CXCR4/YY1 inhibition impairs VEGF network and angiogenesis during malignancy.

Tumor growth requires neoangiogenesis. VEGF is the most potent proangiogenic factor. Dysregulation of hypoxia-inducible factor (HIF) or cytokine stimuli such as those involving the chemokine receptor 4/stromal-derived cell factor 1 (CXCR4/SDF-1) axis are the major cause of ectopic overexpression of VEGF in tumors. Although the CXCR4/SDF-1 pathway is well characterized, the transcription factors executing the effector function of this signaling are poorly understood. The multifunctional Yin Yang 1 (YY1) protein is highly expressed in different types of cancers and may regulate some cancer-related genes. The network involving CXCR4/YY1 and neoangiogenesis could play a major role in cancer progression. In this study we have shown that YY1 forms an active complex with HIF-1alpha at VEGF gene promoters and increases VEGF transcription and expression observed by RT-PCR, ELISA, and Western blot using two different antibodies against VEGFB. Long-term treatment with T22 peptide (a CXCR4/SDF-1 inhibitor) and YY1 silencing can reduce in vivo systemic neoangiogenesis (P < 0.01 and P < 0.05 vs. control, respectively) during metastasis. Moreover, using an in vitro angiogenesis assay, we observed that YY1 silencing led to a 60% reduction in branches (P < 0.01) and tube length (P < 0.02) and a 75% reduction in tube area (P < 0.001) compared with control cells. A similar reduction was observed using T22 peptide. We demonstrated that T22 peptide determines YY1 cytoplasmic accumulation by reducing its phosphorylation via down-regulation of AKT, identifying a crosstalk mechanism involving CXCR4/YY1. Thus, YY1 may represent a crucial molecular target for antiangiogenic therapy during cancer progression.

Immunosensitization with a Bcl-2 small molecule inhibitor.

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x(L), was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.

Regulation of apoptosis-related genes by nitric oxide in cancer.

Nitric oxide (NO) is a simple molecule with a complex and pleiotropic biological activity. NO or related species have been implicated in the regulation of many genes that participate in many diverse biological functions including programmed cell death or apoptosis. Apoptosis is a process that may potentially be disrupted in cancer cells conferring a survival advantage. In addition, malignant tumor cells can develop an intricate system of resistance to apoptotic stimuli. NO or related species have been shown to play a dual role in the regulation of apoptosis in malignant cells either promoting cell death or protecting cells from pro-apoptotic induction. However, the specific role of NO in the regulation of apoptosis/survival-related genes expression seems to tilt the balance toward the promotion of pro-apoptotic and the suppression of anti-apoptotic genes. Herein we have reviewed the most relevant aspects involving NO and/or reactive intermediates in the regulation of apoptosis-related genes--mainly--at the transcriptional level. We described the basic apoptotic molecules that potentially are affected by NO and how NO-mediated signaling gets transmitted to the transcriptional machinery that governs the expression of these genes. In addition, we discussed some of the fundamental functional consequences of the regulation of apoptosis-related genes by NO in cancer biology and its potential therapeutic implications.

Aromatase inhibitors in human lung cancer therapy.

Lung cancer is the most common cancer in the world. It is a highly lethal disease in women and men, and new treatments are urgently needed. Previous studies implicated a role of estrogens and estrogen receptors in lung cancer progression, and this steroidal growth-stimulatory pathway may be promoted by tumor expression and activity of aromatase, an estrogen synthase. We found expression of aromatase transcripts and protein in human non-small cell lung cancer (NSCLC) cells using reverse transcription-PCR and Western immunoblots, respectively. Aromatase staining by immunohistochemistry was detected in 86% of archival NSCLC tumor specimens from the clinic. Further, biological activity of aromatase was determined in NSCLC tumors using radiolabeled substrate assays as well as measure of estradiol product using ELISA. Significant activity of aromatase occurred in human NSCLC tumors, with enhanced levels in tumor cells compared with that in nearby normal cells. Lung tumor aromatase activity was inhibited by anastrozole, an aromatase inhibitor, and treatment of tumor cells in vitro with anastrozole led to significant suppression of tumor cell growth. Similarly, among ovariectomized nude mice with A549 lung tumor xenografts, administration of anastrozole by p.o. gavage for 21 days elicited pronounced inhibition of tumor growth in vivo. These findings show that aromatase is present and biologically active in human NSCLCs and that tumor growth can be down-regulated by specific inhibition of aromatase. This work may lead to development of new treatment options for patients afflicted with NSCLC.

G2A Attenuates Propionibacterium acnes Induction of Inflammatory Cytokines in Human Monocytes.

