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Inhibition of mevalonate pathway is involved in alendronate-induced cell growth inhibition, but not in cytokine secretion from macrophages in vitro
European Journal of Pharmaceutical Sciences 19 (2003) 223–230 www.elsevier.com / locate / ejps
Inhibition of mevalonate pathway is involved in alendronate-induced cell growth inhibition, but not in cytokine secretion from macrophages in vitro ¨ ¨ *, Jouko Ollikainen, Markku Taskinen, Jukka Monkkonen ¨ ¨ Anu Toyras Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland Received 1 October 2002; received in revised form 13 March 2003; accepted 10 April 2003
Abstract Bisphosphonates are antiresorptive drugs used for the treatment of metabolic bone diseases. They can be divided into two different pharmacological classes: nitrogen-containing and non-nitrogen-containing bisphosphonates. Non-nitrogen-containing bisphosphonates, like clodronate, are metabolised to a toxic ATP-analogue preventing osteoclast mediated bone resorption. Nitrogen-containing bisphosphonates, including alendronate, prevent osteoclast function by inhibiting the mevalonate pathway. Clodronate is known to have anti-inflammatory properties while alendronate induces cytokine secretion from lipopolysaccharide- (LPS) induced macrophages. This study investigates whether the cytotoxicity and cytokine production induced by alendronate and LPS could be counteracted by clodronate or products of mevalonate pathway: oxidized low density lipoprotein (ox-LDL), farnesol and geranylgeraniol. Treatment with alendronate increased LPS-induced secretion of IL-1b, IL-6 and TNF-a from RAW 264 macrophages 2.4-, 1.4- and 1.8-fold, respectively. This treatment was cytotoxic for macrophages as indicated by lowered cell viability. Clodronate and ox-LDL both counteracted the cytokine secretion and cytotoxicity of alendronate. Farnesol and geranylgeraniol did neither reverse the cytokine secretion nor reduce the cytotoxicity of alendronate. Clodronate and ox-LDL were able to counteract the effects of alendronate on macrophages in vitro, probably by their known ability to inhibit DNA binding activity of transcription factors, nuclear factor-kB (NF-kB) and activating protein-1 (AP-1). These findings suggest that inhibition of mevalonate pathway is not the mechanism responsible for the proinflammatory response caused by alendronate, as it is in alendronate-induced apoptosis and prevention of osteoclast function. 2003 Elsevier B.V. All rights reserved. Keywords: Bisphosphonate; Mevalonate pathway; Cytokines; Farnesol; Geranylgeraniol; Oxidized low density lipoprotein
1. Introduction Bisphosphonates are antiresorptive drugs used for the treatment of metabolic bone diseases. They can be divided into two different pharmacological classes: nitrogen-containing and non-nitrogen-containing bisphosphonates. Clodronate and other non-nitrogen-containing bisphosphonates, closely resembling an inorganic pyrophosphate, are metabolised to a toxic ATP-analogue preventing osteoclast mediated bone resorption (Frith et al., 1997; Rogers et al., 2000). Inhibition of the mitochondrial ADP/ATP translocase by the metabolite of clodronate, AppCCl 2 p (adenosine-59-[b,g-dichloromethylene]triphosphate), is likely the route by which clodronate causes primary apoptosis of osteoclasts (Benford et al., 2001; Lehenkari et al., 2002). *Corresponding author. Tel.: 1358-17-163-461; fax: 1358-17-162252. ¨ ¨ E-mail address: [email protected] (A. Toyras). 0928-0987 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0928-0987(03)00108-8
Nitrogen-containing bisphosphonates, such as alendronate, inhibit the mevalonate pathway, which is responsible for the production of cholesterol and isoprenoid lipids. The exact molecular target is considered to be farnesyl pyrophosphate synthase (van Beek et al., 1999b). The consequent loss of prenylated proteins, particularly geranylgeranylated, seems to be the main reason for the inhibition of osteoclast function and secondary apoptosis caused by nitrogen-containing bisphosphonates (van Beek et al., 1999a; Fisher et al., 1999; Coxon et al., 2000; Benford et al., 2001). The cell-permeable products of mevalonate pathway, farnesol and geranylgeraniol, have been described to suppress the apoptosis caused by nitrogen-containing bisphosphonates (Benford et al., 1999). Clodronate and its metabolite are known to have antiinflammatory properties while alendronate increases lipopolysaccharide- (LPS) induced cytokine secretion of macrophages in vitro (Makkonen et al., 1999). In vivo, alendronate has been reported to exacerbate the arthritis in mice, whereas clodronate was able to suppress the arthritis
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(Nakamura et al., 1996). An acute phase response, to which increased level of interleukin-6 (IL-6) is typically related, is developed for some patients treated with nitrogen-containing bisphosphonate for the first time (Schweitzer et al., 1995). Clodronate has been found to inhibit alendronate-induced inflammatory reaction in vivo, suggesting that combined administration of clodronate and nitrogen-containing bisphosphonate could prevent the acute phase response (Endo et al., 1999). The aim of this study was to investigate the ability of clodronate to counteract the cytotoxic and inflammatory effects of alendronate, in vitro. Intermediates of mevalonate pathway, farnesol and geranylgeraniol, are known to be capable of preventing apoptosis caused by alendronate (Benford et al., 1999). In this study their possible capability to reverse cytokine secretion and macrophage growth inhibition induced by alendronate was investigated. Oxidized LDL (ox-LDL) contains cholesterol, the end product of mevalonate pathway, and induces murine macrophage growth and survival (Biwa et al., 1998; Hamilton et al., 1999). It is also known to inhibit expression of tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) (Ohlsson et al., 1996). The ability of ox-LDL to counteract proinflammatory and cytotoxic effects of alendronate was also examined.
2. Materials and methods
2.1. Materials Alendronate was provided by Merck Sharp and Dohme (Rahway, NJ, USA) and clodronate was obtained from Leiras Oy (Turku, Finland). Stock solutions (100 mM alendronate, 110 mM clodronate) were prepared in water and pH 7.02 was adjusted with NaHCO 3 . Ox-LDL was obtained from Dr Pauliina Lehtolainen, A.I. Virtanen Institute, University of Kuopio, Finland. LDL was oxidized by overnight incubation with copper ions (20 mmol / ¨ l) as described earlier (Yla-Herttuala et al., 1989). Farnesol was purchased from Sigma–Aldrich (Steinheim, Germany). Geranylgeraniol, LPS (lipopolysaccharide from E. coli) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were purchased from Sigma (St. Louis, MO, USA). Recombinant mouse IL-1b, IL-6, TNFa / TNFSF2, anti-mouse IL-1b and IL-6 monoclonal antibodies, anti-mouse TNF-a / TNFSF2 polyclonal antibody and biotinylated anti-mouse IL-1b, IL-6, TNF-a antibodies were bought from R&D Systems (Minneapolis, MN, USA).
(Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum and 100 IU / ml penicillin–streptomycin in 7% CO 2 atmosphere at 37 8C. In growth inhibition studies cells were dispensed with a density of 4310 3 cells / well on 96-well plates and allowed to grow overnight. Cells were exposed to 3–1000 mM alendronate combined with 15 mM farnesol or 20 mM geranylgeraniol for 46 h. Cell growth was measured by using an MTT assay (Hansen et al., 1989).
2.2.2. Cytokine induction In the cytokine experiments cells were dispensed to 96-well plates as monolayer (2310 5 cells / well) and the cells were allowed to adhere for 4–5 h. The cells were treated with 100 mM alendronate combined with 1000 mM clodronate, 15 mM farnesol, 15 mM geranylgeraniol or 100 mg / ml ox-LDL for 20 h. These media were removed and cells were instantly treated with LPS in serum free DMEM (10 mg / ml) for 24 h in order to induce the cytokine secretion. Cell culture media were collected (four wells were pooled), centrifuged and stored at 280 8C. The cell viability was evaluated with MTT assay both after drug treatment and after LPS treatment. In contrast to other cell experiments, which were carried out four times, ox-LDL experiments were reproduced three times. 2.2.3. Time-resolved fluoroimmunoassay for IL-1b, IL-6 and TNF-a Principle of time-resolved fluoroimmunoassay has been described earlier (Pennanen et al., 1995a). Briefly, the microtiter strips were coated with the capture antibody, 4 mg / ml of IL-1b, 1 mg / ml of IL-6 or 0.8 mg / ml of TNF-a in PBS over night in room temperature. After three washings with 0.05% Tween 20 in PBS the non-specific binding was blocked for 1 h with PBS solution containing 1% bovine serum albumin (BSA) and 5% saccharose. Subsequently, the washings were repeated. The samples and the standards (diluted in 0.1% BSA, 0.05% Tween 20, 20 mM Tris, 150 mM NaCl pH 7.3) were incubated for 2 h and washed as earlier. The biotinylated detection antibodies (200 ng / ml of IL-1b and IL-6, 300 ng / ml of TNF-a) were incubated for 2 h followed by four washings. Eu-labelled streptavidin (100 ng / ml) in Delfia assay buffer (Wallac Oy, Turku, Finland) was dispensed to wells and incubated for 30 min with gently shaking. Strips were washed six times before adding of Delfia enhancement solution. After 20 min incubation the fluorescence was measured with Victor 2 1420 Multilabel counter (Wallac Oy, Turku, Finland). 2.3. Statistical analysis
2.2. Methods 2.2.1. Cell culture and growth inhibition studies RAW 264 murine macrophage cells were grown in Dulbecco’s modified Eagle’s medium 21885 (DMEM)
SPSS 9.0.1 software (SPSS, Chicago, IL, USA) was used for statistical analyses. The statistical differences between treatments were investigated by using nonparametric Mann–Whitney U-test. Different treatments
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were compared with combined treatment with alendronate and LPS.
3. Results and discussion
3.1. Growth inhibition studies In this study the ability of farnesol and geranylgeraniol to enhance growth of alendronate treated RAW 264 macrophage cells was investigated. Exposure to 100 mM alendronate diminished macrophage cell growth to 81% of that of the untreated controls, while treatment with 300 mM alendronate decreased the growth to only 1% (Fig. 1). Farnesol and especially geranylgeraniol were able to partly counteract the growth inhibitory effect of alendronate. Cell growth was 58 and 23% of that of control when 300 mM alendronate exposure was combined with 20 mM geranylgeraniol or 15 mM farnesol treatment, respectively (Fig. 1). The capability of farnesol and geranylgeraniol to inhibit macrophage cell growth was also investigated. Farnesol (15 mM) and geranylgeraniol (15 mM) reduced macrophage cell growth to 83 and 90% of that of control, respectively. These concentrations of farnesol and geranygeraniol were used in further cytokine experiments. Bisphosphonates are known to inhibit growth of amoebae of the slime mold (Dictyostelium discoideum) (Rogers et al., 1994, 1995) and prevent macrophage ¨ ¨ proliferation (Cecchini et al., 1987; Monkkonen et al., 1994b). Nitrogen-containing bisphosphonates inhibit mevalonate pathway leading to absence of prenylated proteins (Luckman et al., 1998a,b). Our data demonstrates that growth of macrophages was considerably inhibited by alendronate, and farnesol (15 mM) and particularly geranylgeraniol (20 mM), intermediates of mevalonate pathway, counteracted this effect (Fig. 1). Suri et al. (2001) have also found that ox-LDL, farnesol and particularly
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geranylgeraniol suppress anti-proliferative effects of nitrogen-containing bisphosphonates in Caco-2 cells. Biwa et al. (1998) have reported that granulocyte macrophage colony stimulating factor (GM-CSF) is involved in ox-LDL-induced murine macrophage growth. According to Chisolm and Chai (2000), lysophosphatidylcholine, and structurally related lipids, are among the growth-promoting constituents of ox-LDL. However, oxLDL did not have any effect on alendronate-induced growth inhibition in the present study (data not shown). This is probably due to its inability to counteract the alendronate-induced absence of prenylated proteins. OxLDL contains cholesterol, one of the end products of mevalonate pathway, which, in contrast to farnesol and geranylgeraniol, can not replace the prenylated proteins. Clodronate was not included in growth inhibition studies as 1000 mM clodronate itself strongly inhibits the growth of RAW 264 cells and the IC 50 value for clodronate is 980 ¨ ¨ mM (Monkkonen et al., 1994b). Raiteri et al. (1997) found that proliferation of arterial smooth muscle cells was reduced after exposure of drugs affecting different steps of mevalonate pathway. Furthermore, they found that the reduction was prevented by treatment with 5 mM all-trans geranylgeraniol and in most cases by treatment with 10 mM all-trans farnesol or 100 mM mevalonate. They suggested that farnesylated and geranylgeranylated proteins are involved in the control of cell proliferation. According to Tatsuno et al. (1997) mevalonate pathway and its metabolite, geranylgeranylpyrophosphate, play a critical role in cell cycle progression in human peripheral blood mononuclear cells stimulated by phytohemagglutinin. However, mevalonate pathway might be linked to the control of cell proliferation also through the regulation of phosphatidylcholine biosynthesis (Miquel et al., 1998). Fisher et al. (1999) found that 10 mM geranylgeraniol, but not 10 mM farnesol, restored rabbit osteoclast-me-
Fig. 1. The effect of alendronate (•), alendronate1farnesol (h) and alendronate1geranylgeraniol (^) on the growth of macrophages. Cells were exposed to 3–1000 mM alendronate combined with 15 mM farnesol or 20 mM geranylgeraniol for 46 h. Values are the mean6S.D. expressed as % of untreated control (n53).
