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.     

Mechanism of elemene enhancing radiosensitivity of human glioma U251 cells

AIM To investigate the effect of elemene on the radiosensitivity of human glioma U251 cells and its mechanism. METHODS The U251 cells were used as a glioma model in vitro, and were exposed to different concentrations of elemene and different doses of radiation. The cell viability was measured by MTT assay, the apoptosis and cell cycle distribution were analyzed by flow cytometry, and the related protein levels were determined by Western blot. RESULTS Elemene inhibited the viability of U251 cells in vitro and enhanced the radiosensitivity of the cells. The cells in radiotherapy combined with elemene group had higher rates of early apoptosis, secondary necrosis and total cell death than those in radiation group. Elemene induced G₂/M phase arrest in the U251 cells. Elemene reduced the protein expression of cell division cycle protein 2 （Cdc2）, which resulted in the decrease in cyclin B1 expression induced by radiotherapy, thereby inhibiting the formation of cyclin B-Cdc2 complex. Elemene reduced Cdc2 activity by inhibiting the phosphorylation of Cdc2 protein at threonine 161, thereby inducing G₂/M phase arrest in the cells. It also mediated apoptosis by down-regulating survivin expression. CONCLUSION Elemene may increase the sensitivity of U251 cells to radiotherapy by down-regulating Cdc2 protein, decreasing cyclin B1 expression, inhibiting the formation of cylcin B-Cdc2 complex and down-regulating the expression of survivin.     

CHK1 inhibitor SCH900776 suppresses proliferation and migration of U251 cells

AIM: To investigate the effect of SCH900776, an inhibitor of checkpoint kinase 1 (CHK1), on the proliferation and migration abilities of human glioma U251 cells.METHODS: The cell viability was detected by MTT assay, and cell proliferation was determined by cell colony formation assay. Cell cycle distribution was analyzed by flow cytometry. Wound healing assay was used to determine the cell migration ability. The protein levels were determined by Western blot.RESULTS: SCH900776 inhibited the growth of U251 cells in a dose-dependent manner (P<0.05). SCH900776 treatment substantially induced U251 cell cycle arrest in S and G₂/M phases by decreasing the level of cell division cycle protein 2 (Cdc2) and p-Cdc2. Moreover, SCH900776 inhibited the cell migration. Western blot results indicated that SCH900776 increased the phosphorylation level of p38 MAPK and inhibited the activation of Akt.CONCLUSION: SCH900776 inhibits the proliferation and migration abilities in human U251 cells by promoting the phosphorylation of p38 MAPK and suppressing the activation of Akt.     

Effect of sorcin expression on sensitivity of human glioma cells to cisplatin

AIM：To investigate the effect of sorcin expression on the sensitivity of human glioma cells to cisplatin. METHODS：pSilencerTM 3.1-H1-sorcin siRNA recombinant plasmid was constructed, and transfected into human glioma U251 cells. RT-PCR and Western blotting were used to analyze the expression of sorcin at mRNA and protein levels after transfection. The viability of U251 cells was measured by MTT assay. The protein expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) in U251 cells was detected by Western blotting. RESULTS：The plasmid pSilencerTM 3.1-H1-sorcin siRNA was successfully constructed, and was confirmed by restriction enzyme digestion and sequence analysis. The expression of sorcin at mRNA and protein levels was significantly decreased after sorcin siRNA was transfected into U251 cells (P<0.05). Inhibition of sorcin expression significantly decreased the viability of U251 cells treated with cisplatin (P<0.05), and the expression of P-gp and MRP1 proteins was also inhibited (P<0.05). CONCLUSION：Inhibition of sorcin expression increases the sensitivity of U251 cells to cisplatin by decreasing the expression of resistance-related proteins P-gp and MRP1, suggesting that sorcin may be associated with the resistance of glioma cells to cisplatin.     

