Effects of Substrate Stiffness on Epithelial-Mesenchymal Transition of Colon Cancer Cells under Simulated Hypoxia Environment / 医用生物力学

Objective To explore the effects from the synergy of substrate stiffness and hypoxia on epithelial mesenchymal transition (EMT) of colon cancer cells SW480 by simulating the microenvironment of human colon cancer tissues. Methods Polyvinyl alcohol gels with different stiffness ( 4. 5, 20, 40 kPa) were prepared to simulate the stiffness of each part of colon cancer tissues. The morphological change of cells on substrate with different stiffness was detected under simulated hypoxia ( CoCl2 ) environment. The expression of hypoxia inducible factor (HIF-1α), and EMT markers E-cadherin, Vimentin, Snail 1 were detected by Western blot. The mRNA expression of E-cadherin, Vimentin, Snail 1, matrix metalloproteinase-2 ( MMP-2), and MMP-9 was detected by quantitative real-time PCR ( qRT-PCR). Results Under simulated hypoxia environment, with the increase of substrate stiffness, the SW480 cells spreading area increased, and transformed from round shape into irregular polygon. The EMT of SW480 could be enhanced through up-regulating expression of Vimentin, Snail 1, MMP-2, MMP-9, and down-regulating expression of E-cadherin. Conclusions This study is important for exploring the synergistic effect of substrate stiffness and hypoxia on the EMT of colon cancer cells as well as the molecular mechanism.

The Effect of Saccharin on the Gene Expression of NF-κB and Inflammatory Cytokines in LPS-Stimulated SW480 Colon Cancer Cells

There have been no published studies concerning the anti-inflammatory effects of corn silk on colon cancer cells. Thus, this study was conducted to investigate the effect of corn silk extract containing high levels of maysin on inflammation and its mechanism of action in colon cancer cells. SW 480 human colon cancer cells were treated with 1 µg/mL of lipopolysaccharide (LPS) to induce inflammation, and next they were treated with different concentrations of corn silk extract (0, 5, 10 and 15 µg/mL). The concentrations of nitric oxide (NO) were determined. The mRNA expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β) and interleukin-6 (IL-6), were determined. Western blot analysis was performed to determine the protein expressions of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases, and the latter consists of extracellular signal-related kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 MAP kinase (p38). The concentration of NO and the mRNA expression of iNOS were significantly and dose-dependently decreased in the corn silk-treated groups (P<0.05). The mRNA expression of TNF-α, IL-1β and IL-6 were significantly increased in the LPS-treated group (P<0.05), but these expressions were significantly and dose-dependently decreased in the corn silk treated groups (P<0.05). The protein expressions of NF-κB (in a dose-dependent fashion), ERK (at 10 and 15 µg/mL), JNK (at 15 µg/mL) and p38 (at 10 and 15 µg/mL) were significantly decreased with corn silk treatments (P<0.05). In conclusion, corn silk extract containing high levels of maysin seems to inhibit the LPS-induced inflammatory responses in SW480 colon cancer cells via the NF-κB pathway.

Study on Synthesis and Anti-tumor Activity of 6 Kinds of Tectorigenin Mannich Base Derivatives / 中国药房

