HIF-1alpha Levels Research Articles

Observation of the effect of clopidogrel combined with aspirin in the treatment of ischemic cerebrovascular disease and its influence on serum HIF-1 alpha and caspase-3

Objective To compare the effect of clopidogrel combined with aspirin and dipyridamole combined with aspirin in the treatment of ischemic cerebrovascular disease, and to analyze its influence on the levels of serum hypoxia-inducible factor 1 alpha(HIF-1 alpha), cysteine proteinase 3(caspase-3). Methods 96 patients with ischemic cerebrovascular disease were selected, and they were randomly divided into A group(n=48) and B group(n=48) according to the digital table.A group was treated with clopidogrel and aspirin, B group was treated with dipyridamole and aspirin.The clinical efficacy, adverse reactions and serum HIF-1 alpha, caspase-3 expression before and after treatment were compared between the two groups. Results The total effective rate of A group was 93.8%, which was significantly higher than 83.3% of B group (χ2=12.760, P 0.05). After 4 weeks of treatment, the MESSS score of A group was (5.07±1.26)points, which of B group was (6.12±1.48)points, the MESSS scores of the two groups were decreased significantly than those before treatment, and the MESSS score of A group after treatment was significantly lower than that of B group(t=7.306, P 0.05). After 4 weeks of treatment, the levels of HIF-1 and caspase-3 in A group were (248.55±12.44)ng/mL, (7.34±1.77)ng/mL, respectively, which in B group were (301.63±14.86)ng/mL, (8.85±1.94)ng/mL, respectively.The levels of HIF-1 alpha and caspase-3 levels in A group were significantly lower than those in B group (t=8.778, 9.156, all P<0.05). The incidence rate of adverse reaction of A group was 4.2%, which was significantly lower than 10.4% of B group(χ2=8.461, P<0.05). Conclusion Compared with dipyridamole combined with aspirin, clopidogrel combined with aspirin in the treatment of ischemic cerebrovascular disease can effectively improve the patients' neurological impairment, inhibit the expression of serum HIF-1, caspase-3, with less adverse reaction, and it is worthy of application and promotion. Key words: Cerebrovascular disorders; Clopidogrel; Aspirin; Hypoxia-inducible factor 1; Cysteine Proteases

The Influence of HIF-1&amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt; Expression on Apoptosis and Number of T Lymphocyte in Peyer’s Patches after Burn with Delayed Fluid Resuscitation in Rats at Plateau

Objective: To research the expression of hypoxia inducible factor-1 alpha (HIF-1 alpha) on the apoptosis and number of T lymphocyte in Peyer’s patches after severe burn on plateau in rats. Methods: Wistar rats (n = 130) were subjected to deep thickness burn injury (30% TBSA, III degree), at two different altitudes. 60 of them were given delayed fluid resuscitation (DFR, n = 30 at each altitude) 6 h after burn at different altitude; 60 of them were carried out immediate fluid resuscitation (IFR, n = 30 at each altitude); 10 rats were subjected to 37°C warm water as sham burn (SG, n = 10). The Peyer’s patches were harvested from the ileum of rats at different time point after burn respectively. The expression of HIF-1 alpha, CD3(+) and the apoptosis and number of T lymphocyte in Peyer’s patches were detected by tissue microarray technology and immunohistochemistry. Results: The apoptosis was higher in DFR group than that in IFR group. The increase in HIF-1 alpha expression was observed mainly on cell nucleus in T lymphocytes. The expression levels of HIF-1 alpha in Peyer’s patches were much higher in DFR group and IFR group than those in SG, and they were higher at high altitude (3848 metres) than those at lower altitude (1517 metres), and also higher in DFR group compared with IFR group (all P + in Peyer’s patches were much lower in DFR group and IFR group than those in sham group, and the lowest value appeared at 12 hours after burn (all P

MDM2 and HIF1alpha expression levels in different histologic subtypes of malignant pleural mesothelioma: correlation with pathological and clinical data.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited treatment options. Sarcomatoid/biphasic mesotheliomas are characterized by more aggressive behaviour and a poorer prognosis compared with the epithelioid subtype. To date prognostic and tailored therapeutic biomarkers are lacking. The present study analyzed the expression levels of MDM2 and HIF1alpha in different histologic subtypes from chemonaive MPM patients. Diagnostic biopsies of MPM patients from four Italian cancer centers were centrally collected and analyzed. MDM2 and HIF1alpha expression levels were investigated through immunohistochemistry and RT-qPCR. Pathological assessment of necrosis, inflammation and proliferation index was also performed. Molecular markers, pathological features and clinical characteristics were correlated to overall survival (OS) and progression free survival (PFS). Sixty MPM patients were included in the study (32 epithelioid and 28 non-epithelioid). Higher levels of MDM2 (p < 0.001), HIF1alpha (p = 0.013), necrosis (p = 0.013) and proliferation index (p < 0.001) were seen mainly in sarcomatoid/biphasic subtypes. Higher levels of inflammation were significantly associated with epithelioid subtype (p = 0.044). MDM2 expression levels were correlated with HIF1alpha levels (p = 0.0001), necrosis (p = 0.008) and proliferation index (p = 0.009). Univariate analysis showed a significant correlation of non-epithelioid histology (p = 0.04), high levels of necrosis (p = 0.037) and proliferation index (p = 0.0002) with shorter PFS. Sarcomatoid/biphasic and epithelioid mesotheliomas showed different MDM2 and HIF1alpha expression levels and were characterized by different levels of necrosis, proliferation and inflammation. Further studies are warranted to confirm a prognostic and predictive role of such markers and features.

