Effect Of SLC22A18 Over-expression On Sensitivity To Chemotherapeutic Agents In Human Breast Cancer Of MCF-7

Chemotherapy is one of the important means of the treatment to breast cancer,however,quite a few cases of chemotherapy failed because tumor cells have developed Multidrug Resistance(MDR) to chemotherapeutics.Thus,the researches on the emergence mechanisms of MDR have great clinical significance of improving the curative effeciency of the breast cancer chemotherapy.Gene SLC22A18 is a newly discovered candidate anti-oncogene.The proteins encoded by this gene,which is similar to transmembrance transporter,may influence the sensitivity of chemotherapuetics,the metabolism and growth of cells,and probably have some effect on the development of breast cancer MDR.Gene SLC22A18,also called IMPT1/BWR1A/TSSC5,is a patrilineal imprinting gene.The occurrence and development of many cancer cases have been proved to be related to the abnormal expression of imprinting genes.Researches in recent years have demonstrated that the loss of gene expression,the mutation of SLC22A18,or the abnormal gene-imprinting are relevant to the occurrence and development of many kinds of tumors.The proteins encoded by SLC22A18 may probably have effect on the excretion of chemotherapuetics inside tumor cells, and lead the tumor cells to develop MDR,because these proteins have ten transmembrance areas which illustrates the homology between these proteins and transmembrance transporter proteins.The so-called classical MDR mechanism is that high expression of transmembrance transporter proteins leads to rise of excretion of intracellular drugs,and decay of intracellular drug accumulation.The major role in the classical MDR mechanism is played by the expression of a large class of genes called ATP-binding cassette(ABC) superfamily.The common point of ABC proteins is that all of them have a ATP-binding cassette structure, which can pump specific substrates out of cells by binding and hydrolyzing ATP in the structure.Many researches have shown that some members of the ABC superfamily play an important role in the occurrence of MDR in tumor cells, especially in breast cancers.These genes or proteins related to MDR include P-gp,multi-drug resistance protein 1(MRP1),breast cancer resistance protein (BCRP),lung resistance related protein(LRP),etc..It is speculated that SLC22A18,belonging to the same transmembrance transporter protein family as P-gp,has something to do with drug resistance of many chemotherapuetics in tumor cells similar to P-gp.In this research,we increased the expression of SLC22A18 gene in MCF-7 cells by transient transfection,detected the effect on the sensitivity of chemotherapuetics of MCF-7 cells by CCK-8 method,and used flow cytometry to study the effect of the expression on cell cycle and adriamycin accumulation inside the cells.Partâ… Construction of eukaryotic expression plasmid PIRES2-EGFP-SLC22A18Objective To obtain the full-length gene fragment of SLC22A18 from organization,and to construct the eukaryotic expression plasmid pIRES2-EGFP-SLC22A18.Methods To design the restriction site containing primers,and to obtain the full-length gene fragment of SLC22A18 from organization by RT-PCR.The gene fragments were amplified by PCR and connected to vector pIRES2-EGFP which was digested by restriction enzymes.The recombinant plasmid pIRES2-EGFP-SLC22A18 was identified by electrophoresis and sequencing.Results The full-length gene fragment of SLC22A18 was obtained from organization and connected to vector pIRES2-EGFP successfully.The correctness of SLC22A18 fragment was proved by electrophoresis and sequencing.Partâ…¡Up-regulation of SLC22A18 expression on breast cancer MCF-7 cellsObjective To up-regulate the expression of SLC22A18 on breast cancer MCF-7 cells,and to identify the over-expression of SLC22A18.Methods The plasmids were amplified by transfecting the competent cells. The concentration and purity of plasmids solution were detected by UV spectrophotometer.The recombinant plasmid pIRES2-EGFP-SLC22A18 was transfected into MCF-7 cells by Lipofectamine2000 to up-regulate the SLC22A18 expression.The efficiency of transfection was identified by the inverted fluorescence microscope.The mRNA expression of SLC22A18 gene in MCF-7 was detected by Real-time RT-PCR.Results The plasmid solution of high concentration and purity was obtained. The transfection efficiency of pIRES2-EGFP-SLC22A18 was over 40%,while which of pIRES2-EGFP was about 