Effect of interfering TGF-β receptor Ⅱ expression on viability,differentiation and apoptosis of NB4 cells

AIM: To evaluate the effect of interfering TGF-β receptor Ⅱ (TβRⅡ) expression on the viability and differentiation of human acute promyelocytic leukemia NB4 cells induced by all-trans retinoic acid (ATRA) and their apoptosis induced by arsenic trioxide (ATO). METHODS: The technique of lentivirus-mediated RNA interference was used to obtain stable NB4 cells with TβRⅡ knockdown, named TβRⅡ-shRNA NB4 cells. CCK-8 assay was used to detect the viability of TβRⅡ-shRNA NB4 cells. The expression level of CD11b was analyzed by flow cytometry, and Wright-Giemsa staining was used to detect the effects of ATRA on the differentiation of TβRⅡ-shRNA NB4 cells. Double staining (Annexin V-FITC/PI) and AO/EB staining were used to detect the effects of ATO on the apoptosis of TβRⅡ-shRNA NB4 cells. RESULTS: The viability of TβRⅡ-shRNA NB4 cells was significantly higher than that of NB4 parental cells. The differentiation was induced in TβRⅡ-shRNA NB4 cells and NB4 parent cells by treatment with ATRA at different concentration (0.01, 0.02, 0.04, 0.08, 0.1 μmol/L) for 96 h. The differentiation rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells in a dose-dependent manner. ATO induced apoptosis of TβRⅡ-shRNA NB4 cells and NB4 parent cells at different concentrations (2, 4 and 8 μmol/L) for 24 h. The apoptotic rate of TβRⅡ-shRNA NB4 cells was lower than that of NB4 parental cells dose-dependently. At the concentration of 8 μmol/L for 24 h, the apoptotic rates in TβRⅡ-shRNA NB4 cells and NB4 cells were (49.15±2.05)% and (66.85±2.41)%, respectively (P<0.01). CONCLUSION: Down-regulation of TβRⅡ increases the viability of NB4 cells, inhibits NB4 cell differentiation induced by ATRA, and also inhibits apoptosis induced by ATO.     

Expression of telomerase inhibitor Pinx1 in leukemia cells and its correlation with telomerase activity

AIM:To study the expression of telomerase inhibitor Pinx1 in acute leukemia cells and during the differentiation of acute promyelocytic leukemia cells,and to realize its effect on telomerase activity.METHODS:Realtime quantitative PCR with fluorescence probe hybridization was used to measure the expression of Pinx1 and hTERT mRNA in acute leukemia cells and during differentiation of NB4 cells induced by ATRA.The correlations between Pinx1 and hTERT expression were also analyzed.RESULTS:Pinx1 mRNA expression in acute leukemia samples (0.00312,5.42×10-4-0.024) was significantly higher than that in normal bone marrow mononuclear cells (7.89×10-4,0-0.00863,P<0.01).The expression of Pinx1 mRNA had significant positive correlation with hTERT mRNA expression (r=0.296,P<0.05).Pinx1 mRNA expression decreased during differentiation,its expression was positive correlated with hTERT mRNA expression (r=0.900,P<0.05).CONCLUSIONS:As an inhibitor of telomerase,however,Pinx1 also had the same direction of regulation with telomerase activity in acute leukemia cells,suggesting its expression variation may be a subsequent reaction induced by that of hTERT to stabilize telomerase activity.The exact mechanisms remained to be verified.     

Effect of all-trans-retinoic acid on the proliferation and expression of α-SMA in human embryonic lung fibroblasts