Blocking autophagy magnifies MK-2206-induced DNA damage in SGC-7901 cells

AIM: To investigate the effect of MK-2206, an inhibitor of protein kinase B(Akt), on the DNA damage of SGC-7901 cells.METHODS: SGC-7901 cells were treated with different concentrations of MK-2206, and phosphorylated histone H2AX(γ-H2AX) foci formation was detected by immunofluorescence staining. Western blot analysis was used to exam the levels of DNA damage-related protein. The expression of LC3-Ⅱ was determined to evaluate the change of autophagy.RESULTS: MK-2206 treatment increased the formation of γ-H2AX foci and histone H2AX phosphorylation in the SGC-7901 cells. The levels of DNA damage response protein were also increased. In addition, MK-2206-treated SGC-7901 cells increased the expression of LC3-II, a hallmark of autophagy. Inhibition of autophagy significantly enhanced MK-2206-mediated histone H2AX phosphorylation.CONCLUSION: MK-2206 induces DNA damage and autophagy in SGC-7901 cells. Blocking autophagy potentiates the response of MK-2206-induced DNA damage.     

miR-130b reverses temozolomide resistance in glioma

AIM:To investigate the expression level of microRNA-130b (miR-130b) and the molecular me-chanisms of miR-130b in temozolomide (TMZ)-resistant glioma. METHODS:The relative levels of miR-130b in 3 glioma cell lines (U251, SHG-44 and U87) were assessed by RT-qPCR. The half maximal inhibitory concentration (IC₅₀) of TMZ for the glioma cell lines was analyzed. To establish the TMZ-resistant glioma cell line, U251 cells were exposed to gradually increasing concentrations of TMZ. The IC₅₀ and resistance index (RF) were calculated with GraphPad Prism software. miR-130b-overexpressing U251/TR cells and miR-130b-knockdown U251 cells were established by transient transfection with miR-130b mimics and miR-130b inhibitor, respectively. The viability of the glioma cells was measured by CCK-8 assay. The apoptosis of glioma cells was analyzed by Annexin V/PI apoptosis assay. Bioinformatics software was used to predict the potential target gene of miR-130b, and such prediction was validated by luciferase reporter assay. Electrophoretic mobility shift assay was performed to detect the DNA binding ability of NF-κB. Western blot was used to determine the protein levels of tumor necrosis factor-α (TNF-α), Bcl-2, X-linked inhibitor of apoptosis protein (XIAP) and survivin in the glioma cells. RESULTS:The IC₅₀ values of TMZ for the giloma cell lines U251, SHG-44 and U87 were 54.8, 94.8 and 149.6 μmol/L, respectively. U251/TR cells were approximately 8.1 times resistant to TMZ as compared with its parental cells. Up-regulation of miR-130b significantly reduced the resistance of U251/TR cells to TMZ. On the contrary, down-regulation of miR-130b dramatically increased the tolerance of U251 cells to TMZ. The overexpression of miR-130b promoted apoptosis induced by TMZ in the U251/TR cells. However, the knockdown of miR-130b expression decreased the percentage of apoptotic cells in the U251 cells induced by TMZ (P<0.05). Luciferase reporter assay confirmed that TNF-α was a direct target gene of miR-130b. Knockdown of miR-130b in the U251 cells significantly promoted, while overexpression of miR-130b in the U251/TR cells reduced the DNA binding ability of NF-κB as well as the levels of TNF-α, Bcl-2, XIAP and survivin. Furthermore, NF-κB inhibitor Bay 11-7082 enhanced TMZ-induced apoptosis in the U251/TR cells. CONCLUSION:The expression of miR-130b is significantly decreased in TMZ-resistant glioma cells. miR-130b inhibits resistance of glioma to TMZ by targeting TNF-α/NF-κB pathway.     

