Biological characteristics of side population cells sorted from hepatoma SMMC-7721 cell line

AIM: To study the biological characteristics of side population (SP) cells sorted from hepatoma SMMC-7721 cell line. METHODS: Fluorescence-activated cell sorter (FACS) was used to sort SP cells and non-SP (NSP) cells from SMMC-7721 cell line. The colony-formation ability and proliferation ability between SP cells and NSP cells were compared in terms of plate colony assay and growth curve. The migratory and invasive properties of SP cells and NSP cells were tested by Transwell method. The cell cycle and apoptosis were analyzed by flow cytometry. The oncogenicity of the cells was analyzed by nude mouse transplantation tumor experiment in vivo. RESULTS: The results of FACS analysis indicated that (9.2±0.2)% of the SMMC-7721 cells were SP cells. The proportion of G₀/G₁ phase of SP cells was higher, and the apoptotic rate was lower than those of NSP cells (P<0.05). The proliferation ability and colony-forming ability and migratory and invasive properties of SP cells were significantly higher than those of NSP cells (P<0.05). The nude mouse transplantation tumor experiment displayed that the oncogenicity of SP cells was higher than that of NSP cells. CONCLUSION: The SP cells sorted from SMMC-7721 cell line may enrich tumor stem cells.     

Effects of 2-deoxy-D-glucose and oxaliplatin on proliferation and apoptosis of human hepatoma cell line SMMC-7721

AIM: To investigate the effects of 2-deoxy-D-glucose (2-DG) or oxaliplatin (L-OHP) alone and the combination of both on the proliferation and apoptosis of hepatoma cell line SMMC-7721 in vitro. METHODS: Human hepatoma cell line SMMC-7721 was treated with 2-DG or L-OHP alone, or both. The inhibitory effect on the proliferation of SMMC-7721 cells was estimated by MTT method. The q value, which represents synergistic effect, was determined. Apoptotic rate and cell cycle were assayed by flow cytometry. The activity of caspase-3 was detected by a caspase-3 activity assay kit. RESULTS: 2-DG or L-OHP at different concentrations inhibited the growth of SMMC-7721 cells obviously and the inhibitory effect on SMMC-7721 cell growth strongly depended on the exposure time and dose. When the 2 drugs worked together, the inhibitory effect was improved (P<0.05). 2-DG induced cell apoptosis and arrested the cells at G₂/M phase. When combined with L-OHP,the 2 drugs induced more severe apoptosis and arrested the cells at S and G₂/M phase. Meanwhile, the activity of caspase-3 increased when the 2 drugs used together. CONCLUSION: 2-DG inhibits the growth of hepatoma cell line SMMC-7721. The combination of 2-DG and L-OHP improves the ability of L-OHP to attack the tumor cells. The mechanism might be related to increasing the activity of caspase-3.     

miR-15a-5p inhibits proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells

AIM: To observe the influence of high expression of miR-15a-5p on the proliferation and migration of human hepatocellular carcinoma SMMC-7721 cells.METHODS: The miR-15a-5p oligonucleotide, which was reconstructed with additional restriction sites of EcoR Ⅰ and Hind Ⅲ, was chemically synthesized and confirmed by sequencing. The miR-15a-5p eukaryotic expression system was constructed by pcDNA6.2-GW/Em-GFP-pre-miR-15a-5p plasmid. The miR-15a-5p was transfected into the SMMC-7721 cells transiently by plasmid, and quantified by quantitative real-time PCR at the mRNA level. The cell viability was measured by CCK-8 assay, and the living cell counting was performed by the method of Trypan blue exclusion. The migration ability of the SMMC-7721 cells with high expression of miR-15a-5p was detected by wound healing test.RESULTS: The sequence of miR-15a-5p oligonucleotide 100% matched the designed sequence. Compared with control group, the miR-15a-5p expression was increased significantly (P<0.05). The viability, the living cell number and the migration ability of the SMMC-7721 cells were decreased in high expression of miR-15a-5p group with statistically significant difference (P<0.05).CONCLUSION: The abilities of proliferation and migration in human hepatocellular carcinoma SMMC-7721 cells are decreased by high expression of miR-15a-5p.     