Abstract 3846: DNA double-strand breaks in hypoxic but not in normoxic conditions determine different radiosensitivity of tumors

Abstract Purpose: While only few biomarkers like tumor histology and stage are used in routine clinical practice to predict tumor cure probability after chemo-radiotherapy, sustained efforts are made to establish more precise predictors for individual treatment. Recently chemotherapy corresponded with radiotherapy (CCR) to oral cancer has developed treatment efficiency of killing tumor cells. However we can't distinguish the number of tumor cells that may cause recurrences after radiotherapy. Then we hypothesized that hypoxic condition may have some influence of cell death by irradiation, and we tried to demonstrate how the correlation of phosphorylated H2AX (γH2AX) was influenced by oxygenation status of the tumor regions to predict radiation treatment response. Results: We used oral squamous cell carcinoma (OSCC) cell lines and divided them into two groups that showed hypoxic response or not. HSC-2 cells increased the expression level of HIF-1 alpha under hypoxia. No difference was observed in G1 arrest to irradiation of 6Gy in these cell lines. While the expression revel of γH2AX decreased in HSC-2, its revel didn't change under hypoxic condition in HSC-3 and SAS. From these findings, we postulate that one of the mechanism by which hypoxic cells become resistant to irradiation may involve the expression of HIF-1 alpha. Conclusion: The γH2AX is highly dependent on tumor cell oxygenation. Further studied are ongoing to establish the potentially important role of γH2AX under hypoxia. Citation Format: Kazuhiro Ogi, Mayumi Kawata, Takafumi Nakagaki, Kousuke Takeda, Miyazaki Akihiro, Hiratsuka Hiroyoshi. DNA double-strand breaks in hypoxic but not in normoxic conditions determine different radiosensitivity of tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3846. doi:10.1158/1538-7445.AM2015-3846

Left-Biased Spermatogenic Failure in 129/SvJ Dnd1Ter/+ Mice Correlates with Differences in Vascular Architecture, Oxygen Availability, and Metabolites.

Homozygosity for the Ter mutation in the RNA-binding protein Dead end 1 (Dnd1(Ter/Ter)) sensitizes germ cells to degeneration in all mouse strains. In 129/SvJ mice, approximately 10% of Dnd1(Ter/+) heterozygotes develop spermatogenic failure, and 95% of unilateral cases occur in the left testis. The first differences between right and left testes were detected at Postnatal Day 15 when many more spermatogonial stem cells (SSCs) were undergoing apoptosis in the left testis compared to the right. As we detected no significant left/right differences in the molecular pathway associated with body axis asymmetry or in the expression of signals known to promote proliferation, differentiation, and survival of germ cells, we investigated whether physiological differences might account for asymmetry of the degeneration phenotype. We show that left/right differences in vascular architecture are associated with a decrease in hemoglobin saturation and increased levels of HIF-1alpha in the left testis compared to the right. In Dnd1 heterozygotes, lower oxygen availability was associated with metabolic differences, including lower levels of ATP and NADH in the left testis. These experiments suggest a dependence on oxygen availability and metabolic substrates for SSC survival and suggest that Dnd1(Ter/+) SSCs may act as efficient sensors to detect subtle environmental changes that alter SSC fate.

Heat-shock-protein 90 protects from downregulation of HIF-1α in calcineurin-induced myocardial hypertrophy

Aim of the studyCapillary/myocyte mismatch is a hallmark of maladaptive myocardial hypertrophy, but the exact mechanisms of this phenomenon remain unknown. We therefore aimed to evaluate the role of calcineurin A in the regulation of hypoxia-inducible factor-1 alpha (HIF-1 alpha) in a calcineurin overexpressing mouse model of myocardial hypertrophy. Methods and resultsMice overexpressing calcineurin A (CnATg) showed persistent upregulation of HIF-1 alpha protein without evidence of a reduction in capillary density despite progressive myocardial hypertrophy. Likewise, overexpression of calcineurin A in isolated cardiomyocytes induced upregulation of HIF-1 alpha protein. In contrast, NFAT-overexpression had no such effect, implying that NFAT-independent mechanisms were responsible for increased HIF-1 alpha levels. In addition, inhibition of HSP90 via the HSP90-inhibitor 17-AAG or siRNA abolished calcineurin A-induced upregulation of HIF-1 alpha. Consequently, upregulation of HIF-1 alpha target genes like VEGF-A, BNIP-3 or PGK-1 was also inhibited by either 17-AAG or siRNA directed against HSP90. Finally, when CnATg mice were treated with 17-AAG, they demonstrated reduced left ventricular function and capillary density. ConclusionsWe describe here for the first time that overexpression of the phosphatase calcineurin A prevents the development of a capillary/myocyte mismatch despite progressive myocardial hypertrophy. This effect was mediated by HSP-90 induced stabilization of HIF-1 alpha. Further work is needed to understand this unexpected cardioprotective effect of calcineurin A.

