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Abstract: Upon antigenic stimulation, CD1d-restricted NKT cells quickly secrete large amounts of cytokines. This prompt response demonstrates that CD1d-restricted NKT cells may potentially prove to be useful therapeutic agents for the treatment of many diseases. Despite the clinical importance of CD1d-restricted NKT cells, the regulating mechanisms of this unique T cell population remain to be defined. We found murine LAG-3 is inducible on CD1d-restricted NKT cells as the result of a variety of stimulants such as concanavalin A (con A) and anti-CD3. Also, antigen-specific CD1d stimulation can elicit LAG-3 in CD1d-restricted NKT cells. Moreover, ectopic LAG-3 expression on CD1d-restricted NKT cells results in cell cycle arrest in the S phase. These results show that LAG-3 signaling on activated CD1d-restricted NKT cells may down-modulate NKT cell proliferation. [Copyright &y& Elsevier]

Arsenic trioxide (As2O3) inhibits cell growth and induces apoptosis in certain types of cancer cells including acute promyelocytic leukemia, prostate and ovarian carcinomas, but its effect on response of tumor cells to ionizing radiation has never been explored before. Here we demonstrate that As2O3 can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo. As2O3 in combination with ionizing radiation have a synergistic effect in decreasing clonogenic survival and in the regression of established human cervical tumor xenografts. Pretreatment of the cells with As2O3 also synergistically enhanced radiation-induced apoptosis. Apoptosis of the cells by combined treatment of As2O3 and radiation was associated with reactive oxygen species generation and loss of mitochondrial membrane potential, resulting in the activation of caspase-9 and caspase-3. The combined treatment also resulted in an increased G2/M cell cycle distribution at the concentration of As2O3 which did not alter cell cycle when applied alone. These results indicate that As2O3 can synergistically enhance radiosensitivity of human cervix carcinoma cells in vitro and in vivo, suggesting a potential clinical applicability of combination treatment of As2O3 and ionizing radiation in cancer therapies. [Copyright &y& Elsevier]

Otitis media (OM) is a group of inflammatory diseases of the middle ear (ME), regardless of cause or pathological mechanism. Among the molecular biological studies assessing the pathology of OM are investigations into the expression of C-type lectin receptors (CLR) in the ME and Eustachian tube (ET). To date, nine studies have evaluated CLR expression in the ME and ET. The expression of individual CLRs in mammalian ME and ET varies by species and model of OM. Assessments have shown that the patterns of CLR expression in the ME and ET vary; that CLR expression may vary by type of OM; and that the distribution and levels of expression of CLRs may depend on the presence or absence of inflammation, with variations even within the same species and same tissue. Infection of the ME and ET with various pathogens is a common cause of all types of OM, with host responses to pathogens mediated initially by the innate immune system. CLRs are important factors in the innate immune system because they act as both adhesion molecules and as pathogen recognition receptors. The expression of CLRs in OM tissues suggests that CLRs are associated with the pathogenesis of various types of OM. [ABSTRACT FROM AUTHOR]

Arsenic trioxide (As2O3) inhibits cell growth and induces apoptosis in certain types of cancer cells including acute promyelocytic leukemia, prostate and ovarian carcinomas, but its effect on response of tumor cells to ionizing radiation has never been explored before. Here we demonstrate that As2O3 can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo. As2O3 in combination with ionizing radiation have a synergistic effect in decreasing clonogenic survival and in the regression of established human cervical tumor xenografts. Pretreatment of the cells with As2O3 also synergistically enhanced radiation-induced apoptosis. Apoptosis of the cells by combined treatment of As2O3 and radiation was associated with reactive oxygen species generation and loss of mitochondrial membrane potential, resulting in the activation of caspase-9 and caspase-3. The combined treatment also resulted in an increased G2/M cell cycle distribution at the concentration of As2O3 which did not alter cell cycle when applied alone. These results indicate that As2O3 can synergistically enhance radiosensitivity of human cervix carcinoma cells in vitro and in vivo, suggesting a potential clinical applicability of combination treatment of As2O3 and ionizing radiation in cancer therapies. [Copyright &y& Elsevier]

