Captopril inhibits thioacetamide-induced chronic liver injury associated with the suppression of inflammation / hypoxia- inducible factor 1-alpha / profibrogenic axis-mediated hepatotoxicity in rats / Captopril inhibe la lesión hepática crónica inducida por tioacetamida asociada con la supresión de la inflamación/factor 1-alfa inducible por hipoxia/hepatotoxicidad mediada por el eje profibrogénico en rata

SUMMARY: Liver transplantation is the only available method to treat liver failure induced by chronic liver injury. We sought to determine whether the angiotensin-converting enzyme inhibitor, captopril, can inhibit the development of chronic liver injury induced by the hepatotoxic agent thioacetamide (TAA) in association with the suppression of inflammation (hsCRP, TNF-α, and IL-6) / hypoxia- inducible factor 1-alpha (HIF-1α) / profibrosis (TIMP-1, MMP-9, and α-SMA) axis that mediates liver injury. Therefore, the model group of rats was injected for eight weeks with 200 mg/kg TAA starting at week two. The protective group was pretreated with 150 mg/ kg captopril daily for two weeks prior to TAA injections and continued receiving both capropril and TAA agents until being humanely scrificed at week 10. We observed a substantial damage to liver tissue in the model group as demonstrated by a significant (p<0.0001) increase in blood and hepatic tissue levels of high sensitivity C-reactive protein (hsCRP), tumor necrosis factor-a (TNF-α), interleukin- 6 (L-6), HIF-1α, tissue inhibitor of metalloproteinases-1 (TIMP-1), matrix metalloproteinase-9 (MMP-9), alpha-smooth muscle actin (α-SMA), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). All these parameters were significantly (p<0.0244) protected by captopril. Also, a significant (p<0.0001) positive correlation was observed between a-SMA (profibrosis) and the serum and tissue levels of hsCRP, TNF-α, HIF-1α, TIMP-1, MMP-9, and ALT. Thus, these findings suggest that the induction of chronic liver injury by the hepatotoxic compound, TAA is associated with the upregulation of inflammation/HIF-1α/profibrosis, with captopril exhibiting beneficial hepatic pleotropic effects.

El trasplante de hígado es el único método disponible para tratar la insuficiencia hepática inducida por una lesión hepática crónica. Buscamos determinar si el inhibidor de la enzima convertidora de angiotensina, captopril, puede inhibir el desarrollo de lesión hepática crónica inducida por el agente hepatotóxico tioacetamida (TAA) en asociación con la supresión de la inflamación (hsCRP, TNF-α e IL-6) / factor inducible por hipoxia 1-alfa (HIF-1α) / profibrosis (TIMP-1, MMP-9 y α- SMA) eje que media la lesión hepática. Por lo tanto, al grupo modelo de ratas se le inyectó durante ocho semanas 200 mg/kg de TAA a partir de la semana dos. El grupo protector fue pretratado con 150 mg/kg de captopril al día durante dos semanas antes de las inyecciones de TAA y continuó recibiendo capropril y agentes TAA hasta que fue sacrificado en la semana 10. Observamos un daño sustancial en el tejido hepático en el grupo modelo, como lo demuestra un aumento significativo (p<0,0001) de los niveles en sangre y tejido hepático de proteína C reactiva de alta sensibilidad (hsCRP), factor de necrosis tumoral-α (TNF-a), interleucina-6 (L-6), HIF-1α, inhibidor tisular de metaloproteinasas-1 (TIMP-1), metaloproteinasa de matriz-9 (MMP-9), actina de músculo liso alfa (α-SMA), alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST). Todos estos parámetros estaban significativamente (p<0,0244) protegidos por captopril. Además, se observó una correlación positiva significativa (p<0,0001) entre α-SMA (profibrosis) y los niveles séricos y tisulares de hsCRP, TNF-α, HIF-1α, TIMP- 1, MMP-9 y ALT. Por lo tanto, estos hallazgos sugieren que la inducción de daño hepático crónico por el compuesto hepatotóxico, TAA, está asociada con la regulación al alza de la inflamación/HIF-1α/profibrosis, con captopril exhibiendo efectos pleotrópicos hepáticos beneficiosos.

