Effect of NS-398 on invasion of colon cancer HT-29 cells in vitro and its regulation by CD44v6 and nm23-H1 genes

AIM: To study the anti-invasive effect of NS-398 on colon cancer cell line HT-29 in vitro an its regulation by CD44v6 and nm23-H1 genes. METHODS: Flow cytometry was used to detect the expression of COX-2 and CD44v6 in HT-29 cells. MTT was used for cell survival rate tests. The modified Boyden chamber model was used for quantitative invasion assay. RT-PCR was used to detect the expression of nm23-H1 mRNA. RESULTS: Flow cytometry analysis showed that COX-2 was positive in HT-29 cells. NS-398 had significant inhibitory effects on invasion of HT-29 cells, which had no relation with its cytotoxicity. NS-398 down-regulated the expression of CD44v6 and up-regulated the expression of nm23-H1 mRNA. CONCLUSION: NS-398 has an anti-invasive effect on HT-29 cells in vitro. Down-regulation of CD44v6 and up-regulation of nm23-H1 may be its underlying mechanisms.     

In vitro enhancement of radiation-induced apoptosis in human hepatoma cell line HepG2 by a selective inhibitor of cyclooxygenase-2 enzyme NS-398

AIM:To investigate the possible role of NS-398, a selective inhibitor of cyclooxygenase-2 enzyme, in radiation-induced apoptosis of human hepatoma cell line HepG2 in vitro. METHODS:Hepatoma cell line HepG2 was treated with various concentrations (25, 50, 100, 200 μmol/L) of NS-398 before MTT assay was used to evaluate the cytotoxicity of NS-398. Transmission electron microscopy (TEM) was used to observe the changes of apoptosis in morphology. FCM was performed to quantify the apoptotic percentage. Real-time PCR was used to detect the expression of bcl-2, bax and caspase-3 mRNA, Western blotting was used to measure the expression of Bcl-2 and bax protein, and colorimetric method was provided to analyze the change of caspase-3 activity. RESULTS:The cytotoxicity of NS-398 increased in time-dependent and dose-dependent manners. NS-398 significantly enhanced radiation-induced apoptosis (P<0.01), increased the expression of bax mRNA, Bax protein, caspase-3 mRNA and enhanced caspase-3 activity, whereas no significant change in Bcl-2 expression was found (P>0.05). CONCLUSION:NS-398 enhances radiation-induced apoptosis in hepatoma cell line HepG2. The mechanism may be associated with the up-regulation of the expression of Bax, caspase-3 and enhancement of the activity of caspase-3, which ultimately induce apoptosis in HepG2.     

Effects of NS-398 on the proliferation and apoptosis of HepG2 cells

AIM: To evaluate the effects of NS-398, a cyclooxygenase-2(COX-2) inhibitor, on the proliferation and apoptosis in HepG2 cells. METHODS: The effects of NS-398 on the proliferation of HepG2 cells was evaluated by MTT. DNA fragmentation gel analysis was used to analyze the apoptotic cells; DNA ploidy and apoptotic cell percentage were examined by flow cytometry. Furthermore, the expression of COX-2 and Bcl-2 mRNA was identified by competitive RT-PCR. RESULTS: NS-398 inhibited cell proliferation and induced apoptosis in HepG2 in a concentration-dependent manner. DNA ploidy analysis showed that S phase cells were significantly decreased with NS-398 concentration increasing. The quiescent G₀/G₁ phase was accumulated with decreasing of Bcl-2 mRNA. Whereas NS-398 had no effect on the expression of COX-2 mRNA, no correlations were found between COX-2 mRNA and the HepG2 cell proliferation and apoptosis induced by NS-398 (r=0.056 and r=0.119, respectively). CONCLUSION: NS-398 significantly inhibits the proliferation and induces apoptosis in HepG2. Mechanisms may be involved in accumulation of quiescent G₀/G₁ phase and decrease in Bcl-2 mRNA expression, but independent to COX-2 mRNA expression.     

