Effect of decitabine on proliferation and differentiation of K562 cells

AIM: To investigate the effect of decitabine (Dacogen, DAC) on the proliferation and differentiation of K562 cells. METHODS: The K562 cells were treated with different concentrations of DAC. The colony formation ability of the cells was detected by the colony formation assay with semi-solid culture. The cell viability was detected with MTT assay. The morphologic features were observed under inverted microscope with Wright's staining. The changes of the cell cycle distribution and the expression of CD11b and CD42b were analyzed with flow cytometry. The protein expression of CDK2, cyclin E1, P27, GATA-1 and PU.1 in the K562 cells was determined by Western blot. RESULTS: DAC significantly decreased the colony number of the cells and cell viability in a dose-dependent manner. The morphological changes of the cells displayed partial differentiation. After treated the K562 cells with DAC for 72 h, the cell proportion in S phase was obviously decreased, while the cell proportion in G₂/M phase was obviously increased in a dose-dependent manner. After treated the K562 cells with DAC for 7 d, the percentage of CD11b and CD14 positive cells was further elevated, and the protein expression of P27, GATA-1 and PU.1 was increased. However, the protein expression of CDK2 and cyclin E1 was decreased. CONCLUSION: DAC inhibits the proliferation and induces differentiation of the K562 cells via regulation of cell cycle.     

Knockdown of RALA by siRNA inhibits cell proliferation and induces apoptosis in human leukemic K562 cells

AIM: To investigate the effect of siRNA-induced knockdown of v-ral simian leukemia viral oncogene homolog A(RALA) on proliferation and apoptosis of chronic myelogenous leukemia(CML) K562 cells. METHODS: The chemically synthesized siRNA targeting to RALA gene was transfected into K562 cells using Lipofectamine^TM 2000. The proliferation and viability of K562 cells were detected by MTT assay and trypan blue dye exclusion. The expression levels of RALA mRNA and protein were determined by quantitative real-time PCR and Western blotting,respectively. The cell apoptosis was analyzed using flow cytometry by double staining with annexin V and propidium iodide, and the apoptotic morphological changes were detected by Hoechst 33258 staining. RESULTS: RALA siRNA significantly down-regulated RALA mRNA and protein expression in K562 cells(P<0.05). The proliferation of K562 cells in RALA siRNA group was inhibited compared with control group(P<0.05). The apoptotic rate was much higher in RALA siRNA group than that in negative control group(P<0.05). The apoptotic morphological changes were observed in the nuclei of K562 cells transfected with RALA siRNA. CONCLUSION: The siRNA-mediated knockdown of RALA results in inhibition of proliferation and induction of apoptosis in K562 cells, indicating that RALA might be used as a potential therapeutic target in chronic myelogenous leukemia.     

Effect of miR-16 on megakaryocytic differentiation of K562 cells

AIM: To observe the effect of microRNA-16 (miR-16) on the megakaryocytic differentiation of K562 cells, and to explore the potential mechanism.METHODS: miR-16 was over-expressed or silenced by transfection with miR-16 mimics or inhibitor in K562 cells. The level of miR-16 was detected by real-time PCR. The expression of CD41, CD42b and CD61, as megakaryocytic differentiation markers, was detected by flow cytometry. The effect of miR-16 on the expression of myeloblastosis oncogene (MYB) was measured by Western blotting, and flow cytometry was performed to confirm whether the effect of miR-16 on expression of CD41, CD42b and CD61 was mediated by MYB.RESULTS: Transfection with miR-16 mimics dramatically elevated the level of miR-16 and the expression of CD41, CD42b and CD61 in the K562 cells. Transfection with miR-16 inhibitor decreased the level of miR-16 and the expression of CD41, CD42b and CD61 in the K562 cells (P<0.05). The expression of MYB was regulated by miR-16, and MYB silencing reversed the regulation of CD41, CD42b and CD61 induced by miR-16.CONCLUSION: miR-16 regulates the megakaryocytic differentiation of K562 cells by targeting MYB.     

