ABSTRACT
To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G₀/G₁ or G₂/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.
ABSTRACT
Objective To establish and evaluate intestinal epithelial barrier model using Caco-2 cell so as to play a foundation for next study of barrier permeability.Methods Caco-2 cells were cultured in vitro then seeded into Transwell cell culture inserts.The permeability of the intestinal epithelial barrier was detected by transepithelial electrical resistance(TEER)and lucifer yellow flux,and verified by transmission electron micro-scope.Different concentrations of PAF(0,50,100,and 200 nmol /L)were exposed for 24 hours to Caco-2 mono-layer when cultured 21 days.The tight junction was observed under transmission electron microscope.Assess-ment of ZO-1 protein localization and expression were detected by immunofluorescence and Western blot analy-sis.Results Cultured Caco-2 cell confluencd as monolayer with time passed.From 5th day,TEER increased, then reached 600Ω?cm2 at 15th day and lasted to 21 st day,there was little flux of lucifer yellow,transmission e-lectron microscopy also found cells differentiated better,had well-arranged villi and polarity alined as monolayer, forming completed tight junction which was the marker of intestinal epithelial barrier model in vitro.TEER de-creased and lucifer yellow flux increased in cells exposed to PAF.The permeability reached the peak when ex-posed to 100 nmol /L PAF(P <0.01 ),tight junction disrupted,ZO-1 protein expression downregulated,abnor-mal localization and distribution was assessed by immunofluorescence staining.Conclusion Cultured Caco-2 cells for 2-3w can be used to study intestinal epithelial barrier as a model in vitro.PAF increased intestinal epi-thelial permeability,which would correlate to the decreased protein expression and abnormal distribution of ZO-1.
ABSTRACT
AIM:To check the antiproliferative and apoptosis-inducing effect of Compound Changtai Granule(Radix et Rhizoma ginseng,Semen coicis,Rhizoma curcumae,etc)(CCG)on human colon carcinoma cell line(SW480).METHODS:Methy thiazolyl tetrazolium chromatomerry was used to observe the influence of several concentration of CCG on SW480 cell proliferation cultured in vitro at the various times,and flow cytometry was applied to examining the change in SW480 cell proliferation cycle and apoptosis rate.RESULTS:CCG could successfully prevent SW480 cell from entering G_0/G_1 phase and G_2/M phase,decrease cell numbers in S phase at the same time.CONCLUSION:CCG has an obvious dose-response relationship in the range of 3.51 to 7.81 mg/mL.
ABSTRACT
To investigate the effect of meloxicam, a selected NSAIDs, on cell growth, expression of VEGF and angiopointin-2 (Ang-2) protein in HT-29 cell line, cultured HT-29 cells were treated with meloxicam of various concentrations for various lengths of time. The proliferation of HT-29 was detected by cell counting kit-8 (CCK-8), the cell cycle was determined by flow cytometer and the levels of VEGF and Ang-2 protein in supernatants were examined by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of VEGF and Ang-2 in cultured HT-29 were determined by real-time quantitative reverse-transcription polymerase chain reaction. Our results showed that treatment of meloxicam of different concentrations and for various lengths of time had a cytotoxicic effect on the cell proliferation of HT-29 cells in a concentration-dependant and time-dependant manner. Cell cycle analysis showed that the cells were mainly blocked in G0/G1 phase. The VEGF and Ang-2 protein levels in supematants of the culture medium were decreased gradually in a concentration-dependent or time-dependent fashion. The mRNA expression of cox-2, VEGF and Ang-2 showed a gradual and concentration-dependent reduction. It is concluded that meloxicam can reduce the expression of VEGF and Ang-2 at the protein and mRNA level in colon carcinoma cell line.
