Synergistic effect of decitabine and valproic acid on apoptosis and cell cycle arrest at G0/G1 phase in gastric cancer MGC-803 cells

AIM: To investigate the synergistic effect of decitabine (DCA) and valproic acid (VPA) on apoptosis and cell cycle arrest at G₀/G₁ phase in gastric cancer MGC-803 cells. METHODS: Gastric cancer MGC-803 cells were used in the study and divided into the following groups according to the treatment with different drugs for 72 h: DCA 1.5 μmol/L,DCA 3.0 μmol/L, VPA 1.5 mmol/L, DCA 1.5 μmol/L+VPA 1.5 mmol/L and DCA 3.0 μmol/L+VPA 1.5 mmol/L. The early and late apoptotic rates were detected by annexin V and PI staining. The cell cycle was also determined by flow cytometry. The relative nm23-H1 mRNA expression level was measured by real-time quantitative PCR. RESULTS: The apoptotic rates in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (early: 33.58%±3.88%; late: 31.52%±4.20%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (early: 42.61%±4.23%; late: 38.01%±3.86%), the percentages of the cells in G₀/G₁ phase in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (61.55%±2.38%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (66.75%±2.48%), and the relative nm23-H1 mRNA expression levels in VPA 1.5 mmol/L +DCA 1.5 μmol/L group (1.84±0.46) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (3.02±0.36) were all significantly higher than those in the corresponding concentrations of single drug treatment groups (P<0.01). CONCLUSION: Synergistic effect of VPA and DCA on apoptosis and cell cycle arrest in gastric cancer MGC-803 cells is possibly via inactivation of nm23-H1 gene expression.     

Photoactivation promotes curcumin to induce apoptosis of human gastric cancer MGC-803 cells

AIM: To investigate the effect of photoactivated curcumin on apoptosis of human gastric cancer MGC-803 cells. METHODS: The effect of photoactivated curcumin on the growth inhibitory rate of gastric cancer MGC-803 cells was detected by MTT assay. The changes of nuclear morphology were observed under optical microscope with Hoechst 33258 fluorescent staining. The apoptotic rate, mitochondrial membrane potential, intracellular reactive oxygen species and Ca²⁺ level was determined by flow cytometry. The activity of caspase-3, caspase-8 and caspase-9 was detected by colorimetry. The protein levels of cytochrome C, Bcl-2, Bax and heat-shock protein 70 (HSP70) were analyzed by Western blotting. RESULTS: The growth inhibitory rate of MGC-803 cells treated with curcumin at concentration of 5.0 μmol/L was (29.74±2.30)%.Some apoptotic cells were observed under optical microscope, and the apoptotic rate was (12.54±1.75)%. The growth inhibitory rate of MGC-803 cells treated with photoactivated curcumin was (44.93±3.61)%.Significant morphological changes in the nucleus, such as chromatin condensation and apoptotic body formation, were observed under light microscope, and the apoptotic rate was (26.58±2.67)%. The cell cycle was arrested at G₀/G₁ phase. Compared with curcumin group, significant reduction in mitochondrial membrane potential,significant increase in cytochrome C, intracellular reactive oxygen species, Ca²⁺ level and the activity of caspase-3, caspase-8 and caspase-9 were observed in photoactivated curcumin group (P<0.01). Photoactivated curcumin also significantly inhibited the protein expression of Bcl-2 and HSP70 in the cells. CONCLUSION: Photoactivated curcumin enhances the apoptosis of gastric cancer MGC-803 cells by inhibiting Bcl-2 expression and promoting the mitochondrial pathway.     

Salinomycin inhibits proliferation and induces apoptosis of Gleevec-resistant chronic myeloid leukemic cell line K562/Glv

AIM: To study the effect of salinomycin on inhibiting proliferation and inducing apoptosis of Gleevec-resistant chronic myeloid leukemia cell line K562/Glv. METHODS: The inhibitory effect of salinomycin on the growth of K562/Glv cells was detected by CCK-8 assay in vitro. Flow cytometry was used to observe apoptosis, mitochondria membrane potential (ΔΨ_m), reactive oxygen species (ROS) and the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) in K562/Glv cells. The activity of caspase-3, -8 and -9 was measured 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 determined by Western blotting. RESULTS: Salinomycin inhibited the growth of K562/Glv cells in a dose-dependent manner. Salinomycin at concentration of 0.2 μmol/L inhibited the growth of the cells with the inhibitory rate of (36.70±2.31)%. The cell apoptotic rate was (19.66±2.23)%. Salinomycin at concentration of 0.2 μmol/L decreased the level of ΔΨ_m, and increased the levels of ROS, cytochrome C and[Ca²⁺]_i in the cells. Salinomycin also increased the activity of caspase-3, -8 and -9 in the cells, reduced the ratio of Bcl-2/Bax, and attenuated the levels of β-catenin and p-LRP6. CONCLUSION: Salinomycin induces the apoptosis of Gleevec-resistant myeloid leukemia cell line K562/Glv via Bcl-2/Bax and mitochondria-dependent pathways, and inhibits the cell growth through Wnt signal pathway.     