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diated bone resorption prevented by alendronate in vitro. Furthermore, geranylgeraniol cancelled alendronate-induced inhibition of osteoclast formation in vitro. The inhibitory effects of nitrogen-containing bisphosphonates on osteoclastic resorption can be reversed by 50 mM geranylgeraniol and only slightly by 50 mM farnesol (van
Beek et al., 1999a), and the apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates can be reversed both by 50 mM geranylgeraniol and 50 mM farnesol (Benford et al., 1999). It is most probable that geranylgeraniol, and in some extent also farnesol, can block most of the inhibitory effects of alendronate on cells
Fig. 2. The effect of 1000 mM clodronate (CLO), 15 mM farnesol (FOH), 15 mM geranylgeraniol (GGOH) and 100 mg / ml ox-LDL on (A) IL-1b, (B) IL-6, and (C) TNF-a secretion induced by combined exposure to 100 mM alendronate and 10 mg / ml LPS. The cells were exposed for 20 h and subsequently treated with LPS for 24 h. Values are the mean6S.E.M. expressed as % of LPS control (n54, except that for ox-LDL n53). * P,0.05, Mann–Whitney U-test (treatments were compared with alendronate1LPS treatment).
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at low concentrations (5–20 mM). Concentration of 50 mM was not used in the present study since it was cytotoxic to RAW 264 macrophages.
3.2. Cytokine induction In this study, 100 mM alendronate clearly increased LPS-stimulated cytokine secretion from RAW 264 macrophages. The secretion of IL-1b increased 2.4-fold compared to LPS treated control (Fig. 2A). The productions of IL-6 and TNF-a were also increased 1.4- and 1.8-fold, respectively (Fig. 2B and C). We have previously reported that alendronate augments LPS-stimulated release of IL-1b by increasing the binding of nuclear factor-kB (NF-kB) to DNA (Makkonen et al., 1999). Pietschmann et al. (1998) have also reported that alendronate augments production of proinflammatory cytokines (IL-1b, TNF-a and IFN-g) of human peripheral blood mononuclear cells. In fact, other nitrogen-containing bisphosphonates, like incadronate, ibandronate, pamidronate and zoledronate, have also reported to possess proinflammatory activity (Nakamura et ´ ¨ ¨ al., 1996; Thiebaud et al., 1997; Monkkonen et al., 1998). In vivo, IL-1 seems to be involved in the inflammatory actions of nitrogen-containing bisphosphonates in mice (Yamaguchi et al., 2000). Interestingly, alendronate is reported to enhance LPS-induced IL-1 production in mice, but decrease the production of TNF-a. However, the same decrease of TNF-a has not been seen in in vitro studies (Sugawara et al., 1998; Makkonen et al., 1999). This is consistent with our in vitro findings: IL-1b and TNF-a secretions were increased 2.4- and 1.8-fold, respectively. In addition, nitrogen-containing bisphosphonates are associated with acute phase response in some patients (Adami ´ et al., 1987; Schweitzer et al., 1995; Thiebaud et al., 1997). ¨ ¨ Clodronate (Monkkonen et al., 1994a) and another non-nitrogen-containing bisphosphonate, tiludronate ¨ ¨ (Monkkonen et al., 1998), are capable of inhibiting cytokine production in vitro. Clodronate is metabolised to a toxic ATP-analogue in vitro and in vivo (Frith et al., 1997, 2001) and this metabolite is responsible for the prevention of LPS-stimulated release of cytokines and nitric oxide (NO) from RAW 264 macrophages (Makkonen et al., 1999). In vitro results obtained in the present study clearly demonstrate, that clodronate totally counteracts the secretion of all studied cytokines induced by alendronate and LPS (P,0.015, Fig. 2). This is probably due to the ability of clodronate to inhibit DNA binding activity of transcription factors, NF-kB and activating protein-1 (AP-1) (Makkonen et al., 1999). Endo et al. (1999) have reported that clodronate inhibits inflammatory reactions induced by alendronate, incadronate or ibandronate in mice in vivo. Furthermore, clodronate is described to suppress arthritis in mice and in rabbits in vivo (Dunn et al., 1993; Nakamura et al., 1996; Ceponis et al., 2001). It is known that the patients treated with clodronate do not get the acute phase response and that pre-treatment with
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clodronate enhances the tolerance for acute phase response induced by administration of nitrogen-containing bisphosphonate (Adami et al., 1987). Our findings support the idea presented by Endo et al. (1999), that simultaneous administration of clodronate and nitrogen-containing bisphosphonate could also suppress the acute phase response. Ox-LDL had similar influence on the cytokine production as clodronate: the secretion of IL-6 and TNF-a stimulated by alendronate and LPS was significantly decreased by ox-LDL (P50.025, Fig. 2B and C). Also, the production of IL-1b was diminished, although the effect was not statistically significant (P50.053, Fig. 2A). OxLDL has been shown to prevent mRNA expressions of IL-1b (Fong et al., 1991) and TNF-a (Hamilton et al., 1990) in LPS-stimulated macrophages. Ohlsson et al. (1996) have also revealed that ox-LDL inhibits LPSinduced binding of transcription factors, NF-kB and AP-1, to DNA and the subsequent expression of TNF-a and IL-1b in macrophages. The inhibitory effect of ox-LDL was found both in mRNA and protein levels. Ox-LDL contains different oxysterols and many of them, especially 25-hydroxycholesterol, decrease LPS-induced TNF-a secretion and mRNA expression (Englund et al., 2001). In general, farnesol and geranylgeraniol did not significantly decrease the cytokine secretion induced by alendronate and LPS. However, farnesol reduced secretion of IL-6 (P50.027, Fig. 2). Even though farnesol and geranylgeraniol can prevent alendronate-induced apoptosis (Benford et al., 1999) and alendronate-induced growth inhibition (Fig. 1), we found that they fail to inhibit the inflammatory effects of alendronate. Inhibition of osteoclastic resorption can also be counteracted by geranylgeraniol and slightly by farnesol (van Beek et al., 1999a; Fisher et al., 1999). On the other hand, farnesol and geranylgeraniol did not affect anti-inflammatory properties of clodronate in the present study (data not shown). This suggests that the inhibition of mevalonate pathway is not the mechanism responsible for the proinflammatory response caused by alendronate, as it is in alendronateinduced apoptosis, inhibition of proliferation and prevention of osteoclast function.
