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.     

Xanthorrhizol inhibits 12-O-tetradecanoylphorbol-13-acetate-induced acute inflammation and two-stage mouse skin carcinogenesis by blocking the expression of ornithine decarboxylase, cyclooxygenase-2 and inducible nitric oxide synthase through mitogen-activated protein kinases and/or the nuclear factor-kappa B

Xanthorrhizol is an active component isolated from Curcuma xanthorrhiza Roxb. (Zingiberaceae) that is traditionally used in Indonesia for medicinal purposes. In the present study, we found that the topical application of xanthorrhizol before 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment significantly inhibits TPA-induced mouse ear edema and TPA-induced tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated ICR mouse skin. The topical application of xanthorrhizol following the induction of papillomas with TPA-induced hyperplasia and dysplasia also reduced tumor multiplicity and incidence in DMBA-initiated mouse skin. To further elucidate the molecular mechanisms underlying the antitumor-promoting activity of xanthorrhizol, its effect on the TPA-induced expression of ornithine decarboxylase (ODC), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) and the upstream signaling molecules controlling these proteins were explored in mouse skin. The pre-treatment with xanthorrhizol inhibited the expression of ODC, iNOS and COX-2 proteins and nuclear factor-kappaB (NF-kappaB) activation in both mouse skin with TPA-induced acute inflammation and DMBA-initiated mouse skin promoted by TPA for 19 weeks. When mouse skin was treated after TPA-induced production of papillomas, xanthorrhizol remarkably suppressed the expression of ODC, iNOS and COX-2 and inhibited the activation of NF-kappaB. Furthermore, western blot analysis showed that xanthorrhizol suppressed the activation of extracellular signal-regulated protein kinase, p38, c-Jun-N-terminal kinase and Akt in mice after topical application for 6 weeks following the induction of papillomas. Taken together, the present study demonstrates that xanthorrhizol not only delays or inhibits tumor formation, but also reverses the carcinogenic process at pre-malignant stages by reducing the protein levels of ODC, iNOS and COX-2 regulated by the NF-kappaB, mitogen-activated protein kinases and/or Akt.     

Timosaponin AIII inhibits melanoma cell migration by suppressing COX‐2 and in vivo tumor metastasis

Melanoma is the leading cause of death from skin disease, due in large part to its propensity to metastasize. We examined the effects of timosaponin AIII, a compound isolated from Anemarrhena asphodeloides Bunge, on melanoma cancer cell migration and the molecular mechanisms underlying these effects using B16‐F10 and WM‐115 melanoma cells lines. Overexpression of COX‐2, its metabolite prostaglandin E₂ (PGE₂), and PGE₂ receptors (EP2 and EP4) promoted cell migration in vitro. Exposure to timosaponin AIII resulted in concentration‐dependent inhibition of cell migration, which was associated with reduced levels of COX‐2, PGE₂, and PGE₂ receptors. Transient transfection of COX‐2 siRNA also inhibited cell migration. Exposure to 12‐O‐tetradecanoylphorbal‐13‐acetate enhanced cell migration, whereas timosaponin AIII inhibited 12‐O‐tetradecanoylphorbal‐13‐acetate‐induced cell migration and reduced basal levels of EP2 and EP4. Moreover, timosaponin AIII inhibited activation of nuclear factor‐kappa B (NF‐κB), an upstream regulator of COX‐2 in B16‐F10 cells. Consistent with our in vitro findings, in vivo studies showed that timosaponin AIII treatment significantly reduced the total number of metastatic nodules in the mouse lung and improved histological alterations in B16‐F10‐injected C57BL/6 mice. In addition, C57BL/6 mice treated with timosaponin AIII showed reduced expression of COX‐2 and NF‐κB in the lung. Together, these results indicate that timosaponin AIII has the capacity to inhibit melanoma cell migration, an essential step in the process of metastasis, by inhibiting expression of COX‐2, NF‐κB, PGE_2, and PGE₂ receptors.     

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.     

Antitumor effect of nuclear factor‐κB decoy transfer by mannose‐modified bubble lipoplex into macrophages in mouse malignant ascites

