NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP)

NADPH oxidase/dual‐oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa‐B (NFκB)‐mediated inflammation and inflammation‐associated pathologies. We sought to examine, for the first time, the role of Nox/Duox and NFκB in rats treated with the cooked meat heterocyclic amine carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP). In the PhIP‐induced colon tumors obtained after 1 year, Nox1, Nox4, NFκB‐p50 and NFκB‐p65 were all highly overexpressed compared with their levels in adjacent normal‐looking colonic mucosa. Nox1 and Nox4 mRNA and protein levels also were markedly elevated in a panel of primary human colon cancers, compared with their matched controls. In HT29 human colon cancer cells, Nox1 knockdown induced G1 cell cycle arrest, whereas in Caco‐2 cells there was a strong apoptotic response, with increased levels of cleaved caspase‐3, ‐6, ‐7 and poly(ADP‐ribose)polymerase. Nox1 knockdown blocked lipopolysaccharide‐induced phosphorylation of IκB kinase, inhibited the nuclear translocation of NFκB (p50 and p65) proteins, and attenuated NFκB DNA binding activity. There was a corresponding reduction in the expression of downstream NFκB targets, such as MYC, CCND1 and IL1β. The results provide the first evidence for a role of Nox1, Nox4 and NFκB in PhIP‐induced colon carcinogenesis, including during the early stages before tumor onset. Collectively, the findings from this investigation and others suggest that further work is warranted on the role of Nox/Duox family members and NFκB in colon cancer development.     

Low molecular weight heparin suppresses receptor for advanced glycation end products‐mediated expression of malignant phenotype in human fibrosarcoma cells

The receptor for advanced glycation end products (RAGE) is a pattern‐recognition receptor and its engagement by ligands such as high mobility group box 1 (HMGB1) is implicated in tumor growth and metastasis. Low molecular weight heparin (LMWH) has an antagonistic effect on the RAGE axis and is also reported to exert an antitumor effect beyond the known activity of anticoagulation. However, the link between the anti‐RAGE and antitumor activities of LMWH has not yet to be fully elucidated. In this study, we investigated whether LMWH could inhibit tumor cell proliferation, invasion, and metastasis by blocking the RAGE axis using in vitro and in vivo assay systems. Stably transformed HT1080 human fibrosarcoma cell lines were obtained, including human full‐length RAGE‐overexpressing (HT1080^RAGE), RAGE dominant‐negative, intracellular tail‐deleted RAGE‐overexpressing (HT1080^dnRAGE), and mock‐transfected control (HT1080^mock) cells. Confocal microscopy showed the expression of HMGB1 and RAGE in HT1080 cells. The LMWH significantly inhibited HMGB1‐induced NFκB activation through RAGE using an NFκB‐dependent luciferase reporter assay and the HT1080 cell lines. Overexpression of RAGE significantly accelerated, but dnRAGE expression attenuated HT1080 cell proliferation and invasion in vitro, along with similar effects on local tumor mass growth and lung metastasis in vivo. Treatment with LMWH significantly inhibited the migration, invasion, tumor formation, and lung metastasis of HT1080^RAGE cells, but not of HT1080^mock or HT1080^dnRAGE cells. In conclusion, this study revealed that RAGE exacerbated the malignant phenotype of human fibrosarcoma cells, and that this exacerbation could be ameliorated by LMWH. It is suggested that LMWH has therapeutic potential in patients with certain types of malignant tumors.     

Sustained NF‐kappaB activity in chronic lymphocytic leukemia is independent of genetic and epigenetic alterations in the TNFAIP3 (A20) locus

Inappropriate nuclear factor (NF) κB activity is one major hallmark of B‐cell malignancies and chronic lymphocytic leukemia (CLL). NFκB‐dependent genes are involved in antiapoptosis, cell proliferation and metastasis and are responsible for survival and proliferation of tumors. However, the mechanisms of NFκB activity in CLL still need to be elucidated. Previously, we identified translocations in a region on chromosome 6q that encodes tumor necrosis factor alpha‐induced protein 3, which is a key player in negative feedback loop regulation of NFκB. Inactivation of this ubiquitin‐editing enzyme is involved in immunopathologies and in tumorigenesis. Frequent mutations in the A20 locus—leading to sustained NFκB activity—could be shown to play a dominant role in development of different B‐cell malignancies. To check if A20 is involved in upregulation of NFκB activity in CLL, we sequenced Exons 2–9 of the A20 gene in 55 CLL DNA samples. Furthermore, we determined the methylation status of the promoter region in 63 CLL DNA samples and compared to 10 control DNAs of B cells from healthy donors. Contrary to reports from other B‐cell malignancies, the A20 region showed neither mutations nor aberrant DNA methylation. Moreover, its expression could be confirmed by immunoblotting and showing comparable results to healthy B cells. These results indicate that malignant development in CLL differs from most of other B‐cell malignancies, which show frequent inactivation of A20.     

