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Antiproliferative Effect of Two Novel Palmitoylcarnitine Analogs

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Antiproliferative Effect of Two Novel Palmitoylcarnitine Analogs Eur. J. Biochem. 266, 855±864 (1999) q FEBS 1999 Anti-proliferative effect of two novel palmitoyl-carnitine analogs, selective inhibitors of protein kinase C conventional isoenzymes Tea Garcia-Huidobro, Enrique Valenzuela, Andrea V. Leisewitz, Jaime Valderrama and Miguel Bronfman Departamento de BiologõÂa Celular y Molecular, Facultad de Ciencias BioloÂgicas y Facultad de QuõÂmica, P. Universidad CatoÂlica de Chile, Santiago, Chile Previous studies have shown that palmitoyl-carnitine is an anti-proliferative agent and a protein kinase C inhibitor. Two new palmitoyl-carnitine analogs were synthesized by replacing the ester bond with a metabolically more stable ether bond. An LD50 value in the nm range was found in anti-proliferative assays using HL-60 cells and was dependent on the alkyl-chain length. The inhibitory action of these water-soluble compounds on protein kinase C in vitro was greatly increased with respect to palmitoyl-carnitine and was dependent on the length of the alkyl chain. Its effect was mediated by an increase in the enzyme's requirement for phosphatidylserine. Inhibition of the in situ phosphorylation of a physiological platelet protein kinase C substrate and of phorbol ester-induced differentiation of HL-60 cells was also observed. Finally, to test for isoenzyme selectivity, several human recombinant protein kinase C isoforms were used. Only the Ca2+-dependent classic protein kinase Cs (a, bI, bII and g) were inhibited by these compounds, yet the activities of casein kinase I, Ca2+/calmodulin-dependent kinase and cAMP-dependent protein kinase were unaffected. Thus, these novel inhibitors appear to be both protein kinase C and isozyme selective. They may be useful in assessing the individual roles of protein kinase C isoforms in cell proliferation and tumor development and may be rational candidates for anti-neoplasic drug design. Keywords: anti-proliferative drugs; palmitoyl-carnitine; protein kinase C isoenzymes. Protein kinase C (pkC) isoenzymes play a major role in signal isoforms and are not selective for pkC because other transduction pathways affecting proliferation, differentiation intracellular receptors for phorbol esters have been identified and tumor development [1±4]. In this context, the design of [12]. Similarly, staurosporine [13], the most potent pkC selective pkC inhibitors is of interest because of their potential inhibitor described to date (IC50 = 6±10 nm), is also the most role as anti-proliferative and anti-neoplasic agents. The unspecific, as it inhibits other kinases with similar efficiencies classical isoforms of pkC (cpkC-a, cpkC-bI, cpkC-bII and [14,15]. The search for more selective pkC inhibitors has cpkC-g) are activated by Ca2+ and diacylglycerol or phorbol yielded many structurally distinct compounds directed against ester, and phosphatidylserine (PtdSer), whereas the novel either the catalytic or the regulatory domains of the enzyme [7], isozymes (npkC-d, npkC-:, npkC-u, npkC-h/L and npkC-m) although few of these compounds are truly isoenzyme selective. 2 respond to diacylglycerol and phorbol esters but not to Ca . In early work on pkC inhibitors, alkyl-lysophospholipids [16] Finally, the atypical isoforms (apkC-z and apkC-zl/i) are were shown to possess selective anti-neoplasic activity in many activated only by anionic phospholipids such as PtdSer [2]. The cell cultures and in vivo [16±18]. Much of this work placed various pkC isoforms exhibit marked differences in tissue and special emphasis on ether analogs because of their higher subcellular distribution [5], compartmentalization [6], substrate metabolic stability and longer half-life [19]. Ether lysophos- specificity [7,8], protein±protein interactions [9,10] and pholipids were also found to be more toxic in cell cultures susceptibility to translocation and down-regulation [11]. than their respective ester analogs [19]. In the present study, These differences are suggestive of functional divergence we wished to combine this precedent with previous work on between individual pkC isotypes, although the exact roles of l-palmitoyl-carnitine (palm-car) in order to generate a new particular isoforms, particularly in tumor promotion, are as yet pkC inhibitor. Palm-car is an intermediate with long-chain unclear, in part because of the lack of pkC-isozyme-selective fatty acid metabolism and is capable of inhibiting cellular activators and inhibitors. Although phorbol esters are potent proliferation, differentiation and tumor promotion induced by pkC activators, they do not distinguish between individual phorbol esters, as well as pkC activity in vitro [20±23]. However, the in vivo inhibitor potential of palm-car is limited Correspondence to M. Bronfman, Departamento de BiologõÂa Celular by its rapid metabolic turnover, which is largely facilitated by y Molecular, Facultad de Ciencias BioloÂgicas, P. Universidad CatoÂlica its ester linkage and the breakdown of the acyl chain. Here we de