Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

Effects of propofol on biological characteristics of colorectal cancer cells

AIM: To investigate the effect of propofol on the viability, invasion ability and apoptosis of colorectal cancer cells.METHODS: Propofol at 10, 25, 50 and 100 μmol/L was used to treat LoVo cells for 72 h, and propofol at 100 μmol/L was used to treat the LoVo cells for 12, 24, 48 and 72 h. The cell viability was measured by CCK-8 assay. The invasion ability of the LoVo cells treated with propofol at 100 μmol/L for 72 h was detected by Transwell assay. The cell cycle distribution and cell apoptotic rate were analyzed by flow cytometry. The protein levels of matrix metalloproteinase (MMP)-2, MMP-9, cleaved caspase-3, Notch1 and hairy and enhancer of split 1 (Hes1) were determined by Western blot.RESULTS: Propofol inhibited LoVo cell viability. The cell invasion ability, S stage cells, and the protein levels of MMP-2, MMP-9, Notch1 and Hes1 in propofol group were significantly lower than those in control group, and the apoptotic rate, G₀/G₁ cells and the protein level of cleaved caspase-3 were significantly higher than those in control group (P<0.01).CONCLUSION: Propofol inhibits the viability and invasion ability of colorectal cancer LoVo cells, blocks cell cycle and induces apoptosis. The mechanism is related to down-regulation of Notch1 signaling pathway.     

Effects of folic acid and vitamin B12 on human umbilical vein endothelial cells against homocysteine-induced apoptosis

AIM: To investigate the effect of folic acid and vitamin B₁₂ on homocysteine (Hcy)-induced apoptosis of human umbilical vein endothelial cells (HUVECs) through mammalian sterile 20-like kinase 1 (MST1). ME-THODS: HUVECs were cultured in the absence (control group), or presence of 100 μmol/L Hcy alone (Hcy group) or 100 μmol/L Hcy plus 30 μmol/L folic acid and vitamin B₁₂ (intervention group) for 72 h. The effect of Hcy on the apoptosis of HUVECs was analyzed by flow cytometry. The transfection efficiency of DNA methyltransferase 1 (DNMT1)-overexpressing adenovirus was observed under fluorescence inverted microscope. The mRNA and the protein levels of DNMT1 and MST1 were determined by RT-qPCR and Western blot. The DNA methylation level of MST1 promoter was detected by methylation-specific PCR. RESULTS: Compared with control group, the apoptotic rate (P<0.01) and the expression of MST1 at mRNA (P<0.01) and protein (P<0.05) levels in the HUVECs were significantly increased, while the mRNA levels of DNMT1 was decreased in Hcy group (P<0.01). In addition, folic acid and vitamin B₁₂ treatment significantly inhibited Hcy-mediated apoptosis of HUVECs (P<0.01), increase in MST1 mRNA level (P<0.01) and decrease in DNMT1 mRNA level (P<0.01). Meantime, the mRNA level of MST1 was positively correlated with the apoptotic rate of the HUVECs (r=0.943 9, P<0.001). The expression of DNMT1 at mRNA and protein levels was significantly increased after the transfection of DNMT1-overexpressing adenovirus into HUVECs (P<0.01), and a large amount of green fluorescent protein expression was observed. Meanwhile, the DNA methylation level of MST1 promoter was increased (P<0.01), while the protein level of MST1 was decreased (P<0.01).CONCLUSION: Up-regulation of MST1 promotes Hcy-induced apoptosis of HUVECs, while folic acid and vitamin B₁₂ exert an anti-apoptosis effect, which might be regulated by hypermethylation of MST1 promoter region.     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Farrerol inhibits nicotine-induced proliferation of pulmonary vascular smooth muscle cells by enhancing Kv1.5 and Kv2.1 expression

