THE VINCA ALKALOIDS: FROM BIOSYNTHESIS AND ACCUMULATION IN PLANT CELLS, TO UPTAKE, ACTIVITY AND METABOLISM IN ANIMAL CELLS
MARIANA SOTTOMAYOR^ AND ALFONSO ROS BARCELO^
^ Department of Botany of Faculty of Sciences and Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre, 823, 4150-
180 Porto, Portugal, ^ Department of Plant Biology (Plant Physiology), University ofMurcia,
E-30100 Murcia, Spain,
ABSTRACT: The leaves of Catharanthm roseus (L.) G. Don (formerly Vinca rosea L.) were used in traditional medicine as an oral hypoglycemic agent and investigation of this activity ultimately led to the serendipitous discovery of the cytostatic terpenoid indole alkaloids vinblastine and vincristine. These compounds were the first natural anticancer agents to be clinically used and, together with a number of semisynthetic derivatives, are universally known as the Vinca alkaloids. Due to its important pharmaceutical alkaloids, C. roseus has now become one of the most extensively studied medicinal plants and much has been discovered about the biosynthetic pathway of terpenoid indole alkaloids, the regulation and compartmentation of the pathway, and the mechanisms of accumulation of those compounds inside the plant cell. The biosynthesis of vinblastine involves more than twenty enzymatic steps, nine of which are now well characterized at the enzyme and gene level and, recently, regulatory genes of the initial part of the pathway (ORCAs) have been cloned, in what consists a highly promising strategy for the manipulation of the pathway. On the other hand, the activity of vinblastine and vincristine in human cells has been thoroughly studied. The cytostatic activity has been shown to result from interference with tubulin, but the precise mechanism of action is still not perfectly understood. Uptake and extrusion in human cells has been characterized, specially the extrusion mechanism responsible for resistance to the drugs, and their metabolism in the human body has also been studied. Together, the above mentioned studies enable to establish some interesting evolutionary links between the enzymes involved in plant biosynthesis of the anticancer alkaloids and the enzymes involved in animal metabolism of the drugs, and also, possibly, between their vacuolar transport in plant cells and multidrug resistance in human cancer cells.
INTRODUCTION
The so-called Vinca alkaloids are dimeric terpenoid indole alkaloids well known by their antimitotic activity, which has made them extremely
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useful drugs in cancer therapy for more than thirty years. This designation includes the natural products vinblastine and vincristine, Fig. (1), extracted from the leaves of the plant Catharanthus roseus (L.) G. Don (previously Vinca rosea L.), and a number of semi-synthetic derivatives like vindesine, vinorelbine and the recently developed vinflunine. Fig. (1).
0COCH3
Fig. (1). Structure of natural and semi synthetic Vinca alkaloids. Shaded areas indicate the structural differences from vinblastine.
Catharanthus roseus, known as the Madagascar periwinkle, was used in traditional medicine as an oral hypoglycemic agent in the treatment of diabetes mellitus, and investigation on this activity ultimately led to the discovery of the anticancer alkaloids, almost simultaneously, by two totally independent groups: the group of Noble and collaborators in Canada [1,2] and the group of Svoboda and collaborators from the Eli
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Lilly Company, Indianapolis, in the United States [3]. The chain of events leading to the discovery of vinblastine by the group of Noble is particularly interesting and represents a paradigmatic example of the role serendipity often plays in scientific discoveries. A fiill description of the discovery, including the casual details that started the whole process, was published by Robert Noble in 1990 [4].
After their discovery, the Vinca alkaloids became the first natural anticancer agents to be clinically used, and they are still an indispensable part of most curative regimens used in cancer chemotherapy nowadays. On the other hand, the plant producing these alkaloids, C roseus, has become one of the most extensively studied medicinal plants. The levels of vincristine and vinblastine in the plant revealed to be extremely low and, for pharmaceutical production, approximately half a ton of dry leaves is needed to obtain 1 g of vinblastine [4]. This fact stimulated intense investigation in alternative methods for the production of vinblastine and vincristine, namely chemical synthesis and plant cell cultures. However, chemical synthesis showed not to be viable due to the high number of transformations involved, and the anticancer alkaloids were never detected in cell cultures, which express alkaloid metabolism very poorly [5, 6]. The biosynthetic pathway of terpenoid indole alkaloids in C roseus has also been intensively studied with the objective of developing a manipulation strategy to improve the levels of the anticancer alkaloids in the leaves of the plant [5, 7-10].
This review intends to put together what is known about the biosynthesis and accumulation of Vinca alkaloids in the plant -Catharanthus roseus^ with what is known about their uptake, mechanism of action stnd metabolism in animal cells. This will enable to highlight the curious matching of some of the enzymes involved in alkaloid biosynthesis in plant cells with some of the enzymes involved in alkaloid metabolism in animal cells, as well as to highlight the likely relation between the putative alkaloid accumulation mechanism in the vacuole of plant cells, and the transport mechanism responsible by multidrug resistance in animal cells. These similarities suggest that, during plant/herbivore co-evolution, plants have developed toxic chemical defenses against herbivores, like the Vinca alkaloids in C roseus, by recruiting the same type of enzymes and transport mechanism that animal herbivores have, on their side, recruited for the defense against the very same compounds.
