FAK gene silencing enhances chemotherapeutic drug sensitivity in leukemic cells XU Lü-hong1,

AIM: To investigate the effect of focal adhesion kinase (FAK) gene silencing on chemotherapeutic drug sensitivity in leukemic cells. METHODS: Lentiviral-FAK-shRNA was transfected into BCR/ABL-BaF3 leukemic cells. The protein expression of FAK was detected by Western blotting. BCR/ABL-BaF3 leukemic cells were treated with different concentrations of imatinib in vitro, and the apoptosis was determined by labeling with Annexin V. A murine model of leukemia was established and the mice were treated with FAK shRNA and imatinib. Survival time and distribution of leukemic cells in bone marrow and spleen of the mice were monitored. RESULTS: FAK shRNA was successfully constructed and effectively inhibited FAK gene expression. With 5 μmol/L imatinib treatment, the percentages of apoptotic cells in vector control group and FAK shRNA group were (9.76±1.97)% and (21.90±3.20)%, respectively, and significant difference between these 2 groups (P<0.05) was observed. With 50 μmol/L imatinib treatment, the percentages of apoptotic cells in vector control group and FAK shRNA group were (56.10±6.00)% and (82.10±5.70)%, respectively,also with significant difference between these 2 groups (P<0.05). Compared with vector control group, the mice in FAK gene silencing group displayed significantly prolonged survival time. Moreover, 60 days after injection of leukemic cells, the percentages of leukemic cells in bone marrow and spleen of the mice were significantly decreased in FAK gene silencing group as compared with those in vector control group. CONCLUSION: FAK gene silencing promotes the efficacy of chemotherapeutic drug in leukemic cells, indicating that FAK gene silencing might be considered as a new therapeutic strategy for leukemia.     

CREG gene silencing induces spontaneous apoptosis of ESCs-derived Ebs

AIM: To investigate the effect of cellular repressor of E1A-stimulated genes (CREG) on the spontaneous apoptosis of murine embryonic stem cells(ESCs)-derived embryoid bodies (EBs).METHODS: The murine ESCs R1 were infected with pDS-shRNA-CREG and pDS-shRNA-GFP retrovirus, respectively. R1, R1-GFP and R1-shCREG were cultured on STO feeder cells in DMEM supplied with leukemia inhibitory factor(LIF). Alkaline phosphatase(AKP) staining and teratoma formation assay inoculated into mouse myocardium were used to detect stemness of transfected ESCs. R1/EB, R1-GFP/EB and R1-shCREG/EB were produced by liquid suspension method. The expression of CREG and cleaved caspase-3 were analyzed by Western blotting and quantitative RT-PCR on day 7. The apoptotic rates of the 3 kinds of EBs were analyzed by flow cytometry(FCM) analysis with Annexin V/PI dual staining. RESULTS: The stably-transfected ES cells (R1-shCREG and R1-GFP) were obtained by screening the G418-resistant clones. R1-GFP and R1-shCREG had the stem cell properties similar to those of R1 detected by AKP staining. However, it was found that R1-shCREG didn't show the same almighty differentiation function as of R1 and R1-GFP by tumor experiments in mouse myocardium that it couldn't form teratomas analogue and had the lower ability of cell differentiation. Observation under phase-contrast microscope showed that the cell differentiation was inhibited while the number of cell death was increased in R1-shCREG/EB group. Compared with R1/EB and R1-GFP/EB, Western blotting analysis demonstrated that the protein expression of CREG was decreased to (78.0±1.3)% and (84.0±2.4)% on day 7, respectively. The mRNA expression of CREG was also decreased, but the expression of cleaved caspase-3 at the mRNA and protein levels was increased obviously. Annexin V/PI FCM assay indicated that the apoptotic rate of R1-shCREG/EB was significantly higher those that in other 2 groups on day 7. CONCLUSION: The down-regulation of CREG inhibits ESCs differentiation and promotes cell apoptosis.     

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

AIM: To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A(RALA) on proliferation and apoptosis of chronic myelogenous leukemia(CML) K562 cells. METHODS: The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using Lipofectamine^TM 2000. The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion. The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively. The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide, and the apoptotic morphological changes were detected by Hoechst 33258 staining. RESULTS: RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells(P<0.05). The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group(P<0.05). The apoptotic rate was much higher in RALA siRNA group than that in negative control group(P<0.05). The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA. CONCLUSION: The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells, indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.     

