S-phase kinase-associated protein 2 is involved in epithelial-mesenchymal transition in methotrexate-resistant osteosarcoma cells

Abstract

Osteosarcoma (OS), a common worldwide primary aggressive bone malignancy, arises from primitive transformed cells of mesenchymal origin and usually attacks adolescents and young adults. Methotrexate (MTX) is the anti-folate drug used as a pivotal chemotherapeutic agent in the treatment of OS. However, patients with OS often develop drug resistance, leading to poor treatment outcomes. In the present study, in order to explore the underlying mechanisms responsible for MTX resistance, we established MTX‑resistant OS cells using the U2OS and MG63 cell lines and examined whether MTX‑resistant OS cells underwent epithelial-mesenchymal transition (EMT) by Transwell assay, wound healing assay, MTT assay, RT-PCR and western blot analysis. We found that the viability of the MTX‑resistant cells remained relatively unaltered following further treatment with MTX compared to the parental cells. The resistant cells appeared to possess a mesenchymal phenotype, with an elongated and more spindle‑like shape, and acquired enhanced invasive, migratory and attachment abilities. The measurement of EMT markers also supported EMT transition in the MTX‑resistant OS cells. Our result further demonstrated that the overexpression of S-phase kinase-associated protein 2 (Skp2) was closely involved in the resistance of OS cells to MTX and in the acquirement of EMT properties. Thus, the pharmacological inhibition of Skp2 may prove to be a novel therapeutic strategy with which to overcome drug resistance in OS.

Figure 1

Methotrexate (MTX)-resistant cells acquire the epithelial-mesenchymal transition (EMT) phenotype. (A) MTX-resistant osteosarcoma cells were observed to have an increased cell proliferation (thus great resistance to MTX), as shown by MTT assay. (B) Cell morphology was observed by phase contrast microscopy in parental and MTX-resistant osteosarcoma (OS) cells. (C) Upper panel: Cell invasion assay was performed to measure the invasive capacity of the parental and MTX-resistant OS cells. Lower panel: Results of quantitative analysis are shown for the findings in the upper panel. ^*P<0.05 vs. control (parental cells). (D) Cell attachment and detachment assays were carried out in the parental and MTX-resistant cells. ^*P<0.05 vs control (parental cells).

Figure 2

Methotrexate (MTX)-resistant cells exhibit an enhanced migration and acquire epithelial-mesenchymal transition (EMT)-like characteristics. (A) Upper panel: Wound healing assays were performed using cell monolayers to detect the motility of MTX-resistant osteosarcoma (OS) cells. Cells that migrated into the wounded region were photographed. Lower panel: Results of quantitative of the findings shown in the upper panel by comprehensive migrated distance. ^*P<0.05 vs. control (parental cells). (B) RT-qRCR revealed that the mRNA expression levels of the mesenchymal markers, Vimentin, Slug and N-cadherin, were upregulated, whereas those of the epithelial molecules, ZO-1 and E-cadherin, were markedly decreased in the MTX-resistant OS cells. ^* P<0.05 vs. control (parental cells).

Figure 3

Methotrexate (MTX)-resistant osteosarcoma (OS) cells undergo epithelial-mesenchymal transition (EMT)-like changes. (A) Left panel: The expression levels of E-cadherin, N-cadherin, Slug, Vimentin, Nanog, Oct4, ABCB1 and ZO-1 were detected by western blot analysis in the U2OS and U2OS MTX-resistant cells. Right panel: Results of quantitative of the blots shown in the left panel. ^*P<0.05 vs. control (parental cells). (B) Left panel: The expression levels of the above-mentioned EMT-related and stem cell markers were measured by western blot analysis in the MG63 and MG63 MTX-resistant cells. Right panel: Results of quantitative of the blots shown in the left panel. ^*P<0.05 vs. control (parental cells).

Figure 4

Skp2 is overexpressed in methotrexate (MTX)-resistant osteosarcoma (OS) cells and the downregulation of Skp2 suppresses cell invasion. (A) The mRNA expression of Skp2 was detected by RT-qPCR assay in the parental and MTX-resistant OS cells. ^*P<0.05 vs. control (parental cells). (B) The protein expression levels of Skp2 and its targets, Foxo1 and p21, were detected by western blot analysis. (C) RNAi efficiency was determined by western blot analysis in the MTX-resistant OS cells transfected with Skp2 shRNA. (D) Cell morphology was observed under a phase contrast microscope. (E) Upper panel: Cell invasion assay were performed in the MTX-resistant OS cells in which Skp2 was knocked down. Lower panel: Results of quantitative of the findings shown in the upper panel. ^*P<0.05 vs. control. CS, control shRNA-transfected MTX-resistant OS cells; SS, Skp2 shRNA-transfected MTX-resistant OS cells.

Figure 5

Stable knockdown of Skp2 inhibits motility and restores sensitivity to methotrexate (MTX) in MTX-resistant osteosarcoma (OS) cells. (A) Upper panel: Cell monolayers were scratched with a clean tip. Cells that had migrated into the wounded region were photographed. Lower panel: Results of quantitative analysis of the findings shown in the upper panel. ^*P<0.05 vs. control. (B) Cell attachment and detachment ability were measured in the MTX-resistant OS cells following the knockdown of Skp2. ^*P<0.05 vs. control. (C) MTT assay revealed that the stable knockdown of Skp2 restored the sensitivity of the MTX-resistant cells to MTX, as evidenced by the decrease proliferation of the cells in which MTX was knocked down. CS, control shRNA-transfected MTX-resistant OS cells; SS, Skp2 shRNA-transfected MTX-resistant OS cells. ^*P<0.05 vs. control.

Figure 6

Stable knockdown of Skp2 abrogates epithelial-mesenchymal transition (EMT) induced by methotrexate (MTX) resistance in osteosarcoma (OS) cells. (A) Left panel: Stable knockdown of Skp2 in MTX-resistant U2OS cells promoted the expression of epithelial markers, such as Zo-1 and E-cadherin, whereas it suppressed the expression of mesenchymal markers, including N-cadherin, Slug and Vimentin. Right panel: Results of quantitative analysis of the findings shown in the left panel. ^*P<0.05 vs. control. (B) Left panel: Western blot analysis was performed to measure the expression of EMT markers in the MTX-resistant MG63 cells after Skp2 knockdown. Right panel: Results of quantitative analysis of the findings shown in the left panel. ^*P<0.05 vs. control. CS, control shRNA-transfected MTX-resistant OS cells;SS, Skp2 shRNA-transfected MTX-resistant OS cells.