Chile, Casilla 114-D, Santiago, Chile. Tel.: +56 2 686 2833, synthesized and evaluated two new palm-car analogs in which Fax: +56 2 635 2499, E-mail: [email protected] the ester linkages were replaced by an ether bond, to augment Abbreviations: CaMKII, Ca2+/calmodulin-dependent kinase II; CHO, the compounds' metabolic stability. We also verified the Chinese hamster ovary cells; CKI, casein kinase I; DO, diolein; importance of the acyl moiety by testing ether carnitines of palm-car, palmitoyl-carnitine; palm-CoA, palmitoyl-CoA; PtdIns(3,4,5)P3, varying alkyl-chain lengths, as well as nafenopin and phosphatidylinositol-3,4,5-triphosphate; pkA, cyclic AMP-dependent ciprofibrate acyl carnitines, two carboxylic acid-containing protein kinase; pkC, protein kinase C; PMA, 4b-phorbol 12-myristate carcinogenic peroxisome proliferators. The two novel palm-car 13-acetate; PtdSer, phosphatidylserine. analogs, POC-16 and AM-44, showed increased inhibitory (Received 7 September 1999, accepted 4 October 1999) capacity with respect to palm-car, both in proliferation studies 856 T. Garcia-Huidobro et al. (Eur. J. Biochem. 266) q FEBS 1999 using human leukemia cell cultures (HL-60), in which AM-44 penicillin and 100 mg´mL21 streptomycin. For proliferation showed greater potency, and pkC activity assays in vitro,in inhibition experiments, cells were plated in fresh medium at a which several pkC isoforms were tested. Similarly, the carnitine density of 1 105 cells´mL21 per well in 24-well plates for analogs inhibited the thrombin-induced phosphorylation of a HL-60, and at 5 105 cells´mL21 in 35 mm plates for CHO, physiological pkC substrate in intact human platelets and to which the indicated drug concentrations were applied in phorbol ester-induced differentiation of HL-60 cells. Both duplicate. Cells were then incubated at 37 8C in a humidified derivatives were found to be both pkC selective and isoenzyme 5% CO2 atmosphere for 24, 48 or 72 h, and cell viability selective. Only the Ca2+-dependent cpkCs (a, bI, bII and g) was calculated using the Trypan Blue exclusion method. The were inhibited by these compounds, while other second- HL-60 differentiation assay was performed as described messenger kinases were not. previously [25]. EXPERIMENTAL PROCEDURES Incorporation of 32P orthophosphate into platelet proteins Materials Platelets were obtained from freshly EDTA-anticoagulated Human recombinant pkC isozymes were obtained from Pan human blood, as described in Orellana et al. [24], and diluted to Vera Corp. (Madison, WI, USA) and the epsilon substrate 2 109 platelets´mL21 in 15 mm Tris/HCl pH 7.4 containing peptide from QCB Inc. (Hopkinton, MA, USA). Histone V-S 140 mm NaCl, 5.5 mm glucose and 1 mg´mL21 of bovine (calf thymus), l-a-phosphatidylserine (P5660), diolein (DO), serum albumin. For 32P protein-labeling experiments, platelets ATP, 4b-phorbol 12-myristate 13-acetate (PMA) and cipro- 32 32 were incubated in the same medium as above plus P fibrate were purchased from Sigma Chemicals and [g- P]ATP 21 21 32 ortophosphate (50 mCi´mL ) for 1 h at 25 8C with gentle (10.0 mCi´mL ) and P phosphate from NEN Research agitation. Platelets were then incubated for 3 min at 37 8C and Products (Boston, MA, USA). Chemicals for drug synthesis a further 2 min in the presence of the acyl-carnitine derivatives were purchased from Aldrich (Milwaukee, WI, USA) and at the desired concentrations. Stimulation with thrombin nafenopin was provided by CIBA GEIGY (Basel, Switzerland). (0.25 U´mL21 final concentration) was carried out for 1 min All other reagents were obtained from commercial sources in a total volume of 60 mL. The incubation was terminated unless stated otherwise. The bisindolylmaleimide selective pkC by the addition of 40 mL SDS buffer [0.25 m Tris/HCl inhibitor Ro 31-8220 was donated by Roche Pharmaceuticals pH 6.8 containing 40% glycerol (v/v), 20% 2-mercaptoetha- (Herstfordshire, UK). nol (v/v), 10% SDS, and 0.2 mg´mL21 of bromophenol blue] and heating at 100 8C for 2 min. SDS/PAGE was then performed according to Laemmli [26] using 11% (w/v) Cell cultures acrylamide gels, that were stained with Coomassie Brilliant Human leukemia (HL-60) cells were maintained in RPMI- Blue R, dried and exposed to Kodak X-Omat film for 21 1640 medium supplemented with 2 mg´mL NaHCO3, autoradiography. Densitometric analysis of the autoradiograph 100 U´mL21 penicillin, 100 mg´mL21 streptomycin and either was performed using a CS-9000 scanning densitometer 2% or 5% heat-inactivated fetal bovine serum. Chinese hamster (Shimadzu, Kyoto, Japan), and the relative content of phos- ovary (CHO) cells were maintained in HamF12 medium phorylated proteins was expressed in arbitrary densitometric supplemented with 5% fetal bovine serum, 100 U´mL21 units. Fig. 1. Structure of acyl-carnitines and alkyl-carnitines. Nafenopin-carnitine (I) and ciprofibroyl-carnitine (II) were synthesized as described previously [27]. Alkyl-carnitine ethers were synthesized from 1±2 epoxides of varying chain lengths and l-carnitine (see Experimental procedures) yielding a mixture with proportions of 65±70% compound III and 30±35% compound IV.
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