AIM:To study the effect of farrerol (Far) on nicotine-induced proliferation of rat pulmonary smooth muscle cells (PASMCs), and further to explore its relationship with voltage-dependent potassium channels (Kv) 1.5 and Kv2.1. METHODS:Firstly, the effect of nicotine on the proliferation of PASMCs was detected by cell counting method, and the optimal concentration of nicotine was selected. Primary cultured PASMCs were randomly divided into 5 groups:normal control group, nicotine (1 μmol/L)group, nicotine (1 μmol/L) + Far (10^-6 mol/L, 10^-5 mol/L and 10^-4 mol/L) Far group. The activity of caspase-3 was measured by apoptosis kit, the cell viability was measured by CCK-8 assay, the apoptotic rate was analyzed by flow cytometry. The expression of Kv1.5 and Kv2.1, and apoptosis-related factors Bcl-2 and Bax at mRNA and protein levels was determined by RT-qPCR and Western blot respectively. RESULTS:Nicotine at 1 μmol/L increased the number of PASMCs to the maximum extent (P<0.01). Nicotine at 1 μmol/L significantly reduced the caspase-3 activity and enhanced the cell viability of the PASMCs (P<0.01). Farrerol at 10^-6~10^-4 mol/L eliminated the effect of PASMCs induced by nicotine in a concentration dependent manner. Compared with control group, nicotine at 1 μmol/L significantly increased the proliferation and inhibited the apoptotic rate of rat PASMCs (P<0.01). The apoptotic rate of PASMCs in farrerol intervention group was significantly higher than that in nicotine group (P<0.01). Nicotine at 1 μmol/L significantly inhibited the expression of Kv1.5, Kv2.1 and Bax but increased the expression of Bcl-2 in PASMCs (P<0.01). Farrerol at 10^-5 mol/L obviously inhibited the effect of PASMCs induced by nicotine. CONCLUSION:Farrerol eliminates nicotine-induced inhibition of caspase-3 and Bax, and enhancement of Bcl-2 in PASMCs by enhancing Kv1.5 and Kv2.1 expression.     

Aldolase A level in malignant pleural effusion and its effects on proliferation,migration and invasion of human lung adenocarcinoma cells

AIM：
To investigate the levels of aldolase A (ALDOA), carcinoembryonic antigen (CEA) and lactate dehydrogenase (LDH) in malignant pleural effusion (MPE) from patients with lung cancer and tuberculous pleural effusion (TBPE) from patients with tuberculous pleurisy, and to explore the effects of ALDOA on the proliferation, migration and invasion of human lung adenocarcinoma A549 cells. METHODS：Pleural effusion samples including 65 cases of MPE and 35 cases of TBPE were collected, and the levels of ALDOA, CEA and LDH were detected by ELISA and chemiluminescence assay. After A549 cells were treated with different concentrations of ALDOA, the proliferation, migration and invasion of the cells were investigated by MTT assay, scratch test, Matrigel assay and Transwell invasion assay. RESULTS：The levels of ALDOA, CEA and LDH in MPE were (46.8±21.4) μg/L, (82.2±56.6) μg/L and (755.8±382.5) U/L, respectively, which were significantly higher than those in TBPE ［(23.9±17.2) μg/L, (12.6±9.7) μg/L and (388.4±163.9) U/L, respectively; P<0.01］. The concentration of ALDOA in MPE from adenocarcinoma patients ［(71.7±32.1) μg/L］ was significantly higher than that in MPE from squamous-cell carcinoma patients ［(21.3±14.6) μg/L, P<0.05］. The concentrations of ALDOA in MPE and TBPE were positively correlated with the concentrations of CEA and LDH (P<0.01 or P<0.05). ALDOA enhanced the proliferation, migration and invasion of A549 cells in a concentration-dependent manner. CONCLUSION：The expression level of ALDOA in MPE is significantly higher than that in TBPE, especially in MPE from lung adenocarcinoma patients. There are highly positive correlations between ALDOA and CEA, ALDOA and LDH in pleural effusion. ALDOA concentration-dependently promotes the proliferation, migration and invasion of A549 cells.     

Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells

AIM: To investigate the effect of plumbagin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the apoptosis of leukemic Kasumi-1 cells. METHODS: Kasumi-1 cells were treated with plumbagin alone, recombinant soluble TRAIL(rsTRAIL) alone or the combination of plumbagin with rsTRAIL to induce apoptosis. The cell proliferation was analyzed by CCK-8 assay. Apoptosis was determined by flow cytometry with AnnexinⅤ/PI double staining and TUNEL staining. The expression of DR4 and DR5 at mRNA level was measured by real-time PCR. The expression of signal transduction proteins, such as DR5, caspase-3, caspase-8, caspasep-9, Bid, Bax and c-FLIP was detected by Western blotting. RESULTS: Both rsTRAIL and plumbagin induced the apoptosis in Kasumi-1 cells, and combination of plumbagin with rsTRAIL enhanced the apoptosis. The ratios of Annexin V-positive Kasumi-1 cells were (27.7±2.9)%, (25.6±3.1)% and (52.1±3.3)% in 100 μg/L rsTRAIL group, 2 μmol/L plumbagin group and the combination group, respectively, and the positive rate in combination group was significantly higher than those in other 2 groups. TUNEL assay demonstrated that the number of apoptotic cells in combination group was higher than that in the cells treated with rsTRAIL or plumbagin alone. Plumbagin up-regulated the expression of DR5 at mRNA level in Kasumi-1 cells, and up-regulation of DR5, activation of caspase-8 and down-regulation of c-FLIP at protein level were detected in the cells treated with plumbagin alone and the combination of plumbagin with rsTRAIL. CONCLUSION: Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells by up-regulating DR5, activating caspase-8 and down-regulating c-FLIP.     

Effect of Vaccinium vitis procyanidin on regulation of glioma cell growth

AIM: To explore the effect of Vaccinium vitis procyanidin on the growth of glioma cells. METHODS: Glioma C6 cells were cultured and divided into control and 10, 20 and 40 μg/L Vaccinium vitis procyanidin groups. The influence of Vaccinium vitis procyanidin on the growth of C6 cells was measured by MTT assay and the observation under inverted microscope. The apoptotic rate was detected by Annexin V/PI staining. The protein expression of Bcl-2 and Bax was determined by immunocytochemistry. The protein levels of Bcl-2, Bax and caspase-3 were also examined by Western blotting. RESULTS: The growth of C6 glioma cells was inhibited by Vaccinium vitis procyanidin at concentrations of 10, 20 and 40 μg/L. The growth was significantly inhibited in 40 μg/L Vaccinium vitis procyanidin group at 24 h and 48 h, and in 20 and 40 μg/L Vaccinium vitis procyanidin groups at 72 h (P<0.01). The density of the cells was decreased when the concentration of Vaccinium vitis procyanidin increased. The apoptotic rate was increased when the concentration of Vaccinium vitis procyanidin increased either. The expression of Bcl-2 was decreased and Bax was increased after 10, 20 and 40 μg/L Vaccinium vitis procyanidin treatments. The ratio of Bax/Bcl-2 was increased when the dose of Vaccinium vitis procyanidin increased (P<0.05 or P<0.01). The expression of Bcl-2 was decreased (P<0.01), and Bax and caspase-3 were increased after 10, 20 and 40 μg/L Vaccinium vitis procyanidin treatments. The ratio of Bax/Bcl-2 was increased when the dose of Vaccinium vitis procyanidin increased (P<0.01). CONCLUSION: Vaccinium vitis procyanidin inhibits the growth of glioma cells by down-regulating Bcl-2 protein and up-regulating Bax protein to activate caspase-3, thus inducing apoptosis.     

