Parthenolide enhances the apoptosis induced by 4-hydroxyphenyl-retinamide in human hepatoma cells

AIM:To detect the signal pathway of apoptosis induced by 4-hydroxyphenyl-retinamide (4-HPR) and the biological effect of parthenolide-induced apoptosis. METHODS:TUNEL staining, FCM analysis, electrophoretic mobile shift assay (EMSA) were used to determine the actual effects and its mechanism of parthenolide on the 4-HPR-induced apoptosis in human hepatoma Hep-3B and SK-Hep-1 cells. RESULTS:The results of TUNEL and PI staining showed that parthenolide selectively enhanced 4-HPR-induced apoptosis in Hep-3B and SK-Hep-1 cells. Subsequent observations using EMSA assay indicated that parthenolide effectively inhibited NF-κB activation during fenretinide-induced apoptosis. CONCLUSION:These findings indicate that parthenolide suppresses 4-HPR-induced apoptosis via inhibition of NF-κB activation and that NF-κB activation during fenretinide-induced apoptosis might have an anti-apoptotic effect.     

Effects of angiotensin-converting enzyme 2 gene transfection on the expression of macrophage migration inhibitory factor in human endothelial cells

AIM: To implore the effects of recombinant angiotensin- converting enzyme 2 (ACE2) gene transfection on the expression of macrophage migration inhibitory factor (MIF) induced by angiotensin (Ang) II in cultured human endothelial cells. METHODS: A recombinant plasmid encompassing human ACE2 gene (pACE2) was constructed and transfected into human endothelial cells. Endothelial cells were stimulated with Ang II or Ang IV in the presence and absence of pACE2 gene transfer. The mRNA and protein levels of MIF in endothelial cells were determined by real-time PCR and Western blotting, respectively. RESULTS: The mRNA and protein expressions of MIF were strikingly enhanced after exposures of endothelial cells to 100 nmol/L Ang Ⅱ and 100 nmol/L Ang Ⅳ (P<0.01, respectively). However, significant downregulations of MIF mRNA and protein expression were observed in endothelial cells pretreated with pACE2 gene transfer (P<0.05, respectively). CONCLUSION: ACE2 gene overexpression contributes to diminishments of inflammation mediator MIF expression in endothelial cells, suggesting that ACE2 gene has anti-inflammatory properties to some extent and may provide novel therapeutic strategies for the inflammation-related diseases such as atherosclerosis.     

Proliferation and apoptosis of human umbilical vein endothelial cells induced by oxidized low density lipoprotein

AIM:To investigate proliferation and apoptosis of cultured endothelial cell (ECV-304 cell line) induced by varied concentrations of oxidized low density lipoprotein (ox-LDL). METHODS:Cell morphology, Typan blue test, MTT test, LDH release test, flow cytometry and micro-molecular weight DNA fragment gel electrophoresis of apoptosis were used for the detection of the cytotoxic effects of ox-LDL on ECV-304 cell line.RESULTS:0.1, 1, 10 mg/L ox-LDL could promote proliferation of ECV-304 cells. When the concentration of ox-LDL reached up to 100 mg/L and above, the distinct cytotoxic effect appeared. Further study showed that the apoptosis rate of endothelial cells, induced by ox-LDL of 150 mg/L and 200 mg/L for 12 hours, are 15.86% and 21.89%, respectively. 18 h and above hours after incubation, the apoptosis rate began to decrease and rate of necrosis increased. CONCLUSION:ox-LDL has strong cytotoxic effects on endothelial cells and could give rise to different pathologic process, such as proliferation, apoptosis prophase, apoptosis and necrosis.     

