PPARγ mediates effects of diosgenin on proliferation and apoptosis in human glioblastoma U87MG cells

AIM:To investigate the effect of diosgenin (Dio) on the proliferation, apoptosis and expression of peroxisome proliferator-activated receptor γ (PPARγ) in human glioblastoma U87MG cells and its possible mechanism. METHODS:Human astrocytes (HA) and U87MG cells were cultured in vitro and treated with Dio (0, 10, 20, 30, 40 and 50 μmol/L) and GW9662 (5 μmol/L) for 48 h, and then the cell viability was detected by CCK-8 assay. Cell colony formation assay was used to assess the proliferation potential. Flow cytometry was used to analyze the cell cycle distribution and apoptosis. The mRNA expression level of PPARγ was measured by RT-PCR. Western blot was used to determine the protein levels of PPARγ, cyclin D1, cyclin E1, Bcl-2 and Bax. RESULTS:Dio had no significant influence on the viabi-lity of HA (P>0.05). However, Dio remarkably reduced the viability of U87MG cells in a dose-dependent manner (P<0.05) with IC₅₀ of 24.31 μmol/L. Meanwhile, Dio remarkably diminished colony formation ability (P<0.05), induced G₀/G₁ phase arrest of the cell cycle and apoptosis (P<0.05), up-regulated the expression of PPARγ at mRNA and protein levels, increased the protein level of Bax (P<0.05), and down-regulated the protein levels of cyclin D1, cyclin E1 and Bcl-2 (P<0.05) in a dose-dependent manner. However, these effects induced by Dio were inhibited by GW9662 (P<0.05), a specific inhibitor of PPARγ. CONCLUSION:Dio may inhibit proliferation and induce apoptosis in human glioblastoma U87MG cells most likely via up-regulating the expression of PPARγ, and then down-regulating the protein levels of cyclin D1, cyclin E1 and Bcl-2, and up-regulating the protein level of Bax.     

Hypoxia promotes apoptosis of neural stem cells and down-regulates miR-26a

AIM: To investigate the effect of cobalt chloride (CoCl₂) on the apoptosis of neural stem cells (NSCs) and the expression of microRNA-26a (miR-26a) in vitro, and to explore the mechanisms of NSC apoptosis induced by CoCl₂. METHODS: NSCs were exposed to CoCl₂ at different doses (200~600 μmol/L) for 24 h. The cell viability and apoptosis were measured by CCK-8 assay and TUNEL method. The expression of miR-26a-3p, miR-26a-5p, GSK-3β, caspase-3, Bcl-2 and Bax was examined by real-time PCR. The protein levels of Bcl-2 and Bax were detected by Western blotting. RESULTS: The cell viability was inhibited and the apoptosis of NSCs was increased significantly by CoCl₂ in a dose-dependent manner (P<0.05). CoCl₂ at concentration of 400 μmol/L for 24 h was used to induce apoptosis and the expression of miR-26a was down-regulated compared with control (P<0.05). Exposure to CoCl₂ at concentration of 400 μmol/L up-regulated the expression of GSK-3β, caspase-3 and Bax, down-regulated the expression of Bcl-2 and Bcl-2/Bax (P<0.05). CONCLUSION: CoCl₂ at concentration of 400 μmol/L induces the apoptosis of NSCs obviously. CoCl₂ may induce the NSC apoptosis by mitochondrial apoptotic pathway. Declining miR-26a may be related to NSC apoptosis.     

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 down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Effect of Notch1/Hes1 signaling pathway on proliferation and differentiation of AECⅡ by regulating expression of C/EBPα