Effects of salinomycin combined with L-asparaginase on proliferation and apoptosis of human acute T-cell leukemia Jurkat cells

AIM:To investigate the effects of salinomycin alone or in combination with L-asparaginase on the growth and apoptosis of human acute T-cell leukemia Jurkat cells, and the possible mechanism. METHODS:The growth of Jurkat cells was tested by Cell Counting Kit-8 in vitro. The levels of cytochrome C, Bcl-2, caspase-3, caspase-8 and caspase-9 were measured by Western blotting. Flow cytometry was used to assay cell apoptosis. RESULTS:Salinomycin or L-asparaginase alone inhibited the growth of Jurkat cells in a dose-dependent manner. The IC50 value of L-asparaginase was 8.12 IU/L, while that of salinomycin was 0.75 μmol/L. Salinomycin combined with L-asparaginase induced more significant inhibition of cell proliferation (P<0.05). Western blotting showed that the expression of Bcl-2 protein in combination group was significantly reduced, and the expression of caspase-3, caspase-8, caspase-9 and cytochrome C was significantly increased (P<0.05). Flow cytometry showed that the apoptotic rates of Jurkat cells incubated with salinomycin (0.5 μmol/L), L-asparaginase (2.5 IU/L) and both drugs for 48 h were (7.11±0.23)%, (25.43±0.47)% and (39.12±1.97)%, respectively, and significantly higher than that in control group ［(6.67±0.13)%, P<0.05］.CONCLUSION: Salinomycin synergizes with L-asparaginase-induced cytotoxicity in vitro, and the combined treatment with salinomycin and L-asparaginase induces the apoptosis of Jurkat cells.     

中国园艺学会第七届青年学术讨论会

中国园艺学会第九届第8次常务理事扩大会决定,“中国园艺学会第七届青年学术讨论会”由山东农业大学园艺科学与工程学院和山东省园艺学会承办,将于2006年7月或8月在山东泰安举行。会议交流主题:(1)园艺作物种质资源、遗传育种与生物技术;(2)园艺作物有机、无公害及标准化安全生     

Ethyl acetate extract of Pleione bulbocodioides (Franch.) Rolfe induces apoptosis of human leukemia K562 and HL-60 cells through intrinsic mitochondrial apoptosis pathway

AIM:To investigate the effects of ethyl acetate (EtOAc) extract of Pleione bulbocodioides (Franch.) Rolfe on proliferation and apoptosis of human leukemia K562 and HL-60 cells and the possible apoptosis pathway. METHODS:Human leukemia cell lines were treated with EtOAc extract of Pleione bulbocodioides at different concentrations. XTT method was used to evaluate the viability of K562 cells and HL-60 cells. The cell growth inhibition was calculated by Trypan blue exclusion test. The percentage of apoptotic cells was determined by flow cytometry, and 4',6-diamidino-2-phenylindole (DAPI) was used to observe morphological changes of the cells. The cell cycle was observed by propidium iodide (PI) staining. The protein expression of Bcl-2, Bax, cleaved poly(ADP-ribose) polymerase (PARP), cleaved caspase-3, cytochrome C and apoptosis-inducing factor (AIF) wase determined by Western blot. RESULTS:The cell viability and proliferation were inhibited by EtOAc extract of Pleione bulbocodioides with IC₅₀ of (42.14±2.54) mg/L for HL-60 cells and (51.28±3.12) mg/L for K562 cells at 24 h. The results of Annexin V-FITC/PI and DAPI staining showed that EtOAc extract of Pleione bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased compared with control group (P<0.05). The G₂ phase increased with typical cell apoptosis-induced morphological changes. The levels of pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 were increased, while Bcl-2 was down-regulated (P<0.05). Cytochrome C and AIF in cytosol, characteristic proteins of intrinsic mitochondrial apoptosis pathway, also increased with the concentration of EtOAc extract of Pleione bulbocodioides increasing (P<0.05). CONCLUSION:EtOAc extract of Pleione bulbocodioides significantly inhibits cell proliferation and induces cell apoptosis in human leukemia cell lines HL-60 and K562 through intrinsic mitochondrial apoptosis pathway.     