Effect of abnormal expression of miR-141 on malignant biological behaviors of human hepatocarcinoma cells

AIM: To investigate the expression of microRNA-141 (miR-141) in human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal hepatocyte line HL-7702, and to analyze the effect of abnormal expression of miR-141 on the malignant biological behaviors of human hepatocarcinoma cells. METHODS: The RNA from SMMC-7721 cells and HL-7702 cells was extracted. SYBR Green real-time PCR was performed to detect the expression of miR-141. Synthetic miR-141 mimic and its negative control were transfected into the SMMC-7721 cells, and miR-141 inhibitor and its negative control were transfected into the HL-7702 cells by the method of Lipofectamine. After transfection, MTS assay and BrdU-ELISA were employed to evaluate the effect of miR-141 on the cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. The changes of migration ability were investigated by Transwell invasion assay. RESULTS: The expression of miR-141 in the SMMC-7721 cells was significantly lower than that in the HL-7702 cells (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of the SMMC-7721 cells transfected with 25 nmol/L miR-141 mimic was significantly inhibited in a time-dependent manner (P < 0.05). The percentages of G₁ phase cells and early apoptotic rate were significantly increased when miR-141 was up-regulated, but the migration ability was inhibited (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of HL-7702 cells transfected with 50 nmol/L miR-141 inhibitor was significantly increased in a time-dependent manner (P < 0.05). When miR-141 was down-regulated, the percentages of G₁ phase cells and early apoptotic rate were significantly decreased, but the migration ability was enhanced (P < 0.05). CONCLUSION: miR-141 is down-regulated in human hepatocarcinoma cell line. Up-regulation of miR-141 will not only inhibit cell proliferation and migration ability, but also affect the cell cycle and apoptosis of SMMC-7721 cells. miR-141 may function as a tumor suppressor gene during HCC development.     

Effect of Beclin 1 RNA interference on Vit K3 injured SMMC-7721 cells

AIM: To observe the effect of Beclin 1 silencing by RNA interference (RNAi) technique to the injury of SMMC-7721 hepatoma cells by vitamin K3 (Vit K3).METHODS: The recombinant plasmid Psilencer 3.1-siRNA-Beclin 1 was transfected into SMMC-7721 hepatoma cells by eukaryotic cell transfection technique. Plasmid vector and cell culture medium were used as negative and control, respectively. The cells were collected 48 h later to extract cell RNA and total protein and to detect Beclin 1 gene expression by RT-PCR and Western blotting. 40 μmol/L Vit K3 was used to treate the Beclin 1-siRNA cells, Hoechst33342 staining was used for the determination of the percentage of cell apoptosis.RESULTS: Compared with the control group, the synthetic siRNA of Beclin 1 significantly decreased the levels of Beclin 1 mRNA and protein expressions. Beclin 1 mRNA was up-regulated in 40 μmol/L Vit K3 treated SMMC-7721 hepatoma cells, the percentage of apoptosis cells increased (P﹤0.01). In beclin 1-siRNA cells, Beclin 1 mRNA was down-regulated obviously, the percentage of apoptosis cells increased significantly compared with the 40 μmol/L Vit K3 group (P﹤0.01).CONCLUSION: The transfection of SMMC-7721 hepatoma cells by Psilencer3.1-siRNA-Beclin 1 effectively inhibits the expressions of Beclin 1 mRNA and protein, inhibits the activation of Beclin 1 dependent autophagic signaling pathway, and aggravates the apoptosis induced by Vit K3.     

Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.     

