Effects of curcumin on migration and invasion of myeloma cells

AIM：To explore the effect of curcumin on the migratory and invasive abilities of myeloma cells. METHODS：shRNA expression plasmid was transfected into RPMI8226 cells to knock down IQ motif-containing GTPase-activating protein 1 (IQGAP1). The expression of IQGAP1 in RPMI8226 cells tranfected with shIQGAP1 or shRNA negative control, and in un-transfected RPMI8226 cells was detected by Western blotting. All the cells in RPMI8226-shIQGAP1 group, RPMI8226-shRNA negative control group and un-transfected RPMI8226 group were treated with curcumin at various concentrations. The migratory and invasive abilities of the RPMI8226 cells were measured by Transwell chamber and Matrigel assays. The expression of IQGAP1 at mRNA and protein levels in the RPMI8226 cells treated with curcumin was also determined by RT-PCR and Western blotting. RESULTS：The expression of IQGAP1 decreased when IQGAP1 gene was knocked down by shRNA. The migration and Matrigel invasion tests showed that the number of cells moving into under chamber of Transwell decreased in RPMI8226-shIQGAP1 group in comparison with the other 2 groups. Curcumin decreased the migratory and invasive abilities of RPMI8226-shRNA negative control cells and un-transfected RPMI8226 cells, which was not related to the curcumin concentratory. The migratory and invasive abilities of RPMI8226-shIQGAP1 cells showed no significant difference when treated with curcumin at various concentrations. The expression of IQGAP1 at mRNA and protein levels decreased in the RPMI8226 cells treated with curcumin. CONCLUSION：Curcumin decreases the migratory and invasive abilities of myeloma cells via inhibition of IQGAP1 expression.     

Effects of fucoidan on angiogenesis of human multiple myeloma RPMI 8226 cells

AIM: To investigate the effects of fucoidan on the angiogenesis of multiple myeloma cells in vitro, and its related mechanisms. METHODS: The human multiple myeloma RPMI 8226 cells and human endothelial cells were cultured in vitro. The growth inhibition rate of RPMI 8226 cells was examined by MTT assay. The cell cycle and apoptosis rate were measured by flow cytometry. RPMI 8226 cells were treated with fucoidan for 72 h, and the cell culture supernatant was collected. The VEGF concentration was examined by ELISA, and the tube formation assay was applied to assess the angiogenic activity. After treatment with fucoidan for 72 h at different concentrations, the protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 were detected by Western blot. RESULTS: Fucoidan inhibited the growth of RPMI 8226 cells in a dose- and time-dependent manner. After treatment with fucoidan for 72 h, the cell cycle was arrested at G₁ phase, and the apoptotic rate of RPMI 8226 cells was increased with the increasing concentration of fucoidan, which was much higher than that in control group (P<0.05). The VEGF concentration was significantly decreased with the increa-sing concentration of fucoidan. The numbers and areas of the capillary-like structures decreased while the concentration of fucoidan increased, and those at 100 mg/L were less than those in the control (P<0.05). The protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 in fucoidan group were significantly lower than those in control group (P<0.05). CONCLUSION: Fucoidan inhibits the secretion of VEGF in multiple myeloma cells, and reduces angiogenesis induced by multiple myeloma cells. It inhibits the protein expression of HIF-1α and VEGF, which may be related to inhibiting the phosphorylation of AKT and ERK1/2.     

Effects of sodium valproate on RPMI8226 and U266 cell proliferation and IL-6/JAK/STAT signaling pathway

AIM: To investigate the effects of sodium valproate (VPA) on the proliferation of multiple myeloma cell lines RPMI8226 and U266 and the regulation of IL-6/JAK/STAT signaling pathway. METHODS: The cells were treated with different concentrations of VPA for 12 h and 24 h. The growth of RPMI8226 cells and U266 cells was detected by MTT assay. Apoptotic rates and cell cycle were analyzed by flow cytometry. The mRNA expression of STAT3, STAT5 and STAT target genes Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF was measured by RT-PCR. Western blotting analysis was used to determine the total proteins and protein phosphorylation levels of JAK2 and STAT5. RESULTS: VPA inhibited the growth and induced the apoptosis of RPMI8226 cells and U266 cells in a concentration- and time-dependent manner. The levels of IL-6 in the culture supernatants of RPMI8226 cells and U266 cells treated with VPA were significantly higher than that in negative control group. VPA down-regulated the mRNA expression of STAT3, STAT5, Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF. After treated with VPA, the protein levels of p-JAK2, JAK2, p-STAT5 and STAT5 in RPMI8226 cells and U266 cells were significantly lower than those in control group. CONCLUSION: VPA inhibits the proliferation of PRMI8226 cells and U266 cells in vitro. The modulation of IL-6/JAK/STAT signaling pathway may be involved in its potential mechanisms.     

