IQGAP1 is overexpressed and interacts with ERK in human myeloma cells

AIM:To explore the role of IQ motif-containing GTPase-activating protein 1 (IQGAP1) in the cell proliferation of multiple myeloma and its mechanism. METHODS:RT-PCR and Western blotting were performed to detect the expression of IQGAP1 in human myeloma cell lines RPMI8226 and U266. shRNA-expressing plasmids were used to transfect into RPMI8226 cells to knock down the expression of IQGAP1. MTT assay was used to examine the proliferation of RPMI8226-shIQGAP1 (clone 1) cells, RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells with or without VEGF/IL-6 treatment. The protein levels of p-ERK1/2, ERK1/2, AKT, p-AKT, STAT3 and p-STAT3 in RPMI8226-shIQGAP1 (clone 1) cells, RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells were measured by Western blotting. The interaction between IQGAP1 and ERK was determined by the method of co-immunoprecipitation. RESULTS:IQGAP1 was overexpressed in human myeloma cell lines RPMI8226 and U266 as compared with normal lymphocytes. Transfection with shRNA targeting to IQGAP1 decreased the expression levels of IQGAP1 in RPMI8226 cells. The proliferation of RPMI8226 cells decreased when IQGAP1 was knocked down by shRNA with or without VEGF/IL-6 treatment. IQGAP1 affected the proliferation of RPMI8226 cells by regulation of MAP kinase (ERK1/2) pathway. The level of p-ERK1/2 in RPMI8226-shIQGAP1 (clone 1) cells decreased by 70.2% as compared with RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells. The interaction between IQGAP1 and ERK in RPMI8226 cells was observed. CONCLUSION: IQGAP1 plays an important role in the cell proliferation of multiple myeloma via MAP kinase (ERK) pathway.     

Angiogenic effect of HIF-1α on extranodal nasal-type NK/T-cell lymphoma

AIM: To study the angiogenic effect of hypoxia inducible factor 1α(HIF-1α) and its significance on human extranodal nasal-type NK/T-cell lymphoma. METHODS: The protein levels of HIF-1α, vascular endothelial growth factor(VEGF) and VEGF receptor 2(VEGFR2) in human extranodal nasal-type NK/T-cell lymphoma were detected by immunohistochemistry. Microvessel density (MVD) of the tumor tissues was determined by labeling of microvessel endothelium with CD34 antibody. The correlation between the expression of HIF-1α, VEGF and VEGFR2 and MVD was analyzed with SPSS 13.0 statistical software. RESULTS: The positive expression of HIF-1α was observed in 39 cases (39/50, 78%) and the positive expression of VEGFR2 was 27 cases (27/50, 54%) of human extranodal nasal-type NK/T-cell lymphoma. A statistical difference of HIF-1α and VEGFR2 expression between tumor tissues and normal lymphocytes in lymph node was observed (P<0.05). In the tumor tissues, the co-expression of VEGF or VEGFR2 with HIF-1α was 72% and 64%, respectively, significantly higher than that without HIF-1α co-expression (P<0.05). The expression of HIF-1α, VEGFR and VEGFR2 was positively correlated with MVD of the tumor tissues (P<0.01). HIF-1α was expressed in all 15 cases of extranodal nasal-type NK/T-cell lymphoma with angiocentric infiltration.CONCLUSION: HIF-1α may promote angiogenesis of extranodal nasal-type NK/T-cell lymphoma through VEGF/VEGFR2 signaling pathway.     