Effects of marrow stromal cell-mediated microenvironment on human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the effects of marrow stromal HS-5 cells on hepatocellular carcinoma SMMC-7721 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the proliferation, migration and invasion abilities of SMMC-7721 cells were detected by MTT, wound-healing and Transwell assays. After co-culture of SMMC-7721 cells with HS-5 cells in the Transwell chamber, the expression of chemokine CCL5 and its receptor CCR5 at mRNA and protein levels in SMMC-7721 cells was examined by quantitative real-time PCR (qRT-PCR), ELISA or Western blotting. Akt and p-Akt473 protein levels in SMMC-7721 cells treated with PI3K inhibitor LY294002 were observed by Western blotting. RESULTS: HS-5-CM promoted the proliferation, migration and invasion abilities of SMMC-7721 cells. The expression of CCL5 and CCR5 at mRNA and protein levels in SMMC-7721 cells was increased after co-cultured with HS-5 cells. PI3K inhibitor LY294002 inhibited the activation of PI3K-Akt signaling pathway and the secretion of CCL5 in SMMC-7721 cells after co-cultured with HS-5 cells. CONCLUSION: HS-5 cells significantly promote the proliferation, migration and invasion abilities of SMMC-7721 cells. Co-culture of SMMC-7721 cells with HS-5 cells activates PI3K-Akt signaling pathway to increase the secretion of CCL5 in SMMC-7721 cells.     

Alteration of AQP2 expression in kidney tissues of emphysema model rats

AIM: To observe the expressions of aquaporin 2 (AQP2) in kidney tissues and the contents of endotoxin (ET), interleukin-1 β (IL-1β), tumor necrosis factor-α (TNF-α) in serum in emphysema model rats, and to investigate the relationship between lungs and kidney in humoral metabolism. METHODS: The rats of emphysema were treated by injecting lipopolysaccharide into the trachea with cigarette smoking. Immunohistochemistry and Western blotting analysis were used to observe the expression of AQP2 in kidney tissues. RT-PCR was applied to detect the expression of AQP2 mRNA in kidney tissues. Blood sample and lung tissue were taken and the levels of ET, IL-1β and TNF-α were measured by radioimmunoassay. RESULTS: AQP2 expression in the kidney tissue in model group was greater than that in control group, and the expression of AQP2 mRNA showed the same results (P<0.01). ET, IL-1β and TNF-α levels in serum and lung tissue in model group were markedly higher than those in control group (P<0.01). CONCLUSION: In the emphysema model rats, AQP2 expression is up-regulated in the kidney tissue. The mechanism of emphysema may be related to increasing the levels of ET, IL-1β and TNF-α in the serum and lung tissue obviously.     

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 NF-κB inhibitor pyrrolidine dithiocarbonate on the proliferation and apoptosis in K562 cells

AIM: To investigate the effect of pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-κB on the proliferation and apoptosis of K562 cells and to explore the anti-tumor mechanism of PDTC.METHODS: Trans AMTM NF-κB p65 kit was used to detect the activity of p65 in K562 cells treated by PDTC. The effect of PDTC on the proliferation of K562 cells was measured by WST-1 method. DNA damage was detected by single cell gel electrophoresis (comet assay). The procaspase-3 and activated protein level of caspase-3 were detected by Western blotting.RESULTS: The activity of p65 in K562 cells was inhibited after treated by PDTC (P<0.01). Simultaneously the cell proliferation was significantly inhibited in a dose-and time-dependent manner (P<0.01). The degree of DNA damage in K562 cells treated with PDTC at concentrations of 25 μmol/L, 50 μmol/L or 100 μmol/L was more severe than that in control. The rates of comet cells in the PDTC-treated groups (43.50％, 84.00％, 95.63％) were significantly higher than those in control (9.75％, P<0.01), and it was also dose-dependent. The expression of procaspase-3 and activated caspase-3 protein were detected in the cytoplasm of the K562 cells treated by PDTC by Western blotting.CONCLUSION: NF-κB plays an important role in regulating cell proliferation and apoptosis in K562 cells. PDTC inhibits NF-κB activity and elevates the expression of caspase-3, which is related to increase in cell apoptosis.     

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.     