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.     

Lovastatin-induced G1 phase synchronization in human endometrial cancer JEC cells

AIM: To investigate the method of inducing G₁ phase synchronization in human endometrial cancer JEC Cells by lovastatin and the cell cycle progress of JEC cells after desynchronization. METHODS: The doubling time of JEC cells was detected by Cell Counting Kit-8 (CCK-8) assay. To determine the best lovastatin concentrations for G₁ synchronization, JEC cells were treated with lovastatin at concentrations of 10, 20, 30 and 40 μmol/L for 1× doubling time, and the cell cycle was detected using flow cytometry (FCM). To determine the best period of lovastatin treatment to achieve G₁ synchronization, JEC cells were treated with lovastatin at the best concentration for 0.5× to 2× doubling time, and the cell cycle was detected every 4 h using FCM. Furthermore, the cell cycle progress of JEC cells after desynchronization was also observed. RESULTS: The doubling time of JEC cells was almost 24 h. Treatment with lovastatin at the concentration of 20 μmol/L for 24 h achieved maximum G₁ arrest in JEC cells. Minimum G₁ phase and maximum S phase were observed after desynchronization for 16 h. CONCLUSION: Maximum G₁ synchronization of JEC cells is induced by lovastatin at the concentration of 20 μmol/L for 24 h. The JEC cells show minimum G₁ phase and maximum S phase after desynchronization for 16 h.     

Effect of Bmi-1 gene overexpression on proliferation of human gastric mucosal epithelial cell line GES-1

AIM: To investigate the effect of B lymphoma Moloney murine leukemia virus insertion region 1 ( Bmi-1 ) gene overexpression on the proliferation of a human normal gastric epithelial cell line GES-1. METHODS: The plasmid containing Bmi-1 gene or empty plasmid was transfected into GES-1 cells by retroviral mediation. The expression of Bmi-1 at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) analysis and Western blotting, respectively. The effect of Bmi-1 gene overexpression on the cell cycle of GES-1 cells was evaluated by flow cytometry. The proliferation of the stably transfected cells was measured by Cell Counting Kit-8. RESULTS: The results of qRT-PCR analysis and Western blotting demonstrated that stably transfected cell line was successfully established. The results of flow cytometry analysis showed that overexpression of Bmi-1 reduced the G₀/G₁ phase, arrested the cells in G₂/M phase and S phase. The growth curve showed that overexpression of Bmi-1 resulted in increased growth speed. CONCLUSION: Increase in Bmi-1 gene expression regulates the cell cycle and promotes the proliferation of GES-1 cells.     

Role of ERK1/2-STAT3 pathway in adaptive cytoprotection induced by H2O2 preconditioning

AIM:To explore the role of extracellular signal-regulated kinases ERK1/2-STAT3 pathway in adaptive cytoprotection induced by H₂O₂ preconditioning in PC12 cells. METHODS:In PC12 cells, the experimental model of cytoprotection by H₂O₂ preconditioning against oxidative stress-induced injury was set up. The morphological changes in the apoptotic cells were observed by using of chromatin dye Hoechst 33258. The percent of apoptotic cells was determined by flow cytometry (FCM) with propidium iodide staining. The levels of p-ERK1/2 and p-STAT3 expression were detected by Western blotting assay. RESULTS:Preconditioning with H₂O₂ at concentration of 100 μmol/L for 90 min obviously inhibited apoptosis induced by 300 μmol/L H₂O₂, and both ERK1/2 and STAT3 were activated. UO126 (10 μmol/L, an inhibitor of ERK1/2) or AG-490 (10μmol/L, an inhibitor of JAK2) significantly blocked the cytoprotection effect of H₂O₂ preconditioning. Moreover, UO126 (10 μmol/L) also markedly inhibited the up-regulation of p-STAT3 expression by H₂O₂ preconditioning. CONCLUSION:H₂O₂ preconditioning activates ERK1/2-STAT3 signal pathway, which may be one of the mechanisms underlying H₂O₂ preconditioning-induced cytoprotection.     

