Bisphosphonates induce apoptosis in mouse macrophage-like cells by a mechanism that does not involve...
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Bone Vol. 17, No. 6 Abstracts 605 December 1995:597-618 29 BISPHOSPHONATES INDUCE APOPTOSIS IN MOUSE MACROPHAGE-LIKE CELLS BY A MECHANISM THAT DOES NOT INVOLVE CHANGES IN THE LEVEL OF NITRIC OXIDE OR p53 M,J, Ro~ers. K.M. Chilton. S. Suri and R.G.G. Russell. Department of Human Metabolism& Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, SI0 2RX, UK. Although the exact mechanisms by which bisphosphonates inhibit bone resorption have not been identified, one likely mechanism is a toxic effect on osteoclasts that have resorbed bisphosphonate-coated bone mineral. This toxic effect has recently been ascribed to induction of apoptosis. Maerophages, like osteoclasts, are also particularly sensitive to the toxic effects of bisphosphonates in vitro. We have recently demonstrated (on the basis of changes in nuclear morphology and internucleosomal DNA fragmentation) that this, too, is due to induction of apoptotie cell death. Nitric oxide (NO) appears to be one mediator of apoptosis in mouse macrophages, following expression of the inducible isofonn of NO synthase after macrophage activation. Also, NO-induced apoptosis has recendy been reported to be associated with increased levels of the apoptosis-promoting factor p53. Since NO is produced by osteoclasts and other bone cells and, like bisphosphonates, also inhibits bone resorption and causes morphological changes in osteoclasts, we have examined whether induction of apoptosis by bisphosphonates in macrophages (and hence in osteoclasts) could be mediated by increased synthesis of NO, and whether bisphosphonate-induced apoptosis also involves changes in the level of p53. Using the Greiss assay, the concentration of nitrite (a stable oxidation product of NO) was determined in conditioned medium from the mouse macrophage-like cell lines J774 and RAW264 that had been treated with lmM clodronate or etidronate, or 100p.M pamidronate, alendronate or ibandronate. While substantial apoptosis was evident after 24-48 hours, none of the bisphosphonates caused an increase in the formation of nitrite above that in control cultures (about ll.tM). By contrast, LPS+IFN-7 treatment increased nitrite formation by almost 100 fold. In addition, 500I~M L-NIO or lmM L-NAME, potent inhibitors of NO synthases, did not prevent bisphosphonate-induced apoptosis. Immuooprecipitation and Western blotting of p53 using a pantropic p53 monoclonal antibody indicated that there was no apparent difference in the level of p53 between cultures of control cells and apoptotic, alendronate-treated cells. These results indicate that bisphosphonate-induced apoptosis in macrophages does not involve increased levels of NO or p53. 30 LIPOSOME-ENCAPSULATED BISPHOSPHONATES ARE MORE POTENT GROWTH INHIBITORS OF DICTYOSTELIUM AMOEBAE THAN FREE BISPHOSPHONATES M,,I, l~o~ers. D.J. Watts. R.G.O. Russell and J. MOnkktJnen*. Dept. of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, SI0 2RX, UK, and *Dept. of Pharmaceutics, University of Kuopio, Finland. There is increasing evidence that bisphosphonates have an intracellular mechanism of action, since ceils that are highly phagocytic or endocytic, such as ostooclasts and maerophages, appear to be most susceptible to the toxic or anti-proliferative effects of these compounds. Liposomal encapsulation of bisphosphonates also enhances the potency of the drugs towards phagocytic cells, since a high intracellular concentration of bisphosphonate is achieved by ingestion of the liposomes and the subsequent release of bisphosphonate. We have previously shown that amoebae of the eukaryotic micro- organism Dictyostelium discoideum are also particularly sensitive to bisphosphonates. Furthermore, the order of potency of bisphosphonates as inhibitors of growth of Dictyostelium closely matches the order of potency as inhibitors of bone resorption, suggesting that bisphosphonates act by a similar mechanism in Dictyostelium and in osteoclasts (Rogers et al, J. Bone Min. Res. 9, 1029-1039, 1994). As with osteoelasts and macrophages, the toxic and growth-inhibitory effect of bisphosphonates towards Dictyostelium is dependent on the cellular uptake of bisphospbonates, which in Dictyostelium appears to occur initially by fluid-phase pinocytosis. Since Dictyostelium amoebae are also phagocytic, we have now examined whether the amoebae are also affected by bisphosphonates contained within liposomes. Both ciodronate and pamidronate were found to be considerably more potent towards Dictyostelium when contained within negatively-charged DSPG liposomes. Following encapsulation, the IC50 for inhibition of growth by clodronate was reduced from 5001.tM to less than 251,tM, while the IC50 for pamidronate was reduced from 150I~M to about 101~M.Empty liposomes had no effect on cell growth or viability. These observations support the view that bisphosphonates exert their anti-proliferative and cytotoxie effects by an intracellular mechanism. Since we have previously reported that free clodronate may act intracellularly in Dictyosteliton due to the metabolism of the bisphosphonate into a potentially toxic, non*hydrolysable analogue of ATP, we are investigating whether liposomal clodronate is also metabolised in the same manner. 