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THE VINCA ALKALOIDS AND THEIR CLINICAL USES
The first Vinca alkaloid to be discovered was isolated in 1957 by Noble and collaborators from the Western University of Ontario, London, Canada, who named the alkaloid vincaleukoblastine, in view of its origin and its effect on immature white cells - leukoblasts [1, 2]. Later, the name was shortened to the less cumbersome vinblastine. Almost at the same time, the group of Svoboda and collaborators, at the Eli Lilly Research Division in the United States, detected two compounds with antitumour activity in C. roseus [3]. One of them was the already identified vinblastine, the other one was named leurosine. The two groups came in touch in a conference held by the New York Academy of Sciences in 1958, and worked in close collaboration thereafter.
Clinical trials confirmed the usefiilness of vinblastine in the treatment of Hodgkin's disease, lymphoma and other cancers, and the drug was introduced in the clinic shortly after. Leurosine was proved to be unsuitable for cancer therapy due to its toxicity, but Svoboda later isolated another alkaloid, which was also cytostatic and suitable for therapy [11]. This compound was first named leurocristine, then vincaleukocristine and finally vincristine.
Vinblastine and vincristine have now earned a place among the most valuable agents used in cancer chemotherapy. They are dimeric terpenoid indole alkaloids differing only in that vincristine has a formyl group at a position where vinblastine has a methyl group. Fig. (1), but, although their chemical structure is very similar, they differ markedly in the type of tumors they affect and in their toxicity. The basic structure of terpenoid indole alkaloids includes an indole nucleus derived from tryptophan, via tryptamine, and a versatile C9 or CIO unit arising from the monoterpenoid secologanin (see biosynthesis below). The anticancer alkaloids are built from two different terpenoid indole units derived from the precursors vindoline and catharanthine, this later suffering a rearrangement during the dimerization reaction to give rise to the so called velbenamine or cleavamine part of the dimeric molecule. Fig. (2). The direct product of the dimerization reaction is the dimer a-3',4'-anhydrovinblastine whose potential for cancer therapy is also being investigated [12]. However, apart from the reference cited, no report about the anticancer action of anhydrovinblastine was found in indexed
scientific publications.
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CH3
Vindoline
Vinblastine
Fig. (2). Biosynthesis of vinblastine from the monomeric precursors catharanthine and vindoline. AnhydrovinbJastine is the direct product of the dimerization reaction and the precursor of the anticancer drugs. Shaded areas indicate the structural diflFerences between the precursor catharanthine and the cieavamine part of anhydrovinblastine.
Vinblastine, with the commercial names Velbe®, Velban® and Vinblastine , is used alone and as a component of combined regimens with other anticancer drugs in the treatment of Hodgkin's disease and other lymphomas, in advanced carcinoma of the testis, in Kaposi's sarcoma and histiocytosis X. It can also be used in the treatment of breast carcinoma and choriocarcinoma. The use of vinblastine is mainly limited by its hematological toxicity due to destruction of the bone marrow [13-15].
Vincristine, with the commercial names Oncovin , Vincasar , Vincrisul®, Pericristine®, and Kyocristine®, is used as a component of combination therapy in the treatment of Hodgkin's disease and lymphomas, and also in acute leukemias, sarcomas and carcinomas. Because of its relative lack of hematologic toxicity it is widely used as a component of many chemotherapeutic regimens. Combined with prednisone it produces complete remission in up to 90% of children with
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acute lymphocytic leukemia. The major and dose-limiting adverse eflFect of vincristine is neurotoxicity, specially to the peripheral nervous system [13-15].
Soon after the introduction of vinblastine and vincristine in clinical usage, during the 1970's, intensive chemical research was undertaken in order to try to obtain semi-synthetic derivatives of Vinca alkaloids showing higher activity, lower toxicity, and a wider spectrum of anticancer eflBcacy (for reviews see [16, 17]).
The Eli Lilly company developed several series of derivatives with modifications in the vindoline part of the dimeric structure, culminating with the approval of vindesine. Fig. (1), for clinical treatments. Vindesine, with the commercial name Eldisine and Enisone , has a vincristine-like spectrum of activity, and is used mainly in the treatment of melanoma, acute lymphoblastic leukaemia and advanced non-small cell lung cancer [13, 14, 16]. Vindesine is approved in Europe and other areas but, in the United States, vindesine is approved only for investigational use [15].
In 1975, Potier and collaborators proposed that, inplanta, the dimeric vinblastine type alkaloids resulted fi^om the coupling of catharanthine and vindoline and, in light of this hypothesis, they reported for the first time the chemical synthesis of a dimer with the natural configuration through a modified Polonovski reaction [18, 19], This reaction resulted in the formation of an iminium dimer which, after reduction with NaBH4, yielded a-3',4'-anhydrovinblastine. Fig. (2), later proved to be the first dimeric biosynthetic precursor of vinblastine in the plant. The group of Potier investigated possible modifications of anhydrovinblastine and produced vinorelbine. Fig. (1), which was the first active derivative with an altered cleavamine (catharanthine) moiety [20, 21].
