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Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of osteosarcoma cells.

Ory B, Moriceau G, Trichet V, Blanchard F, Berreur M, Rédini F, Rogers M, Heymann D - J. Cell. Mol. Med. (2008)

Bottom Line: Furthermore, the Zol-resistant cells were sensitive to conventional anti-cancer agents demonstrating that this resistance process is independent of the multidrug resistance phenotype.The transfection of Zol-resistant cells with FPPS siRNA strongly increased their sensitivity to Zol.This study demonstrates for the first time the induction of metabolic resistance after prolonged Zol treatment of osteosarcoma cells confirming the therapeutic potential of Zol for the treatment of bone malignant pathologies, but points out the importance of the treatment regimen may be important in terms of duration and dose to avoid the development of drug metabolic resistance.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, ERI 7, Nantes, France.

ABSTRACT
We recently demonstrated original anti-tumor effects of zoledronic acid (Zol) on osteosarcoma cell lines independently of their p53 and Rb status. The present study investigated the potential Zol-resistance acquired by osteosarcoma cells after prolonged treatment. After 12 weeks of culture in the presence of 1 microm Zol, the effects of high doses of Zol (10-100 microm) were compared between the untreated rat (OSRGA, ROS) and human (MG63, SAOS2) osteosarcoma cells and Zol-pretreated cells in terms of cell proliferation, cell cycle analysis, migration assay and cytoskeleton organization. Long-term treatment with 1 microm Zol reduced the sensitivity of osteosarcoma cells to high concentrations of Zol. Furthermore, the Zol-resistant cells were sensitive to conventional anti-cancer agents demonstrating that this resistance process is independent of the multidrug resistance phenotype. However, as similar experiments performed in the presence of clodronate and pamidronate evidenced that this drug resistance was restricted to the nitrogen-containing bisphosphonates, we then hypothesized that this resistance could be associated with a differential expression of farnesyl diphos-phate synthase (FPPS) also observed in human osteosarcoma samples. The transfection of Zol-resistant cells with FPPS siRNA strongly increased their sensitivity to Zol. This study demonstrates for the first time the induction of metabolic resistance after prolonged Zol treatment of osteosarcoma cells confirming the therapeutic potential of Zol for the treatment of bone malignant pathologies, but points out the importance of the treatment regimen may be important in terms of duration and dose to avoid the development of drug metabolic resistance.

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Osteosarcoma cell lines develop Zol-resistance after long-term of continuous treatment with low doses of Zol. (A) rat (OSRGA, ROS) and human (MG63, SAOS2) sensitive and resistant (corresponding cell Name-res) osteosarcoma cell lines were treated with increasing concentrations of Zol (0.1–100 μm) for 72 hrs. The number of viable cells was then measured using the XTT assay. Graphs represent the mean values of three independent experiments performed in triplicate. ***P < 0.001. Statistical evaluation of the data was performed using the ANOVA test. (B) Cell cycle distribution of OSRGA and OSRGAres, treated or not with 10 μm Zol for 48 hrs was analyzed by propidium iodide staining and FACS analysis. G1/S and G2/M DNA checkpoints were analyzed by western blot and compared between sensitive and resistant OSRGA osteosarcoma cell lines in the presence or absence of 10 μm Zol for 24, 48 and 72 hrs. All experiments were repeated three times and a representative blot is shown. (C) Zol effects on organization of actin stress fibres were observed by confocal microscopy after phalloïdine staining. The actin network reorganization was associated with membrane ruffling (white arrow) in Zol-sensitive OSRGA cell line (Original magnification: × 1000). Zol effects on cell migration were also analyzed by time-lapse microscopy. The horizontal bars represent the limit of the slit cut performed on the cell monolayer at the start of the experiment (Original magnification: × 100). (D) Farnesyl diphosphate synthase (FPPS) transcription level was determined by semi quantitative RT-PCR in OSRGA sensitive and resistant cell lines. The 18S was used as a control.
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fig01: Osteosarcoma cell lines develop Zol-resistance after long-term of continuous treatment with low doses of Zol. (A) rat (OSRGA, ROS) and human (MG63, SAOS2) sensitive and resistant (corresponding cell Name-res) osteosarcoma cell lines were treated with increasing concentrations of Zol (0.1–100 μm) for 72 hrs. The number of viable cells was then measured using the XTT assay. Graphs represent the mean values of three independent experiments performed in triplicate. ***P < 0.001. Statistical evaluation of the data was performed using the ANOVA test. (B) Cell cycle distribution of OSRGA and OSRGAres, treated or not with 10 μm Zol for 48 hrs was analyzed by propidium iodide staining and FACS analysis. G1/S and G2/M DNA checkpoints were analyzed by western blot and compared between sensitive and resistant OSRGA osteosarcoma cell lines in the presence or absence of 10 μm Zol for 24, 48 and 72 hrs. All experiments were repeated three times and a representative blot is shown. (C) Zol effects on organization of actin stress fibres were observed by confocal microscopy after phalloïdine staining. The actin network reorganization was associated with membrane ruffling (white arrow) in Zol-sensitive OSRGA cell line (Original magnification: × 1000). Zol effects on cell migration were also analyzed by time-lapse microscopy. The horizontal bars represent the limit of the slit cut performed on the cell monolayer at the start of the experiment (Original magnification: × 100). (D) Farnesyl diphosphate synthase (FPPS) transcription level was determined by semi quantitative RT-PCR in OSRGA sensitive and resistant cell lines. The 18S was used as a control.

