The Reversal Effect Of Mechanism Of Compound WS-10 On ABCB1-mediated Drug Resistance In Colon Cancer Cells

The drug resistance and multidrug resistance(MDR)of tumor cells is an important factor leading to the failure of chemotherapy,which seriously affects the efficacy of clinical chemotherapy.There are many reasons which resulting in the development of multidrug resistance.And the ABC transporter family(ATP-bindingcassette transporter,ABC)has a transport effect on chemotherapeutic agents,which may be an important factor that caused the occurrence and development of multidrug resistance(MDR).ATP-Bonded box transporter B-family member 1(ABCB1),a member of ABC Transporter,is a crucial reason for the emergence of multidrug resistance as well.Based on this,it is possible to reverse the multidrug resistance of chemotherapeutic drugs with ABCB1 as substrate by finding compounds that not only inhibit ABCB1 protein with highly effective but also low in cell toxicity.Therefore,it is the key to search for selective ABCB1 inhibitors.This study explored the reversal ability of compound WS-10 to multidrug resistance of drug-resistant tumor cells.It also studied the mechanism of compound WS-10 reversing drug resistance.The specific research contents are as follows:1.MTT assay to detect the cytotoxicity of compound WS-10 to colon cancer cells,lung cancer cells,esophageal cancer cells,gastric cancer cells and other cancer cell lines.When the concentration of compound WS-10 was 20μM,the inhibition rate of cell proliferation was less than 20%,which suggested that the compounds had no cytotoxicity to the above several cell lines.2.Use of colon cancer cell SW620 and its selected drug-resistant cell line SW620/Ad300 as the basis.The drug-resistance reversal effect of paclitaxel,doxorubicin and cisplatin was studied by MTT assay with compound WS-10 in non-toxic concentrations.paclitaxel and doxorubicin are ABCB1 as substrates of chemotherapeutic drugs.And chemotherapy cisplatin is not ABCB1 substrate of chemotherapeutic drugs,can be used as a control.The results showed that the compound WS-10 could reverse the drug-resistant effect of paclitaxel and doxorubicin in the ABCB1 as substrate,and the compound had no reversal effect on the chemotherapeutic drugs of ABCB1 substrate.3.Further we use the tool cell HEK293 T and lentivirus infection techniques to establish an ABCB1-overexpressing cell line HEK293T/ABCB1.Using the HEK293T/ABCB1 cell line Repeat the above MTT assay.The experimental results are the same as those of SW620 and SW620/Ad300,which further proves that the compound WS-10 may be the effect of reversing the drug resistance by influencing the protein ABCB1.4.Observing the morphological changes of drug-resistant cell SW620/Ad300 after adding paclitaxel and WS-10 we found that after adding paclitaxel or paclitaxel with WS-10,the morphological changes were observed.We also found that the shape of the polygon was transformed from the neatly arranged and the size rules to the round with irregular order and size.However,the cell morphology did not change significantly before and after the addition of WS-10 alone.5.Using ultra-high performance liquid chromatography(UPLC)to detect intracellular paclitaxel accumulation when parental cell line SW620 and its drug-resistant cell line SW620/Ad300 with or without a certain concentration of WS-10 incubation 72 h with adding the same concentration of paclitaxel for the same time.The accumulation of paclitaxel in drug-resistant cells incubated by compound WS-10 was significantly higher than that of drug-resistant cells without WS-10 incubation.This proves that WS-10 can influence the function of ABCB1 to increase the accumulation of paclitaxel in the cell,obtaining the effect of reversing drug resistance.6.Using flow cytometry technique to detect the effect of compound WS-10 on drug efflux in resistant cells.The results showed that compound WS-10 could decrease the efflux of chemotherapeutic drugs in ABCB1 high expression cells.This indicated that compound WS-10 might influencing the function of ABCB1 to reduce intracellular chemotherapeutic drug efflux,achieving the effect of reversing drug resistance.7.Detected by the Western blot technique,the expression of ABCB1 protein was not significantly altered with different concentration and time of compound WS-10 treatment.Detected by Q-PCR technique,the compound WS-10 could not change the expression level of ABCB1 m RNA.It is indicated that the effect of compound WS-10 reversal resistance is not achieved by altering the express level of ABCB1 protein or m RNA.8.The protein location of ABCB1 were detected by immunofluorescence technique.Detecting the protein location of ABCB1 after different concentrations of compound WS-10 were treated with parental cell line SW620 and its drug-resistant cell line SW620/Ad300 for 72 h.The results illustrated that compound WS-10 did not change the protein location of ABCB1.9.The cellular thermal shift assay(CETSA)was used to be a tool for validation of drug target engagement.After the ABCB1 protein incubated with or without the compound WS-10,with different temperatures for the same time,the amount of protein was detected by Western blot assay.Therefore,the results demonstrate that WS-10 binding to ABCB1 and it would be the important reason of WS-10 has reversal effect.This study found that pyrimidine and triazole compounds WS-10 as selective ABCB1 inhibitors,which can reverse the ABCB1 mediated tumor cell resistance in a non-toxic concentration.We further study the mechanism of compound WS-10 reversal of drug resistance,the results showed that WS-10 reverse the drug resistance by inhibiting the ABCB1 transfer pump,without affecting the expression of ABCB1 protein.Further study,we found that compound WS-10 can be combined with ABCB1 protein to increase the accumulation of chemotherapeutic drugs in cells,so as to improve the sensitivity of drug-resistant tumor cells to chemotherapeutic agents and to produce reverse drug resistance.The results show that compound WS-10 may open a new breakthrough point for the treatment of drug-resistant tumors with high ABCB1 protein expression,and provide a new research plan and model for solving multidrug resistance of tumor.