Protective effect of sodium selenite on human keratinocytes from ultraviolet-B irradiation

AIM: To investigate the protective effect of sodium selenite (Na₂SeO₃) on human keratinocytes under ultraviolet-B (UVB) irradiation. METHODS: The cultured HaCaT cells were divided into 4 groups: (1) normal control group; (2) Na₂SeO₃ group: pretreated with Na₂SeO₃ at doses of 10 nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L and 1 μmol/L for 24 h; (3) UVB group: irradiated with UVB at doses of 300, 600 and 900 J/m² ; (4) Na₂SeO₃+UVB group: after pretreated with Na₂SeO₃ for 24 h, irradiated with UVB at doses of 300, 600 and 900 J/m² . The cell proliferation was detected by MTT assay. The apoptotic rates of HaCaT cells treated with UVB at dose of 300 J/m² were assessed by flow cytometry. RESULTS: Compared with normal control group, the cell proliferation activity in UVB group decreased significantly (P<0.05). The cell activity was inversely correlated with the irradiation intensity. No significant difference of the cell activity between Na₂SeO₃ group and normal control group was observed. The cell proliferation in Na₂SeO₃+UVB group was higher than that in UVB group significantly (P<0.05). Na₂SeO₃ at concentration of 100 nmol/L showed the strongest activity to promote cell proliferation. After 300 J/m² UVB irradiation, the apoptotic rate in Na₂SeO₃+UVB group decreased significantly (P<0.05) compared with UVB group. The inhibitory effect of Na₂SeO₃ at concentration of 100 nmol/L on apoptosis was the strongest.CONCLUSION: The damage of human keratinocytes by UVB irradiation is in a dose-dependent manner. The photoprotection performance of Na₂SeO₃ reduces the damage of human keratinocytes induced by UVB irradiation.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.     

Effect of microtubule-destabilizing protein stathmin gene silencing on proliferation and apoptosis of nasopharyngeal carcinoma cell line 5-8F

AIM: To investigate the effects of stathmin gene silencing on nasopharyngeal carcinoma cell line 5-8F. METHODS: Double-strand siRNA targeting to stathmin gene was obtained by chemical synthesis and annealing, and was sub-cloned into the vector pGenesil-1.1. The plasmid was introduced into 5-8F cells by liposome-mediated transfection. The gene expression of stathmin, and the proliferation, morphology and apoptosis of the cells were analyzed by Western blotting, MTT assay and flow cytometry. RESULTS: The cell suppression rate in stathmin gene silencing group was (53.01?1.12)%, significantly higher than that in transfection reagent group and in negative control group. The cell apoptotic rate in stathmin gene silencing group was (8.75?0.67)%, also significantly higher than that in transfection reagent group and in negative control group (P<0.05). CONCLUSION: Silencing of stathmin gene in nasopharyngeal carcinoma cells inhibits the cell proliferation and induces cell apoptosis.     

Leptin protects rat cardiomyocytes from H2O2-induced apoptosis

AIM: To investigate the effect of leptin on H₂O₂-induced apoptosis in rat cardiomyocytes (H9c2 cells) and the underlying mechanisms.METHODS: Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to determine H₂O₂-induced apoptosis in H9c2 cells in the absence or presence of leptin. The activities of caspase-3 and extracellular signal-regulated kinase (ERK) were examined by Western blotting.RESULTS: (1)Leptin significantly inhibited H₂O₂-induced apoptosis in H9c2 cells (P<0.01). This effect of leptin was opposed by MEK inhibitor PD98059. (2)H₂O₂ inhibited basal ERK activity. Leptin activated ERK and partially inhibited H₂O₂-induced caspase-3 activation.CONCLUSION: Leptin protects H9c2 cells from H₂O₂-induced apoptosis possibly by activating ERK.     

