Antimalarial drug mefloquine inhibits nuclear factor kappa B signaling and induces apoptosis in colorectal cancer cells

Nuclear factor kappa B (NF‐κB) signaling pathway is activated in many colorectal cancer (CRC) cells and in the tumor microenvironment, which plays a critical role in cancer initiation, development, and response to therapies. In the present study, we found that the widely used antimalarial drug mefloquine was a NF‐κB inhibitor that blocked the activation of IκBα kinase, leading to reduction of IκBα degradation, decrease of p65 phosphorylation, and suppressed expression of NF‐κB target genes in CRC cells. We also found that mefloquine induced growth arrest and apoptosis of CRC cells harboring phosphorylated p65 in culture and in mice. Furthermore, expression of constitutive active IKKβ kinase significantly attenuated the cytotoxic effect of the compound. These results showed that mefloquine could exert antitumor action through inhibiting the NF‐κB signaling pathway, and indicated that the antimalarial drug might be repurposed for anti‐CRC therapy in the clinic as a single agent or in combination with other anticancer drugs.     

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.     

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.     

3,3′‐diindolylmethane suppresses 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced inflammation and tumor promotion in mouse skin via the downregulation of inflammatory mediators

3,3′‐Diindolylmethane (DIM) is a major acid‐condensation product of indole‐3‐carbinol and is present in cruciferous vegetables. In this study, we evaluated the effects of DIM on antiinflammatory and antitumor promotion activity in mouse skin and explored the relevant mechanisms. When 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was applied topically to the mouse ear to induce inflammation, DIM pretreatment effectively inhibited TPA‐induced ear edema formation. To evaluate the mechanisms underlying DIM's antiinflammatory effects, DIM was topically treated to the shaved backs of mice 30 min before TPA treatment. DIM inhibited the TPA‐induced increases in the expression of cyclooxygenase (COX)‐2, inducible nitric oxide synthase (iNOS), chemokine (C‐X‐C motif) ligand (CXCL) 5, and interleukin (IL)‐6 in mouse skin. DIM also inhibited nuclear factor‐kappa B (NF‐κB)'s DNA binding activity, the nuclear translocation of p65, and the degradation of inhibitor of κB (IκB) α in TPA‐stimulated mouse skin. Furthermore, DIM reduced TPA‐induced increases in the activity of extracellular signal regulated protein kinase (ERK)‐1/2 and IκB kinase (IKK). When mouse skin papillomas were initiated via the topical application of 7,12‐dimethylbenz[α]anthracene (DMBA) and promoted with repeated topical applications of TPA, repeated topical applications of DIM prior to each TPA treatment significantly suppressed the incidence and multiplicity of the papillomas. DIM also reduced the expression of COX‐2 and iNOS, ERK phosphorylation, and the nuclear translocation of p65 in papillomas. Collectively, these results show that DIM exerts antiinflammatory and chemopreventive effects in mouse skin via the downregulation of COX‐2, iNOS, CXCL5, and IL‐6 expression, which may be mediated by reductions in NF‐κB activation. © 2010 Wiley‐Liss, Inc.     

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     

Effect of verbascoside on apoptosis and metastasis in human oral squamous cell carcinoma

Despite significant advances in therapy, the 5‐year survival rates for patients with advanced stage oral cancers still remains poor as an appropriate treatment has not been found yet, due to side effects of chemo/radiotherapy. Verbascoside (VB), a major bioactive constituent of the Tsoong herb, displays pharmacological properties by exhibiting anti‐oxidative, anti‐inflammatory and anti‐cancer activities. However, the underlining function and mechanism of VB in human oral squamous cell carcinoma (OSCC) remains unclear. In this study, we show that VB significantly decreased the viability and metastasis of HN4 and HN6 tumor cells, while promoting apoptosis. A xenograft OSCC mouse model further showed that intraperitoneal injection of VB strongly inhibited growth and lung metastasis of implanted tumor cells. Immunoblot analysis confirmed that VB effectively suppressed nuclear factor (NF)‐κB activation and downstream Bcl‐2/Bcl‐XL expression, resulting in increased OSCC cell apoptosis. In addition, VB suppressed mRNA and protein expression of matrix metalloproteinase‐9 via suppression of NF‐κB activation, thereby inhibiting tumor cell metastasis. Inspiringly, compared to cisplatin‐treated group, VB is a biocompatible agent without signficant side effects in vivo. Collectively, our results demonstrate that VB effectively inhibits OSCC tumor cell growth and metastasis via suppression of IκB kinase complex (IKK)/NF‐κB‐related signaling activation, suggesting that VB has potential use as a potent anticancer agent in OSCC therapeutic strategies.     

