Resistance In Tumor Cells (MCF-7/adr) By Macrocyclic Lactone Compounds

Cancer is one of the important causes of human death at present, and chemotherapy plays a very important role in cancer treatment. But chemotherapy drugs are difficult efficacy or even invalid because the tumor cells produce multi-drug resistance (MDR) during the progress of clinical chemotherapy. Therefore, the successful reversal of MDR could improve the effects on the patients. So far the drugs reversing MDR have been researched, but they have the side effects or the limited efficacy, there is not still an ideal reversal drug. In recent years, with more and more extensive applications of macrolide drugs, and they have no toxic side effects, well efficacy and structural features, they may be an ideal MDR reversal agents. In this study, we chose the five kinds of macrolides to investigate the effects on reversing to the tumor cells resistance to adriamycin in human breast cancer cell line (MCF-7/adr) in vitro and in vivo, and the corresponding mechanisms, to develop an effective tumor MDR reversal drugs.In vitro study, firstly, we established the MCF-7/adr cell line with increasing concentrations of adriamycin (ADR) gradient and the high concentration gradient way by repeating intermittent administration of adriamycin, then tested the resistance index, and evaluated the efficacy of doramectin, milbemycin A4, nemadectin, milbemycinβ14 and secomilbemycin D overcoming MDR of MCF-7/adr cells via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] approach. Secondly, we detected whether the five drugs blocked drug pump function of P-gp by detected the dose of adriamycin and Rh123 since they are good P-gp substrates with an autofluorescence capacity. We also employed the RT-PCR and immunofluorescence flow cytometry to check the expressions of MDR 1 and MRP 1 mRNA and P-gp. Finally, before the experiment in vivo, we measured the effect of tested drugs on CYP1A2 (one of P450 enzymes) activity and the toxic effect on the normal human fibroblasts and peripheral blood mononuclear cells. Then we established human cancer MCF-7/adr xenograft in nude mice, and selected doramectin, the best performing drug in vitro, as experimental agent in vivo. Combining with ADR, we tested the mean surviving time (MST) of xenografts nude mice and the volume of the transplanted tumor.The results showed that the resistance index of MCF-7/adr cell line resistant is up to 61 times in vitro. And our findings also demonstrated that 5μM doramectin, milbemycin A4, nemadectin, milbemycinβ14 and secomilbemycin D could reverse the MDR, and fold-reversal was 28.93,22.23,16.64,13.50 and 10.59, respectively, and they were better than VRP on reversing MDR. In addition, the five drugs in the experimental groups, especially doramectin with the highest hydrophobic, could concentration-dependent enhance the accumulation of intracellular adriamycin and Rh123, but did not affect that of parental MCF-7 cells (P<0.05). In the efflux assay, these compounds could inhibit the efflux of Rh123 from treated MCF-7/adr cells in a time-dependent manner. At the same time, The data showed that doramectin, milbemycin A4 and nemadectin could decrease the expressions of MDR 1 gene, MRP 1 gene and P-gp protein (P< 0.05). For the future study, our results showed that low concentrations of tested drugs had no effect on the CYP1A2 metabolic activity (P>0.05) and no obvious toxic effects can be observed on the normal human fibroblasts and peripheral blood mononuclear cells (IC 10:4.545±0.876μmol/L,4.029±0.638μmol/L). Moreover, doramectin in combination with ADR can significantly extended the mean surviving time (MST) of xenografts nude mice and inhibit the growth of transplanted tumor (P<0.05)In summary, we successfully established MCF-7 cells in multi-drug resistant cell line, and provided abundant evidences here revealing that the five drugs could effectively reverse MDR of MCF-7/adr cells in vitro through two main mechanisms:via inhibiting the P-gp pump function and down regulating expressions of MDR1 gene and P-gp and MRP 1 gene. Among them doramectin containing sugar moiety especially appeared more effective as MDR modulator than the others, therefore, we detected its effect of reversal MDR in vivo and provided a theoretical basis for the studies of other milbemycins reversing MDR in vivo. In conclusion, the five drugs might be candidates as effective MDR reversing agents in cancer chemotherapy, and take the hope for cancer patients undergoing the chemotherapy.