Maternal omega-3 fatty acid supplementation to a vitamin B12 deficient diet normalizes angiogenic markers in the pup brain at birth

Vitamin B12 and omega-3 fatty acids are critical for normal brain development and function and their deficiencies during pregnancy could have adverse effects on cognitive performance in children. Our earlier studies indicate that both maternal vitamin B12 and omega-3 fatty acids influence brain development by regulating the levels of neurotrophins. Literature suggests that there exists a cross talk between neurotrophins like nerve growth factor (NGF) and angiogenic factors like vascular endothelial growth factor (VEGF). It remains to be established whether maternal nutrients like vitamin B12 and omega-3 fatty acids influence the levels of angiogenic markers like VEGF and NGF in the brain of the offspring. Therefore the present study examines the effect of maternal vitamin B12 and omega-3 fatty acids on protein and mRNA levels of VEGF, HIF-1 alpha (hypoxia inducible factor alpha) and NGF in the pup brain at birth. Pregnant Wistar rats were divided into five dietary groups (n=8 each): control, vitamin B12 deficient, vitamin B12 deficient+omega-3 fatty acid, vitamin B12 supplemented, vitamin B12 supplemented+omega-3 fatty acid. At birth the pups were dissected to collect the brain tissue. Maternal vitamin B12 deficiency showed lower (p<0.05) pup brain mRNA and protein levels (p<0.01) of VEGF, higher (p<0.01) HIF-1 alpha protein levels, lower (p<0.05) NGF protein levels while NGF mRNA levels were not altered. Omega-3 fatty acid supplementation to a vitamin B12 deficient group normalized the VEGF mRNA levels, NGF protein levels and HIF-1 alpha protein levels. Vitamin B12 supplementation showed similar protein and mRNA levels of VEGF and NGF as well as HIF-1 alpha protein levels as compared to control. Omega-3 fatty acid supplementation to the vitamin B12 supplemented group showed higher (p<0.01) protein and mRNA levels of NGF but the protein and mRNA levels of VEGF were comparable to control. In conclusion maternal vitamin B12 and omega-3 fatty acids both influence the levels and expression of neurotrophins and angiogenic factors in the offspring brain suggesting a possible benefit of combined maternal supplementation of these vital nutrients.

Abstract 727: Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis

Abstract Humans are unable to synthesise ascorbate (vitamin C) due to a mutation in the gene for gulono-lactone oxidase (Gulo), the terminal enzyme in the synthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including its action as a vital cofactor for Cu- and Fe-containing enzymes. As enzyme cofactor for HIF-hydroxylases, it is important for the regulation of hypoxia-inducible factor-1 (HIF-1), which governs cancer growth and spread. A lack of ascorbate increases both the level and activity of HIF-1 in cell culture. This study aimed to restore ascorbate synthesis to human cells and to determine the effect of intracellular ascorbate biosynthesis on HIF-1 levels. HepG2 cells were gene modified using Lipofectamine LTX with a plasmid encoding the mouse Gulo cDNA and a control plasmid encoding the green fluorescent protein (GFP). Modified HepG2 cells were tested for genomic incorporation by PCR, for Gulo enzyme production by Western blotting, and ascorbate synthesis by high performance liquid chromatography with electrochemical detection (HPLC-ECD). Protein levels of HIF-1alpha in hypoxic cells were measured by Western blot analysis. Parental HepG2 cells accumulated a mean of 120 nmol ascorbate per 1 million cells when loaded for 24h with 0.5 mM ascorbate. Gene-modified HepG2Gulo cells demonstrated a change in morphology and an increase in adherence, compared to HepG2GFP and parental HepG2 cells. A PCR-positive stable clone was able to synthesise ascorbate but only when the Gulo substrate, L-gulono-1,4-lactone, was supplied. Intracellular ascorbate levels then reached 5% of saturation levels. Hypoxic HIF-1 accumulation was reduced by addition of gulonolactone to this clone. However, gulonolactone also reduced HIF-1 levels in parental HepG2 cells, suggesting that it may similarly act as cofactor for the HIF-hydroxlases. This study indicated that the ascorbate biosynthesis pathway in human cells is significantly modified and may contain numerous non-functional members besides gulono-lactone oxidase. Future biochemical studies will determine which other enzymes are non-functional. Citation Format: Teresa Flett, Elizabeth Campbell, Elisabeth Phillips, Margreet CM Vissers, Gabi U. Dachs. Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2014-727