Effect of acupuncture on HIF-1α/NLRP3 inflammatory signaling pathway in the rats with cerebral ischemia-reperfusion injury / 中国针灸

OBJECTIVE@#To observe the effects of Xingnao Kaiqiao (regaining consciousness and opening orifices) acupuncture therapy on the expression of hypoxia-inducible factor 1α (HIF-1α) and Nod-like receptor protein 3 (NLRP3) in cerebral ischemia-reperfusion rats, and to explore the mechanism of acupuncture against cerebral ischemia-reperfusion injury.@*METHODS@#Seventy-two male SD rats were randomly divided into a sham-operation group, a model group, an acupuncture group and a non-point acupuncture group, with 18 rats in each one. Using modified Longa thread embolization method, the rat model of acute focal cerebral ischemia was prepared; and after 2 h ischemia, the reperfusion was performed to prepared the model of cerebral ischemia-reperfusion. Immediately after reperfusion, Xingnao Kaiqiao acupuncture method was applied to bilateral "Neiguan" (PC 6) and "Shuigou" (GV 26) in the acupuncture group, while in the non-point acupuncture group, acupuncture was delivered at non-points and all of the needles were retained for 30 min in these two groups. The samples were collected 24 h after reperfusion in the rats of each group. Zea-Longa neurological deficit score was used to evaluate the degree of cerebral neurological impairment, TTC staining was adopted to observe the volume percentage of cerebral infarction, HE staining was provided to observe the morphological changes of brain, and Western blot was applied for detecting the expression of HIF-1α and NLRP3 proteins in the cerebral cortex on the right side.@*RESULTS@#Compared with the sham-operation group, neurological deficit score and volume percentage of cerebral infarction were increased in the model group (P<0.01), and HIF-1α and NLRP3 protein expression was elevated (P<0.01). Compared with the model group, neurological deficit score and volume percentage of cerebral infarction were decreased (P<0.01), and HIF-1α and NLRP3 protein expression was lower (P<0.01) in the acupuncture group. There was no significant difference in above indexes in the non-point acupuncture group compared with the model group (P>0.05). Compared with the sham-operation group, the brain tissue of the rats in the model group and the non-point acupuncture group was loose and edema, and the nuclei were shriveled. The brain tissue morphology in the acupuncture group was similar to that of the sham-operation group.@*CONCLUSION@#Acupuncture can alleviate cerebral ischemia-reperfusion injury, and its mechanism may be related to the regulation of HIF-1α/NLRP3 signaling pathway to attenuate inflammatory response.

Mechanism study of Curcumin on inhibiting experimental choroidal neovascularization <i>in vitro</i> by AKT/HIF-1α/VEGF pathway / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effect and mechanism of curcumin on inhibiting choroidal neovascularization(CNV)<i>in vitro</i>. METHODS: Human retinal pigment epithelial(ARPE-19)cells chemical hypoxia model was established by cobalt chloride(CoCl2). CCK-8 method was used to detect the effect of curcumin on the activity of ARPE-19 cells induced by CoCl2. RT-qPCR and Western blot were used to detect the expression of AKT, HIF-1α, VEGF mRNA and protein in ARPE-19 cells hypoxia model induced by CoCl2. Cell scratch test, transwell chamber migration test, transwell chamber invasion test and matrigel matrix hose lumen formation test were used to observe the effects of conditioned medium of curcumin in ARPE-19 cells on the proliferation, migration, invasion and lumen formation of human umbilical vein endothelial cells(HUVEC)in non-contact condition. RESULTS:Chemical hypoxia model of ARPE-19 cells can successfully establish by CoCl2 at 100μmol/L. CoCl2 at the final concentration of 100μmol/L can promote the expression of AKT, HIF-1α and VEGF mRNA and p-AKT, HIF-1α and VEGF protein in ARPE-19 cells. Curcumin at the final concentration of 100μmol/L can reduce the expression of AKT, HIF-1α and VEGF mRNA in ARPE-19 hypoxia model. Curcumin at the final concentration of 100μmol/L can reduce the expression of AKT, HIF -1α and VEGF proteins in ARPE-19 hypoxia model. The conditioned medium of low(6.25μmol/L), medium(25μmol/L)and high dose(100μmol/L)curcumin in ARPE-19 cells can significantly inhibit the level migration of HUVEC. The conditioned medium in high dose group can significantly inhibit the vertical migration and cell invasion of HUVEC. The conditioned medium of middle and high dose curcumin in ARPE-19 cells can inhibit the lumen formation of HUVEC. CONCLUSION:Curcumin at 100μmol/L can protect ARPE-19 cells from hypoxia induced by CoCl2. Curcumin can inhibit the formation of blood vessels at the cellular level.

