Cytochrome C induces apoptosis in HL-60 cells through inhibiting bcl-2,bcl-xl mRNA expression

AIM:To study the effect of cytochrome C on HL-60 cells in vitro and the expression of relevant apoptotic genes.METHODS:HL-60 cells were treated with different concentrations of cytochrome C for 24 h.The suppressing rate was assayed by MTT.The morphology of cell was observed by microscope and fluorescence microscope.The apoptosis was assayed by flow cytometry (FCM) and DNA electrophoresis.The expression changes of bcl-2 and bcl-xl mRNA was examined by RT-PCR.RESULTS:The suppressing rate increased with the increase in the cytochrome C concentrations.When treated with 0-37.5 mg/L cytochrome C for 24 h,the percentage of apoptotic HL-60 cells increased in a dose-dependent manner,and the typical cells and the appearance of apoptotic DNA ladder were observed.At the same time,within this range of concentration,the expression of bcl-2 and bcl-xl mRNA decreased gradually.When treated with cytochrome C at concentration higher than 37.5 mg/L,the percentage of apoptotic HL-60 cells did not increase,but decreased,while the cell necrosis was observed.CONCLUSIONS:It suggested from the results that at certain range of concentration,cytochrome C induces apoptosis or necrosis in HL-60 cells.The percentage of apoptosis,the changes of expression of bcl-2 and bcl-xl depend on the dose of cytochrome C.The mechanism that cytochrome C induces apoptosis in HL-60 cells may be related to suppressing the expression of bcl-2 and bcl-xl.     

Sodium valproate induces cycle arrest,apoptosis and elevates p21WAF1 mRNA expression in chronic myeloid leukemia cell line K562

AIM:To investigate the effects of sodium valproate (VPA) on the cell cycle and apoptosis of chronic myeloid leukemia cell line K562, and to explore the possible mechanisms. METHODS:K562 cells were treated with VPA. Cell cycle and apoptosis were analyzed by flow cytometry. The expression of p21WAF1 mRNA was detected by RT-PCR. RESULTS:After treatment with VPA, cell cycle was arrested obviously at G0/G1 phase ［(82.30±9.41)% vs (40.13±2.12)%, P<0.05］. The apoptotic rate was significantly higher in the cells treated with VPA than that in untreated cells ［(11.47%±0.25%) vs (4.77%±0.40%), P<0.05］. The level of p21WAF1 mRNA was increased ［(1.65±0.91) vs (0.25±0.04), P<0.05］. CONCLUSION:VPA induces elevated expression of p21WAF1 mRNA in K562 cells, resulting in G0/G1 phase arrest and apoptosis in vitro.     

Inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in two human ovarian carcinoma cell lines

AIM: To investigate the inhibitory effects of recombinant human Mullerian inhibiting substance on cell proliferation in human ovarian carcinoma cells (OVCAR8 and SKOV3 cell lines). METHODS: The expression of MISIIR protein and the localization of MISIIR protein were analyzed by Western blotting and confocal spectral microscopy, respectively. Cell apoptosis and cell cycle were detected by flow cytometry (FCM). Cell viability was determined via MTT method. Clone formation test was used to detect oncogenicity in vitro.RESULTS: The MISIIR protein expression in OVCAR8 cells but not in SKOV3 cells was observed. MISIIR expression was seen on the OVCAR8 cell surface and in the cytoplasm with both antibodies. After treated with rhMIS for 48 h, the cell viability was significantly decreased in OVCAR8 cells. rhMIS inhibited the oncogenicity of OVCAR8 cells greatly. The cell apoptosis of OVCAR8 cell exposed to 10 mg/L rhMIS was (31.3±2.1)%, and OVCAR8 cells in the G1 phase were increased by (70.4±3.0)%. Compared to SKOV3 cells the differences were significant (P<0.01). CONCLUSION: Recombinant human Mullerian inhibiting substance suppresses the growth of MISIIR-positive ovarian cancer cells by inducing apoptosis and cell cycle arrest. We predict that rhMIS might be a new target to treat human ovarian malignancies.     