Recombinant human endostatin regulates proliferation and related protein expression in human multiple myeloma cell line RPMI 8226

AIM: To investigate the effects of recombinant human endostatin (Endostar) on proliferation, cell cycle constitution and regulation of related protein expression in RPMI 8226 cells. METHODS: CCK-8 assay was used to explore the effect of Endostar on the proliferation of RPMI 8226 cells. The action of Endostar on apoptosis and cell cycle distribution were determined by flow cytometry. The expression of Bcl-2 and caspase-3 was evaluated by Western blotting analysis. The production of vascular cell adhesion molecule 1 (VCAM-1), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) in response to Endostar was measured by real-time PCR and Western blotting. The influence of Endostar on the secretion of IL-6 and VEGF in the culture supernatants was detected by ELISA. RESULTS: Endostar inhibited the proliferation of RPMI 8226 cells with the highest inhibitory rate of 59.5±5.6% at dose of 250 mg/L, which was associated with slight up-regulation in the proportion of G1 phase. No significant modulation of apoptotic rate was observed with Endostar, as well as the expression of Bcl-2 or caspase-3. Endostar declined the expression of VCAM-1 and also attenuated the production and secretion of IL-6 and VEGF in RPMI 8226 cells. CONCLUSION: Endostar suppresses the proliferation of RPMI 8226 cells by enhancing the proportion of G1 phase and decreasing VCAM-1 expression and self-secretion of IL-6 and VEGF. No apoptosis of RPMI 8226 cells is induced by Endostar.     

Genistein inhibits proliferation of human oral cancer TCA8113 cells through suppression of VEGF expression

AIM: To investigate the effect of genistein on the proliferation of human oral cancer TCA8113 cells and to explore the underlying mechanisms.METHODS: The cell proliferation was examined by MTT assay, cell counting and colony formation assay. Western blotting was employed to examine the protein levels of vascular endothelial growth factor(VEGF), extracellular signal-regulated kinase(ERK) and p-ERK. RESULTS: Genistein significantly inhibited the proliferation of TCA8113 cells in a concentration-dependent fashion. Moreover, genistein dose-dependently decreased the protein levels of VEGF, ERK and p-ERK. The expression of VEGF was also blunted by U0126, a specific inhibitor of ERK. U0126 and axitinib, a VEGF receptor antagonist, both significantly inhibited the proliferation of TCA8113 cells. CONCLUSION: Genistein inhibits the proliferation of TCA8113 cells, which may be related to its inhibitory effect on ERK expression and activation, thus subsequently decreasing the expression of VEGF.     

Effect of conditioned medium of human multiple myeloma RPMI 8226 cells on osteoclastogenesis of RAW264.7 cells

AIM: To investigate the osteoclastogenic effect of conditioned medium of human multiple myeloma RPMI 8226 cells on preosteoclast RAW264.7 cells. METHODS: The protein expression of soluble receptor activator of NF-κB ligand (sRANKL) was detected by Western blotting. The morphological changes of RAW264.7 cells were observed after tartrate-resistant acid phosphatase (TRAP) staining. The mRNA expression of TRAP and cathepsin K was evaluated by RT-PCR. RESULTS: The result of Western blotting showed that conditioned medium of RPMI 8226 cells contained sRANKL. RPMI 8226 cell conditioned medium induced RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppressed the differentiation of preosteoclasts in a dose-dependent manner, which was induced by 30% RPMI 8226 cell conditioned medium. RPMI 8226 cell conditioned medium increased the mRNA expression of TRAP and cathepsin K in RAW264.7 cells. CONCLUSION: The sRANKL in conditioned medium of human multiple myeloma RPMI 8226 cells has the bioactivity to induce preosteoclast RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppresses the differentiation of preosteoclasts induced by sRANKL in a dose-dependent manner.     

