- Email: [email protected]
Water-soluble HPMA copolymer–wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo
Journal of Controlled Release 74 (2001) 275–281 www.elsevier.com / locate / jconrel
Water-soluble HPMA copolymer–wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo L. Varticovski a , *, Zheng-Rong Lu b , Kahlil Mitchell a , Isabel de Aos a , ˇ b Jindrich Kopecek b
a Department of Medicine, St. Elizabeth’ s Medical Center, TUSM, Boston, MA 02135, USA Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UH 84112, USA
Abstract Phosphoinositide kinases and ATM-related genes play a central role in many physiological processes. Activation of phosphoinositide 3-kinase (PI 3-kinase) is essential for signal transduction by many growth factors and oncogenes and may contribute to tumor progression. In the nanomolar range, Wortmannin (WM), a fungal metabolite, is a potent inhibitor of type I PI 3-kinase; it covalently modifies its catalytic subunit. Because WM is soluble only in organic solvents and unstable in water, there are difficulties in its use in vivo. To generate a water-soluble WM derivative, we used a conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has a slightly lower inhibitory activity than WM. We covalently attached DAWM to HPMA copolymer containing oligopeptide (GFLG) side-chains. The final product had an estimated molecular mass of 20 kDa and contained 2 wt.% of DAWM. The HPMA copolymer (PHPMA)–DAWM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo; it possessed approximately 50% of the inhibitory activity of DMSO solubilized WM. The specificity and stability of the PHPMA–DAWM conjugate is currently under investigation. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo. 2001 Elsevier Science B.V. All rights reserved. Keywords: Angiogenesis; HPMA copolymer; PI 3-kinase; Wortmannin
1. Introduction The role of polyphosphoinositides in intracellular signaling was not recognized until the early 1980s and the nature of the protein–phospholipid interactions has only been appreciated in the last few years *Corresponding author. Tel.: 11-617-789-3107; fax: 11-617789-5021. E-mail address: [email protected] (L. Varticovski).
[1]. Phosphatidylinositol (PI) [1] is the simplest component of the phosphorylated derivatives of inositol-containing phospholipids and has an inositol ring attached by D-1-OH group to phosphatidic acid. The free hydroxyl groups of inositol ring, except those at the D-2 and D-6 positions, can be phosphorylated in different combinations in living cells. Of particular interest, are phosphatidylinositol 3kinases (PI 3-kinases), the enzymes that generate the 39 phosphorylated polyphosphoinositides. PI 3-ki-
0168-3659 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00349-2
276
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
nase lipid products are not substrates for the PIspecific phospholipases C (PLC) [2,3]. This separates PI 3-kinase signaling from the PLC–PI 4,5-P2 pathway, a pathway that leads to Ca 21 release and activation of protein kinase C (PKC). Nine mammalian genes have been cloned to date and their protein products are part of the PI 3-kinase family that has been grouped into three classes (I–III) based on their structure, in vitro substrate specificity and their probable mechanism of regulation [4]. The minimally phosphorylated PI 3-kinase lipid product, PI 3-P, constitute a small fraction (,0.25%) of total inositol-containing phospholipids. PI 3,4-P2 and PI 3,4,5-P3 , generated by Class I and Class II PI 3kinases, are not detectable in quiescent cells and appear only in transformed or growth factor-stimulated cells [5,6]. The participation of PI 3-kinase lipid products in intracellular signals is based on protein–protein and protein–lipid interactions provided by lipid binding domains in target proteins. Two lipid-binding domains have been identified that specifically recognize inositol-containing phospholipids: the FYVE and PH domains. To date, over 40 FYVE domain-containing proteins have been described in mammals and most of them have been implicated in intracellular trafficking. PH domains are found in more than 100 proteins with a wide range of cellular functions, including kinases, phospholipases, nucleotide-exchange factors and adaptor proteins and proteins, which mediate protein–phospholipid and protein–protein interactions [7,8]. These recognition motifs are responsible for the central role of PI 3-kinase lipid products in many physiological processes. PI 3-kinase-dependent activation of Akt is required for its role in cell survival. Akt participates in counteracting the apoptotic pathway directly or indirectly on several levels. Akt phosphorylates and inactivates BAD, Caspase 9 [9,10], forkhead transcription factor [11–14] and activates I-kB kinases [15–17]. The effect on cell survival may depend on specific apoptotic pathway activated in different cells. Although activation of PI 3-kinase has a clear function in cell survival, only endothelial cells actively undergo apoptosis in the presence of PI 3-kinase inhibitors [18,19] which suggest that most normal cells have alternative mechanisms for survival.
There has been an intense search for inhibitors of PI 3-kinase because of potential role of 39-polyphosphoinositides in tumorigenesis, cell survival and other intracellular processes. There are two cellpermeable inhibitors of PI 3-kinase activity: wortmannin and LY294002 [20–22]. Wortmannin is an irreversible inhibitor and binds covalently to Class I PI 3-kinase catalytic subunits with nanomolar specificity [23], whereas LY294002 is a competitive inhibitor of the ATP binding site in micromolar range [22]. Formation of an Schiff-base between the p110a and Wortmannin involves a conserved Lys 802 , a residue that is also required for ATP hydrolysis [23]. At higher concentrations, these compounds loose specificity. Class II PI 3-kinases are less sensitive to both agents and the ubiquitously expressed PI 3-K-C2a isoform is ten-fold more resistant to Wortmannin [24,25]. The mammalian Class III PI 3-kinase are also less sensitive to Wortmannin [26], whereas their S. cerevisiae and S. pombe counterpart, Vps34, is completely resistant [27,28]. Wortmannin and LY29002 are soluble only in organic solvents, which limits their use in vivo. To generate a water-soluble Wortmannin derivative, we used an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has slightly lower inhibitory activity than Wortmannin [29]. The HPMA copolymer is a hydrophilic biocompatible polymer, which is developed as a water-soluble polymeric drug carrier [30]. The HPMA copolymer–drug conjugates enter cells by endocytosis and accumulate in the lysosomes where the active drug can be released from the carrier by hydrolysis of the linker peptide by lysosomal enzymes. The HPMA copolymer–WM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo.
