at 4 for 45 min with 20 l of reagent R-PE conjugated anti-human EGFR. After this incubation, the cells were washed twice with PBS OSU-03012 PDK-1 inhibitor and the supernatant was discarded. Closing Lich cells were resuspended in 400 l of PBS for analysis by flow cytometry. Samples contr the isotype were identical to the PE-labeled mouse antibody body κ IgG2b treated. For HER2 expression analysis, 10 l PE-conjugated and Dai al. Cancer Res page 6 Author manuscript, increases available in PMC 2009 1 October. PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript NIH anti-human ErbB2 regents was mixed with 25 l of cells. After incubation for 45 minutes at 4, cells were washed twice with PBS and resuspended in 400 l PBS for analysis by flow cytometry. Samples contr the isotype were identical to the PE-labeled mouse antibody body IgG2B treated.
Statistical analysis All experiments were repeated at least three times and statistical significance using the Student’s r test. Significance was determined at P0.05. Results Effect of lapatinib and chemotherapeutic agents in various MDR cells and their parental cells, the cytotoxic effects of lapatinib, we examined only in different cell lines using the MTT XAV-939 Wnt/beta-catenin inhibitor assay. over 90% of cells were present in concentrations of lapatinib up to 2.5 m in MCF-7, MCF 7/adr, S1 and S1 80 M1 cells lebensf compatibility available. In contrast, lapatinib to 10 M almost no cytotoxic effect on HEK293 cells. The cytotoxic effects of chemotherapeutic agents in MCF-7, MCF 7/adr, S1, M1 and H1 80 cells in the presence of 0.625, 1.25, 2.50 M or lapatinib were tested.
The mean IC 50 of chemotherapeutic agents in different pairs of sensitive and resistant cells to various concentrations of lapatinib are shown in Table 1. ABCB1 in cells overexpressing MCF 7/adr, lapatinib produced a significant increase in dose- Independent Cytotoxicity t 7/adr of doxorubicin in MCF. In contrast, lapatinib alone produced sensitization to doxorubicin 2 times in the parental MCF-7 cells. Importantly, lapatinib, the lowest concentration tested was capable of resistance to doxorubicin to 6.5 times in MCF 7/adr reverse. When MCF-7 and MCF cells were transfected with specific ABCG2 inhibitor FTC 7/adr incubated at 2.5 M, we found that the FTC did not significantly influence the toxicity of t of doxorubicin in both MCF-7 cell lines or MCF 7 / adr.
This result shows that lapatinib resistance of MCF 7/adr interaction with ABCB1 versa. Lapatinib has also been reduced fa Is considerable resistance to mitoxantrone and topotecan in ABCG2-overexpressing S1-80 M1 cells. In addition, a small synergistic effect of combining lapatinib with either topotecan or mitoxantrone in the parental S1 cells was observed, but the FTC did not significantly dependent Ngig Be the toxic effects of mitoxantrone in the parental cells S1. These results suggest that lapatinib increased Ht ABCB1 and ABCG2, the sensitivity of cells overexpressing MDR to Herk Mmlichen chemotherapeutic agents, but has little effect in the parental cells. Recent studies have shown that mutations affecting the amino Acid 482 in ABCG2 substrate and antagonist specificity t ABCG2. Therefore, we investigated whether lapatinib reverse ABCG2-mediated resistance to mitoxantrone in cells with wild-type or mutant forms of ABCG2 transfected. As shown in Table 2, the IC50 shown val