Overexpression of Par‐4 sensitizes TRAIL‐induced apoptosis via inactivation of NF‐κB and Akt signaling pathways in renal cancer cells

The prostate‐apoptosis‐response‐gene‐4 (Par‐4) is up‐regulated in prostate cells undergoing programmed cell death. Furthermore, Par‐4 protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor‐mediated cell death pathways. In this study, we investigated how Par‐4 modulates TRAIL‐mediated apoptosis in TRAIL‐resistant Caki cells. Par‐4 overexpressing cells were strikingly sensitive to apoptosis induced by TRAIL compared with control cells. Par‐4 overexpressing Caki cells treated with TRAIL showed an increased activation of the initiator caspase‐8 and the effector caspase‐3, together with an enforced cleavage of XIAP and c‐FLIP. TRAIL‐induced reduction of XIAP and c‐FLIP protein levels in Par‐4 overexpressing cells was prevented by z‐VAD pretreatment. In addition, the surface DR5 protein level was increased in TRAIL‐treated Par‐4 overexpressing cells. Interestingly, even though a deletion of leucine zipper domain in Par‐4 recovered Bcl‐2 level to basal level induced by wild type Par‐4, it partly decreased sensitivity to TRAIL in Caki cells. In addition, exposure of Caki/Par‐4 cells to TRAIL led to reduction of phosphorylated Akt levels, but deletion of leucine zipper domain of Par‐4 did not affect these phosphorylated Akt levels. In conclusion, we here provide evidence that ectopic expression of Par‐4 sensitizes Caki cells to TRAIL via modulation of multiple targets, including DR5, Bcl‐2, Akt, and NF‐κB. J. Cell. Biochem. 109: 885–895, 2010. © 2010 Wiley‐Liss, Inc.     

Imiquimod‐induced apoptosis of melanoma cells is mediated by ER stress‐dependent Noxa induction and enhanced by NF‐κB inhibition

Melanoma is characterized by dysregulated intracellular signalling pathways including an impairment of the cell death machinery, ultimately resulting in melanoma resistance, survival and progression. This explains the tumour's extraordinary resistance to the standard treatment. Imiquimod is a topical immune response modifier (imidazoquinoline) with both antiviral and antitumour activities. The mechanism by which imiquimod triggers the apoptosis of melanoma cells has now been carefully elucidated. Imiquimod‐induced apoptosis is associated with the activation of apoptosis signalling regulating kinase1/c‐Jun‐N‐terminal kinase/p38 pathways and the induction of endoplasmic stress characterized by the activation of the protein kinase RNA‐like endoplasmic reticulum kinase signalling pathway, increase in intracellular Ca²⁺ release, degradation of calpain and subsequent cleavage of caspase‐4. Moreover, imiquimod triggers the activation of NF‐κB and the expression of the inhibitor of apoptosis proteins (IAPs) such as, X‐linked IAP (XIAP) together with the accumulation of reactive oxygen species (ROS). Also, imiquimod triggers mitochondrial dysregulation characterized by the loss of mitochondrial membrane potential (Δψm), the increase in cytochrome c release, and cleavage of caspase‐9, caspase‐3 and poly(ADP‐ribose) polymerase (PARP). Inhibitors of specific pathways, permit the elucidation of possible mechanisms of imiquimod‐induced apoptosis. They demonstrate that inhibition of NF‐kB by the inhibitor of nuclear factor kappa‐B kinase (IKK) inhibitor Bay 11‐782 or knockdown of XIAP induces melanoma apoptosis in cells exposed to imiquimod. These findings support the use of either IKK inhibitors or IAP antagonists as adjuvant therapies to improve the effectiveness topical imiquimod in the treatment of melanoma.     

Lysyl oxidase propeptide sensitizes pancreatic and breast cancer cells to doxorubicin‐induced apoptosis