2. Materials and methods
2.1. Synthesis of HPMA copolymer–wortmannin Wortmannin was obtained from Developmental
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
Therapeutics Program, NCI. Wortmannin was derivatized to obtain DAWM as described [29] and linked to a lysosomally degradable tetrapeptide spacer, MA–Gly–Phe–Leu–Gly, to obtain a polymerizable drug conjugate. The HPMA copolymer–wortmannin conjugate was prepared by copolymerization of HPMA and the drug containing monomer [31].
2.2. PI 3 -kinase assay PI 3-kinase activity was measured in whole cell extracts from BCR /ABL transformed BaF 3 cells (250 ng of protein) by in vitro kinase reaction in presence of PI (Avanti Polar Lipids) and PI 4,5-P2 (Sigma) as substrates and 32 PATP as described [5]. PS was used as a carrier phospholipid for micellar presentation of the lipids. The products were separated overnight by TLC in n-propanol–1 M acetic acid (65:35) and quantified by scraping and counting the radioactivity incorporated in to fully phosphorylated product, PI 3,4,5-P3 .
2.3. Activation of PI 3 -kinase in cultured cells Activation of PI 3-kinase was monitored by phosphorylation of its immediate downstream target, Akt. Human umbilical cord endothelial cells in passage 3 were grown to 80% confluency on gelatincoated tissue culture plates and induced to quiescence in 1% BSA for 14 h. Cells were treated with Wortmannin (Sigma) or HPMA copolymer–WM conjugate for 20 min. The media was removed, cells rinsed twice with ice-cold PBS and cell extracts prepared in 1% Triton-containing buffer as described [32]. Proteins were fractionated on 12% SDS–PAGE, transferred to nitrocellulose and probed with phospho-Akt antibody (NEB) and goat anti-rabbit-HRP secondary antibodies (Gibco BRL). The reactive bands were detected by ECL reagent (Promega) as per manufacturer’s instructions.
3. Results
3.1. Generation of HPMA copolymer–WM conjugate Wortmannin was derivatized to obtain DAWM as
277
described [29] and linked to a lysosomally degradable tetrapeptide spacer, MA–Gly–Phe–Leu–Gly, to obtain a polymerizable drug conjugate. The HPMA copolymer–wortmannin conjugate was prepared by copolymerization of HPMA and the drug containing monomer as described [31]. The final product had an estimated molecular mass of 20 000 Da and contained approximately 2% by weight of DAWM (Fig. 1).
3.2. Effect of HPMA copolymer–WM conjugate on class I PI 3 -kinase activity in vitro To test the effect of HPMA copolymer–WM conjugate on Class I PI 3-kinase, we used an assay previously developed in our laboratory [33]. This method allows detection of highly phosphorylated PI 3-kinase lipid product, PI 3,4,5-P3 by Class I catalytic subunits in whole cell lysates. PI 3-kinase activity was measured using 250 ng of cell protein by in vitro kinase reaction in presence of PI and PI 4,5-P2 (Avanti) as substrates and 32 PATP as described [5]. The reaction was carried out for an optimal time (20 min) and products were extracted twice in to chloroform and separated by TLC in n-propanol–1 M acetic acid (65:35) overnight. The amount of 32 P labeled PI 3,4,5-P3 was quantified by scraping the corresponding spot and counting in liquid scintillation counter and compared to the activity of PI 4-kinases by the formation of PIP composed of a mixture of PI 3-P and PI 4-P. The results shown in Fig. 2 indicate that WM conjugated to the HPMA copolymer is active in vitro and inhibits Class I PI 3-kinase with one hundred times lower activity than the free WM in vitro. No effect was seen on activity of PI 4-kinases.
3.3. Effect of HPMA copolymer–WM conjugate on activation of Akt in cultured cells PI 3-kinase dependent phosphorylation of Akt is a hallmark of activation of Akt serine–theronine kinase activity. Activation of Akt in response to endothelial growth factors, VEGF and angiopoietin-1, are required for survival of human endothelial cells [18,19,34]. We tested whether exposure of human umbilical cord endothelial cells (HUVECs) to HPMA copolymer–WM conjugate interferes with
278
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
Fig. 1. HPMA copolymer–Wortmannin conjugate. (A) Structure of Wortmannin. (B) Structure of Wortmannin ester, 11-O-desacetylwortmannin (DAWM). (C) HPMA copolymer–Wortmannin conjugate.
