TNF-alpha-inducing protein, a carcinogenic factor secreted from H. pylori, enters gastric cancer cells

TNF-alpha inducing protein (Tip alpha) is secreted from Helicobacter pylori (H. pylori): it is a potent inducer of TNF-alpha and chemokine genes, mediated through NF-kappaB activation, and it also induces tumor-promoting activity in Bhas 42 cells. To investigate the carcinogenic mechanisms of H. pylori with Tip alpha, we first examined how Tip alpha acts on gastric epithelial cells. We found that fluorescent-Tip alpha specifically bound to, and then entered, the cells in a dose- and temperature-dependent manner, whereas deletion mutant of Tip alpha (del-Tip alpha), an inactive form, neither bound to nor entered the cells, suggesting the presence of a specific binding molecule. Mutagenesis analysis of Tip alpha revealed that a dimer formation of Tip alpha with a disulfide bond is required for both specific binding and induction of TNF-alpha gene expression. A confocal laser scanning microscope revealed some Tip alpha in the nuclei, but del-Tip alpha was not present, which indicated that an active form of Tip alpha can penetrate the nucleus and may be involved in the induction of TNF-alpha gene expression. Examination of Tip alpha production and secretion in 28 clinical isolates revealed that H. pylori obtained from gastric cancer patients secreted Tip alpha in significantly higher amounts than did H. pylori from patients with chronic gastritis, suggesting that Tip alpha is an essential factor in H. pylori inflammation and cancer microenvironment in the human stomach. Tip alpha is thus a new carcinogenic factor of H. pylori that can enter the nucleus through a specific binding molecule, and its mechanism of action is completely different from that of CagA.     

NF-kappaB activity blockade impairs the angiogenic potential of human pancreatic cancer cells

The effect of blockade of NF-kappaB activity on human pancreatic cancer angiogenesis was determined in an orthotopic xenograft model. Highly metastatic L3.3 human pancreatic cancer cells, which expressed an elevated level of constitutive NF-kappaB activity, were transfected with a mutated IkappaBalpha (IkappaBalphaM). After implantation in the pancreas of nude mice, parental (L3.3) and control vector-transfected (L3.3-Neo) cells produced rapidly growing tumors and liver metastases, whereas IkappaBalphaM-transfected (L3.3-IkappaBalphaM) cells had decreased tumorigenicity and metastatic potential. NF-kappaB signaling blockade significantly inhibited the in vitro and in vivo expression of the major proangiogenic molecules vascular endothelial growth factor and interleukin-8 and decreased tumor vascular formation. These events were correlated with retarded tumor growth and suppression of metastasis. Collectively, these data suggest that suppression of tumorigenicity and metastasis by NF-kappaB blockade is due to impaired angiogenic potential of tumor cells.     

Overexpression of PKCepsilon sensitizes LNCaP human prostate cancer cells to induction of apoptosis by bryostatin 1

Phorbol 12-myristate 13-acetate (PMA)-induced apoptosis of androgen sensitive LNCaP human prostate cancer cells is a well known phenomenon that involves prolonged translocation of multiple protein kinase C (PKC) isozymes to nonnuclear membranes. We have shown recently that PMA-induced death of C4-2 cells, androgen hypersensitive derivatives of LNCaP cells, requires both PKCdelta and a redundant pathway that includes PKCs alpha and epsilon. In contrast, it has been reported that overexpression of murine PKCepsilon in LNCaP cells renders those cells resistant to PMA-induced death, as well as androgen insensitive. Here we report that inducible or constitutive overexpression of human PKCepsilon does not alter the sensitivity of LNCaP cells to either PMA or androgen, nor does it alter expression of caveolin-1 or phosphorylated Rb, reported effects of overexpression of murine PKCepsilon. Moreover, overexpression of very high amounts of PKCepsilon sensitized LNCaP cells to induction of apoptosis by bryostatin 1, a non tumor-promoting activator and down-regulator of PKC isozymes that blocks PMA-induced apoptosis of parental LNCaP cells, mimicked our previous results with overexpression of PKCalpha in LNCaP cells. Given reports that overexpression of PKCepsilon is frequent in human prostate tumors, our results may have important implications for a potential prostate cancer therapy.     