OBJECTIVE： To conduct structural modification of tectorigenin to search for new compounds with anti-tumor activity. METHODS： Tectorigenin was used as a lead compound， and then added into amine reagents as ethanolamine， methylamine， ethylamine， dimethylamine， diethylamine， n-propylamine and formaldehyde solution. Tectorigenin Mannich base derivatives were synthesized by mannich reaction with as the lead compound. The structures of the derivatives were identified according to IR， UV， MS and NMR data. Solubility of tectorigenin and its derivatives were investigated by solubility test method. MTT assay was used to investigate the inhibitory effects of tectorigenin and its derivatives on the proliferation of human colon cancer cell line HCT116， human lung cancer cell line A549 and human hepatoma cell line HepG2， and half inhibitory concentration （IC50） was calculated. The inhibition rate of tectorigenin and its derivatives （100 mg/kg） on H22 hepatoma-bearing mice in vivo was studied. RESULTS： Totally of 6 kinds of tectorigenin mannich base derivatives were synthesized， such as 8-（N-hydroxyethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-methyl）-methyleneamino-5，7，4′-trihydroxy-6- methoxyisoflavone， 8-（N， N-diethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N， N-dimethyl）-methyleneamino- 5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-ethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-propyl）- methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone （compounds 1-6 in turn）. Compared with tectorigenin， the water solubility of six derivatives was significantly improved， and the solubility was 5-20 times higher than that of tectorigenin. IC50 of compounds 1， 3 and 5 to HCT116 cells were （34.82±3.27）， （16.21±4.13）， （33.12±3.25） μmol/L， which were stronger than that of tectorigenin [（45.23±5.74） μmol/L]； IC50 of compounds 1， 3 and 5 to A549 cells were （37.05±5.74）， （26.88±4.52）， （30.13±6.23） μmol/L， which were stronger than that of tectorigenin [（53.24±6.34） μmol/L]； IC50 of compounds 1， 3 and 5 to HepG2 cells were （23.74±1.45）， （18.96±2.34）， （30.95±2.87） μmol/L， which were stronger than that of tectorigenin [（48.98±2.58） μmol/L]. Compounds 1， 3 and 5 showed higher inhibition rates （55.51％， 57.20％ and 49.15％） than tectorigenin （33.05％） on H22 hepatoma-bearing mice， respectively. The other three compounds had no obvious advantage over tectorigenin in anti-tumor activity. CONCLUSIONS： In this study， compounds 1， 3 and 5 of six tectorigenin mannich base derivatives synthesized in this study have stronger antitumor activity than tectorigenin.

Effect of miR-126 on proliferation and apoptosis in colon cancer cells via targeting regulation of SOX2 expression / 中国比较医学杂志

Objective To explore the effect of miR-126 on proliferation and apoptosis in colon cancer cells via targeting regulation of SOX2 expression. Methods miR-126 mimics and miR-126 NC were transfected into SW480 cells by liposome LipofectamineTM2000. The expression of miR-126 was detected by RT-PCR. Cell viability was determined by MTT staining. Cell apoptosis and cell cycle were detected by flow cytometry. The expression of SOX2 protein and mRNA was measured by western blot and RT-PCR. Luciferase reporter analysis was performed. Results Compared with miR-126 NC, the expression of miR-126 was upregulated(P< 0.01),cell viability was reduced(P< 0.01),early cell apoptosis rate and late apoptosis rate were increased(P< 0.01), cell cycle was arrested at G1 phase(P< 0.01), meanwhile, miR-126 mimics targeted downregulation of the expression of SOX2 protein and mRNA(P< 0.01). Conclusions miR-126 mimics can inhibit SW480 cell proliferation and induce cell apoptosis by targeting downregulation of expression of SOX2.

The effect and underlying mechanisms of down-regulated CDX2 on migration and invasion of colon cancer cells / 西安交通大学学报(医学版)

Objective To observe the effect of down-regulated CDX2 gene on the migration and invasion abilities of colon cancer cells (SW480 and HT29) and investigate the role and mechanisms of CDX2 gene in occurrence and development of colon cancer metastasis.Methods CDX2 gene in HT29 and SW480 cells was down-regulated using lentivirus RNA interference (RNAi) vector.The interference efficiency of CDX2 was detected by qRT-PCR and Western blotting.The effect of down-regulated CDX2 expression on colon cancer cells'migration and invasion was determined by Transwell and wound heal methods.Then the effects of down-regulated CDX2 on the expressions of epithelial-mesenchymal transition (EMT)-related genes (E-cadherin,ZEB-1,Vimentin,Twist and Snail) were detected by RT-PCR and Western blotting.Results The constructed CDX2 siRNA expression vector could significantly inhibit the expression of CDX2 in HT29 and SW480 cells.Compared with those of the cells transfected with empty vector (LV-NT-shRNA) and non-transfected cells,the migration and invasion abilities of cells transfected with LV-CDX2-shRNA were markedly enhanced (P ＜ 0.05).E-cadherin expression was reduced while expressions of ZEB-1,Vimentin,Twist,and Snail were significantly increased (all P＜0.05).Conclusion Down-regulating the expression of CDX2 can induce the occurrence of EMT,thus enhancing the invasion and migration of colon cancer cells.