Akt activation. HUVECs were induced to quiescence and treated with 30 ng / ml VEGF, 100 ng / ml Angiopoietin-1 with WM or HPMA copolymer–WM conjugate for 20 min. As shown in Fig. 3, activation of Akt was induced by angiopoietin-1. In contrast, treatment of cells with HPMA copolymer–WM conjugate at 25–100 nM or with 100 nM free WM blocked activation of Akt as detected by phosphospecific Akt antibodies. A similar effect was seen in cells treated with VEGF (data not shown). Previous data indicate that internalization of HPMA copolymer into the cells by endocytosis results in slow and
sustained release of conjugated drug, such as Daunomycin. The rapid downregulation of phosphorylated Akt in intact cells is unexpected and suggest that the intracellular pool of PI 3-kinase is highly sensitive to WM conjugated to HPMA copolymer. This effect could be due to co-localization of HPMA copolymer–WM conjugate to the endosomal compartment which may contain PI 3-kinase bound to recycling active growth factor receptors. The kinetics of release of free WM in the intracellular compartment will require further investigation. The intracellular localization of PI 3-kinase and role
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
279
Fig. 2. Inhibition of Class I PI 3-kinase activity by HPMA copolymer–WM conjugate in vitro. (A) PI 3-kinase activity in cell extracts was tested by in vitro kinase reaction in pressure of PI and PI 4,5-P2 as substrates and 32 PATIP [32]. PS was used as a carrier phospholipid for micellar presentation of the lipids. The products represent formation of 39 polyphosphoinositides, PI 3-P and PI 3,4,5-P3 separated by TLC in n-propanol–1 M acetic acid (65:35) overnight. The results are from one of three independent experiments performed in duplicate samples. (B) 32 P incorporated in to the highly phosphorylated Class I PI 3-kinase product, PI 3,4,5-P3 was removed from TLC plate and results quantified by scintillation counting. The results are shown6S.D. from three independent experiments performed in duplicates.
of specific isoforms in mediating distinct cellular functions have been elusive. HPMA copolymer–WM conjugate could be further derivatized to be targeted to specific intracellular compartments and could be a useful reagent for study the role of PI 3-kinase lipid products in intracellular trafficking and other biological functions.
4. Conclusion HPMA copolymer–WM conjugate is a cell-permeable water-soluble derivative that is active in vivo and in vitro and blocks Class I PI 3-kinase activity in the nanomolar range. The inhibitory activity of HPMA copolymer–Wortmannin conjugate is approx-
280
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
Fig. 3. HPMA copolymer–wortmannin conjugate PI 3-kinase-dependent activation of Akt in human endothelial cells. HUVECs in passage 3 were grown to 80% confluency on gelatin-coated tissue culture plates and induced to quiescence in serum-free media with 1% BSA for 14 h. PI 3-kinase-dependent activation of Akt was induced by angiopoietin-1 [18] in the presence or absence of WM or HPMA copolymer–WM conjugate as indicated. Phosphorylation of Akt in whole cell lysates was detected by phosphospecific anti-Akt antibodies (30 mg of whole cell extract protein was loaded in each well). The results from one of two independent experiments are presented here.
imately ten-fold lower than the activity of free WM in vitro but has 50% of the effect in intact cells. The specificity and stability of the HPMA copolymer– WM conjugate is currently under investigation. The new HPMA copolymer–WM conjugate can be further modified to allow cell-specific targeting and may be useful in investigations of the role of PI 3-kinase in angiogenesis, tumor progression and specific intracellular functions in vivo.
Acknowledgements The authors thank Done Marie Myronchuck for preparation of the figures and Tatehiro Kagawa for advice in preparation of the manuscript. This work was supported, in part, by NIH grants CA53094 and CA51578 (JK) and DK54785 (LV).
References [1] N. Divecha, R.F. Irvine, Phospholipid signaling, Cell 80 (1995) 269–278. [2] L.A. Serunian, M.T. Haber, T. Fukui, J.W. Kim, S.G. Rhee, J.M. Lowenstein, L.C. Cantley, Polyphosphoinositides produced by phosphatidylinositol 3-kinase are poor substrates for phospholipases C from rat liver and bovine brain, J. Cell Biol. 264 (1989) 17809–17815.