Biological and chemical inhibitors of NF-kappaB sensitize SiHa cells to cisplatin-induced apoptosis

Cisplatin, a chemotherapeutic agent, is known to induce apoptosis of cancer cells. We examined the role of NF-kappaB during cisplatin-induced apoptosis in two human cervical cancer cell lines, HeLa and SiHa, known to differ in their response to cisplatin treatment. We found that SiHa cells were relatively more resistant than HeLa cells to the cytotoxic effects induced by cisplatin as measured by MTT assays. HeLa cells were more sensitive to the apoptotic effects induced by cisplatin as shown by increases in annexin staining, DNA fragmentation, and loss of mitochondrial membrane potential. Similarly the activities of caspases 3, 8, and 9 and cleavage of PARP induced by cisplatin were more in HeLa than SiHa cells. Cisplatin induced NF-kappaB DNA binding activity in HeLa and SiHa cells but not in primary cervical cells and the active DNA binding complex in SiHa cells consists of p50 and RelA heterodimers. However, when NF-kappaB DNA binding activity was blocked by chemical (curcumin, PDTC, or salicylic acid) or biological inhibitors (NIK-KM or IKK-beta DN), the cell viability was less in SiHa cells with cisplatin treatment, but these effects were not observed in HeLa cells. Similarly upon treatment with cisplatin SiHa cells had more activation of caspases compared to that seen in HeLa cells under conditions of NF-kappaB inhibition by biological or chemical inhibitors. These results suggest that NF-kappaB may contribute to the resistance of human cervical cancer cells to cisplatin and highlight the potential use of combination therapy involving cisplatin and NF-kappaB inhibitors.     

Independent activation of Akt and NF-kappaB pathways and their role in resistance to TNF-alpha mediated cytotoxicity in gliomas

Tumor associated macrophages (TAMs) constitute a substantial mass in gliomas. The activated macrophages secrete various cytokines that affect diverse functions of tumors. The aim of this study was to elucidate the role of Akt and NF-kappaB pathways in resistance to TNF-alpha mediated cell death in human gliomas using monolayers and multicellular spheroids (MCS) as in vitro models. Akt and NF-kappaB are constitutively expressed and intimately involved in progression of gliomas. The activation of these pathways also renders the tumors resistant to conventional treatments including chemotherapy. While PI3K/Akt is shown to regulate the NF-kappaB activation in diverse systems, other studies place NF-kappaB upstream of Akt activation. Using a stable IkappaBalpha mutant LN-18 cell line and pharmacological inhibitors to PI3K/Akt (LY294002) and Akt (Akt2), we provide evidence that Akt and NF-kappaB are activated independently on stimulation with TNF-alpha and both the pathways contribute towards resistance to TNF-alpha mediated cell death. TNF-alpha-induced NF-kappaB activation independent of PI3K/Akt pathway was also confirmed in human glioma cell lines-LN-229 and U373MG. We also show that NF-kappaB and Akt are activated during spheroidogenesis and their expression is further enhanced on stimulation with TNF-alpha implicating their involvement in resistance to cell death. The findings thus underscore the relevance of spheroids as appropriate in vitro models for studying the signaling pathways in drug induced resistance.     

A novel NF-kappaB inhibitor DHMEQ selectively targets constitutive NF-kappaB activity and induces apoptosis of multiple myeloma cells in vitro and in vivo

Multiple myeloma (MM) is a fatal lymphoid malignancy that is incurable with conventional modalities of chemotherapy. Strong and constitutive activation of nuclear factor kappa B (NF-kappaB) is a common characteristic of MM cells. In our study we successfully target NF-kappaB with a novel NF-kappaB inhibitor dehydroxymethylepoxyquinomycin (DHMEQ). DHMEQ completely abrogates constitutive NF-kappaB activity and induces apoptosis of MM cells, whereas control peripheral blood mononuclear cells (PBMC) are resistant to NF-kappaB inhibition and apoptosis by DHMEQ treatment. DHMEQ inhibition of NF-kappaB triggers activation of caspases 8 and 9, as well as G0/G1 cell cycle arrest accompanied by downregulation of antiapoptotic genes Bcl-XL and c-FLIP and cell cycle progression gene cyclins D1 and D2. DHMEQ-mediated inhibition of vascular endothelial growth factor (VEGF) production in MM cells raises the possibility that DHMEQ abrogates the autocrine VEGF loop and enhances its antitumor effects by inhibiting neovascularization in the bone marrow. Using an in vivo NOD/SCID/gammac(null) (NOG) mice model, we show that DHMEQ has a potent inhibitory effect on the growth of MM cells. Compared to other compounds having the potential to inhibit NF-kappaB, DHMEQ is a unique compound that blocks the translocation of NF-kappaB p65 into the nucleus and selectively targets NF-kappaB activated in tumor cells. Therefore, our study presents a new molecular target therapy in MM.     