Effect of glutamine deprivation on growth and differentiation of primary APL cells

AIM:To investigate the effect of Gln deprivation on growth and differentiation of primary APL cells. METHODS: The primary APL cells were collected from 18 cases of APL patients’ periphery blood, the patients had not been treated. The cells were incubated in RPMI 1640 without or with different contents of glutamine and with 10% fetal calf sera, at 37℃,5% CO 2 and saturation humidity for 4 days.The initial living cell density was 5×10⁸/L. After 4 days incubation,the cells were counted,collected, smeard and stained with Wright-Giemsa, DNA, POX, NAE and NaF inhibition,gulping ink and NBT etc. cytochemical technology. The stained cells were investigated under oil immersion lens. RESULTS: The living cell density became (54.28±4.28)% of the initial in glutamine deprivation group, but the living cell density in control with 4 mmol/L Gln was (108.56±12.27 )% of the initial ( P <0.01) after 4 days incubation. The cells differentiated into mature granulocytes in glutamine deprivation group.CONCLUSION: Deprivation of glutamine inhibited primary APL cell growth and induced cell differentiation toward mature granulocyte.     

Effect of tanshinone IIA on expression of cell cycle regulators and proliferation of pancreatic cancer cells

AIM：To observe the effect of tanshinone IIA on the expression of cell cycle regulators and the proliferation of pancreatic cancer cell line BX-PC-3. METHODS： The pancreatic cancer cell line BX-PC-3 was treated with tanshinone ⅡA at various concentrations for 48 h. The inhibition of proliferation was measured by MTT method. The change of the cell cycle was detected by flow cytometry. The protein levels of cyclin A and cyclin D2 were determined by Western blotting. RESULTS：Tanshinoone IIA significantly inhibited the proliferation of BX-PC-3 cells in a dose-dependent manner. The cancer cells were arrested in stage G0/G1 after treated with tanshinone IIA at low dose. The protein levels of cyclin A and cyclin D2 were decreased after drug intervention. CONCLUSION：Tanshinone IIA inhibits the proliferation of human pancreatic cancer cell line BX-PC-3 and the expression of cell cycle-promoting factors (cyclin A and cyclin D2), which may be the mechanism of attenuating the proliferation of pancreatic cancer cells.     

Inhibitory effect of propolis on proliferation of K562 cells in vitro

AIM: To study the effect of propolis on the proliferation of K562 cells.METHODS: K562 cells were cultured in vitro. Cell proliferation was measured by MTT method. The apoptotic rate was determined by flow cytometry. RT-PCR was applied to detect mRNA expression of Nup98. The protein level of Nup98 was determined by Western blotting. RESULTS: The inhibitory rates of proliferation induced by propolis at the concentrations of 2 mg/L, 20 mg/L and 200 mg/L were obviously higher than that in control cells in a time-and dose-dependent manner. The apoptotic rate was increased in a dose-dependent manner. High concentration of propolis down-regulated the expression of Nup98 at mRNA and protein levels. CONCLUSION: Propolis inhibits the proliferation and induces apoptosis in K562 cells. The mechanism may be related with down-regulation of Nup98.     

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.     

Effects of resveratrol on proliferation of ARPE-19 cells

AIM:To investigate the effects of resveratrol (Res) on the proliferation of ARPE-19 cells and to explore the possible mechanisms. METHODS:After ARPE-19 cells were treated with Res at concentrations of 0, 50, 100, 150, 200 and 300 μmol/L for 24 h, 48 h and 72 h, the effects of Res on the proliferation of the cells were tested by CCK-8 assay. The ARPE-19 cells were treated with Res at concentrations of 0, 100, 150 and 200 μmol/L for 48 h. The effects of Res on the cell cycle and apoptosis were detected by flow cytometry with Annexin V-FITC/PI staining. The protein expression of proliferating cell nuclear antigen (PCNA) was detected by immunofluorescent assay. The mRNA expression of PCNA, P21 and P27 was determined by real-time PCR. RESULTS: The results of CCK-8 assay showed that Res inhibited the proliferation of ARPE-19 cells in a time- and dose-dependent manner. The treatment with Res for 48 h resulted in an arrest of cell cycle at S phase without increasing cell apoptosis. Res inhibited the protein expression of PCNA in ARPE-19 cells. The results of real-time PCR showed that Res increased the mRNA expression of P21 and P27, and decreased the mRNA expression of PCNA. CONCLUSION: Res inhibits the proliferation of ARPE-19 cells and induces the cell cycle arrest at S phase. The mechanism may be related to up-regulation of P21 and P27, and down-regulation of PCNA.     