[3] D.L. Lips, P.W. Majerus, F.R. Gorga, A.T. Young, T.L. Benjamin, Phosphatidylinositol 3-phosphate is present in normal and transformed fibroblasts and is resistant to hydrolysis by bovine brain phospholipase C II, J. Biol. Chem. 264 (1989) 8759–8763. [4] J. Domin, M.D. Waterfield, Using structure to define the function of phosphoinositide 3-kinase family members, FEBS Lett. 410 (1997) 91–95. [5] L. Varticovski, B. Druker, D. Morrison, L.C. Cantley, T. Roberts, The CSF-1 receptor associates with and activates PI 3-kinase, Nature 342 (1989) 699–702. [6] K.R. Auger, L.A. Serunian, S.P. Soltoff, P. Libby, L.C. Cantley, PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells, Cell 57 (1989) 167–175. [7] L.E. Rameh, L.C. Cantley, The role of phosphoinositide 3-kinase lipid products in cell function, J. Biol. Chem. 274 (1999) 8347–8350. [8] R.A. Christensen, I. DeAos, L. Varticovski, Synthesis of and signaling through 39-phosphoinositides, in: I.M. Arias. (Ed.), The Liver, Biology and Pathobiology, 4th Edition, Lippincott, Williams and Wilkins, Philadelphia, 2001, p 495–510. [9] M.H. Cardone, N. Roy, H.R. Stennicke, G.S. Salvesen, T.F. Franke, E. Stanbridge, S. Frisch, J.C. Reed, Regulation of cell death protease-9 by phosphorylation, Science 282 (1998) 1318–1321. [10] E.S. Alnemri, Hidden powers of the mitochondria, Nat. Cell. Biol. 1 (1999) E40–E42. [11] A. Brunet, A. Bonni, M. Zigmond, M.Z. Lin, P. Juo, L.S. Hu, M.J. Anderson, K.C. Arden, J. Blenish, M.E. Greenberg, Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor, Cell 96 (1999) 857–868. [12] G. Rena, S. Guo, S.C. Cichy, T.G. Unterman, P. Cohen, Phosphorylation of the transcription factor forkhead family
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
[13]
[14]
[15] [16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
member FKHR by protein kinase B, J. Biol. Chem. 274 (1999) 17179–17183. G.J. Kops, N.D. de Ruiter, A.M. De Vries-Smits, D.R. Powell, J.L. Bos, B.M. Burgering, Direct control of the Forkhead transcription factor AFX by protein kinase B, Nature 398 (1999) 630–634. W.H.r. Biggs, J. Meisenhelder, T. Hunter, W.K. Cavenee, K.C. Arden, Protein kinase B /Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1, Proc. Natl. Acad. Sci. USA 96 (1999) 7421– 7426. J.A. Romashkova, S.S. Makarov, NF-kB is a target of AKT in anti-apoptotic PDGF signalling, Nature 401 (1999) 86–90. O.N. Ozes, L.D. Mayo, J.A. Gustin, S.R. Pfeffer, L.M. Pfeffer, D.B. Donner, NF-kB activation by tumour necrosis factor requires the Akt serine–threonine kinase, Nature 401 (1999) 82–85. L.P. Kane, V.S. Shapiro, D. Stokoe, A. Weiss, Induction of NF-kB by the Akt / PKB kinase, Curr. Biol. 9 (1999) 601– 604. K. Fujikawa, I. DeAos, S.K. Jain, E. Presman, L. Varticovski, Role of PI 3-kinase in angiopoietin-1-mediated migration and attachment-dependent survival of endothelial cells, Exp. Cell Res. 253 (1999) 663–672. H.P. Gerber, A. McMurtrey, J. Kowalski, M. Yan, B.A. Keyt, V. Dixit, N. Ferrara, Vascular endothelial growth factor regulates endothelial cell survival trough the phosphatidylinositol 39-kinase /Akt signal transduction pathway, J. Biol. Chem. 273 (1998) 30336–30343. A. Arcaro, M.P. Wymann, Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses, Biochem. J. 296 (1993) 297–301. H. Yano, S. Nakanishi, K. Kimura, N. Hanai, Y. Saitoh, Y. Fukui, Y. Nonomura, Y. Matsuda, Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells, J. Biol. Chem. 268 (1993) 25846–25856. C.J. Vlahos, W.F. Matter, K.Y. Hui, R.F. Brown, A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), J. Biol. Chem. 269 (1994) 5241–5248. M.P. Wymann, G. Bulgarelli-Leva, M.J. Zvelebil, L. Pirola, B. Vanhaesebroeck, M.D. Waterfield, G. Panayotou, Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction, Mol. Cell Biol. 16 (1996) 1722–1733.
281
[24] J. Domin, F. Pages, S. Volinia, S.E. Rittenhouse, M.J. Zvelebil, R.C. Stein, M.D. Waterfield, Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin, Biochem. J. 15 (1997) 139–147. [25] J.V. Virbasius, A. Guilherme, M.P. Czech, Mouse p170 is a novel phosphatidylinositol 3-kinase containing a C2 domain, J. Biol. Chem. 271 (1996) 13304–13307. [26] S. Volinia, R. Dhand, B. Vanhaesebroeck, L.K. MacDougall, R. Stein, M.J. Zvelebil, J. Domin, C. Panaretou, M.D. Waterfield, A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p–Vps15p protein sorting system, EMBO J. 14 (1995) 3339–3348. [27] J.H. Stack, S.D. Emr, Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3kinase activities, J. Biol. Chem. 269 (1994) 31552–31562. [28] K. Takegawa, D.B. DeWald, S.D. Emr, Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology, J. Cell Sci. 108 (1995) 3745–3756. [29] L.C. Creemer, H.A. Krist, C.J. Vlahos, R.N. Schultz, Synthesis and in vitro evaluation of new wortmannin esters: potent inhibitors of phosphatidylinositol 3-kinase, J. Med. Chem. 39 (1996) 5021–5024. ˇ ˇ ´ T. Minko, Z.-R. Lu, HPMA [30] J. Kopecek, P. Kopeckova, copolymer–anticancer drug conjugates: design, activity and mechanism of action, Eur. J. Pharm. Biopharm. 50 (2000) 61–81. ˇ ´ J. Kopecek, ˇ [31] T. Minko, P. Kopeckova, Efficacy of the chemotherapeutic action of HPMA copolymer-bound Doxorubicin in a solid tumor model of ovarian carcinoma, Int. J. Cancer 86 (2000) 108–117. [32] L. Varticovski, G.Q. Daley, P. Jackson, D. Baltimore, L.C. Cantley, Activation of phosphatidylinositol 3-kinase in cells expressing abl oncogene variants, Mol. Cell. Biol. 11 (1991) 1107–1113. [33] M. Susa, M.L. Keeler, L. Varticovski, Platetet-derived growth factor activates membrane-associated phosphatidyinositol 3kinase and mediates its translocation from the cytosol, J. Biol. Chem. 267 (1992) 22951–22956. [34] Y. Fujio, K. Walsh, Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner, J. Biol. Chem. 274 (1999) 16349–16354.
