Synergistic anti-proliferation effect of aspirin and 5-fluorouracil on colon cancer cells and its mechanism

AIM：To investigate the synergistic anti-proliferation effect of aspirin and 5-fluorouracil on the colon cancer cells and its mechanism. METHODS：Colon cancer cells were divided into 4 groups: control group, aspirin group, 5-fluorouracil group and aspirin+5-fluorouracil group. Synergistic anti-proliferation effect of aspirin and 5-fluorouracil on the colon cancer cells was observed by MTT assay. Apoptosis-inducing effect and mechanism were detected by Hoechst 33258 staining, caspase activity assay and flow cytometry analysis. The mRNA and protein levels of apoptosis-related proteins were evaluated by real-time PCR and Western blotting. RESULTS：5-Fluorouracil inhibited proliferation of HCT116 and SW620 colon cancer cells effectively, and low concentration of aspirin exerted synergistic inhibitory effect. 5-Fluorouracil induced apoptotic morphology and increased caspase activity and sub-G1 phase in HCT116 cells. The synergistic effect of aspirin obviously enhanced apoptotic ratio and caspase activity. Moreover, 5-fluorouracil inhibited the mRNA and protein expression of Bcl-2, which was amplified by low concentration of aspirin. CONCLUSION：Aspirin and 5-fluorouracil had a synergistic anti-proliferation effect on the colon cancer cells through apoptosis pathway.     

Antitumor effect of allicin on esophageal cancer EC109 cells compared with chemotherapeutics in vitro

AIM: To investigate the inhibitory effect of allicin on human esophageal cancer cell line EC109 and its possible mechanism by comparison with chemotherapeutic drugs. METHODS: The EC109 cells were treated with different concentrations of allicin, 5-fluorouracil and cisplatin. The cell viability was detected by CCK8 assay and the activity of lactic dehydrogenase (LDH) was analyzed at 6 h, 12 h, 24 h, 48 h and 72 h. The apoptosis of the EC109 cells induced by Z-VAD-FMK, allicin, allicin+Z-VAD-FMK, 5-fluorouracil and cisplatin was analyzed by flow cytometry with Annexin V-FITC and PI double staining. The enzyme activity changes of caspase-3, caspase-8 and caspase-9 were detected by spectrophotometry. RESULTS: Allicin had inhibitory effect on the growth of the EC109 cells and killed them in concentration-dependent and time-dependent manners. LDH activity was decreased compared with 5-fluorouracil and cisplatin. The increased activity of caspase-3 and caspase-8 in allicin-treated cells was statistically significant, but caspase-9 activity changed without statistical significance. CONCLUSION: Allicin inhibits the growth of EC109 cells in concentration-and time-dependent manners through extrinsic apoptosis pathways activated by caspase-8, and the side effects are weaker compared with 5-fluorouracil and cisplatin.     

Construction of siArid2 recombinant adenovirus and its effect on proliferation of hepatocarcinoma cells

AIM: To observe the effects of Arid2gene on cell proliferation and cell cycle by interference of endogenous Arid2 expression in hepatoma cells. METHODS: Three pairs of shRNA targeting Arid2gene were cloned into a shuttle vector to construct recombinant adenovirus plasmids. HEK293 cells were transfected with the recombinant adenovirus plasmids. After several rounds of the package and amplification, the high-titer adenoviruses AdsiArid2-1~3 were obtained. To verify the inhibitory effects of AdsiArid2 adenoviruses, Western blotting was used to detect the endogenous Arid2 protien expression in SMMC-7721 cells. Cell growth and cell cycle analysis were carried out by MTS assay and flow cytometry. RESULTS: High- titer recombinant adenovirus of siArid2 were successfully obtained, and named AdsiArid2-1~3, among which the AdsiArid2-3 had the best inhibitory effects. MTS assay showed that the absorbance values at 490 nm were increased at 72 h and 96 h after transduction compared with the mock and Adsicontrol groups. These data indicated that knockdown of Arid2 promoted the proliferation rate of SMMC-7721 cells(P<0.05). Moreover, the flow cytometry analysis revealed that the G1-phase distribution at 72 h in AdsiArid2 group was lower than that in mock group and Adsicontrol group. In contrast, the S-phase distribution in AdsiArid2 group was much higher than that in mock group and Adsicontrol group. CONCLUSION: The recombinant plasmids and recombinant adenovirus were successfully constructed. shRNA-mediated knockdown of Arid2 promotes the proliferation and the transition from G1 phase to S phase of hepatoma cells.     