Caspase-3 gene silencing attenuates apoptosis in alveolar epithelial cells after Streptococcus pneumoniae infection

AIM: To investigate the influence of Streptococcus pneumoniae on the apoptosis of alveolar epithelial cell and the modulation of caspases-3 on apoptosis. METHODS: A study was undertaken to determine whether Streptococcus pneumoniae infection induced the apoptosis of A549 and facilitated the expression of pro-inflammative cytokine. After inhibited by siRNA, the expression of caspase-3 and apoptosis grade in A549 were determined. RESULTS: Streptococcus pneumoniae contributed to alveolar epithelial cell apoptosis in a time-dependent manner. Streptococcus pneumoniae facilitated the expression of caspase-3 and IL-6 in A549, while depressed IL-10 concentration in culture medium. SiRNA directly decreased the expression of caspase-3 to rescue A549 from apoptosis but not affected IL-6 and IL-10 concentration. CONCLUSION: caspase-3 gene silencing rescues epithelial cells from apoptosis initiated by Streptococcus pneumoniae infection.     

Doxycycline induces apoptosis in THP-1 cells

AIM: This study was designed to investigate the apoptotic effect of doxycycline in THP-1 cells.METHODS: After differentiated by PMA, THP-1 cells were treated with doxycycline at different concentrations ranging from zero to 80 mg/L. The morphological changes of THP-1 cells were observed under light microscope. MTT assay were used to examine the effects of doxycycline on proliferation of THP-1 cells. Apoptotic THP-1 cells were measured by Annexin-V flow cytometry analysis and TdT-mediated dUTP nick end labeling assay.RESULTS: Treated with a certain concentration of doxycycline, differentiated THP-1 cells contracted and turn round, a number of cells were dead. MTT assay and positive Annexin V-FITC on cell membrane and TUNEL assay showed that doxycycline induced apoptosis in THP-1 cells in a dose-dependent manner.CONCLUSION: Doxycycline induces apoptosis in THP-1 cells in a dose-dependent manner.     

Genistein downregulates survivin gene expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To probe into the role of 1, 4, 5-trisphosphate inositol (IP3) and survivin protein in apoptosis of HepG2 cells induced by genistein. METHODS: HepG2 cells were treated with 60 μmol/L genistein for 12 h, 24 h, 48 h and 72 h. IP3, survivin and apoptosis rate were assayed by IP3-［3H］ Birtrak assay, Western blotting and flow cytometry, respectively. RESULTS: IP3 in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein were significantly lower than that in control (P<0.01) ［(12.0±1.4) pmol/106cells, (7.5±0.8) pmol/106 cells, (5.6±0.5) pmol/106cells, (3.3±0.6) pmol/106 cells， vs (29.2±0.6) pmol/106 cells］. V-survivin/ V-β-actin, which was the gray degree multiply area of survivin/the gray degree multiply area of β-actin in groups incubated for 12 h, 24 h, 48 h and 72 h with 60 μmol/L genistein, were significantly lower than that in control (P<0.01) ［（0.36±0.13, 0.33±0.03, 0.23±0.04, 0.18±0.04）， vs 0.63±0.06］. The apoptosis rate in groups incubated with 60 μmol/L genistein for 24 h, 48 h and 72 h was significantly higher than that in control （P<0.01） ［(7.4%±0.5%, 20.5%±2.0%, 30.7%±1.6%) vs 2.6%±0.1%］. CONCLUSION: Genistein induces apoptosis in HepG2 cells by reducing IP3 production and survivin protein expression.     

Effects of Smo gene silencing on cell activity and apoptosis of human cervical carcinoma HeLa cells

AIM: To investigate the effect of Hedgehog (Hh) signaling pathway on the viability and apoptosis of cervical carcinoma cells by shRNA technique to knock down Smoothened (Smo) gene. METHODS: Smo shRNA was used to transfect the cervical carcinoma HeLa cells. The expression of Smo and Gli1 at mRNA and protein levels in the HeLa cells was determined by RT-PCR and Western blot, respectively. The effect of Smo gene silencing on the growth of the cells was measured by MTT assay. The apoptosis and cell cycle were determined by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression of Smo and Gli1 were evenly reduced obviously after transfected with Smo shRNA for 72 h (P<0.05). The viability of HeLa cells transfected with Smo shRNA was significantly inhibited. The percentages of the cells in G₀/G₁ phase and early apoptosis rate were obviously higher in Smo shRNA transfection group than those in control group. CONCLUSION: Smo gene silencing effectively inhibits the cell growth and induces the apoptosis of human cervical carcinoma cells.     