Water-soluble HPMA copolymer–wortmannin conjugate retains phosphoinositide 3-kinase inhibitory activity in vitro and in vivo L. Varticovski a , *, Zheng-Rong Lu b , Kahlil Mitchell a , Isabel de Aos a , ˇ b Jindrich Kopecek b
a Department of Medicine, St. Elizabeth’ s Medical Center, TUSM, Boston, MA 02135, USA Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UH 84112, USA
Abstract Phosphoinositide kinases and ATM-related genes play a central role in many physiological processes. Activation of phosphoinositide 3-kinase (PI 3-kinase) is essential for signal transduction by many growth factors and oncogenes and may contribute to tumor progression. In the nanomolar range, Wortmannin (WM), a fungal metabolite, is a potent inhibitor of type I PI 3-kinase; it covalently modifies its catalytic subunit. Because WM is soluble only in organic solvents and unstable in water, there are difficulties in its use in vivo. To generate a water-soluble WM derivative, we used a conjugate of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has a slightly lower inhibitory activity than WM. We covalently attached DAWM to HPMA copolymer containing oligopeptide (GFLG) side-chains. The final product had an estimated molecular mass of 20 kDa and contained 2 wt.% of DAWM. The HPMA copolymer (PHPMA)–DAWM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo; it possessed approximately 50% of the inhibitory activity of DMSO solubilized WM. The specificity and stability of the PHPMA–DAWM conjugate is currently under investigation. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo. 2001 Elsevier Science B.V. All rights reserved. Keywords: Angiogenesis; HPMA copolymer; PI 3-kinase; Wortmannin
1. Introduction The role of polyphosphoinositides in intracellular signaling was not recognized until the early 1980s and the nature of the protein–phospholipid interactions has only been appreciated in the last few years *Corresponding author. Tel.: 11-617-789-3107; fax: 11-617789-5021. E-mail address: [email protected] (L. Varticovski).
[1]. Phosphatidylinositol (PI) [1] is the simplest component of the phosphorylated derivatives of inositol-containing phospholipids and has an inositol ring attached by D-1-OH group to phosphatidic acid. The free hydroxyl groups of inositol ring, except those at the D-2 and D-6 positions, can be phosphorylated in different combinations in living cells. Of particular interest, are phosphatidylinositol 3kinases (PI 3-kinases), the enzymes that generate the 39 phosphorylated polyphosphoinositides. PI 3-ki-
0168-3659 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 01 )00349-2
276
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
nase lipid products are not substrates for the PIspecific phospholipases C (PLC) [2,3]. This separates PI 3-kinase signaling from the PLC–PI 4,5-P2 pathway, a pathway that leads to Ca 21 release and activation of protein kinase C (PKC). Nine mammalian genes have been cloned to date and their protein products are part of the PI 3-kinase family that has been grouped into three classes (I–III) based on their structure, in vitro substrate specificity and their probable mechanism of regulation [4]. The minimally phosphorylated PI 3-kinase lipid product, PI 3-P, constitute a small fraction (,0.25%) of total inositol-containing phospholipids. PI 3,4-P2 and PI 3,4,5-P3 , generated by Class I and Class II PI 3kinases, are not detectable in quiescent cells and appear only in transformed or growth factor-stimulated cells [5,6]. The participation of PI 3-kinase lipid products in intracellular signals is based on protein–protein and protein–lipid interactions provided by lipid binding domains in target proteins. Two lipid-binding domains have been identified that specifically recognize inositol-containing phospholipids: the FYVE and PH domains. To date, over 40 FYVE domain-containing proteins have been described in mammals and most of them have been implicated in intracellular trafficking. PH domains are found in more than 100 proteins with a wide range of cellular functions, including kinases, phospholipases, nucleotide-exchange factors and adaptor proteins and proteins, which mediate protein–phospholipid and protein–protein interactions [7,8]. These recognition motifs are responsible for the central role of PI 3-kinase lipid products in many physiological processes. PI 3-kinase-dependent activation of Akt is required for its role in cell survival. Akt participates in counteracting the apoptotic pathway directly or indirectly on several levels. Akt phosphorylates and inactivates BAD, Caspase 9 [9,10], forkhead transcription factor [11–14] and activates I-kB kinases [15–17]. The effect on cell survival may depend on specific apoptotic pathway activated in different cells. Although activation of PI 3-kinase has a clear function in cell survival, only endothelial cells actively undergo apoptosis in the presence of PI 3-kinase inhibitors [18,19] which suggest that most normal cells have alternative mechanisms for survival.
There has been an intense search for inhibitors of PI 3-kinase because of potential role of 39-polyphosphoinositides in tumorigenesis, cell survival and other intracellular processes. There are two cellpermeable inhibitors of PI 3-kinase activity: wortmannin and LY294002 [20–22]. Wortmannin is an irreversible inhibitor and binds covalently to Class I PI 3-kinase catalytic subunits with nanomolar specificity [23], whereas LY294002 is a competitive inhibitor of the ATP binding site in micromolar range [22]. Formation of an Schiff-base between the p110a and Wortmannin involves a conserved Lys 802 , a residue that is also required for ATP hydrolysis [23]. At higher concentrations, these compounds loose specificity. Class II PI 3-kinases are less sensitive to both agents and the ubiquitously expressed PI 3-K-C2a isoform is ten-fold more resistant to Wortmannin [24,25]. The mammalian Class III PI 3-kinase are also less sensitive to Wortmannin [26], whereas their S. cerevisiae and S. pombe counterpart, Vps34, is completely resistant [27,28]. Wortmannin and LY29002 are soluble only in organic solvents, which limits their use in vivo. To generate a water-soluble Wortmannin derivative, we used an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer and 11-O-desacetylwortmannin (DAWM), which has slightly lower inhibitory activity than Wortmannin [29]. The HPMA copolymer is a hydrophilic biocompatible polymer, which is developed as a water-soluble polymeric drug carrier [30]. The HPMA copolymer–drug conjugates enter cells by endocytosis and accumulate in the lysosomes where the active drug can be released from the carrier by hydrolysis of the linker peptide by lysosomal enzymes. The HPMA copolymer–WM conjugate inhibited type I PI 3-kinase activity in vitro and growth factor-stimulated activation of Akt in vivo. The new water-soluble form of WM may be useful in investigations of the role of PI 3-kinase in tumor progression and other cellular biological functions in vivo.