P01.06 * HIF-1 AND HIF-2 AS POSIBLE NEW TARGETS IN THE TREATMENT OF VON HIPPEL-LINDAU DISEASE HEMANGIOBLASTOMAS

BACKGROUND: Von Hippel-Lindau disease (VHL)is a genetic condition predisposing to the development of multiple specific tumors, mainly CNS and retinal hemangioblastomas and clear cell renal cancer (CCRC). Protein pVHL, codified by VHL gene, is an important component of the functional complex responsible for degrading hypoxia inducible factors (HIF1alpha and HIF2alpha). In hypoxic condition, or in absence of functional protein VHL, an accumulation of HIF results, and thus an activation of gene transcription related to angiogenesis, and cell proliferation and survival. A number of works supports a role of unregulated amplification of HIF activity and of proteins codified by HIF-inducible genes as oncogenic, promoting the development of some tumors as CCRC, in VHL patients. Their role is still unknown in the appearance of hemangioblastomas, as there is no information about the level of synthesis and transcription of HIF proteins in these tumors. The main objective of our study is to determine the levels of proteins HIF-1alpha and HIF-2alpha and the eventual associated VEGF variation. MATERIAL AND METHODS: Tissue from nine hemangioblastomas (cerebellar, spinal cord, brainstem, brain) resected from VHL patients was obtained immediately after each tumor resection, and cultured for growing. Six primary cultures were obtained, and analyzed by Flow Cytometry for cell population component evaluation and by Western-Blot in order to measure the levels of proteins HIF-1alpha, HIF-2alpha and of VEGF. RESULTS: Cellular characterization of the 6 primary cultures obtained from 9 hemangioblastomas has shown they are composed of stromal cells (more than 50% CD99 + cells), endothelial cells (15% CD34+ cells), and pericytes (30% NG2+ cells). In the study, 25-40% of stromal cells, 3-15% of endothelial cells, and 5-30% of pericytes had increased levels of HIF-1alpha and HIF-2alpha. Furthermore, Western-Blot assay showed a very high concentration of VEGF in hemangioblastoma cells, 2 to 4 times more than in cell lines characterized by over-expression of VEGF (lines derived from colon cancer and from retinal pigmentary epithelium). No correlation of HIF expression in culture and cell proliferation in the original tumor (Mib-1) has been found. CONCLUSIONS: Our results show that proteins HIF-1alpha and HIF-2alpha are over-expressed in hemangioblastomas from VHL patients, suggesting a role of these proteins in the development and growth of these tumors. The results support the exploration of possible treatments directed to inactivate HIF1alpha and HIF-2alpha proteins as new targets, in order to control de proliferation of hemangioblastomas in these patients.

Activity and expression pattern of NF-κB /P65 in peripheral blood from hepatocellular carcinoma patients - link to hypoxia inducible factor -1α.

Hepatocellular carcinoma is a complex and heterogeneous tumor with poor prognosis due to frequent intrahepatic spread and extrahepatic metastasis. The molecular mechanisms underlying HCC pathogenesis still remain obscure. We aimed to investigate the abundance and the DNA binding activity of nuclear factor kappa B/p65 subunit in peripheral blood mononuclear cells from patients with HCC and to assess its prognostic significance and association with hypoxia inducible factor one alpha (HIF-1α) in blood. This study was carried out on 40 patients classified equally into liver cirrhosis (group I) and HCC (group II), in addition to 20 healthy volunteers (group III). All groups were subjected to measurement of NF-κB /P65 subunit expression levels by real time-PCR, and DNA binding activity was evaluated by transcription factor binding immunoassay. Serum HIF-1α levels were estimated by enzyme-linked immunosorbent assay (ELISA). Significant increase of both the expression level and DNA binding activity of NF-κB /P65 subunit together with serum HIF-1 alpha levels was noted in HCC patients compared to liver cirrhosis and control subjects, with significant positive correlation with parameters for bad prognosis of HCC. In conclusion, NF-κB signaling is activated in HCC and associated with disease prognosis and with high circulating levels of HIF-1 alpha.

Cell density-mediated pericellular hypoxia and the local dynamic regulation of VEGF-a splice variants in ovine ovarian granulosa cells.