Expression and significance of EPO and HIF-1α in serum and aqueous humor of patients with acute anterior uveitis / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To detect the expression of erythropoietin(EPO)and hypoxia-inducible factor-1α(HIF-1α)in serum and aqueous humor of patients with acute anterior uveitis(AAU), and to explore their clinical significance. <p>METHODS: From January 2018 to December 2020, 60 patients with AAU in our hospital were prospectively selected as the research objects, and 60 patients with proliferative vitreoretinopathy in the same period were taken as control(control group). The serum and aqueous humor of two groups were collected, enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of EPO and HIF-1α in serum and aqueous humor; the self-rating anxiety scale(SAS)and the self-rating depression scale(SDS)were used to evaluate the AAU patients, and the severity of the disease was scored; Pearson method was used to analyze the correlation between SAS score, SDS score and levels of EPO and HIF-1α in serum and aqueous humor, and the correlation between levels of EPO and HIF-1α in the serum and aqueous humor. Spearman was used to analyze the correlation between the disease severity score of AAU patients and the levels of EPO and HIF-1α in serum and aqueous humor. <p>RESULTS: Compared with the control group, the levels of EPO and HIF-1α in the serum and aqueous humor of the study group were higher(<i>P</i><0.01). Among AAU patients, 23 were negative of SAS score and 37 were positive, and 29 were negative of SDS score and 31 were positive. Compared with patients with negative SAS score, the level of HIF-1α in serum and the level of EPO in the aqueous humor were higher in patients with positive SAS score(<i>P</i><0.05); compared with patients with negative SDS score, the level of EPO in serum and the levels of EPO and HIF-1α in aqueous humor were higher in patients with positive SDS score(<i>P</i><0.01). There were 26 mild patients and 34 severe patients with AAU. Compared with mild patients with AAU, the levels of EPO and HIF-1α in serum and aqueous humor were increased in severe patients(<i>P</i><0.01). Pearson analysis showed that the SAS and SDS scores of AAU patients were not significantly correlated with the levels of EPO and HIF-1α in serum and aqueous humor(<i>P</i>>0.05), there was a positive correlation between EPO and HIF-1α in serum(<i>P</i><0.05), and between EPO and HIF-1α in aqueous humor(<i>P</i><0.05). Spearman analysis showed that the disease severity score of AAU patients was positively correlated with the levels of EPO and HIF-1α in serum and aqueous humor(<i>P</i><0.05). <p>CONCLUSION: EPO and HIF-1α are highly expressed in serum and aqueous humor of AAU patients, and they are closely related. The two are closely related to the disease severity score, and should be paid attention to clinically.

Protective effect of complex thrombolysis capsule on high altitude retinopathy in rat models / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the protective effect of complex thrombolysis capsule on high altitude retinopathy in rat models and its possible mechanism.<p>METHODS: Twenty-four adult male SD rats were randomly divided into 6 groups and were put into plateau environment simulation experimental chamber at simulated altitude of 5 000m for 2, 4, 6, 10, 24, 72h respectively. Retinal pathology, HIF-1α and the b wave amplitudes of Max-R of flash ERG were examined by HE,IHC and flash ERG. Twenty-four adult male SD rats were randomly divided into 4 groups and were given respectively placebo, rhodiolarosea, inosine tablet and compound xueshuantong capsule by gavage for 7d. They were put into plateau environment simulation experimental chamber at simulated altitude of 5 000m for 10h. Retinal pathology, HIF-1α and the b wave amplitudes of Max-R of flash ERG were examined by HE, IHC and flash ERG.<p>RESULTS: In the SD rat model of high-altitude retinopathy, with the increase of experimental time, the ganglion cell layer of rat's retina showed obvious edema and HIF-1α expression increased in the cytoplasm of ganglion cells and core cells. All of them were most obvious at 10h. Compared with the self-comparison of b wave amplitudes of Max-R of flash ERG in each group of SD rats before and after entering in plateau environment simulation experimental chamber, the b wave amplitudes of Max-R in 4h, 6h, 10h and 72h were dramatically decreased(<i>P</i>﹤0.05). And the 2h, 4h(<i>P</i>=0.007), 6h(<i>P</i>=0.008), 10h(<i>P</i>=0.002)were statistically significant differences, the 24h and 4h(<i>P</i>=0.035), the 6h(<i>P</i>=0.040)and 10h(<i>P</i>=0.012)were also statistically significant differences. In the study of protective effect of complex thrombolysis caps on high altitude retinopathy in rat models, the results showed that the rat retinal edema of rhodiolarosea group, inosine tablet group and compound thrombosis capsule group and HIF-1α expression in ganglion cell layer of compound thrombosis group and rhodiolarosea group were significantly reduced comparing with the placebo group. Test for homogeneity of variance and one-way ANOVA were used to test the difference of b wave amplitudes of Max-R of flash ERG in four groups of SD rats after entering in the plateau environment simulation experimental chamber, the results showed the complex thrombolysis caps group(<i>P</i>=0.032)and rhodiola rose group(<i>P</i>=0.001)was significantly lower than placebo group.<p>CONCLUSION: Compound thrombosis caps may have a protective effect on highaltitude retinopathy in rats by inhibiting the expression of HIF-1α, however, the specific mechanism needs to be further studied.

lncRNALUCAT1 facilitates the proliferation and metastasis of clear cell renal cell carcinoma 786-O cells via regulating miR-199a-5p/HIF-1α axis / 中国肿瘤生物治疗杂志