31 CLODRONATE INHIBITS MMP-1 AND MMP-8 IN GCF AND PISF. O, Teronen*. YT. Konttinen. T. Salo. T. In~,man and T. Sorsa. Departments of Oral and Maxillofacial Surgery*, Periodontology and Anatomy, Universities of Helsinki and Oulu, Finland. * Institute of Dentistry, University of Helsinki, PB 41 (Marmerheimintie 172), FIN-00014, University of Helsinki, Finland Proteolytic enzymes, especially matrix metaUoproteinases are known to take part in the periodontal tissue remodelling and destruction during periodontitis and peri-implantitis. Clodronate is a well tolerated drug in the group of bisphosphonates currently used in the treatment of some bone related diseases in humans, due to its inhibitory effect on osteoclastic bone resorption. However, the basic mechanism is not yet known. We studied the effect of clodronate on purified MMP-1 and -8 and on the collagenolytic activity in gingival crevicular fluid (GCF) and pefi-implant sulcus fluid (P1SF) collected with filter paper strips. Purified MMP-8, -1, GCF and PISF were each incubated with type I collagen in a concentration series of clodronate. The collagen degradation was observed using SDS-PAGE/Laser Densitometry techniques. Effect of clodronate on gelatinases, MMP-9 and -2, was also studied using 1251-labelled gelatin as a substrate. Degradation of gelatin was measured using a gamma scintillation counter. Collagenolytic activity of purified MMP-1, MMP-8, PISF and GCF was effectively inhibited in dose dependent manner at clinically attainable concentrations <I00 I.tM. However, clodronate did not inhibit MMP-2 or MMP-9 at even higher concentrations. The results su~,~est that cloclronate can inhibit MMP-1 and MMP- 8 in human inflammatory exudates (GCF. PISP-'3 and may be used lueallv or systemically in the treatment of a variety of diseases characterized by excessive colla~enolvtic activity. Supported by ITI- Foundation and The Academy of Finland. 32 DISSOCIATION OF BINDING AND ANTIRESORPTIVE PROPERTIES OF HYDROXYBISPHOSPHONATES BY AN AMINO SUBSTITUTION OF THE HYDROXYL GROUP. E. van Beck. C.W.G.M. Ltwik and S.E.Paoaooulos. Department of Endocrinology, University Hospital, 2333 AA Leiden, The Netherlands. Geminal bisphosphonates contain a P-C-P bond and two side chains of variable length (R1 and R2 respectively). Although no clear structure- function relations of bisphosphonates have been demonstrated, R1 is generally believed to be involved in the binding of bisphosphonates to bone mineral and not to contribute to their cellular effects. Bisphosphonates with a OH group at R 1 have high affinity for bone mineral. This is attributed to the tridendate binding of this structures to crystals surfaces. Tridendate binding can be also obtained by substituting the OH with an NH2 group, as suggested previously. If, therefore, R1 is not important for cellular activity then such substitution will theoretically not affect the antiresorptive effect of hydroxybisphosphonates. To test this hypothesis we substituted the OH group of 3 clinically useful bisphosphonates (etidronate, pamidronate, olpadronate) with an NH2 group leaving the R2 intact. The effects of these bisphosphonates were compared in in vitro assays some of which are representative for the in vivo effects of bisphospbonates (JBMR 1994; 12: 1875-82). All three aminosubstituted bisphosphonates bound to bone mineral and inhibited the growth of calcium oxalate crystals to a similar degree as their hydroxy- counterparts. There were, however, marked differences in their antiresorptive potencies. The antiresorptive effect of olpadronate was totally abolished by the aminosubstitution while this of pamidronate was reduced by about six-fold. Etidronate and its aminosubstituted analog had identical antiresorptive potencies. In conclusion, the structure of R1 can greatly affect the antiresorptive potency of bisphosphonates without altering their affinity for bone. These studies demonstrate that the whole bisphosphonate molecule is important for cellular activity and provide a model compound (the aminosubstituted olpadronate) for the study of molecular mechanisms involved in bisphosphonate action.
Transcript of Bisphosphonates induce apoptosis in mouse macrophage-like cells by a mechanism that does not involve...
