Repression of NF‐κB and activation of AP‐1 enhance apoptosis in prostate cancer cells

TNFα and TRAIL, 2 members of the tumor necrosis factor family, share many common signaling pathways to induce apoptosis. Although many cancer cells are sensitive to these proapoptotic agents, some develop resistance. Recently, we have demonstrated that upregulation of c‐Fos/AP‐1 is necessary, but insufficient for cancer cells to undergo TRAIL‐induced apoptosis. Here we present a prostate cancer model with differential sensitivity to TNFα and TRAIL. We show that inhibition of NF‐κB or activation of AP‐1 can only partially sensitize resistant prostate cancer cells to proapoptotic effects of TNFα or TRAIL. Inhibition of NF‐κB by silencing TRAF2, by silencing RIP or by ectopic expression of IκB partially sensitized resistant prostate cancer. Similarly, activation of c‐Fos/AP‐1 only partially sensitized resistant cancer cells to proapoptotic effects of TNFα or TRAIL. However, concomitant repression of NF‐κB and activation of c‐Fos/AP‐1 significantly enhanced the proapoptotic effects of TNFα and TRAIL in resistant prostate cancer cells. Therefore, multiple molecular pathways may need to be modified, to overcome cancers that are resistant to proapoptotic therapies. © 2008 Wiley‐Liss, Inc.     

Functional roles of tumor necrosis factor‐related apoptosis‐inducing ligand–DR5 interaction in B16F10 cells by activating the nuclear factor‐κB pathway to induce metastatic potential

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) has been recognized as a promising target for cancer therapy because it can induce apoptotic cell death in tumor cells but not normal cells. Although TRAIL shows specific tumoricidal activity, resistance to TRAIL‐induced apoptosis in some tumor cells has been considered a clinical obstacle of its application. It has been shown that TRAIL provides inflammatory signals that may contribute to the TRAIL‐resistance of cancer cells; however, it is not known whether TRAIL itself is involved in malignant cancer cell behavior. In the present study, we examined the functional role of TRAIL in B16F10 mouse melanoma cells, which are totally insensitive to TRAIL‐induced apoptosis. By establishing B16F10 cells stably expressing the nuclear factor‐κB (NFκB)‐luciferase reporter gene, we found that TRAIL can activate NFκB through its death receptor DR5 in B16F10 cells. Furthermore, TRAIL–DR5 interaction not only promoted malignant behaviors of B16F10 cells, such as cell proliferation and MMP‐9 production, but also induced lung metastasis of B16F10 cells in vivo. These findings may imply a contrary role for the TRAIL–DR5 pathway in the inflammatory tumor microenvironment, in its ability to induce the metastatic potential of B16F10 melanoma cells instead of inducing apoptosis.     

Endoplasmic reticulum stress pathway PERK‐eIF2α confers radioresistance in oropharyngeal carcinoma by activating NF‐κB

Endoplasmic reticulum stress (ERS) plays an important role in the pathogenesis and development of malignant tumors, as well as in the regulation of radiochemoresistance and chemoresistance in many malignancies. ERS signaling pathway protein kinase RNA‐like endoplasmic reticulum kinase (PERK)‐eukaryotic initiation factor‐2 (eIF2α) may induce aberrant activation of nuclear factor‐κB (NF‐κB). Our previous study showed that NF‐κB conferred radioresistance in lymphoma cells. However, whether PERK‐eIF2α regulates radioresistance in oropharyngeal carcinoma through NF‐κB activation is unknown. Herein, we showed that PERK overexpression correlated with a poor prognosis for patients with oropharyngeal carcinoma (P < 0.01). Meanwhile, the percentage of the high expression level of PERK in oropharyngeal carcinoma patients resistant to radiation was higher than in patients sensitive to radiation (77.7 and 33.3%, respectively; P < 0.05). Silencing PERK and eIF2α increased the radiosensitivity in oropharyngeal carcinoma cells and increased radiation‐induced apoptosis and G2/M phase arrest. PERK‐eIF2α silencing also inhibited radiation‐induced NF‐κB phosphorylation and increased the DNA double strand break‐related proteins ATM phosphorylation. NF‐κB activator TNF‐α and the ATM inhibitor Ku55933 offset the regulatory effect of eIF2α on the expression of radiation‐induced cell apoptosis‐related proteins and the G2/M phase arrest‐related proteins. These data indicate that PERK regulates radioresistance in oropharyngeal carcinoma through NF‐kB activation‐mediated phosphorylation of eIF2α, enhancing X‐ray‐induced activation of DNA DSB repair, cell apoptosis inhibition and G2/M cell cycle arrest.     

