Rb1 protects human umbilical vein endothelial cells from hydrogen peroxide-induced senescence:involvement of caveolin-1

AIM: Endothelial cell senescence has been proposed to be involved in endothelial dysfunction and atherogenesis. This study aims to investigate the effects of ginsenoside Rb1, a major constituent of ginseng, on hydrogen peroxide (H₂O₂)-induced endothelial cell senescence, and to explore the expression and production of caveolin-1 in H₂O₂-induced premature senescence.METHODS: The senescence of primary human umbilical vein endothelial cells (HUVECs) was induced by H₂O₂ as judged by morphology inspection, senescence-associated β-galactosidase (SA-β-Gal) staining and cell cycle detection. The protein expression of caveolin-1 was determined by Western blot and confocal laser-scanning microscopy.RESULTS: Treatment of the HUVECs with H₂O₂ at 60 μmol/L induced premature senescence, as judged by enlarged, flattened cell morphology, increased SA-β-Gal activity and sustained growth arrest. H₂O₂ effectively increased caveolin-1 level. Pretreatment of the HUVECs with Rb1 was found to reverse endothelial cell senescence, as witnessed by a significant decrease in senescent cell numbers and a decreased percentage of G₀/G₁ phase cells. Furthermore, Rb1 administration reversed the H₂O₂-increased protein level of caveolin-1.CONCLUSION: Ginsenoside Rb1 antagonizes H₂O₂-induced endothelial cell senescence through caveolin-1 modulation.     

Effects of aspirin on cell cycle in pancreatic carcinoma cells

AIM: To observe the effects of aspirin and prostaglandin E2 (PGE2) on the cell viability and cell cycle in SW1990 human pancreatic carcinoma cell lines, and to investigate the mechanisms of aspirin-induced growth inhibition and cell cycle arrest. METHODS: After incubated with aspirin or PGE2 and their combination, the viability of SW1990 cells was measured by MTT assay. The levels of intracellular PGE2 were determined by ELISA. The effects of aspirin or PGE2 on cell cycle were investigated by flow cytometry （FCM）. The expression of p21Wafl/cipl and p27Kipl/pic2 (the cyclin-dependent kinase inhibitors) were analyzed by Western blotting. RESULTS: Aspirin could inhibit the growth of cells and level of intracellular PGE2 in a dose-dependent manner. Aspirin enhanced the expression of p21Wafl/cipl and p27Kipl/pic2 and induced cell cycle arrest at G0/G1 phase. PGE2 increased the cell viability of SW1990 cells. However, it couldn't antagonize the changes of cell viability and cell cycle that induced by aspirin. CONCLUSIONS: The inhibitory effects of aspirin on growth and cell cycle of pancreatic carcinoma cells might not be mediated by a COX-dependent pathway completely. Cell cycle arrest induced by aspirin might be associated with up-regulation of p21Wafl/cipl and p27Kipl/pic2.     

Establishment of arsenic trioxide-resistant K562/AS2 cell line and the mechanism of multidrug resistance

AIM:To establish a arsenic trioxide (As₂O₃ )-resistant leukemic cell line to explore the mechanism of resistance to As₂O₃, and the relationship between the resistant cell line and the multidrug resistance was also investigated. METHODS:The arsenic trioxide (As₂O₃ )-resistant leukemic cell line was established by exposing the cells to the increasing concentration of As₂O₃. MTT assay was used to detect the cytotoxicity. Cell cycle was detected by PI assay. Flow Cytometry was used to detect the P-glycoprotein on the surface of the cells, the intracellular concentration of DNR, and the immuetype of the cells. RESULTS:The cell doublings time and the cell cycle of the arsenic trioxide (As₂O₃ )-resistant leukemic cell line, K562/AS2, is similar to that of K562. The relative resistant fold of K562/AS2 to As₂O₃, DNR, VP16 and Ara-C was 7.4, 2.9, 3.8 and 1.1, respectively. The relative resistant fold of multidrug resistant cell line, K562/ A02, to As₂O₃, DNR, VP16 and Ara-C was 0.8、94、2.5 and 0.9, respectively. The fluorescence of the P-glycoprotein on the surface or of the DNR inside the cells detected was not significantly different between the K562 and the K562/AS2 cell lines. CONCLUSIONS:A cell line, K562/AS2, resistant to clinical achieving level (2 μmol/L) of As₂O₃ has been established. The relative resistant fold of K562/ AS2 to As₂O₃ is about 7.4 fold to the parent K562 line sensitive to As₂O₃. Partial resistance of K562/AS2 to DNR and VP16 is observed , the mechanism of which is unrelated to the P-gp, the expression product of multidrug resistance gene 1 (mdr1).     