Crossregulation of NF-kappaB by the APC/GSK-3beta/beta-catenin pathway

Glycogen synthase kinase-3beta (GSK-3beta) and adenomatous polyposis coli (APC) play an important role in the regulation of beta-catenin. Inhibition of or defects in their functions can lead to activation of beta-catenin. beta-catenin has been recently found to interact with and inhibit nuclear factor kappa B (NF-kappaB). However, the regulatory roles of GSK-3beta/APC on the NF-kappaB signaling pathway are unknown because of their diverse effects. In this study, we investigated whether GSK-3beta/APC might regulate NF-kappaB activity through beta-catenin. We found that inhibition of GSK-3beta suppressed NF-kappaB activity, whereas reexpression of APC restored NF-kappaB activity in APC mutated cells. The regulatory effects were through beta-catenin because depletion of beta-catenin with small interfering RNA (siRNA) in the same systems reversed the effects. The regulatory relationship was further supported by the analysis of primary breast tumor tissues in vivo in which NF-kappaB target TRAF1 was inversely correlated with activated beta-catenin. Thus, APC/GSK-3beta, through beta-catenin, may crossregulate NF-kappaB signaling pathway.     

Arsenic trioxide sensitizes CD95/Fas-induced apoptosis through ROS-mediated upregulation of CD95/Fas by NF-kappaB activation

CD95/Fas is a cell surface protein that belongs to the tumor necrosis factor receptor family. Signals through CD95/Fas are able to induce apoptosis in sensitive cells. Therefore, modalities to regulate the CD95/Fas expression level in tumor cells are called for. In the present study, we show that sublethal doses of arsenic trioxide (As2O3) sensitized CD95/Fas-induced apoptosis in human cervical cancer cells, and the sensitizing effects resulted from As2O3-mediated increase in the expression of the CD95/Fas. N-acetyl-L-cysteine, a specific scavenger of reactive oxygen species, abrogated As2O3-induced upregulation of CD95/Fas and enhancement of CD95/Fas-mediated apoptosis. Furthermore, inhibition of NF-kappaB by transient transfection of IkappaBalpha supersurppessor blocked the increase of CD95/Fas expression following As2O2 treatment. Antisense oligonucleotide of CD95/Fas and ZB4, an antibody that blocks the binding of CD95/Fas ligand to CD95/Fas, reduced the amount of As2O3-sensitized CD95/Fas-induced apoptosis, demonstrating the specificity of CD95/Fas-binding ligands in the As2O3-sensitized CD95/Fas-induced apoptosis. These findings demonstrate that sensitization of human cervical cancer cells to CD95/Fas-mediated apoptosis by As2O3 can be partly due to induction of ROS and subsequent upregulation of CD95/Fas gene expression by NF-kappaB activation.     

Inhibition of nuclear factor kappab activity by genistein is mediated via Notch-1 signaling pathway in pancreatic cancer cells

Pancreatic cancer remains the fourth most common cause of cancer related death in the United States. Therefore, novel strategies for the prevention and treatment are urgently needed. Genistein is a prominent isoflavonoid found in soy products and has been proposed to be responsible for lowering the rate of pancreatic cancer in Asians. However, the molecular mechanism(s) by which genistein elicits its effects on pancreatic cancer cells has not been fully elucidated. We have previously shown that genistein induces apoptosis and inhibits the activation of nuclear factor kappaB (NF-kappaB) pathway. Moreover, Notch signaling is known to play a critical role in maintaining the balance between cell proliferation, differentiation and apoptosis, and thereby may contribute to the development of pancreatic cancer. Hence, in our study, we investigated whether there is any cross talk between Notch and NF-kappaB during genistein-induced apoptosis in BxPC-3 pancreatic cancer cells. We used multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, Western blotting and EMSA for measuring DNA binding activity of NF-kappaB. We found that genistein inhibits cell growth and induces apoptotic processes in BxPC-3 pancreatic cancer cells. This was partly due to inhibition of Notch-1 activity. BxPC-3 cells transfected with Notch-1 cDNA showed induction of NF-kappaB activity, and this was inhibited by genistein treatment. From these results, we conclude that the inhibition of Notch-1 and NF-kappaB activity and their cross talk provides a novel mechanism by which genistein inhibits cell growth and induces apoptotic processes in pancreatic cancer cells.     

