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.     

Effect of NF-κB inhibitor pyrrolidine dithiocarbamate on proliferation and apoptosis of human multiple myeloma U266 cells

AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G₂ phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways.     

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.     

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.     

Effect of 3 isoforms of Ikaros on proliferation of human ovarian cancer SKOV3 cells

AIM: To investigate the effect of Ikaros isoforms on the proliferation of human ovarian cancer SKOV3 cells. METHODS: Three isoforms of Ikaros, IK1, IK2 and IK6, were transfected into ovarian cancer SKOV3 cells. CCK-8 assay and cell counting were used to detect the effects of Ikaros isoforms on the proliferation of SKOV3 cells. The cell cycle was analyzed by flow cytometry. The cell cycle-related proteins were detected by Western blot. RESULTS: IK1 and IK2 expression inhibited SKOV3 cells proliferation. Flow cytometry analysis indicated that IK1 and IK2 induced SKOV3 cell cycle arrest at the G₁ phase. IK6 isoform exerted no obvious effect on the proliferation or cell cycle of SKOV3 cells. Compared with control EV group, IK1 group and IK2 group showed a dramatic elevation in the expression of the cell cycle inhibitor p21, along with a substantial decrease in the expression of the cell cycle inducers cyclin D1 and cyclin D2, which did not change in IK6 group. CONCLUSION: IK1 and IK2 significantly inhibit the proliferation of ovarian cancer SKOV3 cells and induce cell cycle arrest at G₁ phase by regulation of cell cycle-related proteins cyclin D1, cyclin D2 and p21, while IK6 isoform exerts no obvious effect on the proliferation and cell cycle of SKOV3 cells.     

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.     

PCDH10 inhibits proliferation of breast cancer MDA-MB-231 cells by NF-κB/cyclin D1 signaling pathway

AIM To investigate the inhibitory effect of protocadherin 10 （PCDH10） on proliferation of human breast cancer cells and its possible mechanism. METHODS RT-PCR was used to measure the mRNA expression levels of PCDH10 in 4 human breast cancer cell lines and normal mammary epithelial MCF-10A cells. The breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed by recombinant lentivirus （pLV-PCDH10） infection, and blank control （blank） group and negative control （pLV-NC） group were also set up. The cell proliferation ability was detected using methyl thiazolyl tetrazolium （MTT） and colony formation experiments. The cell cycle was detected by flow cytometry. Western blot was used to determine the expression of cyclin D1, cyclin-dependent kinase 4 （CDK4）, nucear factor-κB p65 subunit （NF-κB p65） and NF-κB inhibitor α （IκBα）. RESULTS The mRNA expression levels of PCDH10 in 4 breast cancer cell lines were lower than that in normal mammary epithelial MCF-10A cells （P<0.05）. A breast cancer cell line MDA-MB-231 with stable PCDH10 overexpression was constructed successfully. Compared with negative control group, PCDH10 overexpression significantly inhibited cell proliferation, induced cell cycle arrest at G₁ phase, down-regulated the expression of cyclin D1 and CDK4, and decreased phosphorylation of NF-κB p65 and IκBα（P<0.05）. CONCLUSION PCDH10 inhibits the proliferation and blocks cell cycle progression of breast cancer cells by targeting NF-κB/cyclin D1 signaling pathway.     

Inhibitory effect of madecassoside on LPS-stimulated microglia

AIM: To observe the inhibitory effect of madecassoside on the LPS-stimulated microglia and to investigate its possible mechanism. METHODS: Microglia cells of neonatal Sprague-Dawley (SD) rats were cultured, isolated and purified. Microglia cells were activated with lipopolysaccharide (LPS). The inhibitory effect of madecassoside on microglia was measured by MTT assay. Tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β) were detected by ELISA. Cell cycle and apoptotic rate were evaluated by flow cytometry. The expression of TLR4 was detected by Western blotting. The expression of NF-κB was detected by RT-PCR. RESULTS: LPS induced the proliferation of microglia and release inflammatory cytokines significantly. Compared with LPS group, madecassoside inhibited the proliferation of microglia induced by LPS in a dose dependent manner. The IC₅₀ value of madecassoside was 10.97 nmol/L to microglia after incubation for 48 h. Madecassoside also decreased the levels of TNF-α and IL-6, increased the ratios of microglia at the G₂ phase and the apoptotic rate, decreased the expression of TLR4 and NF-κB significantly (P<0.05). CONCLUSION: Madecassoside has inhibitory effects on the proliferation of LPS-stimulated microglia, by which the mechanism may be related to inhibition of the expression of TLR4 and NF-κB, change of cell cycle distribution and induction of microglia apoptosis.     