Effect of biological clock gene Timeless silencing on apoptosis and invasion of ovarian cancer SKOV3 cells

AIM:To evaluate the effect of biological clock gene Timeless (TIM) silencing on the apoptosis and invasion ability of human ovarian cancer SKOV3 cells. METHODS:The protein expression of TIM in the ovarian cancer tissues and normal ovarian tissues was detected by immunohistochemistry, and the correlation between the protein expression of TIM in ovarian cancer tissues and the pathological features was analyzed. The ovarian cancer SKOV3 cells were transfected with PBS (blank control group), control siRNA (siRNA control group) or TIM siRNA (TIM siRNA group). The protein expression of TIM, Bcl-2, Bax, MMP-2, MMP-9, caspase-3 and caspase-9 was determined by Western blot. The apoptosis was detected by flow cytometry. The invasion ability was measured by Transwell chamber test. RESULTS:The positive expression rate of TIM in the ovarian cancer tissues (84.0%) was significantly higher than that in the normal ovarian tissues (10.0%; P<0.01). TIM expression was associated with ovarian cancer differentiation, depth of invasion, lymph node metastasis and TNM stage (P<0.05), but was not associated with age and pathological type (P>0.05). The protein expression levels of TIM, MMP-2, MMP-9 and Bcl-2 in TIM siRNA group were significantly decreased as compared with control group and siRNA control group (P<0.01), and the protein expression of Bax, caspase-3 and caspase-9 in TIM siRNA group was significantly increased as compared with blank control group and siRNA control group (P<0.01). No significant difference of the protein expression of TIM, MMP-2, MMP-9, Bcl-2, Bax, caspase-3 and caspase-9 between blank control group and siRNA control group was observed (P>0.05). The apoptotic rate in TIM siRNA group was significantly higher than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). The penetrated cell number in TIM siRNA group was significantly less than that in blank control group and siRNA control group (P<0.01), and that in blank control group and siRNA control group was not significantly different (P>0.05). CONCLUSION:Silencing of TIM gene in ovarian cancer SKOV3 cells by siRNA promotes apoptosis, and inhibits cell invasion.     

Oxidative stress induces apoptosis in HepG2 cells

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved.METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis. The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader.RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2-treated cells appeared green fluorescence as early as 4 h, which represents de-energized mitochondria, the untreated cells appeared red fluorescence, a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase-3 activity increased by 6.7-fold (P<0.01) at 8 h and caspase-9 activity increased by 3.6-fold (P<0.01) at 12 h, respectively, however, the activity of caspase-8 remained unchanged.CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase-9 and-3, but not caspase-8.     

Effect of lentinan on apoptosis and PI3K/AKT signaling pathway in leukemic HL-60 cells in vitro

AIM:To study of the regulatory effect of lentinan on human leukemic HL-60 cell apoptosis and its effect on PI3K/AKT signaling pathway in HL-60 cells in vitro.METHODS:Lentinan at concentrations of 0 mg/L, 15 mg/L, 30 mg/L and 45 mg/L was applied to HL-60 cells cultured to the logarithmic phase in vitro, and the inhibitory effect of lentinan on the viability of HL-60 cells was measured by MTT assay after 24 h, 48 h and 72 h. The apoptosis induced by lentinan was analyzed by flow cytometry. The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3, cleaved caspase-8, cytochrome C, PI3K, AKT and p-AKT were determined by Western blot. After treatment with PI3K inhibitor LY294002 at 5 mg/L for 72 h, the apoptosis of HL-60 cells was analyzed by flow cytometry. RESULTS:The viability of HL-60 cells was inhibited after treatment with lentinan at concentrations of 15 mg/L, 30 mg/L and 45 mg/L for 24 h, 48 h and 72 h in concentration-dependent and time-dependent manners (P<0.05). The apoptosis of HL-60 cells was promoted after treatment with lentinan (15 mg/L, 30 mg/L and 45 mg/L) for 72 h in a concentration-dependent manner (P<0.05). The protein levels of cleaved PARP, cleaved caspase-9, cleaved caspase-3 and cytoplasmic cytochrome C in the HL-60 cells induced by 30 mg/L lentinan were increased significantly with the increase in the treatment time (P<0.05), but caspase-8 did not show any change. The protein levels of PI3K, AKT and p-AKT were decreased obviously with the increase in the lentinan concentration (P<0.05). Treatment of HL-60 cells with LY294002, a PI3K pathway inhibitor, produced apoptosis-inducing effect similar to lentinan (P<0.05). CONCLUSION:Lentinan induces HL-60 cell apoptosis by inhibiting PI3K/AKT signaling pathway.     

