Systematic screen with kinases inhibitors reveals kinases play distinct roles in growth of osteoprogenitor cells

Abstract

Cancer treatment-related bone loss has become growing problematic, especially in breast and prostate cancer treated with hormone/endocrine therapy, chemotherapy and radiotherapy. However, bone loss caused by targeted therapy in cancer patients is largely unknown yet. In present study, a kinase inhibitors screen was applied for MC3T3-E1, a murine osteoprogenitor cell line, and seven kinase inhibitors (GSK1838705A, PF-04691502, Dasatinib, Masitinib, GDC-0941, XL880 and Everolimus) were found to suppress the cell viability with dose- and time-dependent manner. The most interesting is that many kinase inhibitors (such as lapatinib, erlotinib and sunitinib) can promote MC3T3-E1 cell proliferation at 0.01 μM. 4 out of 7 inhibitors were selected to perform the functional study and found that they lead to cell cycle dysregulation, treatments of PF-04691502 (AKT inhibitor), Dasatinib (Src inhibitor) and Everolimus (mTOR inhibitor) lead to G1 arrest of MC3T3-E1 cells via downregulation of cyclin D1 and p-AKT, whereas XL880 (MET and VEGFR inhibitor) treatment results in increase of sub-G1 and G2/M phase by upregulation of p53 protein. Our work provides important indications for the comprehensive care of cancer patients treated with some targeted drugs.

Keywords: Cancer treatment-related bone loss; kinases inhibitors screening; osteoprogenitor cells.

Figure 2

Seven positive hits impair MC3T3-E1 proliferation in a dose- and time-dependent manner. A: The dose-effect of 7 inhibitors on cell viability. MC3T3-E1 cells were left untreated or treated with 7 inhibitors at 3 distinct concentrations, 1 × average cellular IC50 dose, 5 × average cellular IC50 dose, and 25 × average cellular IC50 dose, respectively, for 72 h. B: The time-effect of 7 inhibitors on cell viability. MC3T3-E1 cells were left untreated or treated with the 7 drugs at the corresponding highest doses used in (A), respectively, for 24 or 48 h. NC represents negative control, the untreated cells. The bar represents the standard error (SD).

Figure 1

Systematic screening with 44 kinases inhibitors on proliferation of MC3T3-E1 cells. All the 44 inhibitors were applied to MC3T3-E1 cells at four concentrations, 0.01, 0.1, 1.0 and 10.0 μM, respectively. The cell viabilities following inhibitors treatment were plotted in (A) and that treated with the seven positive hits was plotted in (B). All the names of inhibitors were briefly represented with the Catalogue No., which can be readily found in Table 1. NC represents negative control, the untreated cells. The bar represents the standard error (SD).

Figure 3

Treatment of 4 kinases inhibitors leads to cell cycle dysregulation. A: Treatment of PF-04691502, Dasatinib, XL880 and Everolimus resulted in different patterns of cell cycle dysregulation. Cells were treated with 0.5 μM of the 4 inhibitors for 48 h. Cell cycle profiles were assessed by propidium iodide (PI) staining and fluorescence-activated cell scanning (FACS) from cell aliquots. B: Histogram represented the percentage of cells in each phase of the cell cycle. Significantly increase in G1 phase following treatment of PF-04691502, Dasatinib, and Everolimus, respectively, were observed, indicating G1 arrest by these three inhibitors. While for XL880, its treatment resulted in increase of sub-G1 and G2/M phase.

Figure 4

Immunoblotting experiments showed that the underlying signaling were different following the treatment of the 4 inhibitors. A: CCND1 was repressed by PF-04691502, Dasatinib, and Everolimus treatment whereas induced by XL880 treatment. The number 1, 2 represent the two repeated samples. B: Dasatinib treatment caused AKT inactivation, while XL880 treatment led to p53 upregulation.