TM‐233, a novel analog of 1′‐acetoxychavicol acetate,induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities

Although the introduction of bortezomib and immunomodulatory drugs has led to improved outcomes in patients with multiple myeloma, the disease remains incurable. In an effort to identify more potent and well‐tolerated agents for myeloma, we have previously reported that 1′‐acetoxychavicol acetate (ACA), a natural condiment from South‐East Asia, induces apoptotic cell death of myeloma cells in vitro and in vivo through inhibition of NF‐κB‐related functions. Searching for more potent NF‐κB inhibitors, we developed several ACA analogs based on quantitative structure–activity relationship analysis. TM‐233, one of these ACA analogs, inhibited cellular proliferation and induced cell death in various myeloma cell lines with a lower IC₅₀ than ACA. Treatment with TM‐233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti‐apoptotic Mcl‐1 protein, but not Bcl‐2 and Bcl‐xL proteins. In addition, TM‐233 rapidly decreased the nuclear expression of NF‐κB and also decreased the accumulation of cytosolic NF‐κB. We also examined the effects of TM‐233 on bortezomib‐resistant myeloma cells that we recently established, KMS‐11/BTZ and OPM‐2/BTZ. TM‐233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS‐11/BTZ and OPM‐2/BTZ cells. Interestingly, the combination of TM‐233 and bortezomib significantly induced cell death in these bortezomib‐resistant myeloma cells through inhibition of NF‐κB activity. These results indicate that TM‐233 could overcome bortezomib resistance in myeloma cells mediated through different mechanisms, possibly inhibiting the JAK/STAT pathway. In conclusion, TM‐233 might be a more potent NF‐κB inhibitor than ACA, and could overcome bortezomib resistance in myeloma cells.     

Enhancement of chemotherapeutic agent‐induced apoptosis by inhibition of NF‐κB using ursolic acid

NF‐κB activation is known to reduce the efficiency of chemotherapy in cancer treatment. Ursolic acid, a minimally toxic compound, has shown the capability to inhibit NF‐κB activation in living cells. Here, for the first time, we investigated the effects and mechanisms of NF‐κB inhibition by ursolic acid on chemotherapy treatment (Taxol or cisplatin) of cancer. ASTC‐a‐1 (human lung adenocarcinoma), Hela (human cervical cancer) cells, primary normal mouse cells of lung and liver and mouse in vivo model were used. Activity of signal factors (NF‐κB, Akt, Fas/FasL, BID, Bcl‐2, cytochrome c and caspase‐8, 3) was used to analyze the mechanisms of ursolic acid‐chemo treatment. Ursolic acid‐mediated suppression of NF‐κB drastically reduced the required dosage of the chemotherapeutic agents to achieve identical biological endpoints and enhanced the chemotherapeutic agent‐induced cancer cells apoptosis. Chemosensitization by ursolic acid in cancer cells was dependent on the amplified activation of intrinsic pathway (caspase‐8‐BID‐mitochondria‐cytochrome c‐caspase‐3) by augmentation of BID cleavage and activation of Fas/FasL‐caspase‐8 pathway. Prolonged treatment with relatively low doses of ursolic acid also sensitized cancer cells to the chemotherapeutic agents through suppression of NF‐κB. Chemosensitization by ursolic acid was observed only in cancer cells, but not in primary normal cells. The inhibitive effect of ursolic acid on NF‐κB was reversible, and the reversal was not accompanied by a loss in cells viability. By supplementing chemotherapy with minimally toxic ursolic acid, it is possible to improve the efficacy of cancer treatment by significantly reducing the necessary drug dose without sacrificing the treatment results.     