Resveratrol-induced apoptosis in MCF-7 human breast cancer cells involves a caspase-independent mechanism with downregulation of Bcl-2 and NF-kappaB

Resveratrol (RES), a chemopreventive molecule, inhibits the proliferation of tumor cells of different etiologies. We previously showed that RES alters the cell cycle and induces apoptosis in MCF-7 breast tumor cells by interfering with the estrogen receptor (ERaalpha)-dependent phosphoinositide 3-kinase (PI3K) pathway. Here, we analyzed signaling downstream of PI3K, to understand the mechanisms of RES-induced apoptosis. Apoptotic death by RES in MCF-7 was mediated by Bcl-2 downregulation since overexpression of this protein abolished apoptosis. Decreased Bcl-2 levels were not related to cytochrome c release, activation of caspases 3/8 or poly(ADP-ribose) polymerase proteolysis. However, RES decreased mitochondrial membrane potential and increased reactive oxygen species and nitric oxide production. NF-kappaB, a regulator of Bcl-2 expression, and calpain protease activity, a regulator of NF-kappaB, were both inhibited by RES. The patterns for NF-kappaB and calpain activities followed that of PI3K and were inhibited by LY294002. NF-kappaB inhibition coincided with diminished MMP-9 activity and cell migration. These data suggest that RES-induced apoptosis in MCF-7 could involve an oxidative, caspase-independent mechanism, whereby inhibition of PI3K signaling converges to Bcl-2 through NF-kappaB and calpain protease activity. Therefore, Bcl-2 and NF-kappaB could be considered potential targets for the chemopreventive activity of RES in estrogen-responsive tumor cells.     

Lysophosphatidic acid stimulates PC-3 prostate cancer cell Matrigel invasion through activation of RhoA and NF-kappaB activity

This study was performed to determine the relationship of lysophosphatidic acid (LPA) stimulation and increased Ras homolog A (RhoA) activity to nuclear factor kappa B (NF-kappaB) activity, and the role of these factors in regulating prostate cancer cell invasion. PC-3 high invasive cells demonstrated constitutively increased RhoA, NF-kappaB, and in vitro Matrigel invasion which were further induced by LPA stimulation or transfection with constitutively active RhoA Q63E mutant. LPA treatment rapidly and transiently induced RhoA activity followed by maximally increased DNA binding of NF-kappaB at 1 h and AP-1 at 4 h. The LPA-induced NF-kappaB DNA binding was preceded by transient IkappaBalpha phosphorylation, and decreased total IkappaBalpha levels. Further demonstrating the relationship between RhoA and NF-kappaB activation, PC-3 cells stably transfected with constitutively active RhoA Q63E demonstrated constitutively increased phospho-IkappaBalpha, while PC-3 cells transfected with dominant negative RhoA N19 exhibited decreased phospho-IkappaBalpha levels. The LPA-induced Matrigel invasion and NF-kappaB DNA binding activity were both inhibited by expression of the RhoA inhibitor C3 exoenzyme or dominant negative mutant NF-kappaB inhibitor IkappaBalpha S32/36A. Similarly, transfection with dominant negative IkappaBalpha S32/36A inhibited PC-3 RhoA Q63E cell in vitro invasion. Treatment of PC-3 high invasive and RhoA Q63E cells with sodium salicylate or lactacystin inhibited NF-kappaB and invasion, while pyrrolidine dithiocarbamate (PDTC) treatment of PC-3 high invasive cells inhibited NF-kappaB only. Each inhibitor blocked LPA-induced invasion while PDTC inhibited LPA-induced NF-kappaB and invasion to the greatest extent. These results point to a model where LPA stimulates RhoA and increased PC-3 prostate cancer cell invasion activity through an NF-kappaB-dependent pathway.