Up-regulation of mRNA expressions of fas,bcl-2, bim,bax, caspase-3, caspase-9, and bcl2l12 in K562 treated with bortezomib

AIM: To detect the treatment of K562 leukemia cells with bortezomib altering the expression of genes fas, bcl-2, bcl2l12, bim, bax, caspase-9 and caspase-3.METHODS: MTT assay was used to detect the inhibition of proliferation. Apoptosis was detected by Annexin-V staining and mitochondrial transmembrane potential (Δψm). RT-PCR was used to analyze the mRNA expressions of fas, bcl-2, bcl2l12, bim, bax, caspase-3 and caspase-9.RESULTS: Bortezomib caused a time- and dose-dependent inhibition of cell proliferation and IC50 of 24 h and 48 h were 161.41 nmol/L and 96.33 nmol/L, respectively. At the concentration of 104 nmol/L, bortezomib induced apoptosis in a time-dependent manner, including increasing annexin-V positivity and decreasing the Δψm. RT-PCR showed that bortezomib up-regulated the mRNA expression of fas, bcl2l12, caspase-9 and caspase-3, but mRNA expressions of bcl-2, bim and bax did not changed obviously.CONCLUSION: Bortezomib inhibits the proliferation of K562 and induces apoptosis, in which fas, bcl2l12, caspase-9 or caspase-3 gene is one of the main genes taking part in.     

NKX3.1 down-regulates bcl-2 gene expression in prostate cancer PC-3 cells

AIM: To investigate the effect of NKX3.1 on the gene expression of bcl-2 and apoptosis in prostate cancer PC-3 cells. METHODS: The eukaryotic expression plasmid pcDNA3.1- NKX3.1 was transiently transfected into PC-3 cells. RT-PCR and Western blotting were used to detect the effects of NKX3.1 on the expression of bcl-2 gene. Down-regulatory effect of NKX3.1 on bcl-2 gene was detected by EMSA. Flow cytometry and apoptotic body staining were carried out to study the effects of NKX3.1 on apoptosis of PC-3 cells. RESULTS: The mRNA and protein expression of bcl-2 in PC-3 cells was down-regulated by over-expression of NKX3.1. The EMSA result showed that NKX3.1 interacted with the NKX3.1 binding elements in upstream regulatory region of bcl-2 gene. The results of flow cytometry showed that the number of apoptotic PC-3 cells increased by 1.41-fold after NKX3.1 transfection to PC-3 cells. NKX3.1 increased the apoptotic bodies stained by Hoechst 33258 significantly. CONCLUSION: NKX3.1 down-regulates the expression of anti-apoptotic gene bcl-2 and induces the apoptosis of prostate cancer PC-3 cells.     

Effect of shock lymph on apoptosis relative gene expression in pulmonary micro-vascular endothelial cells

AIM:To observe the effects of shock lymph on apoptosis relative gene expressions of pulmonary micro-vascular endothelial cells (PMVECs), and explore its mechanism.METHODS:The model of severe hemorrhagic shock was established by maintaining the blood pressure of rats in the condition of sepsis, mesentery lymph and shock portal vein blood was taken out. As control, mesentery lymph, portal vein blood of normal rats was taken out. The primary PMVECs of passages 3 were treated by different treatment factors, respectively. The apoptosis rate was analyzed by flow cytometry, and the expressions of relative genes of apoptosis such as fas, fas L, bcl-2 and bax were detected by RT-PCR. RESULTS:The apoptosis rate of PMVECs was 9.86%±3.24% after exposed to shock lymph at the final concentration of 4% for 4 hours and significantly higher than that in control (P<0.01). The expression levels of fas, fas L and bax mRNA were higher and bcl-2 mRNA was lower in shock lymph group than those in control group.CONCLUSION:The results demonstrated that the apoptosis of PMVECs of rats was induced by shock lymph, and its mechanism relate to high expression of apoptosis accelerative genes such as fas, fas L, bax mRNA and low expression of apoptosis inhibitory gene bcl-2.     

Effects of tetrahydroxystilbene-2-O-β-D-glucoside on apoptosis and expression of bcl-2/bax/caspase-3 in HUVECs treated with homocysteine

