Study On The Relationship Between Polylactosamine-type N-glycans And The Drug Resistance Of Colon Cancer Cells SW620

Objectives5-fluorouracil (5-FU) is most commonly used in clinical treatment of colon cancer.We aim to investigate the expression variation of polylactosamine, simultaneously studythe expression changes in the mRNA and protein level of glycosyltransferases β3GnT8after colon cancer cells SW620treated with5-FU. To futher clarify the relationshipbetween polylactosamine or glycosyltransferases β3GnT8and the5-FU resistance ofcolon cancer cells SW620,5-FU resistant SW620cells were used as research object andto detect the expression levels of polylactosamine and glycosyltransferases β3GnT8.Preliminarily we detect whether polylactosamine or glycosyltransferases β3GnT8isrelated to the drug resistance of colon cancer cells SW620.Methods1. After SW620cells treated with5-fluorouracil (5-FU), MTT assay was used todetect IC50of SW620cells; The nuclear fluorochrome Hoechst33258and flowcytometry were used to measure cell apoptosis; Flow cytometry was used to detect thecell cycle at the same time; The expression level of polylactosamine in the SW620cellstreated with5-FU was tested by flow cytometry and lectin blot. After the SW620cellstreated with5-FU, alkaline phosphatase assay kit was used to detect alkalinephosphatase activity which has been used to monitor the differentiation. Also theWestern-blot detection of glycosyltransferase β3GnT8and CD147protein levelsexpression was used after the SW620cells treated with5-FU. 2.5-FU was added to SW620cells and the concentrations of5-FU were stepwiselyincreased to develop a drug resistant cell subline. MTT assay, Real-time PCR, apoptosisassay were used to identify the5-FU resistant SW620cell subline (SW620/5-FU); Theprotein expression level of CD133which has been used to monitor the cancer stem cellswas detected by Western-blot; Flow cytometry and Lectin blot were used to test theexpression level of polylactosamine in the SW620/5-FU.Results1. The apoptosis and differentiation of SW620treated with5-FU increasedobviously. A significant increase in the percentage of SW620cells in S phase and adecrease in G0/G1and G2/M phases following incubation with5-FU were both detected.These phenomenons were time dependent. The expression levels of glycosyltransferaseβ3GnT8and CD147protein were increased obviously.2. In the5-FU resistant cell SW620/5-FU, the expression level of P-gp mRNA, theprotein expression levels of glycosyltransferase β3GnT8, CD147and polylactosamineand the degrees of apoptosis were all increased obviously compaired with colon cancercells SW620.Conclusion1.5-FU has a role in the regulation of apoptosis,5-FU can induce apoptosis inSW620treated with5-FU.5-FU caused a significant increase in the percentage ofSW620cells in S phase and a decrease in G0/G1and G2/M phases.2.5-FU can decrease the synthesis of polylactosamine and the expression levels inprotein of glycosyltransferase β3GnT8and CD147. In conclusion, the present studyindicates that polylactosamine may be a potential target that contributes to the5-FUresistance in colon cancer cells. Thus it may be a novel drug target candidate for thetreatment of colon cancer.