Doxorubicin Induces Drug Resistance In Human Breast Cancer MCF-7 Cells And A New CD44st Expression Via NF-κB

Multidrug resistance cell line cells(MCF-7/Adr) were obtained from the human breast cancer cell line cells MCF-7 induced by doxorubicin. During inducing process, the expression of MDR-1 and CD44 st were detected to investigate the relationship between the acquired multidrug resistance induced by doxorubicin and the expression of the gene as well as protein related to tumor invasiveness. 2. During the process of inducing multidrug resistance, the expression of NF-κB transcription factor were observed to explore the regulated effect on the expression of MDR1 and CD44 st. 3.In order to investigate the influence of NF-κB specific inhibitor on HA inducing tumor invasion ability, the induced resistance model were pretreated with NF-κB specific inhibitor followed with hyaluronan(HA) for detecting the expression of MMP-2 ﹑ MMP-9 and the change of the invasion ability of the cells.Methods: The inhibition of doxorubicin at various concentrations on the growth of human breast cancer cell line MCF-7 was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, so as to investigate the appropriate doxorubicin concentration for the induction of drug resistance. The expression of the multidrug resistance protein 1(MDR1), nuclear factor κB(NF-κB), CD44 st, matrix metalloproteinase-2(MMP-2) and MMP-9 m RNA was detected using quantitative real-time polymerase chain reaction(q RT-PCR) and semi-quantitative reversetranscription PCR, and the gene sequence of the standard CD44(CD44st) was confirmed by sequencing. The percentage of P-glycoprotein(P-gp)-positive MCF-7 cells was detected using flow cytometry(FCM), and P-gp activity was assessed using a Rhodamine-123(Rho-123) retention assay. Western blotting analysis was performed to determine the expression of CD44 and NF-κB protein, and the NF-κB DNA-binding activity was measured using an electrophoretic mobility shift assay(EMSA). The changes of the MMP-2 and MMP-9 expression in the cell culture supernatants of MCF-7 cells weredetected using gelatin zymography, and the changes of the invasive ability of MCF-7 cells following doxorubicin treatment were measured using a Transwell invasion assay.Results: The 50% inhibitory concentration(IC50 value) for doxorubicin against MCF-7 cells was 0.68 ± 0.04 μg/ml. Treatment with doxorubicin at concentrations of 0.01,0.03, 0.06 and 0.12 μg/ml for 7 d significantly inhibited the growth of the MCF-7 cells as compared to those without doxorubicin treatment(P < 0.05). The MCF-7 cells treated with doxorubicin at concentrations of 0.03 and 0.06 μg/ml for 7 d only developed mild swelling,with a good cell state, while the cells treated with 0.12 μg/ml doxorubicin for 7 d exhibited a poor state and obvious swelling, with apparent cell debris seen. Therefore, 0.06 μg/ml doxorubicin was employed for the subsequent induction of drug resistance. The expression of MDR1, CD44 st and NF-κB m RNA was low in MCF-7 cells, and the m RNA expression of these genes gradually increased in the MCF-7 cells at a dose-dependent manner following treatment with doxorubicin at concentrations of 0.01, 0.03 and 0.06 μg/ml. There were significant differences in the expression of MDR1, CD44 st and NF-κB m RNA between the doxorubicin-treated MCF-7 cells and the untreated cells(P < 0.05).Sequencing revealed that the CD44 st gene sequence was consistent with the sequence of gene(Gen Bank accession number: FJ216964) we identified previously. The percentage of P-gp-positive MCF-7 cells was significantly different from those of P-gp-positive doxorubicin-treated MCF-7 cells(P < 0.05). Rho-123 retention assay revealed that the intracellular fluorescence intensity gradually reduced with the elevation of P-gp expression(P < 0.05). Western blotting analysis revealed that the CD44 and NF-κB protein expression coincided with the CD44 and NF-κB m RNA expression, and EMSA showed that the NF-κB DNA-binding activity enhanced in a dose-dependent manner with the increasing concentration of doxorubicin. RT-PCR and gelatin zymography showed that HA induced the production of MMP-2 and MMP-9 in MCF-7 cells treated with 0.06 μg/ml doxorubicin,which could be blocked by the specific NF-κB inhibitor BMS-345541. Transwell invasion assay revealed that HA increased the invasive ability of the MCF-7 cells treated with 0.06μg/ml doxorubicin, which could be blocked by BMS-345541.Conclusions: MCF-7 cells subjected to drug pressure may develop multidrug resistance to doxorubicin, and the expression of MDR1, CD44 st and NF-κB m RNA and protein is gradually up-regulated in a dose-dependent manner in MCF-7 cells treated with doxorubicin. HA increases the secretion of MMP-2 and MMP-9 in multidrug resistantMCF-7 cells and affects the invasive ability of MCF-7 cells through the up-regulation of CD44 st expression, and such an effect can be blocked by the specific NF-κB inhibitor BMS-345541.