20%-30%.Real-time RT-PCR showed that the relative expression of SLC22A18 mRNA in MCF-7 cells was 26.85(18.38-39.21) after transfection of pIRES2-EGFP-SLC22A18,which means the transfection increased the gene's expression by 26.85 times compared to the control group.Partâ…¢Effects of SLC22A18 over-expression on sensitivity to chemotherapeutic agents in breast cancer cell MCF-7Objective To study the effects of SLC22A18 over-expression on sensitivity to paclitaxel,adriamycin,cyclophosphamide,mitoxantone and DDP in human breast cancer cell MCF-7.Methods The sensitivity to chemotherapeutic agents of MCF-7 cells was studied with CCK-8 assay.Results CCK-8 assay showed that the over-expression of SLC22A18 reduced the sensitivity while increasing the IC₅₀ to paclitaxel,cyclophosphamide,and mitoxantone respectively in MCF-7 cells,but increased the sensitivity and decreased the IC₂₀ to DDP.The sensitivity and IC₅₀ to adriamycin was not changed significantly.Compared to control group,the IC₅₀ to adriamycin was raised from 1.82±0.16mg/L to 2.11±0.30mg/L(P=0.22),the IC₅₀ to paclitaxel was raised from 5.38±0.63mg/L to 7.36±0.89mg/L(P＜0.05),the IC₅₀ to cyclophosphamide was raised from 1160.80±167.73mg/L to 1736.73±217.34(P＜0.05),the IC₅₀ to mitoxantone was raised from 5.76±0.52mg/L to 7.68±0.53mg/L(P＜0.05);the IC₅₀ to DDP was decreased from 3.35±0.45mg/L to 2.08±0.25mg/L(P＜0.05). Partâ…£Effects of SLC22A18 over-expression on intracellular accumulation of adriamycin in breast cancer cell MCF-7Objective To study the Effects of SLC22A18 over-expression on intracellular accumulation of adriamycin in breast cancer cell MCF-7.Methods Flow cytometry was used to analysis the adriamycin fluorescence intensity in MCF-7 cells with SLC22A18 over-expression.Results FCM showed that the relative fluorescence intensity of adriamycin in MCF-7 cells with SLC22A18 over-expression was 38.31±1.93,while which of the control group was 44.04±2.60.The results showed that the accumulation of adriamycin in MCF-7 cells with SLC22A18 over-expression was significantly decreased compared to control(P＜0.05).Partâ…¤The proliferation of breast cancer MCF-7 cells after transfection of SLC22A18Objective To research the effect of over-expression of SLC22A18 on the proliferation of breast cancer MCF-7 cells.Methods The cell proliferation was observed by inverted microscope and CCK-8 method.FCM was used to detect the cell cycle of MCF-7 cells after transfection.Results The down-regulation of MCF-7 cells proliferation ability was observed by inverted microscope.CCK-8 method showed that the absorptance of MCF-7 cells 48h after pIRES2-EGFP-SLC22A18 transfected was 0.72±0.02,while which of pIRES2-EGFP transfected cells was 0.83±0.04.FCM showed that the over-expression of SLC22A18 in MCF-7 cells led to an increase of G₀/G₁ cell population(59.71%to 56.03%,P＜0.05),and a decrease of S cell population(37.20%to 40.30%,P＜0.05) and G₂/M cell population(3.08%to 3.67%, P＜0.05) compared to control,which showed that SLC22A18 may inhibit the proliferation of cancer cells.ConclusionIn this research,we constructed the eukaryotic expression plasmid pIRES2-EGFP-SLC22A18 and transfected it into MCF-7 cells by Lipofectamine2000 to up-regulate the expression of SLC22A18.In this way we studied the effect of SLC22A18 over-expression on the proliferation and the sensitivity to chemotherapeutic agents of MCF-7 cells.The study showed that the over-expression of SLC22A18 in MCF-7 cells may lead to a down-regulation of cell proliferation,and it also brought a decrease of the sensitivity to paclitaxel,cyclophosphamide,and mitoxantone respectively in MCF-7 cells, but increased the sensitivity to DDP.The intracellular adriamycin accumulation was also decreased in the MCF-7 cells with SLC22A18 over-expression.The characteristic of MDR led by SLC22A18 was similar to that led by the members of ABC family,which supported that the protein encoded by SLC22A18 may have a transmembrane transporter like function,and the gene may play a role in the development of MDR in breast cancers.In this study we showed direct evidence to the inhibition function of SLC22A18.For the first time the various and representative specific chemotherapeutics were researched on MDR in breast cancer led by SLC22A18, which supplied the clinical treatment direct directing information.For the first time we researched the MDR mechanism of SLC22A18,and proved that the SLC22A18 over-expression may lead to an excretion of adriamycin in cells.