AIM: To investigate the effects of all-trans-retinoic acid (ATRA) on the proliferation and differentiation of transforming growth factor β₁(TGF-β₁)-stimulated human embryonic lung fibroblasts (HFL-I).METHODS: The HFL-I cells were cultured in vitro and were pretreated with ATRA for 3 days at the concentrations of 0.1 μmol/L, 1 μmol/L and 10 μmol/L. The proliferation of HFL-1 cells was detected by MTT method. The mRNA expression of α-smooth muscle actin(α-SMA) in HFL-I cells stimulated with TGF-β₁ for 0 h, 6 h, 12 h, 24 h, 48 h and 72 h was detected by RT-PCR and the protein expression of α-SMA at the time points of 1,3 and 5 days was detected by Western blotting. The mRNA expression of α-SMA in HFL-I cells pretreated with different concentrations of ATRA for 24 h was detected the by RT-PCR and the protein expression at time point of 3rd day was detected by Western blotting. RESULTS: Different concentration of ATRA inhibited the proliferation of HFL-I in a dose-dependent manner (P<0.05). Both mRNA and protein expression of α-SMA in HFL-I cells pretreated with TGF-β₁ was up-regulated (P<0.05). ATRA down-regulated the mRNA and protein expression of α-SMA induced by TGF-β₁ in a dose-dependent manner (P<0.05). CONCLUSION: ATRA inhibits the proliferation and TGF-β₁-stimulated differentiation in HFL-I cells by down-regulating the mRNA and protein expression of α-SMA.     

Effects of alkyl-lysophospholipids on proliferation and differentiation in HL-60cells

AIM: To explore the effect of alkyl-lysophospholipids (ALP) on the proliferation, apoptosis and differentiation of HL-60cells. METHODS: Proliferative potential was measured by colony formation assays. Apoptotic cells were detected by morphology, DNA gel electrophoresis and flow cytometry analysis. Both morphological criteria and NBT dye reduction were utilized to determine the extent of differentiation. RESULTS: After 9 h of incubation wit15 mg/L of ALP, apoptotic cells, identified by condensened and fragmented nuclei, were present. After 6 days of incubation wit1 mg/L of ALP, the NBT reduction rate in HL-60cells increased to 84.2±2.6%. CONCLUSION: ALP can induce apoptosis and differentiation, and inhibit growth in HL-60cells.     

Inhibitory effect of 5-fluorouracil on ultra-microstructure and insulin secretion of pancreatic β cells

AIM: To investigate the molecular mechanisms of β cell dysfunction induced by 5-fluorouracil (5-FU) in islet β cell line (NIT-1 cells). METHODS: The NIT-1 cells were treated with different concentrations of 5-FU. The content of insulin in the culture medium was determined by radioimmunoassay. Cell apoptosis was observed by flow cytometry with annexin V/PI staining. The ultra-microstructural changes of NIT-1 cells were observed under transmission electron microscope. The expression of pancreatic and duodenal homeobox protein 1(PDX-1) at mRNA and protein levels in NIT-1 cells was examined by RT-PCR and Western blotting, respectively. RESULTS: Exposed to the low glucose concentration (5.6 mmol/L), insulin secretion in NIT-1 cells was not significantly decreased following a 24 h treatment with 5.0 to 40.0 mg/L 5-FU (P>0.05). On the contrary, the high glucose (16.7 mmol/L)-stimulated insulin secretion in NIT-1 cells was inhibited by 5.0 to 40.0 mg/L of 5-FU in a dose-dependent manner after 24 h of incubation (P<0.01). The apoptosis rate of NIT-1 cells was significantly increased as compared to those in the control levels(P<0.05). The structural changes of mitochondria were the main apoptotic changes under transmission electron microscope. Significant down-regulation of PDX-1 expression at mRNA and protein levels was observed in NIT-1 cells treated with 5-FU at the concentration of 10.0 mg/L to 40.0 mg/L(P<0.05).CONCLUSION: 5-FU inhibits the insulin secretion in islet β cell induced by high glucose. A relative deficiency in insulin secretion following 5-FU treatment is related to the changes of β cell ultra-microstructure and the reduction of β cell numbers, by which an increase in apoptosis of pancreatic β cells is induced. Down-regulation of PDX-1 expression may play a pivotal role in increasing the apoptosis of pancreatic β cells induced by 5-FU in high-glucose condition.     