Mutations in the superoxide dismutase 1 (SOD1) gene are linked to glutamate excitotoxicity in familial amyotrophic lateral sclerosis (fALS), but the underlying mechanism remains unclear. We investigated whether nuclear factor-kappaB (NF-kappaB) activation is involved in glutamate excitotoxicity by using motor neuron-neuroblastoma hybrid cells that expressed a mutant (G93A) SOD1 (mtSOD1) or wild-type SOD1 (wtSOD1). MtSOD1 cells were more vulnerable to glutamate excitotoxicity than wtSOD1 cells and showed higher NF-kappaB activity, higher nuclear cRel expression, and lower nuclear RelA expression under basal conditions. Glutamate treatment increased NF-kappaB activation along with nuclear expressions of RelA and cRel in wtSOD1 cells but induced only weak nuclear RelA expression in mtSOD1 cells. Suppression of NF-kappaB activation using transfection of the superrepressive mutant form of IkappaBalpha (mIkappaBalpha) inhibited nuclear RelA expression in both types of SOD1 cells, which increased glutamate excitotoxicity in wtSOD1 cells but not in mtSOD1 cells. Furthermore, immunohistochemistry confirmed stronger RelA immunoreactivity in the nuclei of motor neurons of spinal cord in wild-type SOD1 transgenic mice than in those in SOD1 G93A transgenic mice. In addition, we found that glutamate treatment decreased XIAP expression and increased caspase-3 activity in mtSOD1 cells and mIkappaBalpha-overexpressing wtSOD1 cells. Our results suggest that glutamate excitotoxicity in motor neurons of SOD1-linked fALS is attributable, at least in part, to the impairment of IkappaBalpha-dependent RelA activation and subsequent apoptosis mediated by XIAP inhibition and caspase-3 activation., ((c) 2010 Wiley-Liss, Inc.)

Sensitization of cancer cells to TRAIL could improve the effectiveness of TRAIL as an anticancer agent. We explored whether TRAIL in combination with phytosphingosine could sensitize cancer cells to TRAIL. The combined treatment enhanced synergistic apoptotic cell death of Jurkat T cells, compared to TRAIL or phytosphingosine alone. Enhanced apoptosis in response to the combination treatment was associated with caspase-8 activation-mediated Bax and Bak activation and mitochondrial dysfunction. The combination treatment also resulted in synergistic up-regulation of TRAIL receptor R1 (DR4) and R2 (DR5). siRNA targeting of DR5 significantly attenuated the combination treatment-induced caspase-8 activation, mitochondrial dysfunction, and apoptotic cell death. Upon stimulation of cells with the combination treatment, NF-kappaB was activated. Moreover, siRNA targeting of NF-kappaB significantly attenuated the combination treatment-induced DR4 and DR5 expression and receptor-mediated caspase-8 activation. These results indicate that phytosphingosine sensitizes cancer cells to TRAIL through the synergistic up-regulation of DR4 and DR5 in an NF-kappaB-dependent fashion resulting in caspase-8 activation and subsequent mitochondrial dysfunction. These findings support the potential application of combination treatment with TRAIL and phytosphingosine in the treatment of cancers that are less sensitive to TRAIL.

In this study, we show that dexamethasone treatment increases ionizing radiation-induced cell death by inducing the inhibitory kappaBalpha (IkappaBalpha) pathway in mice. The effect of dexamethasone on radiation-induced cell death was assessed by changes in total spleen cellularity and bone marrow colony-forming unit-granulocyte-macrophage (CFU-GM) contents after total body irradiation. While in vivo treatment of mice with dexamethasone alone (1 mg/kg/day, for 2 days) failed to elicit cell death in spleen cells, the combined treatment with dexamethasone (1 mg/kg/day, for 2 days) and gamma-rays (1 or 5 Gy) caused a 50-80% reduction in total cellularity in spleen and CFU-GM contents in bone marrow. These results demonstrate that dexamethasone has a synergistic effect on radiation-induced cellular damages in vivo. Immunoblot analysis showed that dexamethasone treatment significantly increases IkappaBalpha expression in the spleens of irradiated mice. In addition, the dexamethasone treatment significantly reduced radiation-induced nuclear translocation of the nucleus factor-kappaB in the spleens of irradiated mice. These results indicate that dexamethasone treatment in vivo may increase radiation-induced cell damages by increasing IkappaBalpha expression in hematopoietic organs such as spleen and bone marrow.

