Smac mimetic promotes TNF-α to induce apoptosis of gallbladder carcinoma cells

Gallbladder carcinoma has a high degree of malignancy. No effective treatment exists for patients with advanced tumors. The second mitochondria-derived activator of caspases (Smac) is the antagonist of the inhibitors of apoptosis protein. Smac mimetics are a class of effective tumor-targeted drugs undergoing clinical trials. However, studies on the effect of Smac mimetics on gallbladder cancer are unavailable. In this study, Smac mimetics can promote tumor necrosis factor-α (TNF-α) to inhibit the proliferation of gallbladder cancer cells and activate the apoptotic pathway, thereby promoting the ubiquitination of Lys48 on Receptor interacting protein kinase-1 (RIPK1) and leading to proteasomal degradation that causes damage to RIPK1 protein integrity. The formation of complex I (RIPK1, tumor necrosis factor 1-associated death domain protein, and TNF receptor-associated factor 2) is inhibited. Then, nonubiquitinated RIPK1 binds with the Fas-associated death domain and caspase-8 to form complex II and promotes the death receptor pathway of apoptosis. Animal experiments further verify that TNF-α combined with Smac mimetics can inhibit the growth of transplanted tumors and induce the apoptosis of transplanted tumor cells. This research provides a new direction for the targeted therapy of gallbladder cancer.     

Over-expressed estrogen receptor-α up-regulates hTNF-α gene expression and down-regulates β-catenin signaling activity to induce the apoptosis and inhibit proliferation of LoVo colon cancer cells

TAK-778 has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which TAK-778 exerts its effects is still unclear. There is evidence that TAK-778 acts via estrogen-receptor (ER)-mediated signaling; this study therefore aimed to investigate the roles that ERα, ERβ, and membrane ER play in the osteogenic effect of TAK-778. To this end, human bone marrow mesenchymal cells were cultured with TAK-778 in the presence of either ICI182,780 (ERα and ERβ antagonist) or MPP (ERα antagonist) or PD98059 (an extracellular-regulated kinase inhibitor that acts on the membrane ER pathway). The following parameters were evaluated: cell proliferation, collagen content, alkaline phosphatase (ALP) activity and bone-like formation. Data were compared using ANOVA. The effect of TAK-778 on expression of ERα and ERβ was investigated by immunolabeling. In order to investigate whether TAK-778 binds to ER, an ER binding assay was performed. Both immunolabeling and binding assays were conducted using cells from human alveolar bone. The osteogenic effect of TAK-778 was inhibited by ICI182,780 and MPP; however, it was not affected by PD98059. The expression of both ERα and ERβ was not affected by TAK-778. The competition curve obtained from the binding assay using TAK-778 showed maximal displacement when 10⁻⁵ M TAK-778 was used. This study's results show that TAK-778 enhances osteoblast differentiation through an ERα-dependent pathway by binding to this receptor and not by increasing the expression of ER. (Mol Cell Biochem xxx: 1–9, 2005)     

Phase I study of TNFα AutoVaccIne in Patients with metastatic cancer

We evaluated the safety and immunogencity of a novel vaccine directed against autologous TNF in a Phase I fixed dose escalation trial. The vaccine consisted of two recombinant TNF proteins, with specific peptides replaced by foreign immunodominant T cell epitopes from tetanus toxoid. The main objectives were to establish a safe dose and evaluate the vaccines ability to raise neutralising TNF antibodies. Secondary objectives were improvements in body weight and tumour response. Thirty-three patients were vaccinated with three doses (20, 100, or 400 g) of TNF vaccine at 2-weekly intervals adjuvanted with aluminium hydroxide. Anti-TNF antibody titres were measured by both a RIA, using soluble native TNF as the antigen, and by an ELISA using immobilized partly denatured TNF. Eleven patients (33%) had mild grade1/2 injection site reactions at the higher doses. In 10 of 20 patients, serum antibodies recognize denatured TNF in the ELISA, whereas, antibody titres against native TNF in the RIA were undetectable. This suggests that the production process had partly denatured the vaccine preventing the formation of cross-reacting antibodies to native TNF. In conclusion, TNF vaccine was able to elicit vaccine specific antibodies. However, since the antibodies were only able to cross-react with partly denatured TNF, evaluation of safety and tumour responses to the TNF vaccine was compromised.     