The primary aims of this study were to utilize a specialized culture system to further elucidate the functional significance of pericellular hypoxia within the granulosa cell (GC) compartment of growing follicles, and to clarify its effects on the production of vascular endothelial growth factor (VEGF)-A isoforms and steroid hormones. Multilaminar clusters formed rapidly in ovine GCs seeded at high density (HD), and Hypoxyprobe-1 protein adducts appeared markedly more abundant and HIF-1 activation significantly (P < 0.001) greater than in cells seeded at low density (LD). Four proangiogenic VEGF mRNA transcript variants were identified in cultured GCs. Most abundant were VEGF120 and VEGF164, but VEGF182 and VEGF188 were also detected. Total VEGF mRNA was shown to be up-regulated transiently in the HD cells (P < 0.001) and VEGF164 mRNA appeared to contribute most to this. The hypoxia mimetic cobalt chloride also induced marked increases in HIF-1 activation (P < 0.01) and total VEGF mRNA (P < 0.01) production. HD cells increased levels of HIF-1alpha (P < 0.001) and VEGF receptor type 1 (P < 0.05), but not VEGF receptor type 2 mRNA, compared to LD cells or cells grown under chemically induced hypoxia. Both 17beta-estradiol (E2) and progesterone (P4) were markedly lower (P < 0.001) in the HD, cells but though cobalt chloride treatment accompanied significantly reduced P4 production (P < 0.05), E2 levels remained similar to those in untreated cells. These outcomes suggest that pericellular hypoxia may be an important mediator of VEGF production in the GCs of growing follicles, but that local regulation is complex and may involve multiple mechanisms such as mediation by steroid hormones and differential variant mRNA production.

Mechanisms of skeletal muscle deformity in hyperhomocysteinemia (1102.24)

Hyperhomocysteinemia (HHcy) is associated with elderly frailty, skeletal muscle malfunction and injury, reduced vascular integrity and function and mortality. Although HHcy was implicated in impairment of vasculogenesis after hind limb ischemia in murine models, the underlying mechanisms are unclear. We hypothesize that HHcy compromises perfusion, collateral formation and vasculogenesis through diminishing post‐ischemic vasculogenic responses in the muscle fibers. To test this hypothesis, we created femoral artery ligation in wild type (WT) and CBS+/‐ mice (a model for HHcy) and assessed tissue perfusion, collateral formation and skeletal muscle function using Laser Doppler perfusion imaging, barium angiography and fatigue tests. In addition, we evaluated post‐ischemic levels of VEGF, and levels of its muscle specific regulators: HIF‐1α and PGC‐1α in the skeletal muscle fibers. The observations indicated that dysregulation of VEGF, HIF‐1 alpha and PGC‐1 alpha levels in the CBS+/‐ mice ischemic skeletal muscles. Moreover, there was enhanced FOXO dependent atrogene transcription in the muscles and reduced muscle function after ischemia in the CBS+/‐ mice. These results suggest that HHcy adversely affects muscle specific ischemic responses and contribute to muscle frailty.Grant Funding Source: 1RO1HL108621

Differential and Combined Effects of Simvastatin and Vildagliptin on Angiogenic Markers and Oxidative Stress in Hind Limb Model of Ischemia in Diabetic Rats

Background: This study investigated the potential therapeutic effect of simvastatin and vildagliptin and their combination on angiogenesis in diabetic hind-limb ischemia. Methods: 60 Sprague-dawely rats were divided into 5 groups, group A (normal control), group B (diabetic ischemic control), group C (simvastatin treated diabetic ischemic group), group D (vildagliptin treated diabetic ischemic group) and group E (combined sim. + vild. diabetic ischemic group). Parameters of angiogenesis as well as oxidative stress markers were evaluated. Results: Increased capillary density in ischemic gastrocnemius tissue of diabetic rats treated with either simvastatin or vildagliptin with marked increase in its combination. This effect was accompanied by up-regulated plasma levels of HO-1, nitric oxide, vascular endothelial growth factor (VEGF) and expression of HIF-1 alpha levels. Tissue SOD and Catalase enzymes activities were normalized in groups treated with simvastatin or their combination with vildagliptin with concomitant decrease of lipid peroxidation. Conclusion: Both vildagliptin and simvastatin has antioxidant and angiogenic effects and their combination could be a promising strategy in the management of diabetes associated peripheral arterial disease. Original Research Article British Journal of Medicine & Medical Research, 4(35): 5587-5603, 2014 5588

Toxic damage increases angiogenesis and metastasis in fibrotic livers via PECAM-1.

Excessive ethanol consumption is one of the main causes of liver fibrosis. However, direct effects of ethanol exposure on endothelial cells and their contribution to fibrogenesis and metastasis were not investigated. Therefore we analysed whether ethanol directly affects endothelial cells and if this plays a role during fibrogenesis and metastasis in the liver. Murine and human endothelial cells were exposed to ethanol for up to 72 hours. In vitro, effects on VEGF, HIF-1alpha, PECAM-1, and endothelial cell functions were analysed. In vivo, effects of continuous liver damage on blood vessel formation and metastasis were analysed by PECAM-1 immunohistochemistry. Ethanol increased HIF-1alpha and VEGF levels in murine and human endothelial cells. This resulted in enhanced intracellular signal transduction, and PECAM-1 expression as well as tube formation and wound healing. In vivo, toxic liver damage increased angiogenesis during fibrogenesis. Metastasis was also enhanced in fibrotic livers and located to PECAM-1 positive blood vessels compared to nonfibrotic mice. In conclusion, ethanol had strong effects on endothelial cells, which—at least in part—led to a profibrotic and prometastatic environment mediated by PECAM-1. Blockade of increased PECAM-1 expression could be a promising tool to inhibit fibrogenesis and metastasis in the liver.