Effects of antioxidant and NF-κB on the induction of IL-8 in human colon cancer cell line HT-29 cells in vitro

AIM: To investigate the role of nuclear factor κB (NF-κB) in the induction of IL-8 gene by TNF-α in colon cancer cells and the effect of antioxidant on the induction of IL-8. METHODS: ELISA was used to detect the concentrations of IL-8. IL-8 mRNA was analyzed by using RT-PCR. NF-κB in the cell nuclei was detected with electrophoretic mobility shift assay. RESULTS: (1) IL-8 production and IL-8 mRNA expression induced by TNF-α was blocked by pyrrolidine dithiocarbamate (PDTC). (2) TNF-α triggered the activation and translocation of NF-κB and PDTC inhibited the activation of NF-κB induced by TNF-α. CONCLUSION: The induction of IL-8 gene and protein by TNF-α is dependent on the activation of NF-κB. Antioxidants may inhibit the induction of IL-8 gene and protein through inhibiting NF-κB activation.     

Construction of an immunoconjugate targeting tissue factor and its effect on colorectal cancer cells

AIM: To construct a recombinant eukaryotic expression vector pcDNA3.1(+)-hFVII-LC+hIgG1-Fc, and to produce and purify the immunoconjugate hFVII-LC+hIgG1-Fc protein. METHODS: The target sequences were amplified by RT-PCR from hepatic tissue and lymphocyte RNA, and cloned into eukaryotic expression vector pcDNA3.1(+). After confirmed by restriction endonuclease digestion and DNA sequencing, the recombinant plasmid was transfected into CHO-K1 cells by lipofectamine 2000. The transfectant clones were selected by G418 screening. The positive monoclonals were grown in CHO-K1 serum-free medium Excel 301 and the culture medium was collected. The hFVII-LC+hIgG1-Fc protein was purified by affinity Ni-NTA resin. The immunoconjugate was identified by ELISA with tissue factor (TF) affinity and specificity. Induction of NK cell-mediated antibody-dependent cell cytotoxicity(ADCC) was examined in HT-29 colorectal cancer cell line. RESULTS: Human liver tissue and lymphocytes from Han population were used as template for amplification of hFVII-LC and hIgG1-Fc DNA fragments, which were confirmed by sequencing and were exactly the same as those GenBank reported. The eukaryotic expression vector pcDNA3.1(+)-hFVII-LC+hIgG1-Fc was successfully constructed, and 1.3 mg of hFVII-LC+hIgG1-Fc protein could be prepared from 1 liter of Excel 301 serum-free culture medium through Ni-NTA affinity chromatography. The immunoconjugate was specially bound to TF and induced a significant ADCC response in HT-29 cells. CONCLUSION: The human hFVII-LC+hIgG1-Fc recombinant plasmid and the hFVII-LC+hIgG1-Fc immunoconjugate are obtained, which provide the basis for further study of cancer-targeted therapy.     

