Adipophilin accumulates cellular cholesteryl ester by PKC and ACAT1 in THP-1 macrophages

AIM: To investigate the mechanism of adipophilin accumulating cellular cholesteryl ester in THP-1 macrophages. METHODS: New Zealand rabbit atherosclerotic model was made with high cholesterol diet for 12 weeks. The expressions of adipophilin and PKCα were determined by Western blotting and immunochemical staining in aortic arteries. Cholesteryl ester-loading cells (CE-loading cells) were made from THP-1 macrophages incubated with oxidized low density lipoprotein. In CE-loading cells, expressions of adipophilin and ACAT1 were analyzed by RT-PCR, and the activity of PKC was determined by PepTag assay and spectrophotometry. When the CE-loading cells were incubated with PKC activator PMA and inhibitor calphostin C, expression of adipophilin was observed with RT-PCR, and cellular lipid was measured with oil red O staining and HPLC. The pcDNA3.1-HA-adi vector was transfected to THP-1 macrophage for making adipophilin over expression cells. After the CE-loading adipophilin over expression cells were incubated with or without ACAT inhibitor, the ACAT1 expression and cellular cholesteryl ester were analyzed. RESULTS: Compared with control, both adipophilin and PKCα expression increased in aortic arteries of atherosclerotic animal. In CE-loading THP-1 macrophages, adipophilin and ACAT1 highly were expressed and PKC activity was augmented also. PMA enhanced the high expression of adipophilin and cellular cholesteryl ester in CE-loading THP-1 macrophages, but calphostin C inhibited the effect. ACAT1 expression and cellular cholesteryl ester increased in adipophilin over expression cells, the effect was impaired by incubating with ACAT inhibitor. CONCLUSION: The results suggest that adipophilin increases ACAT1 activity through enhancing PKC activity, resulting in cellular cholesteryl ester accumulation in THP-1 macrophages.     

Effect of MIF antisense oligonucleotides on expression of MIF in macrophages

AIM: To investigate the effect of antisense oligonucleotides on expression of macrophage migration inhibitory factor (MIF) in macrophages. METHODS: MIF phosphorothioate oligonucleotides was designed and synthesized. The phosphorothioate antisense, sense and missense oligonucleotides of mouse MIF was transfected into macrophages, separately. After that, macrophages were incubated with LPS. Cell culture medium was collected for MIF protein detection by EIA. Cellular RNA was extracted and the expression of MIF mRNA was examined by RT-PCR analysis. RESULTS: LPS stimulation resulted in a specific time-dependent expression of MIF derived from macrophages. MIF mRNA and MIF protein level increased at 6 h and reached a plateau at 9-12 h after LPS stimulation. The macrophages treated with antisense oligonucleotides showed a significant decrease in MIF mRNA and MIF protein after LPS stimulation than those with LPS stimulation only and LPS plus sense or missense oligonucleotides (P<0.05). CONCLUSION: Antisense oligonucleotides of MIF inhibit the expression of MIF mRNA and MIF protein in macrophages treated with LPS.     

The effect of antisense bcr-abl oligonucleotides on the growth of K562 cells

AIM:To study the effect of bcr- abl gene antisense phosphorothioate oligonucleotides(Aspo) on K562 cell line and explore its significance in chrenic myelogeneous leukemia (CML) gene therapy.METHODS:Cells were exposed to oligomeis, observed by inverted microscope.Cells inhibitory rate were determined by 0.4 trypan blue exclusion . CFU-K562 were cultured in 0.8 % methylcellulose . P210 was measured by flow cytomety RESULTS: K562 cells were treated with Aspo, they still grew in clone state and show antisense sequence specific and dose dependent. When the concentration of Aspo was more than Spznol/L, the growth of cells was inhibited and P210 was down regulated or completely suppressed, and the greatest growth inhibition was at 120h . There was signifi-cant inhibition of cell proliferation in a rang‘cells number from 1×10⁴/mL to 5×10⁴/mL after treatment with 10unol/L Aspo. b2a2 Aspo was also effect on K562 cells which expressing b3a2 mRNA.CONCLUSION: bcr-abl Aspo has a specific growth inhibition effect on K562 cells, and worths further study in CML gene therapy.     