2. Materials and methods
2.1. Synthesis of HPMA copolymer–wortmannin Wortmannin was obtained from Developmental
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
Therapeutics Program, NCI. Wortmannin was derivatized to obtain DAWM as described [29] and linked to a lysosomally degradable tetrapeptide spacer, MA–Gly–Phe–Leu–Gly, to obtain a polymerizable drug conjugate. The HPMA copolymer–wortmannin conjugate was prepared by copolymerization of HPMA and the drug containing monomer [31].
2.2. PI 3 -kinase assay PI 3-kinase activity was measured in whole cell extracts from BCR /ABL transformed BaF 3 cells (250 ng of protein) by in vitro kinase reaction in presence of PI (Avanti Polar Lipids) and PI 4,5-P2 (Sigma) as substrates and 32 PATP as described [5]. PS was used as a carrier phospholipid for micellar presentation of the lipids. The products were separated overnight by TLC in n-propanol–1 M acetic acid (65:35) and quantified by scraping and counting the radioactivity incorporated in to fully phosphorylated product, PI 3,4,5-P3 .
2.3. Activation of PI 3 -kinase in cultured cells Activation of PI 3-kinase was monitored by phosphorylation of its immediate downstream target, Akt. Human umbilical cord endothelial cells in passage 3 were grown to 80% confluency on gelatincoated tissue culture plates and induced to quiescence in 1% BSA for 14 h. Cells were treated with Wortmannin (Sigma) or HPMA copolymer–WM conjugate for 20 min. The media was removed, cells rinsed twice with ice-cold PBS and cell extracts prepared in 1% Triton-containing buffer as described [32]. Proteins were fractionated on 12% SDS–PAGE, transferred to nitrocellulose and probed with phospho-Akt antibody (NEB) and goat anti-rabbit-HRP secondary antibodies (Gibco BRL). The reactive bands were detected by ECL reagent (Promega) as per manufacturer’s instructions.
3. Results
3.1. Generation of HPMA copolymer–WM conjugate Wortmannin was derivatized to obtain DAWM as
277
described [29] and linked to a lysosomally degradable tetrapeptide spacer, MA–Gly–Phe–Leu–Gly, to obtain a polymerizable drug conjugate. The HPMA copolymer–wortmannin conjugate was prepared by copolymerization of HPMA and the drug containing monomer as described [31]. The final product had an estimated molecular mass of 20 000 Da and contained approximately 2% by weight of DAWM (Fig. 1).
3.2. Effect of HPMA copolymer–WM conjugate on class I PI 3 -kinase activity in vitro To test the effect of HPMA copolymer–WM conjugate on Class I PI 3-kinase, we used an assay previously developed in our laboratory [33]. This method allows detection of highly phosphorylated PI 3-kinase lipid product, PI 3,4,5-P3 by Class I catalytic subunits in whole cell lysates. PI 3-kinase activity was measured using 250 ng of cell protein by in vitro kinase reaction in presence of PI and PI 4,5-P2 (Avanti) as substrates and 32 PATP as described [5]. The reaction was carried out for an optimal time (20 min) and products were extracted twice in to chloroform and separated by TLC in n-propanol–1 M acetic acid (65:35) overnight. The amount of 32 P labeled PI 3,4,5-P3 was quantified by scraping the corresponding spot and counting in liquid scintillation counter and compared to the activity of PI 4-kinases by the formation of PIP composed of a mixture of PI 3-P and PI 4-P. The results shown in Fig. 2 indicate that WM conjugated to the HPMA copolymer is active in vitro and inhibits Class I PI 3-kinase with one hundred times lower activity than the free WM in vitro. No effect was seen on activity of PI 4-kinases.
3.3. Effect of HPMA copolymer–WM conjugate on activation of Akt in cultured cells PI 3-kinase dependent phosphorylation of Akt is a hallmark of activation of Akt serine–theronine kinase activity. Activation of Akt in response to endothelial growth factors, VEGF and angiopoietin-1, are required for survival of human endothelial cells [18,19,34]. We tested whether exposure of human umbilical cord endothelial cells (HUVECs) to HPMA copolymer–WM conjugate interferes with
278
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
Fig. 1. HPMA copolymer–Wortmannin conjugate. (A) Structure of Wortmannin. (B) Structure of Wortmannin ester, 11-O-desacetylwortmannin (DAWM). (C) HPMA copolymer–Wortmannin conjugate.