Receptor activator for nuclear factor‐κB ligand signaling promotes progesterone‐mediated estrogen‐induced mammary carcinogenesis

Breast cancer is a leading cause of cancer‐related death in women. Prolonged exposure to the ovarian hormones estrogen and progesterone increases the risk of breast cancer. Although estrogen is known as a primary factor in mammary carcinogenesis, very few studies have investigated the role of progesterone. Receptor activator for nuclear factor‐κB (NF‐κB) ligand (RANKL) plays an important role in progesterone‐induced mammary carcinogenesis. However, the molecular mechanism underlying RANKL‐induced mammary carcinogenesis remains unknown. In our current study, we show that RANKL induces glioma‐associated oncogene homolog 1 (GLI‐1) in estrogen‐induced progesterone‐mediated mammary carcinogenesis. In vivo experiments were carried out using ACI rats and in vitro experiments were carried out in MCF‐7 cells. In ACI rats, mifepristone significantly reduced the incidence of mammary tumors. Likewise, mifepristone also inhibited the proliferation of MCF‐7 cells. Hormone treatments induced RANKL, receptor activator of NF‐κB (RANK), and NF‐κB in a protein kinase B‐dependent manner and inhibited apoptosis by activation of anti‐apoptotic protein Bcl2 in mammary tumors and MCF‐7 cells. Mechanistic studies in MCF‐7 cells reveal that RANKL induced upstream stimulatory factor‐1 and NF‐κB, resulting in subsequent activation of their downstream target GLI‐1. We have identified that progesterone mediates estrogen‐induced mammary carcinogenesis through activation of GLI‐1 in a RANKL‐dependent manner.     

Association between activation of atypical NF‐κB1 p105 signaling pathway and nuclear β‐catenin accumulation in colorectal carcinoma

Recent studies have demonstrated that increased expression of coding region determinant‐binding protein (CRD‐BP) in response to β‐catenin signaling leads to the stabilization of β‐TrCP1, a substrate‐specific component of SCF E3 ubiquitin ligase complex, resulting in an accelerated degradation of IκBα and activation of canonical nuclear factor‐κB (NF‐κB) pathway. Here, we show that the noncanonical NF‐κB1 p105 pathway is constitutively activated in colorectal carcinoma specimens, being particularly associated with β‐catenin‐mediated increased expression of CRD‐BP and β‐TrCP1. In the carcinoma tissues exhibiting high levels of nuclear β‐catenin the phospho‐p105 levels were increased and total p105 amounts were decreased in comparison to that of normal tissue indicating an activation of this NF‐κB pathway. Knockdown of CRD‐BP in colorectal cancer cell line SW620 resulted in significantly higher basal levels of both NF‐κB inhibitory proteins, p105 and IκBα. Furthermore decreased NF‐κB binding activity was observed in CRD‐BP siRNA‐transfected SW620 cells as compared with those transfected with control siRNA. Altogether, our findings suggest that activation of NF‐κB1 p105 signaling in colorectal carcinoma might be attributed to β‐catenin‐mediated induction of CRD‐BP and β‐TrCP1. © 2009 Wiley‐Liss, Inc.     

Anti‐inflammatory effects of caffeic acid phenethyl ester (CAPE), a nuclear factor‐κB inhibitor,on Helicobacter pylori‐induced gastritis in Mongolian gerbils

