Anti-cancer effect of cytotoxic T lymphocytes activated by sensitized dendritic cells

AIM: To investigate the anti-cancer effect of cytotoxic T lymphocytes (CTL) activated by sensitized dendritic cells (DCs). METHODS: Immature DCs were induced in vitro from peripheral blood monocytic cells (PBMC) and sensitized by adding tumor cells antigen extract. DCs were identified by their morphology and surface markers. MTT assay was used to evaluate the killing activity of CTL activated by sensitized DCs. The effects of specific CTL cells on inhibiting transplanted tumor HT-29 growth and on preventing HT-29 tumor generation were evaluated by injecting CTL into nude mice. RESULTS: After cultured for seven days, a large number of activated DCs were obtained with typical morphology, extensive stimulatory proliferation capacity and high CD80 (63.5%), CD83 (67.6%) and CD3/HLA-DR (83.2%) expressions. The killing activity of CTL at 20∶1 ratio of effective cells to target cells was more than 75% to tumor cells, 35%-45% to homologous cell line and weaker to other germ cell line (P<0.01). Injection of CTLs activated by HT-29 cell antigen sensitized DCs inhibited HT-29 transplanted tumor growth and prevented HT-29 tumor occurring in nude mice (P<0.05). PCNA expression level in tumor cells in CTL therapy group was dramatically lower than that in control (P<0.05). CONCLUSION: CTL activated by sensitized DCs kill tumor cells specifically, inhibit transplanted tumor growth and prevent tumor transplantation in nude mice.     

Induction of tumor-specific cytotoxic T lymphocytes in vitro by dendritic cells pulsed with different glioma antigens

AIM: The goal of this study was to compare different methods for tumor antigen preparation, to observe the induction of tumor-specific cytotoxic T lymphocytes in rats by dendritic cells (DCs) pulsed with different tumor antigens. METHODS: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thaw, boiling or total protein extracted from ultrasonic crushed tumor cell. Subsequently primed DCs were cocultured with T lymphocytes isolated from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-γ release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test. RESULTS: DCs pulsed with boiled tumor cell in vitro induced an enhanced ability of T-cell proliferation and cytotoxic T lymphocyte activity.CONCLUSION: Our results demonstrated that DCs primed with boiled tumor cell may represent a method for inducing immune responses against the entire repertoire of tumor antigens of malignancies.     

Antigen-loaded dendritic cells and CD40L triggers the killing effects of cytotoxic T lymphocytes on K562 cells in vitro

AIM:To investigate the anti-tumor effects of special cytotoxic T lymphocytes (CTLs) activated by dendritic cells (DCs) loaded with antigens and CD40L in vitro.METHODS:Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation from normal human heparinized blood.The adherent cells were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF),interleukin-4 (IL-4),alpha tumor necrosis factor (TNF-α),DCs were co-cultured with frozen-thawed antigen of K562 cells and CD40L,then triggered T cells into specific CTLs.RESULTS:Most suspended cells exhibited distinctive morphological features of DCs which expressed CD40 96%,CD86 97%,CD80 77%,CD1a 69%,and gained the powerful capacity to stimulate proliferation of allogenic lymphocytes.Under the effector∶target ratio of 20∶1,CTLs derived from cultures with DCs and frozen-thawed antigen of K562 cells were showed 71.3% cytotoxicities against K562 cells.CTLs derived from cultures with DCs loaded with frozen-thawed antigen and CD40L were showed 86.9% cytotoxicities against K562 cells.Cytotoxicities by CTLs derived from cultures with unloaded DCs against K562 cells were 37.6% and cytotoxicities by monocytes were 21.1%.Cytotoxicities by CTLs derived from experiment groups were stronger than control groups (P<0.05).CONCLUSIONS:The tumor antigen-pulsed DCs induces efficient and specific anti-tumor immunity,CTLs derived from cultures containing DCs pulsed with CD40L show the strongest cytolytic activities on K562 cells.     