RAS mutations or its activation by upstream receptor tyrosine kinases are frequently associated with poor response of carcinomas to chemotherapy. The 18 kDa propeptide domain of lysyl oxidase (LOX‐PP) released from the secreted precursor protein (Pro‐LOX) has been shown to inhibit RAS signaling and the transformed phenotype of breast, pancreatic, lung, and prostate cancer cells in culture, and formation of tumors by Her‐2/neu‐driven breast cancer cells in a mouse xenograft model. Here, we tested the effects of LOX‐PP on MIA PaCa‐2 pancreatic cancer cells, driven by mutant RAS. In MIA PaCa‐2 cells in culture, LOX‐PP attenuated the ERK and AKT activities and decreased the levels of the NF‐κB p65 and RelB subunits and cyclin D1, which are activated by RAS signaling. In mouse xenograft growth, LOX‐PP reduced growth of tumors by these pancreatic cancer cells, and the nuclear levels of the p65 NF‐κB subunit and cyclin D1 proteins. While biological agents attenuate tumor growth when used alone, often they have additive or synergistic effects when used in combination with chemotherapeutic agents. Thus, we next tested the hypotheses that LOX‐PP sensitizes pancreatic and breast cancer cells to the chemotherapeutic agent doxorubicin. Purified LOX‐PP enhanced the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells, as judged by ATP production, Cell Death ELISA assays, caspase 3 activation, PARP cleavage, and Annexin V staining. Thus, LOX‐PP potentiates the cytotoxicity of doxorubicin on breast and pancreatic cancer cells, warranting additional studies with a broader spectrum of current cancer treatment modalities. J. Cell. Biochem. 111: 1160–1168, 2010. © 2010 Wiley‐Liss, Inc.     

Attenuating Smac mimetic compound 3‐induced NF‐κB activation by luteolin leads to synergistic cytotoxicity in cancer cells

Smac mimetics are potential anticancer therapeutics selectively killing cancer cells through autocrine tumor necrosis factor (TNF)‐mediated apoptosis pathway. Our recent study reveal that the Smac mimetic compound 3 (SMC3)‐activated NF‐κB protects cancer cells against apoptosis, thus blunting SMC3's anticancer activity. Based on our previous observations that the nutrient flavonoid luteolin potently blocks TNF‐induced NF‐κB activation in cancer cells, we investigated if the combination of SMC3 and luteolin would achieve a synergistic anticancer activity. The results show that luteolin had no effect on autocrine TNF but it effectively blocked SMC3‐induced nuclear factor kappa B (NF‐κB) activation and expression of anti‐apoptotic NF‐κB targets. When SMC3 and luteolin were combined in treating cancer cells derived from lung and liver tumors, the activation of TNF‐dependent apoptosis was markedly sensitized and a synergistic cytotoxic effect was achieved. In addition, the SMC3 and luteolin co‐treatment had marginal effect on immortalized normal human bronchial epithelial cells. The results suggest that combination of SMC3 and luteolin is an effective approach for improving the anticancer value of SMC3, which has implications in cancer prevention and therapy. J. Cell. Biochem. 108: 1125–1131, 2009. © 2009 Wiley‐Liss, Inc.     

Disabled‐1 is down‐regulated in clinical breast cancer and regulates cell apoptosis through NF‐κB/Bcl‐2/caspase‐9

Disabled‐1 (Dab1) is best known as an adaptor protein regulating neuron migration and lamination during development. However, the exact function of Dab1 in breast cancer is unknown. In this study, we examined the expression of Dab1 in 38 breast cancer paraffin sections, as well as 60 paired frozen breast cancer and their adjacent tissues. Our results showed that Dab1 was reduced in breast cancer, and its compromised expression correlated with triple negative breast cancer phenotype, poor differentiation, as well as lymph node metastasis. Functional analysis in breast cancer cell lines demonstrated that Dab1 promoted cell apoptosis, which, at least partially, depended on its regulation of NF‐κB/Bcl‐2/caspase‐9 pathway. Our study strongly suggests that Dab1 may be a potential tumour suppressor gene in breast cancer.     

MiR‐802 causes nephropathy by suppressing NF‐κB‐repressing factor in obese mice and human

Obesity is associated with significant microvascular complications including renal injuries and may induce end‐stage renal disease. Emerging studies have demonstrated microRNAs (miRNAs) are potential mediators in the pathophysiological process of nephropathy. The present study aimed to investigate the role of miR‐802 in obesity‐related nephropathy and potential molecular mechanisms. Through utilizing obese mouse model and human subjects, we explored the therapeutic benefits and clinical application of miR‐802 in protecting against nephropathy. Renal miR‐802 level was positively correlated with functional parameters, including blood urea nitrogen and creatinine in obese mice. Specific silencing of renal miR‐802 improved high fat diet (HFD)‐induced renal dysfunction, structural disorders and fibrosis. The up‐regulated inflammatory response and infiltrated macrophages were also significantly decreased in miR‐802 inhibitor‐treated obese mice. Mechanistically, miR‐802 directly bond to 3?‐UTR of NF‐κB‐repressing factor (NRF) and suppressed its expression. In clinical study, the circulating miR‐802 level was significantly increased in obese subjects, and positively correlated with plasma creatinine level but negatively correlated with creatinine clearance. Taken together, our findings provided evidence that miR‐802/NRF signalling was an important pathway in mediating obesity‐related nephropathy. It is a possible useful clinical approach of treating miR‐802 inhibitor to combat nephropathy.     