Propofol inhibits LPS-induced inflammatory response in microglia via TRPV1 ion channels

AIM:To investigate the effects of propofol (P) on the inflammatory response of microglia induced by lipopolysaccharide (LPS) and the mechanisms. METHODS:Mouse microglia BV2 cells were treated with LPS at 100 μg/L to establish a neuroinflammatory injury model. The BV2 cells were divided into 4 groups:control group (C group), model group (L group), L+P group and LPS+AMG517 group (L+A group). The level of tumor necrosis factor-α (TNF-α) in the cell culture supernatant was measured by ELISA. The mRNA expression of transient receptor potential cation channel subfamily V member 1 (TRPV1) was detected by real-time PCR. The protein levels of TRPV1, TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and phosphorylated calcium/calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ) were determined by Western blot. The content of free Ca²⁺ in the microglia BV2 cells was detected by Fluo-3 AM assay. RESULTS:Compared with C group, the level of TNF-α was significantly increased in L group (P<0.01), but that in P group was not changed. Compared with L group, the level of TNF-α was significantly lower than that in L+P group within 4 h (P<0.01). Compared with C group, the mRNA expression of TRPV1 was significantly increased in L group (P<0.01). Compared with L group, the mRNA expression of TRPV1 was significantly down-regulated in L+P group (P<0.01).Compared with L group, the protein levels of TNF-α, IL-1β, IL-6 and p-CaMKⅡ and intracellular Ca²⁺ concentration were significantly lower than those in L+P group and L+A group (P<0.01). CONCLUSION:Propofol inhibits the inflammatory response of microglia by reducing the expression of TNF-α, IL-1 and IL-6, which may be related to the down-regulation of TRPV1 and p-CaMKⅡ and the reduction of intracellular Ca²⁺ concentration.     

Effects of leptin on hypoxia-reoxygenation induced apoptosis in human L02 cells and expression of Fas and FasL

AIM: To observe the effect of leptin (LEP) on hypoxia-reoxygenation induced apoptosis in L02 cells.METHODS: In the experiment, L02 cell injury was induced by hypoxic air (95%N2 and 5% CO2). The cultured L02 cells were divided into hypoxic 12 h group (IR group) alone, normal control group and the hypoxic plus leptin (100 μg/L, 200μg/L, 400 μg/L, 800 μg/L and 1 600 μg/L) treatment groups in vitro. Flow cytometry, terminal dUTP nick-end labeling (TUNEL) assay and fluorescent quantitative PCR were used to measure the changes of apoptosis in L02 cells and expression of Fas/FasL mRNA.RESULTS: (1) The percentage of L02 cells apoptosis and positive TUNEL cells significantly increased in IR group compared to control group (P<0.01). When L02 cells were treated with LEP, the percentage of cell apoptosis and positive TUNEL cells were decreased compared to IR group. (2) Compared to control group, the Fas/FasL mRNA expression significantly increased in IR group (P<0.01). When L02 cells were treated with LEP, the Fas/FasL mRNA expression decreased compared to IR group, the effect of LEP at concentration of 400 μg/L was obviously (P<0.05). CONCLUSION: LEP decreases the apoptosis of hypoxic-reoxygenation L02 cells by down-regulation of Fas/FasL mRNA expression in L02 cells.     

CTLA4Ig induces immune tolerance of T cells to oxidized-low density lipoprotein in vitro

AIM: Recently,it is widely accepted that atherosclerosis (AS) is an auto-immune related disease and the oxidized-low density lipoprotein (ox-LDL) is the most important AS-related antigen.In order to prevent immune injuries in AS and find new strategies to prevent AS,the immune tolerance of T cells to ox-LDL in vitro was induced in this study.METHODS: Human monocytes were separated from peripheral blood to induce dendritic cells (DCs).DCs were treated with LPS (30 μg/L),ox-LDL (10 mg/L) and LDL (10 mg/L) for 48 h.Then DCs were mixed with allogenic T lymphocytes to carry out mixed lymphocytes reaction (MLR).CTLA4Ig in different concentrations was added in the MLR of ox-LDL group.MTT method was used to assay the proliferation of T cells and expressed in stimulation index (IS).The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry.The excretion of IL-2,IFN-γ and IL-4 was assayed by ELISpot method.RESULTS: SI in ox-LDL group was higher than that in LDL group significantly (P<0.05) and CTLA4Ig inhibited the SI in ox-LDL group with dose-dependent effect (P<0.05,P<0.01).CTLA4Ig decreased the CD25 expression (P<0.05,P<0.01) and induced apoptosis of T cells in MLR (P<0.05,P<0.01).CTLA4Ig decreased the ELISpot counts of IL-2 and IFN-γ (P<0.01),while increased that of IL-4 (P<0.05).CONCLUSION: CTLA4Ig induces T cells tolerance to ox-LDL in vitro.CTLA4Ig inhibits T cells activation,promotes T cells apoptosis and Th1/Th2 immune deviation,which is the important mechanism in it′s induction of tolerance.     