Effects of melatonin on apoptosis of lymphocytes induced by ionizing radiation in mice

AIM: To explore the effect of melatonin (MLT) on the apoptosis of thymocytes and splenocytes in mice induced by ionizing radiation and its mechanism. METHODS: The percentages of apoptotic bodies and the DNA lytic rates of thymocytes and splenocytes in mice in vitro and in vivo were detected with flow cytometry and fluorospectrophotometry, respectively. RESULTS: The apoptosis of mouse thymocytes and splenocytes in vitro increased with significant dose-dependence in 0.5-6.0 Gy X-irradiation. When MLT of 2 mmol·L^-1 was added into thymocytes or splenocytes in vitro before irradiation with 0.5-6.0 Gy X-rays, the percentages of apoptotic bodies and the DNA lytic rates all decreased significantly as compared with those in the irradiation group. The percentages of apoptotic bodies in these two kinds of cells were 86.25% and 89.22% of those in the irradiation group, respectively, and the DNA lytic rates were 87.23% and 89.16%, respectively. When MLT was injected into intraperitonium in mice 60 min before whole-body irradiation with 2 Gy X-rays, the percentages of apoptotic bodies and the DNA lytic rates were significantly lower than those in the irradiation group, and near or lower than those in the sham-irradiation group. MLT of 0.1-2.5 mg/kg decreased the lymphocyte apoptosis, but without significant dose-dependence. CONCLUSION: The protective effects of MLT on mouse lymphocytes damaged by irradiation in vivo were obvious than those in vitro.     

The potential role of staphylococcal enterotoxin B in the early intestinal injury in postburn Staphylococcus aureus sepsis

AIM: To investigate the role of staphylococcal enterotoxin B (SEB) in early intestinal injury in scald rats with Staphylococcus aureus sepsis. METHODS: 86 male Wistar rats were randomly divided into four groups as folows: normal controls (n=10), scald control group(n=10), postburn sepsis group (n=50) and SEB monoclonal antibody (MAb) treatment group (n=16). Plasma samples were collected to determine SEB, endotoxin, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). RESULTS: After scald injury followed by Staphylococcus aureus challenge, the levels of SEB, TNF-α and IFN-γ in plasma were significantly higher than those of normal controls, peaking at 2-6 h (P<0.05 or P<0.01), while the intestinal diamine oxidase (DAO) activity declined constantly (P<0.05). It was shown that plasma SEB levels were significant negatively correlated with intestinal DAO activity (r=-0.4398, P=0.0170), and SEB MAb pretreatment could ameliorate the intestinal injury to certain extent. Moreover, Staphylococcus aureus challenge could increase the endotoxin levels in plasma and various tissues, which were attenuated by SEB MAb pretreatment. CONCLUSION: In postburn sepsis, SEB might be involved in the development of intestinal barrier dysfunction, which in turn resulting in gut-derived endotoxin translocation and aggravating the pathophysiologic changes caused by Staphylococcus aureus challenge.     

Clonal expansion and specific cytotoxicity of autologous or allogeneic T cells induced by M2a cells

AIM: To investigate clonal expansion and specific cytotoxicity of TCR Vβ subfamily T cells from acute myelogenous leukemia M_2a subtype (AML-M_2a)patients and normal individuals induced by AML-M_2a cells, respectively. METHODS: Complementarity determining region 3(CDR3) of TCR β with variable region genes was amplified in autologous or allogeneic T cells from mixed lymphocyte and tumor culture (MLTC) using RT-PCR. The positive products were further analyzed to identify the clonality of T cells by genescan. The specific cytotoxicity of T cells was analyzed by MTT. RESULTS: T cells from both M_2a patients and normal individuals after MLTC showed high response to M_2a cells with 4-17 TCR Vβ subfamily dominant utilization, one or two clonal expansion of T cells were identified in some predominant TCR Vβ subfamilies. Difference of distribution and clonal expansion of TCR Vβ subfamily T cells were related to source of T cells and the phase during MLTC. Compared with LAK cell, most of T cells from MLTC were CD3⁺CD8⁺T cells with higher and more specific cytoxicity to the induced cells, M_2a cells, but not HL60 or K562 cell line. CONCLUSION: Clonal expansion of TCR Vβ subfamily T cells stimulated selectively by M_2a cells may be a specific immune response of autologous and allogeneic T cells to M_2a cells associated antigen. The T cells induced by M_2a cells have the ability of specific cytoxicity to the AML-M_2a cells.     