Antitumor effects of HSV-tk and cytosine deaminase double suicide gene mediated by adenovirus on cholangiocarcinoma cells in vitro

AIM: To study the antitumor effects of HSV-tk and cytosine deaminase double suicide gene system on cholangiocarcinoma cells in vitro. METHODS: Recombinant adenovirus vector carring HSV-tk and cytosine deaminase double suicide gene was constructed and was transfected into QBC939 huamn cholangiocarcinoma cells. The antitumor effects were observed in vitro in comparison with a single suicide gene system. RESULTS: By transfection, HSV-tk and (or) cytosine deaminase double suicide gene gained high expression in tumor cells. Compared with single suicide gene system, this double gene system presented higher sensitivity, stronger antitumor effect and bystander effect. CONCLUSION: HSV-tk and cytosine deaminase double suicide gene system has a powerful antitumor effect on cholangiocarcinoma cells.     

miR-496 over-expression inhibits growth and metastasis in colon cancer cells

AIM: To investigate the effect of miR-496 over-expression on the growth and metastasis of colon cancer cells and its molecular mechanism.METHODS: The proteins interacting with miR-496 were screened by bioinformatic method. The levels of miR-496, CTNNB1 mRNA and β-catenin protein in colon cancer cell lines, HT29, HCT116 and SW480, and normal colonic epithelial cell line NCM460 were detected by real-time PCR and Western blot. HT29, HCT116 and SW480 cells were transfected with miR-496 mimics using Lipofectamine 2000 and named as HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimics cells, respectively, and the cells transfected with the scramble served as negative control. The cell viability, lactate dehydrogenase (LDH) leakage, and colony formation and metastatic abilities were determined by MTT assay, LDH assay, colony formation assay and Transwell method, respectively. The promoter activity of miR-496 was measured using luciferase reporter gene assay. The protein levels of β-catenin, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), p-4E-BP1, low-density lipoprotein receptor-related protein 6(LRP6), p-LRP6, MMP-7, MMP-9, MMP-13 and TIMP-2 were monitored by Western blot.RESULTS: Endogenous miR-406 interacted with β-catenin was found in the colon cancer cells. Low miR-496 expression in the HT29, HCT116 and SW480 cells and high miR-496 expression in NCM460 cells were detected. In contrast, high β-catenin expression was found in the HT29, HCT116 and SW480 cells and low β-catenin expression was observed in the NCM460 cells. Compared with control group, the cell viability, colony formation rate and the number of metastatic cells remarkably decreased in the HT29-miR-496 mimics, HCT116-miR-496 mimics and SW480-miR-496 mimic cells (P<0.05). The promoter activity of miR-496 was significantly increased in colon cancer cells transfected with miR-496 mimics, and was 1.75, 2.04 and 1.61 times as high as control group. miR-496 over-expression inhibited β-catenin levels, and p-4E-BP1 and p-LRP6 protein levels were also reduced. siRNA- or over-expressed miR-496-mediated β-catenin down-regulation inhibited MMP-7 and MMP-9 expression, but promoted TIMP-2 expression.CONCLUSION: The expression level of miR-496 in the colon cancer cells is low, but in the normal colonic epithelial cells is high. miR-496 over-expression inhibits the protein levels of MMP-7 and MMP-9, and promotes the protein expression of TIMP-2 via inhibiting Wnt/β-catenin pathway, thus suppressing malignant phenotype in the colon cancer cells.     

Inhibitory effect of propolis on proliferation of K562 cells in vitro

AIM: To study the effect of propolis on the proliferation of K562 cells.METHODS: K562 cells were cultured in vitro. Cell proliferation was measured by MTT method. The apoptotic rate was determined by flow cytometry. RT-PCR was applied to detect mRNA expression of Nup98. The protein level of Nup98 was determined by Western blotting. RESULTS: The inhibitory rates of proliferation induced by propolis at the concentrations of 2 mg/L, 20 mg/L and 200 mg/L were obviously higher than that in control cells in a time-and dose-dependent manner. The apoptotic rate was increased in a dose-dependent manner. High concentration of propolis down-regulated the expression of Nup98 at mRNA and protein levels. CONCLUSION: Propolis inhibits the proliferation and induces apoptosis in K562 cells. The mechanism may be related with down-regulation of Nup98.     