AIM：To explore the effects of tetrahydroxystilbene-2-O-β-D-glucoside (TSG) from Polygonum multiflorum on the apoptosis and the mRNA expression of bcl-2, bax and caspase-3 in human umbilical vein endothelial cells (HUVECs) treated with homocysteine (Hcy). METHODS：Cultured HUVECs were treated with Hcy (3 mmol/L) to establish a Hcy-damaged model. HUVECs in TSG treated groups were pre-incubated with TSG at concentrations of 1 μmol/L and 10 μmol/L for 2 h before treated with Hcy. Cell nuclear damage was detected by Hoechst 33342 staining. Cell apoptosis was determined by flow cytometry. The mRNA expression of bcl-2, bax and caspase-3 was measured by real-time fluorescence quantitative RT-PCR. RESULTS： After treatment with Hcy at concentration of 3 mmol/L, the nuclear damage and apoptotic rate of HUVECs were higher than that in normal group. The expression of bcl-2 was lower, and the expression of Bax and caspase-3 was higher than that in normal group. On the other hand, pre-incubation with TSG at concentrations of 1 μmol/L and 10 μmol/L decreased the nuclear damage and cell apoptosis, increased the expression of bcl-2, and decreased the expression of bax and caspase-3 as compared with the cells only treated with Hcy. CONCLUSION：TSG reduces the apoptosis of HUVECs induced by Hcy, and the mechanism might be associated with regulating the expression of bcl-2, bax and caspase-3.     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.     

C-reactive protein induces apoptosis in human renal tubular epithelial cells

ATM: To explore whether the C-reactive protein (CRP) level in microinflammation state induces the apoptosis of renal tubular epithelial cells. METHODS: HK-2 cells were stimulated with recombinant human CRP. Annexin-FITC-PI staining and flow cytometry were used to detect the percentage of apoptotic cells. Morphology observation of apoptosis was assessed by Hoechst 33258 staining. Caspase-3 activity was measured by a colorimetric assay. The expression of apoptotic gene bax and anti-apoptotic gene bcl-2 at mRNA levels was determined by real-time PCR. RESULTS: CRP induced apoptosis of HK-2 cells in a time- and dose-dependent manner. The maximal apoptotic effect of CRP concentration was 10 mg/L CRP at concentration of 20 mg/L. CRP treatment was associated with the characteristic morphological features of apoptosis such as condensation, fragmentation or margination of nuclear chromatin. CRP exposure increased caspase-3 activity, up-regulated the mRNA expression of Bax and down-regulated the mRNA expression of Bcl-2. CONCLUSION: Slightly increased CRP level has the potential to induce apoptosis of renal tubular cells.     

Inhibitory effects of a cyclooxygenase-2 inhibitor,NS398,on cancer cells

AIM:To study the mechanism of growth inhibitory effect of a selective cyclooxygenase-2 (COX-2) inhibitor,NS-398,on cancer cells.METHODS:The esophageal cancer cell line (EC9706),which expresses COX-2 constitutively,and hepatocellular carcinoma cell line (SMMC7721),which expresses no COX-2,were studied.The cell lines were incubated with NS-398 at doses of 10,20,50,100 μmol/L for 24 h,48 h and 72 h.Antiproliferation effect was measured by ［3H］-TdR incorporation.The cell apoptosis were determined by flow cytometry (FCM) and DNA fragmentation analysis.Survivin was detected by immunocytochemical technique.RESULTS:The growth inhibition was induced by NS398 in a dose- and time-dependent manners in both cell lines.FCM analysis revealed a high sub-G1 cell peak in EC9706 group and agarose electrophoresis showed marked apoptosis ladder pattern.However,no apoptosis was observed in SMMC7721 cells treated with NS-398.The difference of apoptosis percentage in EC9706 and SMMC7721 was (45.23±1.08)% and (3.05±0.15)% (P<0.01).After 24 h incubation with NS-398 at concentration of 100 μmol/L,the expression of survivin was markedly reduced in EC9706,no change was observed in SMMC7721.CONCLUSION:NS-398 suppresses cell growth in cancer cell lines by different mechanism.NS-398 suppresses cell growth and increases apoptosis in the cancer cells that expresses COX-2.     