Effects of dendritic cells co-cultured with CIK cells on renal carcinoma cells

AIM: To study the effects of CIK cocultured with DC that pulsed with RCC antigen on renal carcinoma cells.METHODS: DC and CIK cells were generated respectively by cytokines from PBMC of healthy blood donor.Cell surface markers were analyzed by flow cytometry.Then CIK were cocultured with autologous DC that was (or not) pulsed with RCC antigen (786-0 cells).Cytotoxic activity against 786-0 or PC3 cells was measured by MTT assay under three different conditions: CIK cocultured with DC which was pulsed with 786-0 antigen (group A);CIK cocultured with DC which is not pulsed with 786-0 antigen (group B);CIK without DC (group C).RESULTS: The cytotoxic activity of three groups against 786-0 cells was (70.64±8.26)%, (53.40±7.33)%, (46.64±6.01)%, respectively (E/T=20∶〖KG-*2〗1).Significant differences between group A and group B or between group A and group C were observed (P<0.05).There was a significant difference in cytotoxic effects of group A against 786-0 and PC3 cells (P<0.05).CONCLUSION: Coculture of CIK with autologous tumor-pulsed DC leads to a significant increase in cytotoxic activity against renal carcinoma cells, and cytotoxicity mediated by RCC lysate antigen possess stronger specificity.     

The molecular mechanism of tolegenic dendritic cells induced by IL-10 in mouse

AIM:To investigate the relation of tolerogenic dendritic cells (DC) induced by interleukin-10 (IL-10) and the paired immunoglobin-like receptor (PIR) A and B (PIR-A and PIR-B) in mouse. METHODS:The mouse dendritic cell line, DC2.4 cells were cultured with the IL-10 to develope the IL-10-DC and were stimulated by lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (LPS-DC). Special small inference RNA (siRNA) molecule of PIR-B was chemically synthesized and was transfected into IL-10-DC by Lipofectamine 2000 (Si-DC). The expression of PIR A and PIR B on DC2.4 cells were measured by semi-quantitative RT-PCR and flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using ［3H］-thymidine incorporation test. The concentration of IFN-γ in supernatants of MLR from distinct groups was analyzed by ELISA. RESULTS:IL-10 up-regulated the PIR-B and down-regulated the PIR-A by semi-quantitative RT-PCR. On the contrary, LPS down-regulated the PIR-B expression and up-regulated the PIR-A expression. The expression of PIR, which is the common extra-membrane of PIR-A and PIR-B, was increased in both the IL-10-DC and the LPS-DC groups by FCM detection, but the higher expression was found in IL-10-DC group than that in LPS group. The IL-10 induced the higher PIR-B expression, inhibited allogenetic T cell proliferation and down-regulated the IFN-γ secretion. Special siRNA molecules of PIR-B in IL-10 group promoted the T cell proliferation and enhanced the IFN-γ secretion in MLR. CONCLUSION:IL-10 up-regulates the PIR-B expression and makes DC tolerance. Up-regulated PIR-B expression may be the molecular mechanism of tolerogenic dendritic cells induced by IL-10 in mouse.     