Effect of fucoidan on inhibition of proliferation and induction of apoptosis in human breast carcinoma cell line MCF-7

AIM: To investigate the effects and related mechanism of fucoidan on the proliferation and apoptosis in breast carcinoma cell line MCF-7. METHODS: MCF-7 cells were treated with different concentrations of fucoidan (100 mg/L, 300 mg/L, 500 mg/L, 1 000 mg/L) for 48 h. Cell viability was measured by MTT assay. Apoptosis morphological and biochemical changes were detected by Hoechst 33258 staining and agarose gel electrophoresis. The expression of 〖STBX〗bcl-2〖STBZ〗 and bax was examined by RT-PCR and Western blotting. RESULTS: Fucoidan at different concentrations (100 mg/L, 300 mg/L, 500 mg/L, 1 000 mg/L) effectively inhibited the proliferation of MCF-7 cells (P<0.01). The inhibitory ratio and apoptosis rate increased in a concentration-dependent manner. Agarose gel electrophoresis of DNA revealed the characteristic “ladder” pattern of apoptosis. Fucoidan down-regulated the expression of 〖STBX〗bcl-2〖STBZ〗 and up-regulated bax in the levels of mRNA and protein. The ratio of Bcl-2 to Bax decreased as the concentrations of fucoidan increased (P<0.05). CONCLUSION: Fucoidan inhibits the cell proliferation by inducing cell apoptosis, and the apoptosis is related to the down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of apoptotic protein Bax.     

Effect of oxidized LDL on expression of connexin43 in cultured human umbilical vein endothelial cells

AIM: To investigate the effect of oxidized LDL (ox-LDL) on the expression of gap junction protein connexin43 in cultured human umbilical vein endothelial cells (HUVECs) in vitro. METHODS: Human umbilical vein endothelial cells cultured in normal condition were divided into blank control group, 50 mg/L,100 mg/L and 200 mg/L ox-LDL intervention groups. The mRNA expression of connexin43 in cultured HUVECs was detected with RT-PCR method; while the protein level of connexin43 was determined by the method of immunocytochemistry in the control and 100 mg/L ox-LDL intervention groups 24 h after ox-LDL was given. RESULTS: Different concentrations (50 mg/L, 100 mg/L, 200 mg/L) of ox-LDL up-regulated mRNA expression of connexin43 in cultured HUVECs after 24 h intervention (P<0.01). The protein level of connexin43 in cultured HUVECs intervened with 100 mg/L Ox-LDL for 24 h was up-regulated as compared to the control cells (P<0.01).CONCLUSION: Ox-LDL may up-regulate the expression of connexin43 at mRNA and protein levels in cultured human umbilical vein endothelial cells within short time, indicating that connexin43 plays an important role in the pro-atherosclerotic effect of Ox-LDL.     

Effects of Scutellaria barbata flavonoids on abnormal expression of NOS,HSP70 and apoE induced by Aβ 25-35 in rat astrocytes