@# Objective: To investigate the effect of long non-coding RNA (lncRNA) lung cancer associated transcript 1 (LUCAT1) on proliferation and migration of clear cell renal cell carcinoma (ccRCC) 786-O cells and the underlying mechanism. Methods: A total of 40 pairs of pathologically confirmed tumor tissues and corresponding adjacent normal tissues from ccRCC patients, who underwent surgical resection in the Department of Urology, the First People's Hospital of Yichang during June 2013 and June 2017, were selected for this study. ccRCC cell lines (786-O, ACHN, UM-RC-2) and normal renal epithelial KiMA cells were also used in this study. qPCR was used to detect the mRNA expressions of LUCAT1, miR-199a-5p and hypoxia inducible fator 1α (HIF-1α) in above mentioned tissues and cell lines; CCK-8 assay was used to evaluate the proliferation of 786-O cells; Transwell assay was used to evaluate the migration of 786-O cells; Dual luciferase reporter gene assay was performed to validate the relationship between LUCAT1 and miR-199a-5p; and Western blotting was conducted to detect the effect of LUCAT1 and miR-199a-5p on the protein expression of HIF-1α. Results: LUCAT1 was significantly up-regulated in ccRCC tissues and cell lines (all P<0.01), and its knockdown significantly inhibited the proliferation and migration of 786-O cells (all P<0.01). miR-199a-5p was low-expressed in ccRCC tissues and cell lines (all P<0.01), StarBase analysis showed that LUCAT1 contained a conserved target site for miR-199a-5p. miR-199a-5p exerted significant suppression on the luciferase activity of LUCAT1-Wt (P<0.01), and LUCAT1 knockdown significantly reduced miR-199a-5p expression (P< 0.01). LUCAT1 was low-expressed in 786-O cells transfected with miR-199a-5p mimics, however, it was attenuated after co-transfection with LUCAT1. The mRNA and protein expressions of HIF-1α in 786-O cells transfected with miR-199a-5p mimics were up-regulated, which was then reversed by LUCAT1 over-expression (P<0.05 or P<0.01). miR-199a-5p over-expression suppressed the proliferation and migration of 786-O cells, which was partially attenuated by LUCAT1 transfection (P<0.05 or P<0.01). Conclusion: LUCAT1 exerts oncogenic function in ccRCC via regulating miR-199a-5p/HIF-1α axis.·

miR-377-5p inhibits proliferation and invasion of hepatocellular carcinoma HepG2 cells by down-regulating HIF-1α and related VEGFsignaling pathway / 中国肿瘤生物治疗杂志

@# Objective：To explore the targeting relationship between miR-377-5p and hypoxia inducible factor-1 (HIF-1α), and investigate the regulatory effect of miR-377-5p on proliferation, invasion and epithelial-mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) cells through vascular endothelial growth factor (VEGF) signaling pathway. Methods： ：The expression of miR-377-5p in 35 pairs of human HCC tissues and para-cancerous tissues was detected by qPCR. Then, HepG2 cells were divided into control group, mimic-NC group and miR-377-5pmimicgroup.qPCRwasusedto detect the transfection efficiency; the effects of miR-377-5p over-expression on proliferation and invasion of HepG2 cells were examined by EdU staining and Transwell assay, respectively; and the effect of miR-377-5p over-expression on the expressions of proliferation-related protein Ki-67, proliferating cell nuclear antigen (PCNA) and epithelial-mesenchymal transition (EMT) markers (E-cadherin and N-cadherin) were detected by Western blotting (WB); the effect of miR-377-5p over-expression on the expression of hypoxia inducible factor-1α (HIF-1α) in HepG2 cells was detected by qPCR and WB; and the targeting relationship between miR-377-5p and HIF-1α gene was determined by Luciferase reporter gene assay. Results: The expression of miR-377-5p in HCC tissues was significantly lower than that in para-cancerous tissues (P<0.01). Compared with the control group, the expression of miR-377-5p in HepG2 cells of miR-377-5p mimic group elevated significantly, and the proliferation, invasion and the expression of N-caderin proteins decreased,significantly (all P<0.01), while the expression of E-caderin increased significantly (P<0.01). At the same time, the mRNA and protein expressions of HIF-1α in miR-377-5p mimic group decreased significantly (P<0.01 or P<0.05). miR-377-5p targetedly inhibited the expression of HIF-1α gene and suppressed the activation of VEGF pathway (all P<0.05). Conclusion: miR-377-5p inhibits the proliferation, invasion and EMT of HepG2 cells via targetedly inhibiting HIF-1α expression and suppressing the activation of VEGF signaling pathway.