Foam cells can be induced by oxidized low density lipoprotein in human monocyte-derived dendritic cells

AIM: The purpose of this study is to investigate the mechanisms related to oxidized low-density lipoprotein (ox-LDL) and dendritic cells (DCs) in the process of atherosclerosis.METHODS: Human DCs were prepared from human CD14+ peripheral blood monocytes using rhGM-CSF (100 μg/L) and rhIL-4 (40 μg/L).Cells were incubated with 100 mg/L native or oxidized LDL for 72 h.The formation of foam cells was investigated by electron microscopy and oil red O staining.Phenotypic and immune functional assays were used with FACS, FITC-dextran phagocytosis, allogeneic mixed T lymphocytes reaction and secretion of Th1/Th2 (IL-12/IL-2) cytokines were also conduced.RESULTS: DCs treated with ox-LDL, but not native LDL were induced into foam cells after cultured for 72 h.Compared with native LDL, ox-LDL-treated DCs were less potent in FITC-dextran phagocytosis.ox-LDL promoted allogeneic T cells proliferation.Moreover, ox-LDL upregulated CD80 (72.4± 9.6 vs 89.5±10.1, P<0.01), CD86 (67.2±8.8 vs 80.2±11.6, P<0.01), HLA-DR (80.6±9.8 vs 86.6±10.8, P<0.01) and CD1a (40.2±10.3 vs 60.2±9.3, P<0.01) expressions, increased IL-12 secretion ［(44.3±8.9)ng/L vs (65.1±10.4)ng/L, P<0.05］ in DCs.However, the secretion of IL-2 was decreased ［(43.6±7.8)ng/L vs (10.0±4.5 )ng/L, P<0.01］ significently.CONCLUSION: DCs were induced into foam cells by ingesting ox-LDL with some functional characteristic of mature DC.DCs seem to be a new source of foam cells and play a key role in immunopathogenesis of atherosclerosis.     

Effects of CpG ODN on dendritic cells and its mechanisms

Oligodeoxynucleotide containing unmethylated cytosine phosphate-guanosine motif(CpG ODN) may induce high expression of CD80, CD86, CD83, HLA I and HLAⅡ molecules on dendritic cells(DC) and stimulate DC to produce high level of IL-6, IL-12, TNF-α and IFN-α. CpG ODN is demonstrated in vivo to be a very potent adjuvant for Th1 cells, regulating Th0 cells to develop toward Th1 cells. Its role for DC is characteristics of CpG ODN sequence specificity and species specificity. CpG ODN is, at present, considered as a pathogen associated molecular pattern which binds its specific receptor，Toll-like receptor 9，then functions through TLR/IL-1R signaling pathway. It may represent a new therapeutic drug for broad applications in infectious disease, autoimmune disease, allergy and cancer therapy.     

Effects of CpG ODN on dendritic cells in anti-hepatocarcinoma action

AIM: To study the effect of oligonucleotides containing unmethylated CpG motif (CpG ODN) on mouse bone marrow-derived dendritic cells (BMDCs) in anti-HcaF cytotoxicity. METHODS: BMDCs were stimulated by CpG ODN in combination with tumor antigen (TAg). The expression of CD80 on BMDC surface was analyzed by FCAS. Level of IL-12 (p70) in supernatants of BMDC culture was detected by ELISA. The proliferation of T cells was examined by MTT assay. Cytotoxicity of CTL induced by CpG ODN combining with TAg was detected by MTT assay. RESULTS: CpG ODN combining or not combining with TAg up-regulated the expression of CD80 on BMDC surface and stimulated BMDCs to produce a high level of IL-12. CpG ODN-activated BMDC promoted the proliferation of T cells. CTL induced by CpG ODN in combination with TAg appeared strong specific cytotoxicity on Hca-F cells. CONCLUSION: CpG ODN may effectively induce the functional maturation of mouse BMDC in vitro. CpG ODN in combination with TAg can enhance the anti-HcaF cytotoxicity of CTL.     

