ABSTRACT
ObjectiveTo investigate the effect of curcumin on the cycle arrest of human colon cancer HCT116 cells and decipher the possible molecular mechanism. MethodThe methyl thiazolyl tetrazolium （MTT） method was employed to examine the effects of curcumin （0， 12.5， 25， 50， 75， 100 μmol·L-1） and 5-fluorouracil （5-FU， 600 μmol·L-1） on the proliferation of HCT116 cells at different time points （24， 48， 72 h）. Flow cytometry was employed to examine the cycle of HCT116 cells treated with curcumin （0， 25， 50， 75 μmol·L-1） and 5-FU. Western blot was employed to determine the expression of proteins in the Janus kinase 1 （JAK1）/signal transducer and activator of transcription 1 （STAT1） /cyclin-dependent kinase inhibitor 1A （p21） pathway in HCT116 cells. The binding of STAT1 to p21 promoter region was detected by chromatin immunoprecipitation （ChIP）. Small interfering RNA （siRNA） was employed to measure the role of STAT1 in regulating the expression of p21 and that of JAK1 in regulating the activation of STAT1 by Western blot and cellular immunofluorescence， respectively. ResultCompared with the blank group， the HCT-116 cells treated with curcumin and 5-FU showed decreased viability （P<0.05）， increased proportions of cells in the G0/G1 phase （P<0.05）， decreased proportions of cells in the S phase and G2/M phase （P<0.05）， down-regulated protein level of phosphorylated p21 （P<0.05）， and up-regulated protein level of p21 （P<0.05）. Compared with the curcumin group， the p21 siRNA+ curcumin group presented decreased proportion of cells in G0/G1 phase （P<0.05）. Compared with the blank group， curcumin elevated the level of phosphorylated STAT1 （p-STAT1） （P<0.05）. Compared with the curcumin group， the curcumin + STAT1 siRNA group showcased up-regulated protein level of p21 in HCT116 cells （P<0.05）. The mechanism study showed that curcumin treatment enhanced the enrichment of STAT1 in the p21 promoter region （P<0.05） compared with the blank group. Compared with the blank group， curcumin up-regulated the level of phosphorylated JAK1 （p-JAK1） （P <0.05）. Compared with the curcumin group， the curcumin + STAT1 siRNA group demonstrated up-regulated protein levels of p-STAT1 and p21 in HCT116 cells （P<0.05）. ConclusionCurcumin may induce the cycle arrest of human colon cancer HCT116 cells by activating the JAK1/STAT1/p21 signaling pathway.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Humans , Apoptosis , Bacillus , Bacteria , Blotting, Western , Carcinoma, Hepatocellular , Caspase 8 , Caspase 9 , Caspases , Cell Line , Colon , Colonic Neoplasms , Colorectal Neoplasms , Cytochromes c , Dietary Fiber , Fibroblasts , Incidence , Korea , Membrane Potential, Mitochondrial , Membranes , Mitochondria , Probiotics , Glycine max , Strikes, EmployeeABSTRACT
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 .
ABSTRACT
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 .
ABSTRACT
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.
ABSTRACT
Cancer tissues are characterized by increased glucose uptake. 18F-fluorodeoxyglucose(FDG), a glucose analogue is used for the diagnosis of cancer in PET studies. This study was aimed to compare the glucose uptake and glucose transporter l(GLUT1) expression in various human colon cancer cells. We measured FDG uptake by cell retention study and expression of GLUTI using Western blotting. Human colon cancer cells, SNU-C2A, SNU-C4 and SNU-C5, were used. The cells were incubated with 1micro Ci/ml of FDG in HEPES-buffered saline for one hour. The FDG uptake of SNU-C2A,SNU-C4 and SNU-C5 were 16.8+/-1.36, 12.3+/-5.55 and 61.0+/-2.17cpm/microgram of protein, respectively. Dose-response and time-course studies represent that FDG uptake of cancer cells were dose dependent and time dependent. The rate of FDG uptake of SNU-C2A, SNU-C4 and SNU-C5 were 0.29+/-0.03, 0.21+/-0.09 and 1.07+/-0.07cpm/min/microgram of protein, respectively. Western blot analysis showed that the GLUT1 expression of SNU-C5 was significantly higher than those of SNU-C2A and SNU-C4. These results represent that FDG uptake into human colon cancer cells are different from each other. In addition, FDG uptake and expression of CLUT1 are closely related in human colon cancer cells.