Regulatory effect of hsa-miR-150-5p on malignant behaviors of glioblastoma U-251MG cells by targeting HIF1α / 中国肿瘤生物治疗杂志

@#Objective: To explore the effect and possible mechanisms of has-miR-150-5p targeting HIF1α to regulate malignant biological behaviors of glioblastoma (GBM) U-251MG cells. Methods: Real-time quantitative PCR (RT-PCR) was used to detect the expression of miR-150-5p and hypoxia inducible factor 1 (HIF1α) in U-251MG cells. Luciferase report assay was carried out to verify the biological relationship between miR-150-5p and HIF1α and their biological functions in U-251MG cells. The protein expressions of miR150-5pand HIF1α in U-251MG cells were detected by western blotting. The ability of cell migration was detected by wound healing test and cell invasion ability was detected by transwell test. Results: After miR-150-5p mimic transfection, the mRNA expression of HIF1α was significantly reduced in U-251MG cells (P<0.01). Bioinformatics prediction and luciferase reporter assay demonstrated that miR-150-5p down-regulated HIF1α through directly binding to HIF1α 3’-untranslated region (3’-UTR) (all P<0.05). In U-251MG cells, miR-150-5p over-expression significantly inhibited HIF1α expression, cell invasion and migration (all P<0.05). Conclusion: miR150-5p inhibits cell invasion and metastasis through negative regulation of HIF1α, indicating that miR-150-5p and HIF1α were both potential therapeutic targets for glioblastoma.

Inhibition of HIF1α and PDK Induces Cell Death of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain tumors. GBMs, like other tumors, rely relatively less on mitochondrial oxidative phosphorylation (OXPHOS) and utilize more aerobic glycolysis, and this metabolic shift becomes augmented under hypoxia. In the present study, we investigated the physiological significance of altered glucose metabolism and hypoxic adaptation in the GBM cell line U251 and two newly established primary GBMs (GBM28 and GBM37). We found that these three GBMs exhibited differential growth rates under hypoxia compared to those under normoxia. Under normoxia, the basal expressions of HIF1α and the glycolysis-associated genes, PDK1, PDK3, and GLUT1, were relatively low in U251 and GBM28, while their basal expressions were high in GBM37. Under hypoxia, the expressions of these genes were enhanced further in all three GBMs. Treatment with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), induced cell death in GBM28 and GBM37 maintained under normoxia, whereas DCA effects disappeared under hypoxia, suggesting that hypoxic adaptation dominated DCA effects in these GBMs. In contrast, the inhibition of HIF1α with chrysin suppressed the expression of PDK1, PDK3, and GLUT1 and markedly promoted cell death of all GBMs under both normoxia and hypoxia. Interestingly, however, GBMs treated with chrysin under hypoxia still sustained higher viability than those under normoxia, and chrysin and DCA co-treatment was unable to eliminate this hypoxia-dependent resistance. Together, these results suggest that hypoxic adaptation is critical for maintaining viability of GBMs, and targeting hypoxic adaptation can be an important treatment option for GBMs.

Thymosin Beta-4, Actin-Sequestering Protein Regulates Vascular Endothelial Growth Factor Expression via Hypoxia-Inducible Nitric Oxide Production in HeLa Cervical Cancer Cells

Vascular endothelial growth factor (VEGF) is an important regulator of neovascularization. Hypoxia inducible nitric oxide (NO) enhanced the expression of VEGF and thymosin beta-4 (Tbeta4), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by Tbeta4 expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of Tbeta4. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(beta-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of Tbeta4 expression with Tbeta4-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in Tbeta4 expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-Tbeta4 plasmids for Tbeta4 overexpression. Taken together, these results suggest that Tbeta4 could be a regulator for the expression of VEGF via the maintenance of NOS activity.

The Role of TWIST in Ovarian Epithelial Cancers

BACKGROUND: Epithelial-mesenchymal transition (EMT) is associated with tumor hypoxia. EMT is regulated, in part, by the action of TWIST, which inhibits of E-cadherin expression and may interfere with the p53 tumor-suppressor pathway. METHODS: We examined the expression of TWIST, E-cadherin, hypoxia-inducible factor 1alpha (HIF1alpha), and p53 by immunohistochemistry in 123 cases of ovarian epithelial cancers (OEC) to evaluate the role of TWIST in OEC. We assessed the association between protein expression and clinicopathologic parameters. RESULTS: The expression of TWIST, E-cadherin, HIF1alpha, and p53 proteins was found in 28.5%, 51.2%, 35.0%, and 29.3% of cases, respectively. TWIST expression was associated with higher histologic grade and unfavorable survival. TWIST expression was correlated with HIF1alpha expression and reduced E-cadherin expression. The altered HIF1alpha/TWIST/E-cadherin pathway was associated with lower overall survival (OS), while the co-expression of TWIST and p53 was correlated with lower progression-free survival. In the multivariate analyses, TWIST expression was an independent prognostic factor for OS. CONCLUSIONS: Our data imply that TWIST expression could be a useful predictor of unfavorable prognosis for OEC. TWIST may affect the p53 tumor-suppressor pathway. Moreover, hypoxia-mediated EMT, which involves the HIF1alpha/TWIST/E-cadherin pathway may play an important role in the progression of OEC.

Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state

With the increasing use of culture-expanded mesenchymal stromal cells (MSCs) for cell therapies, factors that regulate the cellular characteristics of MSCs have been of major interest. Oxygen concentration has been shown to influence the functions of MSCs, as well as other normal and malignant stem cells. However, the underlying mechanisms of hypoxic responses and the precise role of hypoxia-inducible factor-1alpha (Hif-1alpha), the master regulatory protein of hypoxia, in MSCs remain unclear, due to the limited span of Hif-1alpha stabilization and the complex network of hypoxic responses. In this study, to further define the significance of Hif-1alpha in MSC function during their self-renewal and terminal differentiation, we established adult bone marrow (BM)-derived MSCs that are able to sustain high level expression of ubiquitin-resistant Hif-1alpha during such long-term biological processes. Using this model, we show that the stabilization of Hif-1alpha proteins exerts a selective influence on colony-forming mesenchymal progenitors promoting their self-renewal and proliferation, without affecting the proliferation of the MSC mass population. Moreover, Hif-1alpha stabilization in MSCs led to the induction of pluripotent genes (oct-4 and klf-4) and the inhibition of their terminal differentiation into osteogenic and adipogenic lineages. These results provide insights into the previously unrecognized roles of Hif-1alpha proteins in maintaining the primitive state of primary MSCs and on the cellular heterogeneities in hypoxic responses among MSC populations.

Effects of the knockdown of hypoxia inducible factor-1alpha expression by adenovirus-mediated shRNA on angiogenesis and tumor growth in hepatocellular carcinoma cell lines / 대한간학회지

BACKGROUND/AIMS: Hypoxia-inducible factor-1alpha (HIF-1alpha) is a central transcriptional factor involved in the cellular responses related to various aspects of cancer biology, including proliferation, survival, and angiogenesis, and the metabolism of the extracellular matrix in hypoxia. This study evaluated whether adenovirus-mediated small hairpin RNA (shRNA) against HIF-1alpha (shHIF-1alpha) inhibits cell proliferation and angiogenesis in hepatocellular carcinoma (HCC) cell lines. METHODS: Knockdown of HIF-1alpha expression was constructed by adenovirus-mediated RNA interference tools, and HCC cell lines infected with shHIF-1alpha coding virus were cultured under a hypoxia condition (1% O2) for 24 hours. Following infection, the expression levels of HIF-1alpha, angiogenesis factors, and matrix metalloproteinase (MMP) were examined using Western blotting. Cell proliferation and angiogenesis were measured by a cell proliferation assay (MTT assay) and an angiogenesis-related assay (invasion and tube-formation assay), respectively. RESULTS: Adenovirus mediated inhibition of HIF-1alpha induced suppression of tumor growth in HCC cell lines. It also down-regulated the expression of angiogenesis factor and MMP proteins. Angiogenesis as well as mobility of vascular cells to tumor was suppressed by adenovirus-mediated shHIF-1alpha-infected groups in human umbilical vein endothelial cells (HUVECs). CONCLUSIONS: These data suggest that adenovirus-mediated inhibition of HIF-1alpha inhibits the invasion, tube formation, and cell growth in HUVECs and HCC cells.

Induction of hypoxia-inducible factor-1alpha inhibits drug-induced apoptosis in the human leukemic cell line HL-60

BACKGROUND: Leukemic cells originate from hypoxic bone marrow, which protects them from anti-cancer drugs. Although many factors that cause drug resistance in leukemic cells have been studied, the effect of hypoxia on drug-induced apoptosis is still poorly understood. METHODS: In this study, we examined the effect of hypoxia on anti-leukemic drug resistance in leukemic cell lines treated with cobalt chloride (CoCl2), a hypoxia-mimetic agent. Cellular proliferation was evaluated using the methyl thiazolyl tetrazolium (MTT) assay. Flow cytometry analysis and western blots were performed to investigate apoptosis-related proteins. RESULTS: Unlike its previously known apoptotic effect, the expression of HIF-1alpha increased the survival rate of human promyelocytic leukemia HL-60 cells when these cells were exposed to anti-leukemic drugs; these effects were mediated by heat-shock protein HSP70 and the pro-apoptotic protein Bax. CONCLUSION: These findings may provide new insights for understanding the mechanisms underlying hypoxia and for designing new therapeutic strategies for acute myeloid leukemia.