AIM:To study the effects of Scutellaria barbata flavonoids (SBF) on abnormal expression of nitric oxide synthase (NOS), heat-shock protein 70 (HSP70) and apolipoprotein E (apoE) induced by Aβ 25-35 in rat astrocytes. METHODS:The third generation of cultured rat astrocytes was divided into 5 groups. The cells in 3 drug treatment groups were given SBF at dose of 17.5 mg/L, 35 mg/L and 70 mg/L for 24 h, and then the cells in model group and 3 doses of SBF groups were exposed to Aβ 25-35 at concentration of 100 μmol/L for 24 h. The expression of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and neuronal nitric oxide synthase (nNOS) in the cultured cells was assayed by immunohistochemical method. The expression of HSP70 was estimated by Western blotting and the mRNA expression of apoE was assessed by RT-PCR. RESULTS:Compared with control group, the protein level of eNOS were significantly decreased and the protein level of iNOS increased (P<0.01) in model group. The protein expression of HSP70 and mRNA expression of apoE were notably increased (P<0.01) in model group. However, these disturbances were attenuated by SBF at dose of 17.5, 35 and 70 mg/L (P<0.01). CONCLUSION:SBF has an obvious protective effect on damaged astrocytes induced by Aβ 25-35, suggesting that SBF may be helpful for the treatment of Alzheimer disease.     

Effect of luteolin on TGF-β1-induced epithelial-mesenchymal transition in lung cancer A549 cells

AIM:To investigate the effects of luteolin on the invasion and epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β1 (TGF-β1) in lung cancer A549 cells. METHODS:The effect of luteolin at 5, 10, 20, 40, 80 and 160 μmol/L on the viability of A549 cells was measured by MTT assay. The invasion ability was analyzed by Transwell method. The morphological changes of the A549 cells were observed under microscope.The protein expression of E-cadherin and vimentin in the A549 cells were determined by Western blot. RESULTS:The viability of the A549 cells was significantly inhibited by luteolin in a dose-time dependent manner (P<0.05). The IC₅₀ of luteolin for the A549 cells (24 h) was 68.79 μmol/L, while that (48 h) was 47.86 μmol/L. TGF-β1 induced morphological alteration of the A549 cells from epithelial to mesenchymal forms. Luteolin significantly inhibited TGF-β1-induced invasion of the A549 cells (P<0.01). The protein expression of E-cadherin was significantly down-regulated and the protein expression of vimentin was significantly up-regulated in the presence of TGF-β1 at 5 μg/L (P<0.01). However, luteolin reversed TGF-β1-induced EMT, up-regulation of E-cadherin and down-regulation of vimentin (P<0.01). CONCLUSION:Lu-teolin reverses TGF-β1-induced EMT in the lung cancer A549 cells.     

Proapoptotic mechanism and changes of endogenous TGF-β1 in NB4 cells induced by exogenous TGF-β1

AIM: To study the effects of transforming growth factor-β1 (TGF-β1) on cell apoptosis, cell cycle, production of endogenous TGF-β1, expressions of P27Kip1, cyclin E and bcl-2 mRNA levels in NB4 cells. METHODS: Apoptotic morphological changes were observed by Wright-Giemsa staining. Cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine the mRNA levels of endogenous TGF-β1, P27Kip1, cyclin E and bcl-2. RESULTS: TGF-β1 significantly restrained the growth and promoted the apoptosis of NB4 cells. The blockage of NB4 cells treated by TGF-β1 at concentration of 5 μg/L was in G1 phase. Endogenous TGF-β1 mRNA expression in NB4 cells was up-regulated when the concentration of exogenous TGF-β1 was <5 μg/L. Meanwhile, the expression of endogenous TGF-β1 mRNA was down-regulated when the concentration of exogenous TGF-β1 was 10 μg/L. After treated with TGF-β1 at concentration of 5 μg/L, P27Kip1 mRNA expression in NB4 cells was up-regulated, cyclin E and bcl-2 were reduced. CONCLUSION: TGF-β1 is able to induce apoptosis and cell cycle distribution abnormally in NB4 cells by (1) Up-regulation of endogenous TGF-β1, so that NB4 cells was induced into apoptosis through consequently high expression of P27Kip1. (2) TGF-β1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by inhibiting the activity of cyclin E through the increased expression of P27Kip1. (3) Down-regulation of bcl-2 induces apoptosis of NB4 cells.     