Resveratrol induces apoptosis in human primary gastric carcinoma cells

AIM: To investigate the apoptosis in primary gastric cancer cells induced by resveratrol, and the relation between this apoptosis and expression of bcl-2 and bax. METHODS: In in vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission electron microscopy and TUNEL staining were used to quantitatively and qualitively detect the apoptosis of primary gastric cancer cells before and after the resveratrol treatment. Immunohistochemical staining and RT-PCR was used to detect the expression of apoptosis-regulated gene bcl-2 and bax. RESULTS: Resveratrol inhibited the growth of primary gastric cancer cells in a dose- and time-dependent manner. Resveratrol induced primary gastric cancer cells to undergo apoptosis with typically apoptotic characteristics. TUNEL assay showed that after the treatment of primary gastric cancer cells with resveratrol for 24, 48, 72, 96 hours, the apoptotic indexs were 4.93%±0.19%, 16.74%±0.43%, 27.88%±0.36%, 36.84%±1.07% respectively. Immunohistochemical staining showed that after the treatment of primary gastric cancer cells with resveratrol for 24, 48, 72, 96 hours, the positive rates of Bcl-2 proteins were 20.68%±0.49%, 10.84%±0.33%, 6.80%±0.34%, 3.91%±0.15% and the positive rates of Bax proteins were 19.79%±0.98%, 30.74%±0.85%, 40.14%±1.17%, 60.08%±1.64%. After exposed to resveratrol for 24 h, 48 h, 72 h and 96 h, the density of bcl-2 mRNA decreased progressively with elongation of time and the density of bax mRNA increased progressively with elongation of time by RT-PCR. CONCLUSION: Resveratrol is able to induce the apoptosis in primary gastric cancer. This apoptosis may be mediated by down-regulation of Bcl-2 and up-regulation of Bax.     

The anticancer activity of genistein on implanted tumor of human primary gastric carcinoma cells in nude mice

AIM: To investigate the apoptosis of implanted tumor of primary human gastric cancer cells in nude mice induced by genistein and the relation between this apoptosis and expression of bcl-2 and bax.METHODS: Establishing a transplanted tumor model by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice.The different doses of genistein (0.5mg/kg,1mg/kg and 1.5 mg/kg ) were directly injected beside tumor body respectively,for six times at an interval of two days.Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated.We observed the morphologic alteration by electron microscope,measured the apoptotic rate by TUNEL staining method,detected the expression of apoptosis-regulated gene bcl-2 and bax by immunohistochemical staining and RT-PCR.RESULTS: Genistein could significantly inhibit carcinoma growth when it was injected near the carcinoma.Genistein induced implanted tumors cells to undergo apoptosis with apoptotic characteristics by transmission electron microscope.The apoptosis index of above three groups was increased progressively.Positive rate of Bcl-2 protein of above three groups was decreased progressively and positive rate of Bax protein of above three groups was increased progressively by immunohistochemical staining.The density of bcl-2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT-PCR.CONCLUSION: Genistein is able to induce the apoptosis of transplanted tumor cells.This apoptosis may be mediated by down-regulating bcl-2 and up-regulating bax mRNA and its protein.     

Apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol

AIM: To investigate the molecular mechanism of the apoptosis of implanted tumor of human primary gastric cancer cells in nude mice induced by resveratrol. METHODS: Human primary gastric cancer cells were planted into nude mice to establish the cancer model. Resveratrol at different doses were injected near the carcinoma on the nude mice. After treatment, transmission electron microscope and TUNEL staining method were used to detect the apoptosis of implanted tumor cells. Immunohistochemical staining and RT-PCR were used to detect the expression of apoptosis-related genes bcl-2 and bax in implanted tumor. RESULTS: Resveratrol significantly inhibited carcinoma growth when it was injected near the carcinoma. The apoptotic cells in implanted tumor induced by resveratrol were detected by transmission electron microscope and TUNEL staining, immunohistochemical staining and RT-PCR showed resveratrol inhibited bcl-2 expression and increased bax expression in human primary gastric cancer cells. CONCLUSION: Resveratrol inhibits implanted tumor of human primary gastric cancer cells in nude mice through inducing apoptosis. This apoptosis may be mediated by down-regulation of bcl-2 expression and up-regulation of bax expression.     

HAPLN1 induces resistance to MTX in human colorectal cancer HT-29 cells though IKK/p65 pathway

AIM:To investigate the changes of hyaluronan and proteoglycan link protein 1 (HAPLN1) expression before and after resistance to methotrexate (MTX) in human colorectal cancer HT-29 cells and its effect on this drug resistance, and to explore the molecular mechanism in the process. METHODS:The drug-resistant HT-29/MTX cells were established by stepwise exposure of the cells to MTX, and then the HT-29/MTX cells were stably transfected with specific shRNA interference plasmid vectors targeting HAPLN1 and multidrug resistance-associated protein 2 (MRP2). The mRNA expression levels of HAPLN1 and MRP2 were measured by RT-PCR. CCK-8 assay was used to detect the viability of HT-29/MTX cells. The apoptosis rate was analyzed by flow cytometry. The protein levels of HAPLN1, MRP2, IκB kinase (IKK) α/β, p-IKKα/β (Ser176/Ser177), p65 and p-p65 (Ser536) were determined by Western blot. RESULTS:The HT-29/MTX cells had significantly higher mRNA and protein levels of HAPLN1 and MRP2 than HT-29 cells (P<0.05) with resistant factor of 463.756. HAPLN1 and MRP2 gene silencing significantly increased the cytotoxicity and apoptosis of HT-29/MTX cells induced by MTX (P<0.05). The IC₅₀ value was decreased from 15.304 μmol/L to 6.119 μmol/L and 7.801 μmol/L, respectively, and their reversal folds were 2.501 and 1.962, respectively. Silencing of HAPLN1 and IKK inhibitor IKK16 inhibited the phosphorylation of IKKα/β and p65 (P<0.05), and down-regulated the protein level of MRP2 in the HT-29/MTX cells (P<0.05). However, IKK16 did not affect the protein level of HAPLN1 in the HT-29/MTX cells.CONCLUSION:Knock-down of HAPLN1 gene expression reverses the resistance to MTX in human colorectal cancer HT-29/MTX cells possibly by blocking the IKK/p65 signaling pathway and thus down-regulating the expression of MRP2.     

Changes of bcl-2, bax expression and neuron apoptosis in lymphostatic encephalopathy of rats

AIM: To investigate the changes of bcl-2, bax expression and neuron apoptosis of cerebral cortex in lymphostatic encephalopathy of rats. METHODS: The model of lymphostatic encephalopathy was established by occluding and removing both the shallow and deep cervical lymph nodes in rats. The animals were sacrificed at 1, 2, 3, 5, 7 and 14 days after operation. HE staining was used to observe the structure of brain tissues and TUNEL staining was used to detect in situ cell apoptosis. The expressions of bcl-2 and bax were examined by RT-PCR. RESULTS: Cerebroedema appeared at the second day and was the most serious at the 5th day after blockage of cervical lymphatics. The number of TUNEL positive cells and the expression of bax began to increase at the 2nd day, reached a peak at the 5th day and dropped to control level at the 14th day. The expression of bcl-2 began to increase at the 1st day, reached a peak at the 5th day and dropped to control level at the 7th day. The increasing extent of bax was higher than that of bcl-2. CONCLUSION: The blockage of cervical lymphatics can lead to lymphostatic encephalopathy. Apoptosis is the main form of neuron death in the cortex and has relation to the increasing expression of bcl-2 and bax.     

Effect of sodium ferulate on bcl-2 and bax gene expression of apoptotic hippocampal neurons induced by sodium ferulate

AIM: To investigate the protective effects of sodium ferulate (SF) on apoptosis in cultured hippocampal neurons induced by sodium nitroprusside (SNP), and the effect of SF on expression of bcl-2 and bax. METHODS: The primary cultured hippocampal neurons were exposed to 50 μmol SNP, a nitric oxide-donor, for 24 h after pretreatment with different concentrations of SF (10-160 μmol/mL) for 6 h. Then neuronal viability was tested by MTT assay. Fluorescent staining with Hoechst 33258 and agarose gel electrophoresis was used to analyze apoptosis. The expressions of bcl-2, bax mRNA and protein were tested by RT-PCR and Western blotting. RESULTS: Pretreatment with SF(10-160 μmol/L) for 6 h increased the survival rate of neurons. SF prevented the neuronal nuclei from shrinkage, condensation and cleavage and blocked neuronal nuclear DNA fragmentation induced by SNP. SF also increased the expressions of bcl-2 mRNA and Bcl-2 protein and decreased the expressions of bax mRNA and Bax protein. CONCLUSION: SF prevents the cultured hippocampal neurons against SNP neurotoxicity. The mechanism of protection is related to the increase in Bcl-2 level and the decrease in Bax level. As a result, the ratio of Bcl-2/Bax is changed.     

Protective effects and mechanisms of losartan on apoptosis of H9c2 cells induced by β-adrenergic stimulation in vitro

AIM: To investigate the protective effect of losartan (Los) on apoptosis of H9c2 cells induced by isoprenaline (ISO), and to discover its related mechanism. METHODS: H9c2 cells cultured on plastic plates were divided into control, ISO, ISO+Los, ISO+Los+LY294002 and DMSO groups. Cell apoptosis was evaluated by flow cytometery and agarose gel electrophoresis. The mRNA levels of bax, bcl-2 and caspase-9 were detected by RT-PCR and the expressions of phosphorylated and total Akt (p-Akt and t-Akt) were assessed by Western blotting. The cAMP was measured by radioimmunoassay. RESULTS: ISO at concentration of 10 μmol/L induced apoptosis of H9c2 with an increase in bax/bcl-2, caspase-9 and cAMP. Addition of 10 μmol/L losartan inhibited apoptosis obviously with a decrease in bax/bcl-2, caspase-9 and cAMP. A significant increase in p-Akt was observed, and its protein level was elevated. LY294002 at concentration of 1 μmol/L abolished the protective effects of losartan on ISO-induced apoptosis in H9c2 cells. CONCLUSION: ISO might induce H9c2 cell apoptosis through stimulation of β-adrenergic receptor (β-AR). Los inhibits downstream signaling of β-AR, and promotes the activation of Akt. Subsequently it might attenuate the apoptosis induced by β-adrenergic stimulation of ISO.     

αvβ6-ERK pathway participates in norcantharidin-induced apoptosis of HT-29 colon cancer cells

AIM: To investigate the pharmacological mechanism of norcantharidin (NCTD)-induced apoptosis of HT-29 colon cancer cells. METHODS: Hoechst 33258 staining was used to analyze the apoptosis of HT-29 cells treated with NCTD. The effects of NCTD on the expression of integrin in HT-29 cells were determined by flow cytometry. The effects of several functional blocking antibodies on HT-29 cells were detected by MTT method. The expression and the phosphorylation of mitogen activated protein kinases (MAPKs) in HT-29 cells were measured by Western blotting. Co-immunoprecipitation assay was used to detect the activity of αvβ6-extracellular signal-regulated kinase (ERK) direct linkage in HT-29 cells.RESULTS: NCTD induced the apoptosis of HT-29 colon cancer cells. The expression of integrin αvβ6 in HT-29 cells treated with NCTD was reduced, but the expression of αvβ3 and αvβ5 was not changed. A function-blocking antibody to αvβ6,10D5,strengthened the growth inhibitory effect of NCTD on HT-29 cells ,but LM609 (a function-blocking antibody to αvβ3) and P1F6 (a function-blocking antibody to αvβ5) did not. The level of phosphorylated ERK (p-ERK) was decreased substantially after treated with NCTD in a dose-and time-dependent manner. NCTD also affected the association of αvβ6 and ERK. CONCLUSION: NCTD decreases the expression of integrin αvβ6 and interferes with the phosphorylation of ERK. As a result, the formation of αvβ6-ERK direct linkage is affected and the signal transduction mediated by αvβ6 is disturbed. The mechanism of NCTD-induced HT-29 cell apoptosis is involved in the αvβ6-ERK signaling pathway.     

BPH-1 induces aromatase expression in prostatic stromal cells through paracrine of PGE2

AIM: To characterize the effect of prostatic epithelial cell paracrine on aromatase expression in prostatic stromal cells.METHODS: Conditioned medium (CM) of prostatic epithelial cell lines (BPH-1, LNCap, DU-145 and PC3) were collected and used to treat prostatic stromal cells. Expression of aromatase was determined by real-time RT-PCR and Western blotting. Expression of cyclooxygenase-2 mRNA in prostatic epithelial cell lines and prostaglandin (PGE2) in CMs were examined by real-time RT-PCR and ELISA, respectively. The CM of BPH-1 cells cultured with NS-398, specific inhibitor of cyclooxygenase-2, were collected, and the effect of NS-398 and PGE2 on aromatase expression was analyzed.RESULTS: CM of human benign prostate hyperplasia epithelial cell line (BPH-1) stimulated expression of aromatase mRNA and protein in stromal cells. But CM of prostate cancer epithelial cell lines (LNCap, DU145, PC3) had no effect on aromatase expression. COX-2 mRNA level in BPH-1 was much higher than that of other cell lines and PGE2 concentration in BPH-1 CM was much higher than that of other CMs. PGE2 concentration of the CM from BPH-1 cultured with NS-398 significantly decreased. CM from BPH-1 cultured with NS-398 failed to stimulate aromatase expression, while PGE2 induced aromatase expression in prostatic stromal cells.CONCLUSION: BPH-1 could induce aromatase expression in prostatic stromal cells through paracrine of PGE2.