Bone Vol. 17, No. 6 Abstracts 605 December 1995:597-618
29 BISPHOSPHONATES INDUCE APOPTOSIS IN MOUSE MACROPHAGE-LIKE CELLS BY A MECHANISM THAT DOES NOT INVOLVE CHANGES IN THE LEVEL OF NITRIC OXIDE OR p53 M,J, Ro~ers. K.M. Chilton. S. Suri and R.G.G. Russell. Department of Human Metabolism& Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, SI0 2RX, UK.
Although the exact mechanisms by which bisphosphonates inhibit bone resorption have not been identified, one likely mechanism is a toxic effect on osteoclasts that have resorbed bisphosphonate-coated bone mineral. This toxic effect has recently been ascribed to induction of apoptosis. Maerophages, like osteoclasts, are also particularly sensitive to the toxic effects of bisphosphonates in vitro. We have recently demonstrated (on the basis of changes in nuclear morphology and internucleosomal DNA fragmentation) that this, too, is due to induction of apoptotie cell death.
Nitric oxide (NO) appears to be one mediator of apoptosis in mouse macrophages, following expression of the inducible isofonn of NO synthase after macrophage activation. Also, NO-induced apoptosis has recendy been reported to be associated with increased levels of the apoptosis-promoting factor p53. Since NO is produced by osteoclasts and other bone cells and, like bisphosphonates, also inhibits bone resorption and causes morphological changes in osteoclasts, we have examined whether induction of apoptosis by bisphosphonates in macrophages (and hence in osteoclasts) could be mediated by increased synthesis of NO, and whether bisphosphonate-induced apoptosis also involves changes in the level of p53.
Using the Greiss assay, the concentration of nitrite (a stable oxidation product of NO) was determined in conditioned medium from the mouse macrophage-like cell lines J774 and RAW264 that had been treated with lmM clodronate or etidronate, or 100p.M pamidronate, alendronate or ibandronate. While substantial apoptosis was evident after 24-48 hours, none of the bisphosphonates caused an increase in the formation of nitrite above that in control cultures (about ll.tM). By contrast, LPS+IFN-7 treatment increased nitrite formation by almost 100 fold. In addition, 500I~M L-NIO or lmM L-NAME, potent inhibitors of NO synthases, did not prevent bisphosphonate-induced apoptosis. Immuooprecipitation and Western blotting of p53 using a pantropic p53 monoclonal antibody indicated that there was no apparent difference in the level of p53 between cultures of control cells and apoptotic, alendronate-treated cells.
These results indicate that bisphosphonate-induced apoptosis in macrophages does not involve increased levels of NO or p53.
30 LIPOSOME-ENCAPSULATED BISPHOSPHONATES ARE MORE POTENT GROWTH INHIBITORS OF DICTYOSTELIUM AMOEBAE THAN FREE BISPHOSPHONATES M,,I, l~o~ers. D.J. Watts. R.G.O. Russell and J. MOnkktJnen*. Dept. of Human Metabolism & Clinical Biochemistry, University of Sheffield Medical School, Beech Hill Road, Sheffield, SI0 2RX, UK, and *Dept. of Pharmaceutics, University of Kuopio, Finland.
There is increasing evidence that bisphosphonates have an intracellular mechanism of action, since ceils that are highly phagocytic or endocytic, such as ostooclasts and maerophages, appear to be most susceptible to the toxic or anti-proliferative effects of these compounds. Liposomal encapsulation of bisphosphonates also enhances the potency of the drugs towards phagocytic cells, since a high intracellular concentration of bisphosphonate is achieved by ingestion of the liposomes and the subsequent release of bisphosphonate.
We have previously shown that amoebae of the eukaryotic micro- organism Dictyostelium discoideum are also particularly sensitive to bisphosphonates. Furthermore, the order of potency of bisphosphonates as inhibitors of growth of Dictyostelium closely matches the order of potency as inhibitors of bone resorption, suggesting that bisphosphonates act by a similar mechanism in Dictyostelium and in osteoclasts (Rogers et al, J. Bone Min. Res. 9, 1029-1039, 1994). As with osteoelasts and macrophages, the toxic and growth-inhibitory effect of bisphosphonates towards Dictyostelium is dependent on the cellular uptake of bisphospbonates, which in Dictyostelium appears to occur initially by fluid-phase pinocytosis.
Since Dictyostelium amoebae are also phagocytic, we have now examined whether the amoebae are also affected by bisphosphonates contained within liposomes. Both ciodronate and pamidronate were found to be considerably more potent towards Dictyostelium when contained within negatively-charged DSPG liposomes. Following encapsulation, the IC50 for inhibition of growth by clodronate was reduced from 5001.tM to less than 251,tM, while the IC50 for pamidronate was reduced from 150I~M to about 101~M. Empty liposomes had no effect on cell growth or viability.