Effect and mechanism of atorvastatin on cytotoxicity of human NK cells to colon cancer cells / 中国免疫学杂志

Objective:To explore the mechanism of the cytotoxicity of human NK cells induced by atorvastatin to colon cancer cell lines. Methods:After colon cancer cells (HCT-116,SW-480,Caco-2) were cultured with different concentrations of atorvastatin, CCK-8 assay was used to assess the effect of atorvastatin on growth of colon cancer cells. The amplification of human NK cells was induced by SCGM medium in vitro. Automatic biochemical analyzer was applied to test the cytotoxicity of NK cells to colon cancer cells which cultured with different concentration of atorvastatin. FCM was used to detect the expression rate of MICA/B on the cells. Results:(1) The cultivation of NK cells:The proportion of NK cells attained to 93. 1% from 4. 5% after cultured for 10 days. (2) The effects of atorvastatin on the growth of the colon cancer cells:After cultured with atorvastatin,the inhibition rate of HCT-116 cells was higher than that in control when the density of atorvastatin increased from 5 μmol/L to 40 μmol/L after 48 h and from 1. 25 μmol/L to 40 μmol/L after 96 h ( P<0. 05 ) . Correlation analysis showed that the concentration of atorvastatin and the growth inhibition rate of HCT-116 cells were positively correlated(r[48 h]=0. 13,r[96 h]=0. 22,P<0. 05). (3) The cytotoxicity of NK cells to colon cancer cells effected after atorvastatin: In different atorvastatin concentrations groups,the cytotoxicity of NK cells to three colon cancer cell lines was all higher than that in control ( P<0. 05 ) . The atorvastatin concentration was from 2. 5 μmol/L to 10 μmol/L for HCT-116 cells,from 5 μmol/L to 20μmol/L for SW-480 cells,and from 2. 5μmol/L to 20μmol/L for Caco-2 cells. Among the three cell lines, the cytotoxicity of NK cells to HCT116 was the highest in the same concentration. (4)NK cells by atorvastatin cutting statins 96 h,the concentration of 20 mmol/L and 40 mmol/L inhibition rate was higher than that of control group,more than other groups on NK cell growth without significant effect. ( 5 ) The impact of atorvastatin on MICA/B expression of colon cancer cells: After cultured with different concentrations of atorvastatin,the expression of MICA/B on colon cancer cells was higher than that in control(P<0. 05). The concentration was 2. 5μmol/L and 5μmol/L for HCT-116 cells,10μmol/L and 20μmol/L for SW-480 cells,and from 2. 5μmol/L to 40 μmol/L for Caco-2 cells. Conclusion:Atorvastatin could inhibit the growth of colon cancer cells (HCT-116,SW-480 and Caco-2) in a dose-dependent manner;and it could enhance the cytotoxicity of NK cells to colon cancer cells;it also could promote the expression of MICA/B of colon cancer cells,and improve the immunogenicity of colon cancer cells.

Bacillus spp. or Bacillus spp.-Derived Membrane Vesicles Induce the Intrinsic Pathways of Apoptosis of Human Colon Cancer Cell Lines