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro., ((c) 2005 Wiley-Liss, Inc.)

Purpose: Because the biological significance of constitutive nuclear factor-kappaB (NF-kappaB) activation in human gastric cancer is unclear, we undertook this study to clarify the regulatory mechanism of NF-kappaB activation and its clinical significance., Experimental Design: Immunohistochemistry for NF-kappaB/RelA was done on 290 human gastric carcinoma specimens placed on tissue array slides. The correlations between NF-kappaB activation and clinicopathologic features, prognosis, Akt activation, tumor suppressor gene expression, or Bcl-2 expression were analyzed. We also did luciferase reporter assay, Western blot analysis, and reverse transcription-PCR using the SNU-216 human gastric cancer cell line transduced with retroviral vectors containing constitutively active Akt or the NF-kappaB repressor mutant of IkappaBalpha., Results: Nuclear expression of RelA was found in 18% of the gastric carcinomas and was higher in early-stage pathologic tumor-node-metastasis (P = 0.019). A negative correlation was observed between NF-kappaB activation and lymphatic invasion (P = 0.034) and a positive correlation between NF-kappaB activation and overall survival rate of gastric cancer patients (P = 0.0228). In addition, NF-kappaB activation was positively correlated with pAkt (P = 0.047), p16 (P = 0.004), adenomatous polyposis coli (P < 0.001), Smad4 (P = 0.002), and kangai 1 (P < 0.001) expression. An in vitro study showed that NF-kappaB activity in gastric cancer cells is controlled by and controls Akt., Conclusions: NF-kappaB activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis and Akt activation. These findings suggest that NF-kappaB activation is a valuable prognostic variable in gastric carcinoma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in some, but not all cancer cells. To assess the regulation of TRAIL-resistance in the human gastric cancer cells, we examined TRAIL sensitivity, TRAIL receptor expression, and intracellular signaling events induced by TRAIL. All the gastric cancer cell lines tested were susceptible to TRAIL to some extent, except for SNU-216 cell line, which was completely resistant. TRAIL receptor expression was not related to the TRAIL-sensitivity. Of the cell lines tested, SNU-216 showed the highest level of constitutively active Akt and the short form of FLICE inhibitory protein (FLIP(S)). Treatment with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or with the protein synthesis inhibitor cycloheximide induced a suppression of constitutive Akt activation in SNU-216 cells and a concomitant decrease in the expression of FLIP(S). The reduction of Akt activity by LY294002 affected the transcriptional level of FLIP(S), but not the mRNA stability. As a result, LY294002 or cycloheximide significantly enhanced TRAIL-induced apoptosis. Moreover, the overexpression of constitutively active Akt in the TRAIL-sensitive cell line, SNU-668, rendered the cell line resistant to TRAIL. In addition, infection of the same cell line with retrovirus expressing FLIP(S) completely inhibited TRAIL-induced apoptosis by blocking the activation of caspase-8. Therefore, our results suggest that Akt activity promotes human gastric cancer cell survival against TRAIL-induced apoptosis via upregulation of FLIP(S), and that the cytotoxic effect of TRAIL can be enhanced by modulating the Akt/FLIP(S) pathway in human gastric cancers.

Host nonspecific beta cell injury by cytokines has been implicated in the process of early islet graft dysfunction. Islet transplant destruction by cytokines released from inflammatory cells that infiltrate the graft is thought to be mainly mediated by NF-kappaB-dependent nitric oxide (NO) production by the islet. In this study, we aimed to evaluate the role of IL-6 in making a beta cell resistant to cytokine-induced apoptosis by decreasing the NF-kappaB-dependent NO production and compared the result with that of the expression of a dominant negative inhibitor of NF-kappaB (DN NF-kappaB). Incubation of MIN6 cells with IL-1beta, IFN-gamma, and TNF-alpha (cytomix) increased production of nitrites, with increased expression of iNOS mRNA. When treated with cytomix, the DN NF-kappaB-transfected mutant demonstrated significantly less nitrite production and apoptosis than parent MIN6. NO production was effectively blocked by IL-6 as well as by N-monomethyl-l-arginine (l-NMMA). Inhibition of the NO production led to decreased rate of apoptosis accompanied by downregulation of the proapoptotic molecule Bax and increased expression of the antiapoptotic molecule Bcl-2 and Bcl-x(L). These data indicate that cytokine-induced cell death in the MIN6 beta cell line involves mechanisms that are, in part, NF-kappaB- and NO-dependent. Inhibition of the NO production by the incubation of the MIN6 cells by the pretreatment of IL-6 or l-NMMA is cytoprotective and can be used as a substitute for the expression of DN NF-kappaB.

