Risedronate inhibits human osteosarcoma cell invasion

Abstract

Background: Osteosarcoma is a highly malignant bone tumor and is the most commonly encountered malignant bone tumor in children and adolescents. Furthermore, significant numbers of patients eventually develop pulmonary metastases and succumb to the disease even after conventional multi-agent chemotherapy and surgical excision. Several solid tumors display enhanced expression of matrix metalloproteinases (MMPs), and recently clinical trials have been initiated on MMP-inhibitors. On the other hand, bisphosphonates (BPs), which have a profound effect on bone resorption, are widely used to treat osteoclast-mediated bone diseases. BPs are also known to inhibit tumor growths and metastases in some tumors such as breast cancer, renal cell carcinoma, and prostate cancer.

Methods: Two osteosarcoma cell lines (SaOS-2 and U2OS) were treated with risedronate (0, 0.1, 1, 10 microM) for 48 hours. Cell viabilities were determined using MTT assay, the mRNA levels of MMP-2 and MMP-9 were analyzed by reverse-transcription polymerase chain reaction, the amount of MMP-2 and MMP-9 protein were analyzed by Westernblot, the activities of MMP-2 and MMP-9 were observed by Gelatin zymography, and Matrigel invasion assays were used to investigate the invasive potential of osteosarcoma cell lines before and after risedronate treatment.

Results: The invasiveness of osteosarcoma cell lines (SaOS-2, U2OS) were reduced in a dose dependent manner follow 48 hour treatment of up to 10 microM of the risedronate at which concentration no cytotoxicity occurred. Furthermore, the gelatinolytic activities and protein and mRNA levels of MMP-2 and MMP-9 were also suppressed by increasing risedronate concentrations.

Conclusion: Given that MMP-2 and MMP-9 are instrumental in tumor cell invasion, our results suggest the risedronate could reduce osteosarcoma cell invasion.

Figure 1

Risedronate at concentrations up to 10 μM had no cytotoxic effect on either SaOS-2 or U2OS cells. Both cell lines in serum-free MEM were treated or not with the indicated concentrations of risedronate and then incubated for 48 h before doing MTT assay for cell growth quantification. The bar graph shows the absorbance (expressed as percentages of controls) measured at 570 nm on an ELISA reader (n = 3 independent experiments; mean ± standard deviation is shown).

Figure 2

Risedronate impedes the invasiveness of SaOS-2 and U2OS cells (A and B). A 10-well chemotaxis chamber was used to measure the effect of risedronate on invasiveness. A Matrigel-coated membrane was inserted between the upper and lower chambers, and stained using a Hemacolor rapid staining kit. Stained areas represented numbers of migrating cells. The numbers in the panels show the concentration of risedronate added. Images are representative of three independent experiments. Bars (C) represent cells number (expressed as percentages of controls) of each image ± standard deviation.

Figure 3

MMP-inhibitor Marimastat (50 μg/mg) impedes the invasiveness of SaOS-2 and U2OS cells. Three different experiments with each cell line were performed. Bars represent the cell numbers (expressed as percentages of controls) of each image ± standard deviation. Abbreviations: C: control; M: Marimastat.

Figure 4

Risedronate inhibited the gelatinolytic activities of MMP-2 and MMP-9. (A) Conditioned media harvested from SaOS-2 and U2OS cells treated for 48 h with the indicated concentrations of risedronate were analyzed by gelatin zymography. The white bands represent MMP-mediated gelatin digestion. The image is representative of three independent experiments. MMPs activities (expressed as percentages of controls) are shown in B (n = 3). Numbers in boxes represent the concentration of risedronate (in μM) added to cells. Bars represent the MMPs activities (expressed as percentages of controls) of each band ± standard deviation.

Figure 5

Risedronate reduced the expressions of MMP-2 and MMP-9 proteins in SaOS-2 and U2OS cells. (A) Cells were treated with the indicated concentrations of risedronate for 48 h, and then cell lysates were Western blotted. Beta-actin was used as a loading control. Images are representative of three independent experiments. B shows MMPs protein levels (expressed as percentages of controls) (n = 3). Numbers in the box represent the concentration of risedronate in μM added to the cells. Bars represent MMPs protein levels (expressed as percentages of controls) of each band ± standard deviation.

Figure 6

Risedronate suppressed the expressions of MMP-2 and MMP-9 mRNA in SaOS-2 and U2OS cells. (A) Cells were treated with the indicated concentrations of risedronate for 48 h and then processed for RT-PCR. Beta-actin was used as a loading control. Images are representative of three independent experiments. MMPs mRNA levers (expressed as percentages of controls) are shown in B (n = 3). Numbers in the box represent the concentration of risedronate in μM added to the cells. Bars represent MMPs mRNA levels (expressed as percentages of controls) of each band ± standard deviation.