Hydrogen sulfide inhibits inflammatory responses induced by acute myocardial ischemia in isolated rat hearts

AIM：To investigate whether hydrogen sulfide (H2S) protects the hearts against inflammatory responses induced by acute myocardial ischemia in isolated rat hearts. METHODS：Rat acute myocardial ischemia injury was induced by ligation of the left anterior descending coronary artery for 4 h, and the normal perfusate was replaced with NaHS (5 μmol/L, 10 μmol/L and 20 μmol/L) perfusate accordingly in NaHS groups 2 h after ischemia. The changes of cardiac function in the myocardial ischemic injury rats were observed. The mRNA expression of TNF-α, IL-1β, IL-6, IL-10 and ICAM-1 was detected by real-time PCR. The protein level of nuclear factor-κB (NF-κB) in the myocardial tissues was detected by Western blotting. RESULTS：The cardiac function in ischemia group was lower than that in sham group (P<0.01). Compared with ischemia group, perfusion of NaHS resulted in the improvement of the cardiac function (P<0.05 or P<0.01). Compared with sham group, the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 in the cardiac tissues was significantly increased, and the mRNA expression of IL-10 in the cardiac tissues was significantly decreased in ischemia group (P<0.01). Compared with ischemia group, the perfusion of NaHS significantly decreased the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 (P<0.05 or P<0.01). The perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly increased the mRNA expression of IL-10 (P<0.01). The protein level of NF-κB in ischemia group was markedly higher than that in sham group (P<0.01). Compared with ischemia group, the perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly decreased the expression of NF-κB (P<0.05 or P<0.01). CONCLUSION：H2S protects the hearts against acute ischemia injury through inhibition of NF-κB activation and subsequent down-regulation of NF-κB-dependent inflammatory gene expression.     

Effect of glutamine deprivation on growth and differentiation of primary APL cells

AIM:To investigate the effect of Gln deprivation on growth and differentiation of primary APL cells. METHODS: The primary APL cells were collected from 18 cases of APL patients’ periphery blood, the patients had not been treated. The cells were incubated in RPMI 1640 without or with different contents of glutamine and with 10% fetal calf sera, at 37℃,5% CO 2 and saturation humidity for 4 days.The initial living cell density was 5×10⁸/L. After 4 days incubation,the cells were counted,collected, smeard and stained with Wright-Giemsa, DNA, POX, NAE and NaF inhibition,gulping ink and NBT etc. cytochemical technology. The stained cells were investigated under oil immersion lens. RESULTS: The living cell density became (54.28±4.28)% of the initial in glutamine deprivation group, but the living cell density in control with 4 mmol/L Gln was (108.56±12.27 )% of the initial ( P <0.01) after 4 days incubation. The cells differentiated into mature granulocytes in glutamine deprivation group.CONCLUSION: Deprivation of glutamine inhibited primary APL cell growth and induced cell differentiation toward mature granulocyte.     

Effect of CKLF1-C19 polypeptide on differentiation of human lung fibroblast into myofibroblast induced by TGF-β

AIM: To investigate the effect of CKLF1-C19 polypeptide (C19) on differentiation of human lung fibroblast (LFB) into myofibroblast (MFB) induced by TGF-β. METHODS: LFBs were cultured and identified. LFBs were treated with TGF-β (5 μg/L) to establish the cell model of LFB differentiate into MFB. The LFBs were divided into 6 experimental groups including control group, TGF-β group, and TGF-β plus different doses (1, 0.1, 0.01, 0.001 mg/L) C19 groups. The cell morphology, cell proliferation rate, and the expression of α smooth muscle actin (α-SMA) and collagen I were observed. RESULTS: Human primary LFB was successfully cultured and was confirmed by the method of immunofluorescence. TGF-β at 5 μg/L induced proliferation and differentiation of LFB. The mRNA levels of α-SMA and collagen I in TGF-β group were higher than that in control group (P<0.05).The cell proliferation rates, mRNA levels of α-SMA and collagen I, and the protein expression of α-SMA in 0.01 mg/L+TGF-β group and 0.001 mg/L+TGF-β group were markedly lower than those in TGF-β group (P<0.05). CONCLUSION: C19 at 0.01 mg/L and 0.001 mg/L effectively inhibits differentiation of LFB into MFB induced by TGF-β, thus inhibiting the process of airway remodeling and fibrosis to some extent.     