ABSTRACT
Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate,which is required for DNA synthesis and repair TS and is an important target for 5-fluorouracil (5-FU). In this study we investigated whether the greater cytotoxicity of irinotecan (CPT-11) followed by 5-FU in HT-29 and SNU-C4 cell lines was related to TSmRNA expression or activity. Thus, cells were exposed for 24 h to each drug, alone or in combination, and assessed for colony formation, TS catalytic activity, TS-mRNA expression and cell cycle distribution. Pre-treatment with CPT-11 at IC20 increased the 5-FU cytotoxicity in HT-29 and SNU-C4 cells. TS catalytic activity and TS-mRNA expression suggested that the differences in sensitivity to 5-FU among the cell lines were not correlated to TS profile. When we exposed cells to CPT-11 at IC20 and subsequently to 5-FU at IC50, the impact on TS activity and mRNA expression were the same as observed with 5-FU alone. CPT-11 induced G2/M arrest, while 5-FU arrested cells in S-phase in both cell lines. When cells were treated with CPT-11 followed by 5-FU we observed an increased in 5-FU-inducible S-phase arrest in both cell lines. Our findings suggested that the greater cytotoxicity of CPT-11/5-FU combination in HT-29 and SNU-C4 cell lines are not related to interference with thymidylate synthase.
Subject(s)
Fluorouracil , Colonic Neoplasms , Thymidylate Synthase , Cell CycleABSTRACT
Objective To investigate the inhibitory effect of ester catechin monomers-EGCG, GCG on the growth of human colon carcinoma cell line SW480 and its potential mechanism. Methods MTT assay, soft-agar colony formation test, Hoechst 33258 stain and flow cytometry analysis were used to determine the inhibitory effect of theasinesin(TS), and its monomers-EGCG, GCG on SW480 growth. Results EGCG, GCG and TS significantly inhibited the growth of human colon carcinoma cell line SW480 in dose-dependent manner, and their half inhibitory concentration (IC 50 ) was 108.88, 183.21, and 83.36?g?ml -1 , respectively. 24 hours after treatment with IC 50 of EGCG, GCG and TS, the colony formation rate of SW480 cells in the experimental group was obviously lower than that in the control group. Hoechst 33258 staining showed typical apoptotic features such as cell shrinkage, nuclear condensation and cell splinter in the experimental group. A subdiploid peak before G 0/G 1 phase in the experimental group was observed by flow cytometry. Conclusion In accord with TS, EGCG and GCG could inhibit the growth of colon carcinoma cell line SW480 cells, the mechanism of which may be related to inducing cell apoptosis.
ABSTRACT
Objective: To study the affection of Diallyl trisulfide on cultured human colon carcinoma cell line HT-29 and its mechanism. Methods: The viability of cells after various treatments were determined using the method of MTT. Flow cytometry were used to analyze the change of cell cycle and Bcl-2, Bax levels. Results: The proliferation of human colon carcinoma HT-29 cells were inhibited by Diallyl trisulfide. The inhibition was associated with dose and time dependence. Flow cytometry results showed that: 1) When cells were treated with Diallyl trisulfide at the concentration of 10 ?g/mL for 12 h and 24 h, the percentage of G0/G1 phase cells was decreased and that of G2/M phase cells was significantly increased compared with those in the control group.2) Diallyl trisulfide down regulated Bcl expression, while up-regulated bax expression. Conclusions: It was suggested that Diallyl trisulfide inhibited HT29 cell proliferation. And these were associated with arrest of cell cycles and down regulation of Bcl2/Bax ratio.
ABSTRACT
AIM:To check the antiproliferative and apoptosis-inducing effect of Compound Changtai Granule(Radix et Rhizoma ginseng,Semen coicis,Rhizoma curcumae,etc)(CCG) on human colon carcinoma cell line(SW480).METHODS:Methy thiazolyl tetrazolium chromatomerry was used to observe the influence of several concentration of CCG on SW480 cell proliferation cultured in vitro at the various times,and flow cytometry was applied to examining the change in SW480 cell proliferation cycle and apoptosis rate.RESULTS:CCG could successfully prevent SW480 cell from entering G0/G1 phase and G2/M phase,decrease cell numbers in S phase at the same time.CONCLUSION:CCG has an obvious dose-response relationship in the range of 3.51 to 7.81 mg/mL.