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     

miR‐203 inhibits cell proliferation,invasion, and migration of non‐small‐cell lung cancer by downregulating RGS17

Involvement of the RGS17 oncogene in the promotion of non‐small‐cell lung cancer (NSCLC) has been reported, but the regulation mechanism in NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand the role of miRNAs in Regulator of G Protein Signaling 17 (RGS17)‐induced NSCLC, we showed that miR‐203 was downregulated during tumorigenesis, and inhibited the proliferation and invasion of lung cancer cells. We then determined whether miR‐203 regulated NSCLC by targeting RGS17. To characterize the regulatory effect of miR‐203 on RGS17, we used lung cancer cell lines, A549 and Calu‐1, and the constructed miR‐203 and RGS17 overexpression vectors. The CCK8 kit was used to determine cell proliferation, and the Transwell® assay was used to measure cell invasion and migration. RT‐PCR, western blots, and immunofluorescence were used to analyze expression of miR‐203 and RGS17, and the luciferase reporter assay was used to examine the interaction between miR‐203 and RGS17. Nude mice were used to characterize in vivo tumor growth regulation. Expression of miR‐203 inhibited proliferation, invasion, and migration of lung cancer cell lines A549 and Calu‐1 by targeting RGS17. The regulatory effect of miR‐203 was inhibited after overexpression of RGS17. The luciferase reporter assay showed that miR‐203 downregulated RGS17 by direct integration into the 3′‐UTR of RGS17 mRNA. In vivo studies showed that expression of miR‐203 significantly inhibited growth of tumors. Taken together, the results suggested that expression of miR‐203 inhibited tumor growth and metastasis by targeting RGS17.     

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     

Hypoxia‐inducible factor‐1α and nuclear factor‐κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non‐small‐cell lung cancer cells

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD‐L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD‐L1 expression regulation in non‐small‐cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD‐L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho‐IκBα (p‐IκBα) and hypoxia‐inducible factor‐1α (HIF‐1α). Flow cytometry results showed stronger membrane co‐expression of EGFR and PD‐L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF‐1α indicated strong accordance among protein levels of PD‐L1, p‐IκBα, and HIF‐1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p‐IκBα, HIF‐1α, and PD‐L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p‐IκBα, HIF‐1α, and PD‐L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non‐small‐cell lung cancer tissues with either p‐IκBα or HIF‐1α positive staining were more likely to possess elevated PD‐L1 expression compared with tissues scored negative for both p‐IκBα and HIF‐1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF‐κB and HIF‐1α in PD‐L1 expression regulation by EGFR mutants in NSCLC.     

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.     

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.     

The side population,as a precursor of Hodgkin and Reed‐Sternberg cells and a target for nuclear factor‐κB inhibitors in Hodgkin’s lymphoma

Although disturbed cytokinesis of mononuclear Hodgkin (H) cells is thought to generate Reed‐Sternberg (RS) cells, differentiation of Hodgkin’s lymphoma (HL) cells is not fully understood. Recent studies indicate that cells found in a side population (SP) share characteristics of cancer stem cells. In this study we identified an SP in the HL cell lines, KMH2 and L428. This SP almost entirely consists of distinct small mononuclear cells, whereas the non‐SP is a mixture of relatively large cells with H or RS cell‐like morphology. Culture of the small mononuclear cells in the SP from KMH2 generated a non‐SP. Single cell culture of the SP cells generated large cells with H or RS cell‐like morphology. We found that CD30 overexpression and constitutive nuclear factor‐κB (NF‐κB) activity, both of which are characteristics of HL cells, are shared between the SP and non‐SP cells for both KMH2 and L428. Inhibition of NF‐κB induced apoptosis in both fractions, whereas the SP cells were resistant to a conventional chemotherapeutic agent doxorubicin. The results show that HL cell lines contain an SP, that is enriched for distinct small mononuclear cells and generates larger cells with H and RS cell‐like morphology. The results also stress the significance of NF‐κB inhibition for eradication of HL cells. (Cancer Sci 2010; 101: 2490–2496)     

MEKK3 expression correlates with nuclear factor κ B activity and with expression of antiapoptotic genes in serous ovarian carcinoma

BACKGROUND:

Constitutively activated nuclear factor κ B (NFκB) contributes to the development of cancer by regulating the expression of genes involved in cell survival, metastasis, and angiogenesis. The authors have demonstrated that MEKK3 plays a critical role in cytokine‐mediated NFκB activation, and that stable expression of MEKK3 in cultured cells leads to increased NFκB activity.

METHODS:

MEKK3 expression in ovarian cancer cells or tumors was assessed by Western blotting and real‐time polymerase chain reaction. NFκB activities were analyzed by electrophoretic mobility shift assay and luciferase reporter assays. Western blot analysis for the survival factors were also performed and correlated with MEKK3 and NFκB activities. Cell survival assays were used to determine the sensitivity of ovarian cancer cells to various chemotherapeutic agents.

