MEK/ERK inhibitor U0126 increases the radiosensitivity of rhabdomyosarcoma cells in vitro and in vivo by downregulating growth and DNA repair signals

Abstract

Multimodal treatment has improved the outcome of many solid tumors, and in some cases the use of radiosensitizers has significantly contributed to this gain. Activation of the extracellular signaling kinase pathway (MEK/ERK) generally results in stimulation of cell growth and confers a survival advantage playing the major role in human cancer. The potential involvement of this pathway in cellular radiosensitivity remains unclear. We previously reported that the disruption of c-Myc through MEK/ERK inhibition blocks the expression of the transformed phenotype; affects in vitro and in vivo growth and angiogenic signaling; and induces myogenic differentiation in the embryonal rhabdomyosarcoma (ERMS) cell lines (RD). This study was designed to examine whether the ERK pathway affects intrinsic radiosensitivity of rhabdomyosarcoma cancer cells. Exponentially growing human ERMS, RD, xenograft-derived RD-M1, and TE671 cell lines were used. The specific MEK/ERK inhibitor, U0126, reduced the clonogenic potential of the three cell lines, and was affected by radiation. U0126 inhibited phospho-active ERK1/2 and reduced DNA protein kinase catalytic subunit (DNA-PKcs) suggesting that ERKs and DNA-PKcs cooperate in radioprotection of rhabdomyosarcoma cells. The TE671 cell line xenotransplanted in mice showed a reduction in tumor mass and increase in the time of tumor progression with U0126 treatment associated with reduced DNA-PKcs, an effect enhanced by radiotherapy. Thus, our results show that MEK/ERK inhibition enhances radiosensitivity of rhabdomyosarcoma cells suggesting a rational approach in combination with radiotherapy.

Figure 1

Clonogenic assay of embryonal rhabdomyosarcoma cell lines, RD, RD-M1 and TE671 at different Gy doses. A) picture of cristal violet stained cultures 15 days after irradiation at indicated doses (1, 2, 4 Gy); B) dose-dependent colonies formation, each point of the three curve is the average ± SEM of three samples. Similar results were obtained in three experiments.

Figure 2

Clonogenic assay of the three embryonal cell lines with (+) or without (-) U0126 and 4 Gy irradiation treatments. A) picture of crystal violet stained cultures 15 days after treatments, U0126 and 4 Gy irradiation or in combination.

Figure 3

Expression of protein kinases (ERK1/2-P04, DNA-PKcs, GSK3-beta-PO4), c-Myc and Cyclin-D1 in untreated (C), irradiated (RT), U0126-treated (U0126) and U0126-pretreated and irradiated (RT+U) RD cells (A) and TE671 (B). GAPDH was blotted as loading control.

Figure 4

(A) Growth curve of tumors volumes from xenografted TE671 cell lines, untreated (), U0126-treated (), irradiated (RT ▼-▼), U0126-pretreated and irradiated (RT+U Δ-Δ). Tumor volumes were evaluated as describes in methods and represent the mean ±.SEM of 8 mice. The upper panel shows the sequential treatments of xenografted mice started when tumor reached a volume of approximate 0,5 cm³. U0126 was administered 1 day before each irradiation. (B) Tumor weights in mice untreated or treated with U0126, radiotherapy or combined treatment.

Figure 5

(A) U0126 with RT combined treatment affects time to progression in vivo xenografted tumors. (B) Box plot diagram comparing median time (days) of tumor progression (TTP) among treatment groups. Box represents interquartile range (IQR), bar median value and whiskers represent data points within IQR.

Figure 6

Expression of protein Kinases (ERK1/2 and DNA-PKcs), cMyc and Cyclin-D1 in tumors untreated (C1,C2), U0126-tretaed (U1, U2), irradiated (RT1, RT2) and U0126-pretreated and irradiated (U+RT1, U+RT2). At the end point mice were sacrificed, tumor excised and extracted as described in methods. GAPDH was blotted as loading control.