Effect of EZH2 regulating Wnt/β-catenin signaling pathway on apoptosis of brain glioma cells

AIM: To investigate the effect of enhancer of zeste homolog 2 (EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87, H4 and U251 and normal human astrocytes (NHA) was detected by RT-qPCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway, the apoptosis rate was measured by flow cytometry, and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87, H4 and U251 were significantly higher than those in NHA (P<0.05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells (P<0.05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells, the apoptotic rate was significantly increased, and the activity of caspase-3 was significantly increased in the cells (P<0.05). Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2, and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Effects of lentivirus-mediated RWDD3 silencing on proliferation and invasion of human glioma U251 cells

AIM: To investigate the effect of RWDD3 gene silencing on the biological characteristics of human glioma U251 cells.METHODS: A lentiviral vector expressing RWDD3 shRNA was constructed and transfeeted into the U251 cells. The expression of RWDD3 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The cell activity was determined by MTT assay. The colony formation ability was detected by the colony formation assay. The cell proliferation ability was detected by BrdU incorporation assay. The cell invasion and migration were evaluated by Transwell assay. Flow cytometry was used to monitor the changes of cell cycle distribution and apoptosis.RESULTS: Recombinant lentivirus was successfully transfected into U251 cells. Compared with the cells transfected with the scrambled shRNA and control cells, the cell activity, colony formation ability, and the invasive and migratory activities were inhibited, the cell cycle was arrested in G₀/G₁ phase, and the apoptosis was increased in the U251 cells transfected with RWDD3 shRNA(P<0.05).CONCLUSION: RWDD3 plays a vital role in proliferation and invasion of glioma cells. It may serve as a potential target of gene therapy for glioma.     

Effect of MCPH1 on irradiation induced DNA damage in esophageal cancer cells

AIM: To discover the effect of MCPH1 on the DNA damage induced by ionizing radiation in esophageal cancer cells. METHODS: ECA109 cancer cells were radiated at dose of 8 Gy. The nuclear foci of relevant factors were detected 1 h after irradiation in the ECA109 cells after silence of MDC 1 gene. A cell line was established that was stable low expression of MCPH 1 . The nuclear foci induced by ionizing radiation after silence of MCPH 1 were determined. RESULTS: The MCPH1 gene silenced ECA109 cell line was successfully constructed. A strong relationship between MDC1, MCPH1 and γ-H2AX was observed 1 h after 8 Gy irradiation. Silence of MDC 1 did not affect the nuclear foci formation of γ-H2AX and MCPH1. The nuclear foci of MDC1 but not γ-H2AX significantly reduced after silencing of MCPH 1 . CONCLUSION: MCPH1 is located in the downstream of H2AX and upstream formation of MDC1, and regulates the nuclear foci formation of MDC1 during DNA damage response.     

Effect of tetrandrine on radiosensitivity of nasopharyngeal carcinoma cells

AIM: To investigate the mechanism of the radiosensitizing effect of maximum non-cytotoxic doses of tetrandrine (Tet) on nasopharyngeal carcinoma cell lines CNE1 and CNE2.METHODS: The cells were treated with ma-ximum non-cytotoxic doses of Tet (for CNE1 cells at 1.5 μmol/L and for CNE2 cells at 1.8 μmol/L), irradiation at 4 Gy, or combination of irradiation and maximum non-cytotoxic doses of Tet. The cell cycle distribution was analyzed by flow cytometry. The protein levels of γ-H2AX, cleaved caspase-3, p-CDC25C, CDK1, p-CDK1, cyclin B1, ERK and p-ERK were determined by Western blot.RESULTS: The expression of γ-H2AX was increased in CNE1 cells and CNE2 cells after combined treatment with irradiation and maximum non-cytotoxic doses of Tet. The percentages of CNE1 cells and CNE2 cells at G₂/M phase in irradiation group were (18.09±0.42)% and (18.48±1.32)%, respectively, which were decreased to (15.88±1.04)% and (13.80±0.82)% in combined treatment group, respectively (P<0.05). Combined treatment enhanced the increase in the protein level of cleaved caspase-3 caused by irradiation. The protein levels of p-CDC25C and p-CDK1 were increased in a dose-dependent manner by Tet treatment (P<0.05), while the expression of CDK1 showed no difference among different doses of Tet treatments. The protein levels of p-CDC25C, p-CDK1 and CDK1 showed no difference after the treatment with maximum non-cytotoxic doses of Tet. The combined treatment with irradiation and the maximum non-cytotoxic doses of Tet decreased the protein levels of p-CDC25C and p-CDK1 (P<0.05), increased the expression of cyclin B1, and had no influence on the expression of CDK1 (P<0.05). The combined treatment resulted in an increase in the protein level of p-ERK1 (P<0.05).CONCLUSION: The maximum non-cytotoxic doses of Tet enhance the DNA damage and apoptosis in CNE1 cells and CNE2 cells caused by irradiation, and the mechanism might be associated with ending of G₂/M arrest via activation of ERK/CDC25C/CDK1/cyclin B1 pathways.     

