Homeostatic response under carcinogen withdrawal, heme oxygenase 1 expression and cell cycle association

BACKGROUND: Chronic injury deregulates cellular homeostasis and induces a number of alterations leading to disruption of cellular processes such as cell cycle checkpoints and apoptosis, driving to carcinogenesis. The stress protein heme oxygenase-1 (HO-1) catalyzes heme degradation producing biliverdin, iron and CO. Induction of HO-1 has been suggested to be essential for a controlled cell growth. The aim of this work was to analyze the in vivo homeostatic response (HR) triggered by the withdrawal of a potent carcinogen, p-dimethylaminoazobenzene (DAB), after preneoplastic lesions were observed. We analyzed HO-1 cellular localization and the expression of HO-1, Bcl-2 and cell cycle related proteins under these conditions comparing them to hepatocellular carcinoma (HC). METHODS: The intoxication protocol was designed based on previous studies demonstrating that preneoplastic lesions were evident after 89 days of chemical carcinogen administration. Male CF1 mice (n = 18) were used. HR group received DAB (0.5 % w/w) in the diet for 78 days followed by 11 days of carcinogen deprivation. The HC group received the carcinogen and control animals the standard diet during 89 days. The expression of cell cycle related proteins, of Bcl-2 and of HO-1 were analyzed by western blot. The cellular localization and expression of HO-1 were detected by immnunohistochemistry. RESULTS: Increased expression of cyclin E/CDK2 was observed in HR, thus implicating cyclin E/CDK2 in the liver regenerative process. p21(cip1/waf1 )and Bcl-2 induction in HC was restituted to basal levels in HR. A similar response profile was found for HO-1 expression levels, showing a lower oxidative status in the carcinogen-deprived liver. The immunohistochemical studies revealed the presence of macrophages surrounding foci of necrosis and nodular lesions in HR indicative of an inflammatory response. Furthermore, regenerative cells displayed changes in type, size and intensity of HO-1 immunostaining. CONCLUSION: These results demonstrate that the regenerative capacity of the liver is still observed in the pre-neoplastic tissue after carcinogen withdrawal suggesting that reversible mechanism/s to compensate necrosis and to restitute homeostasis are involved

In vivo hemin conditioning targets the vascular and immunologic compartments and restrains prostate tumor development

Purpose: Conditioning strategies constitute a relatively unexplored and exciting opportunity to shape tumor fate by targeting the tumor microenvironment. In this study, we assessed how hemin, a pharmacologic inducer of heme oxygenase-1 (HO-1), has an impact on prostate cancer development in an in vivo conditioning model. Experimental Design: The stroma of C57BL/6 mice was conditioned by subcutaneous administration of hemin prior to TRAMP-C1 tumor challenge. Complementary in vitro and in vivo assays were performed to evaluate hemin effect on both angiogenesis and the immune response. To gain clinical insight, we used prostate cancer patient-derived samples in our studies to assess the expression of HO-1 and other relevant genes. Results: Conditioning resulted in increased tumor latency and decreased initial growth rate. Histologic analysis of tumors grown in conditioned mice revealed impaired vascularization. Hemin-treated human umbilical vein endothelial cells (HUVEC) exhibited decreased tubulogenesis in vitro only in the presence of TRAMP-C1-conditioned media. Subcutaneous hemin conditioning hindered tumor-associated neovascularization in an in vivo Matrigel plug assay. In addition, hemin boosted CD8+ T-cell proliferation and degranulation in vitro and antigen-specific cytotoxicity in vivo. A significant systemic increase in CD8+ T-cell frequency was observed in preconditioned tumor-bearing mice. Tumors from hemin-conditioned mice showed reduced expression of galectin-1 (Gal-1), key modulator of tumor angiogenesis and immunity, evidencing persistent remodeling of the microenvironment. We also found a subset of prostate cancer patient-derived xenografts and prostate cancer patient samples with mild HO-1 and low Gal-1 expression levels. Conclusions: These results highlight a novel function of a human-used drug as a means of boosting the antitumor response