The striking increase in colorectal cancer (CRC) has shown the great fatality in Korea for more than 15 years. The leading edge of this rising incidence rate is mainly due to the people's dietary changes in Korea. Some studies have reported that the dietary fiber does not have significant cytotoxic effects on CRC cells, which contrasts to the effects of probiotics. It gives a positive evaluation that the nonpathogenic spore-forming Bacillus species among the probiotics including fermented bacteria might have optimistic effects on CRC incidence rate. Recently, we isolated Bacillus lentus (BL) from Korean soybean fermented food. BL showed the cytotoxic effect on human colon carcinoma cell lines HCT116 and SW480. Interestingly, BL did not have effect on human dermal fibroblast cells and human hepatoma cell line HepG2. It suggested that BL has the target cell-specific cytotoxicity toward human colon carcinoma cells. To clarify the death signaling pathway underlying the BL-induced apoptosis in cancer cells, we analyzed the expression of caspases, Bax and Bcl-2 by western blotting. The apoptotic effects by cytotoxic elements were executed by direct BL contact or membrane-derived vesicles isolated from BL. Treatment of HCT116 with BL activated caspase-9, -3 and increased cleavage form of poly (ADP-ribose) polymerase (PARP). However, caspase-8 activity was not increased by BL. BL-activated intrinsic pathway increased the pro-apoptotic Bax, decreased the anti-apoptotic Bcl-2 proteins on mitochondria, disrupted the mitochondrial membrane potential, and then released the cytochrome c from mitochondria. The membrane-derived vesicles (MVs) from BL induced apoptosis of the HCT116. Here, we propose that BL as a strong candidate for the development of apoptosis-specific anti-tumor agent will give great contribution to the understandings of the tumor-microbe interdisciplinary areas.

Effects of Solanine on the human colon cancer in chicken embryo transplantation model angiogenesis / 重庆医学

Objective To establish chicken embryo transplantation model of human colon cancer and to research the effect of so‐lanine on angiogenesis .Methods Cases with chicken embryos were divided into the low‐,mid‐and high dose solanine group and con‐trol group ,with 10 cases in each groups ,and then the cultured human colon cancer cell line HT‐29 cell lines were inoculated to the chicken embryo villus allantois membrane (CAM ) .We observed the characteristics of the transplanted tumor in CAM angiogenesis by the stereo microscope .Image analysis software of Image‐pro plus 6 .0 and immunohistochemical method were used to observe the effect of different dose of solanine on angiogenesis .Results HT‐29 cell lines were inoculated to CAM 3-5 days ,a large number of blood vessels concentrated in tumors ,growing into or acrossing the surface of tumors .While tumors also rapidly growed .We took photo on the 5th day after receiving medicine and did imaging analysis .Then we calculated the area of angiogenesis in experimental group ,which was significantly lower than that of the control group ,quantitatively in a dose‐dependent manner .There were signifi‐cant differences among the groups(P<0 .01) .Microvascular density of 3 different dose of solanine was significantly lower than that of the control group by immunohistochemical method ;the expression of Ki‐67 antigen index decreased gradually ,which was highest in the control group ,and there were significant differences among the groups (P<0 .01) .Conclusion Solanine could inhibit angio‐genesis induced by human colon cancer HT‐29 cell lines obviously ,thus inhibiting the growth of tumor and providing an important basis for the treatment of anti‐tumor angiogenesis .

Effects of Hrs on Apoptosis and Cell Proliferation in Colon Cancer Cell Lines / 中国医科大学学报

Objective To study the effects of Hrs on apoptosis and cell proliferation in colon cancer cell lines HCT?116 and SW?480. Methods The expression of Hrs in HCT?116 and SW?480 was knocking down by shRNA. Cell apoptosis was detected by FACS and cell proliferation was de?termined by MTT,incucyte and soft agar. Results The apoptosis was significantly increased after Hrs knocking down in both HCT?116 and SW?480,and the cell proliferation was inhibited. Conclusion Hrs is involved in the apoptosis and proliferation of colon cancer cells.

Tannic acid inhibits the proliferation of SW620 cell lines by downregulating TMEM16A expression / 重庆医学

