Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB

Background

Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC) with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.

Methodology/Principal Findings

We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1), with IC₅₀ in the range of 1–2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.

Conclusions/Significance

Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be developed as a new therapeutic agent for hormone-refractory prostate cancer.     

Nuclear factor-kappa B inhibition can enhance apoptosis of differentiated thyroid cancer cells induced by 131I

Objective

To evaluate changes of nuclear factor-kappa B (NF-κB) during radioiodine 131 (¹³¹I) therapy and whether NF-κB inhibition could enhance ¹³¹I-induced apoptosis in differentiated thyroid cancer (DTC) cells in a synergistic manner.

Methods

Three human DTC cell lines were used. NF-κB inhibition was achieved by using a NF-κB inhibitor (Bay 11-7082) or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-κB. Then NF-κB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-κB inhibition could influence radioactive iodide uptake.

Results

The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-κB function and reporter gene activities due to ¹³¹I, yet significant suppression was achieved by NF-κB inhibition. Western blot proved ¹³¹I could increase nuclear NF-κB concentration, while NF-κB inhibition reduced NF-κB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after ¹³¹I therapy. And inhibition of NF-κB could significantly down-regulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements of cleaved caspase 3 and PARP from Western blot, and of Annexin V positively staining from flow cytometry. The iodine uptake assay did not show significant changes when NF-κB was inhibited.

Conclusion

We demonstrated that ¹³¹I could induce NF-κB activation, which would attenuate ¹³¹I efficacy in DTC cells. NF-κB inhibition by Bay 11-7082 or by p65 siRNA transfection was effective in suppressing NF-κB regulated anti-apoptotic changes and in combined regimen apoptosis was achieved synergistically.     

Antitumor Activity of Emodin against Pancreatic Cancer Depends on Its Dual Role: Promotion of Apoptosis and Suppression of Angiogenesis

Background

Emodin has been showed to induce apoptosis of pancreatic cancer cells and inhibit tumor growth in our previous studies. This study was designed to investigate whether emodin could inhibit the angiogenesis of pancreatic cancer tissues and its mechanism.

Methodology/Principal Finding

In accordance with our previous study, emodin inhibited pancreatic cancer cell growth, induced apoptosis, and enhanced the anti-tumor effect of gemcitabine on pancreatic caner cells in vitro and in vivo by inhibiting the activity of NF-κB. Here, for the first time, we demonstrated that emodin inhibited tumor angiogenesis in vitro and in implanted pancreatic cancer tissues, decreased the expression of angiogenesis-associated factors (NF-κB and its regulated factors VEGF, MMP-2, MMP-9, and eNOS), and reduced eNOS phosphorylation, as evidenced by both immunohistochemistry and western blot analysis of implanted tumors. In addition, we found that emodin had no effect on VEGFR expression in vivo.

Conclusions/Significance

Our results suggested that emodin has potential anti-tumor effect on pancreatic cancer via its dual role in the promotion of apoptosis and suppression of angiogenesis, probably through regulating the expression of NF-κB and NF-κB-regulated angiogenesis-associated factors.     

New insights into the role and mechanism of macrophage migration inhibitory factor in steroid-resistant patients with systemic lupus erythematosus

Introduction

Glucocorticoid (GC) therapy remains important in improving the prognosis of patients with systemic lupus erythematosus (SLE). However, some patients do not achieve an effective response with GC treatment, creating an obstacle to the remission of SLE. Identification of the underlying mechanisms responsible for steroid resistance can be significant. Macrophage migration inhibitory factor (MIF) arouses our interest because of its reciprocal relationship with GCs. In the present study, we investigated for the first time whether MIF correlated with steroid resistance in SLE and explored potential mechanisms of action.

Methods

Sixty-two patients with SLE (40 steroid sensitive and 22 steroid resistant) and 21 normal controls were recruited. Serum levels of MIF were measured by ELISA. Cytosolic MIF and IκB expression in peripheral blood mononuclear cells (PBMCs) were determined by western blotting. The electrophoretic mobility shift assay was assessed by NF-κB in nuclear aliquots. Gene silencing was applied to reduce expression of MIF in PBMCs in steroid-resistant patients. PBMCs obtained from steroid-sensitive patients were treated with recombinant human MIF of different concentrations.