Vinorelbine demonstrated important antitumour properties associated with reduced toxic side effects and its application was developed during the 1980's by the French pharmaceutical company Pierre Fabre Medicaments, under the commercial name Navelbine . Vinorelbine is now widely used in the treatment of non-small cell lung cancer and breast cancer, and several other potential indications are under clinical investigation, like lymphoma, esophageal cancer and prostatic carcinoma [16, 22, 23]. Furthermore, it has been proved that vinorelbine is well absorbed orally with no unpredictable toxic effects and an oral formulation of the drug was registered in France in 2001 [13, 16]. The main side effect of vinorelbine is hematological toxicity.
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Pursuing the effort to obtain new useful Vinca alkaloids, the research divisions of Pierre Fabre produced a new family of derivatives using superacidic chemistry, from which vinflunine, a difluorinated derivative of vinorelbine, was selected for detailed preclinical investigations. Results showed that vinflunine is more active than vinorelbine, vinblastine or vincristine against a number of murine tumours and human tumour xenografts, and it entered phase I clinical trials in 1998, phase II in 2000, and is entering phase III in 2003 [16, 23, 24] . For a review on preclinical anticancer properties of vinflunine see [25].
The vinflunine case demonstrated that the Vinca alkaloids remain a drug family where it is still possible to identify new members with unprecedented and promising pharmacological properties. When the ongoing research on the mechanisms of action of Vinca alkaloids unravels the precise relation structure/function of the dimeric molecules, it should be possible to rationally design a new generation of Vinca alkaloids with new therapeutic properties.
BIOSYNTHESIS OF VINCA ALKALOIDS IN CATHARANTHUS ROSEUS
After the discovery of the anticancer properties of vinblastine and vincristine, the elucidation of their structure. Fig. (1), was a natural step achieved in the early 60s [26, 27], and it was shown that they were dimeric terpenoid indole alkaloids - as already stated above. Simultaneously, further studies of the plant C roseus revealed that this plant is an amazing chemical factory, producing more than 100 different terpenoid indole alkaloids, including two other with important pharmacological activity: ajmalicine, used as an antihypertensive, and serpentine, used as sedative [6, 28].
Terpenoid indole alkaloids (TIAs) comprise a large family of secondary metabolites, with around 3000 members identified, including several with important biological activity, like the Vinca alkaloids, the rat poison strychnine, and the antimalarial drug quinine [7], [29, 30]. They are almost restricted to four plant families of dicotyledones: Apocynaceae, Loganiaceae, Rubiaceae and Nyssaceae [9, 31]. In the plant, alkaloids are thought to play a defense role, mainly as deterrent factors against herbivorous pests, and some have been shown to be toxic agaiast certain fimgi and bacteria [32, 33]. A number of reports about the
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antibiotic or antifeedant activity of the TIAs present in C roseus has been published [34-40].
The great pharmacological importance of the dimeric alkaloids, allied with its low availability, stimulated intense research in the biosynthesis of
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TIAs in C roseus and in the regulation of the pathway, with the aim of eventually manipulating plant metabolism in order to obtain higher levels of the anticancer alkaloids. The biosynthesis of vinblastine has shown to be highly complex, involving more than twenty enzymatic steps, and a great deal is already known about the pathway, the enzymes and genes involved, and about their regulation. However, considerable p ^ s of the pathway remain relatively hypothetical, and enzymatic characterization is still lacking for many steps.
The biosynthetic pathway of vinblastine revealed to be highly compartmentalized inside the cell, since a number of enzymes were shown to be localized in different cellular compartments, either experimentally, or by inference from the presence of targeting signal peptides in their aminoacid sequences. Fig. (3) [8, 41]. Enzymes and genes involved in branching points of the early stages of biosynthesis, like geraniol hydroxylase, tryptophan decarboxylase and strictosidine synthase. Fig. (4), have been thoroughly characterized, and the last 6 steps in the path leading to vindoline. Fig. (6), one of the monomeric precursors of vinblastine. Fig. (2), have received much attention as well, with several enzymes/genes of these late steps being characterized. This part of the pathway is not expressed in cell suspension cultures, what possibly accounts for the absence of dimerics in this system. The dimerization step itself has received considerable attention and is thought to be mediated by a class III plant peroxidase [42-44]. In spite of all this, the pathway from primary metabolism to vinblastine and vincristine still includes many transformations that remain to be characterized, even at the level of the biosynthetic intermediates. Previously, the biosynthesis of TIAs has been reviewed in [7, 8, 10, 45].
Biosynthetic pathways
What is known about the biogenetic routes leading to the biosynthesis of the dimeric akaloids vinblastine and vincristine in C. roseus is represented in Fig. (4) to (6). Enzymes and genes that have been characterized are indicated, and the subcellular compartmentalization of the pathway is presented in Fig. (3).
The basic structure of TIAs includes an indole nucleus derived from tryptamine, the decarboxylation product of the aminoacid tryptophan, and a versatile C9 or CIO terpenoid unit arising from the iridoid glucoside secologanin, Fig. (4).
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Tryptophan is a product of the shikimate pathway and is converted into tryptamine by tryptophan decarboxylase (TDC), Fig. (4). TDC is a cytosolic soluble enzyme that occurs as a dimeric protein, and it was shown to exhibit a high substrate specificity and to be under post-translational control [46-52]. A cDNA clone encoding TDC was isolated by DeLuca et al. [53] and the fiill gene was characterized by Gooddijn et al. [54, 55] who found that TDC is encoded by a single copy gene without introns. The Tdc promoter has also been cloned and its regulation characterized [56, 57].