Nuclear factor‐κB (NF‐κB) plays a major role in host inflammatory responses and carcinogenesis and as such is an important drug target for adjuvant therapy. In this study, we examined the effect of caffeic acid phenethyl ester (CAPE), an NF‐κB inhibitor, on Helicobacter pylori (H. pylori)‐induced NF‐κB activation in cell culture and chronic gastritis in Mongolian gerbils. In AGS gastric cancer cells, CAPE significantly inhibited H. pylori‐stimulated NF‐κB activation and mRNA expression of several inflammatory factors in a dose‐dependent manner, and prevented degradation of IκB‐α and phosphorylation of p65 subunit. To evaluate the effects of CAPE on H. pylori‐induced gastritis, specific pathogen‐free male, 6‐week‐old Mongolian gerbils were intragastrically inoculated with H. pylori, fed diets containing CAPE (0–0.1%) and sacrificed after 12 weeks. Infiltration of neutrophils and mononuclear cells and expression of NF‐κB p50 subunit and phospho‐IκB‐α were significantly suppressed by 0.1% CAPE treatment in the antrum of H. pylori‐infected gerbils. Labeling indices for 5′‐bromo‐2′‐deoxyuridine both in the antrum and corpus and lengths of isolated pyloric glands were also markedly reduced at the highest dose, suggesting a preventive effect of CAPE on epithelial proliferation. Furthermore, in the pyloric mucosa, mRNA expression of inflammatory mediators including tumor necrosis factor‐α, interferon‐γ, interleukin (IL)‐2, IL‐6, KC (IL‐8 homologue), and inducible nitric oxide synthase was significantly reduced. These results suggest that CAPE has inhibitory effects on H. pylori‐induced gastritis in Mongolian gerbils through the suppression of NF‐κB activation, and may thus have potential for prevention and therapy of H. pylori‐associated gastric disorders. © 2009 UICC     

Chromosome instability in diffuse large B cell lymphomas is suppressed by activation of the noncanonical NF‐κB pathway

Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in the United States. DLBCL comprises biologically distinct subtypes including germinal center‐like (GCB) and activated‐B‐cell‐like DLBCL (ABC). The most aggressive type, ABC‐DLBCL, displays dysregulation of both canonical and noncanonical NF‐κB pathway as well as genomic instability. Although, much is known about the tumorigenic roles of the canonical NF‐kB pathway, the precise role of the noncanonical NF‐kB pathway remains unknown. Here we show that activation of the noncanonical NF‐κB pathway regulates chromosome stability, DNA damage response and centrosome duplication in DLBCL. Analysis of 92 DLBCL samples revealed that activation of the noncanonical NF‐κB pathway is associated with low levels of DNA damage and centrosome amplification. Inhibiting the noncanonical pathway in lymphoma cells uncovered baseline DNA damage and prevented doxorubicin‐induced DNA damage repair. In addition, it triggered centrosome amplification and chromosome instability, indicated by anaphase bridges, multipolar spindles and chromosome missegregation. We determined that the noncanonical NF‐κB pathway execute these functions through the regulation of GADD45α and REDD1 in a p53‐independent manner, while it collaborates with p53 to regulate cyclin G2 expression. Furthermore, this pathway regulates GADD45α, REDD1 and cyclin G2 through direct binding of NF‐κB sites to their promoter region. Overall, these results indicate that the noncanonical NF‐κB pathway plays a central role in maintaining genome integrity in DLBCL. Our data suggests that inhibition of the noncanonical NF‐kB pathway should be considered as an important component in DLBCL therapeutic approach.     

Antitumor effects of tyropeptin‐boronic acid derivatives: New proteasome inhibitors

The proteasome degrades numerous regulatory proteins that are critical for tumor growth. Thus, proteasome inhibitors are promising antitumor agents. New proteasome inhibitors, such as tyropeptins and tyropeptin‐boronic acid derivatives, have a potent inhibitory activity. Here we report the antitumor effects of two new tyropeptin‐boronic acid derivatives, AS‐06 and AS‐29. AS‐06 and AS‐29 significantly suppress the degradation of the proteasome‐sensitive fluorescent proteins in HEK293PS cells, and induce the accumulation of ubiquitinated proteins in human multiple myeloma cells. We show that these derivatives also suppress the degradation of the NF‐κB inhibitor IκB‐α and the nuclear translocation of NF‐κB p65 in multiple myeloma cells, resulting in the inhibition of NF‐κB activation. Furthermore, we demonstrate that AS‐06 and AS‐29 induce apoptosis through the caspase‐8 and caspase‐9 cascades. In a xenograft mouse model, i.v. administration of tyropeptin‐boronic acid derivatives inhibits proteasome in tumors and clearly suppresses tumor growth in mice bearing human multiple myeloma. Our results indicate that tyropeptin‐boronic acid derivatives could be lead therapeutic agents against human multiple myeloma.     

