Suppression of urokinase receptor expression by thalidomide is associated with inhibition of nuclear factor kappaB activation and subsequently suppressed ovarian cancer dissemination

Thalidomide has been used to treat a variety of diseases ranging from alleviation of autoimmune disorders to prevention of metastasis of cancers. It has been shown previously that increased levels of urokinase-type plasminogen activator receptor (uPAR) correlate well with higher invasive phenotype. We examined whether thalidomide is able to suppress the expression of uPAR mRNA and protein in human ovarian cancer cell line HRA and human chondrosarcoma cell line HCS-2/8. Here, we show that: (a) thalidomide suppresses the expression of constitutive and transforming growth factor-beta1 (TGF-beta1)-induced uPAR mRNA and protein; (b) a nuclear factor kappaB (NF-kappaB) activation system (phosphorylation of IkappaB-alpha and degradation of IkappaB-alpha) is necessary for the TGF-beta1-induced increase in uPAR expression, because L-1-tosylamido-2-phenylethyl chloromethyl ketone, a NF-kappaB inhibitor, reduced the uPAR production as well as mRNA expression; (c) thalidomide failed to further strengthen L-1-tosylamido-2-phenylethyl chloromethyl ketone's action; (d) the once-daily i.p. administration of thalidomide (400 microg/g body weight/d) decreased progressive growth of HRA tumors and ascites formation in an in vivo animal model; and (e) the once-daily i.p. administration of thalidomide in combination with paclitaxel (i.p., 100 microg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. We conclude that thalidomide down-regulates constitutive and TGF-beta1-stimulated uPAR mRNA and protein expression possibly through suppression of NF-kappaB activation. Furthermore, combination therapy with thalidomide plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian cancer dissemination.     

Soluble receptor activator of nuclear factor kappaB Fc diminishes prostate cancer progression in bone

Prostate cancer (CaP) develops metastatic bone lesions that consist of a mixture of osteosclerosis and osteolysis. We have previously demonstrated that targeting receptor activator of nuclear factor kappaB ligand (RANKL) with osteoprotegerin (OPG) prevents the osteolytic activity of CaP and its ability to establish tumor in bone. However, OPG can block tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, suggesting that the clinical use of OPG may prevent apoptosis of tumors mediated by TRAIL. Thus, methods to block RANKL activity, other than OPG, may be important. Accordingly, we evaluated the ability of soluble murine RANK-Fc (sRANK-Fc) to prevent progression of established CaP in a severe combined immunodeficient mouse implanted with fetal human bone. We first confirmed that sRANK did not block TRAIL-mediated apoptosis of LuCaP cells in vitro and that it did block LuCaP-conditioned media-induced osteoclastogenesis in vitro. Then, LuCaP 35 CaP cells were injected into the marrow space of the bone implanted in the severe combined immunodeficient mice implanted with fetal human bone and allowed to develop into tumors for 6 weeks. Either vehicle or sRANK-Fc was then administered for 6 weeks. sRANK-Fc diminished tumor-induced osteoblastic lesions as demonstrated by radiograph, bone mineral density measurement, and bone histomorphometry. sRANK-Fc also reduced systemic bone remodeling markers, including serum osteocalcin and bone-specific alkaline phosphatase and urine N-telopeptide of collagen. Finally, sRANK-Fc decreased serum prostate-specific antigen levels and tumor volume in the bone, which indicates decreased tumor burden. In contrast, sRANK-Fc had no effect on s.c. implanted LuCaP cells. We conclude that sRANK-Fc is an effective inhibitor of RANKL that diminishes progression of CaP growth in bone through inhibition of bone remodeling.     

Suppression of hormone-refractory prostate cancer by a novel nuclear factor kappaB inhibitor in nude mice

We have synthesized and explored the feasibility of using a novel nuclear factor (NF) kappaB inhibitor, a dehydroxymethylepoxyquinomicin designated as DHMEQ, against prostate cancer. The activity of NFkappaB, evaluated by transient transfection of a luciferase reporter DNA containing a specific binding sequence for NFkappaB, was inhibited by DHMEQ in three human hormone-refractory prostate cancer cell lines, DU145, JCA-1, and PC-3. Statistically significant growth inhibition was achieved by 20 micro g/ml of DHMEQ, and marked levels of apoptosis were induced 48 h after DHMEQ administration in vitro. Electrophoretic mobility shift assay showed that DHMEQ completely inhibited NFkappaB DNA binding activity in JCA-1 cells. Furthermore, i.p. administrations of DHMEQ significantly inhibited pre-established JCA-1 s.c. tumor growth in nude mice without any side effects. Our result indicates the possibility of using a novel NFkappaB activation inhibitor, DHMEQ, as a new treatment strategy against hormone-refractory prostate cancer.     

