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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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Involvement of farnesyl diphosphate synthase (FPPS) in the Zol-induced resistance mechanism in osteosarcoma. (A) Farnesyl diphosphate synthase (FPPS) transcription level was determined by semi quantitative RT-PCR in FPPS siRNA transfected cell lines compared to the siRNA control cells. The 18S was used as a control. (B) Western blot analysis of unprenylated RAP1A (unRAP1A) from OSRGA cell lines transfected with FPPS siRNA and control siRNA, treated 24 and 48 hrs with 1 and 10 μm Zol. All experiments were repeated three times, and a representative blot is shown. (C) Photomicrographs of FPPS siRNA transfected cells after 48 hrs with 10 μm Zol compared to control siRNA. Original magnification: × 100. (D) Rat (OSRGA, OSRGAres) and human (MG63, SAOS2) osteosarcoma cell lines were transfected with FPPS siRNA and treated after 24 hrs of culture by increasing concentrations of Zol (0.1–10 μm) for 72 hrs. The number of viable cells was then determined using the XTT assay. Histograms represent the percentage of the increased sensitivity to Zol in the presence of FPPS siRNA compared to control siRNA. Values are mean of three independent experiments performed in triplicate. Error bars represent the standard deviation.
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fig03: Involvement of farnesyl diphosphate synthase (FPPS) in the Zol-induced resistance mechanism in osteosarcoma. (A) Farnesyl diphosphate synthase (FPPS) transcription level was determined by semi quantitative RT-PCR in FPPS siRNA transfected cell lines compared to the siRNA control cells. The 18S was used as a control. (B) Western blot analysis of unprenylated RAP1A (unRAP1A) from OSRGA cell lines transfected with FPPS siRNA and control siRNA, treated 24 and 48 hrs with 1 and 10 μm Zol. All experiments were repeated three times, and a representative blot is shown. (C) Photomicrographs of FPPS siRNA transfected cells after 48 hrs with 10 μm Zol compared to control siRNA. Original magnification: × 100. (D) Rat (OSRGA, OSRGAres) and human (MG63, SAOS2) osteosarcoma cell lines were transfected with FPPS siRNA and treated after 24 hrs of culture by increasing concentrations of Zol (0.1–10 μm) for 72 hrs. The number of viable cells was then determined using the XTT assay. Histograms represent the percentage of the increased sensitivity to Zol in the presence of FPPS siRNA compared to control siRNA. Values are mean of three independent experiments performed in triplicate. Error bars represent the standard deviation.

Mentions: FPPS being the main molecular target of nitrogen containing BPs [23], the FPPS transcript expression was analyzed by RT-PCR and compared in sensitive OSRGA and OSRGAres cells (Fig. 1D). Thus, the Zol-resistant cells expressed a higher level of FPPS mRNA than the sensitive cells. To further determine the involvement of FPPS in the Zol-resistance mechanism of human and rat osteosarcoma cells, the effect of Zol on OSRGA, OSRGAres, MG63 and SAOS2 was analyzed after transfection with FPPS siRNA. Semi-quantitative RT-PCR analysis was used to evaluate the efficacy of FPPS siRNA on FPPS mRNA expression. In all experiments, FPPS mRNA levels were significantly decreased in FPPS siRNA-transfected cell lines compared to the siRNA control (Fig. 3A). Inhibition of FPPS activity was then assessed indirectly by the expression of the unpreny-lated form of the small GTPase Rap1A (unRAP1A) that is expressed after inhibition of FPPS [24, 25] (Fig. 3B). The transfection of Zol-sensitive cells with FPPS siRNA strongly increased their sensitivity to Zol in all osteosarcoma cell lines studied. Indeed, FPPS siRNA transfection modified the unRAP1A expression kinetic in OSRGA, MG63 and SAOS2 cells. In the presence of FPPS siRNA, unRAP1A expression was strongly induced by 1 μm Zol treatment for 24 hrs whereas its expression was only observed with 10 μm Zol treatment for 48 hrs in control siRNA transfected cells (Fig. 3B). In OSRGAres cells, a very weak expression of unRAP1A was observed after Zol treatment. Interestingly, FPPS siRNA re-induced the sensitivity to Zol treatment in these resistant cells to a level comparable to parental OSRGA cells transfected with FPPS siRNA. Thus, the unRAP1A expression was observed after 24 hrs treatment with 1 μm Zol in FPPS siRNA-OSRGAres transfected (Fig. 3B). Similarly, micoscopic observations confirmed the FPPS siRNA effects on the sensitization of osteosarcoma cells to Zol treatment (Fig. 3C). Thus, an increase of floating cell number associated with an inhibition of cell proliferation was observed after transfection of all osteosarcoma cell lines with FPPS siRNA (Fig. 3C).