Effects of microRNA-483-3p on human glioma cells

AIM:To investigate the effects of microRNA (miRNA)-483-3p on the growth and migration of human glioma cell line A172 and its potential mechanisms.METHODS:The abundance of miRNA-483-3p in human embryonic kidney 293 cells and different human glioma cell lines (A172,U251 and SHG44) was measured by RT-qPCR.After down-regulation of miRNA-483-3p by transfection of inhibitor in the A172 cells,the cell viability,cell cycle distribution and cell migration were detected by CCK-8 assay,flow cytometry and Transwell assay,respectively.Furthermore,the protein levels of cell cycle-related molecules and epithelial-mesenchymal transition markers were measured by Western blot.Luciferase reporter assay was used to predict and verify the target gene of miRNA-483-3p.RESULTS:miRNA-483-3p was highly expressed in human glioma cells.Knockdown of miRNA-483-3p inhibited A172 cell viability,arrested cell cycle and decreased cell migration rate.Furthermore,the protein levels of cyclin D1,cyclin-dependent kinase 4,phoshorylated retinoblastoma protein,N-cadherin and vimentin were significantly decreased after knockdown of miRNA-483-3p,accompanied with the up-regulation of E-cadherin and β-catenin protein expression.Luciferase reporter assay demonstrated that Smad4 was a potential target gene of miRNA-483-3p.Down-regulation of Smad4 in the A172 cells transfected with miRNA-483-3p inhibitor partially reversed the effect of miRNA-483-3p on cell viability and migration.CONCLUSION:Knockdown of miRNA-483-3p restrains the growth and migration of A172 cells by targeting Smad4.     

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.     

mTOR kinase inhibitor CC-223 suppresses human breast cancer cell viability

AIM: To investigate the inhibitory effect of CC-223, an inhibitor of mammalian target of rapamycin (mTOR) kinase, on the viability of human breast cancer cells and its mechanism. METHODS: The inhibitory effect of CC-223 on the viability of MCF-7 cells and MDA-MB-231 cells was measured by CCK-8 assay. The cell cycle distribution of breast cancer cells was examined by flow cytometry. The expression of cell cycle-related proteins and oncoproteins c-Myc and survivin was analyzed by Western blot. RESULTS: CC-223 significantly inhibited the viability of both MCF-7 and MDA-MB-231 cells (P<0.05). CC-223 induced cell cycle arrest in both G₁ phase and G₂/M phase in the MCF-7 cells (P<0.05). However, low concentration of CC-223 treatment resulted in the accumulation of MDA-MB-231 cell cycle in the G₂/M phase, and the cell number in G₁ phase was unaffected. Treatment with CC-223 for 24 h clearly inhibited the protein levels of cyclin B1, cyclin D1 and phosphorylated cell division cycle protein 2 in the breast cancer cells (P<0.05). CC-223 suppressed the expression of c-Myc and survivin in both MCF-7 cells and MDA-MB-231 cells (P<0.05). CONCLUSION: CC-223 inhibits cell viability by blocking cell cycle progression and down-regulating expression of c-Myc and survivin in both MCF-7 and MDA-MB-231 cells.     