Nodosin induces apoptosis of HepG2 cells by increasing Apaf-1 expression and activating caspase-3

AIM:To investigate the effects of nodosin on apoptosis of human hepatocellular carcinoma HepG2 cells. METHODS:HepG2 cells were treated with nodosin at different concentrations (1.25 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L and 20 μmol/L) for 24 h. The morphological changes of the HepG2 cells were observed by Hoechst 33258 staining and electron microscopy. The apoptotic rates were analyzed by flow cytometry. The mRNA expression of apoptotic protease-activating factor-1 (Apaf-1) was detected by RT-qPCR. The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were determined by Western blot. RESULTS:HepG2 cells showed obvious cell shrinkage and nucleus drift when treated with nodosin as the concentration was increased. Many apoptotic bodies were observed in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups. The mRNA expression of Apaf-1 was increased in 5 μmol/L, 10 μmol/L and 20 μmol/L nodosin groups as compared with control group (P<0.05). The protein levels of pro-caspase-3, caspase-3 and cleaved caspase-3 were increased with the increasing dose of nodosin (P<0.05). CONCLUSION:Nodosin induces the apoptosis of HepG2 cells. This effect was related to increasing Apaf-1 mRNA expression and subsequently promoting the activation of caspase-3.     

CaMKII aggravates hypoxia/reoxygenation injury in H9c2 cells by activating apoptosis

AIM: To investigate the effect of Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) on hypoxia/reoxygenation (H/R) injury in H9c2 cells. METHODS: H9c2 cells were randomized into 4 groups:control group, KN-93 (an inhibitor of CaMKⅡ; 1 μmol/L) treatment group, H/R group and H/R+KN-93 (1 μmol/L) treatment group. The cells in KN-93 group and KN-93+H/R group were pretreated with KN-93 for 2 h before the other treatment was performed. The viability of H9c2 cells in each group was measured by CCK-8 assay. Lactate dehydrogenase (LDH) activity in the culture medium was detected. The protein levels of phosphorylated CaMKⅡ (p-CaMKⅡ), phosphorylated phospholamban (p-PLN) and cleaved caspase-3 were determined by Western blot. The apoptosis was analyzed by TUNEL staining and the flow cytometry. RESULTS: No significant difference of all indexes tested between control group and KN-93 group was observed. H/R treatment significantly reduced the cell viability, and increased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). KN-93 (1 μmol/L) significantly increased the cell viability, and decreased the activity of LDH (P<0.01), the protein levels of p-CaMKⅡ, p-PLN and cleaved caspase-3 (P<0.05), and the apoptotic rate (P<0.01). CONCLUSION: CaMKⅡ aggravates hypoxia/reoxygenation injury in the H9c2 cells by activating apoptosis.     

Rapamycin enhances chemosensitivity of endometrial cancer cells to adria-mycin

AIM: To explore the therapeutic effect of adriamycin combined with rapamycin on endometrial cancer cells. METHODS: Two endometrial carcinoma cell lines with different PTEN gene states were chosen: HEC-1A (wild type) and Ishikawa (mutant type). Before adriamycin administration, the cells were pretreated with low concentration of rapamycin for 24 h. The cell viability and 50% inhibitory concentration (IC₅₀) of adriamycin at 24 h were determined by MTT assay. Multiple drug effect/combination index (CI) was used to evaluate the interaction between adriamycin and rapamycin. Apoptotic rate was measured by flow cytometry. The effects of the drugs on phosphorylation of PI3K/Akt and apoptosis protein caspase-3 were detected by Western blotting. RESULTS: Both adriamycin and rapamycin showed obvious growth inhibitory effects on the 2 endometrial cancer cell lines in a time- and dose-dependent manner. After pretreated with rapamycin, IC₅₀ of adriamycin decreased sharply. In Ishikawa cells, it decreased from (21.3±3.8) μmol/L to(11.9±1.2) μmol/L,P<0.05. In HEC-1A cells, it decreased from (14.3±2.8) μmol/L to (8.2±0.9) μmol/L,P<0.05. Combination index value of the 2 drugs was more than 1.15 in the 2 endometrial cancer cell lines, indicating synergistic effects. The combination therapy of adriamycin with rapamycin increased apoptotic rates in the 2 cell lines, and induced the down-regulation of phosphorylated Akt and over-expression of caspase-3 as compared with single drug treatment (P<0.05). CONCLUSION: Adriamycin combined with rapamycin significantly enhances the chemosensitivity of endometrial cancer cells and reduces drug resistance, which will become a new trend for treating endometrial cancer.     