Effects of JAG1 gene silencing on proliferation and apoptosis of human breast cancer MDA-MB-231 cells

AIM:To investigate the effects of Jagged 1 (JAG1) gene silencing on the proliferation and apoptosis of human breast cancer MDA-MB-231 cells. METHODS:The specific recombinant vector pRS-JAG1 was transfected into MDA-MB-231 cells with lipofectamine. The protein expression of JAG1 was observed by Western blotting after transfection. MTT assay was used to detect the effect of JAG1 gene silencing on the growth of the cells. The apoptosis and cell cycle were analyzed by flow cytometry. The protein levels of cyclin D1, p21CIP1/WAF1, p27KIP1, p-Rb, Bcl-2, Bax, Bcl-xL and cleaved caspase-3 were determined by Western blotting. RESULTS:Compared with control group, the expression level of JAG1 was reduced by pRS-JAG1 transfection for 72 h (P<0.05). The growth of MDA-MB-231 cells in shJAG1 group was significantly inhibited (P<0.05). The percentages of G 0/G 1-phase cells and early apoptotic rate were obviously higher in shJAG1 group than those in control group (P<0.05). The shRNA-mediated JAG1 silencing decreased the protein levels of cyclin D1, p-Rb, Bcl-2 and Bax, and increased the protein levels of p21CIP1/WAF1, p27KIP1, Bax and cleaved caspase-3 (P<0.05). CONCLUSION:JAG1 silencing effectively inhibits the proliferation and induces the apoptosis of human breast cancer cells, suggesting that JAG1 might serve as a therapeutic target for triple-negative breast cancer.     

Resveratrol induces apoptosis in human primary gastric carcinoma cells

AIM: To investigate the apoptosis in primary gastric cancer cells induced by resveratrol, and the relation between this apoptosis and expression of bcl-2 and bax. METHODS: In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscopy and TUNEL staining were used to quantitatively and qualitively detect the apoptosis of primary gastric cancer cells before and after the resveratrol treatment. Immunohistochemical staining and RT-PCR was used to detect the expression of apoptosis-regulated gene bcl-2 and bax. RESULTS: Resveratrol inhibited the growth of primary gastric cancer cells in a dose- and time-dependent manner. Resveratrol induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of primary gastric cancer cells with resveratrol for 24, 48, 72, 96 hours, the apoptotic indexs were 4.93%±0.19%, 16.74%±0.43%, 27.88%±0.36%, 36.84%±1.07% respectively. Immunohistochemical staining showed that after the treatment of primary gastric cancer cells with resveratrol for 24, 48, 72, 96 hours, the positive rates of Bcl-2 proteins were 20.68%±0.49%, 10.84%±0.33%, 6.80%±0.34%, 3.91%±0.15% and the positive rates of Bax proteins were 19.79%±0.98%, 30.74%±0.85%, 40.14%±1.17%, 60.08%±1.64%. After exposed to resveratrol for 24 h, 48 h, 72 h and 96 h, the density of bcl-2 mRNA decreased progressively with elongation of time and the density of bax mRNA increased progressively with elongation of time by RT-PCR. CONCLUSION: Resveratrol is able to induce the apoptosis in primary gastric cancer. This apoptosis may be mediated by down-regulation of Bcl-2 and up-regulation of Bax.     