Effects of amlodipine on apoptosis of vascular endothelial cells induced by oxyeterols

AIM:To investigate the apoptosis of endothelial cells (EC) induced by oxysterols and to examine the effect of amlodipine on the apoptosis induced by oxysterols.METHODS:Light microscope, electron microscope, DNA agarose gel electrophoresis and flow cytometry were used to detect the apoptosis of EC.RESULTS:The characteristic morphological features of apoptosis were observed under light and electron microscope; DNA electrophoresis showed"DNA Ladder"; Flow cytometry showed the sub-G1 peak, apoptotic rate is 32.25% and 23.04% in Triol-treated and 25-OH-treated groups, respectively. While treated EC with amlodipine at the same time, the apoptotic rate decreased significantly.CONCLUSION:Both Triol and 25-OH can induce apoptosis of EC, which can be inhibited by amlodipine.     

The immunity effect of B7-H1 blockade on immature dendritic cells

AIM: To investigate the immune stimulation capacity of B7-H1 blockade on immature dendritic cells (DCs) in vitro. METHODS: The human monocyte-derived dendritic cells were induced in the presence of cytokine GM-CSF and IL-4. The expression of B7-H1 was detected by FCM. On blockade of B7-H1, the maturation and endocytic activity, T cells stimulatory proliferation capacity, IL-12 production, T cell differentiation effect of DCs were detected by FCM, MTT assay, ELISA and ELISPOT, respectively. RESULTS: The expression of B7-H1 was increased with the induction of DCs. On day 7, the positive expression was 54.12%, and the TNF-α induced mature DCs had the positive expression rate of 83.64%. The blockade of B7-H1 on immature DCs had sharply increased their T cells stimulatory proliferation capacity and IL-12 production, and efficiently induced the development of Th1/Tc1 cells, but had no effect on their maturation and endocytic activity. CONCLUSION: The blockade of B7-H1 on immature DCs increases its immune stimulation activity. It is valuable to investigate the antitumor immune responses of DCs vaccine with B7-H1 blockade.     

Inhibitory effect of proanthocyanidins B1 and B2 on inflammation of BV-2 cells induced by lipopolysaccharide

AIM: To investigate the different inhibitory effects of proanthocyanidins B1 and B2, which are isomers, on the inflammatory response of BV-2 cells induced by lipopolysaccharide (LPS). METHODS: MTT assay was used to detect the effects of proanthocyanidins B1 and B2 on the viability of BV-2 cells. LPS (1 mg/L) was used to promote BV-2 cells to secrete inflammatory factors. ELISA, chemotaxis assay and Western blot were used to detect the influence of proanthocyanidins B1 and B2 on the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), cell chemotaxis and phosphorylation of NF-κB. RESULTS: Proanthocyanidins B1 and B2 did not show cytotoxicity effect on BV-2 cells. Proanthocyanidin B1 and B2 inhibited the cell chemotaxis, phosphorylation of NF-κB, and releases of TNF-α and IL-1β. CONCLUSION: Proanthocyanidins B1 and B2 inhibit the inflammatory response of BV-2 cells induced by LPS, and their action intensity didn't show significant difference.     

Phagocytosis of viable apoptotic cells inhibits the activation of T lymphocytes

AIM: To investigate whether viable apoptotic cells and phagocytosis of them affect the activation of T lymphocytes. METHODS: Ultraviolet irradiation was used to induce apoptotic cells in vitro and the model of phagocytosis of these cells was established. Cytokine TGF_β1 was detected by ELISA. The rate of apoptotic cells and phagocytosis of them were assessed by flow cytometry. Furthermore, flow cytometry was also employed to examine the expression of activation signs, such as CD69, CD25, CD71, of T lymphocytes under the intervention of apoptotic cells and macrophage which ingested apoptotic cells, to reflect whether the apoptotic cells and the phagocytosis of these cells could influence the activation of lymphocytes stimulated by Con A. RESULTS: Ingestion of apoptotic cells increased TGF_β1 secretion. Only the macrophages that had ingested apoptotic cells could suppress the activation of lymphocytes. The expression of the markers of lymphocytes activation such as CD69, CD25, CD71 had been restrained. These inhibition effects were abolished by monoclonal anti-TGF_β1 antibody. CONCLUSION: The macrophages that have ingested apoptotic cells inhibit expression of CD69, CD25 and CD71 of T lymphocytes stimulated by ConA. This effect is dependent on the increase in TGF_β1 secretion in local site.     