Effect of miR-21 over-expression on proliferation,migration and diffe-rentiation of c-Kit+ cardiac stem cells

AIM: To explore the effect of microRNA (miR)-21 on proliferation, migration and differentiation abilities of c-Kit⁺ cardiac stem cells (CSCs). METHODS: c-Kit⁺ CSCs were cultured and selected by the methods of enzyme digestion and magnetic bead separation. miR-21 mimics (50 nmol/L) and mimics negative control (MNC) were transfected into c-Kit⁺ CSCs with Lipofectamine^® 2000. The cells was divided into 3 groups:control group:c-Kit⁺ CSCs without any pretreatment; MNC group:the cells were transfected with MNC for 48 h; mimics group:the cells were transfected with miR-21 mimics for 48 h. qPCR was used to assess the expression of miR-21 in each group. CCK-8 and EdU assays were used to determine the cell proliferation. qPCR and immunofluorescence were used to detect the differentiation in each group. Scratch assay was adopted to explore the migration ability of the cells. RESULTS: The expression of c-Kit in the c-Kit⁺ CSCs were 90.8%, with 0.6% of CD45 and 0.5% of CD34. A significant increase in miR-21 expression was observed when the cells were transfected with miR-21 mimics for 48 h (P<0.05). CCK-8 and EdU assays showed that miR-21 significantly increased cell proliferation as compared with MNC group and control group (P<0.05). No difference in the expression of Nkx2.5, CD31 and α-SMA at mRNA and protein levels was observed, and no difference of the migration ability in 3 groups of the c-Kit⁺ CSCs was found. CONCLUSION: Over-expression of miR-21 significantly promotes the proliferation of c-Kit⁺ CSCs, without any effect on the cell migration and differentiation.     

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.     

CDA-2 induces differentiation and inhibits proliferation of human SWO-38 glioma cells

AIM: To investigate the differentiation-inducing effect of cell differentiation agent-2 (CDA-2) in human SWO-38 glioma cell line in vitro.METHODS: The inhibitory effect of CDA-2 on cell proliferation was assessed by MTT assay and colony formation assay.Cell morphology was determinded by light microscopy observation,and the expression of GFAP (glial fibrillary acidic protein) was detected by immunohistochemistry and Western blotting.Western blotting was also applied to explore the expression of PPARγ and COX-2.RESULTS: The data showed that CDA-2 inhibited proliferation and induced differentiation of SWO-38 cells.The inhibition efficiency was time-dependent and dose-dependent .The IC₅₀ of CDA-2 was (2.33±0.37) g/L and (0.51±0.01) g/L,respectively when cells were treated for 72 h and 10 days.CDA-2 caused differentiation of human glioma cells as indicated by outgrowth of long processes and expression of astrocyte marker GFAP.Simultaneously,the expression of PPARγ increased after 3 h of CDA-2 treatment，while the expression of COX-2 decreased after 48 h of CDA-2 treatment.CONCLUSION: CDA-2 inhibits proliferation and induces differentiation of SWO-38 cells.These effects may be through increasing cellular GFAP,PPARγ level and decreasing COX-2 expression induced by CDA-2.     

Effect of di-indolyl thiozoline on proliferation of lung cancer A549 cells

AIM: To investigate the effect of di-indolyl thiozoline (DIIT) on the proliferation of human lung cancer A549 cells. METHODS: The effects of DIIT on the proliferation of human lung cancer A549 cell line were determined by CCK-8 assay and EdU assay. The effects of DIIT on the expression of cyclin-dependent kinase 4 (CDK4), cyclin D1, and the phosphorylation of Akt and mTOR were determined by Western blot. RESULTS: After the A549 cells were treated with DIIT at 12.5, 25, 50 and 100 mg/L, the cell viability detected by CCK-8 assay was decreased by 12%, 27% (P<0.01), 33% (P<0.01) and 52% (P<0.01), respectively, compared with DMSO control group. The EdU positive cell number determined by EdU assay was decreased by 10%, 21% (P<0.05), 26% (P<0.05) and 34% (P<0.01), respectively, compared with DMSO control group. Compared with DMSO control group, DIIT inhibited the phosphorylation of Akt and mTOR and the expression of cyclin CDK4 and cyclin D1 (P<0.05). CONCLUSION: Di-indolyl thiozoline inhibits the proliferation of A549 cells, which may be related to the decreases in phosphorylation levels of Akt and mTOR and the inhibition of cell cycle-related protein expression.     