Effect of hypoxia-inducible factor-1α knockdown by siRNA on viability and CEACAM1 expression in oral squamous cell carcinoma

AIM: To investigate the relationship between hypoxia-inducible factor-1α (HIF-1α) and viability and apoptosis of oral squamous cell carcinoma and its mechanism. METHODS: The expression of HIF-1α and carcinoembryonic antigen-related cell adhesion molecular 1 (CEACAM1) at mRNA and protein levels in oral squamous cell carcinoma cell lines Tca8113 and CAL27 and normal epithelial cell line NOK was determined by RT-PCR and Western blot. The expression of HIF-1α in CAL27 cells was silenced by RNA interference (RNAi) technique. The cells were divided into blank control group, non-sense control group and siRNA-HIF-1α group. The viability of CAL27 cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry. The protein levels of HIF-1α, P21, vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined by Western blot. RESULTS: The expression of HIF-1α and CEACAM1 in oral squamous cell carcinoma cells was significantly higher than that in normal cells (P<0.05), and the expression of HIF-1α and CEACAM1 was positively correlated. The protein expression of HIF-1α in siRNA-HIF-1α group was significantly lower than that in blank control group (P<0.05). Knockdown of HIF-1α significantly inhibited CAL27 cell viability (P<0.05), promoted apoptosis (P<0.05), increased the protein levels of P21 and Bax (P<0.05), and significantly decreased the levels of VEGF and Bcl-2 (P<0.05). CONCLUSION: HIF-1α is over-expressed in oral squamous cell carcinoma. Knockdown of HIF-1α significantly inhibits cell viability and promotes apoptosis possibly through regulating the expression of HIF-1α downstream target genes and tumor angiogenesis.     

H IF-1αm ediates effect of interm ittent hypoxia on biological behavior of A 549 cells in vitro

ATM: To investigate whether hypoxia-inducible factor 1α (HIF-1α) mediates the effect of intermittent hypoxia on A549 cell viability, apoptosis and invasive ability METHODS: A549 cells were transfected with HIF-1α-siRNA and cultured under intermittent hypoxia. The expression of HIF-1α and its downstream genes, such as Bcl-2, Bax, P53, P21 and VEGF at mRNA and protein levels was determined by real-time PCR and Western blot. The viability of the A549 cells was measured by MTT assay. The apoptosis and cell cycle distribution of the A549 cells were examined by flow cytometry. The invasive ability of the A549 cells was detected by transwell test. RESULTS: The expression levels of HIF-1α, Bcl-2 and VEGF in non-HIF-1α-siRNA transfected A549 cells cultured in intermittent hypoxia environment[blank controlgroup(IH C),empty vector control group (IH E) and negative control group (IH N)] were higher than those in the A549 cells in normoxia group (RA), but the expression levels of Bax and P21 were lower than those in RA group (P<0.05). The siRNA-mediated HIF-1α gene silencing[intermittent hypoxia silenced group (IHS)] resulted in obvious down-regulation of HIF-1α, Bcl-2 and VEGF, and significant increase in the protein expression of P21 and Bax(P<0.05). The expression level of P53 in intermittent hypoxia groups was significantly higher than that in RA group, and no significant difference of P53 expression in different intermittent hypoxia groups was observed. Compared with normoxia, intermittent hypoxia resulted in significantly enhanced cell viability, decreased apoptosis, and enhanced invasive ability of non-HIF-1α-siRNA transfected A549 cells (P<0.05). The siRNA-mediated HIF-1α gene silencing resulted in significant cell viability inhibition, elevated apoptotic rate and decreased invasive ability under hypoxic condition (P<0.05).CONCLUSION: Intermittent hypoxia promotes the viability and invasion of A549 cells by HIF-1α-mediated downstream gene expression. HIF-1α gene silencing inhibits A549 cell growth and invasion under intermittent hypoxia by inhibition of HIF-1α signal pathways in vitro.     

Role of HIF-1α/VEGF pathway in treatment of cerebral ischemia-reperfusion injury by hyperbaric oxygen pretreatment