Autocrine IFN-γ promotes naive CD8 T cell differentiation and synergizes with IFN-α to stimulate strong function

Autocrine IFN-γ signaling is important for CD4 differentiation to Th1 effector cells, but it has been unclear whether it contributes to CD8 T cell differentiation. We show in this paper that naive murine CD8 T cells rapidly and transiently produce low levels of IFN-γ upon stimulation with Ag and B7-1, with production peaking at ～8 h and declining by 24 h. The autocrine IFN-γ signals for upregulation of expression of T-bet and granzyme B and induces weak cytolytic activity and effector IFN-γ production. IFN-α acts synergistically with IFN-γ to support development of strong effector functions, whereas IL-12 induces high T-bet expression and strong function in the absence of IFN-γ signaling. Thus, IFN-γ is not only an important CD8 T cell effector cytokine, it is an autocrine/paracrine factor whose contributions to differentiation vary depending on whether the response is supported by IL-12 or type I IFN.     

Dihydroartemisinin inhibits colon cancer cell viability by inducing apoptosis through up-regulation of PPARγ expression

The present study was aimed to investigate the effect of dihydroartemisinin on the colon cancer cell proliferation and apoptosis. The results from MTT assay revealed a concentration and time dependent relation between the inhibition of SW 948 cell viability and dihydroartemisinin addition. The viability of SW 948 cells was reduced to 45 and 24% on treatment with 30 and 50 µM, respectively concentrations of dihydroartemisinin after 48 h. Morphological examination of SW 948 cells showed attainment of rounded shape and cluster formation on treatment with dihydroartemisinin. Western blot analysis showed a significant increase in the activation of caspase-3 and expression of cleaved PARP by dihydroartemisinin treatment. The activation of PPARγ was increased significantly in SW 948 cells by treatment with dihydroartemisinin. Compared to control, the migration potential of SW 948 cells was reduced significantly (p < 0.005) and the expression levels of MMP-2 and -9 inhibited by dihydroartemisinin at 50 µM concentration. In the dihydroartemisinin treatment group colon tumor formation was significantly inhibited on treatment with 20 mg/kg doses of dihydroartemisinin after 30 days. Therefore, dihydroartemisinin inhibits colon cancer growth by inducing apoptosis and increasing the expression of PPARγ. Thus dihydroartemisinin can be used for the treatment of colon cancer.     

IFN-γ upregulates survivin and Ifi202 expression to induce survival and proliferation of tumor-specific T cells

Background

A common procedure in human cytotoxic T lymphocyte (CTL) adoptive transfer immunotherapy is to expand tumor-specific CTLs ex vivo using CD3 mAb prior to transfer. One of the major obstacles of CTL adoptive immunotherapy is a lack of CTL persistence in the tumor-bearing host after transfer. The aim of this study is to elucidate the molecular mechanisms underlying the effects of stimulation conditions on proliferation and survival of tumor-specific CTLs.

Methodology/Principal Findings

Tumor-specific CTLs were stimulated with either CD3 mAb or cognate Ag and analyzed for their proliferation and survival ex vivo and persistence in tumor-bearing mice. Although both Ag and CD3 mAb effectively induced the cytotoxic effecter molecules of the CTLs, we observed that Ag stimulation is essential for sustained CTL proliferation and survival. Further analysis revealed that Ag stimulation leads to greater proliferation rates and less apoptosis than CD3 mAb stimulation. Re-stimulation of the CD3 mAb-stimulated CTLs with Ag resulted in restored CTL proliferative potential, suggesting that CD3 mAb-induced loss of proliferative potential is reversible. Using DNA microarray technology, we identified that survivin and ifi202, two genes with known functions in T cell apoptosis and proliferation, are differentially induced between Ag- and CD3 mAb-stimulated CTLs. Analysis of the IFN-γ signaling pathway activation revealed that Ag stimulation resulted in rapid phosphorylation of STAT1 (pSTAT1), whereas CD3 mAb stimulation failed to activate STAT1. Chromatin immunoprecipitation revealed that pSTAT1 is associated with the promoters of both survivin and ifi202 in T cells and electrophoresis mobility shift assay indicated that pSTAT1 directly binds to the gamma activation sequence element in the survivin and ifi202 promoters. Finally, silencing ifi202 expression significantly decreased T cell proliferation.

Conclusions/Significance

Our findings delineate a new role of the IFN-γ signaling pathway in regulating T cell proliferation and apoptosis through upregulating survivin and ifi202 expression.     

Human eosinophils exert TNF-α and granzyme A-mediated tumoricidal activity toward colon carcinoma cells

Peripheral blood and tissue eosinophilia is a prominent feature in allergic diseases and helminth infections. In cancer patients, tumor-associated tissue eosinophilia is frequently observed. Tumor-associated tissue eosinophilia can be associated with a favorable prognosis, notably in colorectal carcinoma. However, underlying mechanisms of eosinophil contribution to antitumor responses are poorly understood. We have in this study investigated the direct interactions of human eosinophils with Colo-205, a colorectal carcinoma cell line, and show that eosinophils induce apoptosis and directly kill tumor cells. Using blocking Abs, we found that CD11a/CD18 complex is involved in the tumoricidal activity. Coculture of eosinophils with Colo-205 led to the release of eosinophil cationic protein and eosinophil-derived neurotoxin as well as TNF-α secretion. Moreover, eosinophils expressed granzyme A, which was released upon interaction with Colo-205, whereas cytotoxicity was partially inhibited by FUT-175, an inhibitor of trypsin-like enzymatic activity. Our data present the first demonstration, to our knowledge, that granzyme A is a cytotoxic mediator of the eosinophil protein arsenal, exerting eosinophil tumoricidal activity toward Colo-205, and provide mechanistic evidence for innate responses of eosinophil against tumor cells.     

