Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Inflammation has been recognized as a risk factor for this disease. Heme oxygenase 1 (HO-1), the inducible isoform of the rate-limiting enzyme in heme degradation, counteracts oxidative and inflammatory damage. Here, we investigated the regulated expression of HO-1 and its functional consequences in PCa. We studied the effect of genetic and pharmacologic disruption of HO-1 in the growth, invasion, and migration in androgen-sensitive (MDA PCa2b and LNCaP) and androgen-insensitive (PC3) PCa cell lines. Our results show that HO-1 levels are markedly decreased in PC3 compared with MDA PCa2b and LNCaP. Hemin treatment increased HO-1 at both protein and mRNA levels in all cell lines and decreased cell proliferation and invasion. Furthermore, overexpression of HO-1 in PC3 resulted in markedly reduced cell proliferation and migration. Accordingly, small interfering RNA-mediated silencing of HO-1 expression in MDA PCa2b cells resulted in increased proliferation and invasion. Using reverse transcription-quantitative PCR-generated gene array, a set of inflammatory and angiogenic genes were upregulated or downregulated in response to HO-1 overexpression identifying matrix metalloprotease 9 (MMP9) as a novel downstream target of HO-1. MMP9 production and activity was downregulated by HO-1 overexpression. Furthermore, PC3 cells stably transfected with HO-1 (PC3HO-1) and controls were injected into nu/nu mice for analysis of in vivo tumor xenograft phenotype. Tumor growth and MMP9 expression was significantly reduced in PC3HO-1 tumors compared with control xenografts. Taken together, these results implicate HO-1 in PCa cell migration and proliferation suggesting its potential role as a therapeutic target in clinical settings. (Mol Cancer Res 2009;7(11):1745-55)

The role of oxidative stress in prostate cancer has been increasingly recognised. Acute and chronic inflammations generate reactive oxygen species that result in damage to cellular structures. Haeme oxygenase-1 (HO-1) has cytoprotective effects against oxidative damage. We hypothesise that modulation of HO-1 expression may be involved in the process of prostate carcinogenesis and prostate cancer progression. We thus studied HO-1 expression and localisation in 85 samples of organ-confined primary prostate cancer obtained via radical prostatectomy (Gleason grades 4 -9) and in 39 specimens of benign prostatic hyperplasia (BPH). We assessed HO-1 expression by immunohistochemical staining. No significant difference was observed in the cytoplasmic positive reactivity among tumours (84%), non-neoplastic surrounding parenchyma (89%), or BPH samples (87%) (P ¼ 0.53). Haeme oxygenase-1 immunostaining was detected in the nuclei of prostate cancer cells in 55 of 85 (65%) patients but less often in nonneoplastic surrounding parenchyma (30 of 85, 35%) or in BPH (9 of 39, 23%) (Po0.0001). Immunocytochemical and western blot analysis showed HO-1 only in the cytoplasmic compartment of PC3 and LNCaP prostate cancer cell lines. Treatment with hemin, a well-known specific inducer of HO-1, led to clear nuclear localisation of HO-1 in both cell lines and highly induced HO-1 expression in both cellular compartments. These findings have demonstrated, for the first time, that HO-1 expression and nuclear localisation can define a new subgroup of prostate cancer primary tumours and that the modulation of HO-1 expression and its nuclear translocation could represent new avenues for therapy.

Although platelet-rich plasma (PRP) is used as a source of growth factors in regenerative medicine, its effectiveness remains controversial, partially due to the absence of PRP preparation protocols based on the regenerative role of platelets. Here, we aimed to optimise the protocol by analysing PRP angiogenic and regenerative properties. Three optimising strategies were evaluated: dilution, 4 °C pre-incubation, and plasma cryoprecipitate supplementation. Following coagulation, PRP releasates (PRPr) were used to induce angiogenesis in vitro (HMEC-1 proliferation, migration, and tubule formation) and in vivo (chorioallantoic membrane), as well as regeneration of excisional wounds on mouse skin. Washed platelet releasates induced greater angiogenesis than PRPr due to the anti-angiogenic effect of plasma, which was decreased by diluting PRPr with saline. Angiogenesis was also improved by both PRP pre-incubation at 4 °C and cryoprecipitate supplementation. A combination of optimising variables exerted an additive effect, thereby increasing the angiogenic activity of PRPr from healthy donors and diabetic patients. Optimised PRPr induced faster and more efficient mouse skin wound repair compared to that induced by non-optimised PRPr. Acetylsalicylic acid inhibited angiogenesis and tissue regeneration mediated by PRPr; this inhibition was reversed following optimisation. Our findings indicate that PRP pre-incubation at 4 °C, PRPr dilution, and cryoprecipitate supplementation improve the angiogenic and regenerative properties of PRP compared to the obtained by current methods.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Once a tumor is established it may attain further characteristics via mutations or hypoxia, which stimulate new blood vessels. Angiogenesis is a hallmark in the pathogenesis of cancer and inflammatory diseases that may predispose to cancer. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage and was previously reported to play a key role in prostate carcinogenesis. To gain insight into the anti-tumoral properties of HO-1, we investigated its capability to modulate PCa associated-angiogenesis. In the present study, we identified in PC3 cells a set of inflammatory and pro-angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α and α5β1 integrin. Our results indicated that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stable transfected with HO-1 (PC3HO-1) generated tumours less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Additionally, HO-1 repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, which strongly suggests that HO-1 may regulate angiogenesis through this pathway. Taken together, these data supports a key role of HO-1 as a modulator of the angiogenic switch in prostate carcinogenesis ascertaining it as a logical target for intervention therapy.