Expression of human leukocyte antigen E in hepatocellular carcinoma cell lines

AIM: To investigate the human leukcyte antigen E (HLA-E) expression in hepatocellular carcinoma cell lines. METHODS: The techniques of real-time PCR and Western blotting were used to study the HLA-E expression in the 5 cell lines of hepatocellular carcinoma and a fetal liver cell line at mRNA and protein levels. RESULTS: The results of real-time PCR showed that no statistical difference of HLA-E mRNA level between fetal liver cell L02 and other 4 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC and MHCC97) was observed, and almost absence of HLA-E mRNA expression in Hep3B2.1-7 cells was detected. However, the results of Western blotting showed that there was a significant statistical difference of HLA-E protein levels between L02 cells and the 5 cell lines of hepatocellular carcinoma (HepG2, Bel7402, PLC, MHCC97 and Hep3B2.1-7), and no HLA-E protein in Hep3B2.1-7 cells was detectable. CONCLUSION: Asynchronization of HLA-E expression between mRNA and protein levels was found in hepatocellular carcinoma cell lines.     

Senescence of endothelial cells and gene expression associated with apoptosis induced by angiotensinⅡ

AIM: To study the senescence of human umbilical vein endothelial cells (HUVECs) and Bcl-2, Bax gene expression associated with apoptosis induced by angiotensinⅡ (AngⅡ).METHODS: HUVECs were cultured in vitro and the cell viability was observed by methyl thiazolyl tetrazolium (MTT). HUVECs were intervened by AngⅡ and valsartan (AngⅡ type 1 receptor blocking) and divided into 3 groups: the control group, AngⅡ group (stimulated with AngⅡ10^-6mol/L for 48 h), valsartan group (valsartan was added to cells 1 h before 10^-6mol/L AngⅡ treatment). β-gal staining and cell cycle analysis were used to identify the cell aging status. Morphologic changes and percentage of apoptosis were assayed with Hoechst33258 under fluorescent microscope. The expressions of Bcl-2 and Bax, and the apoptosis-associated genes were detected by immunocytochemical staining, RT-PCR and Western blotting. RESULTS: The cell viability by AngⅡ-induced cells was (81.9%±4.1)%, the positive cell number of β-gal staining was significantly higher in AngⅡ-induced cells (80.10%±6.81)% than that in the control cells. The cell cycle was at G₀-G₁(91.36%±6.45)%, the apoptotic cells significantly increased (31.84±2.86)% under fluorescent microscope. In valsartan group, Bcl-2 mRNA and protein expression increased markedly (P<0.05), but Bax mRNA and protein expression decreased evidently (P<0.05) compared to those in the AngⅡ group.CONCLUSION: Cell apoptosis is possibly an important factor for endothelial cell senescence and vascular aging induced by AngⅡ. One of its molecular mechanisms might be associated with decreasing the expression level of Bcl-2 and increasing that of Bax, which regulate the imbalance between mRNA and protein expression of Bcl-2 and Bax. Valsartan improves endothelial cell aging.     

Acetyl-L-carnitine attenuates H2O2-induced oxidative damage of PC12 cells

AIM: To investigate the effect of acetyl-L-carnitine (ALC) on H₂O₂-induced oxidative damage in PC12 cells and its possible mechanism. METHODS: A moderate oxidative damage PC12 cell model was induced by exposure of the PC12 cells to H₂O₂. ALC at different concentrations (100, 200 and 400 μmol/L) was applied to the PC12 cells cultured in vitro, and CCK8 assay was used to detect the cell viability. The cells were divided into control group, H₂O₂ group, and low-ALC, medium-ALC and high-ALC groups. The apoptosis of the cells was analyzed by flow cytometry. The protein levels of Nrf2 and cleaved caspase-3 were determined by Western blot. The nuclear translocation of Nrf2 was observed by immunofluorescence staining. RESULTS: ALC at different concentrations (100, 200 and 400 μmol/L) significantly inhibited H₂O₂-induced PC12 cell apoptosis, and the medium concentration group had the best effect. Compared with H₂O₂ group, low, medium and high concentrations of ALC significantly increased the viability of the PC12 cells induced by H₂O₂, inhibit cell apoptosis (P<0.05), significantly down-regulated the protein level of cleaved caspase-3 (P<0.05), up-regulated the protein level of Nrf2 (P<0.05), and promoted the translocation of Nrf2 from the cytoplasm to the nucleus. CONCLUSION: Acetyl-L-carnitine attenuates H₂O₂-induced oxidative damage of PC12 cells, inhibits the apoptosis and increases the viability, which is related to the activation of Nrf2 signaling pathway.     