3.3. Cytotoxicity The cell viabilities were measured at the end of the cytokine experiment in order to evaluate the cytotoxicity of different treatments. Combined treatment with alendronate and LPS was cytotoxic for macrophages as manifested by lowered cell viability (42% of LPS control, Fig. 3). Clodronate and ox-LDL completely counteracted the cytotoxicity of alendronate, P50.014 and P50.025, respectively. RAW 264 macrophages are quite resistant to toxic influence of clodronate itself (Pennanen et al., 1995b; Makkonen et al., 1996), but the mechanism responsible for protecting cells from alendronate-induced cell death remains to be studied. Hundal et al. (2001) have also
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Fig. 3. The effect of 1000 mM clodronate (CLO), 15 mM farnesol (FOH), 15 mM geranylgeraniol (GGOH) and 100 mg / ml ox-LDL on cell viability of 100 mM alendronate and LPS treated macrophage cells. The cells were exposed for 20 h and subsequently treated with LPS for 24 h. Values are the mean6S.E.M. expressed as % of LPS control (n54, except that for ox-LDL n53). * P,0.05, Mann–Whitney U-test (treatments were compared with alendronate1LPS treatment).
reported that ox-LDL inhibits macrophage apoptosis, induced by withdrawal of growth factor, by activating the phosphatidylinositol 3-kinase / protein kinase B dependent pathway. Furthermore, Hamilton et al. (1999) have found that treatment of murine bone marrow-derived macrophages with ox-LDL leads to survival, DNA synthesis and enhanced response to the proliferative actions of colony stimulating factor-1 (CSF-1) and (GM-CSF). The effects of ox-LDL were dependent on the degree of oxidation, were found in preloaded cells and occurred even in the absence of endogenous CSF-1 or GM-CSF. However, it remains still unclear, how ox-LDL protects cells from the cytotoxicity of alendronate. In the present study, farnesol and geranylgeraniol did not reduce the cytotoxicity of alendronate. On the contrary, farnesol increased the cytotoxic effect of alendronate (P5 0.027, Fig. 3). These opposite results in growth inhibition and in cytotoxicity studies regarding both ox-LDL and farnesol or geranylgeraniol emphasize, that growth inhibition, cytotoxicity and cytokine production are different processes. The cell viabilities at the end of the cytokine experiment after exposure to alendronate or combination of alendronate and farnesol or geranylgeraniol were very low (Fig. 3). However, the cytokine productions were unchanged or even increased in these treatments (Fig. 2). To conclude, fewer cells produced approximately the same amount of cytokines, possibly indicating potent activation of the remaining macrophages by the cell debris. Nitrogen-containing bisphosphonates cause apoptosis in osteoclasts and in macrophages due to the inhibition of mevalonate pathway and subsequent loss of prenylated proteins (Luckman et al., 1998a,b; Benford et al., 1999, 2001). Suri et al. (2001) have found that ox-LDL, farnesol and particularly geranylgeraniol suppress the apoptotic and anti-proliferative effects of nitrogen-containing bisphosphonates in Caco-2 cells. This is partly consistent with the
macrophage experiments of the present study, since farnesol and especially geranylgeraniol were found to reduce anti-proliferative effect of alendronate (Fig. 1). However, farnesol and geranylgeraniol failed to decrease inflammatory and cytotoxic effects of alendronate (Figs. 2 and 3). In conclusion, farnesol and geranylgeraniol are able to inhibit the anti-proliferative effect of alendronate, but they failed to reverse inflammatory and cytotoxic effects of alendronate and LPS in vitro. Clodronate and ox-LDL were able to counteract the proinflammatory and cytotoxic effects of alendronate, probably by their known ability to inhibit DNA binding activity of transcription factors, NFkB and AP-1. These results suggest that inhibition of mevalonate pathway is not the mechanism responsible for the proinflammatory response caused by alendronate, as it is in alendronate-induced apoptosis, inhibition of proliferation and prevention of osteoclast function. These findings also support the idea, that combined administration of clodronate and nitrogen-containing bisphosphonate could suppress the inflammatory side effects of nitrogen-containing bisphosphonates.
Acknowledgements This work was financially supported by Leiras, Finland, TEKES (the National Technology Agency of Finland) and the Academy of Finland.
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´ Hundal, R.S., Salh, B.S., Schrader, J.W., Gomez-Munoz, A., Duronio, V., Steinbrecher, U.P., 2001. Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase / PKB pathway. J. Lipid Res. 42, 1483–1491. ¨ ¨ Lehenkari, P.P., Kellinsalmi, M., Napankangas, J.P., Ylitalo, K.V., ¨ ¨ ¨¨ ¨ Monkkonen, J., Rogers, M.J., Azhayev, A., Vaananen, H.K., Hassinen, I.E., 2002. Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite. Mol. Pharmacol. 62, 1255–1262. Luckman, S.P., Coxon, F.B., Ebetino, F.H., Russell, R.G., Rogers, M.J., 1998a. Heterocycle-containing bisphosphonates cause apoptosis and inhibit bone resorption by preventing protein prenylation: evidence from structure–activity relationships in J774 macrophages. J. Bone Miner. Res. 13, 1668–1678. Luckman, S.P., Hughes, D.E., Coxon, F.P., Russell, R.G.G., Rogers, M.J., 1998b. Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J. Bone Miner. Res. 13, 581–589. ¨ ¨ Makkonen, N., Hirvonen, M.R., Teravainen, T., Savolainen, K., ¨ ¨ Monkkonen, J., 1996. Different effects of three bisphosphonates on nitric oxide production by RAW 264 macrophage-like cells in vitro. J. Pharmacol. Exp. Ther. 277, 1097–1102. Makkonen, N., Salminen, A., Rogers, M.J., Frith, J.C., Urtti, A., ¨ ¨ Azhayeva, E., Monkkonen, J., 1999. Contrasting effects of alendronate and clodronate on RAW 264 macrophages: the role of a bisphosphonate metabolite. Eur. J. Pharm. Sci. 8, 109–118. ´ F., Selmi, S., Favre, G., 1998. CompetiMiquel, K., Pradines, A., Terce, tive inhibition of choline phosphotransferase by geranylgeraniol and farnesol inhibits phosphatidylcholine synthesis and induces apoptosis in human lung adenocarcinoma A549 cells. J. Biol. Chem. 273, 26179–26186. ¨ ¨ Monkkonen, J., Pennanen, N., Lapinjoki, S., Urtti, A., 1994a. Clodronate (dichloromethylene bisphosphonate) inhibits LPS-stimulated IL-6 and TNF production by RAW 264 cells. Life Sci. 54, 229–234. ¨ ¨ Monkkonen, J., Taskinen, M., Auriola, S.O.K., Urtti, A., 1994b. Growth inhibition of macrophage-like and other cell types by liposomeencapsulated, calcium-bound, and free bisphosphonates in vitro. J. Drug Target. 2, 299–308. ¨ ¨ ¨ J., Rogers, M.J., 1998. Effects of tiludronate and Monkkonen, J., Simila, ibandronate on the secretion of proinflammatory cytokines and nitric oxide from macrophages in vitro. Life Sci. 62, 95–102. Nakamura, M., Ando, T., Abe, M., Kumagai, K., Endo, Y., 1996. Contrast between effects of aminobisphosphonates and non-aminobisphosphonates on collagen-induced arthritis in mice. Br. J. Pharmacol. 119, 205–212. Ohlsson, B.G., Englund, M.C.O., Karlsson, A.