| Background and ObjectiveMalignant Lymphoma(ML),including Hodgkin’s Lymphoma(HD)and non-hodgkin’s Lymphoma(NHL),is a set of derived from the hematopoietic stem/progenitor cells malignant clone disease,come on lymph nodes or outside lymph nodes of lymphatic tissue or organ primarily,the Chinese more occur NHL.NK/T cell lymphoma is derived from T cell NHL,it has high-grade invasive,short term of the disease,poor curative effect,high mortality,it is given priority to with radiation and chemotherapy,but Adriamycin(ADM)on the basis of CHOP scheme is not sensitive for NK/T cell lymphoma treatment.Gemcitabine(nucleoside-derived drugs)is chemotherapy regimens of basic medicine in the treatment of all kinds of solid tumors(such as pancreatic cancer and bile duct cancer and breast cancer,etc.),and have remarkable clinical curative effect.In recent years,on the basis of gemcitabine chemotherapy scheme applied in NK/T cell lymphoma in patients,the clinical treatment effect is significantly better than no gemcitabine chemotherapy regimens,but there are still a part of NK/T cell lymphoma patients appear drug resistance phenomenon.But in solid tumors and lymphoma in clinical research,the determinants of sensitivity and/or resistance to gemcitabine is not yet clear.Cell metabolism is the steps necessary to play a role of the toxicity ofnucleoside-derived drugs,but the premise is a drug must first complete its transport across the membrane,this step is essential to achieve its effectiveness for nucleosidederived drugs.Therefore,the study of membrane transporters is becoming more and more in recent years,research datas suggest that drug effects play without protein mediated drug transport,its transfer efficiency directly determine the therapeutic effect of drugs.hENT1(human equilibrium nucleoside transporter 1)is one of research focuses of membrane transporters in recent years,the main research direction locates in the mediated antitumor and antiviral drugs.At present,the solid tumor research thought,gemcitabine across plasma membrane is an almost entirely mediated process by hENT1,and it is determinants of sensitivity to gemcitabine,but no related research to adriamycin.hENT1(SLC29A1)is the first member found in SLC29 family(the solute carrier 29)in nucleoside transport.hENT1 is the main carrier across the membrane to cells absorb and(or)output of physiological nucleosides and a variety of anticancer and antiviral drugs.According to the NBTI/NBMPR characteristics,hENT1 is divided into NBMPR sensitive type(es)and NBMPR insensitive type(ei).In recent five years,the relationship between hENT1 and anti-cancer drug mostly focused on: a high expression of hENT1 in tumor tissue specimens is associated with the long-term survival of cancer patients;assess the sensitivity and resistance of nucleoside-derived drugs through hENT1 expression in the tumor patients;hENT1 maybe act as therapeutic targets in the tumor patients.most solid tumor research data shows that nucleoside-derived drugs(e.g.,gemcitabine)mediated by hENT1 implement effective drug transport across the membrane,maybe act as one of the biomarkers of cancer patients;but some studies have hold different views on this.So the value of hENT1 as biomarkers in the treatment of solid tumor patients in nucleoside-derived drugs remains controversial.Pub Med search results show the study of hENT1 in protein level and gene level is not much,basic research are more than clinical research,clinical research more concentrated in pancreatic cancer and bile duct ampulla carcinoma.Pub Med,CNKI and Wanfang data failed to retrieve relevant literature data on hENT1 associated with gemcitabine and adriamycin in NK/T lymphoma YTS cell lines.Using the CCK 8 technology,real-time fluorescent quantitative PCR and Western Blot technique,the experiments in this paper explored functional properties of hENT1 in NK/T cell lymphoma YTS cell lines,and tested the correlation between hENT1 and cytotoxic effect of gemcitabine and adriamycin.Materials and Methods1.SubjectsHuman equilibrium nucleoside transporter 1(hENT1,SLC29A1)in NK/T cell lymphoma YTS cell lines.2.Research methodsThis study use CCK 8 technology,real-time fluorescent quantitative PCR and Western Blot technique,detecting the correlation between membrane transporters hENT1 and the cytotoxic effects of gemcitabine and adriamycin in NK/T cell lymphoma YTS cell lines,study functional properties of hENT1 in the level of gene and protein.3.CCK-8 detectionUsing CCK 8 technology,combined with NBMPR(specific small molecule inhibitors of hENT1),study correlation between hENT1 and cytotoxic effects of gemcitabine and adriamycin in NK/T cell lymphoma YTS cell lines,and explore functional properties of hENT1.4.Real-time fluorescent quantitative PCR detectionBy real-time fluorescent quantitative PCR technology,with the correlation between hENT1 and cytotoxic effects of gemcitabine and adriamycin in NK/T cell lymphoma YTS cell lines,study the expression changes of human NK/T cell lymphoma YTS cell lines membrane transporters hENT1 at the genetic