Role of bcl-2 gene expression in inhabition of hepatocellular carcinoma by genistein in nude mice

AIM:To probe into the role of 1, 4, 5 - trisphosphate inositol (IP3) and bcl-2 gene expression in inhibiting hepatocellular carcinoma of nude mice by genistein. METHODS:Animals with hepatocellular carcinoma were treated with genistein 1 mg·kg-1·d-1 (ip) for 3 weeks. The volume and weight of tumaor were measured. IP3, bcl-2 mRNA, Bcl-2 protein were assayed by IP3-［3H］ Birtrak assay, RT-PCR, Western blotting, respectively. RESULTS:The tumor volume and weight of animals treated with genistein were lower than those in control (42.7mm3±27.8mm3 vs 52.3mm3±26.5mm3, 42.7mg±27.8 mg vs 91.3mg±31.4 mg). IP3 content was lower than that in control ［(13.4±1.4)nmol/g protein vs (35.3±6.6)nmol/g protein］. bcl-2 mRNA expression was lower in group treated with genistein than that in control (RI which was the gray degree multiply area of bcl-2 / the gray degree multiply area of β-actin 0.48±0.02 vs 0.56±0.15). Bcl-2 protein expression was lower in group treated with genistein than that in control (RI 1.69±0.52 vs 1.37±0.48). CONCLUSION:Genistein inhibits growth of transplanted hepatocellular carcinoma in nude mouse liver by reducing IP3 production and down-regulating bcl-2 gene expression.     

Effect of CHOP/GADD153 on cardiomyocyte apoptosis induced by angiotensin Ⅱ in vitro

AIM: To investigate the relationship between alteration of CHOP/GADD153 protein expression and cardiomyocyte apoptosis induced by angiotensin Ⅱ (AngⅡ),and inhibitory effects of CHOP/GADD153 antisense oligodeoxynucleotide (anti-ODN) on apoptosis in vitro.METHODS: Cultured neonatal cardiomyocytes were exposed to AngⅡ with or without preincubation of CHOP/GADD153 anti-ODN.Variability of myocytes was measured by MTT assay,the lactate dehydrogenase (LDH) release was also detected,the percentage of annexin V positive myocytes was monitored by flow cytometry as apoptosis rate,CHOP/GADD153,Bcl-2 and Bax expressions were determined by Western blotting.RESULTS: Compared with control group,the expression of CHOP/GADD153 was obviously increased from (0.20±0.02 to 0.75±0.06) in AngⅡ group (P＜0.01).The variability of myocytes was obviously decreased from (100.00%±0.00% to 66.32%±7.16%,P＜0.05).The LDH release was significantly increased from (20.23 U/L±2.83 U/L to 79.36 U/L±5.69 U/L,P＜0.05) and apoptosis rate was significantly increased from (3.33%±0.28% to 16.62%±2.09%,P＜0.05).Bcl-2 expression was obviously decreased from (0.73±0.05 to 0.44±0.05,P＜0.01).Bax expression was obviously increased from (0.69±0.08 to 0.90±0.10,P＜0.01) and Bax/Bcl-2 ratio was obviously increased from (0.93±0.09 to 2.00±0.22,P＜0.01).Preincubation of CHOP/GADD153 anti-ODN in the medium significantly reversed above changes except the expression of Bax,but the Bax/Bcl-2 ratio was lower than that in AngⅡ group significantly (P＜0.01).These effects were not observed in mis-ODN group and there was no significant difference between lipofectin and control group.CONCLUSION: The increased expression of CHOP/GADD153 protein may be one of the mechanisms of AngⅡ-induced cardiomyocyte apoptosis,and CHOP/GADD153 anti-ODN can inhibit the apoptosis.     

早熟大粒无核葡萄新品种‘超级无核’

 ‘超级无核’葡萄系从美国引进葡萄新品种‘Superior Seedless’优选单株培育出的优良品种。无核、大粒、早熟、优质、早实、丰产、生长势强健、耐病、耐不利栽培条件, 是适合高温、高湿、少日照地区栽培的无核葡萄新品种。     

Effects of EBV infection on growth and apoptosis of nasopharyngeal carcinoma cell line