Akt activation. HUVECs were induced to quiescence and treated with 30 ng / ml VEGF, 100 ng / ml Angiopoietin-1 with WM or HPMA copolymer–WM conjugate for 20 min. As shown in Fig. 3, activation of Akt was induced by angiopoietin-1. In contrast, treatment of cells with HPMA copolymer–WM conjugate at 25–100 nM or with 100 nM free WM blocked activation of Akt as detected by phosphospecific Akt antibodies. A similar effect was seen in cells treated with VEGF (data not shown). Previous data indicate that internalization of HPMA copolymer into the cells by endocytosis results in slow and
sustained release of conjugated drug, such as Daunomycin. The rapid downregulation of phosphorylated Akt in intact cells is unexpected and suggest that the intracellular pool of PI 3-kinase is highly sensitive to WM conjugated to HPMA copolymer. This effect could be due to co-localization of HPMA copolymer–WM conjugate to the endosomal compartment which may contain PI 3-kinase bound to recycling active growth factor receptors. The kinetics of release of free WM in the intracellular compartment will require further investigation. The intracellular localization of PI 3-kinase and role
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
279
Fig. 2. Inhibition of Class I PI 3-kinase activity by HPMA copolymer–WM conjugate in vitro. (A) PI 3-kinase activity in cell extracts was tested by in vitro kinase reaction in pressure of PI and PI 4,5-P2 as substrates and 32 PATIP [32]. PS was used as a carrier phospholipid for micellar presentation of the lipids. The products represent formation of 39 polyphosphoinositides, PI 3-P and PI 3,4,5-P3 separated by TLC in n-propanol–1 M acetic acid (65:35) overnight. The results are from one of three independent experiments performed in duplicate samples. (B) 32 P incorporated in to the highly phosphorylated Class I PI 3-kinase product, PI 3,4,5-P3 was removed from TLC plate and results quantified by scintillation counting. The results are shown6S.D. from three independent experiments performed in duplicates.
of specific isoforms in mediating distinct cellular functions have been elusive. HPMA copolymer–WM conjugate could be further derivatized to be targeted to specific intracellular compartments and could be a useful reagent for study the role of PI 3-kinase lipid products in intracellular trafficking and other biological functions.
4. Conclusion HPMA copolymer–WM conjugate is a cell-permeable water-soluble derivative that is active in vivo and in vitro and blocks Class I PI 3-kinase activity in the nanomolar range. The inhibitory activity of HPMA copolymer–Wortmannin conjugate is approx-
280
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
Fig. 3. HPMA copolymer–wortmannin conjugate PI 3-kinase-dependent activation of Akt in human endothelial cells. HUVECs in passage 3 were grown to 80% confluency on gelatin-coated tissue culture plates and induced to quiescence in serum-free media with 1% BSA for 14 h. PI 3-kinase-dependent activation of Akt was induced by angiopoietin-1 [18] in the presence or absence of WM or HPMA copolymer–WM conjugate as indicated. Phosphorylation of Akt in whole cell lysates was detected by phosphospecific anti-Akt antibodies (30 mg of whole cell extract protein was loaded in each well). The results from one of two independent experiments are presented here.
imately ten-fold lower than the activity of free WM in vitro but has 50% of the effect in intact cells. The specificity and stability of the HPMA copolymer– WM conjugate is currently under investigation. The new HPMA copolymer–WM conjugate can be further modified to allow cell-specific targeting and may be useful in investigations of the role of PI 3-kinase in angiogenesis, tumor progression and specific intracellular functions in vivo.
Acknowledgements The authors thank Done Marie Myronchuck for preparation of the figures and Tatehiro Kagawa for advice in preparation of the manuscript. This work was supported, in part, by NIH grants CA53094 and CA51578 (JK) and DK54785 (LV).
References [1] N. Divecha, R.F. Irvine, Phospholipid signaling, Cell 80 (1995) 269–278. [2] L.A. Serunian, M.T. Haber, T. Fukui, J.W. Kim, S.G. Rhee, J.M. Lowenstein, L.C. Cantley, Polyphosphoinositides produced by phosphatidylinositol 3-kinase are poor substrates for phospholipases C from rat liver and bovine brain, J. Cell Biol. 264 (1989) 17809–17815.