AIM: To investigate whether Notch1/Hes1 signaling pathway regulates the expression of CCAAT/enhancer binding protein alpha (C/EBPα), thus affecting the proliferation and differentiation of type Ⅱ alveolar epithelial cells (AECⅡ). METHODS: Human AECⅡ were cultured in vitro, and randomly divided into control group, activator group (adding Notch pathway activator Jagged1 protein, 500 μg/L) and inhibitor group (adding Notch inhibitor DAPT, 10 μmol/L). AECⅡ in each group were collected after 24 h of intervention. The expression of Notch1, Hes1 and C/EBPα at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell proliferation ability of the AECⅡ was measured by living cell counting and CCK-8 assay. The cell cycle distribution and differentiation of the AECⅡ were analyzed by flow cytometry. RESULTS: Compared with control group, the mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly increased in activator group (P<0.05), AECⅡ entered G₂/M phase from S phase, the proliferation of AECⅡ was increased, and the differentiation of AECⅡ was reduced (P<0.05). The mRNA and protein expression levels of Notch1, Hes1 and C/EBPα were significantly reduced in inhibitor group (P<0.05), the cell cycle of AECⅡ cells was arrested in G₀/G₁ phase, the proliferation of AECⅡ cells was reduced, and the differentiation of AECⅡ cells was increased (P<0.05). CONCLUSION: Notch1/Hes1 signaling pathway regulates the expression of C/EBPα and affects the proliferation and differentiation of AECⅡ.     

Effects of human xeroderma pigmentosum group D gene on proliferation of human vascular smooth muscle cells induced by interleukin-6

AIM: To investigate the effects of human xeroderma pigmentosum group D (XPD) gene on the proliferation of human vascular smooth muscle cells (VSMCs) induced by interleukin-6 (IL-6). METHODS: Recombinant plasmid pEGFP-N2/XPD and vacant plasmid pEGFP-N2 were transfected into VSMCs by liposome, and then these cells were incubated with IL-6 at 1×10⁵ U/L for 48 h. The cells were divided into 6 groups: blank control group; pEGFP-N2 group; pEGFP-N2/XPD group; IL-6 group; IL-6 + pEGFP-N2 group; IL-6 + pEGFP-N2/XPD group. The expression of green fluorescent protein was observed under fluorescence microscope. The cell growth was detected by MTT method. The cell cycle and apoptosis rate were examined by flow cytometre. The expression levels of XPD, Bcl-2, Bax and wild type P53 (wt-P53) were detected by RT-PCR and Western blotting.RESULTS: Green fluorescence was observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2, indicating successful transfection MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth, and reduced the positive effects of IL-6 on VSMCs growth. Flow cytometry results showed that the transfection of pEGFP-N2/XPD increased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, decreased the cell numbers in S phase, and reduced the effects that IL-6 decreased the apoptosis rate of VSMCs and the cell numbers in G₀/G₁ phase, and increased the cell numbers in S phase. The results of RT-PCR and Western blotting showed that the transfection of pEGFP-N2/XPD increased the expression of XPD, Bax and wt-P53, decreased the expression of Bcl-2, and reduced the effects that IL-6 decreased the expression of Bax and wt-P53, and increased the expression of Bcl-2. CONCLUSION: XPD gene inhibits VSMCs proliferation, promotes VSMCs apoptosis, and reduces the effects that IL-6 promotes VSMCs proliferation and inhibits VSMCs apoptosis. Therefore, XPD gene is likely to be potential molecular target for treatment of atherosclerosis.     

Effects of all-trans retinoic acid on sensitivity of gastric cancer cells to radiotherapy

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on the viability of gastric cancer cell SGC-7901 and the sensitivity to radiotherapy. METHODS: MTT assay was used to examine the cell viability. Radio-sensitivity and cell cycle were determined by colony formation assay and flow cytometry, respectively. The mRNA levels of Bax, Bcl-2, survivin and NF-κB in the cells were measured by RT-qPCR. RESULTS: ATRA inhibited the viability of SGC-7901 cells in a concentration-dependent manner. The maximal inhibition was at concentration of 8 μmol/L. Colony formation assay revealed that the combination of ATRA with X-ray treatment significantly reduced the values of D0 and Dq, and shifted down the fitting survival curve, as compared with radiotherapy alone. Moreover, ATAR markedly decreased the percentage of G₂/M phase in the SGC-7901 cells (P<0.05). In addition, following ATRA treatment, the mRNA levels of Bcl-2 and survivin were decreased (P<0.05), whereas the mRNA levels of Bax and NF-κB were increased (P<0.05). CONCLUSION: ATRA enhances the sensitivity of SGC-7901 cells to radiotherapy, inhibits G₂/M arrest and regulates the mRNA expression of Bax, Bcl-2, survivin and NF-κB.     