AIM: To explore the effect of EBV infection on growth and apoptosis of nasopharyngeal carcinoma(NPC) cell line.METHODS: NPC cell line CNE1 was directly infected by Epstein Barr virus (EBV). The expression of EBV-latent membrane protein 1 (EBV-LMP1) and bcl-2 were detected by immunohistochemistry method (LSAB). The growth of NPC cells was identified by MTT method. Apoptotic carcinoma cells were detected by flow cytometry analysis and the terminal deoxynucletidyl transferase-medicated dUTP-biotin nick end labeling (TUNEL) methods. RESULTS: EBV-LMP1 was positive in CNE1 infected by EBV(E-CNE1). Compared with CEN1, the growth of E-CNE1 apparently increased (P＜0.01). No apoptotic carcinoma cells were detected and bcl-2 postive cells were 2%~3% respectively in 2 kinds of NPC cells.CONCLUSION: Growth of NPC cells is enhanced by EBV infection and EBV-LMP1 expression, but no influence on expression of bcl-2 and apoptosis of NPC cells.     

Effects of homocysteine on number and activity of endothelial progenitor cells from peripheral blood

AIM: To investigate whether homocysteine (Hcy) has influences on endothelial progenitor cell (EPCs) number and activity from peripheral blood. METHODS: Total mononuclear cells (MNCs) were plated on fibronectin-coated culture dishes and cultured for 7 days, and then attached cells were stimulated with Hcy or vehicle control for 6 h, 12 h, 24 h and 48 h. The adhesion, proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed, respectively. RESULTS: Incubation of isolated human MNCs with Hcy dose and time-dependently decreased the number of EPCs with maximum at 200 μmol/L for 24 hours (35.7±6.7 vs 62.5±10.6， P＜0.01). In addition, Hcy impaired EPC proliferative (0.531±0.061 vs 0.328±0.055， P＜0.05), migratory (26.3±6.4 vs 6.4±3.7， P＜0.01), adhesive (33.1±8.1 vs 17.4±7.5， P＜0.01) and vasculogenesis capacity (25.4±9.1 vs 10.4±4.7， P＜0.01) in a dose and time-dependent manner. CONCLUSION: It is suggested that Hcy may result in the reduction of EPCs and decrease EPC functional activity.     

Effects of hypoxia on the expression of heparanase and invasiveness of SKOV3 ovarian cancer cell line

AIM: To evaluate the effects of hypoxia on the expression of heparanase and the invasiveness of SKOV3 ovarial carcinoma cell line. METHODS: SKOV3 cells were incubated at either normoxia (37 ℃, 5％CO2, 21％O2) or hypoxia (37 ℃, 5％CO2, 1％O2) condition for 12 h, 24 h and 36 h. RT-PCR and Western blotting were used to detect mRNA and the protein expressions of heparanase under different conditions. Cell invasiveness was measured by matrigel invasion assay. RESULTS: Compared to normoxia group, the heparanase mRNA expression level in hypoxia group was increased and in 12 h hypoxia group was the highest. The heparanase protein expression in hypoxia group was also significantly increased (P<0.01) and the expression of heparanase in hypoxia group was also different (P<0.05). Compared to normoxia group, the level of cell invasion was markedly increased in 12 h, 24 h and 36 h groups (P<0.05). During 12-36 h hypoxia period, the increase in hypoxia-induced invasiveness in SKOV3 cell line showed a time-dependent manner (P<0.05). Meanwhile, there was a positive correlation between the expression of HPA and the invasiveness of SKOV3 cells (r=0.8530, P<0.05). CONCLUSION: Invasion of SKOV3 cells in hypoxia condition correlates with heparanase level. Hypoxia plays an important role in the augmentation of the heparanase expression and the invasiveness of human ovarian cancer.     