Islet cells modified with PD-L1-GPI molecule inhibit attack of reactive T cells

AIM: To investigate the immune depressive effect on the reactive T cells and to explore the immunologic injury mechanism of beta cells of islet in type 1 diabetes mellitus (DM-1). METHODS: pAd5/ PD-L1-GPI adenovirus vector with target gene was constructed and transfected into NIT cells which are known as a mouse insuloma cell line. The highly expressed membrane protein of PD-L1-GPI was confirmed by Western blotting. The peripheral blood non-adherence lymph leukocytes and target cells were cultured to detect lymph leukocyte proliferation and the T cell function. The level of IL-2, TNF-α and IFN-γ were detected in the cell culture fluid. RESULTS: Compared with the control group, the NIT cells modified with PD-L1-GPI inhibited the sensitized lymph leukocyte proliferation effectively and down-regulated the level of some cytokine secretions such as IL-2, IFN-γ and TNF-α (P<0.05). CONCLUSION: The islet cells expressed the PD-L1 gene inhibit the reactive responsive T cells, block up the cytotoxic effect of autoimmunity T cells, and induce the immunotolerance, which would be a value direction of the therapy of DM.     

Phenotype and immune activity of dendritic cells under interleukin-18 intervention

AIM: To investigate the phenotype and immune activity of dendritic cells using interleukin-18 as intervent.METHODS: Monocytes were isolated from human peripheral blood and induced into DCs with GM-CSF and IL-4. The cellular morphous was observed under inverted microscope. On the 5th day, 3 groups including IL-18 group, TNF-α group and IL-18+TNF-α group were set. IL-18, TNF-α or IL-18+TNF-α was used as intervents respectively to facilitate cell maturity. Supernatants were collected at 24 h, 48 h and 72 h. IL-12 in the supernatant, CD1a, HLA-DR, CD83 and CD86 were analyzed using flow cytometry. DCs of the 3 groups were co-cultured with T cells respectively on the ratio of 1∶〖KG-*2〗100, 1∶〖KG-*2〗50 and 1∶〖KG-*2〗10. T cell proliferation stimulated by DC was determined using MTT method. DCs were co-cultured with T cells on the ratio of 1∶〖KG-*2〗10, and the supernatant were collected at 24 h, 48 h and 72 h. IFN-γ in the supernatant was detected with ELISA method.RESULTS: Induced by GM-CSF and IL-4, then stimulated by IL-18, TNF-α or IL-18+TNF-α, monocytes showed typical morphous of DC. No morphological difference was observed among DCs of the 3 groups. No statistical difference showed in expression level of CD1a, HLA-DR, CD83 and CD86 between IL-18 group and TNF-α group (P>0.05). The positive rates of CD1a and CD83 in IL-18+TNF-α group were higher than those in other 2 groups. The positive rate of HLA-DR in IL-18+TNF-α group was higher than that in IL-18 group. No difference between IL-18 group and TNF-α group in the potency of stimulating T cell proliferation was found, whereas the stimulating potency in IL-18+TNF-α group was higher than that in IL-18 group and TNF-α group. IL-12 in IL-18+TNF-α group at 48 h and 72 h was higher than that in IL-18 group and TNF-α group (P<0.05). However, there was no difference between the latter 2 groups. There was also no difference between IL-18 group and TNF-α group in IFN-γ secretion. IFN-γ in IL-18+TNF-α group was higher than that in IL-18 group and TNF-α group (P<0.05).CONCLUSION: Using IL-18 as intervent, DC expresses high level of surface molecules, secretes high level of IL-12, stimulates T cell proliferating effectively and produces IFN-γ potently. The actions are stronger when used in combination with TNF-α. It suggests that IL-18 may serve as a promoting agent of DC maturity, or combination with TNF-α in DC induction will strengthen the immune activity of DC.     

Reverse effects of saikoside on multidrug resistance of human leukemic cell line K562/ADM in vitro