Effects of adenovirus-mediated shRNA targeting PTEN on proliferation and apoptosis of activated hepatic stellate cells in vitro

AIM:To investigate the down-regulation of phosphatase and tensin homolog deleted on chromosome 10(PTEN) gene by adenovirus-mediated short hairpin RNA(shRNA) on proliferation and apoptosis of activated hepatic stellate cells(HSCs) in vitro and the related signaling transduction pathways. METHODS:The activated HSCs were cultured in vitro and transfected with recombinant adenovirus expressing shRNA targeting PTEN. The proliferation of HSCs was measured by MTT assay and the apoptosis was assessed by TUNEL and flow cytometry. Western blotting was used to detect the protein levels of PTEN, Bax, Bcl-2, Akt, p-Akt, ERK1/2 and p-ERK1/2 in HSCs, and real-time fluorescent quantitative PCR was applied to detect the mRNA expression of Akt and ERK1. RESULTS:The recombinant adenovirus expressing shRNA targeting PTEN was successfully transfected into activated HSCs in vitro, and significantly promoted the proliferation of HSCs in a time-dependent manner within a certain extent. The apoptotic rate of HSCs was significantly decreased 72 h after transfection(P<0.05). Meanwhile, reduced expression of Bax and elevated expression of Bcl-2 were induced 72 h after transfection(P<0.05). Furthermore, the expression of p-Akt and p-ERK1/2 were increased significantly(P<0.05), while no significant difference in the expression of Akt and ERK1 at mRNA and protein levels was observed(P>0.05). CONCLUSION:Down-regulation of PTEN by adenovirus-mediated shRNA dramatically promotes the proliferation of activated HSCs, and inhibits the apoptosis through Bcl-2/Bax pathway. In addition, the phosphorylation of Akt and ERK1/2 is increased, indicating that PI3K/Akt and ERK1/2 signal transduction pathways may play an important role in the regulation of proliferation and apoptosis of HSCs.     

High mobility group box 1 protein promotes proliferation,migration and invasion of colon cancer cells

AIM: To investigate the expression of high mobility group box 1 protein (HMGB1) in colon can-cer cells, and to determine its regulatory roles in colon cancer cell proliferation, migration and invasion. METHODS: qPCR and Western blot were used to quantify the mRNA and protein expression levels of HMGB1 in human colon cancer SW620 cells and normal colonic epithelial FHC cells. HMGB1 shRNA was transfected into the SW620 cells to establish the stable HMGB1-downregulating colon cancer cells (shHMGB1 group), and negative control (shNC) group and blank control (blank) group were also set up. The proliferation, migration and invasion of the cells were determined by CCK-8 assay, colony formation experiment and Transwell chamber assays. Western blot was used to determine the protein levels of p-ERK, ERK, c-Myc, matrix metalloproteinase (MMP)-2/9, E-cadherin, N-cadherin, Bcl-2 and Bax. RESULTS: Both of the mRNA and protein levels of HMGB1 in colon cancer cells were higher than those in the normal colonic epithelial cells (P<0.05). HMGB1 gene was successfully knocked down in SW620 cells. Compared with blank group and shNC group, the proliferation, migration and invasion abilities of the cells in shHMGB1 group were significantly inhibited (P<0.05). The protein levels of MMP-2, MMP-9, N-cadherin, c-Myc, Bcl-2 and p-ERK were reduced notably, while the expression of Bax protein was increased (P<0.05) in shHMGB1 group compared with shNC group and blank group.CONCLUSION: HMGB1 effectively promotes the proliferation, migration and invasion of colon cancer cells through ERK/c-Myc signaling pathway.     

