Transcript Slide 1 - Elsevier

Chapter 51
Bisphosphonates, bone
metabolism: interest for the
treatment of bone cancers
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
1
FIGURE 51.1 The structure of pyrophosphate, a geminal bisphosphonate, simple bisphosphonates (medronate, clodronate, etidronate),
and nitrogen-containing bisphosphonates (N-BPs; alendronate, risedronate, zoledronate).
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
2
FIGURE 51.2 Cellular uptake of BPs by osteoclasts. (A) After binding to bone mineral, the BP is internalized into bone-resorbing
osteoclasts by endocytosis during the resorption process. (B) Visualization of BP uptake by a resorbing osteoclast cultured on mineral
pre-coated with a fl uorescentlabelled BP (green). The F-actin ring of the osteoclast is shown in red. Reproduced from Coxon et al., Bone
42:848–860 (2008). (C) Transmission electron microscopy shows morphological changes in osteoclasts induced by alendronate
treatment, particularly loss of the ruffl ed border and large intracellular vesicles involved in the resorption process. Arrows indicate sealing
zones. Reproduced from Sato et al., J Clin Invest 88:2095–2105 (1991).
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
3
FIGURE 51.3 (A) The formation of AppCp-type metabolites of BPs is catalysed by aminoacyl-tRNA synthetases. An amino acid
condenses with ATP (Appp) to form an aminoacyl-adenylate (amino acid-AMP), releasing pyrophosphate (pOp) in a reversible reaction
(I). The aminoacyl-adenylate then condenses with a molecule of tRNA to form aminoacyl-tRNA (reaction II). Since simple BPs (eg
clodronate, pCCl 2 p) resemble pyrophosphate in structure, the reverse reaction of (I) can occur with pCCl 2 p in place of pOp, to form an
analogue of ATP (AppCCl 2 p) containing the bisphosphonate. (B) The structure of ATP and the AppCp-type metabolite of clodronate
(AppCCl 2 p). (C) Clodronate treatment induces apoptosis in cultured rabbit osteoclasts. Compared to untreated osteoclasts (left),
clodronate causes the appearance of rounded cells with membrane blebbing (right), characteristic of apoptosis. Treatment with the
clodronate metabolite AppCCl 2 p, encapsulated in liposomes, causes the same morphological changes. Reproduced from Frith et al.,
Arth Rheum 44: 2201–2210 (2001) with permission of the American College of Rheumatology.
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
4
FIGURE 51.4 N-BPs inhibit the mevalonate pathway. N-BPs are potent inhibitors of FPP synthase, thereby preventing the synthesis of
FPP and GGPP required for the C-terminal prenylation of small GTPases. Inhibition of FPP synthase also causes the accumulation of
IPP, which is incorporated into the cytotoxic metabolite ApppI (A). By inhibiting FPP synthase, N-BPs cause accumulation of
unprenylated small GTPases (B) and alter their subcellular distribution. For example, multinucleated osteoclasts were immunostained for
Rab6 (C), which localizes to the perinuclear golgi in the untreated osteoclast (left) but has a cytosolic distribution in the osteoclast treated
for 48 hours with the bisphosphonate risedronate (right) (reproduced from Itzstein et al., Small GTPases 2:3, 117–130 (2011)).
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
5
FIGURE 51.5 Binding of risedronate and zoledronate in the GPP pocket of FPP synthase. Interactions between the nitrogen (blue) of
the bisphosphonate side chain and a conserved Lys200 and Thr201 help to stabilize the binding, hence explaining why the position and
orientation of the nitrogen atom plays a crucial role in determining anti-resorptive potency. Adapted from Russell et al., Osteoporos Int
19:733–759 (2008).
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
6
FIGURE 51.6 (A) Inhibition of protein prenylation in osteoclasts by N-BPs can be demonstrated in vitro by culturing cells with
[14C]mevalonate, which becomes incorporated into 14C-labelled, prenylated proteins. Radiolabelled, prenylated proteins can then be
detected by autoradiography following electrophoretic separation. (B) Both alendronate (ALN) and risedronate (RIS) prevent the
incorporation of [14C]mevalonate into prenylated proteins in purifi ed rabbit osteoclasts, whereas clodronate (CLO) has no effect. (C)
Purified osteoclasts were metabolically-labelled with [14C]mevalonate in the presence of 1–100 mM zoledronate (ZOL). Zoledronate 1
mM inhibits the synthesis of radiolabelled isoprenoid lipids at the dye-front (arrowhead), but 10 mM zoledronate also inhibits
prenylation of small GTPases. Reproduced from Coxon et al., J Bone Miner Res 15: 1467–1476 (2000), with permission of the American
Society for Bone and Mineral Research. (D) Neonatal rabbits were injected with 10 mg/kg alendronate or clodronate. Twenty-four hours
later, osteoclasts were purifi ed using immunomagnetic beads and cell lysates were analysed by western blotting for the presence of
unprenylated Rap1A. Alendronate (but not clodronate) treatment in vivo causes the accumulation of unprenylated protein in osteoclasts.
The lack of effect in non-osteoclast cells demonstrates that alendronate specifi cally affects osteoclasts. Reproduced from Frith et al.,
Arth Rheum 44: 2201–2210 (2001) with permission of the American College of Rheumatology.
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
7
FIGURE 51.7 The mechanism underlying the acute phase reaction to N-BPs. Following infusion of N-BP, internalization of the drug by
peripheral blood monocytes leads to rapid inhibition of FPP synthase and accumulation of the upstream metabolite, IPP. Recognition of
IPP by Vg9Vd2+ T-cells then leads to activation of the gd T-cells and the release of proinflammatory cytokines such as TNFa, which
cause the ‘flu-like symptoms of the acute phase reaction. The manner in which IPP is released or “presented” remains unknown.
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
8
FIGURE 51.8 Possible anti-tumour actions of N-BPs. Although N-BPs have direct effects on cultured tumour cells and endothelial cells,
there is no conclusive evidence for direct effects on these cell types in vivo. Increasing evidence indicates that N-BPs may have indirect
anti-tumour activity by affecting the number or phenotype/function of immune myeloid cells such as tumour-associated macrophages and
myeloid-derived suppressor cells (MDSCs), which normally promote tumour growth and metastasis.
© 2015, Elsevier Inc., Heymann, Bone Cancer, Second Edition
9