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway in hyperbaric oxygen (HO) pretreatment in rats with cerebral ischemia-reperfusion (IR) injury. METHODS: Healthy male SD rats (n=32) were randomly divided into control group IR group, HO-IR group and HO-IR-HIF-1α inhibitor group (HO-IR-I group). The IR model was established by middle cerebral artery occlusion. The corresponding blood vessels of the rats in control group were only exposed. The rats in HO-IR group and HO-IR-I group were treated with HO for 4 weeks before the animal modeling. The rats in HO-IR-I group received 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazol (YC-1) by intraperitoneal injection at 4 mg/kg before HO preconditioning every day. At 1 d and 7 d after modeling, the neurological assessment was evaluated.At the end of the 7 th day, after observation, the rats were sacrificed by anesthesia to measure the infarct volume of the brain tissue. The protein levels of HIF-1α, VEGF and apoptosis-related proteins Bax and Bcl-2 were determined by Western blot. The number of apoptotic cells was detected by TUNEL. RESULTS: Compared with control group, the neurological function score was decreased, while the cerebral infarction volume ratio, the protein levels of HIF-1α, VEGF, Bcl-2 and Bax, and the apoptotic cells were increased in IR group, HO-IR group and HO-IR-I group (P<0.05). Compared with IR group, the neurological function score, and the protein levels of HIF-1α,VEGF and Bcl-2 were increased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were decreased in HO-IR group and HO-IR-I group (P<0.05). Compared with HO-IR group, the neurological function score, and the protein levels of HIF-1α, VEGF and Bcl-2 were decreased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were increased in HO-IR-I group (P<0.05). CONCLUSION: The mechanism of HO preconditioning attenuating cerebral IR injury may be related to the regulation of apoptosis by inducing HIF-1α/VEGF signaling pathway activation.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Effect of AG490 on expression of VEGF and HIF-1α in HEL cells

AIM: To investigate the effect of AG490 on the expression of VEGF and HIF-1α, and the capacity of invasion in human erythroleukemia (HEL) cells. METHODS: The HEL cells were treated with AG490 at different concentrations. The cell viability was detected by CCK-8 assay. The apoptosis was detected by Hoechst staining. The apoptosis and the cell cycle were analyzed by flow cytometry. The capacity of migration was evaluated by Transwell assay. The mRNA expression level of JAK2 was measured by RT-PCR. The protein levels of p-JAK2, VEGF and HIF-1α were determined by Western blot. RESULTS: The HEL cell viabilities were 88%, 75%, 48%, 10% and 0.12% after treated with AG490 at 20, 40, 60, 80 and 100 μmol/L for 48 h, respectively. The results of Hoechst staining showed that brilliant blue cells in 80 μmol/L AG490 group was significantly increased compared with control group for 48 h. The apoptosis rate of 80 μmol/L AG490 group was significantly increased compared with control group at 48 h after AG490 treatment. The number of membrane-permeating HEL cells in 20 μmol/L AG490 group at 24 h after AG490 treatment was significantly lower than that in control group (P<0.05). The mRNA level of JAK2 decreased in a concentration-dependent manner after the HEL cells were treated with different concentrations of AG490 for 48 h. The protein levels of p-JAK2, VEGF and HIF-1α were lower in AG490 treatment groups than those in control group (P<0.05). CONCLUSION: AG490 inhibits the expression of VEGF and HIF-1α in HEL cells by inhibiting JAK2 pathway.     

Regulatory effects of DIS3 expression on colony formation ability in human myeloma cells and tube structure formation of endothelial cells

AIM:To investigate the effects of DIS3 expression on the colony formation ability of 3 kinds of human myeloma cells and tube structure formation of the endothelial cells. METHODS:Human myeloma cell lines NCI-H929, RPMI-8226 and U266 were selected as the study objects, and DIS3 gene over-expression vector and DIS3-siRNA were designed and constructed respectively. The cell experiments were divided into 5 groups:control group, siRNA negative control (siRNA-NC) group, siRNA-DIS3 group, empty vector group and DIS3 over-expression group. The colony formation ability was tested by the plate colony formation assay. Western blot was used to detect the protein expression of hypoxia inducible factor-1α (HIF-1α) and HIF-3α. The expression of angiogenesis-related molecules angiogenin 1 (Ang1), Ang2 and vascubar enelothelial growth factor-A(VEGF-A) at the mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Matrigel method was used to detect the effect of supernatant from each group of the cells on the tube structure formation of HUVECs. RESULTS:The trends of the following indexes in NCI-H929 cells, RPMI-8226 cells and U266 cells were similar. Compared with empty vector group, the colony formation ability of the cells in DIS3 over-expression group was significantly inhibited (P<0.05). Compared with siRNA-NC group, siRNA-DIS3 significantly enhanced the colony formation ability of the cells (P<0.05). DIS3 over-expression significantly reduced the expression of HIF-1α and HIF-3α (P<0.05), while knock-down of DIS3 expression significantly increased the protein levels of HIF-1α and HIF-3α (P<0.05). In addition, DIS3 over-expression significantly reduced the expression of Ang1, Ang2 and VEGF-A at mRNA and protein levels (P<0.05), while siRNA-DIS3 significantly promoted the expression of Ang1, Ang2 and VEGF-A (P<0.05). Compared with empty vector group, the supernatant from DIS3 over-expression group significantly inhibited the tube structure formation of HUVECs (P<0.01). Compared with siRNA-NC group, the supernatant from siRNA-DIS3 group significantly promoted the tube structure formation of HUVECs (P<0.05). CONCLUSION:DIS3 over-expression significantly inhibits the colony formation ability of human myeloma cells and tube structure formation of HUVECs, which may be closely related to the regulation of the expression of HIF-1α and HIF-3α.     