Purpose: Sphingolipid metabolites, such as sphingosine and ceramide, are highly bioactive compounds and are involved in diverse cell processes, including cell-cell interaction, cell proliferation, differentiation, and apoptosis. However, the physiological roles of phytosphingosine are poorly understood. In this study, we report that phytosphingosine can potently induce apoptotic cell death in human cancer cells via caspase activation and caspase-independent cytochrome c release., Experimental Design: Phytosphingosine-induced apoptosis was determined by Hoechst 33258 staining, flow cytometric analysis, and DNA fragmentation assay. Involvement of caspases was determined by immunoblot analysis and cell death detection assays after treatment with synthetic inhibitor z-Val-Ala-Asp-fluoromethyl ketone, z-DEVD-fmk, or z-IETD-fmk. Death receptor (DR) dependency was analyzed by examining expression of DRs (Fas, DR4, DR5, TNFR1, and R2), and interaction of Fas-associated death domain and caspase 8. Involvement of the mitochondria pathway was examined by monitoring of the mitochondria membrane potential, cytochrome c release, and Bax translocation., Results: Phytosphingosine-treated cells displayed several features of apoptosis, including increase of sub-G(1) population, DNA fragmentation, and poly(ADP-ribose) polymerase cleavage. We observed that phytosphingosine cause activation of caspase 8 in a DR-independent fashion. Phytosphingosine also induced activation of caspase 9 and 3, loss of mitochondrial membrane potential, and the cytochrome c release from mitochondria. However, we failed to detect Bid cleavage. Moreover, caspase 8 inhibitor z-IETD-fmk did not affect phytosphingosine-induced cytochrome c release and caspase 9 activation, suggesting that phytosphingosine-induced cytochrome c release is caused by caspase 8-independent manner. Phytosphingosine induced mitochondrial translocation of Bax from the cytosol without changes in the protein levels of Bcl-2, Bcl-xL, and Bax. In addition, Bcl-2/Bax interaction was diminished after addition of phytosphingosine., Conclusion: These findings indicate that phytosphingosine induces apoptotic cell death in human cancer cells by direct activation of caspase 8, and by mitochondrial translocation of Bax and subsequent release of cytochrome c into cytoplasm, providing a potential mechanism for the anticancer activity of phytosphingosine.

We expressed the allogenic class II MHC antigen and B7.1 (CD80) co-stimulatory molecule in A20 beta-lymphoma cells in order to test their efficacy as immuno-stimulating adjuvant agents in inducing tumor-specific immunity. The transduction of the allogenic I-Ab alpha and beta chain genes into A20 cell resulted in a surface expression of the allogenic class II MHC molecules. The expression of the allogenic class II MHC antigen (I-Ab) in A20 cells enhanced the proliferation of T cells in a mixed lymphocyte tumor culture and in vitro cytotoxic T lymphocyte (CTL) generation against parental cells. The B7.1 gene, which is known to be a potent co-stimulatory molecule, was also transduced and expressed in A20 cells, either alone or in combination with I-Ab. The B7.1 transduction alone leads to a similar in vitro immune enhancing effect as I-Ab. When both the I-Ab and B7.1 genes were transduced, the in vitro immunostimulating capacity was further enhanced. Finally, we also tested the A20 cells that were transduced with I-Ab and/or B7.1 for their efficacy as preventive tumor vaccines in vivo. The results indicate that the A20 cells that express both the I-Ab and B7.1 have more potent vaccinating potential, compared to the cells that express only one of the molecules.