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     

C086, a novel analog of curcumin, induces growth inhibition and down-regulation of NFκB in colon cancer cells and xenograft tumors

New analogues of curcumin with improved properties are needed to meet therapeutic requirements. In this study, the effects of C086 on growth inhibition and NFκB pathway regulation were investigated in colon cancer cells and xenograft tumors. C086 exhibited potent antiproliferative activity in all 6 colon cancer cell lines. In a xenograft model of SW480 cells in nude mice, the oral administration of C086 showed significant growth suppression of SW480 tumors, and both Western blot and immunohistochemistry analyses showed decreased NFκB (p65) expression in tumor tissues. Using TNF-α to induce NFκB activation in SW480 cells, it was revealed that C086 inhibited IκBα phosphorylation and its subsequent degradation, and suppressed the nuclear translocation and DNA binding activity of NFκB. C-Myc, cyclin D1, and Bcl-2, NFκB-regulated gene products involving in cellular proliferation and antiapoptosis, were decreased in the C086 treated groups. This effect was accompanied by pro-apoptosis of C086 in colon cancer cells and lower expression of PCNA in C086 treated colon cancer xenografts. Immunostaining for CD31 showed that there were fewer microvessels in C086 treated SW480 tumors, and NFκB-targeted gene products involved in angiogenesis (i.e., vascular endothelial growth factor, matrix metalloproteinase-9) were also downregulated. C086 also inhibited bovine aortic endothelial cell (BAEC) proliferation and tube formation in Matrigel. Overall, our results suggest that C086 is a potent antitumor agent and has promising future in colon cancer. C086 suppressed NFκB activation through inhibition of IκBα phosphorylation. Downregulation of NFκB-regulated gene products contributed to the antiproliferation, pro-apoptosis, and antiangiogenesis effect of C086.     

DCLK1 promotes epithelial‐mesenchymal transition via the PI3K/Akt/NF‐κB pathway in colorectal cancer

Double cortin‐like kinase 1 (DCLK1) plays important roles during the epithelial‐mesenchymal transition (EMT) process in human colorectal cancer (CRC). However, the role of DCLK1 in regulating the EMT of CRC is still poorly understood. In this study, we report evidence that DCLK1 acts as a potent oncogene to drive its extremely malignant character of EMT in an NF‐κB‐dependent manner in CRC cells. Mechanistic investigations showed that DCLK1 induced the NF‐κBp65 subunit expression through the PI3K/Akt/Sp1 axis and activated NF‐κBp65 through the PI3K/Akt/IκBα pathway during the EMT of CRC cells. Moreover, we found that silencing the expression of DCLK1 inhibited the invasion and metastasis of CRC cells in vivo. Collectively, our findings identify DCLK1 as a pivotal regulator of an EMT axis in CRC, thus implicating DCLK1 as a potential therapeutic target for CRC metastasis.     

Up‐regulation of pro‐inflammatory genes as adaptation to hypoxia in MCF‐7 cells and in human mammary invasive carcinoma microenvironment

The role of tumor cells in synthesizing pro‐inflammatory molecules is still controversial. Here we report that hypoxic treatment of the MCF‐7 human mammary adenocarcinoma cell line induced activation of hypoxia‐inducible factor 1α (HIF‐1α) and nuclear factor‐kappa B (NF‐κB). Importantly, hypoxia regulated expression of alarmin receptors such as the receptor for advanced glycation end products (RAGE) and the purinoreceptor (P2X7R), and up‐regulated inflammatory response (IR) genes such as the inducible enzymes nitric oxide synthase (NOS2), cycloxygenase (COX2), and the acute‐phase protein pentraxin‐3 (PTX3). Hypoxia also stimulated chemokine (C‐X‐C motif) receptor 4 (CXCR4) mRNA synthesis. In fact, the CXCR4 ligand stromal‐derived factor‐1α (SDF‐1α) increased invasion and migration of hypoxic MCF‐7 cells. Inhibition of HIF‐1α by chetomin and NF‐κB by parthenolide reduced mRNA and protein expression of the studied molecules and prevented invasion of hypoxic MCF‐7 cells. Moreover, solid invasive mammary tumor microenvironment was analyzed after laser‐capture microdissection (LCMD) comparing tumor versus host normal tissue. Nuclear translocation of HIF‐1α and NF‐κB and up‐regulation of IR, CXCR4, estrogen receptor α (ERα), and epithelial growth factor receptor (EGFR) was observed in tumor but not in host normal tissue in the absence of a local inflammatory leukocyte infiltrate. We conclude that under hypoxic conditions MCF‐7 cells acquire a pro‐inflammatory phenotype, and that solid human mammary carcinoma evidenced a similar activation of HIF‐1α, NF‐κB, and IR genes in malignant tumor cells as compared to the normal host tissues. We suggest a role for IR activation in the malignant progression of transformed cells. (Cancer Sci 2010; 101: 1014–1023)     