Effects of IL-22 on rheumatoid arthritis fibroblast-like synoviocytes

AIM: To determine the effects and mechanisms of interleukin-22(IL-22) on the fibroblast-like synoviocytes(FLSs) from rheumatoid arthritis(RA) patients. METHODS: RA-FLSs were cultured by tissue culture method. RA-FLSs were incubated with different concentrations of IL-22(0,1,10,100 μg/L) for 24 h, 48 h and 72 h. The cell viability was examined by CCK-8 assay. IL-22 at concentration of 10 μg/L was used to stimulate RA-FLSs for 24 h, and the change of cell cycle distribution was identified by flow cytometry. The effects of IL-22 at concentrations of 0, 1, 10, 100 μg/L and/or STA-21(a STAT3 inhibitor at concentrations of 0, 25, 50 μmol/L) on the protein levels of Bcl-2 and p-STAT3 in the RA-FLSs were determined by Western blot.RESULTS: Compared with control group, stimulation of rhIL-22 at different concentrations for 24 h, 48 h and 72 h, the cells viabilityof RA-FLSs were obviously increased(P<0.05). After co-cultured with 10 μg/L rhIL-22 for 24 h, the percentages of RA-FLSs were obviously increased in the G₂/M+S phase and decreased in the G₀/G₁ phase. At the same time, rhIL-22 increased, but STA-21 decreased the protein levels of Bcl-2 but p-STAT3 in the RA-FLSs obviously(P<0.05). Treatment with STAT3 inhibitor STA-21 reversed the effect of IL-22-induced Bcl-2 upregulation in the RA-FLSs(P<0.01). CONCLUSION: STAT3 is critical in the process of IL-22-induced Bcl-2 upregulation in RA-FLSs, indicating that IL-22 may play a role in the apoptosis of RA-FLSs.     

Effect of sodium selenite on proliferation of endometrial cancer cells

AIM: To investigate the effect and mechanism of sodium selenite (Na₂SeO₃) on the proliferation of endometrial cancer cells. METHODS: Endometrial cancer Ishikawa cells and HEC-1A cells were treated with Na₂SeO₃. The effect of Na₂SeO₃ on cell proliferation was determined by MTT assay. The effects of Na₂SeO₃ on cell cycle distribution and apoptosis were tested by flow cytometric analysis. The expression of cyclin A was detected by Western blotting. RESULTS: Na₂SeO₃ inhibited the proliferation of Ishikawa cells and HEC-1A cells. For Ishikawa cells, IC₅₀ was 3.26 μmol/L, and for HEC-1A cells, IC₅₀ was 4.77 μmol/L. After treated with Na₂SeO₃, the cells in G₀/G₁ phase were reduced and the cells in S phase and G₂/M phase were increased. Na₂SeO₃ also increased the percentage of apoptosis cells. The result of Western blotting showed that the expression of cyclin A was increased. CONCLUSION: Na₂SeO₃ inhibits the proliferation of endometrial cancer Ishikawa cells and HEC-1A cells via up-regulating the expression of cyclin A, arresting cell cycle and inducing apoptosis.     

Effect of 7-hydroxyisoflavone on HCT116 cells proliferation by Id1

AIM:To investigated the effect of 7-hydroxyisoflavone (7-HIF) on the proliferation, apoptosis and stem-like cell feature of colorectal cancer cells. METHODS:The effect of 7-HIF on the proliferation of HCT116 cells was detected by WST-1 assay and colony formation assay. The effects of 7-HIF on the cell cycle distribution and apoptosis in the HCT116 cells were analyzed by flow cytometry. The expression of cell-cycle related proteins and the stemness related proteins was determined by Western blot. RESULTS:After treated with 7-HIF (200 μmol/L), the viability of HCT116 cells was inhibited, and the size and number of the colony were decreased as compared with control group (P<0.05). The G₀/G₁ phase of the cell cycle was increased. The proportion of S phase was decreased and the cells were mainly arrested in G₀/G₁ phase. The apoptotic rate of HCT116 cells was 21.4%, which was significantly higher than that in the control group (1.1%). The results of Western blot revealed that the expression of inhibitor of differentiation 1(Id1) was significantly decreased (P<0.05). The expression of cell cycle markers cyclin D1 and cyclin E, the proliferative markers survivin and PCNA, and stem cell markers CD133, ALCAM and EpCAM were all down-regulated by 7-HIF treatment (P<0.05). CONCLUSION:7-HIF inhibits the proliferation and induces the apoptosis of colorectal cancer cells, and inhibits the stem-like cell feature, which may be related to Id1 inhibition.     