Phosphorylation and stabilization of topoisomerase IIα protein by p38γ mitogen-activated protein kinase sensitize breast cancer cells to its poisons

Cancer drugs suppress tumor cell growth by inhibiting specific cellular targets. However, most drugs also activate several cellular nonspecific stress pathways, and the implications of these off-target effects are mostly unknown. Here, we report that p38γ, but not p38α, MAPK is specifically activated by treatment of breast cancer cells with topoisomerase II (Topo II) drugs, whereas paclitaxel (Taxol) does not have this effect. The activated p38γ in turn phosphorylates and stabilizes Topo IIα protein, and this enhances the growth inhibition by Topo II drugs. Moreover, p38γ activity was shown to be necessary and sufficient for Topo IIα expression, the drug-p38γ-Topo IIα axis is only detected in intrinsically sensitive but not resistant cells, and p38γ is co-overexpressed with Topo IIα protein in primary breast cancers. These results reveal a new paradigm in which p38γ actively regulates the drug-Topo IIα signal transduction, and this may be exploited to increase the therapeutic activity of Topo II drugs.     

Aberrant expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice

The Th2 locus control region (LCR) has been shown to be a crucial cis-acting element for Th2 cytokine expression and Th2 cell differentiation. To study the role of Th2 LCR in ifng locus regulation, we examined the expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice. We found IFN-γ to be aberrantly up-regulated. In addition, histone 3(H3)-acetylation and histone 3 lysine 4 (H3-K4)-methylation greatly increased at the ifng locus of the Th2 cells. GATA-3 and STAT6 bound to the ifng promoter in Th2 cells from the wild type but not from the Th2 LCR-deficient mice, and they directly repressed ifng expression in transient reporter assay. Moreover, ectopic expression of GATA-3 and STAT6-VT repressed the aberrant expression of the ifng gene and restored repressive chromatin state at the ifng locus in Th2 cells from Th2 LCR-deficient mice. These results suggest that expression of the ifng gene and chromatin remodeling of the ifng locus are under the control of a Th2 LCR-mediated Th2 differentiation program.     

Stage-specific expression of TNFα regulates bad/bid-mediated apoptosis and RIP1/ROS-mediated secondary necrosis in Birnavirus-infected fish cells

Infectious pancreatic necrosis virus (IPNV) can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. We found that IPNV infection can regulate Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic death pathways via the up-regulation of TNFα in zebrafish ZF4 cells. Using a DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage (0-6 h post-infection, or p.i.), we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then, during the early-middle replication stages (6-12 h p.i.), TNFα level was eight-fold induction and the pro-apoptotic Bcl-2 family members Bad and Bid were up-regulated. Furthermore, specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. Moreover, we used Rip1-specific inhibitors (Nec-1 and Rip1-specific siRNA) to block Rip1 expression. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection (12-18 h p.i.). Taken together, our results indicate that IPNV triggers two death pathways via up-stream induction of the pro-inflammatory cytokine TNFα, and these results may provide new insights into the pathogenesis of RNA viruses.     

1, 9-Pyrazoloanthrones downregulate HIF-1α and sensitize cancer cells to cetuximab-mediated anti-EGFR therapy

Cetuximab, a monoclonal antibody that blocks the epidermal growth factor receptor (EGFR), is currently approved for the treatment of several types of solid tumors. We previously showed that cetuximab can inhibit hypoxia-inducible factor-1 alpha (HIF-1α) protein synthesis by inhibiting the activation of EGFR downstream signaling pathways including Erk, Akt, and mTOR. 1, 9-pyrazoloanthrone (1, 9 PA) is an anthrapyrazolone compound best known as SP600125 that specifically inhibits c-jun N-terminal kinase (JNK). Here, we report 1, 9 PA can downregulate HIF-1α independently of its inhibition of JNK. This downregulatory effect was abolished when the oxygen-dependent domain (ODD) of HIF-1α (HIF-1α-ΔODD, the domain responsible for HIF-1α degradation) was experimentally deleted or when the activity of HIF-1α prolyl hydroxylase (PHD) or the 26S proteasomal complex was inhibited, indicating that the 1, 9 PA downregulates HIF-1α by promoting PHD-dependent HIF-1α degradation. We found that the combination of 1, 9 PA and cetuximab worked synergistically to induce apoptosis in cancer cells in which cetuximab or 1, 9 PA alone had no or only weak apoptotic activity. This synergistic effect was substantially decreased in cancer cells transfected with HIF-1α-ΔODD, indicating that downregulation of HIF-1α was the mechanism of this synergistic effect. More importantly, 1, 9 PA can downregulate HIF-1α in cancer cells that are insensitive to cetuximab-induced inhibition of HIF-1α expression due to overexpression of oncogenic Ras (RasG12V). Our findings suggest that 1, 9 PA is a lead compound of a novel class of drugs that may be used to enhance the response of cancer cells to cetuximab through a complementary effect on the downregulation of HIF-1α.     