Objective To investigate the effects of Tannic acid on the proliferation of human colon cancer SW 620 cell line and the mRNA and protein levels of TMEM16A .Methods Human colon cancer cell line SW620 were divided into the low dose(50 μmol/L) ,high dose(100 μmol/L) ,they were cultured for 48 h or 72 h separately .Control groups were cultured in the medium with DMSO .The proliferation of SW620 cell line was detected by the MTT assay at different time points (48 h or 72 h) .The cell cycle and apoptosis in the Tannic acid-treated groups were detected by flow cytometry .RT-PCR and Western blotting were used to de-termine the mRNA and protein levels of TMEM16A separately .All data were analyzed using the one-way analysis of variance (ANOVA) ,SNK test by the SPSS software .Results Compared with the control group ,the proliferation of SW620 cell line was significantly inhibited after the treatment by Tannic acid at the concentration of 50 μmol/L and 100 μmol/L for 48 h or 72 h(t=15 .35 ,P0 .05) ,and increased apoptotic rate when compared with control group (F=545 .3 ,P<0 .01) .The value of 3H-TdR and 3H-Leucine incorporation of SW620 cells treated with Tannic acid(100 μmol/L) 48 h and 72 h separately ,were obviously decreased as compared with that of control group (P<0 .05) .In the low dose treated groups (50 μmol/L) ,the mRNA levels in 48 h group and 72 h group were(0 .633 ± 0 .009) and(0 .621 ± 0 .011) ,and in the high dose treated groups (100 μmol/L) ,the mRNA levels in 48 h group(0 .64 ± 0 .15) and 72 h group(0 .63 ± 0 .11) ,were lower than the control group(F=7 .645 ,P< 0 .05) .After treating SW620 with Tannic acid for 48 h and 72 h ,in the low dose groups ,the protein expression of TMEM16A were(0 .68 ± 0 .14) and(0 .65 ± 0 .12) ,and in the high dose groups ,the protein expression of TMEM16A were(0 .64 ± 0 .15) and(0 .63 ± 0 .11) were decreased when compared with the control group (1 .28 ± 0 .06)(F=4 .508 ,P<0 .05) .Conclusion Tannic acid arrested SW620 at G1-S phase and decrease the mRNA and protein expression of TMEM16A .

Inhibition of Colon Cancer Cells by Ethanol Extract of Oldenlandia Diffusa / 昆明医科大学学报

Objective To explore the inhibitory effect of the ethanol extract of Oldenlandia diffusa on the proliferation of CT-26 colon cancer cells which come from BALB/c mice. Method We determined the inhibitory effect of different concentrations of ethanol extract of Oldenlandia diffusa on CT-26 cells' proliferation by using methyl thiazolyl tetrazolium (MTT), and calculated the 50% inhibiting concentration (IC50) . Results As to the same concentration, the inhibitory effect of the ethanol extract of Oldenlandia diffusa on CT-26 cells was increased with time, for exsample:after treated with 0.08 mg/mL of ethanol extract of Oldenlandia diffusa for 24 h, 48 h and 72 h, the inhibitory rates of CT-26 cells were (16.67 ±9.35)%, (34.66 ±9.23)%and (40.07 ±9.16)%, respectively. After treating CT-26 cancer cells for 24 h, 48 h and 72 h, the IC50 values of the ethanol extract of Oldenlandia diffusa were 0.315,0.155 and 0.115 mg/mL, respectively. In the same treatment time, the inhibitory effect of the ethanol extract of Oldenlandia diffusa on CT-26 cells was increased with the increase of concentration:after treatment for 72 h with different concentrations (0.06 mg/mL,0.08 mg/mL,0.10 mg/mL,0.12 mg/mL, 0.14 mg/mL,0.16 mg/mL,0.18 mg/mL and 0.20 mg/mL) of the ethanol extract of Oldenlandia diffusa,the inhibitory rates of CT-26 cells were (35.46 ±3.59)%, (40.07 ±9.16)%, (40.77 ±6.92)%, (52.81 ±1.87)%, (54.22±2.35)%, (68.72±3.71)%, (70.04±8.03)%and (71.84±3.12)%, respectively. Conclusion The ethanol extract of olenlandia diffusa can inhibit the proliferation of CT-26 colon cancer cells from BALB/c mice in a time and dose dependent manner.