RESULTS:

The authors found that 63% of the ovarian cancers had higher MEKK3 expression than the normal ovarian epithelial cells. Ovarian cancers with high MEKK3 showed correspondingly high IκB kinase and NFκB activity. Moreover, MEKK3 coimmunoprecipitated with Akt and cooperated with Akt to synergistically activate NFκB. Consistent with increased MEKK3 and NFκB activity in ovarian cancers, Bcl‐2, Bcl‐xL, survivin, and X‐linked inhibitor of apoptosis levels were increased, which correlated with increased resistance to chemotherapeutic agents. Knockdown of MEKK3 with small interfering RNA significantly increased cancer cell sensitivity to paclitaxel.

CONCLUSIONS:

MEKK3 may be aberrantly expressed in ovarian cancers and plays an important role in tumors with constitutively activated NFκB. Cancer 2009; 115:3897–3908. © 2009 American Cancer Society.     

BOK displays cell death‐independent tumor suppressor activity in non‐small‐cell lung carcinoma

As the genomic region containing the Bcl‐2‐related ovarian killer (BOK) locus is frequently deleted in certain human cancers, BOK is hypothesized to have a tumor suppressor function. In the present study, we analyzed primary non‐small‐cell lung carcinoma (NSCLC) tumors and matched lung tissues from 102 surgically treated patients. We show that BOK protein levels are significantly downregulated in NSCLC tumors as compared to lung tissues (p < 0.001). In particular, we found BOK downregulation in NSCLC tumors of grades two (p = 0.004, n = 35) and three (p = 0.031, n = 39) as well as in tumors with metastases to hilar (pN1) (p = 0.047, n = 31) and mediastinal/subcarinal lymph nodes (pN2) (p = 0.021, n = 18) as opposed to grade one tumors (p = 0.688, n = 7) and tumors without lymph node metastases (p = 0.112, n = 51). Importantly, in lymph node‐positive patients, BOK expression greater than the median value was associated with longer survival (p = 0.002, Mantel test). Using in vitro approaches, we provide evidence that BOK overexpression is inefficient in inducing apoptosis but that it inhibits TGFβ‐induced migration and epithelial‐to‐mesenchymal transition (EMT) in lung adenocarcinoma‐derived A549 cells. We have identified epigenetic mechanisms, in particular BOK promoter methylation, as an important means to silence BOK expression in NSCLC cells. Taken together, our data point toward a novel mechanism by which BOK acts as a tumor suppressor in NSCLC by inhibiting EMT. Consequently, the restoration of BOK levels in low‐BOK‐expressing tumors might favor the overall survival of NSCLC patients.     

Molecular mechanism underlying the antiproliferative effect of suppressor of cytokine signaling‐1 in non‐small‐cell lung cancer cells

Lung cancer (LC) is the major cause of death by cancer and the number of LC patients is increasing worldwide. This study investigated the therapeutic potential of gene delivery using suppressor of cytokine signaling 1 (SOCS‐1), an endogenous inhibitor of intracellular signaling pathways, for the treatment of LC. To examine the antitumor effect of SOCS‐1 overexpression on non‐small‐cell lung cancer (NSCLC) cells, NSCLC cells (A549, LU65, and PC9) were infected with adenovirus‐expressing SOCS‐1 vector. The cell proliferation assay showed that A549 and LU65, but not PC9, were sensitive to SOCS‐1 gene‐mediated suppression of cell growth. Although JAK inhibitor I could also inhibit proliferation of A549 and LU65 cells, SOCS‐1 gene delivery appeared to be more potent as SOCS‐1 could suppress focal adhesion kinase and epidermal growth factor receptor, as well as the JAK/STAT3 signaling pathway. Enhanced phosphorylation of the p53 protein was detected by means of phospho‐kinase array in SOCS‐1 overexpressed A549 cells compared with control cells, whereas no phosphorylation of p53 was observed when JAK inhibitor I was used. Furthermore, treatment with adenoviral vector AdSOCS‐1 in vivo significantly suppressed NSCLC proliferation in a xenograft model. These results suggest that the overexpression of SOCS‐1 gene is effective for antitumor therapy by suppressing the JAK/STAT, focal adhesion kinase, and epidermal growth factor receptor signaling pathways and enhancing p53‐mediated antitumor activity in NSCLC.     

Lymphotoxin‐β receptor activation by lymphotoxin‐α1β2 and LIGHT promotes tumor growth in an NFκB‐dependent manner

Lymphotoxin beta receptor (LTβR) activation on mouse fibrosarcoma cells (BFS‐1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro‐angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTβR, namely LTα₁β₂ and LIGHT, are involved in the activation of LTβR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTβR in these fibrosarcoma tumors, we used conditional LTβ‐deficient mice that specifically lack LTβ expression either on T cells (T‐LTβ^?/?) or on B cells (B‐LTβ^?/?). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild‐type mice, indicating the participation of both T and B host lymphocytes in the activation of LTβR in these tumors. Tumor growth was also reduced in LIGHT‐deficient mice, suggesting a contribution of this ligand to the activation of LTβR in BFS‐1 fibrosarcomas. LTβR signaling can involve IκBα and/or NFκB‐inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro‐angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro‐angiogenic signaling after LTβR activation. Our data support the idea that the ablation of LTβR signaling should be considered for cancer treatment.