Effect of injecting immature dendritic cell intratumorally after microwave ablation under different temperature on the activity of cytotoxic T lymphocytes

AIM: To investigate whether the injection of immature dendritic cell (iDC) after ablation induce and upregulate tumor specific cytotoxic T lymphocytes (CTL).METHODS: The model of hepatoma was established and the tumors were ablated with microwave under (45±2) ℃, (50±2) ℃, (55±2) ℃, (60±2) ℃ and void ablation, after which the bone marrow derived iDC was injected intratumorally. The activity of CTL was detected, and the levels of IFN-γ and IL-4 secreted by activated spleic lymphocytes were measured.RESULTS: Compared with iDC injection intratumorally after the ablation of (45±2) ℃, (55±2) ℃, (60±2) ℃ and void ablation, the cytotoxic effect of CTL towards Hepa l-6 was heightened by injecting iDC intratumorally 72 hours after ablation of (50±2) ℃ (P<0.05), so was the secretion of IFN-γ (P<0.05). The level of IL-4 decreased after ablation of (50±2) ℃ subsequently with injecting iDC intratumorally (P<0.05). The cytotoxic effect of CTL towards Hepa l-6 was higher than that towards B16 lymphadenoma cell after iDC injection intratumorally and ablation of (50±2) ℃ and (55±2) ℃ (P<0.05).CONCLUSION: Thermal ablation of (50-55) ℃ and subsequent injection of iDC intratumorally may induce tumor specific CTL by the way of promoting the antigenicity of ablated tumor tissue and augmenting the presentation of TAA to effector cells.     

Effects of adrenomedullin on proliferation of vascular smooth muscle cells induced by urotensin Ⅱ

AIM:To observe the effect of adrenomedullin(ADM)on proliferation of vascular smooth muscle cells(VSMC) induced by urotensin Ⅱ(UⅡ). METHODS:DNA synthesis of cultured rat aortic VSMC was measured by [³H]-TdR incorporation. The activities of mitogen activated protein kinase(MAPK) were determined by isotope tagged with [γ-³²P]-ATP. RESULTS:UⅡ(10^-8mol/L) significantly increased [³H]-TdR incorporation of VSMC and MAPK activities by 38%(P＜0.05) and 260%(P＜0.01) respectively compared with control group. Compared with UⅡ group, 10^-10,10^-9,10^-8mol/L ADM decreased [³H]-TdR incorporation of VSMC by 7%(P＞0.05), 32%(P＜0.05)and 41%(P＜0.01),respectively, and diminished MAPK activities by 24%(P＞0.05), 32%(P＜0.05)and 36%(P＜0.05),respectively. CONCLUSION:ADM inhibits proliferation of VSMC induced by urotensin Ⅱ through inhibiting MAPK activation.     

Effect of activated protein C on apoptosis in human umbilical vein endothelial cells induced by lipopolysaccharide