Norcantharidin induces HeLa cells apoptosis through caspases pathway

AIM: To examine the apoptotic pathway of norcantharidin (NCTD)-induced HeLa cells death. METHODS: MTT, photomicroscopical observation, DNA agarose gel electrophoresis, LDH release and Western blot analysis were used. RESULTS: NCTD induced HeLa cells apoptosis and the apoptosis was partially reversed by the inhibitors of caspase-family (-3, -8, -10). The activities of caspase-3, -8 and -9 were significantly increased after treated with NCTD. The expression of the inhibitor of caspase-3 activated DNase (ICAD) was decreased in a time dependent manner. CONCLUSION: NCTD induces HeLa cells apoptosis through activating caspase pathways.     

FAK shRNA inhibits growth of leukemic cell line BCR/ABL-BaF3

AIM: To investigate the effect of focal adhesion kinase (FAK) shRNA on the growth of leukemic cells.METHODS: Lentiviral-FAK-shRNA was transfected into BCR/ABL-BaF3 cells, while empty vector was transfected into the same cells for control. The proteins of FAK and other molecules were detected by Western blotting. Cell growth was observed by culturing the leukemic cells in RPMI-1640 medium in vitro, and colony formation was observed by culturing the leukemic cells in methylcellulose medium. To establish a murine model of leukemia, BCR/ABL-BaF3 cells were injected into BALB/c mice through tail vein. Survival time of the leukemic mice was monitored, and the distribution of the leukemic cells in spleen of the mice was also detected. RESULTS: FAK shRNA inhibited the protein expression of FAK, reduced STAT5 phosphorylation and induced caspases-3 activation in BCR/ABL-BaF3 cells. FAK shRNA inhibited the cell growth in vitro. Colony formation experiment showed that the number of colony in vector control group and FAK shRNA group was 215.60±13.01 and 125.00±9.06, respectively, and the difference was statistically significant (P<0.01). The mice in vector control group died between day 21 and day 27, while the mice in FAK shRNA group died between day 52 and day 60, and the difference was statistically significant (P<0.05). Moreover, 25 days after injection of leukemic cells, the percentage of leukemic cells in spleen of the leukemic mice in vector control group and FAK shRNA group was (82.40±6.13)% and (14.50±3.70)%, respectively (P<0.01). CONCLUSION: FAK shRNA inhibits the growth of leukemic cells in vitro and in vivo, indicating that FAK gene silencing might be a new therapeutic strategy for leukemia.     

Celastrol induces apoptosis of multiple myeloma H929 cells

AIM: To investigate the effect of celastrol on the apoptosis of human multiple myeloma H929 cells and its molecular mechanism. METHODS: The H929 cells were cultured in vitro and treated with celastrol at different concentrations (0.5, 1, 5 and 10 mg/L). The viability of H929 cells was analyzed by CCK8 assay. Annexin V-PE/7-AAD staining was used to analyzed the effect of celastrol on apoptosis of H929 cells, and mitochondrial membrane potential was observed by flow cytometry. The effect of celastrol on DNA damage was detected by comet assay. The protein levels of apoptosis-related molecules P53, XIAP, cleaved PARP-1 and cleaved caspase-3, and the release of mitochondrial cytochrome C in the H929 cells treated with celastrol were determined by Western blot. RESULTS: The viability of H929 cells was significantly inhibited by different concentrations of celastrol in a concentration-dependent and time-dependent manner. Apoptosis and decreased mitochondrial membrane potential of H929 cells in a concentration-dependent manner were observed after treatment with celastrol (P<0.05). The results of comet assay showed that celastrol induced DNA damage in the H929 cells. The protein levels of apoptotic molecules P53, cleaved PARP-1 and cleaved caspase-3 were significantly increased and the expression level of anti-apoptotic protein XIAP was significantly decreased in the H929 cells treated with celastrol (P<0.05). Celastrol promoted the release of cytochrome C in mitochondria, and activated caspase-3 in dependence on caspase-9. CONCLUSION: Celastrol has an apoptosis-inducing effect on multiple myeloma H929 cells. Its mechamism may be related to activation of mitochondrial apoptosis pathway by inducing DNA damage.     