Effects of curcumin analogue B50 on proliferation and apoptosis of homologous nasopharyngeal carcinoma cells with different radioresistance

AIM: To compare the effects of B50, a mono-carbonyl analogue of curcumin, on the proliferation and apoptosis between homologous nasopharyngeal carcinoma cells CNE-2R and CNE-2 with different radioresistance.METHODS: The effects of B50 on cell viability and cell growth were detected by MTT assay and colony-forming experiment, respectively. The changes of cell cycle, apoptosis and mitochondrial membrane potential (MMP) were determined by flow cytometry.RESULTS: B50 inhibited the cell viability of CNE-2R cells in a time-and dose-dependent manner with the IC₅₀ of (8.06±0.14) μmol/L (24 h), (2.49±0.02)μmol/L (48 h) and (1.42±0.02) μmol/L (72 h), which was more effective than that in CNE-2 cells . The inhibitory effect of B50 on CNE-2R cell growth was more effective than that on CNE-2 cells . After treated with B50 for 48 h, the proportion of CNE-2R cells in G₂/M stage was increased from 7.1% to 34.9%, which was better than that of CNE-2 cells (from 12.4% to 35.7%). After treated with B50 for 24 h, the early apoptotic rate in CNE-2R cells was increased from 3.7% to 19.5%, which was better than that in CNE-2 cells (from 4.4% to 14.8%), and the MMP in CNE-2R cells was decreased by (43.17±3.11)%, which was better than that in CNE-2 cells .CONCLUSION: B50 is more effective on inhibiting the cell viability and cell growth, blocking the cell cycle at G₂/M stage, inducing apoptosis and decreasing MMP in CNE-2R cells than those in CNE-2 cells, indicating that B50 may enhance the radio-sensitivity of CNE-2R cells by blocking the cell cycle and inducing apoptosis through mitochondrial pathway.     

Construction and identification of FRT cell line stably expressing UT-A2

AIM: To obtain an FRT cell line that can stably express urea transporter A2 (UT-A2) and provide a cell model for screening UT-A2 inhibitors. METHODS: FRT cells stably expressing aquaporins 1 (AQP1) and YFP were transfected with the recombinant plasmid pUB6/V5-UT-A2 by eukaryotic expression plasmid-lipoplast mediating pathway. The stable UT-A2-FRT cell line was cloned by selection with BSD and confirmed by Western blotting and immunofluorescence staining. The urea permeability across the plasma membrane was detected by a 2 mol/L urea lysis assay. RESULTS: We have obtained a stable UT-A2-FRT cell line. Western blotting analysis showed that UT-A2 protein was expressed stably in this cell line. The immunofluorescence staining detection indicated UT-A2 expression in the plasma membrane. It was found that there was significant urea permeability in this cell line by 2 mol/L urea lysis assay. CONCLUSION: We constructed an FRT cell line that could stably express UT-A2 in plasma membrane in the non-renal epithelia cell. The cell line will be used to screen UT-A2 inhibitors.     