Effects of sodium valproate on RPMI8226 and U266 cell proliferation and IL-6/JAK/STAT signaling pathway

AIM: To investigate the effects of sodium valproate (VPA) on the proliferation of multiple myeloma cell lines RPMI8226 and U266 and the regulation of IL-6/JAK/STAT signaling pathway. METHODS: The cells were treated with different concentrations of VPA for 12 h and 24 h. The growth of RPMI8226 cells and U266 cells was detected by MTT assay. Apoptotic rates and cell cycle were analyzed by flow cytometry. The mRNA expression of STAT3, STAT5 and STAT target genes Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF was measured by RT-PCR. Western blotting analysis was used to determine the total proteins and protein phosphorylation levels of JAK2 and STAT5. RESULTS: VPA inhibited the growth and induced the apoptosis of RPMI8226 cells and U266 cells in a concentration- and time-dependent manner. The levels of IL-6 in the culture supernatants of RPMI8226 cells and U266 cells treated with VPA were significantly higher than that in negative control group. VPA down-regulated the mRNA expression of STAT3, STAT5, Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF. After treated with VPA, the protein levels of p-JAK2, JAK2, p-STAT5 and STAT5 in RPMI8226 cells and U266 cells were significantly lower than those in control group. CONCLUSION: VPA inhibits the proliferation of PRMI8226 cells and U266 cells in vitro. The modulation of IL-6/JAK/STAT signaling pathway may be involved in its potential mechanisms.     

Effects of curcumin on migration and invasion of myeloma cells

AIM：To explore the effect of curcumin on the migratory and invasive abilities of myeloma cells. METHODS：shRNA expression plasmid was transfected into RPMI8226 cells to knock down IQ motif-containing GTPase-activating protein 1 (IQGAP1). The expression of IQGAP1 in RPMI8226 cells tranfected with shIQGAP1 or shRNA negative control, and in un-transfected RPMI8226 cells was detected by Western blotting. All the cells in RPMI8226-shIQGAP1 group, RPMI8226-shRNA negative control group and un-transfected RPMI8226 group were treated with curcumin at various concentrations. The migratory and invasive abilities of the RPMI8226 cells were measured by Transwell chamber and Matrigel assays. The expression of IQGAP1 at mRNA and protein levels in the RPMI8226 cells treated with curcumin was also determined by RT-PCR and Western blotting. RESULTS：The expression of IQGAP1 decreased when IQGAP1 gene was knocked down by shRNA. The migration and Matrigel invasion tests showed that the number of cells moving into under chamber of Transwell decreased in RPMI8226-shIQGAP1 group in comparison with the other 2 groups. Curcumin decreased the migratory and invasive abilities of RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells, which was not related to the curcumin concentratory. The migratory and invasive abilities of RPMI8226-shIQGAP1 cells showed no significant difference when treated with curcumin at various concentrations. The expression of IQGAP1 at mRNA and protein levels decreased in the RPMI8226 cells treated with curcumin. CONCLUSION：Curcumin decreases the migratory and invasive abilities of myeloma cells via inhibition of IQGAP1 expression.     

Hypoxia-induced caveolin-1 up-regulation is involved in migration and invasion of lung adenocarcinoma A549 cells

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride (CoCl2) on caveolin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcinoma A549 cells. METHODS:The concentrations of Cav-1 and hypoxia-inducible factor (HIF)-1α in pleural effusion of the patients with lung cancer (MPE) or tuberculous pleurisy (TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1α inhibitor YC-1. The concentrations of Cav-1 and HIF-1α in the cell supernatants were measured by ELISA. The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respectively. RESULTS:The levels of Cav-1 and HIF-1α in MPE were significantly higher than those in TBPE. There was a highly positive correlation between Cav-1 and HIF-1α levels in the pleural effusion. CoCl2 induced the generation of Cav-1 and HIF-1α in A549 cells in a concentration- and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration- or time-dependent inhibition was observed. HIF-1α inhibitor YC-1 concentration-dependently inhibited the generation of HIF-1α and Cav-1 induced by CoCl2 in A549 cells. CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1. CONCLUSION: The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells. A possible role for HIF-1α is indicated in Cav-1 generation.     

Expression of hypoxia-inducible factor 1α in human gingival tissues with chronic periodontitis

AIM：To observe the expression of hypoxia-inducible factor 1α (HIF-1α) in human gingival tissues with chronic periodontitis. METHODS：A total of 55 volunteers, including 15 healthy controls, 20 cases of moderate chronic periodontitis and 20 cases of severe chronic periodontitis, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining, and the expression of HIF-1α in gingival tissues was detected by immunohistochemical staining. RESULTS： The proportion of HIF-1α positive cells in gingival tissues was significantly higher in chronic periodontitis groups than that in healthy control group (P<0.01), and that in severe chronic periodontitis group was significantly higher than that in moderate chronic periodontitis group (P<0.05). There was a significantly positive correlation between the severity of chronic periodontitis and the proportion of HIF-1α positive cells in gingival tissues. CONCLUSION：The expression of HIF-1α in human gingival tissues is increased with the severity of chronic periodontitis, suggesting that hypoxia may play an important role in chronic periodontitis.     

Effect of conditioned medium of human multiple myeloma RPMI 8226 cells on osteoclastogenesis of RAW264.7 cells

AIM: To investigate the osteoclastogenic effect of conditioned medium of human multiple myeloma RPMI 8226 cells on preosteoclast RAW264.7 cells. METHODS: The protein expression of soluble receptor activator of NF-κB ligand (sRANKL) was detected by Western blotting. The morphological changes of RAW264.7 cells were observed after tartrate-resistant acid phosphatase (TRAP) staining. The mRNA expression of TRAP and cathepsin K was evaluated by RT-PCR. RESULTS: The result of Western blotting showed that conditioned medium of RPMI 8226 cells contained sRANKL. RPMI 8226 cell conditioned medium induced RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppressed the differentiation of preosteoclasts in a dose-dependent manner, which was induced by 30% RPMI 8226 cell conditioned medium. RPMI 8226 cell conditioned medium increased the mRNA expression of TRAP and cathepsin K in RAW264.7 cells. CONCLUSION: The sRANKL in conditioned medium of human multiple myeloma RPMI 8226 cells has the bioactivity to induce preosteoclast RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppresses the differentiation of preosteoclasts induced by sRANKL in a dose-dependent manner.     

Fucoidan sulfate promotes apoptosis of gastric cancer cells by regulating expression of HDAC1