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)

Involvement of farnesyl diphosphate synthase (FPPS) in the Zol-induced resistance mechanism in osteosarcoma. (A) Farnesyl diphosphate synthase (FPPS) transcription level was determined by semi quantitative RT-PCR in FPPS siRNA transfected cell lines compared to the siRNA control cells. The 18S was used as a control. (B) Western blot analysis of unprenylated RAP1A (unRAP1A) from OSRGA cell lines transfected with FPPS siRNA and control siRNA, treated 24 and 48 hrs with 1 and 10 μm Zol. All experiments were repeated three times, and a representative blot is shown. (C) Photomicrographs of FPPS siRNA transfected cells after 48 hrs with 10 μm Zol compared to control siRNA. Original magnification: × 100. (D) Rat (OSRGA, OSRGAres) and human (MG63, SAOS2) osteosarcoma cell lines were transfected with FPPS siRNA and treated after 24 hrs of culture by increasing concentrations of Zol (0.1–10 μm) for 72 hrs. The number of viable cells was then determined using the XTT assay. Histograms represent the percentage of the increased sensitivity to Zol in the presence of FPPS siRNA compared to control siRNA. Values are mean of three independent experiments performed in triplicate. Error bars represent the standard deviation.
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Related In: Results  -  Collection

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fig03: Involvement of farnesyl diphosphate synthase (FPPS) in the Zol-induced resistance mechanism in osteosarcoma. (A) Farnesyl diphosphate synthase (FPPS) transcription level was determined by semi quantitative RT-PCR in FPPS siRNA transfected cell lines compared to the siRNA control cells. The 18S was used as a control. (B) Western blot analysis of unprenylated RAP1A (unRAP1A) from OSRGA cell lines transfected with FPPS siRNA and control siRNA, treated 24 and 48 hrs with 1 and 10 μm Zol. All experiments were repeated three times, and a representative blot is shown. (C) Photomicrographs of FPPS siRNA transfected cells after 48 hrs with 10 μm Zol compared to control siRNA. Original magnification: × 100. (D) Rat (OSRGA, OSRGAres) and human (MG63, SAOS2) osteosarcoma cell lines were transfected with FPPS siRNA and treated after 24 hrs of culture by increasing concentrations of Zol (0.1–10 μm) for 72 hrs. The number of viable cells was then determined using the XTT assay. Histograms represent the percentage of the increased sensitivity to Zol in the presence of FPPS siRNA compared to control siRNA. Values are mean of three independent experiments performed in triplicate. Error bars represent the standard deviation.
Mentions: FPPS being the main molecular target of nitrogen containing BPs [23], the FPPS transcript expression was analyzed by RT-PCR and compared in sensitive OSRGA and OSRGAres cells (Fig. 1D). Thus, the Zol-resistant cells expressed a higher level of FPPS mRNA than the sensitive cells. To further determine the involvement of FPPS in the Zol-resistance mechanism of human and rat osteosarcoma cells, the effect of Zol on OSRGA, OSRGAres, MG63 and SAOS2 was analyzed after transfection with FPPS siRNA. Semi-quantitative RT-PCR analysis was used to evaluate the efficacy of FPPS siRNA on FPPS mRNA expression. In all experiments, FPPS mRNA levels were significantly decreased in FPPS siRNA-transfected cell lines compared to the siRNA control (Fig. 3A). Inhibition of FPPS activity was then assessed indirectly by the expression of the unpreny-lated form of the small GTPase Rap1A (unRAP1A) that is expressed after inhibition of FPPS [24, 25] (Fig. 3B). The transfection of Zol-sensitive cells with FPPS siRNA strongly increased their sensitivity to Zol in all osteosarcoma cell lines studied. Indeed, FPPS siRNA transfection modified the unRAP1A expression kinetic in OSRGA, MG63 and SAOS2 cells. In the presence of FPPS siRNA, unRAP1A expression was strongly induced by 1 μm Zol treatment for 24 hrs whereas its expression was only observed with 10 μm Zol treatment for 48 hrs in control siRNA transfected cells (Fig. 3B). In OSRGAres cells, a very weak expression of unRAP1A was observed after Zol treatment. Interestingly, FPPS siRNA re-induced the sensitivity to Zol treatment in these resistant cells to a level comparable to parental OSRGA cells transfected with FPPS siRNA. Thus, the unRAP1A expression was observed after 24 hrs treatment with 1 μm Zol in FPPS siRNA-OSRGAres transfected (Fig. 3B). Similarly, micoscopic observations confirmed the FPPS siRNA effects on the sensitization of osteosarcoma cells to Zol treatment (Fig. 3C). Thus, an increase of floating cell number associated with an inhibition of cell proliferation was observed after transfection of all osteosarcoma cell lines with FPPS siRNA (Fig. 3C).

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