Construction of shRNA eukaryotic expression vectors targeting PERK gene and effect of PERK gene knockdown on apoptosis in endoplasmic reticulum stress-induced L02 hepatocytes

AIM: To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting the gene of RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase ( PERK ), and to observe the effect of PERK gene knockdown on apoptosis of human normal hepatic L02 cells treated with thapsigargin. METHODS: Three shRNA expression vectors targeting PERK gene, named PERK1-shRNA, PERK2-shRNA and PERK3-shRNA, and one non-homologous negative control expression vector (HK-shRNA) were constructed based on the nucleotide sequence of PERK and the criteria of designing small interfering RNA (siRNA), and were identified by enzyme digestion and DNA sequencing analysis. After L02 hepatocytes were transfected with the plasmids, the PERK expression was determined by RT-PCR and Western blotting, and the plasmid with the best inhibitory effect on PERK expression was screened. The cell viability and apoptotic rate of L02 hepatocytes transfected with PERK-shRNA under endoplasmic reticulum stress (ERS) were measured by the methods of MTT and flow cytometry,respectively. RESULTS: Four shRNA expression vectors were constructed. The mRNA and protein expression levels of PERK gene decreased significantly in L02 hepatocytes transfected with PERK1-shRNA, PERK2-shRNA and PERK3-shRNA as compared with those in control cells (P<0.05). The interfering effect of PERK1-shRNA on PERK gene expression was the best. PERK knockdown by PERK1-shRNA increased the viability and inhibited the apoptosis of L02 cells under ERS.CONCLUSION: The shRNA expression vectors targeting PERK gene are constructed, and PERK gene knockdown may inhibit apoptosis in L02 hepatocytes under ERS.     

Effect of ROCK1 and ROCK2 silencing by shRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM: To investigate the effects of Rho-associated coiled-coil protein kinase-1 (ROCK1) and ROCK2 on apoptosis induced by hypoxia in rat cardiomyocytes. METHODS: Rat cardiomyocytes were cultured primarily and identified using an antibody targeting α-actin of striated muscle. ROCK1-shRNA and ROCK2-shRNA were transiently transfected into the cells by liposome. After 48 h, these cells were subject to hypoxia for 6 h. The cells were divided into 5 groups: blank control group, hypoxia group, hypoxia+negative control shRNA group, hypoxia+ROCK1-shRNA group and hypoxia+ROCK2-shRNA group. The beating frequency and rhythm of the cardiomyocytes were assessed by microscopy. The activity of lactate dehydrogenase (LDH) in the cell culture supernatants was detected by automatic biochemical analyzer. The cell survival rate was analyzed by the method of MTT. The cell apoptotic rate was assessed by flow cytometry. Western blotting was used to determine the expression of ROCK1, ROCK2, caspase-3 and p-PI3K. RESULTS: The primary culture of the cardiomyocytes was successful. Western blotting results showed that the transfection of ROCK1-shRNA or ROCK2-shRNA decreased the expression of ROCK1 or ROCK2 in the cardiomyocytes. Hypoxia slowed down the beat frequency of the cardiomyocytes, also made the rhythm disorder. Hypoxia increased the release of LDH and decreased the cell survival rate. Flow cytometry results showed that hypoxia increased the cell apoptotic rate. Hypoxia increased the expression of caspase-3 and decreased the expression of p-PI3K. Transfection of ROCK1-shRNA and ROCK2-shRNA into the cardiomyocytes reduced all the effects of hypoxia mentioned above. CONCLUSION: Down-regulation of ROCK1 and ROCK2 expression suppresses the apoptosis of rat cardiomyocytes induced by hypoxia. The mechanism is associated with the inhibition of caspase-3 activation and the up-regulation of p-PI3K expression.     

FOXQ1 gene mediate angiogenesis and proliferation induced by Shh in SW480 cells

AIM: To explore the effect of FOXQ1 gene silencing on angiogenesis and proliferation ability of colon cancer cells induced by Sonic hedgehog(Shh). METHODS: Lentivirus expressing different FOXQ1-shRNA or negative cantrol(NC)-shRNA was used to infect the SW480 cells. The best silencing condition was screened and used in the following experiments. The SW480 cells were divided into interfered group(FOXQ1-shRNA) and control group(NC-shRNA). The MTT assay was used to observe the doubling time and cell activity. Tube formation assay was performed to detect the ability of angiogenesis. Meanwhile, the expression of vascular endothelial growth factor(VEGF)-A, matrix metalloproteinase(MMP) 2 and cyclin D1 at mRNA and protein levels was determined by real-time PCR and Western blot. After induction of the cells by recombinant Shh proteins, the changes of angiogenesis and proliferation ability in each group were detected. At the same time, the transformation of related gene was examined. RESULTS: Compared with control group, the angiogenic ability in interfered group was decreased, and no obvious difference of proliferation ability was observed. The expression of VEGF-A and MMP2 was declined significantly, and the expression of cyclin D1 was not obviously changed. Recombinant Shh proteins improved the expression of FOXQ1 gene. Compared with NC-shRNA group, after induction, the angiogenic ability of FOXQ1-shRNA group was decreased, and the proliferation ability was not obviously changed. CONCLUSION: FOXQ1 gene mediates the angiogenic ability but does not affect the proliferation ability of SW480 cells. Meanwhile, it may be regulated by shh pathway.     