Effects of Norrin gene on the proliferation and cell cycle of human retinal capillary endothelial cells

AIM: To isolate, cultivate and identify human retinal capillary endothelial cells (HRCECs), and to assess the effects of high expression of Norrin gene on the proliferation and cell cycle of HRCECs. METHODS: The cultured cells were identified with anti-factor VIII related antigen. AP-3myc-hNorrin/pRK5 were transfected into cultured HRCECs in vitro by lipofectamine 2000. Their transfection efficiency were measured by RT-PCR, immunohistochemistry and Western blotting，respectively. Its effects on cell proliferation and cell cycle were detected. RESULTS: The cultured cells were identified with immunochemically positive brown staining. In comparison with those of the controls, the Norrin expression in experimental group was significantly increased on mRNA and protein levels (showed by the myc tag) after 48 h. The cell number of experimental group was larger than that in the control group with statistically significant differences. Flow cytometry showed the cells in G2 phase were mainly increased (P<0.01). CONCLUSION: The plasmid AP-3myc-hNorrin/pRK5 is successfully transfected into HRCECs by lipofectamine 2000. Norrin gene improves the proliferation ability of HRCECs by promoting the synthesis of DNA. Norrin may have an important role in the retinal angiogenesis, which may provide a new gene target in the treatment of retinal vascular disorders.     

Role of Daxx in inhibiting megakaryocytic differentiation of K562 cells

AIM: To examine the effects of death domain-associated protein (Daxx) overexpression on the viability and megakaryocytic differentiation of K562 cells. METHODS: Daxx overexpression in the K562 cells was established. The expression of Daxx was detected by fluorescence microscopy, fluorescence quantitative real-time PCR and Western blot after transfection. CCK-8 assay was used to detect the cell viability after overexpression of Daxx. The expression of CD41 and CD61 in phorbol 12-myristate 13-acetate (PMA) induced K562 cells was detected by flow cytometry. The protein levels of Daxx and p-ERK were determined by Western blot. Nitroblue tetrazolium (NBT)-reducing test was used to assess leukemia cell differentiation in Daxx-overexpressing K562 cells and control cells. The expression of CD41 and CD61 induced by PMA in Daxx-overexpressing K562 cells was analyzed by flow cytometry. The protein levels of Daxx and p-ERK were also examined by Western blot. RESULTS: The stable overexpression of Daxx in the K562 cells was established. The viability was reduced in Daxx-overexpressing K562 cells. The expression of CD41 and CD61 was significantly increased after PMA induction in the K562 cells (P < 0.01). The protein expression of Daxx was reduced, but the protein level of p-ERK was increased. The expression of CD41 and CD61 was reduced after PMA induction in Daxx-overexpressing K562 cells (P < 0.01). The protein level of p-ERK was also reduced. CONCLUSION: Daxx overexpression inhibits the growth, megakaryocytic differentiation and production of p-ERK in the K562 cells.     

Effect of sirtuin 6 on proliferation of hepatocellular carcinoma cells

AIM: To illuminate the effect of sirtuin 6 (SIRT6) on the proliferation of hepatocellular carcinoma (HCC) cells. METHODS: The mRNA expression of SIRT6 in the peripheral blood from 200 cases of HCC patients and 50 cases of healthy people was detected by real-time quantitative PCR (RT-qPCR). The mRNA expression levels of SIRT6 in the peripheral blood from 200 cases of HCC patients were combined with multiple clinicopathologic parameters for statistical analysis. The protein expression of SIRT6 in primary hepatocytes, immortalized hepatocytes and 4 hepatoma cell lines were determined by Western blotting. The silencing of SIRT6 was conducted by transfection of vector expressing short hairpin RNA targeting on SIRT6, and the protein level of SIRT6 was measured by Western blotting. The viability of HCC cells was tested by MTS assay. DNA synthesis was analyzed by Cell-Light^TM EdU Apollo® 488 In Vitro Imaging Kit. The abilities of colony formation and anchorage-dependent growth were measured by colony formation assay and soft agar assay, respectively. RESULTS: The mRNA expression of SIRT6 in the peripheral blood of HCC patients was significantly higher than that in the healthy people, and its expression was highly associated with tumor size, tumor grade and vascular invasion. SIRT6 expression in 4 hepatoma cell lines was significantly higher than that in the others. SIRT6 silencing led to a significant decrease in the cell viability as tested by MTS assay. EdU staining revealed that SIRT6 silencing reduced DNA synthesis. SIRT6 silencing reduced the ability of colony formation and anchorage-dependent growth as determined by colony formation assay and soft agar assay, respectively. CONCLUSION: Sirtuin 6 promotes the proliferation and malignant transformation of HCC cells.     