Bombesin stimulates nuclear factor kappa B activation and expression of proangiogenic factors in prostate cancer cells

The majority of deaths from prostate cancer occur in patients with androgen-insensitive metastatic disease. An important early event in the development of the metastatic phenotype is the induction of genes that promote angiogenesis, such as vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), which are released from tumor cells into their microenvironment. Coincident with progression from prostatic carcinoma in situ to metastatic disease is an increase in the number of tumor cells exhibiting neuroendocrine (NE) differentiation. NE cells express a variety of peptide hormones, including the bombesin (BBS)-like peptide, gastrin-releasing peptide (GRP), and its cognate receptor, GRP-R. Although there is a strong positive correlation between the degree of NE differentiation and the metastatic potential of prostate cancers, a mechanistic link between increased expression of peptide hormone receptors, such as GRP-R, and proangiogenic gene expression has not been established. Here we report that BBS stimulates nuclear factor kappa B (NF kappa B) activation and proangiogenic gene expression in the androgen-insensitive prostate cancer cells lines, PC-3 and DU-145. In PC-3 cells, BBS stimulation of GRP-R resulted in the up-regulation of IL-8 and VEGF expression through a NF kappa B-dependent pathway. We show that BBS treatment induced inhibitor of NF kappa B degradation, NF kappa B translocation to the cell nucleus, increased NF kappa B binding to its DNA consensus sequence, and increased IL-8 and VEGF mRNA expression and protein secretion. Treatment with the proteasome inhibitor, MG-132, blocked BBS-stimulated NF kappa B DNA binding, and IL-8 and VEGF expression and secretion. Finally, media collected from PC-3 cell cultures, after BBS treatment, stimulated an NF kappa B-dependent migration of human umbilical vascular endothelial cells in vitro. Together, our data demonstrate a role for BBS and GRP-R in the NF kappa B-dependent up-regulation of proangiogenic gene expression, and suggest a possible molecular mechanism linking NE differentiation and the increased metastatic potential of androgen-insensitive prostate cancers.     

Requirement of RhoA activity for increased nuclear factor kappaB activity and PC-3 human prostate cancer cell invasion

To determine the molecular mechanisms of aggressive prostate cancer behavior, we studied RhoGTPases in high and low invasive variants of PC-3 prostate cancer cells. Prior studies with these cells revealed that elevated nuclear factor kappaB (NF-kappaB) expression and activity were necessary for the highly invasive phenotype. In the current study, increased RhoA expression was found in the PC-3 highly invasive cells as compared with the PC-3 low invasive cells through cDNA array and Western blot analyses. Similarly, RhoA activity, as measured by the Rhotekin binding assay, was elevated in the PC-3 highly invasive cells. Transfection of these highly invasive cells with dominant negative RhoA N19 or treatment with 1.0 micro g/ml RhoA inhibitor C3 exoenzyme demonstrated that RhoA activity was necessary for both NF-kappaB activity and cellular invasion of a Matrigel reconstituted basement membrane. Furthermore, stable transfection of the PC-3 highly invasive cells with constitutively active RhoA Q63L resulted in activation of NF-kappaB activity and Matrigel invasion, effects reversed by treatment of the cells with C3 exoenzyme. RhoA was also shown to act through the motility component of the invasion process. RhoA activity was therefore both necessary and sufficient for the elevated NF-kappaB, invasion, and motility activities of the PC-3 highly invasive cells. These findings suggest molecular targets to control cancer cell invasion and aid in the development of definitive tools for predicting the invasive and metastatic potential of cancer cells.     