Effect of vitamin K3 on apoptosis induced by androgen-independent prostate cancer cell PC-3M

AIM: To study the effect of vitamin K₃ (VK₃) on the induction of apoptosis in androgen-independent prostate cancer cell PC-3M in vitro.METHODS: Cell viability was estimated by MTT assay. AO/EB staining was performed to detect apoptotic cells. Apoptosis and the changes of cell cycle were detected by flow cytometry. NAC was used to observe the effect of growth inhibition by VK₃. RT-PCR was used to confirm the changes in gene expression. Levels of intracellular peroxides were estimated by using an oxidation-sensitive fluorescent probe DCFH-DA. RESULTS: PC-3M cells growth was significantly inhibited by VK₃ (≥60 μmol/L, P<0.05). The inhibitory effect was time and dosage dependent. The result of AO/EB staining showed that apoptosis of PC-3M cells were induced by VK₃. A typical subdiploid peak before G₀/G₁ phase was observed after treated for 12 h with VK₃ (60 μmol/L) by flow cytometry. The effect of growth inhibition treated with VK₃ was antagonized by antioxygen NAC (5, 10, 20, 40, 80 μmol/L). An increase in the level of DCF fluorescence after PC-3M cells were treated for 1-2 h with VK₃ was observed. Antioxidase GSH-Px and CAT were run-down after treated with VK₃. CONCLUSION: The results indicate that apoptosis in PC-3M cells is induced through oxidative stress by VK₃.     

Volume-activated chloride channel exists in CD133+ lung cancer stem cells

AIM: To analyze the possibility that volume-activated chloride channel (VACC) exists in tumor stem cells. METHODS: CD133⁺ cells were purified by magnetic cell separation (MACS) system from non-small-cell lung cancer cell line A549. The purity of the cells was detected by flow cytometry. VACC current was recorded with whole-cell patch-clamp. RESULTS: The purity of CD133⁺ cells isolated from A549 by MACS was 92.14%. VACC current was recorded in the 22/40(55%) CD133⁺ cells of A549 by whole-cell patch-clamp. CONCLUSION: VACC exists in CD133⁺ lung cancer stem cells.     

LY367385 attenuates neuronal apoptosis after cerebral vasospasm in mice by inhibiting extracellular signal-regulated kinase pathway

AIM: To investigate the effects of LY367385,a selective antagonist of metabotropic glutamate receptor 1 (mGluR1), on the apoptosis of neuron, and the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathway after cerebral vasospasm (CVS) in mice. METHODS: The model of subarachnoid hemorrhage (SAH) in mice was established by endovascular perforation without opening cranium. Male ICR mice were randomly divided into 3 groups: sham operation group, NS+SAH group and LY367385+SAH group. Ten minutes after SAH, 5 μL of LY367385 or NS was microinjected into lateral cerebral ventricle, and then the neurological scores of the animals were examined. At different time points (6 h, 24 h, 48 h) after operation, the pathological changes in the brain tissues were observed under light and electron microscopes. The mRNA expression of mGluR1 was detected using RT-PCR, and Western blotting was used to examine the protein levels of mGluR1 and p-ERK1/2. Apoptotic cell number was determined by TdT-mediated dUTP nick end labeling (TUNEL) method. RESULTS: In SAH+NS group, the scores of neurological function were significantly lower, while the mGluR1 mRNA, mGluR1 and p-ERK1/2 proteins and the number of apoptotic cells were significantly higher than those in sham operation group.Some neurons displayed histopathological changes of necrosis, myelin sheath internalization and disconnection. LY367385 decreased the mRNA expression of mGluR1 and protein levels of mGluR1 and ERK1/2.The neuronal apoptosis, neurological score and the ultrastructural changes were also improved. CONCLUSION: The increased expression of mGluR1 in hippocampus may induce apoptosis by activation of ERK1/2 signaling after CVS. LY367385 exerts good therapeutic effect on severe CVS.     

Effects of magnesium on apoptosis and expression of Foxp3 in peripheral CD4+CD25+ regulatory T cells from asthma patients

AIM:To observe the apoptosis and the expression of forkhead box protein 3(Foxp3) induced by magnesium in CD4⁺CD25⁺ regulatory T cells isolated from healthy and asthmatic human peripheral blood. METHODS:Peripheral blood from healthy volunteers and asthma patients was collected. CD4⁺CD25⁺ T cells were separated by Percoll centrifugation and magnetic separation. The cells were cultured for 72 h and treated with magnesium(10 mmol/L) or control solution. The apoptotic rate and the expression of Foxp3 in the cells were analyzed by flow cytometry. RESULTS:The purity of CD4⁺CD25⁺T cells was 77.4%~92.3% in health group, and was 75.2％~93.8％in asthma group. The proportion of CD4⁺CD25⁺ T cells in CD4⁺T cells was 4.12%~7.98% in healthy adults, and 4.51%~8.68% in asthma patients. No significant difference between the 2 groups was observed. Magnesium at concentration of 10 mmol/L up-regulated the apoptotic rate of CD4⁺CD25⁺ T cells(P<0.05) and did not affect the Foxp3 expression in the cells in both health and asthma groups. CONCLUSION:Magnesium plays therapeutic effects on asthma by inducing the apoptosis of peripheral CD4⁺CD25⁺ regulatory T cells.     