-L.K., Knutsen, E., Erixon, C., Skribeck, H., Liu, Y., Bondjers, G., Wiklund, O., 1996. Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kB to DNA and the subsequent expression of tumor necrosis factor-a and interleukin-1b in macrophages. J. Clin. Invest. 98, 78–89. ¨ ¨ Pennanen, N., Lapinjoki, S., Palander, A., Urtti, A., Monkkonen, J., 1995a. Macrophage-like RAW 264 cell line and time-resolved fluoroimmunoassay (TRFIA) as tools in screening drug effects on cytokine secretion. Int. J. Immunopharmacol. 17, 475–480. ¨ ¨ Pennanen, N., Lapinjoki, S., Urtti, A., Monkkonen, J., 1995b. Effect of liposomal and free bisphosphonates on the IL-1b, IL-6 and TNFa secretion from RAW 264 cells in vitro. Pharm. Res. 12, 916–921. Pietschmann, P., Stohlawetz, P., Brosch, S., Steiner, G., Smolen, J.S., Peterlik, M., 1998. The effect of alendronate on cytokine production, adhesion molecule expression, and transendothelial migration of human peripheral blood mononuclear cells. Calcif. Tissue Int. 63, 325–330. Raiteri, M., Arnaboldi, L., McGeady, P., Gelb, M.H., Verri, D., Tagliabue, C., Quarato, P., Ferraboschi, P., Santaniello, E., Paoletti, R., Fumagalli, R., Corsini, A., 1997. Pharmacological control of the mevalonate pathway: effect on arterial smooth muscle cell proliferation. J. Pharmacol. Exp. Ther. 281, 1144–1153.
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Inhibition of mevalonate pathway is involved in alendronate-induced cell growth inhibition, but not in cytokine secretion from macrophages in vitro ¨ ¨ *, Jouko Ollikainen, Markku Taskinen, Jukka Monkkonen ¨ ¨ Anu Toyras Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland Received 1 October 2002; received in revised form 13 March 2003; accepted 10 April 2003
Abstract Bisphosphonates are antiresorptive drugs used for the treatment of metabolic bone diseases. They can be divided into two different pharmacological classes: nitrogen-containing and non-nitrogen-containing bisphosphonates. Non-nitrogen-containing bisphosphonates, like clodronate, are metabolised to a toxic ATP-analogue preventing osteoclast mediated bone resorption. Nitrogen-containing bisphosphonates, including alendronate, prevent osteoclast function by inhibiting the mevalonate pathway. Clodronate is known to have anti-inflammatory properties while alendronate induces cytokine secretion from lipopolysaccharide- (LPS) induced macrophages. This study investigates whether the cytotoxicity and cytokine production induced by alendronate and LPS could be counteracted by clodronate or products of mevalonate pathway: oxidized low density lipoprotein (ox-LDL), farnesol and geranylgeraniol. Treatment with alendronate increased LPS-induced secretion of IL-1b, IL-6 and TNF-a from RAW 264 macrophages 2.4-, 1.4- and 1.8-fold, respectively. This treatment was cytotoxic for macrophages as indicated by lowered cell viability. Clodronate and ox-LDL both counteracted the cytokine secretion and cytotoxicity of alendronate. Farnesol and geranylgeraniol did neither reverse the cytokine secretion nor reduce the cytotoxicity of alendronate. Clodronate and ox-LDL were able to counteract the effects of alendronate on macrophages in vitro, probably by their known ability to inhibit DNA binding activity of transcription factors, nuclear factor-kB (NF-kB) and activating protein-1 (AP-1). These findings suggest that inhibition of mevalonate pathway is not the mechanism responsible for the proinflammatory response caused by alendronate, as it is in alendronate-induced apoptosis and prevention of osteoclast function. 2003 Elsevier B.V. All rights reserved. Keywords: Bisphosphonate; Mevalonate pathway; Cytokines; Farnesol; Geranylgeraniol; Oxidized low density lipoprotein
1. Introduction Bisphosphonates are antiresorptive drugs used for the treatment of metabolic bone diseases. They can be divided into two different pharmacological classes: nitrogen-containing and non-nitrogen-containing bisphosphonates. Clodronate and other non-nitrogen-containing bisphosphonates, closely resembling an inorganic pyrophosphate, are metabolised to a toxic ATP-analogue preventing osteoclast mediated bone resorption (Frith et al., 1997; Rogers et al., 2000). Inhibition of the mitochondrial ADP/ATP translocase by the metabolite of clodronate, AppCCl 2 p (adenosine-59-[b,g-dichloromethylene]triphosphate), is likely the route by which clodronate causes primary apoptosis of osteoclasts (Benford et al., 2001; Lehenkari et al., 2002). *Corresponding author. Tel.: 1358-17-163-461; fax: 1358-17-162252. ¨ ¨ E-mail address: [email protected] (A. Toyras). 0928-0987 / 03 / $ – see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0928-0987(03)00108-8
Nitrogen-containing bisphosphonates, such as alendronate, inhibit the mevalonate pathway, which is responsible for the production of cholesterol and isoprenoid lipids. The exact molecular target is considered to be farnesyl pyrophosphate synthase (van Beek et al., 1999b). The consequent loss of prenylated proteins, particularly geranylgeranylated, seems to be the main reason for the inhibition of osteoclast function and secondary apoptosis caused by nitrogen-containing bisphosphonates (van Beek et al., 1999a; Fisher et al., 1999; Coxon et al., 2000; Benford et al., 2001). The cell-permeable products of mevalonate pathway, farnesol and geranylgeraniol, have been described to suppress the apoptosis caused by nitrogen-containing bisphosphonates (Benford et al., 1999). Clodronate and its metabolite are known to have antiinflammatory properties while alendronate increases lipopolysaccharide- (LPS) induced cytokine secretion of macrophages in vitro (Makkonen et al., 1999). In vivo, alendronate has been reported to exacerbate the arthritis in mice, whereas clodronate was able to suppress the arthritis
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(Nakamura et al., 1996). An acute phase response, to which increased level of interleukin-6 (IL-6) is typically related, is developed for some patients treated with nitrogen-containing bisphosphonate for the first time (Schweitzer et al., 1995). Clodronate has been found to inhibit alendronate-induced inflammatory reaction in vivo, suggesting that combined administration of clodronate and nitrogen-containing bisphosphonate could prevent the acute phase response (Endo et al., 1999). The aim of this study was to investigate the ability of clodronate to counteract the cytotoxic and inflammatory effects of alendronate, in vitro. Intermediates of mevalonate pathway, farnesol and geranylgeraniol, are known to be capable of preventing apoptosis caused by alendronate (Benford et al., 1999). In this study their possible capability to reverse cytokine secretion and macrophage growth inhibition induced by alendronate was investigated. Oxidized LDL (ox-LDL) contains cholesterol, the end product of mevalonate pathway, and induces murine macrophage growth and survival (Biwa et al., 1998; Hamilton et al., 1999). It is also known to inhibit expression of tumor necrosis factor-a (TNF-a) and interleukin-1b (IL-1b) (Ohlsson et al., 1996). The ability of ox-LDL to counteract proinflammatory and cytotoxic effects of alendronate was also examined.