Mentions: Consistent with previous results [11, 12, 19], Zol treatment of Zol-sensitive rat ROS, OSRGA (Fig. 1A) and human MG63, SAOS2 (Fig. 1B) osteosarcoma cells strongly reduced their proliferation and induced the cell death without any caspase 1, 3 or 8 activation (data not shown). Thus, 0.1–100 μM Zol decreased the viable cell number in a dose-dependent manner (IC50: 1–8 μM) as revealed by the XTT assay. After 3 month continuous treatment with 1 μm Zol, rat and human osteosarcoma cells became less sensitive to Zol and resistant cell lines (OSRGAres, ROSres, MG63res, SAOS2res) were then progressively established (Fig. 1A). Indeed, the potency of Zol to affect cell proliferation was strongly reduced on human resistant cell lines and Zol was ineffective on rat resistant cell lines (Fig. 1A).


Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of osteosarcoma cells.

Ory B, Moriceau G, Trichet V, Blanchard F, Berreur M, Rédini F, Rogers M, Heymann D - J. Cell. Mol. Med. (2008)

Osteosarcoma cell lines develop Zol-resistance after long-term of continuous treatment with low doses of Zol. (A) rat (OSRGA, ROS) and human (MG63, SAOS2) sensitive and resistant (corresponding cell Name-res) osteosarcoma cell lines were treated with increasing concentrations of Zol (0.1–100 μm) for 72 hrs. The number of viable cells was then measured using the XTT assay. Graphs represent the mean values of three independent experiments performed in triplicate. ***P < 0.001. Statistical evaluation of the data was performed using the ANOVA test. (B) Cell cycle distribution of OSRGA and OSRGAres, treated or not with 10 μm Zol for 48 hrs was analyzed by propidium iodide staining and FACS analysis. G1/S and G2/M DNA checkpoints were analyzed by western blot and compared between sensitive and resistant OSRGA osteosarcoma cell lines in the presence or absence of 10 μm Zol for 24, 48 and 72 hrs. All experiments were repeated three times and a representative blot is shown. (C) Zol effects on organization of actin stress fibres were observed by confocal microscopy after phalloïdine staining. The actin network reorganization was associated with membrane ruffling (white arrow) in Zol-sensitive OSRGA cell line (Original magnification: × 1000). Zol effects on cell migration were also analyzed by time-lapse microscopy. The horizontal bars represent the limit of the slit cut performed on the cell monolayer at the start of the experiment (Original magnification: × 100). (D) Farnesyl diphosphate synthase (FPPS) transcription level was determined by semi quantitative RT-PCR in OSRGA sensitive and resistant cell lines. The 18S was used as a control.
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Related In: Results  -  Collection