Summary Various immunization assays were used to demonstrate the lack of immunogenicity of three BALB/c tumours of spontaneous origin and of a fourth one resulting from foreign body tumorigenesis. All four tumours inhibited the growth of a second implant of the same tumour into the contralateral flank. In our tumour models "concomitant immunity" (1) was not mediated by macrophage or T-cell dependent immune reactions: both thymectomized BALB/c and nude mice (treated or untreated with silica) gave the same results as intact mice; (2) showed some degree of non-specificity, inhibiting the growth of a different tumour in 3/4 cases; though, the existence of a specific component could not be discarded; (3) was proportional to the volume of the primary tumour at the time of the second challenge; (4) was dependent on actively growing primary tumour, not being obtained with progressively increasing daily inocula of irradiated tumour cells; (5) was detectable in an actively growing secondary tumour: recurrent growth after partial surgical excision was inhibited and (6) involved cytostasis of the secondary tumour: a syngeneic graft of the overlying skin led to tumour growth while histological studies revealed the presence of viable tumour cells. It is postulated that "concomitant immunity" or resistance can be generated without the active participation of the immune system and that tumour-related factors are, in certain cases, responsible for blocking the growth of secondary tumours.

Purpose: Clinical and epidemiologic data suggest that obesity is associated with more aggressive forms of prostate cancer, poor prognosis, and increased mortality. C-terminal-binding protein 1 (CtBP1) is a transcription repressor of tumor suppressor genes and is activated by NADH binding. High calorie intake decreases intracellular NAD þ /NADH ratio. The aim of this work was to assess the effect of high-fat diet (HFD) and CtBP1 expression modulation over prostate xenograft growth. Experimental Design: We developed a metabolic syndrome-like disease in vivo model by feeding male nude mice with HFD during 16 weeks. Control diet (CD)-fed animals were maintained at the same conditions. Mice were inoculated with PC3 cells stable transfected with shCtBP1 or control plasmids. Genome-wide expression profiles and Gene Set Enrichment Analysis (GSEA) were performed from PC3. shCtBP1 versus PC3.pGIPZ HFD-fed mice tumors.Results: No significant differences were observed in tumor growth on CD-fed mice; however, we found that only 60% of HFD-fed mice inoculated with CtBP1-depleted cells developed a tumor. Moreover these tumors were significantly smaller than those generated by PC3.pGIPZ control xenografts. We found 823 genes differentially expressed in shCtBP1 tumors from HFD-fed mice. GSEA from expression dataset showed that most of these genes correspond to cell adhesion, metabolic process, and cell cycle.Conclusions: Metabolic syndrome-like diseases and CtBP1 expression cooperate to induce prostate tumor growth. Hence, targeting of CtBP1 expression might be considered for prostate cancer management and therapy in the subset of patients with metabolic syndromes. Clin Cancer Res; 20(15); 4086-95. Ó2014 AACR.

Concomitant tumor resistance (CR) is a phenomenon originally described in 1906 in which a tumor-bearing host is resistant to the growth of secondary tumor implants and metastasis. Although recent studies have indicated that T-cell-dependent processes mediate CR in hosts bearing immunogenic small tumors, manifestations of CR induced by immunogenic and nonimmunogenic large tumors have been associated with an elusive serum factor. In this study, we identify this serum factor as tyrosine in its meta and ortho isoforms. In three different murine models of cancer that generate CR, both meta-tyrosine and ortho-tyrosine inhibited tumor growth. In addition, we showed that both isoforms of tyrosine blocked metastasis in a fourth model that does not generate CR but is sensitive to CR induced by other tumors. Mechanistic studies showed that the antitumor effects of the tyrosine isoforms were mediated, in part, by early inhibition of mitogen-activated protein/extracellular signal-regulated kinase pathway and inactivation of STAT3, potentially driving tumor cells into a state of dormancy. By revealing a molecular basis for the classical phenomenon of CR, our findings may stimulate new generalized approaches to limit the development of metastases that arise after resection of primary tumors, an issue of pivotal importance to oncologists and their patients. Cancer Res; 71(22); 7113-24. Ó2011 AACR.

Activation of the androgen receptor (AR) is a key step in the development of prostate cancer (PCa). Several mechanisms have been identified in AR activation, among them signal transducer and activator of transcription 3 (STAT3) signaling. Disruption of STAT3 activity has been associated to cancer progression. Recent studies suggest that heme oxygenase 1 (HO-1) may play a key role in PCa that may be independent of its catalytic function. We sought to explore whether HO-1 operates on AR transcriptional activity through the STAT3 axis. Our results display that HO-1 induction in PCa cells represses AR activation by decreasing the prostate-specific antigen (PSA) promoter activity and mRNA levels. Strikingly, this is the first report to show by chromatin immunoprecipitation analysis that HO-1 associates to gene promoters, revealing a novel function for HO-1 in the nucleus. Furthermore, HO-1 and STAT3 directly interact as determined by co-immunoprecipitation studies. Forced expression of HO-1 increases STAT3 cytoplasmic retention. When PCa cells were transfected with a constitutively active STAT3 mutant, PSA and STAT3 downstream target genes were abrogated under hemin treatment. Additionally, a significant decrease in pSTAT3 protein levels was detected in the nuclear fraction of these cells. Confocal microscopy images exhibit a decreased rate of AR/STAT3 nuclear co-localization under hemin treatment. In vivo studies confirmed that STAT3 nuclear delimitation was significantly decreased in PC3 tumors overexpressing HO-1 grown as xenografts in nude mice. These results provide a novel function for HO-1 down-modulating AR transcriptional activity in PCa, interfering with STAT3 signaling, evidencing its role beyond heme degradation.