Effects of phosphorylation-defective retinoic acid receptor α1 on proli-feration of human multiple myeloma cells in vitro

AIM: To investigate the effect of phosphorylation-defective retinoic acid receptor α1 (RARα1) on the proliferation of human multiple myeloma cells. METHODS: The mRNA expression of RARα subtypes in U266 cells was detected by RT-PCR. Lentiviral plasmid construction, viral production, titer determination and cell transfection were carried out by the general methods of molecular biology. Proliferation analysis was performed with CCK-8 assay. The U266 cells were treated with all-trans retinoic acid (ATRA,0~100 μmol/L) or transfected with lentivirus RARαS77A. The expression levels of proliferation-related proteins, P53 and Rb, in U266 cells treated with ATRA or transfected with lentivirus RARαS77A were detected by Western blotting. RESULTS: RARα1 was positively expressed in U266 cells and RARα2 expression was negative. ATRA significantly inhibited the proliferation of U266 cells in a dose- and time-dependent manner. Proliferation of U266 cells was significantly inhibited 48 h after transfection with lentivirus RARαS77A, and the inhibitory rate was 15.16%±3.84%. The up-regulated expression of Rb and down-regulated expression of P53 were detected in U266 cells not only in the cells treated with ATRA, but also in the cells transfected with lentivirus RARαS77A. CONCLUSION: Phosphorylation-defective RARα1 (RARαS77A) mimics the growth inhibitory effect of ATRA on U266 cells that express RARα1 (+) and RARα2 (-) via down-regulating the expression of P53 and up-regulating the expression of Rb, suggesting that the antiproliferative effect of ATRA is mainly mediated by decreasing the phosphorylation of RARα1.     

Influence of maxadilan on human adipose-derived stem cells

AIM: To investigate the effect of maxadilan, which specifically activates pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1 receptor), on the proliferation, apoptosis and differentiation potential of human adipose-derived stem cells (ASCs). METHODS: ASCs from human adipose tissue were isolated by enzymatic digestion and cultured. ASCs were confirmed by the analysis of the markers for cell phenotypes by flow cytometry (FCM) and adipogenic/osteogenic induction. The effect of maxadilan on ASCs viability was analyzed by CCK-8 assay and FCM. ASCs were irradiated by ultraviolet C (UVC) at 254 nm and the absorbance of apoptotic ASCs induced by various doses of UVC was measured by CCK-8 assay. ASCs were exposed to 702 J/m² UVC for 24 h to induce apoptosis. The effect of maxadilan on ASC apoptosis was analyzed by FCM and the determination of caspase 3 and caspase 9 levels. RESULTS: Adipose-derived stem cells were confirmed by the detection of the positive expression of cell phenotypes including CD29, CD44, CD59 and CD105 by FCM. The data of CCK-8 assay revealed that ASCs treated with maxadilan (80 nmol/L) had the strongest ability of proliferation. The data of FCM also demonstrated that the addition of 80 nmol/L maxadilan to ASCs in experimental group markedly improved the proliferation capacity of the cells compared with control group (P<0.05). The apoptosis of ASCs exposed to 702 J/m² UVC was dramatically inhibited by the treatment with maxadilan (80 nmol/L). Such process involved the caspase signaling pathway including caspase 3 and caspase 9. There was statistical significance (P<0.05) between experiment group (ASCs irradiated by UVC and supplemented with maxadilan) and control group (ASCs only irradiated by UVC). Meanwhile, adipogenic and osteogenic differentiation potentials were both positive in experiment group and control group. CONCLUSION: Maxadilan promotes proliferation and inhibits apoptosis of the ASCs. The differentiation potential of ASCs toward adipogenic and osteogenic lineages wouldn't be altered by maxadilan. Maxadilan would benefit to growth and expansion of ASCs in vitro.