Inhibitory Effect of Ethanol Extract of Oldenlandia Diffusa on Colorectal Cancer Angiogenesis in BALB/c Mice / 昆明医科大学学报

Objective To explore the inhibitory effect of Oldenlandia diffusa extract on colorectal cancer angiogenesis in BALB/c mice. Methods Thirty-two BALB/c mice with subcutaneous CT26 colon cancer animal model were randomly equally divided into four groups,including the control group (groupⅠ,saline 0.1 mL/(10. d), O. diffusa ethanol extract of 90 mg/(kg.d) (groupⅡ), O. diffusa ethanol extract of 180 mg/(kg.d) (groupⅢ) and O. diffusa ethanol extract of 360 mg/(kg.d) (group Ⅳ) . Each group of mice were treated with intragastric administration of law administration 12 days after vaccination, then stopped and continue fed to 32 days, and the mice were killed. Micro-vascular dense ( MVD) was observed and countered under the microscopy by immunohistory chemistry. Results The murine colon tumor volumes of GroupⅡ,ⅢandⅣwere significantly less than that of groupⅠ,with significant difference ( <0.05) . The tumor microvessel density values of four groups was (7.83±2.87), (5.32±1.27), (1.77±0.70) and (1.87±0.68),respectively. The number of tumor blood vessels in GroupⅡ,Ⅲ and Ⅳ were significantly less than that of Ⅰ group, with significant difference ( <0.05) . Conclusion Within a certain dose range, the ethanol extract of O. diffusa can significantly inhibit the mouse colon cancer and the mechanism may be realated to inhibiting tumor angiogenesis.

Mechanism of sodium selenite-mediated death of human colon cancer HCT116 cell line / 中国医师进修杂志

Objective To investigate the inhibition mechanism of sodium selenite on HCT116 cells.Methods In the present study,we explored the cytotoxicity induced by sodium selenite and the underlying mechanism by MTS assay,WesternBlot,and small RNA interference technique.Results It was found that the sodium selenite at 5uM concentration could indeed reduce the viability of colon cancer cell line HCT116 by a large margin through increasing the generation of reactive oxygen species (ROS),and that the increased levels of ROS could activate c-Jun Nh2-terninal kinase 1 (JNK1).Additionally,knockdown expression of JNK1 or p53 by using RNAi attenuated the cytotoxicity induced by sodium selenite,indicating that both of JNK1 and p53 are required in the process of cell death induced by sodium selenite.Conclusion The sodium selenite could induces cell death in HCT116 through oxidative stress by involvement of JNK1 and p53,both of which play a critical role in toxicity of sodium selenite.

Proliferation inhibition and apoptosis induction effect of dextran-magnetic layered double hydroxide-fluorouracil drug delivery system on human colon cancer cells SW480 / 中国药学杂志

OBJECTIVE: To explore the proliferation inhibition and apoptosis induction effect of dextran-magnetic layered double hydroxide-fluorouracil (DMF) drug delivery system on colon cancer cell SW480 cultivated in vitro. METHODS: MTT experiment was used to detect the cell proliferation inhibition rates of DET-MLDH-FU, MLDH-FU and MLDH supra-molecules at various concentrations. Light microscopy and Giemsa dyeing methods were applied for characterizing the shape change of the apoptotic cells. Flow cytometric analysis was employed to test the apoptosis rates of SW480 cells treated with different drugs for 24 h. DNA ladder method was used to analyze nuclei breakup of the apoptosis cells. RESULTS: The IC50 of MLDH, MLDH-FU and DMF three-level supra-molecules for SW480 cells were 44.83(25.74, 48.78), 15.16(13.78, 16.66) and 13.33 (11.03, 15.13) μg · mL-1, respectively. The proliferation of colon cancer cells SW480 was obviously restrained after being treated with 5-Fu and equivalent MLDH, MLDH-FU and DMF of different concentrations for 24, 48 and 72 h, respectively. The result revealed that the inhibition rates increased in the sequence of MLDH

Inhibition of proliferation and apoptosis of human colon cancer cell sw480 cell line induced by the co-culture supernatant of Toxoplasma gondii / 中国人兽共患病学报