Inhibitory effect of different target-side antisense oligonucleotides on bFGF expression in SWO-38 cells

AIM:Three different antisense oligonucleotides complementary to basic fibroblast growth factor (bFGF) mRNA were compared in inhibitory effect on gene targeted expression.METHODS:After transfecting bFGF antisense oligonucleotides (asODN) into SWO-38 cells by lipofectin, the proliferation of cells was identified by MTT method, apoptosis was examined by flow cytometric cell cycle analysis and the expression levers of bFGF were detected by Western-blotting.RESULTS:There were 49%, 33%, 51% inhibition of cell growth and 35%, 27%, 18% cell apoptosis after asODN1, asODN2 and asODN3 treatment.In addition, the decrease in bFGF protein was 63%, 42%, 11%, respectively.CONCLUSION:The data suggeste that asODN1 is a potent target to bFGF mRNA, which inhibits cell growth and induces apoptosis in SWO-38 cells.     

Effect of acyl coenzyme A: cholesteryl acyltransferase 1 antisense oligonucleotides on the formation of foam cells

AIM: To study the effect of acyl coenzyme A: cholesteryl acyltransferase 1 (ACAT1) antisense oligonucleotides on the formation of foam cells (FC). METHODS: THP-1 cells were cultured and differentiated into macrophages (MP) by phorbol myristate acetate (PMA). Over-expressing ACAT1 gene THP-1 cells were constructed. The ACAT1 antisense and missense oligonucleotides conducted by LipofectamineTM 2000 were incubated with above cells. Ac-LDL was added 6 h later and incubated for 24 h. The expression of ACAT1 protein was detected by Western blotting. The ACAT activity was measured by quantifying the incorporation of ［1-14C］ oleoyl CoA into cholesteryl esters. The formation of foam cells was detected by oil red O staining. RESULTS: The ACAT1 antisense oligonucleotides inhibited the activity of ACAT in macrophages and over-expressing ACAT1 gene THP-1 cells. It also inhibited the formation of foam cell in macrophages and over-expressing ACAT1 gene THP-1 cells with lipid loading. The missense oligonucleotides did not show the inhibitory effects. CONCLUSION: The ACAT1 antisense oligonucleotides inhibit the activity of ACAT and the formation of foam cells.     

Effect of antisense oligonucleotides of c-sis on cellular cycle and proliferation of vascular smooth muscle cells in pulmonary artery

AIM:To investigate the effect of antisense oligonucleotides(ASON) of c-sis on cellular cycle and proliferation of pulmonary artery vascular smooth muscle cells(VSMC).METHODS:Tissue mass culture was done to get VSMC of pulmonary artery. Different concentrations of antisense oligonucleotides of c-sis were added into the cultures to observe the VSMC proliferation curve using MTT test. The changes of VSMC cellular cycle were also observed by flow cytometry.RESULTS:ASON with mid-to high concentrations restrained the proliferation of VSMC apparently with the peak of cell growth being attenuated or eliminated. Affected by mid-concentration ASON, PDGF-BB showed significant accelerating effect on the proliferation of VSMC. The ratio of G₀／G₁ in cellular cycle was increased significantly in VSMC culture with ASON in comparison with control. The G₀/G₁ ratio also showed significant differences among different concentration of ASON groups(P＜0.05).CONCLUSION:Mid-to high concentration of ASON was a powerful inhibitor of cellular proliferation for pulmonary artery VSMC. ASON increased the ratio of G₀／G₁ significantly and the increase seems to be ASON dosage dependent.     

Adipophilin promotes intracellular lipid accumulation through PI3K/Akt signaling pathway

AIM:To observe the possible mechanism through which adipophilin promotes the accumulation of intracellular lipids, and to provide a reference for controlling atherosclerosis.METHODS:RAW264.7 cells were incubated with oxidized low-density lipoprotein (oxLDL) for different time. qPCR, Western blot and Oil red O staining were used to observe the mRNA and protein levels of Akt, p-Akt and adipophilin and lipid accumulation. The above indexes were measured after the cells were treated with PI3K/Akt signaling pathway inhibitor LY294002. The activation of Akt was analyzed in the HEK293 cells over-expressing adipophilin. Co-immunoprecipitation was applied for analysis of protein-protein interaction between adipophilin and Akt. RESULTS:After incubation with oxLDL, the amount of lipid droplets, Akt activity and adipophilin expression increased in the cells with the extension of time (P<0.05). Moreover, LY294002 inhibited the above changes. The p-Akt levels increased after adipophilin over-expression. No direct interaction between adipophilin and Akt proteins was observed. CONCLUSION:Adipophilin promotes the accumulation of intracellular lipids through PI3K/Akt signaling pathway, but possibly not by direct interaction between adipophilin and Akt proteins.     