An oxidative injury model of human kidney epithelial cells

AIM: To establish an injured cell model using human kidney proximal tubule epithelial cell line (HKC) to mimic the oxidative injury by hydrogen peroxide (H₂O₂). METHODS: The cell viability, the content of malondialdehyde (MDA) in the culture supernatant and the activity of intracellular superoxide dismutase (SOD) were detected to investigate the degree of cell injury. Osteopontin (OPN) expressed on the cell membrane surface were observed by laser confocal microscopy before and after cell injury. The changes of cellular morphology and the ultrastructure of membrane surface were observed under scanning electronic microscope. RESULTS: After HKC cells were treated with H₂O₂ at the concentration of 1 mmol/L for different time, the cell viability and the activity of SOD decreased and the content of MDA increased. The expression level of OPN significantly increased and reached to maximae at 1 h. The injured cells appeared shriveled and rough surface, and the shedding of most flagellae was also observed. CONCLUSION: H₂O₂ induces severer injury in HKC cells, including not only the cell viability and membrane surface ultrastructure, but also the OPN expression on the membrane, which could bind calcium oxalate crystal. Therefore, treatment with H₂O₂ at the concentration of 1 mmol/L for 1 h can be used to establish an oxidative injury model in HKC cells.     

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.     

Effect of P2X7R gene silencing by RNA interference on proliferation and phagocytosis of murine macrophage cell line RAW264.7

AIM: To establish a cell line of stable silencing of P2X₇ receptor (P2X₇R) expression through short hairpin RNA (shRNA)-mediated interference in murine RAW264.7 macrophages, and to investigate the proliferation and apoptosis in the cell line. METHODS: Stable silencing of P2X₇R gene in the RAW264.7 cells was achieved by recombinant shRNA plasmid targeting murine P2X₇R gene via liposome mediated transfection, followed by G418 selection. The efficacy of plasmid transfection and P2X₇R silencing in G418 resistant cells was verified by immunofluorescent microscopy and real-time PCR, respectively. The proliferative activity was analyzed by CCK-8 assay and EdU cell proliferation assay. The cell cycle distribution and apoptosis were evaluated by flow cytometry. RESULTS: The expression of P2X₇R at mRNA and protein levels was down-regulated by 80% in sh P2X₇R group compared with negative control (NC) plasmid transfection. In addition, P2X₇R-silencing cells exhibited higher proliferative activity compared with NC and wild-type RAW264.7 cells (P<0.05). Compared with NC cells, P2X₇R silencing resulted in an increase in the phagocytosis of the cells (P<0.05). CONCLUSION: A cell line RAW264.7 of stable silencing of P2X₇R expression was successfully established. P2X₇R gene silencing stimulates the proliferation, and changes phagocytic function in murine RAW264.7 macrophages.     

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.     

Relationship between EGFR/HER2 gene knocked down and the downstream signal pathway in SPC-A-1

AIM: RNAi technique was applied to explore the relationship between downstream signal pathway in EGFR family and the cell proliferation, cell cycle alteration, apoptosis after EGFR/HER2 RNA interference. METHODS: Sequence-specific siRNA for EGFR and HER2 were designed as literatures described. “Tuschl rules” and BLAST on the full length of EGFR/HER2 cDNA were used to ensure the sequence-specific siRNA for EGFR/HER2 joint interference. Based on the above sequence-specific siRNA, recombinant plasmids with GFP and neomycin resistance marker were constructed. Six groups including mock, negative control, shRNA-EGFR, shRNA-HER2, shRNA-EGFR/HER2 and shRNA-EGFR+shRNA-HER2 were established by transient transfection. Real time quantitative RT-PCR was used to detect the silencing of the EGFR/HER2 gene level. Western blotting was used to measure the levels of EGFR/HER2 protein and protein phosphorylation expression. Transfected cells were stimulated with EGF 15 min before protein extraction. MTT assay and flow cytometry were used to evaluate the cell proliferation, apoptosis and cell cycle distribution after RNAi. The protein expression levels of downstream signaling pathway proteins including Akt, p-Akt, p-Erk1/2, p-p38 were measured by real time quantitative RT-PCR and Western blotting. Randomized block analysis of variance and SNK methods were used to compare the differences between groups. RESULTS: Cell proliferation was inhibited in the groups of shRNA-EGFR, shRNA-HER2, shRNA-EGFR+shRNA-HER2 and shRNA-EGFR/HER by MTT assay. Cell cycle analysis by flow cytometry showed that apoptosis ratio in shRNA-HER2 (P<0.01), shRNA-EGFR/HER2 (P<0.01) and shRNA-EGFR+shRNA-HER2 (P<0.05) groups were significantly higher than those in negative control group, while there was no statistical difference between shRNA-EGFR and negative control (P>0.05), and that the distributions in phase G1 and phase S in shRNA-EGFR (P<0.01), shRNA-HER2 (P<0.01), shRNA-EGFR/HER2 (P<0.01) and shRNA-EGFR+shRNA-HER2 (P<0.01) were significantly different compared with the negative control. The level of EGFR/HER2 protein and protein phosphorylation expression were down regulated. The cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference showed no significant relationship with downstream signal pathway molecular in EGFR family. CONCLUSION: EGFR gene knockdown may not cause significant apoptosis in SPC-A-1 cell line. The variations of cell proliferation, apoptosis and cell cycle alterations induced by EGFR/HER2 RNA interference were not found to have significant relationship with downstream signal pathway molecules in EGFR family.     