Expression of vascular endothelial growth factor and hypoxia-inducible factor-1 alpha during the periovulatory period in goats

To evaluate the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 alpha (HIF-1 alpha) during the periovulatory period, goat ovaries were collected at 0, 4, 8, 12, 24, and 48 h after treatment with 300 IU hCG. Total cellular RNA was isolated and VEGF and HIF-1 alpha were quantified using real-time RT-PCR. The mRNA levels of VEGF in thecal tissue remained unchanged prior to ovulation. However, the mRNA levels of VEGF in thecal tissues at 48 h increased to 7.5-fold of the level at 0 h (P < 0.05). In granulosa tissue, the VEGF mRNA levels were not detected in granulosa cells at 48 h. In thecal tissue, the mRNA level of HIF-1 1 alpha at 4 h was 1.5-fold of the level at 0 h and remained unchanged during 8-24 h; however the levels were significantly increased at 48 h. The mRNA levels of HIF-1 alpha in granulosa at 4 h was not changed but levels were increased (P < 0.05) at 8 h and at 12 h and then decreased at 24 and 48 h, respectively. These data describe the relationships of VEGF- and HIF-1 alpha -dependent angiogenesis during the periovulatory periods by which hypoxia during ovulation is crucial for establishing the thecal new vasculature.

IDH1 p.R132 mutations may not be actively involved in the carcinogenesis of hepatocellular carcinoma

BackgroundRecent studies have identified prevalent isocitrate dehydrogenase 1 (IDH1) codon 132 mutations (p.R132) in gliomas and acute myeloid leukemia (AML). The IDH1 mutations lead to a loss of its normal enzymatic activity and acquisition of neomorphic activity in production of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG), which finally cause alterations of multiple gene expression of tumorigenesis-associated α-KG-dependent enzymes. The aim of this study was to determine whether IDH1 p.R132 mutations are involved in the carcinogenesis of hepatocellular carcinoma.Material/MethodsA total of 87 Han Chinese patients with primary hepatocellular carcinoma (HCC) were analyzed by direct DNA sequencing for IDH1 p.R132 mutations. The expression levels of multiple α-KG-dependent enzymes and associated genes were quantified in HepG2 cells overexpressing IDH1 p.R132 mutants by Western blotting and real-time PCR.ResultsNone of 87 Han Chinese patients with HCC harbored any IDH1 p.R132 mutations. The protein levels of HIF-1α and histone methylation marker (H3K4me3 and H3K79me2) were determined in HepG2 cells overexpressing IDH1 p.R132 mutants, but we discerned no difference. Measurement of mRNA expression levels of VEGF, GLUT1, and HOXA genes also showed no significant difference between cells overexpressing IDH1 wild-type and p.R132 mutants.ConclusionsOur negative results, together with some previous reports of the absence of IDH1 p.R132 mutations in HCC tissues, suggests that IDH1 p.R132 mutations are not actively involved in the development of HCC.

Correlation of Nrf2 and HIF-1α in glioblastoma and their relationships to clinicopathologic features and survival

ObjectivesGlioblastoma (GB) is the most common, aggressive, and proliferative among all brain tumors. The prognosis of GB is still far from satisfactory currently, thus demanding great modification and enhancement, which may be acquired by the help of the molecular target therapy. Nuclear factor E2-related factor 2 (Nrf2), a pivotal transcriptional factor of cellular responses to oxidative stress, was observed to function remarkably in cancer pathobiology. In the current study, we analyzed the correlation between Nrf2 and Hypoxia-inducible factor-1alpha (HIF-1alpha) in GB, together with their association to the features and survival of clinicopathology.Methods:We examined the expression of Nrf2 and HIF-1alpha in 68 specimens of GB by tissue microarray and immunohistochemistry, and correlated this investigation to the outcome of GB patients.Results:Nrf2 and HIF-1alpha were overexpressed in GB tissues. There was significant correlation between the high level of Nrf2 and tumor necrosis on MRI and 1-year survival. There was significant correlation between HIF-1alpha level and Nrf2 status (r = 0·294, P = 0·015). Kaplan–Meier analysis showed that high Nrf2 expression was significantly associated with shorter overall survival (OS) (log-rank test, P = 0·006), and was identified as an independent prognostic factor in multivariate analysis (P = 0·034). HIF-1alpha was another independent factor for both OS and progression-free survival by Cox regression analysis (P = 0·048 and P = 0·032, respectively).Discussion:Mutual association between Nrf2 and HIF-1alpha was found in GB: higher Nrf2 expression and poorer outcome of GB patients. Nrf2 would therefore be a new molecular marker for the targeted treatment of GB.