Effects of SphK1 and FAK on epithelial-mesenchymal transition in colon cancer HCT116 cells

AIM: To investigate the effects of sphingosine kinase l(SphK1) and focal adhesion kinase(FAK) on the epithelial-mesenchymal transition(EMT) of human colon cancer HCT116 cells. METHODS: Human colon cancer HCT116 cells were divided into 3 groups. N, N-dimethylsphingosine(DMS) was used to suppress the activity of SphK1. PF573228 was used to suppress the activation of FAK. The cells treated with equal volume of culture medium severed as control group. The cell viability was measured by MTT assay. The protein expression of SphK1, FAK and the EMT relative protein E-cadherin, N-cadherin, vimentin and matrix metalloproteinase(MMP) 2 was analyzed by Western blot. The mRNA expression of SphK1, sphingosine-1-phosphate(S1P), FAK, E-cadherin and vimentin was detected by real-time PCR. The ability of tumor cell migration was measured by wound-healing assay. RESULTS: The cell viability of HCT116 cells was suppressed by DMS and PF573228 in dose and time dependent manners. DMS significantly suppressed the expression of SphK1, FAK, N-cadherin, vimentin and MMP2, meanwhile enhanced the expression of E-cadherin. PF573228 reduced the expression of FAK, SphK1, N-cadherin, vimentin and MMP2, meanwhile increased the expression of E-cadherin(P<0.01). In addition, the migration ability of HCT116 cells was significantly decreased by treating with DMS and PF573228(P<0.01). Compared with control group, the mRNA expression of FAK, SphK1, S1P and vimentin was decreased, while the expression of E-cadherin was increased significantly in PF573228 group and DMS group(P<0.05). CONCLUSION: SphK1 and FAK signaling pathways may play an important role in the occurrence of EMT in the colon cancer HCT116 cells.     

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro

AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.     

Effect of Wip1 gene silencing on chemotherapy sensitivity of human colon cancer cells

AIM: To observe the inhibitory effect of siRNA targeting to Wip1 gene on the Wip1 gene expression in the colon cancer cells and to investigate the influence of Wip1 gene silencing on the chemotherapy sensitivity of colon cancer cells. METHODS: Wip1-811 siRNA targeting to Wip1 gene was transfected into RKO colon cancer cells with high expression of Wip1 gene. The mRNA expression of Wip1 was measured by real-time PCR. The protein level of Wip1 was detected by Western blotting. The viability of RKO colon cancer cells was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Wip1-811 siRNA efficiently inhibited the expression of Wip1 at mRNA and protein levels. The enhanced chemotherapy sensitivity of RKO colon cancer cells was observed after inhibition of Wip1 gene expression. The viability of RKO colon cancer cells was decreased from (89.4±6.6)% to (74.7±3.9)% after treated with 5-fluorouracil (P<0.05) and decreased from (77.9±2.4)% to (66.7±2.9)% after treated with oxaliplatin (P<0.05). The cell apoptotic rate was increased from (7.7±0.5)% to (12.3±3.2)% and from (14.7±2.1)% to (34.0±2.1)% when RKO colon cancer cells were treated with 5-fluorouracil and oxaliplatin, respectively (P<0.05). CONCLUSION: Wip1 gene silencing enhances chemotherapy sensitivity of colon cancer cells.     

IFN-γ reinforces the inhibitory effect of tumor necrosis factor-related apoptosis-inducing ligand on apoptosis of human colon cancer cell line RKO cells

AIM: To explore the influence of IFN-γ on the role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in inducing apoptosis of RKO cell line. METHODS: Survival fraction and apoptosis were measured by MTT method and flow cytometry (FACS). RESULTS: Survival fraction of IFN-γ group, TRAIL and IFN-γ 72 h+TRAIL group were 99.28%, 85.45%, 52.60%, respectively. The percentage of apoptotic cells of IFN-γ group, TRAIL group, IFN-γ 24 h+TRAIL group, IFN-γ 48 h+TRAIL and IFN-γ 72 h+TRAIL group were 1.51%, 2.38%, 4.97%, 13.30%, 21.00%, respectively. The percentage of apoptotic cells of IFN-γ 24 h+TRAIL group was higher than the sum of IFN-γ group and TRAIL group (P<0.01). The longer the IFN-γ pretreated, the higher the percentage of apoptotic cells was observed (P<0.01). CONCLUSION: IFN-γ can reinforce the effect of TRAIL inducing apoptosis in RKO.     