level.And apply the techniques tested the expression of hENT1 at the level of gene in lymphoma YTS,SNK-6,Jeko-1,ly1,Raji,Karpas and Jurket cell lines.5.Western Blot assaysUsing Western Blot technique,detect expression of hENT1 at the level of protein in lymphoma YTS,SNK-6,1,ly1,Raji,Jeko-the Karpas and Jurket cell lines.6.Statistics analysisStatistical package(SPSS 17.0)was used for processing data.Continuous normally distributed variables were presented as mean ± standard deviation(SD),and were compared using either the t test or One-way analysis of variance.Non-normally distributed variables were presented as medians(ranges),and were compared using rank sum test.Chi-square test was used to verify Hardy-Weinberg equilibrium.The classified variables were analyzed using Chi-square test,Fisher exact test or rank sum test.The detection level is considered statistically α=0.05.Results1.Expression of hENT1 in YTS、SNK-6、Jeko-1、ly1、Raji、Karpas and Jurkat cell linesQ-RT-PCR detection results showed that expression of hENT1 in YTS,SNK-6,Jeko-1,ly-1,Raji,Karpas and Jurkat cell lines exist,lower expression exist in YTS and Jeko-1,expression of the rest is higher.Western Blot test results show that: hENT1 in YTS,SNK-6,Jeko-1,ly1,Raji,Karpas and Jurkat cell lines,expression are inequality.2.hENT1 functional detection in NK/T cell lymphoma YTS cell linesIn gemcitabine,adriamycin group,with the extension of drug effect time their cytotoxicity enhanced obviously,the cytotoxic effects of gemcitabine and adriamycin on time dependence exists;but after joining NBMPR in advance,firstly,the cytotoxic effects of gemcitabine and adriamycin decreased significantly,even doesn’t work,secondly,the cytotoxic effects of gemcitabine and adriamycin on time dependence do not exist.NBMPR+drug group and simple drug group,regardless of gemcitabine or adriamycin,comparing differences between the two groups have statisticalsignificance(P<0.05).Low concentration NBMPR(5 nm/L,10 nm/L and 20 nm/L)can be diminished significantly the cytotoxic effects of gemcitabine and adriamycin.3.The correlation research between hENT1 and cytotoxic effects of gemcitabine in NK/T cell lymphoma YTS cell linesCCK 8 results showed: 1)IC50 value of gemcitabine for YTS cells is 25.63nM/L;2)cytotoxic effects of gemcitabine for YTS cells enhance with the extension of time and increased drug concentration obviously.Comparing differences between different times and different concentration groups were statistically significant(P<0.05,P<0.05);3)after add NBMPR,the toxic effects of gemcitabine change significantly;With the extension of action time,its cytotoxicity hardly appeared.NBMPR+drug group and simple drug group,comparing differences between groups was statistically significant(P<0.05).Q-rt-PCR test results shows the gene expression of hENT1 in YTS cells.Different concentrations of gemcitabine after 3 hours,6 hours,compare the difference between groups was statistically significant(P<0.05);But the 3 hours,12 hours comparative differences between groups have no statistical significance(P>0.05);Namely,with gemcitabine to extend the time,increase concentration,hENT1 gene significantly increased in 6 hours;but then the increasing trend began to decline.4.The correlation research between hENT1 and cytotoxic effects of adriamycin in NK/T cell lymphoma YTS cell linesCCK 8 results showed: 1)IC50 value of adriamycin for YTS cells is 400 nM/L;2)cytotoxic effects of adriamycin for YTS cells enhance with the extension of time and increased drug concentration obviously.Comparing differences between different times and different concentration groups were statistically significant(P<0.05,P<0.05).3)after add NBMPR,the toxic effects of adriamycin change significantly;With the extension of action time,its cytotoxicity hardly appeared.NBMPR+drug group and simple drug group,comparing differences between groups was statistically significant(P<0.05).Q-rt-PCR test results shows the gene expression of hENT1 in YTS cells.Different concentrations of adriamycin after 3 hours,6 hours and 12 hours,comparing the difference between groups was statistically significant(P<0.05,P<0.05);Namely,the gene expression of hENT1 increased with extended time and increased concentration of adriamycin.Conclusions1.hENT1 may be the main channel of protein mediated gemcitabine,adriamycin into YTS cell lines.2.When gemcitabine role in YTS cell lines,expression of hENT1 at the level of gene increased with the extension of time,increased concentration;rising trend in 6 hours began to decline;When adriamycin effects in YTS cell lines,expression of hENT1 at the level of gene increased with the extension of time,increased concentration;rising trend in 12 hours or so are still rising.3.Low concentration of NBMPR can obviously inhibit cytotoxic effects of gemcitabine,doxorubicin in YTS cell lines,hENT1 is NBMPR sensitive type.4.Expression of hENT1 in lymphoma YTS,SNK-6,Jeko-1,ly1,Raji,Karpas and Jurkat cell lines exist,but uneven expression,probably as a new target of lymphoma treatment. |
PDF Full Text Request