Role of PAR-2 in tryptase mediated IEC-6 cell injury

AIM: To investigate the role of protease activated receptor-2 (PAR-2) in the process of tryptase mediated IEC-6 cell injury. METHODS: The rat intestinal epithelial cell line IEC-6 was treated with tryptase at different concentrations (1 μg/L, 10 μg/L, 100 μg/L and 1 000 μg/L) in the presence or absence of PAR-2 antagonist FSLLRY-NH2 for 12 h respectively. The cell survival rate was detected by MTT assay. The protein levels of PAR-2 and cleaved-caspase 3 were determined by Western blotting. The LDH activity was also measured. RESULTS: Compared with control group, the cell survival rates were significantly decreased in 100 μg/L and 1 000 μg/L tryptase treated groups, the LDH activities were significantly increased in 10 μg/L to 1 000 μg/L tryptase treated groups, and the protein levels of PAR-2 and cleaved caspase 3 were significantly increased in 100 μg/L and 1 000 μg/L tryptase treated groups (P<0.05). Compared with 1 000 μg/L tryptase treated group, the LDH activity and cleaved caspase 3 protein level were dramatically decreased while the survival rate was significantly increased in the presence of PAR-2 antagonist FSLLRY-NH2 (P<0.05). CONCLUSION: Tryptase induces IEC-6 cell injury in a dose-dependent manner by activating PAR-2.     

Experimental study on effects of ATP and its derivatives on the proliferation of the immortalized human fibroblasts

AIM: To investigate the inhibitory effects of ATP on proliferation of immortalized human fibroblasts, and learn what subtypes of P-2 purinergic receptors are involved in ATP cytotoxicity.METHODS:The immortalized human fibroblast cell line KMST-6 was cultured with ATP and its derivatives including ADP, β,γ-methyleneadenosine5'-triphosphate(MeATP), 2'&3'-O-(4-benzoylbenzoyl) adenosine, triethylammonium salt (BzATP), adenosine 5'-O-(3-methiotriphosphate)(ATPγS), 2-methylthioadenosine 5'-triphosphate(2-MeATP) and UTP, and the cell cycle analysis was finished using flow-cytometer, the cell apoptosis analysis was made using Hoechst 33258 and examination of DNA ladder.RESULTS: The extent of cytotoxicity induced by these drugs was found to be in order of : ATP=ADP＞ATPγS＞MeATP=BzATP, neither 2-MeSATP nor UTP showed any cytotoxicity. No enhanced expression of P²¹ was observed and the cell cycle was held in G₁/S in the cells treated with 0.4mmol/L;no cell apoptosis was detected in the cells treated with 1mmol/L ATP.CONCLUSION: Connecting with P_2X or P_2Y purinergic receptors,ATP activated some intracellular signals to inhibit cell growth, the growth inhibition caused by ATP was not due to apoptosis or induction of cyclin/CDK kinase inhibitor,P²¹.     

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.     