Induction of apoptosis in human colon carcinoma COLO 205 cells by the recombinant α subunit of C-phycocyanin

The α-subunit of C-phycocyanin (CpcA) was expressed in Escherichia coli and purified. The recombinant CpcA inhibited the growth of human colon carcinoma COLO 205 cells. Typical apoptotic morphological characteristics, such as chromatin condensation and nuclear fragmentation, were observed in CpcA-treated COLO 205 cells by fluorescence microscopy and transmission electron microscopy. Moreover, the apoptotic process was associated with the Bax/Bcl-2 ratio up-regulation, mitochondrial membrane depolarization, cytochrome c release, and caspase-9 activation. These findings indicate that CpcA induced the death of COLO 205 cells through the intrinsic apoptotic pathway.     

TNFα and SOCS3 regulate IRS-1 to increase retinal endothelial cell apoptosis

Rates of diabetes are reaching epidemic levels. The key problem in both type 1 and type 2 diabetes is dysfunctional insulin signaling, either due to lack of production or due to impaired insulin sensitivity. A key feature of diabetic retinopathy in animal models is degenerate capillary formation. The goal of this present study was to investigate a potential mechanism for retinal endothelial cell apoptosis in response to hyperglycemia. The hypothesis was that hyperglycemia-induced TNFα leads to retinal endothelial cell apoptosis through inhibition of insulin signaling. To test the hypothesis, primary human retinal endothelial cells were grown in normal glucose (5 mM) or high glucose (25 mM) and treated with exogenous TNFα, TNFα siRNA or suppressor of cytokine signaling 3 (SOCS3) siRNA. Cell lysates were processed for Western blotting and ELISA analyses to verify TNFα and SOCS3 knockdown, as well as key pro- and anti-apoptotic factors, IRS-1, and Akt. Data indicate that high glucose culturing conditions significantly increase TNFα and SOCS3 protein levels. Knockdown of TNFα and SOCS3 significantly increases anti-apoptotic proteins, while decreasing pro-apoptotic proteins. Knockdown of TNFα leads to decreased phosphorylation of IRS-1(Ser307), which would promote normal insulin signaling. Knockdown of SOCS3 increased total IRS-1 levels, as well as decreased IR(Tyr960), both of which would inhibit retinal endothelial cell apoptosis through increased insulin signaling. Taken together, our findings suggest that increased TNFα inhibits insulin signaling in 2 ways: 1) increased phosphorylation of IRS-1(Ser307), 2) increased SOCS3 levels to decrease total IRS-1 and increase IR(Tyr960), both of which block normal insulin signal transduction. Resolution of the hyperglycemia-induced TNFα levels in retinal endothelial cells may prevent apoptosis through disinhibition of insulin receptor signaling.     

TGF-β1 influence on TNF-α production and sTNF-Rs shedding in a coculture of colon carcinoma cell spheroids with normal cells

In this study, the levels of TNF-α and its soluble receptors sTNF-Rp55 and sTNF-Rp75 were analyzed in cocultures of human colon carcinoma cell spheroids prepared from different grades of tumors with normal human colon epithelium, myofibroblast, and endothelial cell monolayers. Additionally, the influence of exogenously added rhTGF-β1 (2 ng/ml) on the TNF-α and sTNF-Rs levels was tested. Direct interactions of colon carcinoma spheroids with normal cells caused decreases in TNF-α levels and normal cell-dependent changes in sTNF-Rs amounts as compared to normal cells cultured alone. The addition of rhTGF-β1 to the cocultures caused a significant increase in TNF-α levels with a simultaneous decrease in the amounts of both sTNF-Rs. During direct interactions of colon carcinoma cells with normal tissue, paracrine effects are very important. We showed that TGF-β1 acts synergistically with TNF-α and significantly limits sTNF-Rs shedding. Therefore, TNF-Rs bound to cellular membranes, but not their soluble forms, play an important role in tumor/normal cell interactions. TGF-β1 and sTNF-Rs, in turn, may be valuable factors in colon cancer development and metastasis.