Pak1 and Pak2 are activated in recurrent respiratory papillomas,contributing to one pathway of Rac1‐mediated COX‐2 expression

Recurrent respiratory papillomas are premalignant tumors of the airway caused by human papillomaviruses (HPVs), primarily Types 6 and 11. We had reported that respiratory papillomas overexpress the epidermal growth factor receptor (EGFR), the small GTPase Rac1 and cyclooxygenase‐2 (COX‐2), and have enhanced nuclear factor‐κB (NFκB) activation with decreased levels of IκB‐β but not IκB‐α. We also showed that EGFR‐activated Rac1 mediates expression of COX‐2 through activation of p38 mitogen‐activated protein kinase. We have now asked whether the p21‐activated kinases Pak1 or Pak2 mediate activation of p38 by Rac1 in papilloma cells. Pak1 and Pak2 were constitutively activated in vivo in papilloma tissue compared with normal epithelium, and Rac1 siRNA reduced the level of both phospho‐Pak1 and phospho‐Pak2 in cultured papilloma cells. Reduction in Pak1 and Pak2 with siRNA decreased the COX‐2 expression in papilloma cells, increased the levels of IκB‐β and reduced the nuclear localization of NF‐κB, but had no effect on p38 phosphorylation. Our studies suggest that Rac1 → Pak1/Pak2 → NFκB is a separate pathway that contributes to the expression of COX‐2 in HPV‐induced papillomas, independently of the previously described Rac1 → p38 → COX‐2 pathway.     

JNK/SAPK activity is not sufficient for anticancer therapy‐induced apoptosis involving CD95‐L,TRAIL and TNF‐α

We report here that stress stimuli such as γ‐irradiation or the anticancer drug doxorubicin activate expression of the death‐inducing ligands (DILs) CD95‐L, TNF‐α and TRAIL. Apoptosis induced by γ‐irradiation or doxorubicin engages a FADD‐ and caspase‐dependent apoptosis pathway which is inhibited by dominant negative FADD or the caspase inhibitor zVAD. zVAD did not prevent activity of JNK/SAPKs in response to doxorubicin suggesting that JNK/SAPK activity is independent of death receptor triggering during cellular stress‐induced apoptosis. In addition, JNK/SAPKs remained activated by doxorubicin in resistant cell lines in which cleavage of caspases and apoptosis was not observed. These data uncouple JNK/SAPK activation and apoptosis signaling and indicate that cellular stress‐induced apoptosis involves signaling via DILs which is paralleled by activation of JNK/SAPKs. Activation of these kinases may contribute e.g., to the expression of molecules involved in apoptosis but is not sufficient for induction of the apoptosis program following cellular stress. Int. J. Cancer 80:417–424, 1999. © 1999 Wiley‐Liss, Inc.     

α-TEA induces apoptosis of human breast cancer cells via activation of TRAIL/DR5 death receptor pathway

Vitamin E derivative RRR‐α‐tocopherol ether‐linked acetic acid analog (α‐TEA) induces apoptosis in MCF‐7 and HCC‐1954 human breast cancer cells in a dose‐ and time‐dependent manner. α‐TEA induces increased levels of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and death receptor‐5 (DR5) and decreased levels of antiapoptotic factor, cellular FLICE‐like inhibitory protein (c‐FLIP L). DR5/TRAIL induced apoptosis involves downregulation of c‐FLIP (L), caspase‐8 activation, activated proapoptotic mediators tBid and Bax, mitochondrial permeability transition, and activation of caspase‐9. siRNA knockdown of either DR5 or TRAIL blocks the ability of α‐TEA to enhance DR5 protein levels, downregulate c‐FLIP(L) protein levels and induce apoptosis. Combination of α‐TEA + TRAIL acts cooperatively to induce apoptosis, and increase DR5 and decrease c‐FLIP (L) protein levels. siRNA knockdown of c‐FLIP produces a low level of spontaneous apoptosis and enhances α‐TEA‐ and TRAIL‐induced apoptosis. Taken together, these studies show that α‐TEA induces TRAIL/DR5 mitochondria‐dependent apoptosis in human breast cancer cells, and that TRAIL/DR5‐dependent increases in DR5 and decreases in c‐FLIP expression are triggered by TRAIL or α‐TEA treatments. © 2010 Wiley‐Liss, Inc.