2. Materials and methods
2.1. Materials Alendronate was provided by Merck Sharp and Dohme (Rahway, NJ, USA) and clodronate was obtained from Leiras Oy (Turku, Finland). Stock solutions (100 mM alendronate, 110 mM clodronate) were prepared in water and pH 7.02 was adjusted with NaHCO 3 . Ox-LDL was obtained from Dr Pauliina Lehtolainen, A.I. Virtanen Institute, University of Kuopio, Finland. LDL was oxidized by overnight incubation with copper ions (20 mmol / ¨ l) as described earlier (Yla-Herttuala et al., 1989). Farnesol was purchased from Sigma–Aldrich (Steinheim, Germany). Geranylgeraniol, LPS (lipopolysaccharide from E. coli) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) were purchased from Sigma (St. Louis, MO, USA). Recombinant mouse IL-1b, IL-6, TNFa / TNFSF2, anti-mouse IL-1b and IL-6 monoclonal antibodies, anti-mouse TNF-a / TNFSF2 polyclonal antibody and biotinylated anti-mouse IL-1b, IL-6, TNF-a antibodies were bought from R&D Systems (Minneapolis, MN, USA).
(Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum and 100 IU / ml penicillin–streptomycin in 7% CO 2 atmosphere at 37 8C. In growth inhibition studies cells were dispensed with a density of 4310 3 cells / well on 96-well plates and allowed to grow overnight. Cells were exposed to 3–1000 mM alendronate combined with 15 mM farnesol or 20 mM geranylgeraniol for 46 h. Cell growth was measured by using an MTT assay (Hansen et al., 1989).
2.2.2. Cytokine induction In the cytokine experiments cells were dispensed to 96-well plates as monolayer (2310 5 cells / well) and the cells were allowed to adhere for 4–5 h. The cells were treated with 100 mM alendronate combined with 1000 mM clodronate, 15 mM farnesol, 15 mM geranylgeraniol or 100 mg / ml ox-LDL for 20 h. These media were removed and cells were instantly treated with LPS in serum free DMEM (10 mg / ml) for 24 h in order to induce the cytokine secretion. Cell culture media were collected (four wells were pooled), centrifuged and stored at 280 8C. The cell viability was evaluated with MTT assay both after drug treatment and after LPS treatment. In contrast to other cell experiments, which were carried out four times, ox-LDL experiments were reproduced three times. 2.2.3. Time-resolved fluoroimmunoassay for IL-1b, IL-6 and TNF-a Principle of time-resolved fluoroimmunoassay has been described earlier (Pennanen et al., 1995a). Briefly, the microtiter strips were coated with the capture antibody, 4 mg / ml of IL-1b, 1 mg / ml of IL-6 or 0.8 mg / ml of TNF-a in PBS over night in room temperature. After three washings with 0.05% Tween 20 in PBS the non-specific binding was blocked for 1 h with PBS solution containing 1% bovine serum albumin (BSA) and 5% saccharose. Subsequently, the washings were repeated. The samples and the standards (diluted in 0.1% BSA, 0.05% Tween 20, 20 mM Tris, 150 mM NaCl pH 7.3) were incubated for 2 h and washed as earlier. The biotinylated detection antibodies (200 ng / ml of IL-1b and IL-6, 300 ng / ml of TNF-a) were incubated for 2 h followed by four washings. Eu-labelled streptavidin (100 ng / ml) in Delfia assay buffer (Wallac Oy, Turku, Finland) was dispensed to wells and incubated for 30 min with gently shaking. Strips were washed six times before adding of Delfia enhancement solution. After 20 min incubation the fluorescence was measured with Victor 2 1420 Multilabel counter (Wallac Oy, Turku, Finland). 2.3. Statistical analysis
2.2. Methods 2.2.1. Cell culture and growth inhibition studies RAW 264 murine macrophage cells were grown in Dulbecco’s modified Eagle’s medium 21885 (DMEM)
SPSS 9.0.1 software (SPSS, Chicago, IL, USA) was used for statistical analyses. The statistical differences between treatments were investigated by using nonparametric Mann–Whitney U-test. Different treatments
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were compared with combined treatment with alendronate and LPS.