CaMKII Inhibitor KN-62 Blunts Tumor Response to Hypoxia by Inhibiting HIF-1alpha in Hepatoma Cells

In rapidly growing tumors, hypoxia commonly develops due to the imbalance between O2 consumption and supply. Hypoxia Inducible Factor (HIF)-1alpha is a transcription factor responsible for tumor growth and angiogenesis in the hypoxic microenvironment; thus, its inhibition is regarded as a promising strategy for cancer therapy. Given that CamKII or PARP inhibitors are emerging anticancer agents, we investigated if they have the potential to be developed as new HIF-1alpha-targeting drugs. When treating various cancer cells with the inhibitors, we found that a CamKII inhibitor, KN-62, effectively suppressed HIF-1alpha specifically in hepatoma cells. To examine the effect of KN-62 on HIF-1alpha-driven gene expression, we analyzed the EPO-enhancer reporter activity and mRNA levels of HIF-1alpha downstream genes, such as EPO, LOX and CA9. Both the reporter activity and the mRNA expression were repressed by KN-62. We also found that KN-62 suppressed HIF-1alpha by impairing synthesis of HIF-1alpha protein. Based on these results, we propose that KN-62 is a candidate as a HIF-1alpha-targeting anticancer agent.

Clinical Significance of MMP-2, MMP-9 and HIF-1alpha Expression in Thyroid Micropapillary Cancer

PURPOSE: Papillary Thyroid Microcarcinoma (PTMC) is rapidly increasing due to increased interests in the public health care system and improvements in ultrasonographic instruments and fine-needle-aspiration technique. The aim of this study is to investigate relationships between clinicopathologic features and molecular markers of PTMC and to help in developing therapeutic strategies in PTMC. METHODS: Tissue samples from patients with 38 PTMC and 21 benign thyroid tumors that were operated on from Jan. 2006 to Nov. 2008 were used to make microarrays and immunohistochemical staining for ER-alpha, E-CD, VEGF, MMP-2, MMP-9, and HIF-1alpha were performed. Clinicopathologic features of each immunohistochemical staining group were analyzed retrospectively. RESULTS: There is no immunohistochemistry staining in cases with benign thyroid lesions. The expression rate of ER-alpha, E-CD, VEGF, MMP-2, MMP-9, and HIF-1alpha in PTMC group was 66%, 58%, 82%, 66%, 71% and 63%, respectively. Bilateral tumor was statistically significant (48.0% vs 7.7%, P=0.015) related to MMP-2(+) PTMC group than in MMP-2(-) group. Bilateral tumor (44.4% vs 9.1%, P=0.060) and lymphovascular invasion (25.9% vs 0%, P=0.084) seemed to have greater relation to MMP-9(+) PTMC group than to MMP-9(-) group, but there is no statistically significant difference. Bilateral tumor (50.0% vs 7.1%, P=0.012), lymph node metastasis (45.8% vs 0%, P=0.003) and lymphovascular invasion (29.2% vs 0%, P=0.033) were significantly related to HIF-1alpha (+) PTMC group compared to HIF-1alpha(-) group. CONCLUSION: Our findings suggest that MMP-2, MMP-9 and HIF-1alpha expression could be used as a prognostic marker in PTMC. Larger studies are needed to assess its prognostic value in PTMC.

The Effect of Epigallocatechin-3-gallate on HIF-1alpha and VEGF in Human Lung Cancer Cell Line / 결핵및호흡기질환

BACKGROUND: Epigallocatechin-3-gallate (EGCG) is the major catechin in green tea, and has shown antiproliferative, antiangiogenic, antimetastatic and cell cycle pertubation activity in various tumor models. Hypoxia can be induced because angiogenesis is insufficient for highly proliferating cancer. Hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream target, vascular endothelial growth factor (VEGF), are important for angiogenesis, tumor growth and metastasis. The aim of this study was to determine how hypoxia could cause changes in the cellular phenomena and microenvironment in a non-small cell culture system and to examine the effects of EGCG on a HIF-1alpha and VEGF in A549 cell line. METHODS: A549 cells, a non-small cell lung cancer cell line, were cultured with DMEM and 10% fetal bovine serum. A decrease in oxygen tension was induced using a hypoxia microchamber and a CO2-N2 gas mixture. Gas analysis and a MTT assay were performed. The A549 cells were treated with EGCG (0, 12.5, 25, 50 micromol/L), and then examined by real-time-PCR analysis of HIF-1alpha, VEGF, and beta-actin mRNA. RESULTS: Hypoxia reduced the proliferation of A549 cells from normoxic conditions. EGCG inhibited HIF-1alpha transcription in A549 cells in a dose-dependent manner. Compared to HIF-1alpha, VEGF was not inhibited by EGCG. CONCLUSION: HIF-1alpha can be inhibited by EGCG. This suggests that targeting HIF-1alpha with a EGCG treatment may have therapeutic potential in non-small cell lung cancers.