C-reactive protein induces apoptosis in human renal tubular epithelial cells

ATM: To explore whether the C-reactive protein (CRP) level in microinflammation state induces the apoptosis of renal tubular epithelial cells. METHODS: HK-2 cells were stimulated with recombinant human CRP. Annexin-FITC-PI staining and flow cytometry were used to detect the percentage of apoptotic cells. Morphology observation of apoptosis was assessed by Hoechst 33258 staining. Caspase-3 activity was measured by a colorimetric assay. The expression of apoptotic gene bax and anti-apoptotic gene bcl-2 at mRNA levels was determined by real-time PCR. RESULTS: CRP induced apoptosis of HK-2 cells in a time- and dose-dependent manner. The maximal apoptotic effect of CRP concentration was 10 mg/L CRP at concentration of 20 mg/L. CRP treatment was associated with the characteristic morphological features of apoptosis such as condensation, fragmentation or margination of nuclear chromatin. CRP exposure increased caspase-3 activity, up-regulated the mRNA expression of Bax and down-regulated the mRNA expression of Bcl-2. CONCLUSION: Slightly increased CRP level has the potential to induce apoptosis of renal tubular cells.     

Celecoxib induces apoptosis in COX-2 non-expression gastric cancer cells

AIM: To investigate whether celecoxib induces gastric cancer cell apoptosis in a COX-2 non-expression cell line. METHODS: The COX-2 protein was examined by western blotting. Fluorescence microscopy, DNA agarose gel electrophoresis and flow cytometry analysis were used to test apoptosis. RESULTS: COX-2 was expressed in AGS but not MGC-803 gastric cancer cell line; Selective COX-2 inhibitor celecoxib induced MGC-803 cell line apoptosis in a concentration and time-dependent manner. CONCLUSION: Celecoxib induces apoptosis in COX-2 non-expression gastric cancer cells.     

Effect of G-CSF on the apoptosis induced by Ara-c in HL-60 leukemic cells

AIM: To observe the effects of methionine-induced hyperhomocysteinemia on protein C(PC), antithrombin-Ⅲ (AT-Ⅲ) and von willebrand factor (vWF).METHODS:The proliferat ion of HL-60 leukemia cell was observed by hemopoiet ic cell culture.Apoptosis was measured by the morphology of apoptosis cell, the quantitation of DNA fragmentation with the diphenylamine reaction.The change in drug sensitivity was measured by MTT.RESULTS:In group M, the levels of methionine(29.97±5.34 μmol/L) and homocysteine(13.30±2.19 μmol/L) in serum were signifficantly higher than those(14.48±1.97 μmol/L and 5.36±1.19 μmol/L, respectively, P＜0.01) of group C.The levels of AT-Ⅲ and PC of group M were signifficantly lower than those of group C (P＜0.01). The level of vWF in plasma of group M was higher than that of group C (P＜0.01). Immunohistochemistry showed that vWF expression in endothelial cells of aorta was decreased. CONCLUSION:Methionine-induced hyperhomocysteinemia had promotive effects on coagulation and inhibiting effects on antioagulation.     

RNA interfering retinoblastoma-like protein 2 gene induces apoptosis of human cardiac stem cells mediated by DNA methyltransferase

AIM: Retinoblastoma-like protein 2 (Rbl-2) plays an important role in the cell proliferation and DNA methyltransferase (DNMT) may involve in the regulation of differentiation in embryonic stem cells. This study is to investigate the effect of knocking down Rbl-2 by specific siRNA on apoptosis in human cardiac stem cells.METHODS: The siRNA of Rbl-2 (siRbl-2) was transfected into human cardiac stem cells. The mRNA expression of Rbl-2 and DNMT-3B was detected by real-time RT-PCR 48 h after transfection. The DNMT-3B protein expression and the activation of caspase-3 were determined by Western-blotting. The cell apoptosis was analyzed by flow cytometry with annexin V-FITC/PI staining. RESULTS: Knocking down of Rbl-2 gene increased the expression of DNMT-3B in human cardiac stem cells, and induced cell apoptosis. Compared with negative control group, caspase-3 was activated and cleaved caspase-3 was increased in the stem cells transfected with siRbl-2. The cleaved caspase-3 accounted for more proportions of total caspase-3 in transfected cells than that in non-transfected cells (P<0.01). The apoptotic rate was also increased significantly in transfected group (P<0.01).CONCLUSION: Rbl-2 plays an important role in the regulation of survival and apoptosis in human cardiac stem cells. This regulatory mechanism may involve in epigenetic modification, which is mediated by DNMT.