miR-205 inhibits invasion of glioma cells via targeting TBX18

AIM: To explore the expression pattern of microRNA-205 (miR-205) in glioma tissues and its role in the invasion of glioma cells. METHODS: The expression of miR-205 and TBX18 was detected by real-time PCR and immunohistochemical observation, respectively. Transwell assay was used to examine the invasion change of U251 glioma cells after miR-205 overexpression via miR-205 mimics or decrease in miR-205 expression by miR-205 inhibitor. The target of miR-205 was searched by bioinformatics analysis combined with experimental analysis. The protein level of TBX18 was determined by Western blotting after siRNA transfection and Transwell assay was conducted. RESULTS: miR-205 expression was downregulated in 82.6% of detected glioma tissues and TBX18 was significantly overexpressed in glioma tissues compared with normal tissues. miR-205 overexpression remarkably inhibited the invasion potential of U251 glioma cells with a decrease in the invasive cells (P<0.01), while inhibition of miR-205 significantly enhanced the invasion ability of U251 cells. Mechanically, miR-205 directly targeted TBX18 and downregulation of TBX18 also significantly inhibited the invasion potential of U251 cells with a decrease in the invasive cells (P<0.01). CONCLUSION: miR-205 expression is decreased in glioma, and miR-205 inhibits glioma cell invasion via targeting TBX18. Our research contributes to the mechanisms responsible for glioma invasion and provides theoretical base for developing new therapeutic strategy to treat glioma.     

Effects of axitinib on biological behavior of adrenocortical carcinoma cell line SW-13

AIM: To investigate the effects of axitinib on the biological behavior of adrenocortical carcinoma cell line SW-13. METHODS: CCK-8 assay was used to measured the viability of SW-13 cells treated with axitinib at different concentrations. The cell cycle distribution was analyzed by flow cytometry. The apoptotic rate was also analyzed by flow cytometry with Annexin V/PI double staining. Wound healing experiment and Transwell invasion assay were used to observe cell migration and invasion abilities,respectively. The protein levels of vascular endothelial growth factor receptor 2(VEGFR2), extracellular regulated protein kinases 1/2 (ERK1/2) and p-ERK1/2 were determined by Western blot. RESULTS: After treated with axitinib, the viability of SW-13 cells was significantly inhibited, the cell cycle was blocked in G₂/M phase, and the apoptosis rate was increased. The migration and invasion abilities of SW-13 cells were markedly inhibited by axitinib (P<0.01). The protein levels of VEGFR2 and p-ERK1/2 in the SW-13 cells were significantly decreased with axitinib treatment (P<0.01). CONCLUSION: Axitinib inhibits the viability, blocks the cell cycle, promotes cell apoptosis, and inhibits the migration and invasion abilities of SW-13 cells. The mechanism may be related to inhibition of VEGFR2 expression and reduction of ERK1/2 phosphorylation.     

Effects of notch1 gene on proliferation and cell cycle of human glioma U251 cells

AIM: To investigate the effect of notch1 gene on the change of proliferation and cell cycle in human glioma U251 cell line. METHODS: The lentiviral vectors, which express notch1 shRNA or notch1 intracellular domain (NICD), were constructed and transfected into U251 cells, respectively. RT-PCR and Western blotting were applied to monitor the validity of down-regulation of notch1 expression and over-expression of NICD. MTT assay was performed to examine the cell proliferation. Flow cytometric analysis was used to detect the cell cycle. RESULTS: The lentiviral vectors, which expressed notch1 shRNA and NICD, were efficient in silencing notch1 expression and over-expression of NICD. Down-regulation of notch1 gene by RNAi inhibited the cell proliferation remarkably (P<0.01), arrested cell cycle at G1 phase (P<0.01) and decreased the cell number of S phase (P<0.01). Over-expression of NICD enhanced the cell proliferation significantly (P<0.01), promoted the cell cycle at G1 phase (P<0.05) and increased the cell number of S phase (P<0.01). CONCLUSION: notch1 gene, which leads to change the proliferation and cell cycle in human glioma U251 cell line, is likely to be potential molecular target for glioma in gene therapy.     