3. Results and discussion
3.1. Growth inhibition studies In this study the ability of farnesol and geranylgeraniol to enhance growth of alendronate treated RAW 264 macrophage cells was investigated. Exposure to 100 mM alendronate diminished macrophage cell growth to 81% of that of the untreated controls, while treatment with 300 mM alendronate decreased the growth to only 1% (Fig. 1). Farnesol and especially geranylgeraniol were able to partly counteract the growth inhibitory effect of alendronate. Cell growth was 58 and 23% of that of control when 300 mM alendronate exposure was combined with 20 mM geranylgeraniol or 15 mM farnesol treatment, respectively (Fig. 1). The capability of farnesol and geranylgeraniol to inhibit macrophage cell growth was also investigated. Farnesol (15 mM) and geranylgeraniol (15 mM) reduced macrophage cell growth to 83 and 90% of that of control, respectively. These concentrations of farnesol and geranygeraniol were used in further cytokine experiments. Bisphosphonates are known to inhibit growth of amoebae of the slime mold (Dictyostelium discoideum) (Rogers et al., 1994, 1995) and prevent macrophage ¨ ¨ proliferation (Cecchini et al., 1987; Monkkonen et al., 1994b). Nitrogen-containing bisphosphonates inhibit mevalonate pathway leading to absence of prenylated proteins (Luckman et al., 1998a,b). Our data demonstrates that growth of macrophages was considerably inhibited by alendronate, and farnesol (15 mM) and particularly geranylgeraniol (20 mM), intermediates of mevalonate pathway, counteracted this effect (Fig. 1). Suri et al. (2001) have also found that ox-LDL, farnesol and particularly
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geranylgeraniol suppress anti-proliferative effects of nitrogen-containing bisphosphonates in Caco-2 cells. Biwa et al. (1998) have reported that granulocyte macrophage colony stimulating factor (GM-CSF) is involved in ox-LDL-induced murine macrophage growth. According to Chisolm and Chai (2000), lysophosphatidylcholine, and structurally related lipids, are among the growth-promoting constituents of ox-LDL. However, oxLDL did not have any effect on alendronate-induced growth inhibition in the present study (data not shown). This is probably due to its inability to counteract the alendronate-induced absence of prenylated proteins. OxLDL contains cholesterol, one of the end products of mevalonate pathway, which, in contrast to farnesol and geranylgeraniol, can not replace the prenylated proteins. Clodronate was not included in growth inhibition studies as 1000 mM clodronate itself strongly inhibits the growth of RAW 264 cells and the IC 50 value for clodronate is 980 ¨ ¨ mM (Monkkonen et al., 1994b). Raiteri et al. (1997) found that proliferation of arterial smooth muscle cells was reduced after exposure of drugs affecting different steps of mevalonate pathway. Furthermore, they found that the reduction was prevented by treatment with 5 mM all-trans geranylgeraniol and in most cases by treatment with 10 mM all-trans farnesol or 100 mM mevalonate. They suggested that farnesylated and geranylgeranylated proteins are involved in the control of cell proliferation. According to Tatsuno et al. (1997) mevalonate pathway and its metabolite, geranylgeranylpyrophosphate, play a critical role in cell cycle progression in human peripheral blood mononuclear cells stimulated by phytohemagglutinin. However, mevalonate pathway might be linked to the control of cell proliferation also through the regulation of phosphatidylcholine biosynthesis (Miquel et al., 1998). Fisher et al. (1999) found that 10 mM geranylgeraniol, but not 10 mM farnesol, restored rabbit osteoclast-me-
Fig. 1. The effect of alendronate (•), alendronate1farnesol (h) and alendronate1geranylgeraniol (^) on the growth of macrophages. Cells were exposed to 3–1000 mM alendronate combined with 15 mM farnesol or 20 mM geranylgeraniol for 46 h. Values are the mean6S.D. expressed as % of untreated control (n53).
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diated bone resorption prevented by alendronate in vitro. Furthermore, geranylgeraniol cancelled alendronate-induced inhibition of osteoclast formation in vitro. The inhibitory effects of nitrogen-containing bisphosphonates on osteoclastic resorption can be reversed by 50 mM geranylgeraniol and only slightly by 50 mM farnesol (van
Beek et al., 1999a), and the apoptosis of J774 macrophages induced by nitrogen-containing bisphosphonates can be reversed both by 50 mM geranylgeraniol and 50 mM farnesol (Benford et al., 1999). It is most probable that geranylgeraniol, and in some extent also farnesol, can block most of the inhibitory effects of alendronate on cells
Fig. 2. The effect of 1000 mM clodronate (CLO), 15 mM farnesol (FOH), 15 mM geranylgeraniol (GGOH) and 100 mg / ml ox-LDL on (A) IL-1b, (B) IL-6, and (C) TNF-a secretion induced by combined exposure to 100 mM alendronate and 10 mg / ml LPS. The cells were exposed for 20 h and subsequently treated with LPS for 24 h. Values are the mean6S.E.M. expressed as % of LPS control (n54, except that for ox-LDL n53). * P,0.05, Mann–Whitney U-test (treatments were compared with alendronate1LPS treatment).
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at low concentrations (5–20 mM). Concentration of 50 mM was not used in the present study since it was cytotoxic to RAW 264 macrophages.
3.2. Cytokine induction In this study, 100 mM alendronate clearly increased LPS-stimulated cytokine secretion from RAW 264 macrophages. The secretion of IL-1b increased 2.4-fold compared to LPS treated control (Fig. 2A). The productions of IL-6 and TNF-a were also increased 1.4- and 1.8-fold, respectively (Fig. 2B and C). We have previously reported that alendronate augments LPS-stimulated release of IL-1b by increasing the binding of nuclear factor-kB (NF-kB) to DNA (Makkonen et al., 1999). Pietschmann et al. (1998) have also reported that alendronate augments production of proinflammatory cytokines (IL-1b, TNF-a and IFN-g) of human peripheral blood mononuclear cells. In fact, other nitrogen-containing bisphosphonates, like incadronate, ibandronate, pamidronate and zoledronate, have also reported to possess proinflammatory activity (Nakamura et ´ ¨ ¨ al., 1996; Thiebaud et al., 1997; Monkkonen et al., 1998). In vivo, IL-1 seems to be involved in the inflammatory actions of nitrogen-containing bisphosphonates in mice (Yamaguchi et al., 2000). Interestingly, alendronate is reported to enhance LPS-induced IL-1 production in mice, but decrease the production of TNF-a. However, the same decrease of TNF-a has not been seen in in vitro studies (Sugawara et al., 1998; Makkonen et al., 1999). This is consistent with our in vitro findings: IL-1b and TNF-a secretions were increased 2.4- and 1.8-fold, respectively. In addition, nitrogen-containing bisphosphonates are associated with acute phase response in some patients (Adami ´ et al., 1987; Schweitzer et al., 1995; Thiebaud et al., 1997). ¨ ¨ Clodronate (Monkkonen et al., 1994a) and another non-nitrogen-containing bisphosphonate, tiludronate ¨ ¨ (Monkkonen et al., 1998), are capable of inhibiting cytokine production in vitro. Clodronate is metabolised to a toxic ATP-analogue in vitro and in vivo (Frith et al., 1997, 2001) and this metabolite is responsible for the prevention of LPS-stimulated release of cytokines and nitric oxide (NO) from RAW 264 macrophages (Makkonen et al., 1999). In vitro results obtained in the present study clearly demonstrate, that clodronate totally counteracts the secretion of all studied cytokines induced by alendronate and LPS (P,0.015, Fig. 2). This is probably due to the ability of clodronate to inhibit DNA binding activity of transcription factors, NF-kB and activating protein-1 (AP-1) (Makkonen et al., 1999). Endo et al. (1999) have reported that clodronate inhibits inflammatory reactions induced by alendronate, incadronate or ibandronate in mice in vivo. Furthermore, clodronate is described to suppress arthritis in mice and in rabbits in vivo (Dunn et al., 1993; Nakamura et al., 1996; Ceponis et al., 2001). It is known that the patients treated with clodronate do not get the acute phase response and that pre-treatment with
