Letrozole sensitizes breast cancer cells to ionizing radiation

Abstract

Introduction: Radiotherapy (RT) is considered a standard treatment option after surgery for breast cancer. Letrozole, an aromatase inhibitor, is being evaluated in the adjuvant setting. We determined the effects of the combination of RT and letrozole in the aromatase-expressing breast tumour cell line MCF-7CA, stably transfected with the CYP19 gene.

Methods: Irradiations were performed using a cobalt-60 source with doses ranging from 0 to 4 Gy. Cells were incubated with androstenedione in the presence or absence of letrozole. Effects of treatment were evaluated using clonogenic assays, tetrazolium salt colorimetric (MTT) assays, and cell number determinations. Cell-cycle analyses were conducted using flow cytometry.

Results: The survival fraction at 2 Gy was 0.66 for RT alone and was 0.44 for RT plus letrozole (P = 0.02). Growth of MCF-7CA cells as measured by the cell number 6 days after radiotherapy (2 and 4 Gy) was decreased by 76% in those cells treated additionally with letrozole (0.7 microM) compared with those receiving radiotherapy alone (P = 0.009). Growth inhibition, assessed either by cell number (P = 0.009) or by the MTT assay (P = 0.02), was increased after 12 days of the combination treatment. Compared with radiation alone, the combination of radiation and letrozole produced a significant decrease in radiation-induced G2 phase arrest and a decrease of cells in the S phase, with cell redistribution in the G1 phase.

Conclusions: These radiobiological results may form the basis for concurrent use of letrozole and radiation as postsurgical adjuvant therapy for breast cancer.

Figures

Figure 1

Aromatase activity in MCF-7 wild-type (wt) and MCF-7CA transfected cell lines. Aromatase activity was measured by the tritiated water assay. Treatment with letrozole inhibited the 40 nM 1β-3H-androstenedione substrate conversion. Open bars, filled bars, and grey bars represent background radioactivity, aromatase activity without treatment, and aromatase activity after treatment with letrozole (100 nM), respectively.

Figure 2

Growth inhibition with letrozole alone. (a) Seven nanomolar letrozole did not inhibit the growth of MCF-7CA cells during 18 days of incubation, whereas (b) 0.7 μM letrozole resulted in about 50% inhibition after 6, 12, and 18 days of incubation. Results are from cell-count assays.

Figure 3

Potentiation of radiation-induced growth inhibition by letrozole measured by clonogenic assay. With radiation alone the MCF-7CA cell survival fraction decreased in a dose-dependent manner, which was significantly potentiated by the addition of 0.7 μM letrozole. For 2 Gy radiation, the surviving fraction was 0.66 with radiation alone and was 0.46 with the addition of letrozole (P = 0.02). For 3 Gy radiation, the corresponding surviving fractions were 0.4 and 0.18, respectively (P = 0.02).

Figure 4

Potentiation of radiation-induced growth inhibition by letrozole measured by the MTT assay. Growth of MCF-7CA cells, measured 6 days after treatment, was inhibited to a 40% greater extent with letrozole plus 2 Gy radiation, and to a 76% greater extent with letrozole plus 4 Gy radiation, compared with radiation alone.

Figure 5

Potentiation of radiation-induced growth inhibition by letrozole measured by cell-count assay. Growth of MCF-7CA cells, measured for 18 days after treatment, was inhibited to a 76% greater extent with letrozole plus 4 Gy radiation after 12 days, and to an 85% greater extent after 18 days, compared with radiation alone. Solid lines, ■ and ◆ represent radiation alone at 2 Gy and 4 Gy, respectively; dotted lines, ■ and ◆ represent combination of radiation plus letrozole (0.7 μM) at 2 Gy and 4 Gy, respectively.

Figure 6

Effect of letrozole (0.7 μM) or/and radiotherapy (RT) on MCF-7CA cell-cycle progression. (a) Control cells were compared with (b) MCF-7CA cells harvested after exposure to letrozole. In the case of RT treatment, cells were harvested after RT (c) without or (d) with letrozole. Cells were fixed and stained with propidium iodide for flow cytometry analysis as described in Materials and methods. Percentages of the G0/G1 phase, the S phase, and the G2/M phase were determined by CellQUEST analysis software on the basis of DNA content of the histogram. Data represent mean values of duplicate samples. Similar results were obtained in replicate experiments.