Lycium barbarum polysaccharides attenuate oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide

AIM: To study the effect of Lycium barbarum polysaccharides (LBP) on oxidative stress injury of human endothelium-like EA.hy926 cells induced by hydrogen peroxide (H₂O₂). METHODS: The EA.hy926 cell model of oxidative stress injury was established by H₂O₂ treatment. The EA.hy926 cells were divided into 5 groups:control group, damage (H₂O₂ at 50 mmol/L) group, LBP (100 mg/L) group, anti-damage groups (LBP at 50 mg/L, 100 mg/L or 200 mg/L+50 mol/L H₂O₂), and LY294002 (20 μmol/L) group. The effect of LBP at different concentrations on the cell viability of EA.hy926 cells was measured by CCK-8 assay, and the optimum concentration of LBP was screened out. The apoptotic of EA.hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide (AO/EB) staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the cell culture medium were examined. The protein levels of cleaved caspase-3, Bax, Bcl-2, endothelial NO synthase (eNOS), p-eNOS and p-Akt were determined by Western blot. RESULTS: LBP at concentration of 100 mg/L significantly attenuated the injury of EA.hy926 cells induced by H₂O₂, as indicated by improved cell viability (P<0.05) and decreased apoptosis (P<0.05). Pretreatment with LBP elevated the levels of NO and VEGF (P<0.05), and promoted the migration ability of EA.hy926 cells. LBP also increased the Bcl-2/Bax ratio, down-regulated the protein level of cleaved caspase-3, and up-regulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA.hy926 cells with the inhibitor of PI3K (P<0.05). As a result, the protein level of p-Akt was down-regulated, and the level of NO was also significantly reduced. CONCLUSION: LBP has protective effect on H₂O₂ -induced EA.hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.     

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.     

Sensitization of chemotherapy for human triple-negative breast cancer cells by methylseleninic acid

AIM:To observe the chemosensitization effect of methylseleninic acid (MSA) on human triple-negative breast cancer (TNBC) cells. METHODS:MDA-MB-231 cell line was co-cultured with MSA plus paclitaxel or doxorubicin. The inhibitory rate of cell proliferation was detected by CCK-8 assay. The combination index was calculated to explore the impact of MSA on the efficacy of chemotherapeutic drugs. The cell cycle was analyzed by flow cytometry. Annexin V-FITC/PI double staining was applied to detect the cell apoptosis. RESULTS:Compared with single usage of chemotherapeutic drugs, the cell proliferation rates were decreased when the chemotherapeutic drugs was combined with MSA, suggesting that there is a synergistic relationship between MSA and chemotherapeutic drugs. Compared with the single-agent groups, the G2/M-phase cells in paclitaxel combined with MSA group increased significantly (P<005),and the S-phase cells increased significantly in doxorubicin combined with MSA group (P<005). These suggested that MSA enhanced the anticancer effect of the drugs by inducing cell cycle arrest. Compared with single usage of 10 nmol/L paclitaxel, the apoptotic rate increased from 41.1% to 59.3% (P<005) as 10 nmol/L paclitaxel combined with 3.5 μmol/L MSA was used. Compared with single usage of 0.5 μmol/L doxorubicin, the apoptotic rate increased from 30.2% to 51.9% (P<0.01) as 0.5 μmol/L doxorubicin combined with 3.5 μmol/L MSA was used. These suggested that MSA enhanced antitumor effect of the drugs by inducing tumor cell apoptosis. CONCLUSION: MSA enhances the antitumor effects of chemotherapeutic drugs doxorubicin and paclitaxel on TNBC cells. One of the possible mechanisms is the enhancement of inducing tumor cell apoptosis and cell cycle arrest.     