Effects of human growth arrest-specific homeobox gene delivery on proliferation,migration and cell cycle distribution of vascular smooth muscle cells

AIM: To explore a new gene therapeutic strategy for vein graft restenosis by investigating the effects of adenovirus-mediated human growth arrest-specific homeobox (Ad5-hGax) gene delivery on the proliferation, migration and cell cycle distribution of serum-induced rabbit vascular smooth muscle cells (VSMCs). METHODS: The recombinant adenovirus vector containing hGax gene was constructed and transfected into rabbit VSMCs. The expression of hGax in VSMCs was detected by RT-PCR and immunofluorescent staining. Methyl thiazolyl tetrazolium (MTT) assay was used to assess the effect of hGax over-expression on serum-induced proliferation of VSMCs. Wound healing method was applied to examine the distance of serum-induce VSMCs migration. Flow cytometry was employed to analyze the cell cycle distribution. RESULTS: The recombinant adenovirus vector Ad5-hGax was successfully constructed. The results of RT-PCR and immunofluorescent staining revealed that the hGax -transfected cells contained a 174 bp specific fragment of hGax gene and target protein 48 h after transfection. The proliferation of serum-induced VSMCs was significantly inhibited by overexpression of hGax gene as compared with control group. The migration of serum-induced VSMCs was inhibited after hGax gene delivery. Flow cytometry showed that 72 h after serum induction, the cells in G₀/G₁ phase in Ad5-hGax group were significantly increased, whereas the cells in G₂/M+S phase were significantly decreased. CONCLUSION: Overexpression of hGax gene inhibits the proliferation and migration of serum-induced rabbit VSMCs, and arrests the cells in G₀/G₁ phase. It is likely that hGax gene is a potential target for the gene therapy of vein graft restenosis.     

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.     

Celastrol blocks cell cycle of A549 cells by regulating miR-17-5p/miR-155-5p and targeting cyclin D1

AIM: To investigate the effect of celastrol on the cell cycle of human lung adenocarcinoma A549 cells and to probe into its mechanisms.METHODS: A549 cells were exposed to celastrol at gradient concentrations. The cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively, and the median lethal concentration (LC₅₀) of celastrol was screened. The A549 cells were treated with celastrol at LC₅₀, and the cell cycle was detected by flow cytometry. The expression of cyclin D1 was determined by Western blot, and the expression of microRNA (miR)-17-5p and miR-155-5p was detected by real-time PCR. The correlation between cyclin D1 and miR-17-5p or miR-155-5p was predicted by bioinformatics software. After miR-17-5p mimics/miR-155-5p mimics/mutant-miR-17-5p/mutant-miR-155-5p and pcDNA-GFP-cyclin D1-3'UTR were cotransfected into the A549 cells, the changes of GFP expression were evaluated by fluorescence microscopy and flow cytometry. Finally, after miR-17-5p mimics or miR-155-5p mimics were transfeced into the A549 cells, the expression of miR-17-5p and miR-155-5p was detected by real-time PCR, and the protein level of cyclin D1 was determined by Western blot. RESULTS: With the increasing concentration of celastrol, the viability inhibition rate and apoptotic rate of the A549 cells were increased, indicating that celastrol effectively inhibited the growth of A549 cells and induced apoptosis. The LC₅₀ of celastrol was almost 3 μmol/L. After treatment with celastrol at LC₅₀, the A549 cell cycle was arrested at G₁ phase, the protein expression of cyclin D1 was down-regulated (P<0.01), and the expression levels of miR-17-5p and miR-155-5p were significantly increased (P<0.01). The results of bioinformatics software prediction indicated that there were binding sites for miR-17-5p and miR-155-5p in the 3'-UTR of cyclin D1. After cotransfected with miR-17-5p or miR-155-5p and pcDNA-GFP-cyclin D1-3'UTR into the A549 cells, the expression of GFP declined (P<0.05). After miR-17-5p or miR-155-5p mimics were transfected into A549 cells, the results of real-time PCR showed this treatment significantly increased the miRNA expression (P<0.01), and the results of Western blot showed the transfection inhibited cyclin D1 expression (P<0.01).CONCLUSION: Celastrol blocks the A549 cells at G₁ phase, inhibits the viability and induces apoptosis, which may be caused by up-regulating the expression of miR-17-5p and miR-155-5p, and then down-regulating cyclin D1 expression. This study provides a new theoretical basis for the treatment of non-small-cell lung cancer with celastrol.     

