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.     

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.     

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.     

High expression levels of IKKα and IKKβ are necessary for the malignant properties of liver cancer

IKK‐NF‐κB signaling is regarded as an important factor in hepatocarcinogenesis and a potential target for liver cancer therapy. Therefore, in this study, we analyzed the expression of mRNAs encoding components and targets of NF‐κB signaling including IKKα, IKKβ, RANK, RANKL, OPG, CyclinD3, mammary serine protease inhibitor (Maspin), CyclinD1, c‐FLIP, Bcl‐xl, Stat3, Cip1 and Cip2 by real‐time PCR in 40 patients with liver cancer. After statistical analysis, 7 indices including IKKα, IKKβ, RANK, Maspin, c‐FLIP, Cip2 and cyclinD1 were found to show significant differences between tumor tissue and its corresponding adjacent tissue. When IKKα and IKKβ were downregulated in the hepatocellular carcinoma (HCC) cell lines of MHCC‐97L and MHCC‐97H in vitro, the numbers of BrdU positive cells were decreased in both IKKα and IKKβ knockdown cells. Levels of apoptosis were also investigated in IKKα and IKKβ knockdown cells. The growth of HCC was inhibited in the subcutaneous implantation model, and lung metastatogenesis was also significantly inhibited in the kidney capsule transplantation model. Downregulation of IKKα and IKKβ in HCC cultured in vitro revealed that increased Maspin, OPG and RANKL expression was associated with metastasis of HCC. These findings were associated with downregulation of Bcl‐XL and c‐FLIP, which may be the reason for increased apoptosis. The therapeutic effect of IKKα and IKKβ downregulation depends on extent of NF‐κB inhibition and the malignant nature of the HCC. We anticipate that IKK‐targeted gene therapy can be used in the treatment of HCC, a cancer that is notoriously resistant to radiation and chemotherapy.     

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.     

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.     

High IKKα expression is associated with reduced time to recurrence and cancer specific survival in oestrogen receptor (ER)‐positive breast cancer

The aim of our study was to examine the relationship between tumour IKKα expression and breast cancer recurrence and survival. Immunohistochemistry was employed in a discovery and a validation tissue microarray to assess the association of tumour IKKα expression and clinico‐pathological characteristics. After siRNA‐mediated silencing of IKKα, cell viability and apoptosis were assessed in MCF7 and MDA‐MB‐231 breast cancer cells. In both the discovery and validation cohorts, associations observed between IKKα and clinical outcome measures were potentiated in oestrogen receptor (ER) positive Luminal A tumours. In the discovery cohort, cytoplasmic IKKα was associated with disease‐free survival (p = 0.029) and recurrence‐free survival on tamoxifen (p < 0.001) in Luminal A tumours. Nuclear IKKα and a combination of cytoplasmic and nuclear IKKα (total tumour cell IKKα) were associated with cancer‐specific survival (p = 0.012 and p = 0.007, respectively) and recurrence‐free survival on tamoxifen (p = 0.013 and p < 0.001, respectively) in Luminal A tumours. In the validation cohort, cytoplasmic IKKα was associated with cancer‐specific survival (p = 0.023), disease‐free survival (p = 0.002) and recurrence‐free survival on tamoxifen (p = 0.009) in Luminal A tumours. Parallel experiment with breast cancer cells in vitro demonstrated the non‐canonical NF‐κB pathway was inducible by exposure to lymphotoxin in ER‐positive MCF7 cells and not in ER‐negative MDA‐MB‐231 cells. Reduction in IKKα expression by siRNA transfection increased levels of apoptosis and reduced cell viability in MCF7 but not in MDA‐MB‐231 cells. IKKα is an important determinant of poor outcome in patients with ER‐positive invasive ductal breast cancer and thus may represent a potential therapeutic target.     

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.     

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.     

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.     

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.     

Insights on distinct pathways of thiazolidinediones (PPARγ ligand)‐promoted apoptosis in TRAIL‐sensitive or ‐resistant malignant urothelial cells

Thiazolidinediones, including rosiglitazone and troglitazone, are insulin‐sensitizing drugs and high‐affinity ligands for the peroxisome proliferator‐activated receptor γ (PPARγ). Apart from their antidiabetic activity, these molecules possess antitumor properties. We investigated their potential apoptotic effects on RT4 (derived from a well‐differentiated Grade I papillary tumor) and T24 (derived from an undifferentiated Grade III carcinoma) bladder cancer cells. Rosiglitazone induced G2/M or G0/G1 phase cell cycle arrest in RT4 and T24 cells, respectively. Only troglitazone triggered apoptosis via extrinsic and intrinsic pathways in both cell lines. Interestingly, rosiglitazone amplified TRAIL‐induced apoptosis in TRAIL‐sensitive RT4 cells or let TRAIL‐resistant T24 cells to respond to TRAIL. Thiazolidinediones acted through PPARγ activation‐independent mechanisms. The underlying mechanisms involved for the first time in cancer cells the upregulation of soluble and/or membrane‐bound TRAIL. This was associated with increased cell surface death receptor 5 expression and c‐FLIP and survivin downregulation, mediated in part through proteasome‐dependent degradation in troglitazone‐promoted cell death. Therefore, the combination of rosiglitazone and TRAIL could be clinically relevant as chemopreventive or therapeutic agents for the treatment of TRAIL‐resistant high‐grade urothelial cancers.