Brucine induces apoptosis in colon cancer SW480 cells via inhibiting IL-6/STAT3 signaling pathway

AIM: To observe the effects of brucine on the viability and apoptosis of colon cancer SW480 cells.METHODS: The SW480 cells were divided into control group, 1 μmol/L brucine treatment group, 100 μg/L IL-6 treatment group and IL-6＋brucine treatment group. The cell viability was detected by CCK-8 assay. The apoptotic rate was measured by flow cytometry using fluorescein-labeled Annexin V/PI. The changes of apoptosis-related proteins were determined by Western blot. The protein level of p-STAT3 was also detected by immunofluorescence staining. RESULTS: Brucine inhibited SW480 cell growth, and the viability inhibition rate of the SW480 cells treated with brucine alone was more efficient than using brucine combined with IL-6 (P < 0.05). The apoptotic SW480 cells increased significantly after 1 μmol/L brucine treatment as compared with brucine treatment alone (P < 0.05). The apoptotic SW480 cells were significantly reduced in brucine and IL-6 combination treatment group (P < 0.05). Brucine inhibited the protein level of p-STAT3 significantly. The protein level of p-STAT3 was significantly increased in 100 μg/L IL-6 treatment group. Compared with 1 μmol/L brucine treatment alone, the expression of Bcl-2 was increased and the protein levels of p-STAT3, Bax and cleaved PARP were reduced in brucine and IL-6 combination treatment group (P < 0.05).CONCLUSION: Brucine may inhibit the activation of STAT3 phosphorylation in IL-6/STAT3 pathway to exert an antitumor effect on SW480 cells in vitro.     

Inhibitory effect of berberine on the activation and proliferation of T lymphocytes

AIM: To investigate the effect of berberine (Ber) on the activation and proliferation of T lymphocytes and its mechanism of action. METHODS: Whole peripheral blood from normal subjects was stimulated with phytohemagglutinin (PHA) or phorbol ester (PDB) plus ionomycin (Ion) and the expression levels of CD69 and CD25 were evaluated with flow cytometry after the staining with appropriate fluorescent monoclonal antibody. The distribution of cell cycles was analyzed by propidium iodide staining and dead cells by 7-aminoactinomycin live staining. RESULTS: 100 μmol/L and 50 μmol/L of Ber had significant inhibition of the expression of CD69 on T cells stimulated with PDB plus Ion or PHA, while effect of 25 μmol/L Ber was not significant. And as time of action extended, the extent of inhibition decreased. For the expression of CD25, Ber at the concentrations as above all exerted significant inhibitory effect in a dose-dependent manner. Moreover, Ber could block lymphocytes cell cycle progression from G₀/G₁ phase to S and G₂/M phase without phase specificity. Besides, live staining analysis revealed that Ber did not have significant cytotoxicity on lymphocytes. CONCLUSIONS: Ber significantly inhibits the expression of early and mid activation antigens of T cells and also blocks the progression of lymphocytes cell cycles. These results suggest that Ber exerts immunosuppression effect through inhibiting the activation and proliferation of T cells.     

Activation of TLR9 affected chemotherapeutic sensitivity of doctaxel in human non-small cell lung cancer in vitro