Effect of IQGAP1 siRNA on biological characteristics of glioma cells by regulating STAT3 signaling pathway

AIM: To investigate the effect of IQGAP1 gene expression knock-down on invasion, migration and immunosuppression of glioma cells and its mechanism. METHODS: Human glioma U251 cells were randomly divided into blank group, negative control group and si-IQGAP1 group. AG490, an inhibitor of STAT3 signaling pathway, was used to treat the cells for 48 h. The cell viability was measured by MTT assay. The protein levels of IQGAP1, vascular endothelial growth factor (VEGF), transforming growth factor-β1 (TGF-β1), STAT3 and p-STAT3 were determined by Western blot. The cell invasion and migration abilities were detected by Transwell assays. RESULTS: The protein expression of IQGAP1 in si-IQGAP1-1 group and si-IQGAP1-2 group was significantly lower than that in blank group (P<0.05). Compared with blank group, the viability, the invasion ability and the migration ability of the cells in si-IQGAP1 group and AG490 group were significantly decreased, while the protein levels of VEGF, TGF-β1 and p-STAT3 were significantly decreased (P<0.05). Compared with AG490 group, the cell viability, invasion ability and migration ability in AG490+si-IQGAP1 group were significantly decreased, and the protein levels of VEGF and TGF-β1 were significantly decreased (P<0.05). CONCLUSION: Silencing of IQGAP1 gene expression reduces the invasion and migration abilities of glioma cells and decreases the protein expression of cellular immunosuppression molecules VEGF and TGF-β1, which is related to down-regulation of STAT3 signaling pathway.     

RTN1A induces renal tubular epithelial cells to secrete VEGF and IL-8 and promotes diabetic nephropathy renal fibrosis via ERK signaling pathway

AIM:To investigate the effects of reticulon 1A (RTN1A) on the secretion of vascular endothelial growth facter (VEGF) and interleukin-8 (IL-8) in renal tubular epithelial cells, and on the diabetic nephropathy (DN) renal fibrosis, and to explore the underlying mechanism. METHODS:The mouse model of DN was established, and the blood glucose, kidney index, urine microalbumin (UMA) and creatinine clearance (CCr) were measured. The protein levels of RTN1A, p-ERK, ERK, VEGF, IL-8 and renal fibrosis markers α-smooth muscle actin (α-SMA) and fibronectin (FN) were determined by Western blot. Human renal tubular epithelial cell line HK-2 was treated with high glucose, and the ERK signaling proteins, fibrosis markers and secretion of cytokines were detected by Western blot and ELISA. The cells were treated with high glucose combined with RTN1A silencing or ERK inhibitor PD98059 for 24 h, and the ERK signaling proteins, fibrosis markers and secretion of cytokines were also detected by Western blot and ELISA. RESULTS:The blood glucose, kidney index, UMA and CCr in the DN mice were significantly higher than those in control group (P<0.05), suggesting that DN model was successfully constructed. The protein levels of RTN1A and its downstream protein p-ERK, the cytokines VEGF and IL-8, and the fibrosis markers α-SMA and FN were significantly increased in the DN model mice (P<0.05). The protein levels of RTN1A, p-ERK, VEGF, IL-8, α-SMA and FN were also significantly increased in the HK-2 cells after treated with high glucose for 24 h, while these proteins were significantly decreased after silencing of RTN1A expression. CONCLUSION:RTN1A may be associated with the occurrence and development of DN. Silencing of RTN1A expression inhibits DN renal inflammation and fibrosis through ERK signaling. RTN1A may be an effective therapeutic target.     