To investigate the inhibitive effect of the co-culture supernatant of Toxoplasma gondii on the proliferation of human colon cancer cell line sw480 and/or its induced apoptosis and necrosis in vitro,the co-culture model of sw480 cells (1x10~6) was established with different number of Toxoplasma gondii (the concentration of tachyzoites was 2×10~6,4×10~6,8×10~6,16×10~6 respectively),and the co-culture supernatants were gathered 72 hours later.The sw480 cells were treated for different hours with different models of the co-culture supernatant.The parasitism and proliferation of Toxoplasma gondii tachyzoites in sw480 cells was observed by Giemsa staining,and the growth inhibition rates of these cells were investigated by the CKK-8 method.Meanwhile,the apoptosis and necrosis of sw480 cells were detected with transmission electron microscopy (TEM) and fluorescence microscopy with Hochest 33258 staining to observe the morphological changes of sw480 cells.Agarose electrophoresis was used to detect the DNA change of sw480 cells,and flow cytometric analysis was used for investigation of rates of apoptosis and necrosis of these cells stained with Annexin-v-FITC/PI.It was found that the parasitism and proliferation of Toxoplasma gondii tachyzoites in sw480 cells could be observed;and as demonstrated by the CKK-8 method,the inhibitive effect of the co-culture supernatant depended upon the treatment time,and a maximum inhibition ratio of 44.55% was detected at 48 hours.The karyopyknosis and karyorrhexis in the treated sw480 cells were observed under the fluorescence microscope;and the cellular necrosis were observed under TEM.In addition,the DNA fragments in the treated cells were revealed by agarose electrophoresis.In the flow cytometric analysis,a maximum prophase apoptosis rate of 11.54% was detected at 48 hours after treatment with the supernatants,while the rate of prophase apoptosis decreased,and the rates of advanced stage/necrosis increased markedly at the treatment time beyond 48 hours.It is concluded that the co-culture supernatant of Toxoplasma gondii and human colon cancer cell sw480 cell line can induce inhibition of proliferation,apoptosis and necrosis of this cell line in vitro.

Genomic Alterations Detected in Colon Cancer Cell Lines by Using Array-Comparative Genomic Hybridization / 대한해부학회지

Cancer development is accompanied by genetic events like losses, gains and amplification of certain chromosome regions or alterations of chromatin structure. Array-based CGH (Array-CGH) is a highly comprehensive, sensitive and fast technique to allow investigation of general changes in target oncogenes and tumor suppressor genes. Recently, the prevalence of colon cancer is rapidly increasing in Korea and now it is the fourth leading cause of cancer death. So, the purpose of this study is to examine genomic alterations in colon cancer cell lines and to search novel genes which might be related to the development of colon cancer. In this study, genomic alterations are analyzed by using array-CGH in three colon cell lines from Korean, SNU-81, SNU-407 and SNU-1047. We observed numerous chromosomal imbalances from all cell lines. The common chromosomal gains were observed in 1p36.33, 1q22, 1q32.1, 2q35, 8p12, 8q22.3, 14q32.33, 16p13.3, and 16q24. Common chromosomal losses were found in 4q22.1, 9q13, 14q21.1, 14q32.33, 20p12.1, Xq21.1, and Yq11.223. Gains of 1p, 2q, 8p, and 8q or losses of 4q, 14q and 20p are already known to be associated with the colon cancer development. For gene alterations, we could see gains of some genes such as ELF3 and AAMP, which were already reported to be associated with colon cancer. Also, we could find some gene alterations which were known to be associated with other cancer types. These genes were GON4L, RNPEP, TMBIM1, TIMM17A, GPBAR1, PPP1R13B and SOX8. Besides, we found alterations of new genes such as PKND and LEPROTL1. The association of these genes with colon cancer is first demonstrated here. These genes may be the novel candidate genes functioning in the development of colon cancer. In conclusion, array-CGH demonstrated the complexity of genetic aberrations in several colon cell lines. These data about the patterns of genomic alterations could be a basic step for understanding more detailed genetic events in the carcinogenesis and also provide information about possible target genes for diagnosis and treatment in colon cancer.