Molecular analysis in breast cancer subtypes and correlation with pathologic complete response (pCR) to neoadjuvant chemotherapy.

e22119 Background: Although it is know that pCR following neoadjuvant chemotherapy is more frequent in some subtypes of breast cancer such as Triple Negative (TN) or erb2 tumors, the predictive role of gene expression and mutation status is not well defined in this setting. Methods: We analyzed samples from 41 patients (p) prospectively treated with neoadjuvant chemotherapy (sequential AC followed by weekly TXL, or inverse sequence, plus trastuzumab for erb2 positive p). Pathologic response (PR) was classified according to Miller-Payne and RCB criteria. Radiologic evaluation was performed by ultrasonography, dynamic MR and PET-TAC after each chemotherapy sequence. We performed expression analysis of AXL, BRCA1, RAP80, BIM, EZH2, ROR1, FGFR1, PTPN12, YAP, GAS6, beta-TRCP, HIF1 alpha and ZNF217 by RT-PCR, and mutational status of p53 and PI3K genes in pretreatment biopsies. Statistical analysis was performed using Mann-Whitney U and Pearson’s chi-squared tests. Results: pCR was detected in 5 p (3TN, 2 erb2) of 25 p (9 luminal A, 5 luminal B, 6 erb2 and 5 TN) evaluated for PR at time of submitting this abstract. TN tumors had lower levels of RAP80 (p=.0013), PTPN12 (p=.003), beta TRCP (p=.001), ZNF217 (p=.014) and YAP (p=.097). Luminal B tumors had low levels of YAP and the highest levels of FGFR1 (p=.09) and ZNF217 (p=.014). Luminal A tumors had high levels of beta-TRCP (p=.003). We found no differences in BRCA1, AXL, BIM, EZH2, ROR1, GAS6 and HIF1 levels by breast cancer subtype. P with low levels of FGFR1 (p=.087), HIF1alpha (p=.07) or EZH2 (p=.005) had higher probability of pCR. No pCR was observed in p with higher levels of AXL, EZH2, RAP80, GAS6, beta TRCP, HIF alpha. Four p had p53 mutations (1 luminal B, 1 erb2 and 2 TN) and 4 p had PI3K mutations (2 luminal A, 1 erb2, 1 luminal B). There was no correlation between p53 status and PR. P with PI3K mutations did not achieve pCR vs 46% of p with wild type PI3K (p=.23). Conclusions: Gene expression profile varies by breast cancer subtype. Chemosensitivity could be higher in tumors with lower levels of FGFR1, HIF1alpha or EZH2. Further results will be presented.

Abstract 962: Prolonged antiangiogenic therapy with oral metronomic topotecan and pazopanib in pediatric solid tumor mouse model.

Abstract Background: We have previously observed that the combination of daily oral Low Dose Metronomic (LDM) Topotecan (TP) and Pazopanib (PZ) significantly delayed the tumor growth and enhanced the survival in neuroblastoma (NB) mouse models (Kumar et al, 2011). However, this combination (TP+PZ) did not stop tumor growth and the animals eventually died due to disease during the course of therapy. Therefore, we planned to investigate the factors contributing to resistance to this antiangiogenic therapy in NB mouse model. Objectives: To investigate the effect of prolonged TP+PZ therapy on angiogenesis by testing the effect of therapy at different durations in NB mice xenograft model. Methods: Mice bearing subcutaneous SK-N-BE(2) NB xenografts were treated with either of the four regimens, daily, orally: vehicle (untreated), PZ (150 mg pazopanib), TP (1.0 mg/Kg topotecan), TP+PZ (combination of topotecan and pazopanib, same doses as single agents). Durations of treatment planned for each regimen were 28 days, 56 days and 80 days. Animals were sacrificed after these durations or upon end point, whichever was earlier. The criteria for end point are tumor sizes exceeding 2.0 cms in diameter or animals showing signs of morbidity. Results: The animals in control, PZ, TP reached the end point after 23d, 28d and 46d respectively. TP+PZ halted the tumor growth for up to 40 days, after which gradual growth was observed. The mean tumor diameters in the third TP+PZ group reached 1.8 cms at the day 80. CD31 immunofluorescence revealed that all the three durations of TP+PZ significantly lowered microvessel densities compared to the control (P= 0.02, 0.04 and 0.009 respectively for 28, 56 and 80 days respectively). However the ratio of CD31 to α- smooth muscle actin (pericyte marker) stained areas were significantly higher in animals treated with TP+PZ for 56 and 80days, compared to control (P=0.0006; 56d and 0.004; 80d) indicating higher pericyte coverage. TP+PZ (28d) showed signs of necrosis and cell cycle arrest (Ki67 staining), which were not observed in later durations (56 and 80 days) with TP+PZ. Long-term TP+PZ therapy also increased the levels of HIF-1 alpha, VEGF and Oct-4 within the tumor tissue. Conclusion: This study indicates that even though prolonged TP+PZ therapy demonstrates sustained antiangiogenic activity, it encounters resistance in NB, potentially mediated by pericyte coverage. Long-term TP+PZ therapy may also increase tumor stemness. Citation Format: Sushil Kumar, Reza Bayat Mokhtari, Syed Islam, Herman Yeger, Sylvain Baruchel. Prolonged antiangiogenic therapy with oral metronomic topotecan and pazopanib in pediatric solid tumor mouse model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 962. doi:10.1158/1538-7445.AM2013-962