Effect of monocyte/macrophages activated by CpG-oligodeoxynucleotides of bacteria on K562 cells

AIM: To study the effect of monocyte/macrophages treated with CpG-oligodeoxynucleotides on leukemic K562 cells. METHODS: The monocytes/macrophages from peripheral blood cells were isolated and induced. The expressions of CD14 and CD16 on monocytes/macrophages were detected by means of flow cytometry. After treated with synthetic CpG-oligodeoxynucleotides, and nonCpG-oligodeoxynucleotides for 24 hours respectively, the inhibiting effect of monocyte/macrophages on K562 cells were detected using MTT method. The secretions of TNF-α and IL-12 from monocytes/macrophages were determined using ELISA method. RESULTS: The monocytes/macrophages treated with CpG-oligodeoxynucleotides enhanced their antitumor effect on K562 cells and increased the secretion levels of TNF-α and IL-12. Whereas, there was no significant difference between antitumor effect and cytokine secretion of the monocytes/macrophages treated with nonCpG-oligodeoxynucleotide. CONCLUSION: CpG-oligodeoxynucleotides increases the cytotoxicity of macrophages on K562 cells in vitro, as well as facilitates the IL-12 and TNF-α secretion. It provides a new approach for immunologic treatment of leukemia.     

Effect of ZNF281 on proliferation of hepatocellular carcinoma cells

AIM:To investigate the role of zinc finger protein 281 (ZNF281) in the proliferation of hepatocellular carcinoma (HCC) cells. METHODS:The mRNA expression levels of ZNF281 in peripheral blood mononuclear cells from 80 cases of healthy people and 206 cases of HCC patients were determined by real-time PCR. Statistical analysis were used to illustrate the relationship between the mRNA expression levels of ZNF281 in the peripheral blood mononuclear cells and the clinicopathologic parameters of HCC patients. Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of ZNF281 in hepatoma cell lines and immortalized hepatocytes. The silencing of ZNF281 was conducted by transfection of small interfering RNA targeting ZNF281, and then the proliferation of HCC cells was analyzed by MTS assay. The DNA synthesis of HCC cells was tested by Cell-Light^TM EdU Apollo^®488 In Vitro Imaging Kit. The ability of colony formation of the HCC cells was measured by colony formation assay, and the ability of anchorage-indepen-dent growth was detected by soft agar test. RESULTS:The mRNA expression level of ZNF281 in the peripheral blood mononuclear cells from HCC patients was significantly increased compared with the healthy people, and the high expression level was positively correlated with tumor size, tumor stage and tumor vascular invasion. Concurrently, the expression level of ZNF281 in hepatoma cell lines was significantly higher than that in immortalized hepatocytes. More importantly, the silencing of ZNF281 inhibited the proliferation, DNA synthesis, colony formation and anchorage-independent growth of the HCC cells. CONCLUSION:ZNF281 promotes the proliferation of HCC cells.     

Effects of alkyl-lysophospholipids on proliferation and differentiation in HL-60cells

AIM: To explore the effect of alkyl-lysophospholipids (ALP) on the proliferation, apoptosis and differentiation of HL-60cells. METHODS: Proliferative potential was measured by colony formation assays. Apoptotic cells were detected by morphology, DNA gel electrophoresis and flow cytometry analysis. Both morphological criteria and NBT dye reduction were utilized to determine the extent of differentiation. RESULTS: After 9 h of incubation wit15 mg/L of ALP, apoptotic cells, identified by condensened and fragmented nuclei, were present. After 6 days of incubation wit1 mg/L of ALP, the NBT reduction rate in HL-60cells increased to 84.2±2.6%. CONCLUSION: ALP can induce apoptosis and differentiation, and inhibit growth in HL-60cells.     

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.