Effects of recombinant human CD40 ligand on biological behavior of ovarian cancer SKOV3 cells in vitro

AIM: To investigate the effect of recombinated human CD40 ligand (rhCD40L) on the biological behavior of ovarian cancer SKOV3 cell line in vitro.METHODS: After the SKOV3 cells were incubated with different concentrations of rhCD40L for various times, the cell proliferation was determined by MTT assay.The expression of the co-stimulatory molecules or adhesion molecules on SKOV3 cells and the changes of tumor necrosis factor receptor associated factor (TRAFs) inside the cells were measured by flow cytometry and direct immunofluorescence.Annexin V and PI dual color label assay were used to detect cell apoptosis or death in culture contained with rhCD40L.RT-PCR assay was employed to determine the change of apoptosis related gene c-myc, bcl-2 and bcl-xl expression in SKOV3 cells.RESULTS: rhCD40L inhibited proliferation of SKOV3 cells at concentration of 100 μg/L (0.65±0.10 vs 0.81±0.05) and reached a peak at concentration of 10 mg/L (0.13±0.12 vs 0.83±0.15, P＜0.01).The inhibitory effects showed a dose dependent manner.Cell cycle analysis showed that cell division was blocked in G1 phase.Increasing proportion of apoptosis of SKOV3 cells was related to up-regulation of CD95 expression (42.4% vs 59.2%, P＜0.05) and down-regulation of anti-apoptosis genes such as bcl-2 and bcl-xl expressions after incubation with rhCD40L.TRAF 2, 5 and 6 expressed highly in SKOV3 cells.The expression of TRAF 2 (81.3%±9.2% vs 50.4%±5.3%，P＜0.05), TRAF5 (47.2%±7.2% vs 7.2%±2.1%, P＜0.01) and TRAF6 (44.5%±6.3% vs 5.1%±1.1%, P＜0.01) was down-regulated and expression of TRAF 3 (25.2%±6.2% vs 68.8%±5.3%, P＜0.01) was up-regulated after co-culture with rhCD40L, but there was no effects found on the expression of TRAF 1 (4.3%±1.2% vs 5.1%±1.4%) and TRAF4 (7.4%±1.2% vs 8.1%±1.4%).CONCLUSION: By down-regulating expression of bcl-2, bcl-xl and changing expression profile of TRAF, rhCD40L inhibits the growth of SKOV3 cells by blocking the cell cycle progress in G1 and promotes the cells to apoptosis.     

Tumor necrosis factor-related apoptosis-inducing ligand participates in injury of vascular endothelial cells induced by rapamycin in vitro

AIM: To investigate the effects of rapamycin on apoptosis, proliferation, migration ability and tumor related apoptosis inducing ligand(TRAIL) in cultured human umbilical vein endothelial cells(HUVECs). METHODS: Cultured HUVECs were treated with rapamycin at the concentrations of 0, 1, 10 and 100 μg/L for 24 h. The cell proliferation was measured by CCK-8 method. The cell migration ability was detected by Transwell chambers and wound healing test. The apoptotic index of HUVECs was quantitatively determined by measuring the activation of caspase-3. The morphological changes of the apoptotic cells were observed by DAPI staining. The expression of TRAIL was detected by Western blotting. RESULTS: A 24 h-incubation with rapamycin(1-100 μg/L) caused significant cell loss associated with the increase in apoptosis, as quantified by the determination of caspase-3 activity(P<0.01) in HUVECs. Obvious apoptotic morphology was observed by DAPI staining in HUVECs incubated with rapamycin. Rapamycin at the concentrations of 1-100 μg/L also impaired the migration ability of HUVECs(P<0.01). In addition, rapamycin(10-100 μg/L) inhibited the proliferation of HUVECs, whereas rapamycin at 1 μg/L had no such effect(P<0.01). Rapamycin(10-100 μg/L) also induced TRAIL expression in a dose-dependent manner(P<0.01). CONCLUSION: Rapamycin induces apoptosis, and inhibits the proliferation and migration of HUVECs. The up-regulation of TRAIL might be related to the injury of vascular endothelial cells caused by rapamycin.