Effect of activated protein C on apoptosis in human umbilical vein endothelial cells induced by lipopolysaccharide

AIM:To study the effect of activated protein C (APC) at different concentrations on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS).METHODS:The HUVECs were induced by LPS (1.0 mg/L) as apoptotic model that was administered by different concentration of APC (10 μg/L or 50 μg/L). Meanwhile, the control group and induced apoptosis group induced by LPS (1.0 mg/L) stimulation were also set up. The changes of cellular ultrastructures were observed under electron microscope. The DNA ladder and TUNEL fluorescent staining were measured in cells. Annexin-Ⅴ/PI double staining was used to measure the cell apoptosis rate by flow cytometry. Cell survival rate was measured by MTT assay. The proliferating cell nuclear antigen (PCNA) expression levels in cells were also measured by Western blotting to reflect the proliferation of the cells.RESULTS:There were significant apoptotic changes in the cells induced by LPS, but the apoptotic changes were reduced and apoptosis rates were decreased in the cells treated with APC. Meanwhile, cell survival rate and the protein levels of PCNA were increased after APC treatment, particularly at the concentration of 50 μg/L, which showed difference when compared with those induced apoptosis group by LPS (P<0.05).CONCLUSION:APC can inhibit HUVECs apoptosis induced by LPS and promote cell proliferation, thus protect the cells from injury.     

Apoptosis induced by simvastatin in rat vascular smooth muscle cells through calpain and caspase-3-dependent pathways

AIM:Hydroxymethylglutaryl CoA (HMG-CoA) reductase inhibitors, such as simvastatin, have been shown to reduce atherosclerotic cardiovascular morbidity and mortality by mechanisms unrelated to its lipid-lowering effect. Several studies have shown that simvastatin induces apoptosis in a varieties of cell lines including vascular smooth muscle cells (VSMC). The aim of this study was to investigate the signal pathways involved in apoptosis induced by simvastatin.METHODS:Cultured VSMC were treated with simvastatin. Calpain activity was determined by measuring Ca²⁺ ionophore-specific calpain substrate (suc-LLVY-AMC), caspase-3 activation was detected by Western blot, and apoptotic changes were distinguished by annexin Ⅴ binding and DNA laddering.RESULTS:After incubated with 30 μmol/L simvastatin for 8 h, calpain activity had a marked increase (P<0.05, n=4) and reached to more than 3-fold of control at 12 h (P＜0.01). Caspase-3 also activated by simvastatin after 12 h. PD150606, a cell-permeable selective calpain inhibitor, decreased simvastatin-induced apoptosis rate from 24.2%±1.7% to 9.5%±1.9% (P＜0.01) and also prevented simvastatin-induced DNA laddering. Furthermore, 100 μmol/L PD150606 efficiently inhibited simvastatin-induced caspase-3 activation.CONCLUSION:Simvastatin induces apoptosis by activating caspase-3 via calcium-dependent protease calpain.     

Changes and significance of Fas and cFLIP in apoptosis of human rhabdomyosarcoma cells induced by combination of TRAIL and cisplatin

AIM: To investigate the synergistic induction of apoptosis in rhabdomyosarcoma cells by the combination of TRAIL or TRAIL gene with cisplatin. METHODS: Rhabdomyosarcoma cells were treated with TRAIL, Ad／GT-TRAIL, cisplatin, respectively or the combination for 3 days. The cytotoxicity was observed by MTT assay. The apoptotic rates and the expression rates of Fas protein were measured by flow cytometry (FCM). The expression of cFLIP mRNA was determined by RT-PCR. RESULTS: Rhabdomyosarcoma cells were treated with Ad／ GT-TRAIL and TRAIL (100.0 μg/L), the cytotoxicity index were 52.5% and 43.5%, the percentage of apoptotic cells were 12.95% and 10.26%, respectively. Combined with cisplatin, the cytotoxicity index and the percentage of apoptotic cells were increased significantly (P<0.05). The expression of Fas protein in rhabdomyosarcoma cells was up-regulated and the expression of cFLIP was down-regulated with cisplatin, which were paralleled by the apoptotic rates. CONCLUSION: Combinatiion of Ad／GT-TRAIL or TRAIL and cisplatin has synergistic apoptosis-inducing effects on rhabdomyosacoma cells.