OH
Glyceraldehyde / pyruvate pathway
Shikimate pathway
I COOH
NH
L-Tryptophan TOC
ccn. NH
Tryptamine
Geraniol
lO-Hydroxy-II geraniol
OH
Secoioganin
Fig. (4). Early steps of the biosynthesis of terpenoid indole alkaloids in Catharanthus roseus. Triple arrowheads indicate multiple steps. GIOH: geraniol 16-hydroxylase; TDC: tryptophan decarboxylase; STR: strictosidine synthase.
The terpenoid portion of TIAs is derived from secoioganin, whose monoterpene precursor geraniol, is produced by the recently discovered Rommer or triose phosphate / pyruvate pathway, responsible for the synthesis of isoprenes like geraniol in the plastids [58-60].
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The first committed step in the biosynthesis of secologanin is the hydroxylation of the C-10 position of geraniol by geraniol 10-hydroxylase (GIOH), Fig. (4), which was one of the first cytochrome P-450 monooxygenases to be characterized in plants [61-63]. The enzyme and the associated NADPHicytochrome P-450 reductase were purified to homogeneity fi-om cell suspension cultures of C roseus and characterized [64, 65]. GIOH was shown to be able to hydroxylate both geraniol and its cis isomer nerol. The end product alkaloid catharanthine was proved to be a reversible, linear, noncompetitive inhibitor of GIOH, while vindoline and vinblastine were less inhibitory but still interfered with activity [66]. GIOH was found to be localized in pro vacuolar membranes [67], although the same authors state later that what they had characterized as vacuolar membranes could in fact represent a differentiated form of endoplasmic reticulum [61]. In spite of this ambiguity, GIOH is considered by most reviewers to be localized in the vacuolar membrane [5, 8, 41]. The NADPHxytochrome P-450 reductase (CPR) was found to be similar to the mammalian enzyme and both GIOH and CPR were cloned and the genes characterized [68-70]. The pathway leading fi:'om 10-hydroxygeraniol to secologanin has been relatively well characterized [8, 10, 29, 71] and the enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, secologanin synthase (SLS), Fig. (4), was also shown to be a cytochrome P450 [72, 73].
The stereospecific condensation of tryptamine and secologanin under the action of strictosidine synthase (STR), Fig. (4), is the first committed step in TIAs biosynthesis, and it yields the glucoalkaloid 3-a(S)-strictosidine, which is the central biogenetic precursor of all TIAs [74-77].
STR was first purified by Treimer and Zenk [78, 79] and Mizukami et al. [80] fi-om cell cultures of C roseus. The enzyme was found to have a high substrate specificity, and to suffer no inhibition by end-product alkaloids, such as vindoline and catharanthine [78-80]. It was observed that STR occurred as different isoenzymes [81, 82], and the subcellular localization was determined to be the vacuole [83]. The complete mRNA sequence of Sir was determined by Pasquali et al. [84], who also showed that STR is encoded by a single-copy gene, indicating that the above mentioned isoenzymes are formed post-translationally from a single precursor. Comparison of the primary structure of the STR protein with the amino acid sequence deduced from the S/r mRNA showed the
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presence of a signal peptide of 31 amino acids in the amino-terminal sequence. This signal peptide appears to be essential for vacuolar targeting of STR, according to results obtained with transgenic A . tabacum [83]. The promotor of Str has been studied in great detail and has enabled the identification of transcriptional factors involved in the regulation of TIAs biosynthesis [9, 85, 86] (see section "Regulation..." below).
Strictosidine is the general precursor of several divergent pathways leading to the multitude of TIAs accumulated by C roseus. Somewhere, downstream of strictosidine formation, the pathway of TIAs suffers several ramifications mostly uncharacterized. The less ill characterized branches are the ones leading to catharanthine and vindoline, the monomeric precursors of the Vinca alkaloids. Fig. (2). Those branches will be the ones discussed here.
Fig. (5). Biosynthesis of catharanthine and tabersonine from strictosidine, the central precursor of all terpenoid indole alkaloids. SGD: strictosidine P-I>glucosida(k;.
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The &st step following strictosidine synthesis is the removal of its glucose moiety by strictosidine p-D-glucosidade (SGD) with formation of an unstable aglycone, Fig. (5) [87]. SGD is encoded by a single copy gene in C roseus and is most likely associated with the ER, as suggested by in vivo staining and by the presence of a putative ER signal sequence in the protein [88].
Deglucosylated strictosidine is converted via several unstable intermediates into 4,21-dehydrogeissoschizine from which catharanthine and vindoline are believed to derive, Fig. (5). This part of the pathway has been scarcely characterized - it includes an undetermined number of steps, seems to involve the intermediate stemmadenine, and the branching point for the 2 paths giving rise to catharanthine and vindoline has been proposed to be dehydrosecodine by Blasko and Cordell [71], and to be stemmadenine by Verpoorte et al. [89]. The 6 last biosynthetic steps leading to the production of vindoline from the intermediate tabersonine have been thoroughly characterized and are represented in Fig. (6) [45, 90].