Effects of up-regulation of c-myc expression on U937 cell line

AIM:To establish a human monocytic　leukemia cell line U937 stably expressing c-myc gene and to investigate the biological characteristics of this cell line. METHODS:The recombinant plasmid MSCV-c-myc-IRES-GFP (MMIG) was constructed. MMIG and MSCV-IRES-GFP (MIG) were used to package the viruses for infecting U937 cells. Fluorescence-activated cell sorter (FACS) was used for sorting U937/GFP and U937/MYC cells. The GFP-positive cells were detected by fluorescence microscopy and FACS. The protein expression of c-Myc, survivin, X-linked inhibitor of apoptosis protein (XIAP) and Bcl-2 was detected by Western blotting. Cell proliferation was evaluated by MTT assay. Propidium iodide (PI) staining was used to determine the cell cycle distribution. Self-renewal ability was observed by colony- forming assay. RESULTS:The GFP expression in the cells infected with MIG or MMIG virus was observed under fluorescence microscope. The green fluorescent rate of the cells infected with MIG was 26.0%, while that of the cells infected with MMIG was 27.7%. The protein expression of c-Myc in MMIG-infected U937 cells was higher than that in MIG-infected cells. After sorting, the green fluorescent rates of U937/GFP and U937/MYC cells reached 98.7% and 93.7%, respectively. The protein expression of c-Myc in U937/MYC cells was higher than that in U937/GFP cells. In addition, survivin, a downstream protein of c-Myc, was up-regulated, while the protein expression of XIAP and Bcl-2 remained unchanged. Cell cycle analysis showed that the percentage of the cells in S phase increased in U937/MYC cells. Moreover, the proliferation and colony-forming ability of U937/MYC cells were also enhanced. CONCLUSION: U937/MYC cell line stably expressing c-myc gene was successfully established. c-Myc may increase cell viability via enhancing the expression of anti-apoptotic protein survivin, the cell cycle transition and the self-renewal ability.     

LPS induced B7-H1 expression in pancreatic carcinoma Panc-1 cells

AIM: To explore the mechanism of lipopolysaccharide (LPS)-induced B7-H1 expression in pancreatic carcinoma cell line Panc-1. METHODS: The levels of phosphorylated p38 mitogen-activated protein kinase (p-p38), phosphorylated extracellular signal-regulated kinase (p-ERK) and phosphorylated c-Jun N-terminal kinase (p-JNK) after stimulated with LPS or treated with mitogen-activated protein kinases (MAPKs) inhibitors were detected by Western blotting. The expression of B7-H1 in Panc-1 cells after LPS stimulation or MAPKs inhibitor treatment was measured by real-time PCR and Western blotting. RESULTS: The levels of B7-H1, p-p38, p-ERK and p-JNK were up-regulated with LPS stimulation. The promoted p-p38, p-ERK and p-JNK levels induced by LPS were inhibited by the corresponding MAPKs inhibitors. Furthermore, the inhibitors of p38 and ERK attenuated LPS-induced B7-H1 expression. However, JNK inhibitor had very little effect on LPS-induced B7-H1 expression. CONCLUSION: LPS induces B7-H1 expression in pancreatic carcinoma cell line Panc-1. ERK and p38 are involved in this regulation as the inhibitors of ERK and p38 attenuate LPS-induced B7-H1 expression.     

Inhibition of proliferation and influence of proto-oncogenes expression by Tanshinone ⅡA in U251 cells

AIM: To investigate the inhibitory effect of Tanshinone ⅡA on U251 glioma cell line and its mechanism. METHODS: MTT was used to measure the levels of the proliferation in U251 cultured with Tanshinone ⅡA at different concentrations. The effects of Tanshinone ⅡA on cell cycle of U251 were observed by FCM. The expression of proto-oncogene c-myc was measured by RT-PCR. RESULTS: The proliferation of U251 was obviously inhibited by Tanshinone ⅡA in a dose dependent manner. The inhibitory rate came to the peak at (54.2±0.9)%, when cultured with Tanshinone ⅡA at 0.10 g/L. The outcome of FCM showed that the proportion of G₀/G₁ phase cells was increased and the proportion of S phase cells was reduced obviously, when cultured with Tanshinone ⅡA at 0.10 g/L for 3 days. The RT-PCR experiment showed that the expression of proto-oncogene c-myc was notably decreased, when the dose of Tanshinone ⅡA was increased. CONCLUSION: Tanshinone ⅡA inhibited the proliferation of U251 and the expression of proto-oncogene c-myc.