Influence of ginsenoside Re on vascular intima hyperplasia and NF-κB p65 signaling pathway in balloon-injured rats

AIM: To investigate the inhibitory effect of ginsenoside Re on intimal hyperplasia induced by balloon-injury and to explore the role of NF-κB p65 signaling pathway in the process. METHODS: SD rats(n=40) were divided into 5 groups randomly: sham operation group, model group, low-dose ginsenoside Re group, middle-dose ginsenoside Re group and high-dose ginsenoside Re group. The carotid artery intima injury model was established by 2F balloon catheters in all groups except the sham operation group. The day after modeling, the animals in model group and sham operation group were administered intragastrically with distilled water, and the rats in low-dose, middle-dose and high-dose ginsenoside Re groups were given ginsenoside Re at doses of 12.5 mg/kg, 25mg/kg and 50 mg/kg, respectively. After 14 continuous days, the morphological changes of the injured arteries were observed by HE staining and the lumen area, intima area and media area as well as the ratio of intimal area/media area were determined. The expression of tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) were detected by real-time PCR. The proliferating cell nuclear antigen(PCNA) and nuclear factor-kappa B(NF-κB) p65 were examined by immunohistochemistry.RESULTS: Compared with sham operation group, the vessel cavity was narrowed(P<0.01), the mRNA levels of TNF-α and IL-1β, and the protein expression of PCNA and NF-κB p65 were increased in model group(P<0.05). Compared with model group, the vascular intimal hyperplasia was alleviated obviously(P<0.05), and the mRNA levels of TNF-α and IL-1β, and protein expression of PCNA and NF-κB p65 were decreased in medium and high-dose ginsenoside Re groups(P<0.05). CONCLUSION: Ginsenoside Re inhibits the vascular neointimal hyperplasia induced by balloon-injury in rats, and the molecular mechanism may be related to the inhibition of NF-κB p65 signaling pathway.     

Effects of p204 expression on expression of p21 and proliferation of vascular smooth muscle cells in rats

AIM: To observe the effect of interferon-inducible protein 204 (p204) on the expression of p21 and proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Interferon alpha (IFN-α) and small interference RNA (siRNA) targeting p204 gene ( Ifi204 ) was used to intervene cultured VSMCs in vitro instantaneously, then the cell vitality was determined by MTT assay to reflect the cell proliferation. The cell cycle was analyzed by flow cytometry. The expression of p204 and p21 at mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blotting. RESULTS: In rat VSMCs, IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality and the G₁/S phase transition, and up-regulated the expression of p21 at mRNA and protein levels. Transfection of Ifi204 siRNA restrained the expression of p204 and p21, increased the cell vitality and promoted the G₁/S phase transition. CONCLUSION: The expression of p204 restrains the proliferation of rat VSMCs, probably by activating the expression of p21.     

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.     

Inhibition of HMGB1 expression promotes apoptosis of hemangioma endothelial cells

AIM: To investigate the effect of inhibiting high-mobility group box protein 1 (HMGB1) expression on the viability and apoptosis of hemangioma endothelial cells (HemECs). METHODS: Human HemECs were isolated and cultured, and HMGB1 small interfering RNA (HMGB1-siRNA) was transfected into the cells. The cell viability was detected by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) content were analyzed by flow cytometry. The protein levels of HMGB1, NF-κB p65, p-IκBα, cyclin D1 and survivin were determined by Western blot. RESULTS: The protein expression of HMGB1 in the HemECs transfected with HMGB1-siRNA was significantly lower than that in blank control group (P<0.05). Compared with NC group, the cell viability was decreased significantly in the HemECs transfected with HMGB1-siRNA, the apoptotic rate was significantly increased, the content of ROS increased significantly, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were significantly decreased (P<0.05). After exposure to NF-κB signaling pathway inhibitor PDTC, the cell viability was inhibited, the apoptosis was increased, ROS content, and the protein levels of NF-κB p65, p-IκBα, cyclin D1 and survivin were down-regulated significantly, as compared with si-HMGB1 group (P<0.05). CONCLUSION: Inhibition of HMGB1 reduces the viability of HemECs and induces apoptosis by increasing the content of ROS and down-regulating the activity of NF-κB signaling pathway.     

CUDC-907 induces DNA damage and autophagy in glioma cells

AIM:To investigate the effect of CUDC-907, a dual histone deacetylase (HDAC) and phosphatidylinositol 3-kinase (PI3K) inhibitor, on the DNA damage, cell cycle distribution and autophagy in human glioma U251 cells. METHODS:U251 cells were treated with CUDC-907 of different concentrations, and the cell viability was detected by MTT assay. The quantitative γ-H2AX foci were determined by laser scanning confocal microscopy. The cell cycle distribution of U251 cells was examined by flow cytometry. The protein expression was determined by Western blot analysis. RESULTS:CUDC-907 inhibited the cell viability and the phosphorylation of Akt and p70 ribosomal protein S6 kinase (p70s6K) in the U251 cells (P<0.05). In CUDC-907-treated cells, the number of γ-H2AX foci and protein expression of γ-H2AX were increased significantly (P<0.05). CUDC-907 also induced cell arrest in the G₂/M phase by up-regulating the expression of p21, and inhibiting the protein level of cyclin B1 and the phosphorylation of cell division cycle protein 2 (Cdc2). In addition, CUDC-907 triggered cell autophagy, and inhibition of autophagy increased CUDC-907-induced DNA damage of U251 cells. CONCLUSION:CUDC-907 significantly inhibits PI3K/Akt signaling pathway, induces DNA damage and arrests cell cycle in G₂/M phase. Blockage of autophagy promotes CUDC-907-induced DNA damage of U251 cells.     