AIM: To study the reverse effects of saikoside (SS) on the multidrug resistance (MDR) of human leukemic cell line K562/ADM and to investigate the related mechanism. METHODS: K562 cells and K562/ADM cells in the culture were treated with SS at the concentrations of 1~100 mg/L. The inhibitory rate of the cell proliferation was measured by MTT assay. Non-cytotoxic dose of SS was determined. K562/ADM cells were treated with SS at non-cytotoxic doses of 1.25, 2.5 and 5.0 mg/L with different concentrations of adriamycin (ADM,0.05~100 mg/L). The 50% inhibitory concentration (IC₅₀) and the reversal index in all groups were determined. The cell morphology was observed after treated with SS+ADM. The effects of SS on ADM accumulation in K562/ADM cells, the cell cycle profile and apoptosis were examined by flow cytometry. RESULTS: The inhibitory rates were significantly increased in a dose-dependent manner when the cells were treated with different doses of SS (1~100 mg/L). The available reversal concentration of SS was 5.0 mg/L and the reversal index was 21.5 folds for K562/ADM cells. After treated with SS+ADM, the number of tumor cells was decreased and apoptotic cells were increased in a dose-response relationship. ADM accumulation in K562/ADM cells treated with SS was significantly higher than that in control cells (P<0.05). SS may significantly enhanced the apoptosis of K562/ADM cells treated with ADM (P<0.05). K562/ADM cells treated with SS were blocked in the stage of G₀/G₁. CONCLUSION: SS has effect on proliferation inhibition and MDR reversal in K562/ADM cell line. The reversal mechanisms of SS may be due to increasing the accumulation of chemo therapeutics in the cell, inducing the cell apoptosis and arresting the cells in G₀/G₁ phase.     

Valproate enhances imatinib-induced apoptosis and down-regulates Bcr/Abl mRNA and phosphorylated protein kinase B in chronic myeloid leukemia cell line K562

AIM：To investigate the effects of valproate and imatinib on the apoptosis of chronic myeloid leukemic cell line K562. METHODS：K562 cells were divided into 3 groups and treated with valproate, imatinib and cotreatment, respectively. Cell cycle, apoptosis, the mRNA expression of Bcr/Abl, total protein kinase B (PKB) and phosphorylated PKB (p-PKB) were analyzed. RESULTS：The apoptotic rates in valproate group, imatinib group and cotreatment group were (11.47±0.25)%, (28.43±1.70)% and (57.73±4.38)%, respectively (P<0.05). No obvious difference was observed in cell cycle between cotreatment group and monodrug group. Bcr/Abl mRNA and p-PKB in the above 3 groups were (0.00±0.00), (64.17±12.27), and (0.00±0.00) ×10 9 copies/(g total mRNA), respectively (P<005), and 0.25±0.02, 0.17±0.01 and 0.08±0.01, respectively (P<0.05). No apparent difference of PKB was found in the 3 groups. CONCLUSION：Valproate enhances imatinib-induced apoptosis and may link to the down-regulation of Bcr/Abl mRNA and p-PKB in chronic myeloid leukemic cell line K562.     

Combination of sorafenib and daunorubicin has antileukemic activity on K562 cells

AIM: To investigate the synergetic inhibitory effect of sorafenib and daunorubicin (DNR) on K562 and U937 cells. METHODS: The inhibitory rate of sorafenib or daunorubicin alone, and the combined inhibitory rate of sorafenib and IC10 daunorubicin were measured by MTT assay. Apoptotic rate of single drug or combination was assessed by flow cytometry (Annexin Ⅴ/PI staining) and Hoechst 33258 staining assay. p-ERK1/2 level was detected by Western blotting after the cells were treated with sorafenib, daunorubicin and U0126 or combinations. Synergistic or antagonistic effect of proliferation and apoptosis on K562 and U937 was estimated according to the Jins Method. RESULTS: Combination of sorafenib and DNR showed synergistic growth inhibition (q>1.15, P<0.01) and synergistic promotion of apoptosis (q>1.15, P<0.05) in K562 and U937 cells. The level of p-ERK1/2 in K562 cells was obviously higher than that in U937 cells (P<0.01). p-ERK1/2 expression was completely inhibited in sorafenib or U0126 treated K562 cells for 24 h. Combination of U0126 with DNR inhibited the proliferation of K562 cells synergistically. CONCLUSION: Combination of sorafenib with DNR showed synergistic cell growth inhibition and promotion of apoptosis in K562 and U937 cells. U937 cells were more sensitive to DNR than K562 cells while K562 cells were more sensitive to sorafenib. Sorafenib enhances the anti-leukemic activity of DNR in K562 and U937 cells via down-regulation of p-ERK1/2 expression.