The first step in the conversion of tabersonine to vindoline is hydroxylation of the C-16, which is catalyzed by the enzyme tabersonine 16-hydroxylase (T16H), Fig. (6). Characterization of T16H indicated the enzyme is a cytochrome P-450 monooxygenase [91], what was confirmed by the molecular analysis of the isolated cDNA [92]. Southem analysis suggests the presence of at least two T16H genes in C roseus.
The following step in the biosynthesis of vindoline is the O-methylation of 16-hydroxytabersonine to yield 16-methox54abersonine by the enzyme S-adenosyl-L-methionine: 16-hydroxytabersonine-16-0-methyltransferase (OMT), Fig. (6) [45, 93]. Only a preliminary identification of OMT has been carried out in crude desalted extracts from C roseus leaves [91, 94].
O-methylation of 16-hydroxytabersonine is followed by an uncharacterized hydration step, and then by A^-methylation of the A -indole by the enzyme iS-adenosyl - L-methionine : 16-methoxy - 2,3-dihydro - 3-hydroxytabersonine - A^-methyltransferase (NMT), originating desacetoxyvindoline. Fig. (6). NMT has been roughly characterized by DeLuca et al. [95], and partially purified by Dethier and DeLuca [96] from young leaves of 6 months old C roseus plants. Subcellular localization studies indicated that NMT is specifically associated with the membranes of thylakoids [46].
The second to last step in vindoline biosynthesis is hydroxylation of the C4 of desacetoxyvindoline by a 2-oxoglutarate-dependent dioxygenase, desacetoxyvindoline 4-hydroxylase (D4H, EC 1.14.11.11), Fig. (6). D4H has an absolute requirement of 2-oxoglutarate and molecular oxygen, and its activity is enhanced by ascorbate [97]. Two-dimension electrophoresis resolved the purified D4H into three isoforms, all showing a high aflSnity for desacetoxyvindoline. The authors suggested that this may partially explain the low concentration of desacetoxyvindoline found inside the cell. Furthermore, D4H did not show inhibition even by high concentrations of the hydroxylation product deacetylvindoiine [98, 99]. D4H seems to be localized in the cytosol [97]. Molecular characterization of cDNA and genomic clones of D4H showed the presence of a single-copy gene in C. roseus and that D4H belongs to a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fimgal origin [100].
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The last step in vindoline biosynthesis is catalyzed by acetylcoenzyme A : 4-O-deacetylvindoline 4-O-acetyltransferase (DAT, EC 2.3.1), a reversible O-acetyltransferase that transfers acetate from acetylcoenzyme A to deacetylvindoline. Fig. (6) [101, 102]. DAT was purified and characterized and was first thought to consist of two subunits with molecular weights between 20 and 30 kDa [101, 103, 104]. However, more recently, work developed while cloning DAT cDNA proved that the cellular form of the enzyme is actually a single polypeptide of 50 kDa, indicating that the protein had been cleaved during purification [105]. The purified enzyme is strongly inhibited by tabersonine and the product coenzyme A, but not by up to 2 mM vindoline. This means that the rate of this reaction may be regulated by the level of free coenzyme A in the cell, while remaining unaffected by vindoline accumulation. This could again explain why also deacetylvindoline does not accumulate in C roseus leaves [103, 104]. Subcellular localization studies indicated that DAT is a cytosolic enzyme [46].
Vindoline and catharanthine are the last monomeric precursors of the dimeric anticancer alkaloids of C roseus, and they are also the two major alkaloids accumulated in the leaves of the plant [106, 107]. The study of the dimerization biosynthetic step stems in early work on the chemical synthesis of the dimeric alkaloids and it has involved much discussion. Moreover, the chemical dimerization reaction has industrial application in the synthesis of vinorelbine and vinflunine. Due to its potential regulatory importance for the production of the dimeric Vinca alkaloids in the plant, and to the much that is known about the chemical and biosynthetic reactions, the dimerization step will be presented in particular detail here.
The dimerization step
In face of the structural similarities unraveled during the 1960s of vindoline and catharanthine with the dimeric alkaloids, and due to their great abundance in the plant, these two compounds were immediately considered the most likely monomeric precursors of the Vinca alkaloids, although the cleavamine moiety of vinblastine presented some differences from catharanthine, namely a fragmentation of the C5-C18 bond. Fig. (2).
The natural big abundance of vindoline and catharanthine made also a semisynthetic process for the synthesis of the dimerics very attractive and, after several failed attemps by other groups, Potier et aL in 1975 [19] reported for the first time the synthesis of a dimer with the natural
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configuration through a modified Polonovski reaction. In this reaction, catharanthine N-oxide was treated with trifluoracetic anhydride in the presence of vindoline, leading to C5-C18 skeletal fi-agmentation of catharanthine, which was followed by nucleophilic attack of the CI8 position by vindoline, and formation of the coupling bond. The simultaneous presence of vindoline was essential to obtain the natural configuration, and the authors proposed that the reaction leading to the natural epimer proceeded through a concerted process in which vindoline was involved in displacing the C5-C18 bond, originating the natural stereochemistry. The formation of the imnatural epimer, in rates dependent on experimental conditions, was explained as resulting fi*om a stepwise reaction [18, 19, 108, 109].