Effect of ERK1/2/c-Fos signal pathway on angiotensin-(1-7) inhibiting proliferation of rat glomerular mesangial cell strain induced by angiotensin Ⅱ

AIM: To investigate the effect of ERK1/2/c-Fos signal pathway during angiotensin-(1-7) inhibiting proliferation of rat glomerular mesangial cell strain (GMCS) induced by angiotensin Ⅱ. METHODS: Rat glomerular mesangial cells (GMC) were co-cultured with angiotensin Ⅱ and different doses of angiotensin-(1-7). The numbers of GMC were evaluated by crystal violet staining. The amounts of p-ERK1/2 and c-Fos expressions were detected by Western blotting. RESULTS: Angiotensin- (1-7) showed its inhibitory effects on GMC number increasing induced by angiotensin Ⅱ as well as the amounts of p-ERK1/2 and c-Fos expressions in a concentration dependent manner. CONCLUSION: ERK/c-Fos signal pathway is involved in the inhibitory effects of angiotensin-(1-7) on angiotensin Ⅱ -induced GMC proliferation.     

Promoting effect of botulinum neurotoxin serotype A heavy chain on neuritogenesis in cultured Neuro-2a cells

AIM: To observe the neuritogenic actions of botulinum neurotoxin serotype A heavy chain (BoNT/A HC) on cultured Neuro-2a cells and to investigate the related signaling mechanisms for the effect of BoNT/A HC. METHODS: Neuro-2a cells were treated with different doses of BoNT/A HC (0.01, 0.1, 1 and 10 nmol/L), and then the cells were harvested at 24 h, 48 h and 72 h of BoNT/A HC exposure for detecting the neurite length and the percen-tage of the cells with neuronal processes by immunofluorescence staining. The most efficient dose of BoNT/A HC was chosen for exposure to Neuro-2a cells as the above. Whole cell protein was harvested at different time points for detecting the protein levels of phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated Akt (p-Akt) by Western blot. RESULTS: Low doses of BoNT/A HC stimulated the neurite outgrowth, and increased the percentage of the cells with neurites compared with the negative controls (P<0.05), especially in the group with 1 nmol/L of BoNT/A HC treatment. Meanwhile, the phosphorylation of ERK1/2 and Akt was increased after treated with BoNT/A HC. There was an increasing tendency for the phosphorylation of ERK1/2 after the exposure of the cells to BoNT/A HC. The obvious increase in p-ERK1/2 was seen from 60 min to 5 h with 1 nmol/L of BoNT/A HC treatment (P< 0.05), and the increased protein level of p-Akt was mainly observed at 15 min and 60 min (P<0.05). CONCLUSION: BoNT/A HC stimulates the neuritogenesis. The neuritogenic mechanism of BoNT/A HC on Neuro-2a cells might be realized by activation of the phosphorylation of ERK1/2 and Akt.     

Inducing vascular smooth muscle cell proliferation by insulin involves in phosphatidylinositol 3-kinase and ERK1/2

AIM: To investigate the effect of insulin on vascular smooth muscle cells (VSMCs) proliferation and to evaluate the intracellular signaling pathways involved.METHODS: VSMCs separated from Sprague-Dawley rats were used in this study. The proliferation of VSMCs induced by insulin was assayed by ［3H］-thymidin incorporation. The protein expression and activity of p-ERK1/2 were determined by immunblot and ［γ-32P］ATP incorporation.RESULTS: Insulin induced cell proliferation in a concentration-dependent manner. The proliferative effect of insulin on VSMCs was inhibited partly by LY294002 (48.8%), an inhibitor of PI-3 kinase, and the ERK1/2 inhibitor PD98059 (43.6%), respectively. Moreover, phosphorylation of ERK1/2 and activity of ERK1/2 induced by insulin were also inhibited partly by LY294002.CONCLUSION: PI-3 kinase and ERK1/2 are involved in insulin induced VSMCs proliferation.     

