Cholestane-3β, 5α, 6β-triol induces apoptosis of malignant glioma cells

AIM：To investigate the effect of cholestane-3β, 5α, 6β-triol (Triol) on apoptosis of malignant glioma cells. METHODS：C6 cells and A172 cells were incubated with Triol at different concentrations for different time durations. MTT assay was used to detect the cell viability. Hoechst 3f3342 staining and TUNEL assay were used to analyze the cell apoptosis. The caspase activity was measured. The expression of apoptosis-related proteins, Bcl-2 family members, was determined by Western blotting. RESULTS：Triol decreased the cell viability of C6 and A172 cells in a dose- and time-dependent manner and the IC50 values were (17.8±0.6)μmol/L and (20.6±0.2) μmol/L, respectively. Visible nuclei with apoptotic characteristics, significant increase in TUNEL-positive cells, and the activation of apoptotic execution enzyme caspase-3 indicated that cell apoptosis was induced by Triol in both cell lines. After C6 cells were exposed to Triol for 12 h, 24 h and 48 h, the activity of caspase-8 in extrinsic apoptotic pathway and caspase-9 in intrinsic apoptotic pathway was increased time-dependently. Meanwhile, the levels of anti-apoptotic proteins, Bcl-2 and Bcl-xL, was down-regulated, while pro-apoptotic protein Bak was up-regulated in a time-dependent manner. CONCLUSION：Triol induces apoptosis of malignant glioma cells by activating intrinsic and extrinsic apoptotic pathways, and Bcl-2 family members are involved in Triol-induced apoptosis.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

Toll-like receptor 3 signaling pathway plays an important role in cardiomyocyte apoptosis and inflammatory response in human viral myocarditis

AIM：To investigate the role of the signaling pathway mediated by Toll-like receptor 3 (TLR3) in cardiomyocyte apoptosis and inflammatory response in human viral myocarditis (VMC). METHODS：The expression of TLR3, TIR domain-containing adaptor inducing IFN-β(TRIF), NF-κB and casepase-3 in myocardial tissues was examined by the method of immunohistochemistry with SP staining, and the apoptosis of cardiomyocytes was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The differences between VMC and control groups were analyzed. RESULTS： Compared with control group, the expression of TLR3, TRIF, NF-κB and casepase-3, and the apoptotic index in VMC group increased remarkably (P<0.05). The positive correlations between TLR3 and TRIF, between TLR3 and NF-κB, between TRIF and NF-κB, and between NF-κB and caspase-3 were observed. The change of the apoptotic index was in accordance with that of caspase-3. CONCLUSION：Inflammatory response and apoptosis mediated by TLR3 play an important role in the genesis and development of viral myocarditis．     

Expression of Smoothened and its role in endothelial cells in synovial tissues of rheumatoid arthritis

AIM: To investigate the expression of Sonic Hedgehog signaling pathway-associated factor Smoothened (Smo) and its role in endothelial cells in synovial tissue of active rheumatoid arthritis (RA). METHODS: Smo expression in synovial tissue from 4 RA patients and 4 patients with trauma or meniscal injury (without arthritis, used for control) was detected by the method of immunohistochemistry. Human umbilical vein endothelial cell line EA.hy926 was used as the model of synovial vascular endothelial cells. The expression of Smo was detected by Western blotting after TNF-α treatment. The small interfering RNA (siRNA) specifically targeting Smo gene was synthesized and transfected into EA.hy926 cells. The interference efficiency of the siRNA on the production of Smo protein was determined by Western blotting. The cells were treated with TNF-α and actinomycin D (ActD) 24 h after siRNA transfection. The cell survival rate was determined by CCK-8 assay and the apoptotic rate was examined by flow cytometry. RESULTS: Smo was highly expressed in synovial tissue from active RA patients, especially in endothelial cells as compared with control group. TNF-α significantly increased the protein expression of Smo in EA.hy926 cells. EA.hy926 cells transfected with Smo-siRNA showed a significant decrease in the cell viability with the cell survival rate of (24.30±0.45)% and the apoptotic rate of (48.00±1.96)%, as compared with those in negative control group ［(36.86±0.62）% and （31.70±0.82）%, respectively］. CONCLUSION: Smo may play a role in the regulation of apoptosis in endothelial cells in RA synovium.     

Effects of shRNA targeting COL1A1 gene on proliferation and apoptosis of human breast cancer cell line MDA-MB-231

AIM: To investigate the effects of shRNA-mediated collagen type I alpha 1 (COL1A1) gene silencing on the proliferation and apoptosis of human breast cancer cell line MDA-MB-231. METHODS: The specific recombinant vector pSilencer2.1-U6-COL1A1 was transiently transfected into human breast cancer cell line MDA-MB-231 with lipofectamine. RT-PCR and Western blotting were performed to detect the expression levels of COL1A1. MTT assay was employed to evaluate the effect of COL1A1 gene silencing on the cell proliferation. Flow cytometry was used to determine the apoptosis and cell cycle of transfected cells. The morphological characteristics of apoptosis were observed by Hoechst 33258 staining. RESULTS: Compared with mock group and scrambled group, the mRNA and protein levels of COL1A1 were reduced by pshRNA-COL1A1 transfection (P<0.05). The proliferation of MDA-MB-231 cells treated in shRNA-COL1A1 was significantly inhibited in a time-dependent way. The percentages of G₀/G₁ phase cells and early apoptotic rate were significantly higher in pshRNA-COL1A1 group than those in mock and scrambled group (P<0.05). The changes of apoptotic morphology such as cell shrinkage and nuclear condensation were also observed by staining with Hoechst 33258 under fluorescence microscope. CONCLUSION: Transfection of eukaryotic expression vector pshRNA-COL1A1 effectively inhibits the proliferation, induces apoptosis and arrests MDA-MB-231 cells in G0/G1 phase.     