The modified Polonovski reaction first used in [19] and later called the Potier-Polonovski reaction, resulted in the formation of an iminium dimer which, after reduction with NaBKU, yielded a-3',4'-anhydrovinblastine, Fig. (2). Thus, this was the first dimeric Vinca alkaloid with the natural configuration to be synthesized. This method allowed, subsequently, the development of approaches to the synthesis of other natural dimerics like vinblastine, vincristine, leurosidine and leurosine [110-114], and more recently, to the semisynthetic vinorelbine and vinflunine [16, 22].
The chemical coupling of catharanthine and vindoline to yield anhydrovinblastine led to the obvious hypothesis that this compound might also be the first product of dimerization in the plant, and the dimeric precursor of vinblastine and vincristine. For three years it was not possible to find anhydrovinblastine in the plant, until Scott et aL in 1978 [115], by modifying the established methods for extraction and purification of alkaloids, isolated anhydrovinblastine fi'om C roseus plants, with incorporation of radiolabelled catharanthine and vindoline, thus proving that anhydrovinblastine was actually a natural product.
In 1979, Langlois and Potier [116] proposed that anhydrovinblastine could be the precursor of most, if not aU, dimeric alkaloids of C roseus, and feeding studies indicated the enzymatic incorporation of anhydrovinblastine into vinblastine and other dimeric alkaloids [117-120]. Incorporation studies were fiirther confirmed by experiments with cell fi"ee homogenates of C. roseus cell suspension cultures [121, 122]. Several biosynthetic routes were proposed in which either anhydrovinblastine or its iminium were the pivotal intermediates of all dimeric alkaloids [114,
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117, 118, 123, 124] but the reactions that really occur in the plant and the respective enzymes have not been characterized.
The search of the enzyme responsible for the dimerization reaction, i.e. for the biosynthesis of anhydrovinblastine, resulted in the finding that peroxidase-like activities extracted fi-om cell suspension cultures were capable of performing the coupling of catharanthine and vindoline into anhydrovinblastine [125-127]. Horseradish peroxidase, a commercial plant peroxidase, was also capable of performing the coupling reaction [128].
At this point, anhydrovinblastine had been proved to actually be a major alkaloid present in C roseus leaves [106, 107] representing together with catharanthine and vindoline the three major alkaloids of the plant. This indicated the presence of high in vivo anhydrovinblastine synthase activity in leaves and that this was the appropriate biological material to search for the enzyme. Work with leaves started at the laboratory of Prof Frank DiCosmo fi"om the University of Toronto, Canada, and has mostly been developed in our labs, at the University of Murcia and the University of Porto.
We have characterized and purified a basic peroxidase fi"om C roseus leaves with a-3',4'-anhydrovinblastine synthase activity. This enzyme was, on the one hand, the single peroxidase isoenzyme detected in C. roseus leaves and, on the other hand, the single anhydrovinblastine synthase activity detected in C roseus extracts, and was thus considered to be the most likely in vivo responsible for the synthesis of anhydrovinblastine [42, 43]. Moreover, vacuole isolation and peroxidase cytochemical detection showed that the enzyme was localized in the vacuole, the same subcellular compartment where both substrates and product of the dimerization reaction are accumulated [42, 44]. The mechanism of the peroxidase mediated dimerization reaction was investigated and it was shown that both vindoline and catharanthine are suitable electron donors for the oxidizing intermediates of the basic peroxidase, compound I and compound II, and it was proposed that the coupling reaction proceeds by a radical propagated mechanism [43, 44, 129]. Recently, we have discussed the assignment of a specific fimction, such as the synthesis of anhydrovinblastine, to a multifimctional enzyme, such as the basic peroxidase of C. roseus leaves, and we have proposed a channeling mechanism for the peroxidase-mediated-vacuolar synthesis of anhydrovinblastine [44]. We have now characterized the cDNA and
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genomic sequence of this anhydrovinblastine synthase-peroxidase in collaboration with Mark Leech from the John Innes Centre, UK [130].
Regulation of the biosynthesis of terpenoid indole alkaloid biosynthesis in the plant
The TIAs pathway in C roseus has been shown to be under developmental regulation, showing also cell-, tissue-, and organ-specific expression [90, 131]. Particularly interesting is the differential localization of early and late stages of vindoline biosynthesis in particular cells of leaves shown by in situ RNA hybridization and immunocytochemistry studies. Those experiments suggest the involvement of at least two cell types in the biosynthesis of vindoline and the existence of intercellular translocation of a pathway intermediate [131]. The pathway from tabersonine to vindoline, specifically, has been shown to be under developmental and light regulation, a fact that was pointed as connected with the absence of vindoline in cell suspension cultures [90, 93, 132].
On the other hand, and since alkaloids constitute a defense against certain environmental stresses, it is not surprising that the TIA pathway shows induction by several biotic and abiotic stress factors [6, 133-137]. Among those, the induction by fungal elicitors has been particularly well characterized, together with the regulation by the plant stress hormone methyljasmonate [5, 9].