NF-κB、I-κB、Bcl-2在宫颈癌组织中的表达及其与人乳头瘤病毒感染的关系

背景与目的:已表明核因子κB(nuclearfactorκB,NF-κB)及其抑制蛋白κB(inhibitorproteinκB,I-κB)对凋亡基因的调控起重要作用,NF-κB在某些肿瘤中高表达,与肿瘤的发生有关,但在宫颈癌中的表达以及与人乳头瘤病毒(humanpapillomavirus,HPV)感染的关系未见报道。本研究旨在探讨宫颈癌组织中NF-κB、I-κB、bcl-2的表达及其与HPV感染的关系。方法:采用免疫组织化学SP法对46例宫颈癌和26例正常宫颈组织进行NF-κB、I-κB、bcl-2蛋白检测,用PCR技术对组织标本的HPV-DNA进行检测。结果:NF-κB、bcl-2在宫颈癌组织中的表达率分别为60.9%(28/46)和52.2%(24/46),在正常对照组的表达率分别为23.1%(6/26)和0(0/26),宫颈癌组显著高于正常对照组(P<0.01);I-κB在宫颈癌组和正常对照组的表达率分别为30.4%(14/46)和57.7%(15/26),宫颈癌组显著低于正常对照组(P<0.05);NF-κB与bcl-2的表达呈显著性相关(P<0.05)。HPV-DNA阳性的宫颈癌组织中NF-κB的阳性率显著高于阴性组(P<0.05);I-κB、bcl-2的阳性率在HPV-DNA阳性和阴性组之间无显著性差异(P>0.05)。结论:NF-κB、bcl-2的高表达和I-κB的低表达与宫颈癌的发生过程可能有关;HPV感染可能与NF-κB表达有关。     

Large-scale independent validation of the nuclear factor-kappa B p65 prognostic biomarker in prostate cancer

PurposeOver the last decade, we and others have uncovered a robust association between the nuclear localisation of nuclear factor-kappa B (NF-κB) p65, prostate cancer (PCa) aggressiveness and biochemical recurrence (BCR). Our goal was to validate these results in a large independent cohort of PCa patients who underwent radical prostatectomy.Experimental designA set of 1826 fully annotated prostate cancers treated by radical prostatectomy were analysed in a tissue microarray (TMA) format for NF-κB p65 immunohistochemistry-based protein expression. We performed standard Cox proportional hazard regression models for follow-up data, bootstrap procedure for model internal validation, Harrell’s concordance index for model discrimination and graphical assessment of predicted versus actual outcomes for model calibration.ResultsWe observed a significant association between an increase in the nuclear frequency of NF-κB p65 and Gleason score (P < 0.001), overall BCR (P < 0.001) and development of metastases (P = 0.001). NF-κB was found to be an independent predictor of BCR (P < 0.001, Cox regression). However its contribution to the predictive accuracy of a multivariate model, which included preoperative PSA, Gleason score, extraprostatic extension, lymph node invasion, seminal vesicle involvement and surgical margin status, was modest.ConclusionsOur study offers validating results linking NF-κB p65 with disease progression using a large cohort of European men. However, the contribution of NF-κB to a post-surgical predictive model appears modest. Further validating work should focus on evaluating the contribution of NF-κB p65 in pre-treatment models.     

Inhibition of nuclear factor kappaB induces apoptosis following treatment with tumor necrosis factor alpha and an antioxidant in human prostate cancer cells

Transforming growth factor beta-1 (TGFbeta-1) and tumor necrosis factor alpha (TNF-alpha), an activator of nuclear factor kappa B (NF-kappaB), modulate apoptosis and/or cell growth. This study was designed to investigate the activity of NF-kappaB and its regulation of inhibitor of apoptosis gene (c-IAP2) in two human prostate cancer cell lines, DU-145 (which is androgen unresponsive) and ALVA-101 (which is moderately androgen responsive). These cells were treated with and without various concentrations of a strong antioxidant, pyrrolidinedithiocarbamate (PDTC), and TNF-alpha at various time intervals. Following treatments, cell growth and apoptosis were determined by ELISA techniques. NF-kappaB activity was determined by electrophoretic mobility shift assay (EMSA), and c-IAP2 mRNA production was determined with Northern blot analysis. PDTC treatment significantly reduced cell growth up to 80% in both DU-145 and ALVA-101 cells. TNF-alpha and lower but not higher doses of PDTC combined demonstrated an additive inhibition of cell growth in both cell lines. Active NF-kappaB and c-IAP2 was blocked significantly following PDTC treatments, whereas treatments with TNF-alpha alone showed increased NF-kappaB activity and c-IAP2. However, when both PDTC and TNF-alpha were combined, nuclear presence of NF-kappaB and c-IAP2 were reduced significantly (P < 0.05) to levels observed with PDTC alone. In conclusion, the antioxidant, PDTC, appears to initiate apoptosis by blocking cytoplasmic NF-kappaB translocation to the nucleus where it normally activates the production of apoptosis-inhibitory proteins like c-IAP2. Both TNF-alpha and PDTC alone cause apoptosis and reduce cell growth, but their combined effects are additive in reducing cell growth of DU-145 and ALVA-101 human prostate cancer cells.     