172 PRECLINICAL EFFICACY OF AZD8055, A DUAL MTORC1/2 INHIBITOR, IN RCC HARBORING TFE3 GENE FUSIONS

You have accessJournal of UrologyKidney Cancer: Basic Research (I)1 Apr 2013172 PRECLINICAL EFFICACY OF AZD8055, A DUAL MTORC1/2 INHIBITOR, IN RCC HARBORING TFE3 GENE FUSIONS Eric C. Kauffman, Soroush Rais-Bahrami, Gopal N. Gupta, Jeffrey W. Nix, Youfeng Yang, W. Marston Linehan, and Ramaprasad Srinivasan Eric C. KauffmanEric C. Kauffman Bethesda, MD More articles by this author , Soroush Rais-BahramiSoroush Rais-Bahrami Bethesda, MD More articles by this author , Gopal N. GuptaGopal N. Gupta Bethesda, MD More articles by this author , Jeffrey W. NixJeffrey W. Nix Bethesda, MD More articles by this author , Youfeng YangYoufeng Yang Bethesda, MD More articles by this author , W. Marston LinehanW. Marston Linehan Bethesda, MD More articles by this author , and Ramaprasad SrinivasanRamaprasad Srinivasan Bethesda, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1552AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Xp11 chromosomal translocations resulting in gene fusions of the TFE3 transcription factor gene comprise up to 5% of all renal cell carcinomas (RCC). TFE3-fusion RCC (TfRCC) is clinically aggressive with frequent metastasis. However, the key signaling pathways for therapeutic targeting remain under investigation, and no drug to date has shown in vivo efficacy in the preclinical setting. We investigated the preclinical efficacy of a dual mTORC1/mTORC2 inhibitor in TfRCC. METHODS Four genetically confirmed TfRCC patient cell lines established at the National Cancer Institute and one benign renal cortical epithelial cell line were treated in vitro with the mTORC1 inhibitor, sirolimus, or the mTORC1/mTORC2 inhibitor, AZD8055 (AstraZeneca). Cell proliferation and cytotoxicity were measured by MTT assay and lactate dehydrogenase (LDH) quantification, respectively. Athymic nude mice (n=40) with TfRCC cell line xenografts were subjected to 3-week treatments with weekly intraperitoneal (IP) sirolimus, daily oral (PO) AZD8055, or IP/PO vehicle controls. RESULTS AZD8055 had little effect on benign renal cells but potently inhibited TfRCC cell growth in vitro (IC50= 20-50 nM), with maximal inhibition of 80-90% at sub-micromolar concentrations, compared to 30-50% with sirolimus. Immunoblotting indicated more effective suppression of Akt/mTOR signaling with AZD8055 than sirolimus, with significant reductions in levels of phosphorylated Akt(S473) and 4EBP1 and HIF1alpha levels with the former. LDH assays suggested growth arrest, rather than cytotoxicity, as the predominant mechanism of AZD8055 growth suppression. In mouse xenograft studies, AZD8055 treatment significantly reduced mean TfRCC tumor size compared to sirolimus or vehicle controls (FIGURE; p<0.001). Median survival was significantly longer in mice treated with AZD8055 (52 days) compared to those receiving sirolimus (32 days, p<0.001) or vehicle control (33 days, p<0.001). CONCLUSIONS The Akt-mTOR pathway is a promising therapeutic target in TfRCC. In preclinical studies, dual mTORC1/2 inhibition with AZD8055 appears more effective than selective mTORC1 inhibition, possibly by overcoming feedback Akt-activation associated with the latter. These data provide the basis for further exploration of dual mTORC1/2 inhibitors in TfRCC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e70-e71 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eric C. Kauffman Bethesda, MD More articles by this author Soroush Rais-Bahrami Bethesda, MD More articles by this author Gopal N. Gupta Bethesda, MD More articles by this author Jeffrey W. Nix Bethesda, MD More articles by this author Youfeng Yang Bethesda, MD More articles by this author W. Marston Linehan Bethesda, MD More articles by this author Ramaprasad Srinivasan Bethesda, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...