Platycodin D suppresses cisplatin‐induced cytotoxicity by suppressing ROS‐mediated oxidative damage,apoptosis, and inflammation in HEK‐293 cells

Cisplatin, a proven effective chemotherapeutic agent, has been used clinically to treat malignant solid tumors, whereas its clinical use is limited by serious side effect including nephrotoxicity. Platycodin D (PD), the major and marked saponin isolated from Platycodon grandiflorum, possesses many pharmacological effects. In this study, we evaluated its protective effect against cisplatin‐induced human embryonic kidney 293 (HEK‐293) cells injury and elucidated the related mechanisms. Our results showed that PD (0.25, 0.5, and 1 μM) can dose‐dependently alleviate oxidative stress by decreasing malondialdehyde and reactive oxygen species, while increasing the levels of glutathione, superoxide dismutase, and catalase. Moreover, the elevation of apoptosis including Bax, Bad, cleaved caspase‐3,‐9, and decreased protein levels of Bcl‐2, Bcl‐XL induced by cisplatin were reversed after PD treatment. Importantly, PD pretreatment can also regulate PI3K/Akt and ERK/JNK/p38 signaling pathways. Furthermore, PD was found to reduce NF‐κB‐mediated inflammatory relative proteins. Our finding indicated that PD exerted significant effects on cisplatin induced oxidative stress, apoptosis and inflammatory, which will provide evidence for the development of PD to attenuate cisplatin‐induced nephrotoxicity.     

Interleukin‐17‐induced expression of monocyte chemoattractant protein‐1 in cardiac myocytes requires nuclear factor κB through the phosphorylation of p65

IL‐17 plays a key role in a variety of autoimmune diseases. MCP‐1 is involved in the infiltration of mononuclear cells of myocardium in VMC. However, the relationship between IL‐17 and MCP‐1 in myocardial injury remains unclear. In this study, expression of MCP‐1 mRNA and protein in cardiac myocytes was detected with qRT‐PCR and ELISA, respectively. It was found that IL‐17A induced MCP‐1 expression in a dose‐ and time‐dependent manner in cardiac myocytes, which could be blocked by IL‐17A and IL‐17RA neutralizing antibodies. NF‐κB p65 and p‐p65 protein expression in cardiac myocytes was studied with western blotting. Rates of p‐p65 in whole lysates and in nuclear lysates all increased in the first 15 min. Meanwhile, the amount of NF‐κB p65 in whole lysates did not change, but the amount of NF‐κB p65 in nuclear lysates increased in the first 15 min. Then the optimal sequence and concentration of NF‐κB p65 siRNAs was selected. After transfection of 10 nM siRNA‐2 of NF‐κB p65 into cardiac myocytes before stimulation by IL‐17A, expression of MCP‐1 mRNA and protein obviously decreased. In conclusion, expression of MCP‐1 induced by IL‐17 requires NF‐κB through the phosphorylation of p65 in cardiac myocytes, which is meaningful to study the onset of chronic viral myocarditis and will provide a new target for the treatment of viral myocarditis.

     

LIGHT/IFN‐γ triggers β cells apoptosis via NF‐κB/Bcl2‐dependent mitochondrial pathway

LIGHT recruits and activates naive T cells in the islets at the onset of diabetes. IFN‐γ secreted by activated T lymphocytes is involved in beta cell apoptosis. However, whether LIGHT sensitizes IFNγ‐induced beta cells destruction remains unclear. In this study, we used the murine beta cell line MIN6 and primary islet cells as models for investigating the underlying cellular mechanisms involved in LIGHT/IFNγ – induced pancreatic beta cell destruction. LIGHT and IFN‐γ synergistically reduced MIN6 and primary islet cells viability; decreased cell viability was due to apoptosis, as demonstrated by a significant increase in Annexin V⁺ cell percentage, detected by flow cytometry. In addition to marked increases in cytochrome c release and NF‐κB activation, the combination of LIGHT and IFN‐γ caused an obvious decrease in expression of the anti‐apoptotic proteins Bcl‐2 and Bcl‐xL, but an increase in expression of the pro‐apoptotic proteins Bak and Bax in MIN6 cells. Accordingly, LIGHT deficiency led to a decrease in NF‐κB activation and Bak expression, and peri‐insulitis in non‐obese diabetes mice. Inhibition of NF‐κB activation with the specific NF‐κB inhibitor, PDTC (pyrrolidine dithiocarbamate), reversed Bcl‐xL down‐regulation and Bax up‐regulation, and led to a significant increase in LIGHT‐ and IFN‐γ‐treated cell viability. Moreover, cleaved caspase‐9, ‐3, and PARP (poly (ADP‐ribose) polymerase) were observed after LIGHT and IFN‐γ treatment. Pretreatment with caspase inhibitors remarkably attenuated LIGHT‐ and IFNγ‐induced cell apoptosis. Taken together, our results indicate that LIGHT signalling pathway combined with IFN‐γ induces beta cells apoptosis via an NF‐κB/Bcl2‐dependent mitochondrial pathway.     