BACKGROUND: Acne vulgaris is a disease of the pilosebaceous unit characterized by increased sebum production, hyperkeratinization, and immune responses to Propionibacterium acnes (PA). Here, we explore a possible mechanism by which a lipid receptor, G2A, regulates immune responses to a commensal bacterium. OBJECTIVE: To elucidate the inflammatory properties of G2A in monocytes in response to PA stimulation. Furthermore, our study sought to investigate pathways by which lipids modulate immune responses in response to PA. METHODS: Our studies focused on monocytes collected from human peripheral blood mononuclear cells, the monocytic cell line THP-1, and a lab strain of PA. Our studies involved the use of enzyme-linked immunosorbent, Western blot, reverse transcription polymerase chain reaction, small interfering RNA (siRNA), and microarray analysis of human acne lesions in the measurements of inflammatory markers. RESULTS: G2A gene expression is higher in acne lesions compared to normal skin and is inducible by the acne therapeutic, 13-cis-retinoic acid. In vitro, PA induces both the Toll-like receptor 2-dependent expression of G2A as well as the production of the G2A ligand, 9-hydroxyoctadecadienoic acid, from human monocytes. G2A gene knockdown through siRNA enhances PA stimulation of interleukin (IL)-6, IL-8, and IL-1ß possibly through increased activation of the ERK1/2 MAP kinase and nuclear factor kappa B p65 pathways. CONCLUSION: G2A may play a role in quelling inflammatory cytokine response to PA, revealing G2A as a potential attenuator of inflammatory response in a disease associated with a commensal bacterium.

Propionibacterium acnes Induces IL-1ß secretion via the NLRP3 inflammasome in human monocytes.

Propionibacterium acnes induction of inflammatory responses is a major etiological factor contributing to the pathogenesis of acne vulgaris. In particular, the IL-1 family of cytokines has a critical role in both initiation of acne lesions and in the inflammatory response in acne. In this study, we demonstrated that human monocytes respond to P. acnes and secrete mature IL-1ß partially via the NLRP3-mediated pathway. When monocytes were stimulated with live P. acnes, caspase-1 and caspase-5 gene expression was upregulated; however, IL-1ß secretion required only caspase-1 activity. P. acnes induced key inflammasome genes including NLRP1 and NLPR3. Moreover, silencing of NLRP3, but not NLRP1, expression by small interfering RNA attenuated P. acnes-induced IL-1ß secretion. The mechanism of P. acnes-induced NLRP3 activation and subsequent IL-1ß secretion was found to involve potassium efflux. Finally, in acne lesions, mature caspase-1 and NLRP3 were detected around the pilosebaceous follicles and colocalized with tissue macrophages. Taken together, our results indicate that P. acnes triggers a key inflammatory mediator, IL-1ß, via NLRP3 and caspase-1 activation, suggesting a role for inflammasome-mediated inflammation in acne pathogenesis.

Propionibacterium acnes Induces an IL-17 Response in Acne Vulgaris that Is Regulated by Vitamin A and Vitamin D.

Acne vulgaris is the most common skin disorder affecting millions of people worldwide and inflammation resulting from the immune response targeting Propionibacterium acnes has a significant role in its pathogenesis. In this study, we have demonstrated that P. acnes is a potent inducer of T helper 17 (Th17) and Th1, but not Th2 responses in human peripheral blood mononuclear cells (PBMCs). P. acnes stimulated expression of key Th17-related genes, including IL-17A, RORα, RORc, IL-17RA, and IL-17RC, and triggered IL-17 secretion from CD4(+), but not from CD8(+) T cells. Supernatants from P. acnes-stimulated PBMCs were sufficient to promote the differentiation of naive CD4(+)CD45RA T cells into Th17 cells. Furthermore, we found that the combination of IL-1ß, IL-6, and transforming growth factor-ß-neutralizing antibodies completely inhibited P. acnes-induced IL-17 production. Importantly, we showed that IL-17-expressing cells were present in skin biopsies from acne patients but not from normal donors. Finally, vitamin A (all-trans retinoic acid) and vitamin D (1,25-dihydroxyvitamin D3) inhibited P. acnes-induced Th17 differentiation. Together, our data demonstrate that IL-17 is induced by P. acnes and expressed in acne lesions and that both vitamin A and D could be effective tools to modulate Th17-mediated diseases such as acne.

Enhanced antitumor activity induced by adoptive T-cell transfer and adjunctive use of the histone deacetylase inhibitor LAQ824.

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes.

Broad antitumor protection by dendritic cells administered to CD8alpha knock out mice.

Dendritic cell (DC) administration to CD8alpha knock-out (CD8alphaKO) mice results in a strong antigen-non-specific protection to a B16 murine melanoma tumor challenge. This response is mediated by lytic NK cells and cytokine-producing CD4 cells. We aimed to determine the signals that guide tumor targeting of this response. CD8alphaKO mice in the C57BL/6 background received subcutaneous (s.c.) injections of immature DC. Mice were challenged in vivo or assayed for lytic activity in vitro to a panel of syngeneic tumors with different levels of MHC class I expression. These studies support the following conclusions: (1) DC administration to CD8alphaKO mice results in protective in vivo responses to syngeneic tumors from epithelial, neuroectodermal and hematopoietic origin; in vivo protection is independent of the level of MHC classes I and II expression. (2) The in vitro lytic activity of DC-activated NK cells from CD8alphaKO mice has sensitive and insensitive targets, which is independent of the cell lineage or the level of inhibitory self-MHC surface molecules. (3) In sensitive targets a putative activating NK ligand in DC-stimulated NK cells from CD8alphaKO mice signals directly to PI3-K, but is distinct from NKG2D.