Gene transfer and expression of recombined human proinsulin in hepatoma cells of rats

AIM:To explore the recombined human proinsulin gene containing glucose reaction element (GLRE) expression in transfected CBRH7919 cells. METHODS:The packaged retrovirus encoding genetically modified human proinsulin PLXSN-(GLRE)3-BP-1MpINS3 and PLXSN-(GLRE)3-BP-1MpINS2 were transfected into CBRH7919 cells. Insulin values in cells after transient and steady expression screened by G418 at different glucose levels were detected. Chromosome DNA was isolated from transfected and untransfected cells and polymerase chain reaction (PCR) was performed. PCR products were analyzed by electrophoresis. RESULTS:38 h after transfection, at the glucose levels of 0-25 mmol/L, the levels of insulin produced by cells including PLXSN-(GLRE)3-BP-1MpINS3 were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L, respectively (P<0.05). One month after transfection, under above glucose levels, insulin values were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L (P<0.05). CBRH7919 cells including PLXSN-(GLRE)3-BP-1MpINS2 secreted detectable insulin value at the level of 25 mmol/L, they were (2.10±0.23)U/L and (2.05±0.17)U/L, respectively. PCR products of transfected cells showed target band, but control cells did not. CONCLUSIONS:Recombined proinsulin gene was transfected successfully in CBRH7919 cells. The cells combined human proinsulin gene has the ability of producing insulin with increase in glucose concentration in vitro.     

Effect of GPNMB on proliferation,apoptosis and invasion of human hepatoma cells

AIM：To investigate the effect of glycoprotein nonmetastatic melanoma protein B (GPNMB) on the proliferation, apoptosis and invasion of human hepatoma HepG2 cells and its molecular mechanisms. METHODS：The GPNMB siRNA and GPNMB-overexpressing vector were constructed, and then transfected into HepG2 cells. MTT assay, flow cytometry and Transwell chamber were used to determine the effects of GPNMB down-regulation and up-regulation on the proliferation, apoptosis and invasive ability of HepG2 cells. RESULTS：The proliferation of HepG2 cells was obviously promoted by the up-regulation of GPNMB. No effect of GPNMB on the apoptosis of HepG2 cells was observed. The invasion of HepG2 cells was also significantly promoted by the up-regulation of GPNMB. When integrin β1 was silenced by siRNA, the promoting effect of GPNMB on the proliferation and invasive ability of HepG2 cells was significantly suppressed. CONCLUSION: GPNMB may promote the proliferation and invasion of HepG2 cells by the interaction with integrin β1, and may be used as a potential therapeutic target in liver cancer.     

Hindlimb ischemia and granulocyte colony-stimulating factor accelerates mobilization of endothelial progenitor cells in mice peripheral blood

AIM: To evaluate the mobilization of endothelial progenitor cells (EPCs) in mice peripheral blood during hindlimb ischemia alone or in combination with granulocyte colony stimulating factor (G-CSF). METHODS: Hindlimb ischemia was established in mice by surgical excision of both femoral arteries. Fluorescence-activated cell sorting (FACS) was used to detect the expression of cell-surface CD34 and vascular endothelial growth factor recepter-2 (VEGFR2) antigens. The ratio of double-positive cells for CD34 and VEGFR2 was regarded as the level of EPCs in peripheral blood. In G-CSF administration in combination with hindlimb ischemia group, the percentage of double-positive cells was also detected. RESULTS: As compared with control group, hindlimb ischemia increased the percentage of EPCs in mice peripheral blood. The hindlimb ischemia combined with G-CSF administration significantly enhanced the percentage of EPCs. CONCLUSION: Ischemia increases the number of EPC in peripheral blood. It may induce the migration of EPC from barrow to peripheral blood. By mobilizing barrow, G-CSF enhances this effect.     

Expression of macrophage migration-inhibitory factor in human recurrent cervical cancer tissues and its prognostic significance

AIM：To investigate the expression of macrophage migration-inhibitory factor (MIF) in human recurrent cervical cancer tissues and its influence on prognosis. METHODS：Eight-seven cases of recurrent cervical cancer were collected from January 2007 to December 2009 and followed up for at least 36 months. The MIF expression in cervical cancer tissues was tested by immunochemistry. RESULTS：The positive rate of MIF expression in recurrent cervical cancer was 75.7% (55/87). Compared with the patients with negative MIF expression, the patients with positive MIF expression showed worse response to antitumor therapy ［47.3% (26/55) vs 63.6% (21/32), P<0.05］ and shorter survival time (28.8 months vs 38.4 months, P<0.05). CONCLUSION：Positive MIF expression could be a risk factor in recurrence and prognosis of cervical cancer.     