Effects of tumor necrosis factor alpha on proliferation and apoptosis of HSCs

AIM:To explore the role of tumor necrosis factor alpha(TNF-α) in the pathogenesis of liver fibrosis.METHODS:The proliferation and apoptosis of hepatic stellate cells (HSCs) in vitro were detected with flow cytometry, electron microscopy and TUNEL.RESULTS:The flow cytometry analysis showed that the cell proliferation index (PI) in the TNF-α(0.5 μg/L, 2.0 μg/L, 8.0 μg/L) groups was evidently lower than that in the control group (P＜0.05). In the cell cycle distribution, the portion of G₀/G₁ phase in the TNF-α groups was significantly higher than that in the control group(P＜0.05), but the portion of S phase in the TNF-α groups was evidently lower than that in the control group(P＜0.05). These indicated that TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. The apoptotic rate in the TNF-α groups was evidently higher than that in the control group(P＜0.05). The gene expression of bcl-2 and bax was also detected with flow cytometry. The expression of bcl-2 in the TNF-α groups was evidently lower than that in the control group(P＜0.05), but the expression of bax in the TNF-α groups was significantly higher than that in the control group(P＜0.05). TUNEL analysis showed the apoptotic rate of HSCs in the TNF-α(2.0 μg/L) group was 18.7%±2.5% compared with 5.3%±1.2% in the control group(P＜0.05).CONCLUSIONS:TNF-α interfered with HSCs entrance into S phase from G₀/G₁ phase whereupon the proliferation of HSCs was inhibited. TNF-α down-regulated bcl-2 gene expression and up-regulated bax gene expression whereupon the apoptosis of HSCs was induced.     

Effect of γ radioactive stent on proliferation and apoptosis of smooth muscle cells

AIM: To observe effect of γ radioactive ［103Pd］ stent on the proliferation and apoptosis of smooth muscle cells, the mechanism of radioactive stent preventing in-stent restenosis was explored. METHODS: Fifty male New Zealand rabbits were randomized into stent group and ［103Pd］ stent group. Control group was set up. The materials were harvested on 3, 7, 14, 28, 56 days after operation and the following investigation were carried out, including pathomorphology, immunohistochemistry, apoptosis (TUNEL) and in situ hybridization studies.RESULTS: ① The severity of the stenosis in ［103Pd］ stent group was less severe than that in stent group. It was most obvious on 56 th day (P<0.01). ② The expression of proliferating cell nuclear antigen(PCNA) of ［103Pd］ stent group was lower than that in stent group on 3 to 28 days. It was most obvious on 7th day, 16.35%±0.79% vs 24.36±0.55% (P<0.01). ③ TUNEL method showed that the ［103Pd］ stent group had much more apoptosis of VSMC than that in stent group. The highest rate of apoptosis appeared on day 7, 14.72%±0.53% vs 12.42%±1.13% (P<0.01). ④ By calculating the ratio of PCNA/apoptosis (P〖KG*6〗∶〖KG-*2〗A), a much lower ratio was seen in ［103Pd］-stent groups than that in stent group at 3 to 28 days. There was significant statistic difference (P<0.05). ⑤ For bcl-2/bax ratio, the result in ［103Pd］-stent group was lower than that in stent group at 3 to 28 days. There was significant statistic difference (P<0.05).CONCLUSION: γ radioactive stent inhibits the proliferation and accelerates apoptosis of injured media vascular smooth muscle cells. It decreases the ratio of proliferation to apoptosis and relieves the severity of restenosis.     

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.     

Quercetin regulates Fas expression and induces apoptosis in hepatocellular carcinoma HepG2 cells

AIM: To investigate the role of 1, 4, 5- trisphosphate inositol (IP3) and Fas gene expression in apoptosis of HepG2 cells induced by quercetin. METHODS: HepG2 cells were treated with quercetin at different concentrations (including 20, 40, 60, 80 μmol/ L) for 72 h and treated with 60 μmol/ L quercetin for 6 h, 12 h, 24 h, 48 h and 72 h. IP3, Fas mRNA, Fas protein and apoptosis rate were assayed by IP3 - ［3H］ Birtrak assay, RT-PCR, Western blotting and flow cytometry, respectively. RESULTS: When HepG2 cells were incubated with different concentrations of quercetin for 72 h, the IP3 content was lower than those in control. Fas mRNA expression, Fas protein expression and the apoptosis rate were higher than those in control. When HepG2 cells were incubated with quercetin for 6 h, 12 h, 24 h, 48 h, 72 h, the IP3 contents were lower than those in control incubated with 60 μmol/L quercetin for 12 h. Fas mRNA expression was higher than that in control incubated with 60 μmol/L quercetin for 12 h . Fas protein expression was higher than that in control. The apoptosis rate was significantly higher than that in control incubated with 60 μmol/L quercetin for 24 h (P<0.01). CONCLUSION: Quercetin induces apoptosis of HepG2 cells by reducing IP3 production and upregulating Fas gene expression.     