AIM:To study the effects of fucoidan sulfate on the apoptosis of gastric cancer cells, and to explore its relationship with histone deacetylase 1 (HDAC1). METHODS:The effects of fucoidan sulfate on the viability of gastric cancer BGC-823 cells were measured by MTT assay. The cells were divided into si-HDAC1 group, si-NC group and fucoidan+pcDNA 3.1-HDAC1 group and fucoidan+pcDNA 3.1 group. The transfection were performed by liposome method. The apoptosis was analyzed by flow cytometry. The protein expression of HDAC1, Bcl-2 and Bax was determined by Western blot. The mRNA expression of HDAC1 was detected by RT-qPCR. RESULTS:Compared with the control cells, the inhibitory rates of the cell viability by fucoidan sulfate (0.2, 0.4, 0.6, 0.8 and 1.0 g/L) were increased signi-ficantly (P<0.05). The appropriate concentration was 0.6 g/L. Compared with control group, the expression of HDAC1 at mRNA and protein levels in fucoidan sulfate group was significantly down-regulated, while the apoptotic rate was significantly increased (P<0.05). Knock-down of HDAC1 expression significantly up-regulated the apoptotic rate, while over-expression of HDAC1 reversed the apoptosis-promoting effect of fucoidan sulfate on the BGC-823 cells. CONCLUSION:Fucoidan sulfate promotes apoptosis of gastric cancer cells. The mechanism may be related to the direct inhibition of HDAC1, which provides support for the clinical application of fucoidan sulfate.     

Effects of NBP on expression of VEGF and HIF-1α in HUVECs under the condition of oxygen-glucose deprivation

AIM: To investigate the mechanism that dl-3-n-butylphthalide (NBP) protects cells from the induced by oxygen-glucose deprivation (OGD).METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Endothelial injury was assessed by measuring the changes of chromatin morphology and MTT method. The protein levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were determined by immunofluorescence and quantitatively analyzed with the software IPP. Western blotting was applied to verify the results.RESULTS: NBP at the concentrations of 0.01 to 100 μmol/L dose-dependently protected against OGD-induced cell damage. Compared with OGD group, NBP enhanced OGD-induced the expression of VEGF and HIF-1α, and the difference was statistically significant. The expression of VEGF and HIF-1α reached to the peak at the time points of 6 h and 8 h after OGD, respectively.CONCLUSION: Under the condition of OGD, NBP enhances the expression of HIF-1α in HUVECs, subsequently promotes the expression of downstream VEGF, and eventually elevates the survival of the cells.     

Effects of tanshinone IIA on proliferation,apoptosis and expression of HIF-1α, VEGF and wild-type P53 in human hepatoma HepG2 cells under hypoxia

AIM:To investigate the effects of tanshinone IIA (Tan IIA) on proliferation, apoptosis and its molecular mechanism in human hepatoma HepG2 cells under hypoxic condition. METHODS:Hypoxia model was established by treatment with cobalt chloride (CoCl2). The cells were divided into normoxia control group, hypoxia control group and hypoxia combined at different concentrations of Tan IIA groups. After HepG2 cells were incubated with different concentrations of Tan IIA (0.5, 1.0, 2.0, 5.0 and 10.0 mg/L) for 24 h, 48 h and 72 h under hypoxic condition, the cell proliferation was determined by MTT assay. After Tan IIA was added to the media at different concentrations for 24 h and 48 h, the apoptotic cells were observed by Hoechst 33258 staining. The protein levels of hypoxia-inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and wild-type P53 were detected by Western blotting after cultured with different concentrations of Tan IIA for 48 h. RESULTS:Tan IIA inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner. Tan IIA induced the typical morphology of apoptotic cells and increased the apoptotic rate in a dose- and time-dependent manner after treatment with 1.0 mg/L~5.0 mg/L for 24 h and 48 h under hypoxic condition. The protein levels of HIF-1α and VEGF were weakly expressed in HepG2 cells under normoxia but up-regulated after incubated under hypoxia for 48 h. The protein expression of HIF-1α and VEGF were decreased with the increase in the concentration of Tan IIA under hypoxia. The protein expression of wild-type P53 was increased with the increase in the concentrations of Tan IIA under hypoxia. CONCLUSION: Tan IIA significantly inhibits the proliferation and induces the apoptosis of human hepatoma HepG2 cells under hypoxia, which may be related to the down-regulation of HIF-1α and VEGF and up-regulation of wild-type P53.     

Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via ERK-mediated autophagy inhibition

AIM: To explore the effect of Pycnogenol on transforming growth factor-β1 (TGF-β1)-induced hepatic stellate cell activation. METHODS: Cultured LX-2 cells were treated with 5 μg/L TGF-β1 and different concentrations (0, 10, 25 and 50 mg/L) of Pycnogenol. The viability of the LX-2 cells under the conditions with or without autophagy inhibitor 3-MA and ERK inhibitor PD98059 was determined by MTT assay. The protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2 and ERK1/2 were detected by Western blot. RESULTS: Compared with control group, 5 μg/L TGF-β1 treatment elevated the cell viability, and increased the protein levels of α-SMA, ColⅠ, TIMP-1, LC3-Ⅱ/Ⅰ, beclin 1, p-ERK1/2, and ERK1/2 in the LX-2 cells (P<0.05). However, these effects were reversed by Pycnogenol pretreatment in a dose-dependent manner and the inhibitory effect of 50 mg/L Pycnogenol was the most significant in the LX-2 cells (P<0.05). Furthermore, compared with TGF-β1 group, pretreatment with 50 mg/L Pycnogenol, 5 mmol/L 3-MA or 20 μmol/L PD98059 downregulated TGF-β1-induced cell viability and the protein levels of α-SMA and LC3-Ⅱ/Ⅰ in the LX-2 cells (P<0.05). CONCLUSION: Pycnogenol suppresses TGF-β1-induced hepatic stellate cell activation via p-ERK and autophagy inhibition.     

Expression of hypoxia-inducible factor 1α in pulmonary tissues from patients with chronic obstructive pulmonary disease

AIM：To study the expression of hypoxia-inducible factor 1α (HIF-1α) in pulmonary tissues from patients with chronic obstructive pulmonary disease (COPD) μg/L and its effects on the pathogenesis of COPD. METHODS：Pulmonary tissues were obtained from 32 subjects (16 patients with COPD and 16 without COPD as controls) who were undergoing single or bilateral lobectomy or wedge resection for lung cancer. The specimens were obtained as far away from the cancer foci (≥8 cm) as possible. The expression of HIF-1α protein in pulmonary tissues was measured by Western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS：The expression of HIF-1α protein in pulmonary tissues from controls and COPD patients was as follows: (0.96±0.43) μg/L and (0.16±0.07) μg/L (ELISA， P<0.05); 0.71±0.22 and 0.53±0.15 (Western blotting, P<0.05). Furthermore, the level of HIF-1α protein in pulmonary tissues from mild and moderate COPD patients was obviously higher than that from severe COPD patients (P<0.05). CONCLUSION：HIF-1α may play an important role in the progress of COPD.     

Recombinant human endostatin regulates proliferation and related protein expression in human multiple myeloma cell line RPMI 8226

AIM: To investigate the effects of recombinant human endostatin (Endostar) on proliferation, cell cycle constitution and regulation of related protein expression in RPMI 8226 cells. METHODS: CCK-8 assay was used to explore the effect of Endostar on the proliferation of RPMI 8226 cells. The action of Endostar on apoptosis and cell cycle distribution were determined by flow cytometry. The expression of Bcl-2 and caspase-3 was evaluated by Western blotting analysis. The production of vascular cell adhesion molecule 1 (VCAM-1), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) in response to Endostar was measured by real-time PCR and Western blotting. The influence of Endostar on the secretion of IL-6 and VEGF in the culture supernatants was detected by ELISA. RESULTS: Endostar inhibited the proliferation of RPMI 8226 cells with the highest inhibitory rate of 59.5±5.6% at dose of 250 mg/L, which was associated with slight up-regulation in the proportion of G1 phase. No significant modulation of apoptotic rate was observed with Endostar, as well as the expression of Bcl-2 or caspase-3. Endostar declined the expression of VCAM-1 and also attenuated the production and secretion of IL-6 and VEGF in RPMI 8226 cells. CONCLUSION: Endostar suppresses the proliferation of RPMI 8226 cells by enhancing the proportion of G1 phase and decreasing VCAM-1 expression and self-secretion of IL-6 and VEGF. No apoptosis of RPMI 8226 cells is induced by Endostar.