Immunosensitization of tumor cells to dendritic cell-activated immune responses with the proteasome inhibitor bortezomib (PS-341, Velcade).

Proteasome inhibition results in proapoptotic changes in cancer cells, which may make them more sensitive to immune effector cells. We established a murine model to test whether the proteasome inhibitor bortezomib could sensitize established B16 melanoma tumors to dendritic cell (DC)-activated immune effector cells. Day 3-established s.c. B16 tumors had significantly decreased tumor outgrowth when treated with a combination of bortezomib and DC, regardless of whether the DC were loaded or not with a tumor Ag. In vivo Ab-depletion studies demonstrated that the effector cells were NK and CD8+ cells, but not CD4+ cells. NF-kappaB nuclear transcription factor assay and gene-expression profiling of B16 treated with bortezomib was consistent with inhibition of NF-kappaB target genes leading to a proapoptotic phenotype. In vitro lytic assays demonstrated that TNF-alpha, but not perforin, Fas-ligand, or TRAIL, was responsible for bortezomib-sensitized B16 cytotoxicity. In conclusion, the proteasome inhibitor bortezomib can pharmacologically sensitize tumor cells to the lytic effects of DC-activated immune effector cells.

Rapid nitric oxide-mediated S-nitrosylation of estrogen receptor: regulation of estrogen-dependent gene transcription.

Nitric oxide (NO) and estrogen receptor (ER) are both important mediators of signal transduction in cardiovascular and reproductive tissues. In this study, we evaluated NO-mediated S-nitrosylation of ER and assessed the effect of this structural modification on transcription-related functions of ER. We have found selective inhibitory effects of NO on specific binding of ER to specific estrogen-responsive elements (ERE) that can be reversed in the presence of the reducing agent, DTT, thus suggesting that S-nitrosylation of thiolate-zinc centers may occur within the ER molecule. Furthermore, we examined inhibitory effects of NO on ER-dependent transcriptional activity by using an ERE-driven reporter gene system. By monitoring biophysical changes in the structure of NO-treated or untreated human recombinant ERalpha,we obtained evidence for the formation of S-nitrosothiols in the ER molecule. In addition, we have detected specific S-nitrosylation of cysteine residues within the ER molecule by immunodetection of S-nitrosocysteine moieties in ER. Collectively, these findings suggest an important physiological role for NO in modification of human ER structure by S-nitrosylation, an effect that leads, in turn, to impaired DNA-binding activity of ER and subsequent blockade of estrogen-dependent gene transcription. Thus, NO-induced S-nitrosylation of ER can occur at cysteine residues that coordinate Zn2+ within the two major DNA-binding Zn-finger domains of ER, resulting in selective inhibition of DNA-binding at specific ERE. This cross-communication between NO and ER may favor activation of rapid (nongenomic) signaling pathways and subsequent modulation of downstream genomic activity.

Estrogen receptor-mediated vascular responsiveness to nebivolol: a novel endothelium-related mechanism of therapeutic vasorelaxation.

Nebivolol is a highly selective and lipophilic beta1-adrenergic receptor antagonist with vasodilating characteristics attributed mainly to endothelial generation of nitric oxide (NO). Coincidently, rapid vascular vasodilating effects of estradiol are also widely reported and membrane-initiated signaling by estrogen receptor (ER), leading to generation of NO, parallels the vasodilating effects observed for nebivolol. Thus, we hypothesized that the NO-dependent vasodilating effect attributed to nebivolol may be partially mediated through its interaction with the membrane-associated form of ER. The objective of this study was to examine the ER-mediated/endothelium-dependent vascular responses to nebivolol and the specific binding properties of nebivolol to ER. In isolated rat aortic rings, the endothelium-dependent vasodilating effect of nebivolol was significantly blocked by the use of the potent ER antagonist, ICI 182,780. In contrast, in the absence of intact endothelium, nebivolol-induced vasorelaxation was not blocked by ICI 182,780, strongly suggesting that nebivolol-elicited vasorelaxation is partially dependent on ER-mediated pathways. Further, we examined the binding of nebivolol to ER (MCF-7 cells) by radioligand binding assay and revealed specific binding kinetics with an estimated DC50 of 5 x 10 M, coinciding with the approximate EC50 of nebivolol as a vasorelaxant. In conclusion, the endothelium-dependent vascular response to nebivolol is attributed partially to its interaction with ER.

Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase.

Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and beta-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-beta-gal) and "gutless" AdV (AdV-CMV-PnNOS; AdV-CMV-beta-gal) vectors, and injected into the penis of adult (beta-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of beta-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-beta-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 10(6) viral particles (vp) of AdV-CMV-beta-gal, and with 10(7) vp beta-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (10(7) vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads.

Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase.

Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and beta-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-beta-gal) and "gutless" AdV (AdV-CMV-PnNOS; AdV-CMV-beta-gal) vectors, and injected into the penis of adult (beta-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of beta-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-beta-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 10(6) viral particles (vp) of AdV-CMV-beta-gal, and with 10(7) vp beta-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (10(7) vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads.