SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo

AIM:To explore the effect of silent information regulator factor 2-related enzyme 1(SIRT1)on modulating apoptosis of human lens epithelial cells(HLECs)and alleviating lens opacification of rats through suppressing endoplasmic reticulum(ER)stress.METHODS:HLECs(SRA01/04)were treated with varying concentrations of tunicamycin(TM)for 24h,and the expression of SIRT1 and C/EBP homologous protein(CHOP)was assessed using real-time quantitative polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8(CCK-8)assay,respectively.In the SRA01/04 cell apoptosis model,which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation,the expression levels of SIRT1,CHOP,glucose regulated protein 78(GRP78),and activating transcription factor 4(ATF4)were examined.The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid(4-PBA;an ER stress inhibitor)was investigated.In vivo,age-related cataract(ARC)rat models were induced by sodium selenite injection,and the protective role of SIRT1,activated by SRT1720 intraperitoneal injections,was evaluated through morphology observation,hematoxylin and eosin(H&E)staining,Western blotting,and RT-PCR.RESULTS:SIRT1 expression was downregulated in TMinduced SRA01/04 cells.Besides,in SRA01/04 cells,both cell apoptosis and CHOP expression increased with the rising doses of TM.ER stress was stimulated by TM,as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model.Inhibition of SIRT1 by siRNA knockdown increased ER stress activation,whereas SRT1720 treatment had opposite results.4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis.In vivo,SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models.CONCLUSION:SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress.These findings suggest a novel strategy for cataract treatment focused on targeting ER stress,highlighting the therapeutic potential of SIRT1 modulation in ARC development.

Safety evaluation of human umbilical cord-mesenchymal stem cells in type 2 diabetes mellitus treatment:A phase 2 clinical trial

BACKGROUND Progressive pancreaticβcell dysfunction is a fundamental aspect of the pathology underlying type 2 diabetes mellitus(T2DM).Recently,mesenchymal stem cell(MSC)transplantation has emerged as a new therapeutic method due to its ability to promote the regeneration of pancreaticβcells.However,current studies have focused on its efficacy,and there are few clinical studies on its safety.AIM To evaluate the safety of human umbilical cord(hUC)-MSC infusion in T2DM treatment.METHODS An open-label and randomized phase 2 clinical trial was designed to evaluate the safety of hUC-MSC transplantation in T2DM in a Class A hospital.Ten patients in the placebo group received acellular saline intravenously once per week for 3 wk.Twenty-four patients in the hUC-MSC group received hUC-MSCs(1×106 cells/kg)intravenously once per week for 3 wk.Diabetic clinical symptoms and signs,laboratory findings,and imaging findings were evaluated weekly for the 1st mo and then at weeks 12 and 24 post-treatment.RESULTS No serious adverse events were observed during the 24-wk follow-up.Four patients(16.7%)in the hUC-MSC group experienced transient fever,which occurred within 24 h after the second or third infusion;this did not occur in any patients in the placebo group.One patient from the hUC-MSC group experienced hypoglycemic attacks within 1 mo after transplantation.Significantly lower lymphocyte levels(weeks 2 and 3)and thrombin coagulation time(week 2)were observed in the hUC-MSC group compared to those in the placebo group(all P<0.05).Significantly higher platelet levels(week 3),immunoglobulin levels(weeks 1,2,3,and 4),fibrinogen levels(weeks 2 and 3),D-dimer levels(weeks 1,2,3,4,12,and 24),and neutrophil-to-lymphocyte ratios(weeks 2 and 3)were observed in the hUC-MSC group compared to those in the placebo group(all P<0.05).There were no significant differences between the two groups for tumor markers(alpha-fetoprotein,carcinoembryonic antigen,and carbohydrate antigen 199)or blood fat.No liver damage or other side effects were observed on chest X-ray.CONCLUSION Our study suggested that hUC-MSC transplantation has good tolerance and high safety in the treatment of T2DM.It can improve human immunity and inhibit lymphocytes.Coagulation function should be monitored vigilantly for abnormalities.

Human epidermal growth factor receptor 2 expression level and combined positive score can evaluate efficacy of advanced gastric cancer

BACKGROUND Although treatment options for gastric cancer(GC)continue to advance,the overall prognosis for patients with GC remains poor.At present,the predictors of treatment efficacy remain controversial except for high microsatellite instability.AIM To develop methods to identify groups of patients with GC who would benefit the most from receiving the combination of a programmed cell death protein 1(PD-1)inhibitor and chemotherapy.METHODS We acquired data from 63 patients with human epidermal growth factor receptor 2(HER2)-negative GC with a histological diagnosis of GC at the Cancer Hospital,Chinese Academy of Medical Sciences between November 2020 and October 2022.All of the patients screened received a PD-1 inhibitor combined with chemotherapy as the first-line treatment.RESULTS As of July 1,2023,the objective response rate was 61.9%,and the disease control rate was 96.8%.The median progression-free survival(mPFS)for all patients was 6.3 months.The median overall survival was not achieved.Survival analysis showed that patients with a combined positive score(CPS)≥1 exhibited an extended trend in progression-free survival(PFS)when compared to patients with a CPS of 0 after receiving a PD-1 inhibitor combined with oxaliplatin and tegafur as the first-line treatment.PFS exhibited a trend for prolongation as the expression level of HER2 increased.Based on PFS,we divided patients into two groups:A treatment group with excellent efficacy and a treatment group with poor efficacy.The mPFS of the excellent efficacy group was 8 months,with a mPFS of 9.1 months after excluding a cohort of patients who received interrupted therapy due to surgery.The mPFS was 4.5 months in patients in the group with poor efficacy who did not receive surgery.Using good/poor efficacy as the endpoint of our study,univariate analysis revealed that both CPS score(P=0.004)and HER2 expression level(P=0.015)were both factors that exerted significant influence on the efficacy of treatment the combination of a PD-1 inhibitor and chemotherapy in patients with advanced GC(AGC).Finally,multivariate analysis confirmed that CPS score was a significant influencing factor.CONCLUSION CPS score and HER2 expression both impacted the efficacy of immunotherapy combined with chemotherapy in AGC patients who were non-positive for HER2.

Genetic modification of miR-34a enhances efficacy of transplanted human dental pulp stem cells after ischemic stroke

Human dental pulp stem cells(hDPSCs) promote recovery after ischemic stro ke;however,the therapeutic efficacy is limited by the poor survival of transplanted cells.For in vitro expe riments in the present study,we used oxygen-glucose deprivation/reoxygenation in hDPSCs to mimic cell damage induced by ischemia/reperfusion.We found that miRNA-34a-5p(miR-34a) was elevated under oxygen-glucose deprivation/reoxygenation conditions in hDPSCs.Inhibition of miR-34a facilitated the prolife ration and antioxidant capacity and reduced the apoptosis of hDPSCs.Moreove r,dual-luciferase reporter gene assay showed WNT1and SIRT1 as the targets of miR-34a.In miR-34a knockdown cell lines,WNT1 suppression reduced cell prolife ration,and SIRT1 suppression decreased the antioxidant capacity.Togethe r,these results indicated that miR-34a regulates cell prolife ration and antioxidant stress via targeting WNT1 and SIRT1,respectively.For in vivo expe riments,we injected genetically modified hDPSCs(anti34a-hDPSCs) into the brains of mice.We found that anti34a-hDPSCs significantly inhibited apoptosis,reduced cerebral edema and cerebral infarct volume,and improved motor function in mice.This study provides new insights into the molecular mechanism of the cell prolife ration and antioxidant capacity of hDPSCs,and suggests a potential gene that can be targeted to improve the survival rate and efficacy of transplanted hDPSCs in brain after ischemic stroke.

Role of reactive oxygen species in epithelial-mesenchymal transition and apoptosis of human lens epithelial cells

AIM:To investigate the role of reactive oxygen species(ROS)in epithelial–mesenchymal transition(EMT)and apoptosis of human lens epithelial cells(HLECs).METHODS:Flow cytometry was used to assess ROS production after transforming growth factorβ2(TGF-β2)induction.Apoptosis of HLECs after H_(2)O_(2) and TGF-β2 interference with or without ROS scavenger N-acetylcysteine(NAC)were assessed by flow cytometry.The corresponding protein expression levels of the EMT markerα-smooth muscle actin(α-SMA),the extracellular matrix(ECM),marker fibronectin(Fn),and apoptosis-associated proteins were detected by using Western blotting in the presence of an ROS scavenger(NAC).Wound-healing and Transwell assays were used to assess the migration capability of HLECs.RESULTS:TGF-β2 stimulates ROS production within 8h in HLECs.Additionally,TGF-β2 induced HLECs cell apoptosis,EMT/ECM synthesis protein markers expression,and pro-apoptotic proteins production;nonetheless,NAC treatment prevented these responses.Similarly,TGF-β2 promoted HLECs cell migration,whereas NAC inhibited cell migration.We further determined that although ROS initiated apoptosis,it only induced the accumulation of the EMT markerα-SMA protein,but not COL-1 or Fn.CONCLUSION:ROS contribute to TGF-β2-induced EMT/ECM synthesis and cell apoptosis of HLECs;however,ROS alone are not sufficient for EMT/ECM synthesis.

Effect of miR-27b-3p and Nrf2 in human retinal pigment epithelial cell induced by high-glucose

AIM:To determine whether the microRNA-27b-3p(miR-27b-3p)/NF-E2-related factor 2(Nrf2)pathway plays a role in human retinal pigment epithelial(hRPE)cell response to high glucose,how miR-27b-3p and Nrf2 expression are regulated,and whether this pathway could be specifically targeted.METHODS:hRPE cells were cultured in normal glucose or high glucose for 1,3,or 6d before measuring cellular proliferation rates using cell counting kit-8 and reactive oxygen species(ROS)levels using a dihydroethidium kit.miR-27b-3p,Nrf2,NAD(P)H quinone oxidoreductase 1(NQO1)and heme oxygenase-1(HO-1)mRNA and protein levels were analyzed using reverse transcription quantitative polymerase chain reaction(RT-qPCR)and immunocytofluorescence(ICF),respectively.Western blot analyses were performed to determine nuclear and total Nrf2 protein levels.Nrf2,NQO1,and HO-1 expression levels by RT-qPCR,ICF,or Western blot were further tested after miR-27b-3p overexpression or inhibitor lentiviral transfection.Finally,the expression level of those target genes was analyzed after treating hRPE cells with pyridoxamine.RESULTS:Persistent exposure to high glucose gradually suppressed hRPE Nrf2,NQO1,and HO-1 mRNA and protein levels and increased miR-27b-3p mRNA levels.High glucose also promoted ROS release and inhibited cellular proliferation.Nrf2,NQO1,and HO-1 mRNA levels decreased after miR-27b-3p overexpression and,conversely,both mRNA and protein levels increased after expressing a miR-27b-3p inhibitor.After treating hRPE cells exposed to high glucose with pyridoxamine,ROS levels tended to decreased,proliferation rate increased,Nrf2,NQO1,and HO-1 mRNA and protein levels were upregulated,and miR-27b-3p mRNA levels were suppressed.CONCLUSION:Nrf2 is a downstream target of miR-27b-3p.Furthermore,the miR-27b-3p inhibitor pyridoxamine can alleviate high glucose injury by regulating the miR-27b-3p/Nrf2 axis.

Effect of WFDC 2 silencing on the proliferation,motility and invasion of human serous ovarian cancer cells in vitro

Objective:To investigate effect and possible mechanisms of silencing human WFDC2（HE4） gene on biological behavior changes as cell proliferation,apoplosis,movement and invasion of human serous ovarian cancer cell line SKOV3.Methods:Lentiviral WFDC2 gene sequence of small interfering siRNA was stablely transfected into SKOV3 identified by Q-PCR and western-blot. Obtained SKOV3 stable strains with silenced HE4 were measured by proliferation,apoplosis, migration,and invasion.Results:Gene sequencing showed that the oligonucleotides were successfully inserted into the expected site.After silencing HE4 in the SKOV3,proliferation was significandy inhibited（P【0.05）.G0/G1 phase was arrested by the cell cycle（P【0.01） and capacity of the migration and invasion decreased significandy（P【0.01）.Slight early apoptosis ratio and no change of late apoplosis were found without change of Caspase-3 or Bcl-2 protein.Proteins involed in ERK pathway as phosphorylated protein as p-EGFR,p- ERK decreased and protease protein involved in tissue remoding as matrix metalloproteinases MMP-9,MMP-2 and cathepsin B decreased compared with control group.Conclusions:HE4 gene plays an important role in regulating proliferation,apoptosis,migration,invasion of serous ovarian cancer cells by ERK pathway and protease system.Its role in apoptosis needs to be further explored,and it may be a potential target for serous ovarian cancer.

Therapeutic potential of human embryonic stem cells in type 2 diabetes mellitus

AIM:To evaluate the safety and efficacy of human embryonic stem cells(h ESCs)for the management of type 2 diabetes mellitus(T2DM).METHODS:Patients with a previous history of diabetes and its associated complications were enrolled and injected with hE SC lines as per the defined protocol.The patients were assessed using Nutech functional score(NFS),a numeric scoring scale to evaluate the patients for 11 diagnostic parameters.Patients were evaluated at baseline and at the end of treatment period 1(T1).All the parameters were graded on the NFS scale from 1to 5.Highest possible grade(HPG)of 5 was considered as the grade of best improvement.RESULTS:Overall,94.8%of the patients showed improvement by at least one grade of NFS at the end of T1.For all the 11 parameters evaluated,54%of patients achieved HPG after treatment.The four essential parameters(improvement in glycated hemoglobin(HbA 1c)and insulin level,and fall in number of other oral hypoglycemic drugs with and without insulin)are presented in detail.For Hb A1c,72.6%of patients at the end of T1 met the World Health Organization cut off value,i.e.,6.5%of HbA 1c.For insulin level,65.9%of patients at the end of T1 were able to achieve HPG.After treatment,the improvement was seen in 16.3%of patients who required no more than two medications along with insulin.Similarly,21.5%of patients were improved as their dosage regimen for using oral drugs was reduced to 1-2 from 5.CONCLUSION:hE SC therapy is beneficial in patients with diabetes and helps in reducing their dependence on insulin and other medicines.

Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

AIM:To investigate the morphological altering effect of transforming growth factor-β2(TGF-β2) on untransfected human corneal endothelial cells(HCECs)in vitro.METHODS:After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology,cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy,immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2(9 μg/L) altered HCE cell morphology after treatment for 36 h, increased the mean optical density(P 【0.01) and the length of F-actin,reduced the mean optical density(P 【0.01) of the collagen type IV in extracellular matrix(ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72 h.·CONCLUTION: TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

Anti-osteoarthritis effect of a combination treatment with human adipose tissue-derived mesenchymal stem cells and thrombospondin 2 in rabbits

BACKGROUND Osteoarthritis(OA),a chronic age-related disease characterized by the slowly progressive destruction of articular cartilage,is one of the leading causes of disability.As a new strategy for treatment of OA,mesenchymal stem cells(MSCs)have the potential for articular cartilage regeneration.Meanwhile,thrombospondin 2(TSP2)promotes the chondrogenic differentiation of MSCs.AIM To investigate whether TSP2 induces chondrogenic differentiation of human adipose-derived MSCs(hADMSCs)and potentiates the therapeutic effects of hADMSCs in OA rabbits.METHODS We investigated the chondrogenic potential of TSP2 in hADMSCs by analyzing the expression of chondrogenic markers as well as NOTCH signaling genes in normal and TSP2 small interfering RNA(siRNA)-treated stem cells.Anterior cruciate ligament transection surgery was performed in male New Zealand white rabbits,and 8 wk later,hADMSCs(1.7×10^6 or 1.7×10^7 cells)were injected into the injured knees alone or in combination with intra-articular injection of TSP2(100 ng/knee)at 2-d intervals.OA progression was monitored by gross,radiological,and histological examinations.RESULTS In hADMSC culture,treatment with TSP2 increased the expression of chondrogenic markers(SOX9 and collagen Ⅱ)as well as NOTCH signaling genes(JAGGED1 and NOTCH3),which were inhibited by TSP2 siRNA treatment.In vivo,OA rabbits treated with hADMSCs or TSP2 alone exhibited lower degree of cartilage degeneration,osteophyte formation,and extracellular matrix loss 8 wk after cell transplantation.Notably,such cartilage damage was further alleviated by the combination of hADMSCs and TSP2.In addition,synovial inflammatory cytokines,especially tumor-necrosis factor-α,markedly decreased following the combination treatment.CONCLUSION The results indicate that TSP2 enhances chondrogenic differentiation of hADMSCs via JAGGED1/NOTCH3 signaling,and that combination therapy with hADMSCs and TSP2 exerts synergistic effects in the cartilage regeneration of OA joints.

Expression of Bcl-2 inhibited Fas-mediated apoptosis in human hepatocellular carcinoma BEL-7404 cells

Apoptosis plays an important role in embryonic development, tissue remodeling, immune regulation and tumor regression. Two groups of molecules (Bcl-2 family and "Death factor" family) are involved in regulating apoptosis. In order to know about the effect of Bcl-2 on apoptosis induced by Fas, a typical member of "Death factor" family, the transfection experiments with expression vectors pcDNA3-fl and pcDNA3-bcl-2 were performed in BEL-7404 cells, a human hepatocellular carcinoma cell line which expresses endogenous Fas, but not FasL and Bcl-2. The data showed that the expression of FasL in pcDNA3-fl transfected hepatoma cells obviously induced the apoptosis of the cells. However, the overexpression of Bcl-2 in pcDNA3-bcl-2 transfected 7404/b-16 cells counteracted pcDNA3-fl transient transfection mediated apoptosis. Further study by cotransfection experiments indicated that Bid but not Bax (both were pro-apoptotic proteins of Bcl-2 family) blocked the inhibitory effect of Bcl-2 on Fas-mediated apoptosis. These results suggested that Fas-mediated apoptosis in human hepatoma cells is possibly regulated by Bcl-2 family proteins via mitochondria pathway.

Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway

AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a doseand timedependent manner. It did notaffect the cell cycle, but induced apoptosis. PE significantly decreased ΔΨm at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a doseand time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.

Effect of Nimesulide on proliferation and apoptosis of human hepatoma SMMC-7721 cells

AIM: Cyclooxygenase-2 (COX-2) has been suggested to beassociated with carcinogenesis. We sought to investigatethe effect of the selective COX-2 inhibitor, Nimesulide onproliferation and apoptosis of SMMC-7721 human hepatomacells.METHODS: This study was carried out on the culture ofhepatic carcinoma SMMC-7721 cell line. Variousconcentrations of Nimesulide (0、200 μmol/L、300 μmol/L、400μmol/L) were added and incubated. Cell proliferation wasdetected with MTT colorimetric assay, cell apoptosis byelectron microscopy, flow cytometry and TUNEL.RESULTS: Nimesulide could significantly inhibit SMMC-7721cells proliferation dose-dependent and in a dependentmanner compared with that of the control group. Theduration lowest inhibition rate produced by Nimesulide inSMMC-7721 cells was 19.06 %, the highest inhibition ratewas 58.49 %. After incubation with Nimesulide for 72 h, themost highest apoptosis rate and apoptosis index of SMMC-7721 cells comparing with those of the control were 21. 20 %+1.62% vs2.24% +0.26% and21.23+ 1.78 vs2.01+0.23(p＜0.05).CONCLUSION: The selective COX-2 inhibitor, Nimesulide caninhibit the proliferation of SMMC-7721 cells and increaseapoptssis rate and apoptosis index of SMMC-7721 cells. Theapoptoois rate and the apoptosis index are dose-dependent.Under electron microscope SMMC-7721 cells incubated with 300μmol and 400 μmol Nimesulide show apoptotic characteristics.With the clarification of the mechanism of selective COX-2inhibitors, These COX-2 selective inhibitors can become thechoice of prevention and treatment of cancers.

Blockade of cholecystokinin-2 receptor and cyclooxygenase-2 synergistically induces cell apoptosis, and inhibits the proliferation of human gastric cancer cells in vitro

Gastrin and cyclooxygenase-2(COX-2) playimportant roles in the carcinogenesis and progression ofgastric cancer.However,it remains unknown whether the combination of cholecystokinin-2(CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic anti-tumor effects on human gastric cancer.Here,we demonstrated that the combination of AG-041R(a CCK-2 receptor antagonist) plus NS-398(a selective COX-2 inhibitor) treatment had synergistic effects on proliferation inhibition,apoptosis induction,down-regulation of Bcl-2 and up-regulation of Bax expression in MKN-45 cells.These results indicate that simultaneous targeting of CCK-2 receptor and COX-2 may inhibit gastric cancer development more effectively than targeting either molecule alone.(C)2008 Elsevier Ireland Ltd.All rights reserved.

Effects of transforming growth factor β2 and connective tissue growth factor on induction of epithelial mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells

AIM:To Investigate the effects of transforming growth factorβ2(TGF-β2)and connective tissue growth factor(CTGF)on transdifferentiation of human lens epithelial cells(HLECs)cultured in vitro and synthesis of extracellular matrix(ECM).METHODS:HLECs were treated with TGF-β2(0,0.5,1.0,5,10μg/L)and CTGF(0,15,30,60,100μg/L)for different times(0,24,48,72h)in vitro and the expression ofα-smooth muscle actin(α-SMA),the main component of the extracellular matrix typeⅠcollagen(Col-1)and fibronectin(Fn)were measured by using real-time polymerase chain reaction(PCR)and western-blot.RESULTS:TGF-β2 and CTGF significantly increased expression ofα-SMA mRNA and protein(P【0.05,P【0.001),Fn mRNA and protein(P【0.001),Col-1 mRNA and protein(P【0.001).TGF-β2 could induce HLECs expression of CTGF mRNA and protein in dosedependent manner(P【0.05,P【0.001).TGF-β2 and CTGF could induce HLECs to expressα-SMA,Fn and Col-1 in time-dependent manner.Each time of TGF-β2and CTGF induced HELCs expression ofα-SMA,Fn,Col-1 mRNA and protein was significant increase compared with control(P【0.05,P【0.001).CONCLUSION:TGF-β2 and CTGF could induce HLECs epithelial mesenchymal transition and ECM synthesis.

EFFECT OF MATRINE ON EXPRESSION OF HCCR1 AND HCCR2 PROTEINS IN CULTURAL HUMAN HEPATOCELLULAR CARCINOMAS CELLS

Objective： To explore the effects of matrine on HCCR1 and HCCR2 expression in cultural human hepatocellular carcinomas （HCC） cells at the level of gene and protein. Methods： Three methods, representational difference analysis （RDA） of cDNA, microarrays and fluorescence in situ hybridization （FISH） were used to detect levels of mRNA and protein expression of HCCR1 and HCCR-2 before and after treatment of matrine. Results： Matrine had inhibitory effect on the mRNA and protein expression of HCCR1 and HCCR2 in cultural HCC cells. Conclusion： Matrine has inhibitory effect on gene transcription, protein expression of HCCR 1 and HCCR2 in cultural HCC cells.

Preparation and in vitro studies of microencapsulated cells releasing human tissue inhibitor of metalloproteinase-2

Objective: To prepare microencapsulated cells releasing human tissue inhibitor ofmetalloproteinase-2 (TIMP-2), and investigate their biological characteristics in vitro. Methods: Chinese hamster ovary (CHO) cells were stably transfected with a human TIMP-2 expression vector, encapsulated in barium alginate microcapsules and cultured in vitro. Morphological appearance of the microcapsules was observed under a light microscope. Cell viability was assessed using MTT (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) assay. Enzyme linked immunosorbent assay (ELISA) and reverse zymography were used to confirm the release of biologically active TIMP-2 from the microcapsules. Cryopreservation study of the microencapsulated cells was carried out using dimethyl sulfoxide (DMSO) as preservative agent. Results: The microcapsules appeared like a sphere kept proliferating over the 6 weeks observed. No significant difference in TIMP-2 secretion was found between encapsulated and unencapsulated cells. Reverse zymography confirmed the bioactivity of MMP (matrix metalloproteinase) inhibition of TIMP-2.The cryopreservation process did not damage the microcapsule morphology nor the viability of the cells inside. Conclusion:Microencapsulated engineered CHO cells survive at least 6 weeks after preparation in vitro, and secrete bioactive TIMP-2 freely from the microcapsules.

Effect of amlodipine on apoptosis of human breast carcinoma MDA-MB-231 cells

Objective: To elucidate the effects of amlodipine on the proliferation and apoptosis of human breast carcinoma MDA-MB-231 cells. Methods: Light microscopy was used to determine the effects of amlodipine on cell morphology; Flow cytometry was used to quantitate cells undergoing apoptosis; the expression of a cell cycle-related protein, proliferating cell nuclear antigen (PCNA) and an antiapoptosis protein, Bcl-2 were assessed by immunocytochemistry. Results: Amlodipine concentration of 8.25 μmol/L (1/2 of IC50) affected the morphology, decreased the expression of PCNA and Bcl-2 and induced apoptosis of human breast carcinoma MDA-MB-231 cells. Conclusion: The effect of amlodipine on the antiproliferation of human breast carcinoma MDA-MB-231 cells is related to inducement of apoptosis, and the decrease of the expression of Bcl-2 and PCNA may be the possible mechanism for proliferation inhibitory and inducement of apoptosis.

Changes of NF-kB,p53,Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells:role of reactive oxygen species

AIM: To identify whether JTE-522 can induce apoptosis inAGS cells and ROS also involved in the process, and toinvestigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process.METHODS: Cell culture, MTT, Electromicroscopy, agarosegel electrophoresis, lucigenin, Western blot andelectrophoretic mobility shift assay (EMSA) analysis wereemployed to investigate the effect of JTE-522 on cellproliferation and apoptosis in AGS cells and relatedmolecular mechanisms.RESULTS: JTE-522 inhibited the growth of AGS cells andinduced the apoptosis. Lucigenin assay showed the generationof ROS in cells under incubation with JTE-522. The inc eareasedROS generation might contribute to the induction of AGS cellsto apoptosis. EMSA and Westem blot revealed that NF-kBactivity was almost completely inhibited by preventing thedegradation of IkBα. Additionally, by using Western blot weconfirmed that the level of bcl-2 was decreased, whereas p53showed a great increase following JTE-522 treatment. Theirchanges were in a dose-dependent manner.CONCLUSION: These findings suggest that reactive oxygenspecies, NF-kB, p53, bcl-2 and caspase-3 may play animportant role in the induction of apoptosis in AGS cellsafter treatment with JTE-522.

Double potentialities of tumoricide and hematopoiesis of human bone marrow cells activated by IL-2 and IL-3

The biomodulative and hematopoietic potentialities of IL-2 and IL-3 activatedbone marrow(ABM)cells from patients with lung adenocarcinoma were studied in vitro.Human bone marrow(BM)cells could be activated by IL-2 in culture for 7d.TheseIL-2 ABM cells had higher cytolytic activities against cells of H 7402 cell line and freshautologous adenocarcinoma cells and maintained the cytotoxicities longer than IL-2 acti-vated peripheral blood lymphocytes(APBLs),a point of possible importance in adoptiveimmunotherapy for cancer patients.The IL-2 ABM cells also had similar number ofBFU-E and CFU-GM to that had fresh BM cells if 1L-3 was added 48h alter IL-2 inculture.The IL-2 and IL-3 ABM cells might be used to eliminate tumor cells and tosupply reconstitutive elements of BM for autologous bone marrow transplantation.