Effects of αvβ6 integrin-mediated cell adhesion on 5-fluorouracil induced apoptosis in human colon carcinoma cell lines

AIM: To investigate the effects of αvβ6 integrin-mediated cell adhesion on 5-fluorouracil (5-FU) induced apoptosis in colon carcinoma cell lines.METHODS: The expression of the αvβ6 integrin in colon carcinoma cell lines HT-29 and WiDr cells was analyzed by flow cytometry. The apoptosis induced by 5-FU and the effects of αvβ6 integrin-mediated cell adhesion on 5-FU induced cell apoptosis were measured by enzyme-linked immunosorbent assay (ELISA) method and acridine orange-ethidium bromide (AO-EB) double fluorescent dye staining.RESULTS: Both the colon carcinoma cell lines HT-29 and WiDr cells expressed the αvβ6 integrin. The percentages of HT-29 and WiDr cells expression were 80.82% and 82.96%. 5-FU induced the apoptosis of colon carcinoma cell lines HT-29 and WiDr. The result of ELISA method displayed that enrichment factor (EF) of HT-29 and WiDr cells planted on fibronectin (FN)-ligand of αvβ6 integrin was lower significantly than the EF of HT-29 and WiDr cells planted on non-integrin ligand polylisin (1.11±0.04 vs 3.68±0.03, 1.09±0.02 vs 3.72±0.02, P<0.01) after cultured in medium containing 20 mg/L 5-FU for 48 h. When HT-29 and WiDr cells preincubated with αvβ6 integrin blocking antibody were planted on FN again, the EF of HT-29 and WiDr cells was higher significantly than those directly planted on FN without being blocked by αvβ6 integrin antibody (2.12±0.04 vs 1.11±0.04, 2.14±0.03 vs 1.09±0.02, P<0.01). The AO-EB double fluorescent dye staining displayed that the apoptosis percents of HT-29 and WiDr cells planting on FN were (5.6±1.1)% and (5.3±0.7)%, which were lower significantly than those planting on polylisin (37.0±1.4)%, (38.5±0.9)%, P<0.01. When HT-29 and WiDr cells preincubated with αvβ6 integrin blocking antibody were planted on FN again the percents of HT-29 and WiDr cells apoptosis were (19.5±1.2)% and (20.0±0.7)%, which increased significantly compared with those directly planting on FN without being blocked by αvβ6 integrin antibody (P<0.01). CONCLUSION: Colon carcinoma cell lines HT-29 and WiDr cells expressed αvβ6 integrin. The cell adhesion with FN mediated by αvβ6 integrin inhibits 5-FU-induced colon carcinoma cell apoptosis. The results suggest that cell adhesion may enhance drug resistance in colon carcinoma cell lines through inhibiting the cell apoptosis.     

Effect of atorvastatin on blood pressure in spontaneously hypertensive rats

AIM: To explore the mechanisms underlying the effect of atorvastatin on blood pressure in spontaneously hypertensive rats (SHR). METHODS: The effects of atorvastatin on plasma endothelin-1, aortic nitric oxide synthase, aortic smooth muscle cell (ASMC) apoptosis and p27 expression in SHR were evaluated. 12 eight-week-old SHR were randomized into atorvastatin group (ATV, n=6) and SHR group (n=6). 6 age-matched normotensive Wistar-Kyoto rats (WKY) were served as controls. 50 mg·kg-1·d-1 of atorvastatin was administered to ATV by gavage for 10 weeks. Serum cholesterol and triglycerides were measured, and systolic blood pressure of caudal artery was examined. Plasma endothelin-1 and nitric oxide synthase activity of aortic tissue were measured. ASMC apoptosis rate was detected by TUNEL technique, and positive expression rate of P27 in ASMC was analyzed. RESULTS: After 10 weeks, systolic blood pressure in ATV was significantly lower than that in SHR ［(134.17±3.60）mmHg vs （173.33±3.78）mmHg, P<0.01］. Compared with SHR, serum cholesterol and triglycerides were significantly lower (P<0.01, P<0.01) in ATV. Additionally，atorvastatin significantly decreased plasma endothelin-1 ［(130.04±40.07）ng/L vs （196.74±59.69）ng/L, P<0.05］ and increased nitric oxide synthase activity in aortic tissue ［(0.189±0.040）kU/g protein vs (0.124±0.057)kU/g protein, P<0.01］, compared with SHR. ASMC apoptosis rate was higher in ATV than that in SHR (16.94%±3.08% vs 9.01%±2.36%, P<0.01). Compared with WKY, positive expression rate of p27 in ASMC from ATV was higher (33.02%±5.01% vs 24.25%±4.41%, P<0.05), whereas that was lower in SHR (16.08%±7.09% vs 24.25%±4.41%, P<0.01). CONCLUSION: Atorvastatin may reduce the plasma endothelin-1， up-regulate nitric oxide synthase activity and ASMC P27 expression and facilitate ASMC apoptosis，which may effectively reduce blood pressure in SHR.     