Adipophilin induces expression of inflammatory factors through ERK1/2-AP-1 signaling pathway

AIM: To observe the effects of adipose differentiation-related protein (adipophilin) on the expression of inflammatory factors in RAW264.7 macrophage and to clarify the related mechanism. METHODS: The cell models with high expression and low expression of adipophilin were constructed by transfecting PA317 packaging cells with stable high or low expression adipophilin retroviral vectors into the RAW264.7 cells. The concentrations of IL-6, MCP-1 and TNF-α in the cell culture medium were detected by ELISA. The protein levels of AP-1, p-AP-1, ERK1/2 and p-ERK1/2 were measured by Western blot. The protein levels of adipophilin, p-ERK1/2 and p-AP-1 and the releases of the inflammatory factors in the RAW264.7 cells treated with or without ERK1/2 inhibitor PD98059 or AP-1 inhibitor curcumin were determined. RESULTS: The RAW264.7 cells with high expression of adipophilin had higher levels of IL-6, MCP-1 and TNF-α, and higher protein levels of p-AP-1 and p-ERK1/2 than those in the cells with low expression of adipophilin. ERK1/2 inhibitor had no significant effect on the expression of adipophilin, but the protein expression of ERK1/2 and AP-1 was significantly inhibited (P<0.05). The administration of AP-1 inhibitor curcumin had no significant effect on the protein expression of adipophilin and ERK1/2, but the protein expression of AP-1 was significantly inhibited (P<0.05). At the same time, the releases of inflammatory factors IL-6, MCP-1 and TNF-α were significantly decreased. CONCLUSION: Adipophilin may regulate the expression of inflammatory factors through ERK1/2-AP-1 pathway in RAW264.7 macrophages.     

Effects of anti-leukemia by antisense c-myc by retrovirus vector

AIM: To study the role of c-myc oncogene in L6565 leukemia oncogenesis and the effects of therapy by inhibition of its expression with antisense c-myc. METHODS: A recombinant retroviral vector containing antisense c-myc of the murine (pGNCas) was constructed and then transfected into PA317 cells by the method of calcium phosphate precipitation. L6565 clone cells were infected with retrovirus particles. Stable integretion of antisense c-myc was shown by PCR. The change of the malignance and phenotype of L6565as were detected by the examination of the growth, morphology, cells cycle, agar assay and expression of c-myc. RESULTS: The shape of most L6565as cells became spherical. The growth of L6565as was inhibited compared to control cells. The analysis of cells cycle: L6565as cells were arrest in G₀/G₁ phase, decreased in S phase. The ability of L6565as cells to form colony in soft agarose was significantly suppressed. c-myc in L6565as cells was lowly expressed. CONCLUSION: (1)c-myc plays a critical role in L6565 leukemia oncogenesis; (2)Inhibition of expression of c-myc makes partly reversion of malignant phenotype of L6565 murine leukemia clone cells.     

Antiproliferative effect of c-myc antisense oligonucleotide in rat thymus lymphocytes

AIM: To observe the antiproliferative effect of c-myc antisense oligonucleotide in rat thymus lymphocytes. METHODS: Rat thymus lymphocytes were separated by Ficoll-Urografin density gradient centrifugation. Lipofectin was used to introduce antisense, sense and mismatched oligonucleotides for c-myc to rat thymus lymphocytes. The antiproliferative effect was assayed by incorporation of [³H]-TdR and MTS cell proliferation assay. TR-PCR was used to detect the expression of c-myc mRNA. RESULTS: c-myc antisense oligonucleotide inhibited ratthymus lymphocytes proliferation[(0.14±0.03)A vs(0.32±0.16)A,P<0.05],but this ef ect had no relationship with the concentration of c-myc antisense oligonucleot ide.c-myc antisense oligonucleotide decreased the expression of c-myc mRNA in rat thymus lymphocytes. CONCLUSION: c-myc antisense oligonucleotide inhibited rat thymus lymphocyte proliferation.     

Advanced studies on antisense oligonucleotide treatment and target-gene of leukemia

The oncogene expression and growth of leukemic cells could be inhibited by antisense oligonucleotide.The selection of target genes is the key step in the research of antisense oligonucleotide on leukemia.The art icle would review the status and prospect of some target genes of leukemia in the investigation of antisense oligonucleotide.     