AIM:To study the effect of activated protein C (APC) at different concentrations on apoptosis of human umbilical vein endothelial cells (HUVECs) induced by lipopolysaccharide (LPS).METHODS:The HUVECs were induced by LPS (1.0 mg/L) as apoptotic model that was administered by different concentration of APC (10 μg/L or 50 μg/L). Meanwhile, the control group and induced apoptosis group induced by LPS (1.0 mg/L) stimulation were also set up. The changes of cellular ultrastructures were observed under electron microscope. The DNA ladder and TUNEL fluorescent staining were measured in cells. Annexin-Ⅴ/PI double staining was used to measure the cell apoptosis rate by flow cytometry. Cell survival rate was measured by MTT assay. The proliferating cell nuclear antigen (PCNA) expression levels in cells were also measured by Western blotting to reflect the proliferation of the cells.RESULTS:There were significant apoptotic changes in the cells induced by LPS, but the apoptotic changes were reduced and apoptosis rates were decreased in the cells treated with APC. Meanwhile, cell survival rate and the protein levels of PCNA were increased after APC treatment, particularly at the concentration of 50 μg/L, which showed difference when compared with those induced apoptosis group by LPS (P<0.05).CONCLUSION:APC can inhibit HUVECs apoptosis induced by LPS and promote cell proliferation, thus protect the cells from injury.     

Effects of TRAF3 on inhibiting cyst formation induced by NF-κB signaling in kidney

AIM: To investigate the effects of tumor necrosis factor (TNF) receptor associated factor 3 (TRAF3) on the signaling pathway of NF-κB and the expression of Bax and Bid in renal collecting duct epithelial cells of polycystic kidney disease (PKD), and to observe the tubulogenesis of TRAF3 transgenics for exploring the protective effect of TRAF3 on the cystogenesis and development of PKD. METHODS: The signaling changes of NF-κB and expression of Bax and Bid in PKD renal collecting duct epithelial cells and TRAF3 transgenic PKD renal collecting duct epithelial cells were determined by the Western blot. The percentage of apoptotic cells was measured by flow cytometry with Annexin V staining. The caspase-3 activity was also measured. The 3D Matrigel culture was performed to examine abnormal tubulomorphogenesis in vitro. RESULTS: The over-expression of TRAF3 significantly inhibited the signaling pathway of NF-κB in PKD renal collecting duct epithelial cells and significantly downregulated the expression of Bax and Bid, and also decreased the cell apoptosis. More branch structures were observed in the TRAF3 transgenic PKD renal collecting duct epithelial cells. CONCLUSION: TRAF3 is a negative regulatory inhibitor for Bax and Bid by regulating the NF-κB signaling and may be a new target for inhibiting the cyst formation in PKD.     

Effects of AGR2 on proliferation and apoptosis of NCI-H460 cells induced by thapsigargin

AIM:To observe the effects of anterior gradient 2 (AGR2) gene on the proliferation and apoptosis of NCI-H460 cells induced by thapsigargin, an endoplasmic reticulum (ER) stress inducer.METHODS:A stable cell line with knockdown of endogenously expressed AGR2 mediated by lentiviral shRNA was established. The proliferation abi-lity of stable NCI-H460 cells in the presence of thapsigargin was measured by colony formation assay, while the effect of AGR2 gene silencing on apoptosis in the presence of thapsigargin was analyzed by flow cytometry. RESULTS:Lentivirus-mediated AGR2 knockdown stable NCI-H460 cells was successfully constructed and the expression of AGR2 was markedly diminished as revealed by Western blot (P<0.01). Knockdown of AGR2 didn't significantly affect the colony formation rate and apoptosis of NCI-H460 cells without thapsigargin treatment. Colony formation rate of the cells with AGR2 gene knockdown was far lower than that of the control cells after 24 h of thapsigargin treatment (P<0.05). The results of flow cytometry showed that knockdown of AGR2 significantly increased the apoptosis of NCI-H460 cells induced by thapsigargin. CONCLUSION:Knockdown of AGR2 doesn't remarkably affect the proliferation and apoptosis of NCI-H460 cells in the absence of thapsigargin, but significantly decreases cell viability and increases apoptosis after thapsigargin treatment. This study reveals that AGR2 might promote the proliferation and reduce the apoptosis of lung cancer cells under ER stress, and lays the foundation for further study of the AGR2 role in initiation, development and drug resistance of lung cancer.