Effects of phosphorylation-defective retinoic acid receptor α1 on proli-feration of human multiple myeloma cells in vitro

AIM: To investigate the effect of phosphorylation-defective retinoic acid receptor α1 (RARα1) on the proliferation of human multiple myeloma cells. METHODS: The mRNA expression of RARα subtypes in U266 cells was detected by RT-PCR. Lentiviral plasmid construction, viral production, titer determination and cell transfection were carried out by the general methods of molecular biology. Proliferation analysis was performed with CCK-8 assay. The U266 cells were treated with all-trans retinoic acid (ATRA,0~100 μmol/L) or transfected with lentivirus RARαS77A. The expression levels of proliferation-related proteins, P53 and Rb, in U266 cells treated with ATRA or transfected with lentivirus RARαS77A were detected by Western blotting. RESULTS: RARα1 was positively expressed in U266 cells and RARα2 expression was negative. ATRA significantly inhibited the proliferation of U266 cells in a dose- and time-dependent manner. Proliferation of U266 cells was significantly inhibited 48 h after transfection with lentivirus RARαS77A, and the inhibitory rate was 15.16%±3.84%. The up-regulated expression of Rb and down-regulated expression of P53 were detected in U266 cells not only in the cells treated with ATRA, but also in the cells transfected with lentivirus RARαS77A. CONCLUSION: Phosphorylation-defective RARα1 (RARαS77A) mimics the growth inhibitory effect of ATRA on U266 cells that express RARα1 (+) and RARα2 (-) via down-regulating the expression of P53 and up-regulating the expression of Rb, suggesting that the antiproliferative effect of ATRA is mainly mediated by decreasing the phosphorylation of RARα1.     

Role of reactive oxygen species and ERK1/2 signaling pathway in proliferation and apoptosis of hypoxic rat pulmonary arterial smooth muscle cells

AIM:To investigate the change of reactive oxygen species (ROS) production in hypoxic pulmonary arterial smooth muscle cells (PASMCs) of rats, the effect of ROS on the expression of extracellular signal-regulated kinase (ERK)1/2 protein, and the role of ROS and ERK1/2 in the imbalance between proliferation and apoptosis of PASMCs.METHODS: Primary cultures of PASMCs were established and cells between passages 2 to 3 were used for experiments. PASMCs were treated with tiron, a membrane permeable ROS scavenger, and PD98059, an ERK1/2 inhibitor, under normoxia or hypoxia condition. The ROS production was measured by DCFH-DA and NBT reduction. The expression of phosphorylated-ERK1/2 (p-ERK1/2) protein was detected by immunofluorescence. Cell proliferation was examined by MTT colorimetric assay and the expression of PCNA. Cell apoptosis was detected by TUNEL.RESULTS: (1)Compared with control group, the ROS levels in hypoxia group were significantly increased (P<0.01). (2) In hypoxia group, the proliferative capacity was higher and the apoptosis index was lower than those in control group (P<0.01). Tiron significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). (3) The expression of p-ERK1/2 in hypoxia group were higher than that in control group (P<0.01), which were significantly suppressed by tiron (P<0.01)．(4) PD98059 significantly attenuated hypoxia-induced cell proliferation (P<0.05) and also significantly raised the apoptosis index in hypoxia cells (P<0.01). The proliferative capacity and apoptosis index was similar in hypoxia+tiron+PD98059 group to those in hypoxia+tiron group (P>0.05).CONCLUSION:The hypoxia-mediated increase in PASMCs proliferation and the decrease in PASMCs apoptosis are related to the overproduction of intracellular ROS through downstream activation of ERK1/2. ROS and ERK1/2 play important roles in the hypoxic remodeling of pulmonary artery.     

Smad pathway participates the process of proliferation of vascular smooth muscle cells induced by extracellular signal regulated kinase pathway

AIM: To investigate whether Smad pathway participates the process of extracellular signal regulated kinase (ERK) induced the proliferation of vascular smooth muscle cells (VSMCs). METHODS: Human umbilical artery smooth muscle cells (hUASMCs) were divided into four groups: control group, PDGF (platelet derived growth factor) group, ERK blocking agent group and PDGF+ERK blocking agent group. MTT assay was used to detect the proliferation of hUASMCs (A value). Immunohistochemical technique was used to detect the expression of PCNA, phosphorylated ERK (p-ERK) and phosphorylated Smad2/3 (p-Smad2/3) protein in hUASMCs. The expression of Smad2/3 mRNA in hUASMCs was detected by RT-PCR. RESULTS: The proliferation of hUASMCs and the expression of PCNA, p-ERK and p-Smad2/3 proteins in hUASMCs in PDGF group were increased obviously than those in other groups (P<0.01). No difference in the expression of Smad2/3 mRNA in hUASMCs among groups was observed. CONCLUSION: Smad pathway participates the process of ERK pathway that induces the proliferation of hUASMCs at the level of protein.