Mammary tissue microenvironment determines T cell‐dependent breast cancer‐associated inflammation

Although the importance of the host tissue microenvironment in cancer progression and metastasis has been established, the spatiotemporal process establishing a cancer metastasis‐prone tissue microenvironment remains unknown. In this study, we aim to understand the immunological character of a metastasis‐prone microenvironment in a murine 4T1 breast tumor model, by using the activation of nuclear factor‐κb (NF‐κB) in cancer cells as a sensor of inflammatory status and by monitoring its activity by bioluminescence imaging. By using a 4T1 breast cancer cell line stably expressing an NF‐κB/Luc2 reporter gene (4T1 NF‐κB cells), we observed significantly increased bioluminescence approximately 7 days after metastasis‐prone orthotopic mammary fat‐pad inoculation but not ectopic s.c. inoculation of 4T1 NF‐κB cells. Such in vivo NF‐κB activation within the fat‐pad 4T1 tumor was diminished in immune‐deficient SCID or nude mice, or T cell‐depleted mice, suggesting the requirement of host T cell‐mediated immune responses. Given the fat‐pad 4T1 tumor expressed higher inflammatory mediators in a T cell‐dependent mechanism compared to the s.c. tumor, our results imply the importance of the surrounding tissue microenvironment for inflaming tumors by collaborating with T cells to instigate metastatic spread of 4T1 breast cancer cells.     

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.     

The Plasmodium Circumsporozoite Protein, a Novel NF-κB Inhibitor, Suppresses the Growth of SW480

The blocking of NF-κB activation is a promising strategy for the treatment of colorectal cancer. The circumsporozoite protein (CSP), a key component of the sporozoite stage of the malaria parasite, was recently reported to block NF-κB activation in hepatocytes. Thus, we investigated the effect of the CSP on the growth of the human colon cancer cell line, SW480. We demonstrated that transfection with a recombinant plasmid expressing CSP inhibited the proliferation of SW480 in a dose-dependent manner and induced the apoptosis of SW480. A NF-κB gene reporter assay showed that both the CSP and its nuclear localization signal (NLS) motif could equally suppress the activation of NF-κB following the stimulation with human recombinant TNF-α in the SW480. Furthermore, western blot analysis indicated that NLS did not affect the phosphorylation and degradation of IκB, but could sharply inhibit the nuclear translocation of NF-κB in TNF-α stimulated SW480. Hence, our data suggest that the CSP might be explored as a new NF-κB inhibitor for the treatment of colorectal cancer.     

Significance of NF‐κB activation in immortalization of nasopharyngeal epithelial cells

NF‐κB is a key regulator of inflammatory response and is frequently activated in human cancer including the undifferentiated nasopharyngeal carcinoma (NPC), which is common in Southern China including Hong Kong. Activation of NF‐κB is common in NPC and may contribute to NPC development. The role of NF‐κB activation in immortalization of nasopharyngeal epithelial (NPE) cells, which may represent an early event in NPC pathogenesis, is unknown. Examination of NF‐κB activation in immortalization of NPE cells is of particular interest as the site of NPC is often heavily infiltrated with inflammatory cellular components. We found that constitutive activation of NF‐κB signaling is a common phenotype in telomerase‐immortalized NPE cell lines. Our results suggest that NF‐κB activation promotes the growth of telomerase‐immortalized NPE cells, and suppression of NF‐κB activity inhibits their proliferation. Furthermore, we observed upregulation of c‐Myc, IL‐6 and Bmi‐1 in our immortalized NPE cells. Inhibition of NF‐κB downregulated expression of c‐Myc, IL‐6 and Bmi‐1, suggesting that they are downstream events of NF‐κB activation in immortalized NPE cells. We further delineated that EGFR/MEK/ERK/IKK/mTORC1 is the key upstream pathway of NF‐κB activation in immortalized NPE cells. Elucidation of events underlying immortalization of NPE cells may provide insights into early events in pathogenesis of NPC. The identification of NF‐κB activation and elucidation of its activation mechanism in immortalized NPE cells may reveal novel therapeutic targets for treatment and prevention of NPC.