[3] D.L. Lips, P.W. Majerus, F.R. Gorga, A.T. Young, T.L. Benjamin, Phosphatidylinositol 3-phosphate is present in normal and transformed fibroblasts and is resistant to hydrolysis by bovine brain phospholipase C II, J. Biol. Chem. 264 (1989) 8759–8763. [4] J. Domin, M.D. Waterfield, Using structure to define the function of phosphoinositide 3-kinase family members, FEBS Lett. 410 (1997) 91–95. [5] L. Varticovski, B. Druker, D. Morrison, L.C. Cantley, T. Roberts, The CSF-1 receptor associates with and activates PI 3-kinase, Nature 342 (1989) 699–702. [6] K.R. Auger, L.A. Serunian, S.P. Soltoff, P. Libby, L.C. Cantley, PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells, Cell 57 (1989) 167–175. [7] L.E. Rameh, L.C. Cantley, The role of phosphoinositide 3-kinase lipid products in cell function, J. Biol. Chem. 274 (1999) 8347–8350. [8] R.A. Christensen, I. DeAos, L. Varticovski, Synthesis of and signaling through 39-phosphoinositides, in: I.M. Arias. (Ed.), The Liver, Biology and Pathobiology, 4th Edition, Lippincott, Williams and Wilkins, Philadelphia, 2001, p 495–510. [9] M.H. Cardone, N. Roy, H.R. Stennicke, G.S. Salvesen, T.F. Franke, E. Stanbridge, S. Frisch, J.C. Reed, Regulation of cell death protease-9 by phosphorylation, Science 282 (1998) 1318–1321. [10] E.S. Alnemri, Hidden powers of the mitochondria, Nat. Cell. Biol. 1 (1999) E40–E42. [11] A. Brunet, A. Bonni, M. Zigmond, M.Z. Lin, P. Juo, L.S. Hu, M.J. Anderson, K.C. Arden, J. Blenish, M.E. Greenberg, Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor, Cell 96 (1999) 857–868. [12] G. Rena, S. Guo, S.C. Cichy, T.G. Unterman, P. Cohen, Phosphorylation of the transcription factor forkhead family
L. Varticovski et al. / Journal of Controlled Release 74 (2001) 275 – 281
[13]
[14]
[15] [16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
member FKHR by protein kinase B, J. Biol. Chem. 274 (1999) 17179–17183. G.J. Kops, N.D. de Ruiter, A.M. De Vries-Smits, D.R. Powell, J.L. Bos, B.M. Burgering, Direct control of the Forkhead transcription factor AFX by protein kinase B, Nature 398 (1999) 630–634. W.H.r. Biggs, J. Meisenhelder, T. Hunter, W.K. Cavenee, K.C. Arden, Protein kinase B /Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1, Proc. Natl. Acad. Sci. USA 96 (1999) 7421– 7426. J.A. Romashkova, S.S. Makarov, NF-kB is a target of AKT in anti-apoptotic PDGF signalling, Nature 401 (1999) 86–90. O.N. Ozes, L.D. Mayo, J.A. Gustin, S.R. Pfeffer, L.M. Pfeffer, D.B. Donner, NF-kB activation by tumour necrosis factor requires the Akt serine–threonine kinase, Nature 401 (1999) 82–85. L.P. Kane, V.S. Shapiro, D. Stokoe, A. Weiss, Induction of NF-kB by the Akt / PKB kinase, Curr. Biol. 9 (1999) 601– 604. K. Fujikawa, I. DeAos, S.K. Jain, E. Presman, L. Varticovski, Role of PI 3-kinase in angiopoietin-1-mediated migration and attachment-dependent survival of endothelial cells, Exp. Cell Res. 253 (1999) 663–672. H.P. Gerber, A. McMurtrey, J. Kowalski, M. Yan, B.A. Keyt, V. Dixit, N. Ferrara, Vascular endothelial growth factor regulates endothelial cell survival trough the phosphatidylinositol 39-kinase /Akt signal transduction pathway, J. Biol. Chem. 273 (1998) 30336–30343. A. Arcaro, M.P. Wymann, Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses, Biochem. J. 296 (1993) 297–301. H. Yano, S. Nakanishi, K. Kimura, N. Hanai, Y. Saitoh, Y. Fukui, Y. Nonomura, Y. Matsuda, Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells, J. Biol. Chem. 268 (1993) 25846–25856. C.J. Vlahos, W.F. Matter, K.Y. Hui, R.F. Brown, A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), J. Biol. Chem. 269 (1994) 5241–5248. M.P. Wymann, G. Bulgarelli-Leva, M.J. Zvelebil, L. Pirola, B. Vanhaesebroeck, M.D. Waterfield, G. Panayotou, Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction, Mol. Cell Biol. 16 (1996) 1722–1733.
281
[24] J. Domin, F. Pages, S. Volinia, S.E. Rittenhouse, M.J. Zvelebil, R.C. Stein, M.D. Waterfield, Cloning of a human phosphoinositide 3-kinase with a C2 domain that displays reduced sensitivity to the inhibitor wortmannin, Biochem. J. 15 (1997) 139–147. [25] J.V. Virbasius, A. Guilherme, M.P. Czech, Mouse p170 is a novel phosphatidylinositol 3-kinase containing a C2 domain, J. Biol. Chem. 271 (1996) 13304–13307. [26] S. Volinia, R. Dhand, B. Vanhaesebroeck, L.K. MacDougall, R. Stein, M.J. Zvelebil, J. Domin, C. Panaretou, M.D. Waterfield, A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p–Vps15p protein sorting system, EMBO J. 14 (1995) 3339–3348. [27] J.H. Stack, S.D. Emr, Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3kinase activities, J. Biol. Chem. 269 (1994) 31552–31562. [28] K. Takegawa, D.B. DeWald, S.D. Emr, Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology, J. Cell Sci. 108 (1995) 3745–3756. [29] L.C. Creemer, H.A. Krist, C.J. Vlahos, R.N. Schultz, Synthesis and in vitro evaluation of new wortmannin esters: potent inhibitors of phosphatidylinositol 3-kinase, J. Med. Chem. 39 (1996) 5021–5024. ˇ ˇ ´ T. Minko, Z.-R. Lu, HPMA [30] J. Kopecek, P. Kopeckova, copolymer–anticancer drug conjugates: design, activity and mechanism of action, Eur. J. Pharm. Biopharm. 50 (2000) 61–81. ˇ ´ J. Kopecek, ˇ [31] T. Minko, P. Kopeckova, Efficacy of the chemotherapeutic action of HPMA copolymer-bound Doxorubicin in a solid tumor model of ovarian carcinoma, Int. J. Cancer 86 (2000) 108–117. [32] L. Varticovski, G.Q. Daley, P. Jackson, D. Baltimore, L.C. Cantley, Activation of phosphatidylinositol 3-kinase in cells expressing abl oncogene variants, Mol. Cell. Biol. 11 (1991) 1107–1113. [33] M. Susa, M.L. Keeler, L. Varticovski, Platetet-derived growth factor activates membrane-associated phosphatidyinositol 3kinase and mediates its translocation from the cytosol, J. Biol. Chem. 267 (1992) 22951–22956. [34] Y. Fujio, K. Walsh, Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner, J. Biol. Chem. 274 (1999) 16349–16354.