Effects of curcumin on actinomycin D/TNF-α-induced injury in PC12 cells and rat hippocampal neurons

AIM: To study the possible mechanism of curcumin on actinomycin D (ActD)/tumor necrosis factor α (TNF-α)-induced injury in PC12 cells and rat hippocampal neurons. METHODS: PC12 cells were divided into control group, TNF-α group, ActD group, curcumin group, ActD/TNF-α group and curcumin+ActD/TNF-α group. The cells were cultured for 24 h. Inverted fluorescence microscopy was used to observe the morphological changes of the cells in each group. Annexin V/PI double staining was applied to analyze the apoptosis of PC12 cells. The level of intracellular Ca²⁺ was detected by Fluo-3 AM staining. Rat hippocampal slices were prepared and divided into the same groups as the PC12 cells. Extracellular microelectrode recording technique was used to observe and calculate the changes of long-term potentiation (LTP) in different groups. RESULTS: Apoptosis of PC12 cells was induced by ActD/TNF-α. Curcumin protected the PC12 cells from ActD/TNF-α-induced apoptosis (P<0.05). ActD/TNF-α increased the intracellular Ca²⁺ concentration. Curcumin significantly reduced ActD/TNF-α-induced apoptosis by decreasing the intracellular Ca²⁺ concentration (P<0.05), inversed the effect of ActD/TNF-α on LTP in hippocampal slices, and improved the synaptic plasticity (P<0.05). CONCLUSION: Curcumin protects ActD/TNF-α-induced neuronal damage by depressing the intracellular Ca²⁺ concentration and maintaining the homostasis of intracellular calcium.     

How Stallion Sperm Age In Vitro? Scenario for Preservation Technologies

Recent findings on the molecular damage occurring in the stallion spermatozoa are reviewed. Mechanisms leading to cell death or survival are briefly overviewed, and recent discoveries on molecular pathways leading to sperm death and sublethal damage are discussed. Increasing the understanding of this particular area may disclose clues to develop new strategies to improve current sperm conservation methods.     

Proctective effect of microRNA-30a on regulating beclin-1 expression in hypoxia-reoxygenated neonatal rat cardiomyocytes

AIM: To explore the potential mechanism of microRNA-30a (miR-30a) overexpression in neonatal rat cardiomyocytes during hypoxia/reoxygenation (H/R). METHODS: The miR-30a overexpression was induced in primary neonatal rat cardiomyocytes by lentivirus transfection. The cardiomyocytes were divided into 5 groups: normal group, H/R group, LV-GFP+H/R group, LV-GFP-miR-30a+H/R group and 3-methyladenine(3-MA)+H/R group. The expression level of miR-30a after lentivirus transfection and H/R was determined by real-time PCR, while the protein levels of LC3 and Beclin-1 after H/R and lentivirus transfection were detected by Western blotting. The cardiomyocyte death after H/R were measured by TUNEL and PI staining. RESULTS: Compared with LV-GFP group, significant down-regulation of Beclin-1 protein level was observed in cardiomyocytes with miR-30a overexpression, while the protein levels of Beclin-1 and LC3 in the cardiomyocytes with miR-30a overexpression were down-regulated after H/R, and apoptosis of these cells were significantly decreased after H/R. CONCLUSION: The protein level of Beclin-1 is down-regulated in cardiomyocytes with miR-30a overexpression. Inhibition of autophagy decreases the cardiomyocyte death after H/R.     

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.     

Salinomycin inhibited proliferation and induced apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP

AIM: To investigate the effect of salinomycin on the proliferation and apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP. METHODS: The inhibitory effect of salinomycin on the growth of A549/DDP cells was tested by MTT method in vitro . The apoptosis and mitochondrial membrane potential (ΔΨ_m) of A549/DDP cells were assayed by flow cytometry. The activity of caspase-3, 8 and 9 was determined by the method of colorimetry. The levels of cytochrome C, Bcl- 2, Bax, β-catenin, and phosphorylated low-density lipoprotein receptor-related protein 6(p-LRP6) were measured by Western blotting. RESULTS: Salinomycin inhibited the growth of A549/DDP cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L decreased ΔΨ_m level, and increased reactive oxygen species (ROS), cytochrome C and cytosolic Ca²⁺ release in the cells. Salinomycin also increased the acti-vity of caspase-3, 8, and 9 in the cells, reduced the ratio of Bcl-2/Bax, and decreased the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin depresses the cell growth by inhibiting Wnt signaling, and induces the apoptosis of cisplatin-resistant human lung adenocarcinoma cell line A549/DDP via mitochondria-dependent and Bcl-2/Bax pathways.