Effects of XPD on expression of Rb and MAD2 in human hepatoma HepG2 cells

AIM：To investigate the effects of xeroderma pigmentosum group D (XPD) protein on the growth of human hepatoma HepG2 cells and the expression of retinoblastoma (Rb) and mitotic arrest deficient 2 (MAD2) proteins. METHODS：The recombinant plasmid pEGFP-N2-XPD and empty plasmid pEGFP-N2 were transfected into HepG2 cells by LipofectamineTM 2000. The cells were divided into 4 groups including blank control group, liposome group, pEGFP-N2 group (N2 group) and pEGFP-N2-XPD group (XPD group). The expression of XPD, Rb and MAD2 at mRNA and protein levels was detected by RT-PCR and Western blotting. The cell growth was measured by MTT assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS：Overexpression of XPD up-regulated the expression of Rb, and down-regulated the expression of MAD2 at mRNA and protein levels. XPD inhibited the proliferation of HepG2 cells and exacerbated the apoptosis. XPD prevented the hepatoma cells from G1 stage to S stage. CONCLUSION：XPD suppresses the growth of hepatoma cells, up-regulates the expression of Rb, and down-regulates the expression of MAD2.     

Effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells

AIM:To investigate the effect of NANOG silencing on cyclin D1 expression and proliferation in human hepatoma HepG2 cells. METHODS:Transient transfection of NANOG targeting siRNA into HepG2 cells was performed. The expression of NANOG and cyclin D1 at mRNA and protein levels was detected by real-time PCR and Western blotting. Cell proliferation was examined by CCK-8 assay and colony formation assay, and cell cycle was tested by flow cytometry. RESULTS:After transfection with NANOG-targeting siRNA, the inhibition of NANOG expression was observed. Compared with mock group, the mRNA and protein expression levels of NANOG and cyclin D1 were decreased (P<005). In addition, knockdown of NANOG expression inhibited the cell proliferation and increased the proportion of G 0/G 1-phase cells (P<0.05). CONCLUSION:Silencing of NANOG expression in HepG2 cells causes down-regulation of cyclin D1 expression and decreases the cell proliferation ability.     

Advanced glycation end products induce apoptosis of human ovarian granulosa COV434 cells and up-regulate pro-inflammatory cytokine HMGB1 autocrine

AIM:To study whether advanced glycation end products (AGEs) induce the apoptosis of human ovarian granulosa COV434 cells, and to explore the possible mechanism. METHODS:Human ovarian granulosa COV434 cells were treated with AGEs at different concentrations. Flow cytometry was used to observe the apoptotic rate. The protein levels of caspase-3 and cleaved caspase-3 were determined by Western blot. The release of high mobility group box 1 protein (HMGB1) in the culture supernatant was measured by ELISA. RESULTS:Compared with control group, early apoptotic rate and late apoptotic rate in 100 mg/L AGEs group and 200 mg/L AGEs group were significantly increased (P<0.05). No obvious difference of caspase-3 protein level in each group was observed, while the protein levels of cleaved caspase-3 in 100 mg/L AGEs group and 200 mg/L AGEs group were significantly increased compared with control group (P<0.05). In addition, compared with control group, pro-inflammatory factor HMGB1 in the culture medium in 100 mg/L AGEs group and 200 mg/L AGEs group was significantly increased. CONCLUSION:The apoptosis of human ovarian granulosa COV434 cells induced by AGEs may be related to pro-inflammatory reaction.     

Effect of bFGF on glucose regulated protein 78 expression and apoptosis in human ovarian cancer CAOV3 cells

AIM: To evaluate the effect of bFGF on the apoptosis and the expression of glucose regulated protein 78 (GRP78) in ovarian cancer CAOV3 cells.METHODS: Starvation induced cell apoptosis was conducted. After treatment with bFGF, the cell cycle and apoptosis were determined by FACS analysis and Annexin V/PI staining, respectively. The expression of protein kinase B (PKB) and GRP78 were detected by Western blotting and RT-PCR. RESULTS: Compared to starvation group, the cells treated with bFGF were still viable and increased activation of PKB and high expression of GRP78 were observed, which were prevented by PKB inhibitor wortmannin effectively (P<0.01). CONCLUSION: bFGF plays a critical role in anti-apoptosis and proliferation in human ovarian cancer through PKB signal transduction pathway, partly by upregulating the expression of GRP78.     