These observations support the view that bisphosphonates exert their anti-proliferative and cytotoxie effects by an intracellular mechanism. Since we have previously reported that free clodronate may act intracellularly in Dictyosteliton due to the metabolism of the bisphosphonate into a potentially toxic, non*hydrolysable analogue of ATP, we are investigating whether liposomal clodronate is also metabolised in the same manner.
31 CLODRONATE INHIBITS MMP-1 AND MMP-8 IN GCF AND PISF. O, Teronen*. YT. Konttinen. T. Salo. T. In~,man and T. Sorsa. Departments of Oral and Maxillofacial Surgery*, Periodontology and Anatomy, Universities of Helsinki and Oulu, Finland. * Institute of Dentistry, University of Helsinki, PB 41 (Marmerheimintie 172), FIN-00014, University of Helsinki, Finland
Proteolytic enzymes, especially matrix metaUoproteinases are known to take part in the periodontal tissue remodelling and destruction during periodontitis and peri-implantitis. Clodronate is a well tolerated drug in the group of bisphosphonates currently used in the treatment of some bone related diseases in humans, due to its inhibitory effect on osteoclastic bone resorption. However, the basic mechanism is not yet known.
We studied the effect of clodronate on purified MMP-1 and -8 and on the collagenolytic activity in gingival crevicular fluid (GCF) and pefi-implant sulcus fluid (P1SF) collected with filter paper strips. Purified MMP-8, -1, GCF and PISF were each incubated with type I collagen in a concentration series of clodronate. The collagen degradation was observed using SDS-PAGE/Laser Densitometry techniques. Effect of clodronate on gelatinases, MMP-9 and -2, was also studied using 1251-labelled gelatin as a substrate. Degradation of gelatin was measured using a gamma scintillation counter. Collagenolytic activity of purified MMP-1, MMP-8, PISF and GCF was effectively inhibited in dose dependent manner at clinically attainable concentrations <I00 I.tM. However, clodronate did not inhibit MMP-2 or MMP-9 at even higher concentrations.
The results su~,~est that cloclronate can inhibit MMP-1 and MMP- 8 in human inflammatory exudates (GCF. PISP-'3 and may be used lueallv or systemically in the treatment of a variety of diseases characterized by excessive colla~enolvtic activity. Supported by ITI- Foundation and The Academy of Finland.
32 DISSOCIATION OF BINDING AND ANTIRESORPTIVE PROPERTIES OF HYDROXYBISPHOSPHONATES BY AN AMINO SUBSTITUTION OF THE HYDROXYL GROUP. E. van Beck. C.W.G.M. Ltwik and S.E.Paoaooulos. Department of Endocrinology, University Hospital, 2333 AA Leiden, The Netherlands.
Geminal bisphosphonates contain a P-C-P bond and two side chains of variable length (R1 and R2 respectively). Although no clear structure- function relations of bisphosphonates have been demonstrated, R1 is generally believed to be involved in the binding of bisphosphonates to bone mineral and not to contribute to their cellular effects. Bisphosphonates with a OH group at R 1 have high affinity for bone mineral. This is attributed to the tridendate binding of this structures to crystals surfaces. Tridendate binding can be also obtained by substituting the OH with an NH2 group, as suggested previously. If, therefore, R1 is not important for cellular activity then such substitution will theoretically not affect the antiresorptive effect of hydroxybisphosphonates. To test this hypothesis we substituted the OH group of 3 clinically useful bisphosphonates (etidronate, pamidronate, olpadronate) with an NH2 group leaving the R2 intact. The effects of these bisphosphonates were compared in in vitro assays some of which are representative for the in vivo effects of bisphospbonates (JBMR 1994; 12: 1875-82). All three aminosubstituted bisphosphonates bound to bone mineral and inhibited the growth of calcium oxalate crystals to a similar degree as their hydroxy- counterparts. There were, however, marked differences in their antiresorptive potencies. The antiresorptive effect of olpadronate was totally abolished by the aminosubstitution while this of pamidronate was reduced by about six-fold. Etidronate and its aminosubstituted analog had identical antiresorptive potencies. In conclusion, the structure of R1 can greatly affect the antiresorptive potency of bisphosphonates without altering their affinity for bone. These studies demonstrate that the whole bisphosphonate molecule is important for cellular activity and provide a model compound (the aminosubstituted olpadronate) for the study of molecular mechanisms involved in bisphosphonate action.