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clodronate enhances the tolerance for acute phase response induced by administration of nitrogen-containing bisphosphonate (Adami et al., 1987). Our findings support the idea presented by Endo et al. (1999), that simultaneous administration of clodronate and nitrogen-containing bisphosphonate could also suppress the acute phase response. Ox-LDL had similar influence on the cytokine production as clodronate: the secretion of IL-6 and TNF-a stimulated by alendronate and LPS was significantly decreased by ox-LDL (P50.025, Fig. 2B and C). Also, the production of IL-1b was diminished, although the effect was not statistically significant (P50.053, Fig. 2A). OxLDL has been shown to prevent mRNA expressions of IL-1b (Fong et al., 1991) and TNF-a (Hamilton et al., 1990) in LPS-stimulated macrophages. Ohlsson et al. (1996) have also revealed that ox-LDL inhibits LPSinduced binding of transcription factors, NF-kB and AP-1, to DNA and the subsequent expression of TNF-a and IL-1b in macrophages. The inhibitory effect of ox-LDL was found both in mRNA and protein levels. Ox-LDL contains different oxysterols and many of them, especially 25-hydroxycholesterol, decrease LPS-induced TNF-a secretion and mRNA expression (Englund et al., 2001). In general, farnesol and geranylgeraniol did not significantly decrease the cytokine secretion induced by alendronate and LPS. However, farnesol reduced secretion of IL-6 (P50.027, Fig. 2). Even though farnesol and geranylgeraniol can prevent alendronate-induced apoptosis (Benford et al., 1999) and alendronate-induced growth inhibition (Fig. 1), we found that they fail to inhibit the inflammatory effects of alendronate. Inhibition of osteoclastic resorption can also be counteracted by geranylgeraniol and slightly by farnesol (van Beek et al., 1999a; Fisher et al., 1999). On the other hand, farnesol and geranylgeraniol did not affect anti-inflammatory properties of clodronate in the present study (data not shown). This suggests that the inhibition of mevalonate pathway is not the mechanism responsible for the proinflammatory response caused by alendronate, as it is in alendronateinduced apoptosis, inhibition of proliferation and prevention of osteoclast function.
3.3. Cytotoxicity The cell viabilities were measured at the end of the cytokine experiment in order to evaluate the cytotoxicity of different treatments. Combined treatment with alendronate and LPS was cytotoxic for macrophages as manifested by lowered cell viability (42% of LPS control, Fig. 3). Clodronate and ox-LDL completely counteracted the cytotoxicity of alendronate, P50.014 and P50.025, respectively. RAW 264 macrophages are quite resistant to toxic influence of clodronate itself (Pennanen et al., 1995b; Makkonen et al., 1996), but the mechanism responsible for protecting cells from alendronate-induced cell death remains to be studied. Hundal et al. (2001) have also
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Fig. 3. The effect of 1000 mM clodronate (CLO), 15 mM farnesol (FOH), 15 mM geranylgeraniol (GGOH) and 100 mg / ml ox-LDL on cell viability of 100 mM alendronate and LPS treated macrophage cells. The cells were exposed for 20 h and subsequently treated with LPS for 24 h. Values are the mean6S.E.M. expressed as % of LPS control (n54, except that for ox-LDL n53). * P,0.05, Mann–Whitney U-test (treatments were compared with alendronate1LPS treatment).
reported that ox-LDL inhibits macrophage apoptosis, induced by withdrawal of growth factor, by activating the phosphatidylinositol 3-kinase / protein kinase B dependent pathway. Furthermore, Hamilton et al. (1999) have found that treatment of murine bone marrow-derived macrophages with ox-LDL leads to survival, DNA synthesis and enhanced response to the proliferative actions of colony stimulating factor-1 (CSF-1) and (GM-CSF). The effects of ox-LDL were dependent on the degree of oxidation, were found in preloaded cells and occurred even in the absence of endogenous CSF-1 or GM-CSF. However, it remains still unclear, how ox-LDL protects cells from the cytotoxicity of alendronate. In the present study, farnesol and geranylgeraniol did not reduce the cytotoxicity of alendronate. On the contrary, farnesol increased the cytotoxic effect of alendronate (P5 0.027, Fig. 3). These opposite results in growth inhibition and in cytotoxicity studies regarding both ox-LDL and farnesol or geranylgeraniol emphasize, that growth inhibition, cytotoxicity and cytokine production are different processes. The cell viabilities at the end of the cytokine experiment after exposure to alendronate or combination of alendronate and farnesol or geranylgeraniol were very low (Fig. 3). However, the cytokine productions were unchanged or even increased in these treatments (Fig. 2). To conclude, fewer cells produced approximately the same amount of cytokines, possibly indicating potent activation of the remaining macrophages by the cell debris. Nitrogen-containing bisphosphonates cause apoptosis in osteoclasts and in macrophages due to the inhibition of mevalonate pathway and subsequent loss of prenylated proteins (Luckman et al., 1998a,b; Benford et al., 1999, 2001). Suri et al. (2001) have found that ox-LDL, farnesol and particularly geranylgeraniol suppress the apoptotic and anti-proliferative effects of nitrogen-containing bisphosphonates in Caco-2 cells. This is partly consistent with the
macrophage experiments of the present study, since farnesol and especially geranylgeraniol were found to reduce anti-proliferative effect of alendronate (Fig. 1). However, farnesol and geranylgeraniol failed to decrease inflammatory and cytotoxic effects of alendronate (Figs. 2 and 3). In conclusion, farnesol and geranylgeraniol are able to inhibit the anti-proliferative effect of alendronate, but they failed to reverse inflammatory and cytotoxic effects of alendronate and LPS in vitro. Clodronate and ox-LDL were able to counteract the proinflammatory and cytotoxic effects of alendronate, probably by their known ability to inhibit DNA binding activity of transcription factors, NFkB and AP-1. These results suggest that inhibition of mevalonate pathway is not the mechanism responsible for the proinflammatory response caused by alendronate, as it is in alendronate-induced apoptosis, inhibition of proliferation and prevention of osteoclast function. These findings also support the idea, that combined administration of clodronate and nitrogen-containing bisphosphonate could suppress the inflammatory side effects of nitrogen-containing bisphosphonates.
Acknowledgements This work was financially supported by Leiras, Finland, TEKES (the National Technology Agency of Finland) and the Academy of Finland.
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