Establishment Of A Human Lung Adenocarcinoma Drug-resistance Cell Line Anip973/NVB And Investigation On Mechanism Of Drug Resistance

Objective To establish a human lung adenocarcinoma multidrug-resistant cell lineAnip973/NVB and to study its biologic characteristics and mechanism of drugresistance.Methods The human lung adenocarcinoma multidrug-resistant cell line(A973/NVB) was established by inducing human lung adenocarcinoma cell lineAnip973 with step-wise increasing concentrations of NVB,and the biologiccharacteristics were observed.cDNA microarray technique was used to detect genedifference of the two cell lines.The results of microarray were validated and theclassics drug resistance related genes were detected by RT-PCR.2-DE techniquewas used to analyze the difference of proteins of the two cell lines.Results 1.The human lung adenocarcinoma multidrug-resistant cell lineAnip973/NVB was established successfully for 24 months.2.Its biologiccharacteristics were:(1) The cell morphous of A973/NVB cells were more irregularthan A973 cells,and there was significant difference in ultramicrostructure whileobserved by the light microscope and electron microscope.(2) The rate of cellproliferation of Anip973/NVB was no significantly different with that of Anip973cell line.(3) Cell cycle distribution of Anip973/NVB had changed compared withparental cells from flow cytometry.The percentage of cells in G₀-G₁ phase increasedto 62.90%,while the percentage of cells in S phase decreased to 28.32%.Thepercentage of Anip973 in G₀-G₁ and S phase were 53.73 %,38.25 % respectively,and there was no changes in G₂-M phase of the two cell lines.(4) The IC₅₀ of NVB inAnip973/NVB cells was 21.18 times than that of Anip973 cells by MTT.In addition, the Anip973/NVB showed various cross-resistance to CDDP,isofosfamide,pharmorubicin,5-fluorouracil,dacarbazine and etoposide,and the resistance indexeswere 11.89,11.68,6.12,3.95,2.86,2.64 and 2.15,respectively.The ceils remainedsensitive to irinotecan and gemcitabine and the sensitive resistance indexes were 1.38and 1.27 (p＜0.05).(5)Medicine concentration in drug resistance cells was obviouslylower than that in parental cells by high efficiency liquid chromatography.3.The resultsof gene arrays showed that 143 genes of 21571 genes were differentially expressedbetween Anip973 and Anip973/NVB cell lines,of them,55 genes showed＞2 timesup-regulation,88 genes showed＜0.5 times down-regulation and the function ofseventy-seven genes had been identified.21 genes among identified genes had beenidentified function related to cancer,and,of 21 genes,the changes of ANXA1,ABCC3,TIMP-3 and cytoadherence related genes might be the major mechanism ofdrug resistance of NVB.4.The results by RT-PCR showed MRP3 and Bcl-2 wereoverexpressed in Anip973/NVB,and there were no significant expression of MDR,MRP1,ABCG2,GSTÏ€1 in two cell lines.5.Well-resolved,reproducible 2-DEpatterns of control and NVB-treated Anip973 were obtained.In Anip973/NVB group,average spots of 3 gels were 806 with an average matching rate of 77%,in Anip973group,average spots of 3 gels were 711 with an average matching rate of 78.33%.37significantly expressed protein spots were detected,among which,16 wereup-regulated and 21 were down-regulated in the drug-resistance group.17 spotswere identified initially with the data bank.Conclusions 1.A973/NVB is a reliable multi-drug resistant cell line of humanlung adenocarcinoma which has the basic biologic characteristics.2.Up regulationof ANXA1 and ABCC3 result in depression of intra-cellular drug concentration andchange of intra-cellular drug distribution,which may be the major mechanism ofdrug resistance of NVB.Down regulation of cell infiltration and metastasis relatedgenes result in better of cell differentiation and elusion of attack fromchemotherapeutics.3.Overexpression of ABCC3(MRP3) may be one of the major mechanism of MDR,that is consistent with the results of gene arrays.Overexpression of Bcl-2 inhibits apoptosis of drug resistance cells which is one ofthe drug resistance cause.4.Difference of chemokine-like factor superfamily 2,NME2 protein,Cytochrome P450,TIMP-4,laminin receptor,Calumenin,and so on,in the two cells shows NVB affects lung adenocarcinoma cell differentiation andproliferation,and the proteins may play a role in NVB resistance.