Regulation studies of the promotor of Str, Fig (4), enabled the identification of an autonomous jasmonic acid-responsive sequence -JERE (jasmonate- and elicitor-responsive element) present in the promotor [138]. The JERE was shown to interact with three jasmonic acid-responsive transcription factors - the ORCAs (octadecanoid responsive Catharanthus AP2-domain proteins) [85, 138]. ORCAs belong to the AP2/ERF family of transcription factors, which are unique to plants. 0RCA3 was shown to positively regulate the TIAs biosynthesis genes Tdc, Str, Cpr and D4h, belonging to both early and late steps of the pathway [139]. Moreover, 0RCA3 also regulates genes from the primary metabolism pathways involved in the biosynthesis of tryptophan and geraniol, and was thus considered a master regulator of metabolism [9, 139]. However, several genes in the TIA and secologanin pathways are not regulated by ORCA3, meaning that overexpression of this transcription regulator alone is not sufficient to enhance the levels of
831
TIAs in C roseus cell suspension cultures or plants. Nevertheless, the transcriptional factor approach bears a great potential for manipulation of the TIA pathway, and discovery of a few more regulators like 0RCA3 may, in the fiiture, enable to induce/increase the biosynthetic pathway of vinblastiae in cell cultures and/or plants.
ACCUMULATION OF THE VINCA ALKALOIDS IN THE PLANT VACUOLE
Vinca alkaloids and other TIAs produced by C. roseus are toxic, not only to animal cells, but also to plant cells, and even to the plant cells that produce them [140-142]. This is actually the common situation with many secondary metabolites produced by plants. Plant cells are able to accumulate high levels of those compounds because they are removed from the cytosol and sequestered inside the vacuole. The vacuole of plant cells may occupy up to 90% of the cell volume and performs a number of important fimctions like regulation of turgor pressure with a role in cell growth, detoxification of xenobiotics, storage of many useful compounds, ion homeostasis, hydrolysis of various molecules and macromolecules, and accumulation of secondary metabolites that may act in defense against herbivores, pathogens, UV light, etc. [143, 144].
In C roseus, Vinca alkaloids and other TIAs are thus transported across the vacuolar membrane, the tonoplast, and accumulated inside the vacuole, as has been shown in a significant number of reports. Deus Neumann and Zenk [145] showed that serpentine is exclusively stored within the vacuoles of C roseus cells, and that C. roseus vacuoles can uptake and accumulate ajmalicine, catharanthine and vindoline. Ajmalicine and serpentine accumulation inside the vacuoles of suspension cells was confirmed by [146], and immunocytochemical localization of vindoline indicated its major presence in the central vacuole and in small vesicles of mesophyll cells [147].
If TIAs are stored inside the vacuole, they must cross the tonoplast and concentrate inside the organelle. The mechanisms for transport of TIAs across the tonoplast and of their accumulation inside the vacuole have been the subject of much controversy during the end of the 1980s. For critical reviews see [143, 148]. Since that time, hardly any report on TIAs transport across the tonoplast has been published, meaning that this is still an unsolved problem.
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Here, we will review the controversial work done about TIAs tonoplast transport and discuss possible directions for future work.
Two types of mechanisms have been proposed for TIA transport across the tonoplast: i) a highly specific carrier mediated mechanism and ii) a variety of more or less unspecific 'trapping" mechanisms which assume that transport through tonoplast occurs by passive difiusion and that inside the vacuole alkaloids suffer some transformation that lowers their activity or permeability and thus favors their accumulation. It has been further proposed that membrane vesicles may be involved in the biosynthesis and/or packaging of the alkaloids and their transport to the central vacuole [147, 148].
Among trapping mechanisms, the most discussed has been the "ion-trap" model [146, 148-153]. This model is based on the assumption that, being low molecular weight amines, the neutral form of the alkaloids is lipophilic and will thus fi'eely diffuse across the lipidic phase of biologic membranes, while the protonated alkaloid formed in acidic pH will have a much lower permeability coeflBcient. Under these conditions, the ion trap model implies that when several compartments exist, the neutral base will diflRise across membranes and will be present at the same concentration in each compartment, while the cation will remain trapped and will thus accumulate in the more acidic compartment. Accumulation will depend on the pH gradient across the compartment membrane and on the dissociation constant (pKa) of the alkaloid - the higher the ApH and the pKa are, the higher the accumulation will be. This model is supported by a number of experiments performed mainly with ajmalicine and with some alkaloids not present in C roseus^ which confirmed the postulated low specificity of the ion trap model [146, 149, 151-153]. Nicotine, an alkaloid which is not produced by C roseus, accumulated in C roseus vacuoles to concentrations 12 times higher than ajmalicine, in agreement with its higher pKa of 8.0 in comparison with 6.3 for ajmalicine [153]. The low specificity observed, the insensivity of accumulation to ATP, the absence of saturation in short-term assays, the identical pattern of influx and eflQux curves, and above all, the linear dependence of accumulation on the external pH, were interpreted as strong evidence supporting the ion trap model [149,151-153].
Other trapping mechanisms, which have been suggested for indole alkaloids and could work in complementation with the ion trap mechanism, are binding to other vacuole components like phenolics, or to
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the inner side of the tonoplast, which seems to be the case of serpentine [148]. A very eflBcient trapping was observed by Hauser and Wink [154] in vacuoles of Chelidonium majus containing high concentrations of chelidonic acid, which was shown to readily complex alkaloids, including vinblastine. The authors showed that vinblastine could easily cross the tonoplast by diffusion and accumulate against a concentration gradient, apparently due to complexation to chelidonic acid. A similar complexation mechanism with meconic acid has been proposed for accumulation of morphine in Papaver latex vacuoles.