Effect of hTERT antisense oligodeoxynucleotide on Cis-diamminedichicloroplatinum-induced apoptosis in Jurkat cells

AIM:To explore the effect of hTERT antisense phosphorothioate oligodeoxynucleotide (ASODN) on apoptosis induced by chemotherapeutic drugs in Jurkat cell lines. METHODS:Cell viability was determined using the trypan blue dye exclusion assay. Apoptosis was detected by morphological observation, DNA gel electrophoresis and flow cytometry analysis. RESULTS:The survival rates of Jurkat cells cultured with daunorubicin, vincristin, and etoposide, respectively were similar with that cultured with those chemotherapic drugs plus hTERT ASODN. The survival rates of Jurkat cells cultured with cis-diamminedichicloroplatinum(DDP) added 24 hours later were higher than that cultured with hTERT ASODN and DDP added 24 hours later. The survival rates of Jurkat cells cultured with DDP were similar with that cultured with hTERT SODN and DDP. In morphological observation of apoptotic cells using Giemsa staining, cells displayed classic apoptotic changes treated with DDP or DDP combined with hTERT ASODN or SODN at 48 hours. Agarose gel electrophoresis of genomic DNA from Jurkat cells treated with ASODN and DDP combination for 48 hours showed typical DNA "ladder". Neither the DNA from Jurkat cells treated with SODN plus DDP nor the DNA from the cells treated with DDP alone showed ladder pattern. Apoptosis rates of Jurkat cells treated with DDP for 48 hours after 24 hours of exposure to ASODN significantly increased. There was significant difference in the percentage of apoptotic Jurkat cells between hTERT ASODN plus DDP and SODN plus DDP or DDP alone, respectively. CONCLUSION:The hTERT ASODN complementary to the translation initiation region of hTERT mRNA enhanced DDP-induced apoptosis in Jurkat cells.     

Multidrug-resistance antisense inhibits volume-activated chloride current in non-pigmented ciliary epithelial cells

AIM: To study the relationship between multidrug-resistance (MDR1) gene product P-glycoprotein (P-gp) and the volume-activated chloride current. METHODS:The volume-activated chloride current in bovine non-pigmented ciliary epithelial cells was recorded using a whole cell recording technique. An antisense technique was used to inhibit the expression of MDR1 gene. The immunofluorescence of P-gp was monitored with a real-time laser confocal microscope.RESULTS:P-gp immunofluorescence correlated negatively with the concentration of the human MDR1 antisense oligonucleotide. The antisense oligonucleotide inhibited the volume-activated chloride current specifically and partially. The latency of activation of the current increased and the peak current decreased. The percentage of inhibition of peak current correlated positively to the concentration of the antisense oligonucleotide(r＝0.99, P＜0.01) and to the reduction(%) of P-gp immunofluorescence(r=0.99,P＜0.01).CONCLUSION:P-gp, the product of MDR1 gene, plays an important role in the activation pathway of volume-activated chloride current in non-pigmented ciliary epithelial cells.     

Effects of DHEA and G6PD antisense oligodeoxynucleotides on Raji cells

AIM:To investigate the suppressive effects of dehydroepiandrosterone (DHEA) and glucose-6-phosphate dehydrogenase (G6PD) antisense oligodeoxynucleotides on Raji cells. METHODS:Raji cell line was cultured in vitro in the presence of DHEA at different concentrations ranged from 0.05 μmol/L to 500 μmol/L or G6PD antisense oligodeoxynucleotides. The viability and proliferation of the cells pretreated with dehydroepiandrosterone or G6PD antisense oligodeoxynucleotides were evaluated. Meanwhile, intracellular activities and mRNA expression of G6PD were analyzed. RESULTS:DHEA and G6PD antisense oligodeoxynucleotides does not influence the viability of cells in culture. Raji cells treated with DHEA at concentration of 50 μmol/L or 500 μmol/L for 72 h or with 10.0 μmol/L G6PD antisense oligodeoxynucleotides for 48 h had significant lower cell numbers compared with control (P<0.01). Raji cells treated with DHEA at concentration more than 5.0 μmol/L for 72 h had significant decreased G6PD activities (P<0.01) but no change in mRNA expression levels was observed. With 10.0 μmol/L G6PD antisense oligodeoxynucleotides pretreatment for 48 h, the G6PD mRNA expression levels and activities were significantly decreased (P<0.01). CONCLUSION:DHEA or G6PD antisense oligodeoxynucleotides at specific concentration have suppressive effects on G6PD activities and proliferation in Raji cells to a certain extent, but the suppressive mechanisms are different.