AIM: To probe whether CpG oligodeoxyribonucleotides 7909 (CpG ODN7909) combined with Toll like receptor (TLR)9 affected the chemotherapeutic sensitivity of doctaxel (DOC) in human lung cancer A549 and H520 cell lines.METHODS: Sequences of TLR9 siRNAs were designed. A549 and H520 cells were transfected with TLR9 siRNA by lipofectamine. The expression of TLR9 was detected by Western blot. The cell activity was measured by CCK-8 assay. The experiments were divided into blank control group, control siRNA group and TLR9 siRNA interference group. The cell cycle distribution and cell apoptosis were analyzed by flow cytometry. The expression of P38 and Bax was determined by Western blot. The cells in each group were exposed to CpG ODN7909 and/or DOC.RESULTS: In A549 cells and H520 cells, CpG ODN7909 alone had no obvious effect on the cell activity, G₂/M phase arrest and apoptosis, but increased the protein expression of P38 and Bax (P<0.01). In addition, there was no significant changes of the above indexes in CpG ODN7909 treated-TLR9 siRNA group was observed. DOC alone significantly inhibited the cell activity, higher the G₂/M phase fractions, apoptotic rates and Bax expression (P<0.01), but didn't affect the expression of P38 in all 3 groups. Compared with the cells treated with DOC alone, the cells treated with CpG ODN7909 combined with DOC exhibited lower cell activity, higher G₂/M phase fractions, apoptosis rates and more Bax expression (P<0.01), showed no significant change of P38 expression. In addition, there was no significant change of the above indexes in CpG ODN7909 combined with DOC treated-TLR9 siRNA group was observed.CONCLUSION: CpG ODN7909 may enhance the chemotherapeutic sensitivity of DOC in human lung cancer cells by combining with TLR9. The mechanism might be related to enhancing the inhibitory effect and apoptosis of DOC on the cell activity in vitro, arresting the cells at G₂/M phase of the cells.     

In vitro growth inhibition effects of rhHGF/cHGF on SMMC-7721 human HCC cell line

AIM:To examine the effects of recombinant human hepatocyte growth factor(rhHGF) and native calf HGF(cHGF) on SMMC-7721 human hepatocellular carcinoma(HCC) cell line. METHODS:Human HCC cell line culture, photometric assay, and flow cytometric assay were used in this study .RESULTS:A similar type of dose-dependent cell growth inhibition effect on SMMC-7721 human HCC cells by rhHGF(5-20 μg/L) as well as by cHGF(25-100 mg/L) had been found, with the maximal effect at the highest concentration used. Approximately over 50% of the cells treated with rhHGF(5 μg/L, 10 μg/L, 20 μg/L) accumulated in the quiescent G₀/G₁ phase of the cell cycle over incubation periods for 3 d. CONCLUSION:The growth of SMMC-7721 human HCC cells was strongly inhibited by both rhHGF and cHGF. This might be because the cells exposed to HGF became arrested in the G₀/G₁ phase.     

Effects of propofol on biological characteristics of colorectal cancer cells

AIM: To investigate the effect of propofol on the viability, invasion ability and apoptosis of colorectal cancer cells.METHODS: Propofol at 10, 25, 50 and 100 μmol/L was used to treat LoVo cells for 72 h, and propofol at 100 μmol/L was used to treat the LoVo cells for 12, 24, 48 and 72 h. The cell viability was measured by CCK-8 assay. The invasion ability of the LoVo cells treated with propofol at 100 μmol/L for 72 h was detected by Transwell assay. The cell cycle distribution and cell apoptotic rate were analyzed by flow cytometry. The protein levels of matrix metalloproteinase (MMP)-2, MMP-9, cleaved caspase-3, Notch1 and hairy and enhancer of split 1 (Hes1) were determined by Western blot.RESULTS: Propofol inhibited LoVo cell viability. The cell invasion ability, S stage cells, and the protein levels of MMP-2, MMP-9, Notch1 and Hes1 in propofol group were significantly lower than those in control group, and the apoptotic rate, G₀/G₁ cells and the protein level of cleaved caspase-3 were significantly higher than those in control group (P<0.01).CONCLUSION: Propofol inhibits the viability and invasion ability of colorectal cancer LoVo cells, blocks cell cycle and induces apoptosis. The mechanism is related to down-regulation of Notch1 signaling pathway.     

The antisense phosphorothioate oligodeoxynucleotides of bcl- 2 oncogene enhances the apoptotic effect of As2O3 on NB4 leukemic cells

AIM: To study and explore whether the antisense phosphorothioate oligodeoxynucleotides (ASODN) of bcl-2 oncogene would increase the apoptotic effect of As₂O₃ on NB4 leukemic cells. METHODS: The biological and morphological changes in NB4 cells from microculture with As₂O₃, bcl-2 ASODN or both were observed. The changes in DNA content and Bcl-2 protein of NB4 cells from microculture with As₂O₃, bcl-2 ASODN or both were determined by tissue chemistry and flowcytometry. RESULTS: There was much more apoptotic effect of As₂O₃ on NB4 cells while it combined with bcl- 2 ASODN (ASODN 10.0 μmol/L+ As₂O₃ 0.25 μmol/L) than alone(ASODN 10.0 μmol/L or As₂O₃ 0.25μmol/L),and so did inhibitory effect of Bcl-2 protein expression by flow cytometry. CONCLUSION:The bcl-2 ASODN can enhance the apoptotic effect of As₂O₃ on NB4 leukemic cells.     