Phosphoproteomics to analyze PTPLAD1-regulated tyrosine-phosphorylated proteins in colon cancer cells

AIM: To identify and analyze tyrosine-phosphorylated proteins regulated by protein tyrosine phosphatase-like A domain containing protein 1 (PTPLAD1) in colon cancer cells by phosphoproteomics. METHODS: The expression of PTPLAD1 in colon cancer cell line HCT-116 was knocked down by small interfering RNAs, and the differential expression of tyrosine-phosphorylated proteins in response to the konckdown of PTPLAD1 in HCT-116 cells was identified by stable isotope labeling with amino acid in cell culture (SILAC), coupled with the tyrosine phosphorylation antibody immunoprecipitation and LC-MS/MS analysis. The Ingenuity Pathway Analysis (IPA) software was employed for bioinformatics analysis on the differentially-expressed proteins. RESULTS: A total of 20 differentially-expressed tyrosine-phosphorylated proteins were identified by MS, including 8 markedly up-regulated and 10 evidently down-regulated proteins. IPA software suggested that these proteins were mainly associated with the disease of cancer, tissue development and function, and cell death and survival. CONCLUSION: We successfully identified PTPLAD1-regulated differentially-expressed tyrosine-phosphorylated proteins in colon cancer cell line HCT-116. Our analysis suggests that PTPLAD1-regulated proteins in colon cancer are closely correlated with colon cancer.     

Human dendritic cells transfected with MUC1 mRNA induce lethal effect of cytotoxic T-lymphocytes on non-small cell lung cancer in vitro

AIM：To investigate the specific anti-tumor effects of mature dendritic cells (DCs) transfected with amplified mucin 1 (MUC1) mRNA in vitro. METHODS：DCs separated and purified from the peripheral blood mononuclear cells were induced in vitro and then identified by flow cytometry. pcDNA3.1（+）-MUC1 plasmid was constructed and was able to transcribe MUC1 mRNA in vitro. The MUC1 mRNA was transfected into DCs by electroporation. MUC1-transfected DCs were used to induce T cells to be cytotoxic T-lymphocytes. Quantitative real-time PCR was performed to assess MUC1 mRNA expression in transfected DCs. The proliferation of T cells was examined by MTT assay. The proportion of CD8+ cells in the T cells was determined by flow cytometry and the specific cytotoxicity was measured by LDH assay. The secretion of IFN-γ was detected by ELISA. RESULTS：The marker gene expression in the DCs transfected with MUC1 mRNA was significantly increased compared with control group, peaking at 24 h. The transfection group showed the higher capacity to stimulate the proliferation of T cells compared with control group when the ratio of DCs to T cells was 1∶10. The proportion of CD8+ cells in transfection group was higher than that in control group. The lethal effect of special cytotoxic T-lymphocytes on target cells in transfection group was stronger than that in control group. The level of IFN-γ in the cell supernatant of transfection group was higher than that in control group. CONCLUSION：DCs plus MUC1 mRNA by electrical transfection induces specific anti-tumor effects, which provides an experiment evidence of using MUC1 as a target for immunotherapeutic strategy against non-small cell lung cancer.     

Proliferative effect of neonatal mouse brain extracts on neural stem cells in vitro

AIM: To discover specific neural stem cell (NSC) proliferation-promoting factor, which will contribute to study on development of nervous system and treatment of nervous system diseases. METHODS: The extracts of forebrain, midbrain,afterbrain and cerebellum of neonatal mice were prepared, and the NSCs of newborn mice were cultured in vitro. Neurospheres were observed, immunocytochemical staining of characteristic protein, nestin, and MTT assay were performed to identify NSCs and their proliferative properties. RESULTS: A great deal of neurospheres were formed in the presence of the extracts of afterbrain and cerebellum, which were positive for characteristic protein （Nestin） of NSC showed by immunocytochemical staining. CONCLUSION: The afterbrain and cerebellum extracts can increase the total number of NSCs isolated from newborn mice in vitro in a dose-dependent manner.     