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.     

Knockdown of survivin expression using siRNA inhibits the growth of PC-3M cells

AIM: To study the inhibitory effects of survivin siRNAs on the growth of PC-3M cells. METHODS: Two pairs of DNA template coding siRNA against survivin were synthesized to construct two recombinant plasmids, pSi-sur1 and pSi-sur2. The two recombinants and the two controls, lipofectin and vacant plasmid were transfected into PC-3M cells. The expressions of survivin mRNA and protein were detected respectively by RT-PCR and Western blotting. Proliferation abilities were measured by MTT, and the cell cycle and apoptosis were assayed by FCM. RESULTS: After 72 h of transfection, the level of cell survivin mRNA in the two siRNA groups was 48%±6% (n=3) and 30%±5% (n=3) of that in lipofectin group, and expression of survivin protein were 38%±4% (n=3) and 36%±4% (n=3) respectively of that in lipofectin control. The proliferation rate of cells in pSi-sur1 and pSi-sur2 groups was also inhibited according to MTT, about 44.20%±2.08% (n=3) and 39.20%±1.93% (n=3) of that in lipofectin group. Cell numbers of G1 phase in two siRNA groups were significantly higher than that in two controls, while cells of G2 phase and S phase were much lower. Cell apoptosis was found in both siRNA groups. CONCLUSION: The two survivin siRNA significantly inhibit the expression of survivin in mRNA and protein levels, arrest the cell cycle in G1 phase, and suppress the growth of PC-3M cells and induce apoptosis in vitro.     

Inhibitory effect of hypoxia preconditioning on hypoxia/reoxygenation-induced apoptosis in cardiomyocytes

AIM: To study the role of hypoxia preconditioning (HP) in hypoxia-reoxygenation (HR)-induced apoptosis in neonatal rat cardiomyocytes and the possible mechanisms. METHODS: Cultured neonatal rat cardiomyocytes were divided into three groups: normal group, HP+H/R group and H/R group. Acridine orange (AO) staining was performed to detect morphological changes of apoptotic cells. Apoptosis rates of cardiomyocytes were detected by flow cytometry. Colorimetric assay was used to detect caspase-3 activity. Expression of Bcl-2 protein was detected by immunohistochemistry combined with computer image analysis. RESULTS: Apoptotic cells were detected by AO staining after hypoxia of 6 h followed by 3 h-reoxygenation. The hypodiploid apoptotic peak was detected by flow cytometry with the apoptotic rates of (29.7±5.4)%. A significantly reduced apoptotic rates of (7.8±1.3)% was detected in HP group（P＜0.01）. The caspase-3 relative activity of cardiomyocytes induced by H/R was 5.9±0.8, significantly higher than that of control group. HP markedly reduced caspase-3 relative activity to 2.6±0.5 in contrast with H/R group （P＜0.01）. Bcl-2 protein was positive in normal cardiomyocytes with an A value of 119.4±7.1. The A value of H/R group was 99.6±5.0, significantly lower than that in normal group （P＜0.01）. The A value of HP+H/R group was 126.5±6.2, significantly higher than that in H/R group（P＜0.01）. CONCLUSION: HP inhibits H/R-induced apoptosis of cardiomyocytes by improving the expression of Bcl-2 and reducing caspase-3 activity.