a flow cytometer since hydrogen peroxide increases intracellular Ca2+ concentration. Cremophor EL at clinically-relevant concentrations greatly increased the population of dead cells in rat thymocytes simultaneously treated with A23187, a calcium ionophore increasing intracellular Ca2+ concentration. Removal of Ca2+ from external solution diminished the cremophor EL-induced increase in the dead cell population. Result suggests that Ca(2+)-dependent process is involved in the cremophor EL-induced decrease in the cell viability in the simultaneous presence of A23187. The population of cells with hypodiploidal DNA was not increased by the application of cremophor EL and A23187 although the cell viability was greatly decreased, indicating that the type of cell death is necrosis. It is suggested that cremophor EL at clinically-relevant concentrations augments the Ca(2+)-dependent process of necrosis.

Polysorbate 80 increases the susceptibility to oxidative stress in rat thymocytes.

Effect of simultaneous application of polysorbate 80, a nonionic surfactant widely used in pharmaceutical products, and hydrogen peroxide on rat thymocytes was examined to see if polysorbate 80 increases the susceptibility to oxidative stress because this surfactant decreases the cellular content of glutathione. Polysorbate 80 at clinically-relevant concentrations increases the cytotoxicity of hydrogen peroxide under the in vitro condition. Result suggests that polysorbate 80 may increase the susceptibility of cells to oxidative stress.

Cytometric analysis of adverse action of diphenyl ditelluride on rat thymocytes: cell shrinkage as a cytotoxic parameter.

Despite the growing use of organotellurium compounds in the chemical and biomedical fields, there has been no great concern about their toxicity until now. To test the possibility that diphenyl ditelluride (DPDT) and tellurium chloride (TeCl2), organic and inorganic tellurium compounds, may exert adverse action on mammals, their effects on rat thymocytes were examined under in vitro conditions using a flow cytometer with fluorescent probes. Incubation of thymocytes with DPDT at 300 nM or more for 24 h significantly increased the populations of shrunken cells and of cells with hypodiploidal DNA. Z-VAD-FMK, a paninhibitor of caspases, greatly suppressed the DPDT-induced increase in the hypodiploidal cell population, suggesting the involvement of caspase activation in DPDT toxicity. Hence, it is possible that DPDT would increase the population of thymocytes undergoing apoptosis if the blood concentration in mammals reached at least 300 nM or more. TeCl2 was much less potent than DPDT in increasing the population of hypodiploidal cells.

Cremophor EL augments the cytotoxicity of hydrogen peroxide in lymphocytes dissociated from rat thymus glands.

The pharmaceutical uses of cremophor EL, a non-ionic surfactant, are similar to those of polysorbate 80. In our previous study, polysorbate 80 exerted some adverse actions on rat thymocytes under in vitro condition. Therefore, the effects of cremophor EL on thymic lymphocytes were examined using a flow cytometer with appropriate fluorescent dyes. Cremophor EL at 10 microg/ml or more (up to 300 microg/ml) concentration-dependently decreased cellular content of glutathione. The cell viability of thymocytes under control condition was 95.4 +/- 1.2% (n = 7, mean +/- S.D.). The incubation of thymocytes with 300 microg/ml cremophor EL or 3 mM hydrogen peroxide for 2 h, respectively, decreased the cell viability to 90.8 +/- 2.8% or 91.2 +/- 2.6%. However, the simultaneous incubation with cremophor EL and hydrogen peroxide decreased the cell viability to 28.7 +/- 8.2%. Cremophor EL at 100 microg/ml accelerated the process of cell death induced by hydrogen peroxide. Results suggest that cremophor EL increases the susceptibility to oxidative stress. Cremophor EL at clinically relevant concentrations may increase the therapeutic potential of some anticancer agents to produce oxidative stress.

Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes.

The effects of polysorbate 80, a non-ionic surfactant widely used in pharmaceutical products, on rat thymocytes were examined to reveal its toxic property at the cellular level. Polysorbate 80 at concentrations of 1-100 microg/ml did not significantly affect the cell viability. This surfactant at 30 microg/ml or more augmented the intensity of fluo-3 fluorescence, indicating the increase in intracellular Ca(2+) concentration. Such an augmentation of fluo-3 fluorescence by polysorbate 80 was not seen under the Ca(2+)-free condition, suggesting that polysorbate 80 increased membrane Ca(2+) permeability. The concentration-dependent polysorbate 80 at 10 microg/ml or more attenuated the intensity of 5-chloromethylfluorescein, indicating a decrease in cellular content of glutathione by polysorbate 80. Furthermore, the agent at 1 microg/ml or more attenuated the intensity of bis-(1,3-dibutylbarbituric acid) trimethine oxonol fluorescence, being independent from the changes in membrane potential. This phenomenon indicates that polysorbate 80 at 1 microg/ml or more may attenuate the incorporation of anionic compounds into the membranes. It can be suggested that polysorbate 80 modifies some of membranes and intracellular physiological parameters without affecting the cell viability.

Effect of thimerosal, a preservative in vaccines, on intracellular Ca2+ concentration of rat cerebellar neurons.

The effect of thimerosal, an organomercurial preservative in vaccines, on cerebellar neurons dissociated from 2-week-old rats was compared with those of methylmercury using a flow cytometer with appropriate fluorescent dyes. Thimerosal and methylmercury at concentrations ranging from 0.3 to 10 microM increased the intracellular concentration of Ca2+ ([Ca2+]i) in a concentration-dependent manner. The potency of 10 microM thimerosal to increase the [Ca2+]i was less than that of 10 microM methylmercury. Their effects on the [Ca2+]i were greatly attenuated, but not completely suppressed, under external Ca(2+)-free condition, suggesting a possibility that both agents increase membrane Ca2+ permeability and release Ca2+ from intracellular calcium stores. The effect of 10 microM thimerosal was not affected by simultaneous application of 30 microM L-cysteine whereas that of 10 microM methylmercury was significantly suppressed. The potency of thimerosal was similar to that of methylmercury in the presence of L-cysteine. Both agents at 1 microM or more similarly decreased the cellular content of glutathione in a concentration-dependent manner, suggesting an increase in oxidative stress. Results indicate that thimerosal exerts some cytotoxic actions on cerebellar granule neurons dissociated from 2-week-old rats and its potency is almost similar to that of methylmercury.

Increase in number of annexin V-positive living cells of rat thymocytes by intracellular Pb(2+).

Lead is ubiquitous in our environment and lead poisoning is a major public health problem worldwide. In this study, to see if intracellular Pb(2+) induces the exposure of phosphatidylserine in rat thymocyte membranes, we have examined the effect of PbCl(2) on rat thymocytes treated with A23187 using a flow cytometer with appropriate fluorescent indicators under nominally-Ca(2+)-free condition. PbCl(2) at 1-30 µM dose-dependently induced the exposure of phosphatidylserine on outer membranes, associated with increasing the concentration of intracellular Pb(2+). The potency of intracellular Pb(2+) to induce the apoptotic change in thymocyte membranes seems to be greater than those of intracellular Ca(2+) and Cd(2+). Results suggest that intracellular Pb(2+) triggers apoptosis of rat thymocytes. This action of Pb(2+) may be one of mechanisms for the lead-induced changes in immunity.