Enhancement effect of adenovirus mediated antisense c-myc gene and caffeine on chemotherapy of osteosarcoma cells to cisplatin

AIM: The cancer biology has showed that overexpression of oncogenes is responsible for the progression of human malignancies,antisense technology can block a certain gene expression.Caffeine has enhancement effect on chemotherapy of osteosarcoma cells to cisplatin,we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin.METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 base pairs in a reverse direction into adenovirus vector.Ad-Asc-myc and caffeine was used respectively or together to co-operate with cisplatin to treat the osteosarcoma MG-63 cells in vitro,and Western blotting,MTT,flow cytometry (FCM),electron microscope were used to evaluate expression of c-Myc protein,tumor cell proliferation in vitro,apoptosis and cell cycle analysis.RESULTS: Ad-Asc-myc was obtained with the titer of 2×10¹² pfu/L.Ad-Asc-myc down-regulated the expression of c-Myc protein,Ad-Asc-myc or caffeine enhanced the effects of 2.0,5.0 mg/L cisplatin on MG-63 cells.Moreover,Ad-Asc-myc combined with caffeine significantly enhanced this effects,not only on cisplatin-induced apoptosis,but also on tumor cells proliferation in vitro.The expression of bcl-2 was downregulated,bax were upregulated,while there was no change in the expression of E2F-1.FCM analysis showed that cisplatin treatment induced a block in S phase,and caffeine reversed this block and speeded up the progression of cells out of the S phase.Ad-Asc-myc induced obvious G₂/M phase arrest in transfected cells.CONCLUSION: Ad-Asc-myc combined with caffeine may enhance apoptosis-induced and chemotherapy effects of osteosarcoma MG-63 cells to cisplatin.     

5-FU upregulates stem cell marker CD133 expression in colon cancer cells

AIM: To investigate the effect of 5-fluorouracil(5-FU)on the expression of the stem cell marker CD133 on colon cancer stem cells. METHODS: CD133 expression on several colon cancer cell lines was detected by flow cytometry. The CD133 positive cells from DLD1 cells were separated by the method of magnetic activated cell separation. Colony assay was used to measure self-renew ability and MTS assay was used to detect the sensitivity to 5-FU after separation. After 5-FU treatment, the change of CD133 mRNA level was measured by qPCR. RESULTS: CD133 expression on the surface of colon cacner cell lines DLD1, HT29, SW480, HCT116, Lovo, RKO was 30.20%, 82.00%, 0.34%, 91.80%, 85.30%, 0.28% respectively. DLD1 cells had two obvious populations according to CD133 expression. CD133 positive cells were separated from DLD1 cells, the positive purity was 87.21%±5.33% and the negative purity was 84.30%±4.65%. CD133 positive cells formed more colonies with limited dilution colony assay(46.33%±4.44% vs 31.00%±2.00%, P<0.05). CD133 positive cells were less sensitive to 5-FU compared to CD133 negative cells(20% less, P<0.01). 5-FU at concentration of 1 mg/L upregulated CD133 mRNA expression in both DLD1 and HT29 cells, the relative quantity was increased from 1 to 1.684±0.012(P<0.01)and 30.702±0.280 to 49.379±0.460(P<0.01)in HT29 and DLD1, respectively. CONCLUSION: Compared to CD133 negative cells, CD133 positive cells show more ability to form colonies in vitro, and are less sensitive to 5-FU. 5-FU upregulats the mRNA expression of CD133, resulting in the CD133 colon cancer stem cells enrichment during 5-FU treatment.     