Construction of SETD2 gene knockout nasopharyngeal carcinoma cell strains using CRISPR/Cas9 technique and analysis of their proliferation property

AIM:To establish SETD2 gene knockout nasopharyngeal carcinoma (NPC) cell strains based on CRIPSR/Cas9 technique and to analyze their proliferation characteristics. METHODS:Sub-quantitative RT-PCR and Western blot were used to detect the expression of SETD2 in immortalized nasopharyngeal epithelial cell line NP-69, well differentiated NPC cell line CNE1, poorly-differentiated NPC cell line CNE2Z and undifferentiated NPC cell line C666-1, and the SETD2 high expression cell line CNE1 was screened. The proliferation ability of CNE1 cells before and after the SETD2 gene knockout was analyzed by CCK-8 and colony formation assay. The cell cycle distribution was detected by flow cytometry, and the expression of cell cycle-related proteins was detected by Western blot. RESULTS:Compared with NP-69 cells, the expression of SETD2 was decreased gradually in CNE1, CNE2Z and C666-1 cells (P<0.01). Based on the CRISPR/Cas9 technique, 2 monoclonal cell strains with SETD2 gene stable knockout, named CNE1-SETD2-KO-#5 and #9, were successfully screened from total 15 monoclones. The results of CCK-8 and plate colony formation assay confirmed that the proliferation ability of CNE1-SETD2-KO-#5 and #9 cells was significantly enhanced compared with CNE1-WT cells (P<0.05). The results of flow cytometry analysis showed that the G₁ phase of CNE1-SETD2-KO-#5 and #9 cells was decreased, while the G₂/M and S phases were increased significantly (P<0.05). The results of Western blot confirmed the increases in the protein levels of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin B1, cyclin A2, cyclin E1, cyclin-dependent kinases 2 (CDK2) and CDK4, and the decrease in the protein level of p21 after SETD2 gene knockout (P<0.05). CONCLUSION:The NPC cell strains with SETD2 gene knockout were successfully constructed based on CRISPR/Cas9 technique. SETD2 expression correlates with cell differentiation status in the NPC cells. SETD2 gene knockout promotes NPC cell proliferation by up-regulating cyclin D1, cyclin B1, cyclin A2, cyclin E1, CDK2 and CDK4, and down-regulating p21 expression.     

Effects of platelet-derived growth factor and angiotensinⅡ on the expression of cell cycle related genes in vascular smooth muscle cells

AIM: To investigate the different expressions of cell cycle related genes in hyperplastic and hypertrophic vascular smooth muscle cells caused by platelet-derived growth factor (PDGF-BB) and angiotensinⅡ(AngⅡ). METHODS: Rat aorta media smooth muscle cells were cultured. PDGF-BB and AngⅡ were added into serum-free medium at a concentration of 20 μg/L and 10^-6 mol/L , respectively. Vascular smooth muscle cells (VSMCs) were harvested after stimulated for 24 hours. The expression of cell cycle related genes was measured by DNA chips(Atlas cDNA Expression Arrays, Clontech Laboratories, Inc.). RESULTS: The expression of cyclin D3 mRNA ,cyclin G1 mRNA,p57 mRNA,p16 mRNA,E2F-3 mRNA and DP2 mRNA were higher in PDGF-BB than those in AngⅡstimulated VSMCs. p15 mRNA,p19 mRNA,E2F-1 mRNA, E2F-5mRNA,and N-myc mRNA were only detected in PDGF-BB stimulated group. But the expression of p53-associated protein mRNA were higher in AngⅡstimulation group. The expression of PCNA mRNA, c-myc binding protein mRNA,p53-dependent cell growth regulater mRNA,cyclinC mRNA, cyclinB1 mRNA, E2F-3mRNA were similar in the two groups. CONCLUSION: The procession of cell cycle relys on the coordination of many regulater molecules expressed in different phases. Our study preliminarily definite the genes that express during PDGF-stimulated VSMC's hyperplasia and Ang II-stimulated VSMC's hypertrophy.