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fig01: Osteosarcoma cell lines develop Zol-resistance after long-term of continuous treatment with low doses of Zol. (A) rat (OSRGA, ROS) and human (MG63, SAOS2) sensitive and resistant (corresponding cell Name-res) osteosarcoma cell lines were treated with increasing concentrations of Zol (0.1–100 μm) for 72 hrs. The number of viable cells was then measured using the XTT assay. Graphs represent the mean values of three independent experiments performed in triplicate. ***P < 0.001. Statistical evaluation of the data was performed using the ANOVA test. (B) Cell cycle distribution of OSRGA and OSRGAres, treated or not with 10 μm Zol for 48 hrs was analyzed by propidium iodide staining and FACS analysis. G1/S and G2/M DNA checkpoints were analyzed by western blot and compared between sensitive and resistant OSRGA osteosarcoma cell lines in the presence or absence of 10 μm Zol for 24, 48 and 72 hrs. All experiments were repeated three times and a representative blot is shown. (C) Zol effects on organization of actin stress fibres were observed by confocal microscopy after phalloïdine staining. The actin network reorganization was associated with membrane ruffling (white arrow) in Zol-sensitive OSRGA cell line (Original magnification: × 1000). Zol effects on cell migration were also analyzed by time-lapse microscopy. The horizontal bars represent the limit of the slit cut performed on the cell monolayer at the start of the experiment (Original magnification: × 100). (D) Farnesyl diphosphate synthase (FPPS) transcription level was determined by semi quantitative RT-PCR in OSRGA sensitive and resistant cell lines. The 18S was used as a control.
Mentions: Consistent with previous results [11, 12, 19], Zol treatment of Zol-sensitive rat ROS, OSRGA (Fig. 1A) and human MG63, SAOS2 (Fig. 1B) osteosarcoma cells strongly reduced their proliferation and induced the cell death without any caspase 1, 3 or 8 activation (data not shown). Thus, 0.1–100 μM Zol decreased the viable cell number in a dose-dependent manner (IC50: 1–8 μM) as revealed by the XTT assay. After 3 month continuous treatment with 1 μm Zol, rat and human osteosarcoma cells became less sensitive to Zol and resistant cell lines (OSRGAres, ROSres, MG63res, SAOS2res) were then progressively established (Fig. 1A). Indeed, the potency of Zol to affect cell proliferation was strongly reduced on human resistant cell lines and Zol was ineffective on rat resistant cell lines (Fig. 1A).

Bottom Line: Furthermore, the Zol-resistant cells were sensitive to conventional anti-cancer agents demonstrating that this resistance process is independent of the multidrug resistance phenotype.The transfection of Zol-resistant cells with FPPS siRNA strongly increased their sensitivity to Zol.This study demonstrates for the first time the induction of metabolic resistance after prolonged Zol treatment of osteosarcoma cells confirming the therapeutic potential of Zol for the treatment of bone malignant pathologies, but points out the importance of the treatment regimen may be important in terms of duration and dose to avoid the development of drug metabolic resistance.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, ERI 7, Nantes, France.

ABSTRACT
We recently demonstrated original anti-tumor effects of zoledronic acid (Zol) on osteosarcoma cell lines independently of their p53 and Rb status. The present study investigated the potential Zol-resistance acquired by osteosarcoma cells after prolonged treatment. After 12 weeks of culture in the presence of 1 microm Zol, the effects of high doses of Zol (10-100 microm) were compared between the untreated rat (OSRGA, ROS) and human (MG63, SAOS2) osteosarcoma cells and Zol-pretreated cells in terms of cell proliferation, cell cycle analysis, migration assay and cytoskeleton organization. Long-term treatment with 1 microm Zol reduced the sensitivity of osteosarcoma cells to high concentrations of Zol. Furthermore, the Zol-resistant cells were sensitive to conventional anti-cancer agents demonstrating that this resistance process is independent of the multidrug resistance phenotype. However, as similar experiments performed in the presence of clodronate and pamidronate evidenced that this drug resistance was restricted to the nitrogen-containing bisphosphonates, we then hypothesized that this resistance could be associated with a differential expression of farnesyl diphos-phate synthase (FPPS) also observed in human osteosarcoma samples. The transfection of Zol-resistant cells with FPPS siRNA strongly increased their sensitivity to Zol. This study demonstrates for the first time the induction of metabolic resistance after prolonged Zol treatment of osteosarcoma cells confirming the therapeutic potential of Zol for the treatment of bone malignant pathologies, but points out the importance of the treatment regimen may be important in terms of duration and dose to avoid the development of drug metabolic resistance.

Show MeSH
Related in: MedlinePlus