Inhibitory effect of VEGFR-3-targeted siRNA against adherence and invasion of human colon cancer cells / 中国肿瘤生物治疗杂志

Objective:To construct a small interfering RNA(siRNA)expression vector(psiRNA-VEGFR-3)targeting vascular endothelial growth factor receptor 3(VEGFR-3)and to investigate the effects of VEGFR-3 siRNA on the adherence and invasion of human colon cancer cells.Methods:A siRNA expression vector(psiRNA-VEGFR-3)targeting VEGFR-3 were constructed and was used to transfect LoVo cells via lipofectamine 2000.The mRNA and protein expression of VEGFR-3 were examined after transfection by reverse transcriptase polymerase chain reaction(RT-PCR)and Western blotting,respectively.The tumor adhesion ability was detected by cell-matrix adhesion experiment and the invasion ability of tumor cells was evaluated by millicell chamber model.Results:The VEGFR-3 siRNA expression vector was successfully constructed.The expression of VEGFR-3 mRNA and protein was inhibited after psiRNA-VEGFR-3 transfection.Seventy-two hours after psiRNA-VEGFR-3 transfection,Western blotting assay showed that the expression of VEGFR-3 protein was decreased from(1.26?0.19)to(0.39 s0.12)(P

Sulindac-induced Apoptosis without Oligonucleosomal DNA Fragmentation in HT-29 Cells: Its Special References to Mitochondrial Pathway

PURPOSE: This study was undertaken to reveal the molecular mechanism underlying sulindac-induced apoptosis in the human colon cancer cell line HT-29 (mutant p53). METHODS: Apoptosis was determined by using Hoechst 33342 staining, and translocation of proteins was established by using immunofluorescence, immunoelectron microscopy, and Western blotting after ultra- fractionation. RESULTS: This type of apoptosis was associated with decreased mitochondrial membrane potential, a translocation of the apoptosis-inducing factor (AIF) to the nucleus, and morphological evidence of nuclear condensation. However, DNA electrophoresis did not elucidate the ladder pattern of DNA fragments. Instead, a pulse-field gel electrophoresis showed that sulindac led to disintegration of nuclear DNA into-high- molecular-weight DNA fragments of about 100~300 kbp. CONCLUSIONS: Our findings indicate that sulindac induces large-scale DNA fragmentation, suggesting a predominantly AIF-mediated cell-death process, through translocation of the AIF to the nucleus in HT-29 cells.

Short chain fatty acids affect colon cancer cells proliferation and differentiation / 肠外与肠内营养

Short chain fatty acids(SCFA) are produced in the large bowel of humans by anaerobic bacterial fermentation.The main fermentative substrates are undigested dietary carbohydrates,including nonstarch polysaccharides and resistant starch(RS).SCFA are major organic acids in the lumen of the large intestine.They are preferred energy source for colonocytes.Their effects include enhancement of electrolyte uptake,stimulation of colon epithelial cell proliferation and mucosa growth,modulation of colonic immune function,nutritional support and protection of colon mucosa.Butyric acid can suppress colon neoplasm cell proliferation,induce its apoptosis and differentiation,affect proto-onc genes expression,which suggest that butyrate may be an important agent in cancer treatment.

Roles of cFLIPL in Fas-mediated Apoptosis of Colon Cancer

PURPOSE: Substantial numbers of the colon cancer cells have been observed to express Fas/Fas ligands, but are resistant to Fas-mediated apoptosis, suggesting that colon tumors might develop the specific mechanisms to overcome Fas-mediated apoptosis. Recently, cellular FLICE-like inhibitory protein (cFLIP) has been identified as an endogenous inhibitor of Fas- or other receptor-mediated apoptosis and its altered high expression has been suspected to be associated with tumor development or progression. This study investigated the prevalence of cFLIPL alterations in colon carcinomas and possible implications in the progression of colon cancers. METHODS: In order to investigate the function of cFLIPL for the development and progression of the colon cancer, we analyzed the expression of the cFLIPL in 58 colon cancer and 6 colon cancer cell lines. RESULTS: The study results demonstrated that cFLIPL is one of the major determinants for sensitivity to Fas-mediated apoptosis in colon cancer cell lines. In addition, we analyzed cFLIPL expression in 58 cases of adenocarcinoma of the colon and showed that cFLIPL, expression was increased in adenocarcinoma as compared to matched normal mucosa. CONCLUSION: Taken together, our data strongly suggested that abnormal overexpression of cFLIPL is a frequent event in colon carcinomas and might contribute to in vivo tumor transformation.