Interleukin-2 pretreatment improves the contractility of isolated rat ventricular myocytes during hypoxia and reoxygenation

AIM: To investigate the effect and possible mechanisms of interleukin-2 (IL-2) on the cell contractility in cardiomyocytes during hypoxia and reoxygenation.METHODS: Glucose-free Krebs-Henseleit (K-H) solution, gassed with 95% N2 and 5% CO2 for hypoxia, were used. The cell contractility were determined after 20 min of hypoxia and 30 min of reoxygenation by the video tracking system. The parameters of cell contractility included peak velocity of cell shortening (+dL/dtmax), peak velocity of cell relengthening (-dL/dtmax), contraction amplitude (dL) and end-diastolic cell length.RESULTS: It was shown that during hypoxia, the cell contraction was depressed. All the parameters were unable to return to the pre-hypoxia level during reoxygenation. Pretreatment with IL-2 at 2×103 U/L attenuated the inhibitory effect of hypoxia/reoxygenation on contractility in single ventricular myocytes. The effect of IL-2 was reduced in the presence of 10-8 mol/L nor-binaltorphimine (nor-BNI), a selective κ-opioid receptor antagonist. On blockade of protein kinase C with 3×10-6 mol/L chelerythrine, the effect of IL-2 was significantly attenuated. The effect of IL-2 was also blocked by 10-4 mol/L 5-hydroxydecanoate (5-HD), a mitochondrial ATP-sensitive potassium (KATP) channel blocker. CONCLUSIONS: The results of the present study provide evidence that pretreatment with IL-2 at 2×103 U/L attenuates the effect of hypoxia/reoxygenation on cell contraction in the isolated ventricular myocytes. The κ-opioid receptor mediates the effect of IL-2, in which activation of PKC and opening of mitochondrial ATP-sensitive potassium (mito KATP) channel are involved.     

Adiponectin attenuates H2O2-induced SH-SY5Y cell injury and tau hyperphosphorylation via activating PP2A

AIM: To study the effects of adiponectin on H₂O₂-induced cell injury and tau hyperphosphorylation in human neuroblastoma SH-SY5Y cells. METHODS: Cell viability was determined by MTT assay. H₂O₂-induced cell injury and morphological changes in the SH-SY5Y cells with or without adiponectin treatment were observed. The level of tau phosphorylation as well as the activities of protein phosphatase 2A(PP2A) and of glycogen synthase kinase-3β(GSK-3β) were examined by Western blotting. RESULTS: Adiponectin significantly attenuated H₂O₂-induced cell injury(P<0.01). Adiponectin upregulated the activity of PP2A and decreased phosphorylation levels of tau under the stimulation with H₂O₂ (P<0.01). Okadaic acid, a specific inhibitor of PP2A, blocked the protective effects of adiponectin(P<0.01). Adiponectin increased the phosphorylation level of GSK-3β at Ser9 site under H₂O₂ stimulation(P<0.01). CONCLUSION: Adiponectin protects SH-SY5Y cells against H₂O₂-induced cell injury and decreases tau hyperphosphorylation by activating PP2A and inactivating GSK-3β.     

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.     

Tumorigenicity and invasiveness induced by ErbB2 are dependent on FAK

AIM: To explore whether or not ErbB2-induced oncogenic transformation and invasion are mediated by FAK.METHODS: Parental FAK-/- and FAK+/+ cells were used and infected with retrovector particles expressing ErbB2 in order to acquire ErbB2-overexpressed cells,i.e.,FAK-/--ErbB2 and FAK+/+-ErbB2.The role of FAK signaling was explored by analyzing the parameters such as cell survival,invasiveness and tumorigenicity shown by both FAK-/- and FAK+/+ cells in which ErbB2 was overexpressed.RESULTS: ErbB2 was overexpressed and functionally activated in both FAK-/- and FAK+/+ cells upon infection with retrovector particles.The ErbB2-induced anchorage-dependent cell survival,cell invasiveness as well as tumorigenicity in vivo were dependent on FAK.CONCLUSION: FAK is essential for cell survival,tumorigenicity and invasiveness induced by ErbB2,and its possible mechanism involves in ErbB-FAK-Src-MAPK 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.