Inhibition of cell proliferation and arrest of cell cycle progression by blocking Cl- channels in nasopharyngeal carcinoma cells

AIM: The roles of Cl^-channels in regulatory volume decrease (RVD), cell proliferation and cell cycle progression in nasopharyngeal carcinoma cells (CNE-2Z) were investigated. METHODS: Image analysis of living cells was used to detect the volume changes following exposure to hypotonic solutions. Cell viability was determined by the trypan blue assay. MTT method was applied to detected cell proliferation. The effect of the blocker on the cell cycle distribution was monitored by the flow cytometry. RESULTS: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) inhibited RVD and cell proliferation in a dose-dependent manner. NPPB at the concentration of 100 μmol/L arrested cells in G₁ phase (G₁ population increased from 54% to 71% at 48 h after treatments), but did not significantly alter cell viability. CONCLUSION: Block of chloride channels suppressed cell proliferation by arresting cells in G₁ phase. The results suggest that activation of Cl^-channels and RVD is necessary for facilitating cells to proceed to the S phase from G₁ phase and maintaining cell proliferation.     

Effects of IL-32γ on proliferation and cell cycle of rat vascular smooth muscle cells

AIM: To observe the effects of interleukin-32γ (IL-32γ)on the proliferation and cell cycle of rat vascular smooth muscle cells (VSMCs). METHODS: The VSMCs were isolated from the thoracic aorta of SD rats by the method of tissue-piece inoculation. The cells were cultured and treated with different concentrations of IL-32γ. The proliferation of the cells was examined by MTT assay. The cell cycles were analyzed by flow cytometry. The protein levels of NF-κB p65 and cyclin D1 were detected by Western blotting. The expression of proliferating cell nuclear antigen (PCNA)was examined by immunocytochemical staining. RESULTS: Administration of IL-32γ at the concentrations of 10~50 μg/L for 24~48 h significantly promoted the proliferation of VSMCs in a dose- and time-dependent manner. After stimulation with IL-32γ at the concentration of 50 μg/L for 24 h, the cell cycle transition from G₁ phase to S/G₂ phase was accelerated and the expression levels of NF-κB p65, cyclin D1 and PCNA increased as compared with those in control group. CONCLUSION: IL-32γ promotes the proliferation of rat VSMCs and accelerates the cell cycle transition via upregulating the expression of NF-κB p65 and cyclin D1.     

Mollugin inhibits viability and collagen synthesis of rat CFSC-2G cells

AIM: To investigate the effects of mollugin on the viability and collagen synthesis of rat hepatic stellate cell line CFSC-2G. METHODS: The activation of CFSC-2G cells was induced with low concentration (10 μmol/L) of hydrogen peroxide (H₂O₂) for 30 min in the experiment. The viability of the CFSC-2G cells after exposed to mollugin at different concentrations (0, 20, 40, 60 and 120 μmol/L) was detected by MTT assay. The mRNA and protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), nuclear factor-κB (NF-κB) p65, Bcl-2, Bcl-xL, Bax, and hepatic stellate cell activation markers α-smooth muscle actin (α-SMA) and collagen type I (Col Ⅰ) were detected by real-time PCR and Western blot. The phosphorylation level of p38 mitogen-activated protein kinase (p38 MAPK) was determined by Western blot. RESULTS: Mollugin significantly inhibited the viability and collagen synthesis of activated CSFC-2G cells induced by H₂O₂. The expression of Nrf2, HO-1 and Bax at mRNA and protein levels, and the phosphorylation level of p38 MAPK were promoted, while the levels of NF-κB p65, Bcl-2, Bcl-xL, α-SMA and ColⅠwere inhibited by mollugin (P<0.05). CONCLUSION: Mollugin may inhibit H₂O₂-induced viability and collagen synthesis of the CSFC-2G cells by activating Nrf2 and HO-1, and blocking the NF-κB p65 and Bcl-2 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.