Transforming growth factor α promotes the proliferation,migration and adhesion of human endothelial progenitor cells

AIM: To explore the effects of transforming growth factor-α (TGF-α) in the monoclonal formation, proliferation, migration and adhesiveness of human endothelial progenitor cells (EPCs). METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α (final concentrations of 1, 5, 10 μg/L, respectively). At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10 μg/L TGF-α plus 1: 1 000 EGFR-TKI) were set. The effects of TGF-α on monoclonal formation, proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment, thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays, respectively. The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor (VEGF) were measured by Western blotting. RESULTS: Different concentrations of TGF-α all significantly induced the monoclonal formation, proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI. The results of Western blotting showed that TGF-α also induced the expression of EGFR and VEGF with a certain concentration effect (P<0.01). CONCLUSION: By combining with EGFR induced the expression of VEGF, TGF-α significantly promotes the related cell function of monoclonal formation, proliferation, migration, adhesiveness in EPCs.     

Smad pathway participates the process of proliferation of vascular smooth muscle cells induced by extracellular signal regulated kinase pathway

AIM: To investigate whether Smad pathway participates the process of extracellular signal regulated kinase (ERK) induced the proliferation of vascular smooth muscle cells (VSMCs). METHODS: Human umbilical artery smooth muscle cells (hUASMCs) were divided into four groups: control group, PDGF (platelet derived growth factor) group, ERK blocking agent group and PDGF+ERK blocking agent group. MTT assay was used to detect the proliferation of hUASMCs (A value). Immunohistochemical technique was used to detect the expression of PCNA, phosphorylated ERK (p-ERK) and phosphorylated Smad2/3 (p-Smad2/3) protein in hUASMCs. The expression of Smad2/3 mRNA in hUASMCs was detected by RT-PCR. RESULTS: The proliferation of hUASMCs and the expression of PCNA, p-ERK and p-Smad2/3 proteins in hUASMCs in PDGF group were increased obviously than those in other groups (P<0.01). No difference in the expression of Smad2/3 mRNA in hUASMCs among groups was observed. CONCLUSION: Smad pathway participates the process of ERK pathway that induces the proliferation of hUASMCs at the level of protein.     

Transferrin receptor and Fcα/μ receptor are not the major IgA1 receptor on human mesangial cells

AIM: To investigate whether transferrin receptor (TfR) and Fc α/μ R are the major IgA1 receptor on human mesangial cells (HMC). METHODS: Serum IgA1 was isolated by jacalin affinity chromatography and heated to aggregated form (aIgA1). RT-PCR was performed to investigate the expression of TfR mRNA and Fcα/μR mRNA. Binding capacity of aIgA1 to human mesangial cell line (HMCL) was evaluated by radio-ligand binding assay. Binding specificity was determined by competitive inhibition assay and phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot. RESULTS: TfR cDNA and Fcα/μR cDNA products were amplified from HMC but not from HMCL. aIgA1 was found to bind to HMCL in a dose-dependent, saturable manner and the binding was inhibited by BSA. Scatchard analysis revealed a Kd of (6.4±1.7)×10-7 mol/L and the binding sites were (3.0±1.2)×106/cells. Both aIgA1 from patients with IgAN and healthy controls were able to induce the phosphorylation of ERK in a similar time-dependent manner, but the effect of aIgA1 from patients with IgAN was much stronger (P＜0.01) and the duration was much longer (P＜0.05) than those from healthy controls. CONCLUSION: There might be a novel IgA1 receptor on HMCL, but TfR and Fc α/μ R are not the major candidates.     