Patients with malignant ascites (MAs) display several symptoms, such as dyspnea, nausea, pain, and abdominal tenderness, resulting in a significant reduction in their quality of life. Tumor‐associated macrophages (TAMs) play a crucial role in MA progression. Because TAMs have a tumor‐promoting M2 phenotype, conversion of the M2 phenotypic function of TAMs would be promising for MA treatment. Nuclear factor‐κB (NF‐κB) is a master regulator of macrophage polarization. Here, we developed targeted transfer of a NF‐κB decoy into TAMs by ultrasound (US)‐responsive, mannose‐modified liposome/NF‐κB decoy complexes (Man‐PEG bubble lipoplexes) in a mouse peritoneal dissemination model of Ehrlich ascites carcinoma. In addition, we investigated the effects of NF‐κB decoy transfection into TAMs on MA progression and mouse survival rates. Intraperitoneal injection of Man‐PEG bubble lipoplexes and US exposure transferred the NF‐κB decoy into TAMs effectively. When the NF‐κB decoy was delivered into TAMs by this method in the mouse peritoneal dissemination model, mRNA expression of the Th2 cytokine interleukin (IL)‐10 in TAMs was decreased significantly. In contrast, mRNA levels of Th1 cytokines (IL‐12, tumor necrosis factor‐α, and IL‐6) were increased significantly. Moreover, the expression level of vascular endothelial growth factor in ascites was suppressed significantly, and peritoneal angiogenesis showed a reduction. Furthermore, NF‐κB decoy transfer into TAMs significantly decreased the ascitic volume and number of Ehrlich ascites carcinoma cells in ascites, and prolonged mouse survival. In conclusion, we transferred a NF‐κB decoy efficiently by Man‐PEG bubble lipoplexes with US exposure into TAMs, which may be a novel approach for MA treatment.     

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.     

Nitric oxide induces expression of cyclooxygenase-2 in mouse skin through activation of NF-kappaB

Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are frequently overexpressed in tumor tissues or transformed cells. In the present work, we assessed the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of iNOS and COX-2 in mouse skin. Topical application to the dorsal skin of female ICR mice of 10 nmol TPA led to maximal induction of iNOS and COX-2 protein expression at approximately 2 and 4 h, respectively. When applied topically onto shaven backs of mice 30 min prior to TPA, the NOS inhibitor aminoguanidine (AG) inhibited the expression of COX-2 protein at the pharmacologically effective dose. Pretreatment with a more specific iNOS inhibitor, N(G)-nitro-l-arginine-methyl ester, also suppressed TPA-induced COX-2 expression. Immunohistochemical analysis of TPA-treated mouse skin using an anti-nitrotyrosine antibody reveals enhanced levels of nitrotyrosine protein localized in epidermal and dermal layers. Topical application of NO donors, such as sodium nitroprusside (SNP) and S-nitroso-N-acetyl-d,l-penicillamine, induced expression of COX-2 in mouse skin, which was attenuated by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide. SNP treatment stimulated NF-kappaB activation in mouse skin, which was associated with the degradation of IkappaBalpha. Topical application of inhibitors of NF-kappaB, such as pyrrolidine dithiocarbamate or N-alpha-p-tosyl-l-lysine chloromethylketone, inhibited the SNP-induced COX-2 expression. SNP induced a weak but concentration-related increase in COX-2 expression in cultured mouse keratinocytes, which was abolished by treatment with SN50, a specific inhibitor of nuclear translocation of NF-kappaB. Mouse keratinocytes treated with SNP exhibited an elevated NF-kappaB-driven COX-2 promoter activity. Topical application of AG (10 micro mol) prior to each TPA treatment after initiation reduced the multiplicity of papillomas by 44% at 22 weeks. In conclusion, up-regulation of COX-2 by NO may be mediated by activation of NF-kappaB in mouse skin, which provides a molecular mechanism by which COX-2 is induced during tumor promotion.     

Curcumin sensitizes human colorectal cancer to capecitabine by modulation of cyclin D1, COX‐2, MMP‐9, VEGF and CXCR4 expression in an orthotopic mouse model

Because of the poor prognosis and the development of resistance against chemotherapeutic drugs, the current treatment for advanced metastatic colorectal cancer (CRC) is ineffective. Whether curcumin (a component of turmeric) can potentiate the effect of capecitabine against growth and metastasis of CRC was investigated. The effect of curcumin on proliferation of CRC cell lines was examined by mitochondrial dye‐uptake assay, apoptosis by esterase staining, nuclear factor‐kappaB (NF‐κB) by electrophoretic mobility shift assay and gene expression by Western blot analysis. The effect of curcumin on the growth and metastasis of CRC was also examined in orthotopically implanted tumors in nude mice. In vitro, curcumin inhibited the proliferation of human CRC cell lines, potentiated capecitabine‐induced apoptosis, inhibited NF‐κB activation and suppressed NF‐κB‐regulated gene products. In nude mice, the combination of curcumin and capecitabine was found to be more effective than either agent alone in reducing tumor volume (p = 0.001 vs. control; p = 0.031 vs. capecitabine alone), Ki‐67 proliferation index (p = 0.001 vs. control) and microvessel density marker CD31. The combination treatment was also highly effective in suppressing ascites and distant metastasis to the liver, intestines, lungs, rectum and spleen. This effect was accompanied by suppressed expression of activated NF‐κB and NF‐κB‐regulated gene products (cyclin D1,c‐myc, bcl‐2, bcl‐xL, cIAP‐1, COX‐2, ICAM‐1, MMP‐9, CXCR4 and VEGF). Overall, our results suggest that curcumin sensitizes CRC to the antitumor and antimetastatic effects of capecitabine by suppressing NF‐κB cell signaling pathway. © 2009 UICC     

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.