Efficacy of the third-generation bisphosphonate risedronate alone and in combination with anticancer drugs against osteosarcoma cell lines.
BACKGROUND
Bisphosphonates (BPs) are currently the most important class of inhibitors of osteoclast-mediated bone resorption and are widely used in the treatment of osteoporosis and other osteoclast-mediated bone diseases. Recently, there has been increasing evidence that BPs also exhibit direct anti-tumor activity against several cancer cell lines. However, the efficacies of BPs against osteosarcoma have not yet been fully elucidated, and the anti-osteosarcoma activity of the potent new-generation BP risedronate, which is widely used clinically, has not been determined.


METHODS
The anti-proliferative effects of the nitrogen-containing BP risedronate on seven osteosarcoma cell lines (LM8, SaOS2, U2OS, HOS, KHOS, MG63 and OST) were studied. The cell viability was determined by MTT assay. Prenylation of Rap1A and Ras and phosphorylation of Erk1 and 2 were examined by Western blot analysis. Genomic DNA fragmentation and TUNEL staining assay were performed to determine whether risedronate induced apoptosis of the osteosarcoma cells. The anti-tumor activities of risedronate in combination with carboplatin, doxorubicin, vincristine or etoposide were assayed with CalcuSyn (Biosoft).


RESULTS
Risedronate inhibited both prenylation of Rap1A and Ras and phosphorylation of Erk 1 and 2 at a dose of 50 microM for 48 h. Treatment with risedronate induced significant time- and dose-dependent cytotoxicity in the LM8 cell line. Risedronate treatment also increased intranucleosomal genomic DNA fragmentation. The TUNEL assay showed that 10 microM and 50 microM risedronate clearly induced apoptosis of osteosarcoma cells. Risedronate also clearly inhibited LM8, SaOS2 and KHOS cell growth in a time- and dose-dependent fashion, but only weakly inhibited that of MG63 and U2OS cells. Risedronate augmented the effects of carboplatin, doxorubicin, vincristine and etoposide synergistically across a wide range of fractions affected (Fa) values.


CONCLUSIONS
The nitrogen-containing bisphosphonate risedronate induces apoptosis and inhibits the growth of osteogenic sarcoma through a mechanism involving the down-regulation of protein prenylation and may be a candidate for combined use with carboplatin, doxorubicin, vincristine or etoposide.
Murayama
Takashi
T
Department of Orthopaedic Surgery, Kagoshima Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
Kawasoe
Yasuomi
Y
Yamashita
Yoshitaka
Y
Ueno
Yoshinori
Y
Minami
Syusaku
S
Yokouchi
Masahiro
M
Komiya
Setsuro
S