Results

MIF levels in serum and PBMCs were higher in steroid-resistant patients compared with steroid-sensitive patients and controls. In contrast to the steroid-sensitive group, NF-κB levels were significantly higher and IκB levels lower in steroid-resistant patients. After MIF gene silencing, IκB levels in cells from steroid-resistant patients were increased. In steroid-sensitive patients, a decrease in IκB levels and an increase in NF-κB expression from baseline were detected in PBMCs treated with a higher concentration of recombinant human MIF. Treatment with recombinant human MIF did not regulate expression of IκB and NF-κB in PBMCs from patients treated with an anti-MIF monoclonal antibody.

Conclusions

Our results indicated that MIF may play a role in the formation of steroid resistance in SLE by affecting the NF-κB/IκB signaling cascade. As a regulator of glucocorticoid sensitivity, MIF may be a potential target for steroid sparing.     

Nitrosylcobalamin potentiates the anti-neoplastic effects of chemotherapeutic agents via suppression of survival signaling

Background

Nitrosylcobalamin (NO-Cbl) is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO) to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-κB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1) measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2) examine the mechanism of action of NO-Cbl as a single agent and in combination therapy.

Methodology

Using anti-proliferative assays, electrophoretic mobility shift assay (EMSA), immunoblot analysis and kinase assays, we demonstrate an increase in the effectiveness of chemotherapeutic agents in combination with NO-Cbl as a result of suppressed NF-κB activation.

Results

Eighteen chemotherapeutic agents were tested in combination with NO-Cbl, in thirteen malignant cell lines, resulting in a synergistic anti-proliferative effect in 78% of the combinations tested. NO-Cbl pre-treatment resulted in decreased NF-κB DNA binding activity, inhibition of IκB kinase (IKK) enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels.

Conclusion

The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-κB or AKT.     

Vimentin and PSF act in concert to regulate IbeA+ E. coli K1 induced activation and nuclear translocation of NF-κB in human brain endothelial cells

Background

IbeA-induced NF-κB signaling through its primary receptor vimentin as well as its co-receptor PSF is required for meningitic E. coli K1 penetration and leukocyte transmigration across the blood-brain barrier (BBB), which are the hallmarks of bacterial meningitis. However, it is unknown how vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB, which are required for bacteria-mediated pathogenicities.

Methodology/Principal Findings

IbeA-induced E. coli K1 invasion, polymorphonuclear leukocyte (PMN) transmigration and IKK/NF-κB activation are blocked by Caffeic acid phenethyl ester (CAPE), an inhibitor of NF-κB. IKKα/β phosphorylation is blocked by ERK inhibitors. Co-immunoprecipitation analysis shows that vimentin forms a complex with IκB, NF-κB and tubulins in the resting cells. A dissociation of this complex and a simultaneous association of PSF with NF-κB could be induced by IbeA in a time-dependent manner. The head domain of vimentin is required for the complex formation. Two cytoskeletal components, vimentin filaments and microtubules, contribute to the regulation of NF-κB. SiRNA-mediated knockdown studies demonstrate that IKKα/β phosphorylation is completely abolished in HBMECs lacking vimentin and PSF. Phosphorylation of ERK and nuclear translocation of NF-κB are entirely dependent on PSF. These findings suggest that vimentin and PSF cooperatively contribute to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB activation. PSF is essential for translocation of NF-κB and ERK to the nucleus.

Conclusion/Significance

These findings reveal previously unappreciated facets of the IbeA-binding proteins. Cooperative contributions of vimentin and PSF to IbeA-induced cytoplasmic activation and nuclear translocation of NF-κB may represent a new paradigm in pathogen-induced signal transduction and lead to the development of novel strategies for the prevention and treatment of bacterial meningitis.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.