Different Expressions of HIF-1alpha, Bcl-2 and Baxin DU145 Prostate Cancer Cells Transplanted in Nude Mouse between X-Ray and Neutron Irradiation / 대한방사선종양학회지

PURPOSE: To investigate the radiobiologic effects of neutron and X-ray irradiation on DU-145 prostate carcinoma cells by identifying the differences of HIF-1alpha expression and apoptosis. MATERIALS AND METHODS: Nude mice were injected with the human prostate cancer cell line, DU-145, and then irradiated with 2 Gy and 10 Gy X-rays, or 0.6 Gy and 3.3 Gy neutrons, respectively. The mice were sacrificed at 24 hours and 120 hours after irradiation. The expression levels of HIF-1alpha, Bcl-2 and Bax were compared with immunohistochemical staining and western blotting. The apoptotic indexes were compared with the Terminal deoxynucleotidyl biotin-dUTP nick and labeling (TUNEL) assay. RESULTS: At day 1, HIF-1alpha and Bcl-2 expression decreased, while Bax expression and the number of TUNEL positive cells increased in neutron irradiated groups for the control and X-ray irradiated groups. The Bcl-2/Bax ratio was significantly lower in the neutron irradiated groups regardless of dose (p=0.001). The same pattern of the differences in the expressions of the HIF-1alpha, Bcl-2, Bax, Bcl-2/Bax ratio, and apoptotic indexes were indentified at day 5. HIF-1alpha expression was related with Bcl-2 (p=0.031), Bax (p=0.037) expressions and the apoptotic indexes (p=0.016) at day 5. CONCLUSION: Neutron irradiation showed a decrease in HIF-1alpha, Bcl-2 expression, and Bcl-2/Bax ratio, but increased Bax expression regardless of dose. This study suggests that the differences radiobiological responses between photon and neutron irradiation may be related to different HIF-1alpha expression and subsequent apoptotic protein expressions.

HIF-1alpha and VEGF Expression in Fracture Healing / 대한정형외과학회잡지

PURPOSE: To elucidate the relation between fracture healing and angiogenesis, we checked expression of Hypoxia-inducible factor (HIF) and Vascular endothelial growth factor (VEGF) in hypoxic cell cultures and the callus from a rat femur fracture model. MATERIALS AND METHODS: Human osteoblasts, chondrocytes, and rat ST2 cells were cultured in DME/F12 media with 10% FBS. Hypoxic DME/F12 media (PO2<60 mmHg) was generated by bubbling with 95% N2 and 5% CO2 and added to cells. After 2, 6, and 24 hours, RNA and proteins were collected for reverse transcription - polymerase chain reaction (RT-PCR) and Western blot. In addition, immunocytochemistry and siRNA treatment for HIF-1alpha were performed. Next, femurs from 9-week SD rats were fractured after fixation with needles. The rats were sacrificed at post-fracture day (PFD) 3, 5, 7, 10, 14, 21 and calluses were collected for RT-PCR and Western blot. RESULTS: HIF-1alpha and HIF-2alpha expression were not increased in RT-PCR but protein levels were increased. VEGF expression in RT-PCR was increased. Treatment with siRNA directed towards HIF inhibited VEGF expression. In the rat fracture callus, HIF-1alpha and VEGF expression peaked between PFD 5 and 7 and decreased after PFD 10. In contrast to cell culture, mRNA expression of HIF-1alpha was increased at PFD 7. CONCLUSION: HIF-1alpha and VEGF peaked early in fracture healing. With expression decreasing as O2 tension increased. Further study is needed to identify other factors affecting chondrogenic differentiation.

Expression of Hypoxia-inducible Factor-1alpha and Vascular Endothelial Growth Factor in Colon Cancer: Relationship to the Prognosis and Tumor Markers

PURPOSE: Angiogenesis is one of the key steps in solid tumor growth and metastasis. We investigated the prognostic significance of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) expressions as markers of angiogenesis in colon cancer. METHODS: We retrospectively reviewed the medical records of 78 patients with colon or rectal cancer who underwent a surgical resection at Soonchunhyang University Hospital from January 2000 to December 2001, and we evaluated the expression of VEGF and HIF-1alpha in archival tumor tissues by using immunohistochemistry. We recorded the clinical and the pathological characteristics of the patients and analyzed their survival outcomes. RESULTS: Thirty-four (34) patients were male, and the mean age of all the patients was 66.7 years. HIF-1alpha and VEGF were positive in 56% (44 patients) and 53% (42 patients) of the tumors, respectively. HIF-1alpha expression was significantly associated with several pathological parameters, such as TNM stage (P=0.001), lymph node metastasis (P=0.001). HIF-1alpha expression was also associated with VEGF expression (P=0.032). The survival of patients with HIF-1alpha expression was worse than that of patients with no HIF-1alpha expression (P=0.036). However, VEGF expression was not associated with other pathological characteristics. CONCLUSIONS: We suggest that, in cases of colorectal cancer, HIF-1alpha expression may be associated with expression of VEGF, progression of tumors, and poor prognosis.