Involvement of extracellular signal regulated kinase in the regulation of amyloid precursor protein processing in PC12 cells by TPPB

AIM: To explore the signal transduction pathways involved in the regulation of amyloid precursor protein (APP) processing by protein kinase C (PKC) activator TPPB.METHODS: PC12 cells were treated with TPPB (PKC activator) for 3 h and various signal transduction inhibitors were added to the conditioned medium to investigate their effects on α-secretase form of soluble amyloid precursor protein (sAPPα) secretion after TPPB treatment via Western blotting. Extracellular signal regulated kinase (ERK, p42/44MAPK) and phospho-p42/44MAPK were also measured after TPPB treatment.RESULTS: TPPB (1 μmol/L) significantly increased sAPPα secretion as compared with control group. The increase in sAPPα secretion by TPPB was partially blocked by ERK inhibitor U0126, c-Jun N-terminal kinase (JNK) inhibitor SP600125 and protein tyrosine kinase (PTK) inhibitor genistein, but not by p38MAPK inhibitor SB203580. TPPB (1 μmol/L) increased the expression of phospho-p42/44MAPK without altering total p42/44MAPK levels.CONCLUSION: ERK, JNK and PTK may be involved in the regulation of APP processing by TPPB.     

Effect of berberine on the adhesion between human umbilical vein endothelial cells and human pulmonary carcinoma cells

AIM: To study the mechanism of berberine on the adhesion between human pulmonary carcinoma cells (PG cells) and HUVECs. METHODS: The effect of berberine (2.5-40 mg/L) on the proliferation of HUVECs was detected by MTT method. Further, PG cells were treated with berberine at doses of 2.5, 5, 10 mg/L for 6, 12, 24 h. The adhesion between PG cells and HUVECs was determined by rose bengal staining. The expression of CD44s on PG cells were determined by fluorescence antibody staining. Fluorescence anisotropy imaging system was used to assay the fluidity of PG cell membrane. RESULTS: Berberine at concentrations of 2.5, 5, 10 mg/L were the safety doses to the proliferation of HUVECs treated for 6, 12, 24 h. Berberine inhibited the adhesion between PG cells and HUVECs significantly in a dose-dependent manner (P<0.05 or P<0.01). Meanwhile, berberine increased the expression of CD44s on PG cells (P<0.05 or P<0.01). Berberine decreased the fluidity of PG cell membrane in a dose-dependent manner after 24 h incubation. CONCLUSION: Berberine inhibits the adhesion between PG cells and HUVECs by regulating the expression of adhesion molecules and the fluidity of cell membrane on PG cells.     

Effects of eicosapentaenoic acid on expression of nuclear factor κB and release of cytokines in human umbilical vein endothelial cells stimulated by lipopolysaccharide

AIM: To investigate the effects of eicosapentaenoic acid(EPA) on the expression of nuclear factor kappa B(NF-κB) and release of vascular endothelial growth factor(VEGF), IL-1α and IL-6 in cultured human umbilical vein endothelial cells(HUVECs) stimulated by lipopolysaccharide(LPS).METHODS: HUVECs were obtained from cell strain and cultured in vitro. HUVECs were divided into 4 groups: control group, LPS group, 0.030 g/L EPA treatment group and 0.050 g/L EPA treatment group. The cells were cultured with LPS alone in LPS group and incubated with EPA for 1 h in the EPA pretreatment groups at the concentrations of 0.030 g/L and 0.050 g/L before LPS stimulation. Twenty-four hours after stimulated by LPS, the protein expression of NF-κB p65 in HUVECs were assessed by Western blotting analysis at different time points. The production of VEGF, IL-1α and IL-6 in cultured HUVECs was evaluated by ELISA. The effects of EPA on the protein expression of NF-κB p65 and the production of VEGF, IL-1α and IL-6 in HUVECs challenged by LPS were also determined.RESULTS: Compared with control group, the protein expression of NF-κB p65 was significantly increased in HUVECs induced by LPS and was inhibited by EPA. Compared with control group, the protein expression of VEGF, IL-1α and IL-6 was dramatically increased in HUVECs induced by LPS and most of the increase was inhibited by EPA.CONCLUSION: LPS enhances the protein expression of NF-κB and the release of VEGF, IL-1α and IL-6. EPA inhibits the protein expression of NF-κB, and the production of VEGF and the inflammatory cytokines in cultured HUVECs stimulated by LPS, indicating that EPA may be useful for preventing and treating neovascular and inflammatory diseases.