Inhibitory Effect Of Artesunate On Human Colon Carcinoma Cell Line In Vitro

ObjectiveArtemisinin,a sesquiterpene lactone isolated from the plant Artemisia annua L,and its derivatives are presently used in various countries as an antimalarial drug in clinic and have a potent effect on chloroquine-resistant malarial parasites.Its derivatives such as artesunate (ART) and dihydroartemisinin (DHA) , distinguish themselves as a new generation of anti-malarial drugs with high solubility in water and low toxicity to human. Especially, artemisinin derivatives exert remarkable activity against otherwise drug-resistant plasmodium falciparum and plasmodium vivax strains. Thus, they are playing a more important role in the treatment of malarial infection. Recent studies have showed that ART has an anti-tumor effect in vito and in vitro,and it's effective,safe and mild in drug resistance. Some groups around the world have reported the inhibition of human cancer cell lines growth and human umbilical vein endothelial cell angiogenesis by ART. However, the molecular action of ART toward tumor cells is still unexplored. The aim of the present investigation of ART is to observe its effects on human colon carcinoma cell linesand analyze the possible mechanism of its anti-cancer action.This will expand the use of ART and help finding a new way of chemotherapy for colon carcinoma.Two human colon carcinoma cell lines were observed in our study: hypso-metastic cancer SW620 cell lines and hypo-metastatic SW480 cell lines . We analysed the anti-proliferation and inducing apoptosis effects of ART on human colon carcinoma cell lines in vitro ,and assessed the inhibitory effect on expression ofVEGF in SW620 cells. Materials and methods 1 .Cell Culture and Cell Proliferation AssayThe two human cancer cell lines were routinely cultured in DMEM medium supplemented with 10% BCS. And these cells were placed in a standard 37'C incubator under normoxic conditions (95% air and 5% CO2) .The cell lines were seed at a density of 1x10~5 cells per well into 96-well plates.After 24h incubation at 37℃ in a 5% CO2 incubator ART at graded concentrations ranging from 10 to 90ug/ml were added to the wells,and the cells were further cultured for 24h and 48h respectively. The proliferation were measured by MTT assay. 2. Quantification of apoptosis by flow cytometryFor ART-induced apoptosis, SW480 and SW620 cell lines were incubated with ART at various concentrations(20 and 40ug/ml). After 48h of incubation, The apoptotic cells were graded qualitatively by their staining characteristics.Apoptotic cells can be distinguished from necrotic cells and quantified using flow cytometric analysis of the cellular DNA.The apoptotic cell population was identified by the DNA-specific fluorochrome PI. Briefly, Detached cells were combined with adherent cells after lifting with trypsin/EDTA, and RNase was added to the cell sample, which was then mixed with a PI solution. The PI fluorescence of individual nuclei was measured using a flow cytometer.3. RNA isolation and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR)Total RNA was isolated using the Trizol kit according to the manufacturer's instructions. RNA's purity and quantitation were detected by extreme ultraviolet spectroscopy. Total RNA was reverse transcribed and PCR reactions were performed on a PX2 programmable thermal controller. PCR products were examined electrophoretically in 1.5% agarose gels stained with ethidium bromide .The intensities of the bands were measured using image analysis software .The gene expression ratio (intensity of PCR products VEGF / intensity of PCR products (3-actin in each case was calculated.4. Data analysisAll values are expressed as the mean±S.D. and the significant levels between two groups were assessed by t-test with SPSS 13.0 for Windows. P values less than 0.05 were considered to be statistically significant.Results1. ART significantly decreased the growth of the two cell lines in a concentration-and time- dependant manner by the MTT assay. The SW480 cell line can be inhibited 53% by 40ug/ml ART after 48h. The other cell line SW620 can be inhibited 52% by 40ug/ml ART respectively.2. ART significantly increased the proportion of apoptosis in colon carcinoma cells compared to the controls(P<0.05). The proportion of apoptotic SW480 cells after treatment with ART at 20 and 40ug/ml was up to 8.6+1. 09% and 11.49+0. 61%, and the proportion of apoptotic SW620 cells was up to 6.16+0. 57% and 13.08± 1.06% respectively.3. The results of RT-PCR showed fart the expression of VEGF-mRNA decreasedafter the ART treatment compared to the control group.There were significantdifferences among the cells treated with different concentration.ConclusionART can effectively inhibit the proliferation of SW480 and SW620 cells in vitro, which may account for induction of the apoptosis and reduction in VEGF expression.