Growth-inhibitory effects of selective cyclooxygenase-2 inhibitor on colon cancer cells and its possible mechanisms

AIM: To evaluate the growth-inhibitory effects of NS-398, a selective cyclooxygenase-2 inhibitor, in human colon cancer HT-29 cells and its possible mechanisms. METHODS: MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect apoptosis rate and cell cycle. RT-PCR was used to detect the expression of bcl-2 mRNA and bax mRNA. Alteration of cytoskeleton component F-actin was observed by confocal laser scanning microscope. RESULTS: NS-398 could inhibit growth of HT-29 cells in dose-and time-dependent manners. Flow cytometry revealed that NS-398 could induce apoptosis and cause G0/G1 arrest of HT-29 cells in a dose-dependent manner. After 72 h incubation with NS-398 at different concentrations, the expression level of bcl-2 mRNA was lowered and the ratio of bcl-2 to bax was decreased in HT-29 cells. F-actin was mainly distributed around nuclei forming annular structure in HT-29 cells. After exposure to NS-398, the annular structure around nuclei disappeared and fluorescence intensity of F-actin decreased obviously. CONCLUSION: NS-398 can inhibit the growth effectively and induce apoptosis in HT-29 cells in vitro, which is associated with the down-regulation of bcl-2 to bax ratio and the disruption of cytoskeleton.     

Anti-inflammatory effect of huperzine A on protection of rat neural stem cells in vitro

AIM：To explore the anti-inflammatory effect of huperzine A (HupA) and its neuroprotective effect on rat neural stem cells (NSCs). METHODS：The microglia and NSCs were isolated from neonatal rat hippocampal tissues and co-cultured in a Transwell system. The cells were divided into 3 groups：control group, amyloid beta-peptide (Aβ) group and HupA group. The microglia layer in Aβ group was treated with Aβ1-42 (10 μmol/L), while that in HupA group was pretreated with HupA (1 μmol/L) before Aβ1-42 stimulation. The culture supernatant levels of inflammatory mediators, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and macrophage inflammatory protein 1α (MIP-1α), were detected by LiquiChip technique. The apoptosis of NSCs was determined by flow cytometry and Western blotting. RESULTS：The microglia secreted a large number of inflammatory mediators with the stimulation of Aβ. In Aβ group, the levels of IL-6, TNF-α and MIP-1α were significantly higher than those in control group at 72 h (P<0.01), and the apoptotic rate of NSCs was 25.46% (P<0.01). In HupA group, the concentrations of IL-6, TNF-α and MIP-1α decreased significantly as compared with Aβ group (P<0.01), and the apoptotic rate of NSCs was only 8.05% (P<0.01). The Bcl-2/Bax ratio in HupA group was higher than that in Aβ group (P<0.05). CONCLUSION：Huperzine A reduces the secretion of cytokines and chemokines, and attenuates microglia-mediated neuroinflammation, thus protecting NSCs against inflammation-induced apoptosis.     

Antiproliferation effects of 188Re labeled monoclonal antibody on prostate cancer cells in vitro

AIM: To investigate the effect of 188Re labeled monoclonal antibody on prostatic specific membrane antigen 7E11C5.3,radioimmunotherapy for the treatment of human prostate cancer cell line LNCaP in vitro.METHODS: 188Re-7E11C5.3 was prepared by direct 2-mercaptoethanol reduction method.Labeling efficiency and radiochemical purity was measured by paper chromatography.Immunoreactive fraction was determined by linear extrapolation.Cytotoxicity to LNCaP cells was determined by MTT assay.RESULTS: The labeling yield of 188Re-7E11C5.3 was (93.16±2.18)%,the radiochemical purity was (95.62±0.48)%,and the immunoreactive fraction was (74.86±1.86)%.The inhibitory effect of 188Re-7E11C5.3 on cell proliferation of LNCaP cells was significantly higher than that of 188Re-mIgG or 188ReO-4.The 50% inhibitory doses (IC50) of 188Re-7E11C5.3,188Re-mIgG,and 188ReO-4 were (23.38±3.73)×107 Bq/L,(59.21±8.02)×107 Bq/L and (68.89±10.91)×107 Bq/L,respectively.CONCLUSION: 188Re-7E11C5.3 can effectively inhibit the growth of in vitro cultured prostate cancer cells and shows much potential for prostate cancer radioimmunotherapy.