Soluble CXCL16 promotes TNF‐α‐induced apoptosis in DLBCL via the AMAD10‐NF‐κB regulatory feedback loop

We had previously identified that the co‐expression of transmembrane CXCL16 (TM‐CXCL16) and its receptor CXCR6 is an independent risk factor for poor survival in patients with diffuse large B‐cell lymphoma (DLBCL). However, the impact of the soluble form of CXCL16 (sCXCL16) on the pathogenesis of DLBCL remains unknown. In the present study, the synergistic effect of sCXCL16 and tumor necrosis factor α (TNF‐α) on apoptosis in DLBCL cell lines (OCI‐LY8 and OCI‐LY10) was investigated in vitro. sCXCL16 reinforced TNF‐α‐mediated inhibition of DLBCL cell proliferation, as determined by the cell counting kit‐8 assay. The results of annexin V staining showed that sCXCL16 enhanced TNF‐α‐induced apoptosis in OCI‐LY8 and OCI‐LY10 cells through a death receptor‐caspase signaling pathway. The results of gene microarray suggested a significant upregulation of differentially expressed genes in the TNF signaling pathway. sCXCL16 increased the concentration of extracellular TNF‐α by binding to CXCR6 to activate the nuclear factor‐κB (NF‐κB) signaling pathway. TNF‐α also induced the secretion of sCXCL16 by increasing the expression of ADAM10, which is known to cleave TM‐CXCL16 to yield sCXCL16. Moreover, bioinformatics analysis revealed that elevated TNF‐α and ADAM10 expression levels in tumor tissues predicted better survival in patients with DLBCL. Thus, our study suggests that sCXCL16 enhances TNF‐α‐induced apoptosis of DLBCL cells, which may involve a positive feedback loop consisting of TNF‐α, ADAM10, sCXCL16, and members of the NF‐κB pathway. sCXCL16 and TNF‐α may be used as prognostic markers in the clinic, and their combinational use is a promising approach in the context of DLBCL therapy.     

Concurrent inhibition of NF‐κB,cyclooxygenase‐2, and epidermal growth factor receptor leads to greater anti‐tumor activity in pancreatic cancer

Inactivation of survival pathways such as NF‐κB, cyclooxygenase (COX‐2), or epidermal growth factor receptor (EGFR) signaling individually may not be sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by recent clinical trials. 3,3′‐Diindolylmethane (B‐DIM) is an inhibitor of NF‐κB and COX‐2 and is a well‐known chemopreventive agent. We hypothesized that the inhibition of NF‐κB and COX‐2 by B‐DIM concurrently with the inhibition of EGFR by erlotinib will potentiate the anti‐tumor effects of cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo. Inhibition of viable cells in seven PC cell lines treated with B‐DIM, erlotinib, or gemcitabine alone or their combinations was evaluated using 3‐(4,5‐dimetylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Significant inhibition in cell viability was observed in PC cells expressing high levels of COX‐2, EGFR, and NF‐κB proteins. The observed inhibition was associated with an increase in apoptosis as assessed by ELISA. A significant down‐regulation in the expression of COX‐2, NF‐κB, and EGFR in BxPC‐3, COLO‐357, and HPAC cells was observed, suggesting that simultaneous targeting of EGFR, NF‐κB, and COX‐2 is more effective than targeting either signaling pathway separately. Our in vitro results were further supported by in vivo studies showing that B‐DIM in combination with erlotinib and gemcitabine was significantly more effective than individual agents. Based on our preclinical in vitro and in vivo results, we conclude that this multi‐targeted combination could be developed for the treatment of PC patients whose tumors express high levels of COX‐2, EGFR, and NF‐κB. J. Cell. Biochem. 110: 171–181, 2010. © 2010 Wiley‐Liss, Inc.