Heme oxygenase-1 in the forefront of a multi-molecular network that governs cell–cell contacts and filopodia-induced zippering in prostate cancer

Prostate cancer (PCa) cells display abnormal expression of cytoskeletal proteins resulting in an augmented capacity to resist chemotherapy and colonize distant organs. We have previously shown that heme oxygenase 1 (HO-1) is implicated in cell morphology regulation in PCa. Here, through a multi 'omics' approach we define the HO-1 interactome in PCa, identifying HO-1 molecular partners associated with the integrity of the cellular cytoskeleton. The bioinformatics screening for these cytoskeletal-related partners reveal that they are highly misregulated in prostate adenocarcinoma compared with normal prostate tissue. Under HO-1 induction, PCa cells present reduced frequency in migration events, trajectory and cell velocity and, a significant higher proportion of filopodia-like protrusions favoring zippering among neighboring cells. Moreover forced expression of HO-1 was also capable of altering cell protrusions in transwell co-culture systems of PCa cells with MC3T3 cells (pre-osteoblastic cell line). Accordingly, these effects were reversed under siHO. Transcriptomics profiling evidenced significant modulation of key markers related to cell adhesion and cell–cell communication under HO-1 induction. The integration from our omics-based research provides a four molecular pathway foundation (ANXA2/HMGA1/POU3F1; NFRSF13/GSN; TMOD3/RAI14/VWF; and PLAT/PLAU) behind HO-1 regulation of tumor cytoskeletal cell compartments. The complementary proteomics and transcriptomics approaches presented here promise to move us closer to unravel the molecular framework underpinning HO-1 involvement in the modulation of cytoskeleton pathways, pushing toward a less aggressive phenotype in PCa

The MD Anderson prostate cancer patient-derived xenograft series (MDA PCa PDX) captures the molecular landscape of prostate cancer and facilitates marker-driven therapy development

BACKGROUND: Advances in prostate cancer (PCa) lag behind other tumor types partly due to the paucity of models reflecting key milestones in PCa progression. OBJECTIVE: To develop clinically relevant PCa models. DESIGN: Since 1996 we have generated clinically annotated patient-derived xenografts (PDXs) (the MDA PCa PDX series) linked to specific phenotypes reflecting all aspects of clinical PCa. RESULTS: We studied two cell line-derived xenografts and the first 80 PDXs derived from 47 human PCa donors. Of these, 47 PDXs derived from 22 donors are working models and can be expanded either as cell lines (MDA PCa 2a and 2b) or PDXs. The histopathologic, genomic, and molecular characteristics (AR, ERG, and PTEN loss) maintain fidelity with the human tumor and correlate with published findings. PDX growth response to mouse castration and targeted therapy illustrate their clinical utility. Comparative genomic hybridization and sequencing show significant differences in oncogenic pathways in pairs of PDXs derived from different areas of the same tumor. We also identified a recurrent focal deletion in an area that includes the SPOPL gene in PDXs derived from 7 human donors out of 28 studied (25%). SPOPL is a SPOP paralog, and SPOP mutations define a molecular subclass of PCa. SPOPL deletions are found in 7% of TCGA PCas, which suggests that our cohort is a reliable platform for targeted drug development. CONCLUSIONS: The MDA PCa PDX series is a dynamic resource that captures the molecular landscape of PCas progressing under novel treatments and enables optimization of PCa-specific, marker-driven therapy

δ-Aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines

BACKGROUND: Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, δ-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines. RESULTS: We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations

Anandamide Capacitates Bull Spermatozoa through CB1 and TRPV1 Activation

Anandamide (AEA), a major endocannabinoid, binds to cannabinoid and vanilloid receptors (CB1, CB2 and TRPV1) and affects many reproductive functions. Nanomolar levels of anandamide are found in reproductive fluids including mid-cycle oviductal fluid. Previously, we found that R(+)-methanandamide, an anandamide analogue, induces sperm releasing from bovine oviductal epithelium and the CB1 antagonist, SR141716A, reversed this effect. Since sperm detachment may be due to surface remodeling brought about by capacitation, the aim of this paper was to investigate whether anandamide at physiological concentrations could act as a capacitating agent in bull spermatozoa. We demonstrated that at nanomolar concentrations R(+)-methanandamide or anandamide induced bull sperm capacitation, whereas SR141716A and capsazepine (a TRPV1 antagonist) inhibited this induction. Previous studies indicate that mammalian spermatozoa possess the enzymatic machinery to produce and degrade their own AEA via the actions of the AEA-synthesizing phospholipase D and the fatty acid amide hydrolase (FAAH) respectively. Our results indicated that, URB597, a potent inhibitor of the FAAH, produced effects on bovine sperm capacitation similar to those elicited by exogenous AEA suggesting that this process is normally regulated by an endogenous tone. We also investigated whether anandamide is involved in bovine heparin-capacitated spermatozoa, since heparin is a known capacitating agent of bovine sperm. When the spermatozoa were incubated in the presence of R(+)-methanandamide and heparin, the percentage of capacitated spermatozoa was similar to that in the presence of R(+)-methanandamide alone. The pre-incubation with CB1 or TRPV1 antagonists inhibited heparin-induced sperm capacitation; moreover the activity of FAAH was 30% lower in heparin-capacitated spermatozoa as compared to control conditions. This suggests that heparin may increase endogenous anandamide levels. Our findings indicate that anandamide induces sperm capacitation through the activation of CB1 and TRPV1 receptors and could be involved in the same molecular pathway as heparin in bovines

Transcriptional Autoregulation by BRCA1

The BRCA1 gene product plays numerous roles in regulating genome integrity. Its role in assembling supermolecular complexes in response to DNA damage has been extensively studied; however, much less is understood about its role as a transcriptional coregulator. Loss or mutation is associated with hereditary breast and ovarian cancers, whereas altered expression occurs frequently in sporadic forms of breast cancer, suggesting that the control of BRCA1 transcription might be important to tumorigenesis. Here, we provide evidence of a striking linkage between the roles for BRCA1 as a transcriptional coregulator with control of its expression via an autoregulatory transcriptional loop. BRCA1 assembles with complexes containing E2F-1 and RB to form a repressive multicomponent transcriptional complex that inhibits BRCA1 promoter transcription. This complex is disrupted by genotoxic stress, resulting in the displacement of BRCA1 protein from the BRCA1 promoter and subsequent upregulation of BRCA1 transcription. Cells depleted of BRCA1 respond by upregulating BRCA1 transcripts, whereas cells overexpressing BRCA1 respond by downregulating BRCA1 transcripts. Tandem chromatin immmunoprecipitation studies show that BRCA1 is regulated by a dynamic coregulatory complex containing BRCA1, E2F1, and Rb at the BRCA1 promoter that is disrupted by DNA-damaging agents to increase its transcription. These results define a novel transcriptional mechanism of autoregulated homeostasis of BRCA1 that selectively titrates its levels to maintain genome integrity in response to genotoxic insult.Fil: de Siervi, Adriana. National Cancer Institute; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de Luca, Paola. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Byun, Jung S.. National Cancer Institute; Estados UnidosFil: Di, Li Jun. National Cancer Institute; Estados UnidosFil: Fufa, Temesgen. National Cancer Institute; Estados UnidosFil: Haggerty, Cynthia M.. National Cancer Institute; Estados UnidosFil: Vazquez, Elba Susana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Moiola, Cristian Pablo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Longo, Dan L.. National Institute on Aging; Estados UnidosFil: Gardner, Kevin. National Cancer Institute; Estados Unido