An autocrine loop for vascular endothelial growth factor is established in prostate cancer cells generated after prolonged treatment with interleukin 6

Concentrations of interleukin 6 (IL-6) and its receptor are increased in human prostate cancer. Prostate cancer LNCaP-IL-6+ cells, established after prolonged treatment with IL-6, have been found to acquire a growth advantage. Vascular endothelial growth factor (VEGF) may accelerate the growth of various tumours by stimulation of VEGF receptor 2 (VEGFR-2). To understand better the regulation of proliferation of LNCaP-IL-6+ cells, the expression of VEGF and VEGFR-2 was here investigated in the LNCaP-IL-6+ subline. VEGF was measured in cellular supernatants by enzyme-linked immunoassay. The expression of VEGFR-2 was assessed by Western blot. LNCaP-IL-6+ and control LNCaP-IL-6- cells were treated with a neutralising antibody against VEGFR-2. VEGF concentrations were 20-fold higher in LNCaP-IL-6+ than in LNCaP-IL-6- cells. The stimulatory effect of IL-6 on VEGF production was abolished by an inhibitor of the signalling pathway for phosphoinositol 3 kinase in LNCaP-IL-6+ and LNCaP-IL-6- cells. Exogenous VEGF did not stimulate proliferation in either LNCaP-IL-6+ cells or controls. VEGFR-2 was detected only in LNCaP-IL-6+ cells, in which the neutralising antibody caused a partial inhibition of cell proliferation. It was concluded that a VEGF autocrine loop is established in prostate cancer cells generated after chronic treatment with IL-6. Because of the upregulation of IL-6 in patients with prostate cancer, these findings might be clinically relevant.     

Inhibition of nuclear factor kappaB activation in PC3 cells by genistein is mediated via Akt signaling pathway.

Prostate cancer is the second leading cause of cancer-related deaths in menin the United States. Genistein is a prominent isoflavonoid found in soy products and has been proposed to be responsible for lowering the rate of prostate cancer in Asians. However, the molecular mechanism(s) by which genistein elicits its effects on prostate cancer cells has not been fully elucidated. We have previously shown that genistein induces apoptosis and inhibits the activation of nuclear factor kappaB (NF-kappaB) pathway, which could be mediated via Akt signaling pathway, the most important survival pathway in cellular signaling. In this study, we investigated whether there is any cross-talk between Akt and NF-kappaB during genistein-induced apoptosis in PC3 prostate cancer cells. We found that genistein inhibits cell growth and induces apoptotic processes in PC3 prostate cancer cells but not in nontumorigenic CRL-2221 human prostate epithelial cells. Immunoprecipitation, kinase assays, and Western blot analysis showed that genistein specifically inhibits Akt kinase activity and abrogates the epidermal growth factor-induced activation of Akt in prostate cancer cells. NF-kappaB DNA-binding analysis and transfection studies with Akt cDNA and NF-kappaB-Luc constructs revealed that Akt transfection results in the induction of NF-kappaB activation and this is completely inhibited by genistein treatment. Moreover, genistein abrogated the epidermal growth factor-induced activation of NF-kappaB, which was mediated via Akt signaling pathway. From these results, we conclude that the inhibition of Akt and NF-kappaB activity and their cross-talk provide a novel mechanism by which genistein inhibits cell growth and induces apoptotic processes in tumorigenic but not in nontumorigenic prostate epithelial cells.