Further metabolization of alkaloids inside the vacuole is another possibility of trapping. Blom et al. [146] showed that ajmalicine is oxidized by peroxidase into the charged serpentine inside the vacuole, creating a trap that retains alkaloids more efficiently within the organelle. The group of Renaudin [152, 153] observed that two pools of ajmalicine were present in C roseus cells: one pool which could rapidly move to and from the vacuole, corresponding to molecules accumulated by ion trapping (quickly exchangeable pool) and a second pool also exchangeable in both directions but within a much longer time scale (slowly exchangeable pool). The authors hypothesized that this could either be due to the presence of two populations of vacuoles with different transport characteristics, or to binding of ajmalicine to other vacuole components like those referred above, meaning the existence of two different trapping mechanisms.
Possibly related with these results, McCaskill et al. [149] observed that accumulation of vindoline, ajmalicine, tabersonine and vinblastine by C roseus protoplasts was biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation, while accumulation of nicotine was monophasic. The data presented suggested that the initial burst for vindoline and ajmalicine and the accumulation of nicotine were driven by the pH gradient between the vacuole and the external medium, through an ion trap. For ajmalicine, the second phase of uptake was not inhibited by azide and the authors suggested it could be due to complexation with organic counterions or phenolics inside the vacuole. In the case of vindoline, it was observed that azide inhibited the second phase of accumulation and the authors concluded that transport of vindoline across the tonoplast should also involve a specific energy-requiring uptake.
This idea that indole alkaloids transport across tonoplast could be mediated by a highly specific energy dependent carrier was proposed and
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defended by Deus-Neumann and Zenk [145, 155]. In their experiments, Deus-Neximann and Zenk observed vacuolar uptake to be very specific for the endogenous alkaloids [145]. Vindoline, catharanthine and ajmalicine were taken up only by vacuoles of C roseus and not by vacuoles of other alkaloid accumulating plants, like Nicotiana tabacum and Papaver somniferum. Inversely, C. roseus vacuoles did not accumulate nicotine or morphine, in contrast to what has been observed by Renaudin and McCaskill et al. [149, 153]. Moreover, transport was saturable and exhibited dependence on pH with an optimum at pH 6.5; transport was sensitive to temperature and was inhibited by the ATPase inhibitor DCCD (A/,iV-decyclohexylcarbodiimide). Transport was not, however, stimulated by ATP. KM values of 1.5 |LIM for vindoline, 2.5 fiM for catharanthine and 1.67 |iM for ajmalicine were determined. In fiirther experiments with Fumaria capreolata and the isoquinoline alkaloids reticuline and scoulerine, Deus-Neumann and Zenk observed again an absolute specificity for the alkaloids indigenous to the plant, and even for the natural (5)-enantiomeric form of the alkaloids[155]. Stimulation by ATP and inhibition by a protonophore was observed, and alkaloid eflQux was shown to have the same characteristics as uptake. The authors proposed that alkaloid uptake and release through the tonoplast is a highly specific carrier-mediated and energy-dependent proton antiport system, and they postulated that "for every alkaloid group, maybe even for every single alkaloid molecular species, there is a highly specific alkaloid carrier, or specific binding sites of a general carrier, present in the tonoplast membrane". Mende and Wink obtained results similar to Deus-Neumann and Zenk for the uptake of the quinolizidine alkaloid lupanine by Lupinuspolyphyllus vacuoles [156].
In view of the contradictory results and proposals concerning alkaloid transport, Guem et al. [148] tried to reconcile the two opposing concepts by proposing that the ion-trap and carrier models are not necessarily exclusive, but most likely coexist. The relative importance of the two systems would depend on the physicochemical properties of the alkaloid molecule, i.e., its lipophilicity and basicity, and on the physiological conditions concerning the driving forces for vacuolar accumulation, i.e., the trans-tonoplast pH gradient. Under this light, Guem et al. [148] reinterpret some of the opposite results obtained by defenders of the two models. According to these authors, and based on measurement of the vacuolar pH using the ^^P-NMR technique, vacuoles isolated using NaCl
835
as an osmoticum, as done by Deus Neumann and Zenk [145], present a higher pH (6.35) compared to vacuoles isolated in sorbitol (pH 5.33), the technique used by the group of Renaudin. Thus, in the vacuoles isolated by Deus Neumann and Zenk [145], which had presumably lost their acidity, uptake of alkaloids should proceed mainly through the highly specific binding component of transport, while the ion-trap component could not exert its action. Also Wink [143], in his outstanding review about the plant vacuole, stresses that the two mechanisms are compatible.
Nevertheless, the discrepancy of results obtained is still puzzling and the transport of indole alkaloids across the tonoplast and their accumulation in the vacuole is still an unsolved problem. Experiments should be designed in a way that enables the distinction between transmembrane transport and possible subsequent intravacuolar events, like complexation or metabolism, and the pH gradient across the tonoplast should be monitored all through the experiments. Membrane permeability to the neutral and ionized forms of alkaloids should also be precisely determined, since this is a fundamental question in the problem, and data available is controversial. If membranes are significantly more permeable to the neutral form of the indole alkaloids than to the protonated form, it is reasonable to think that at least in cells with a vacuolar pH as acid as 3 [150] even alkaloids with a low pKa as vindoline (5.5) will accumulate by ion-trapping. Interestingly, Yoder and Mahlberg [157] observed that alkaloids accumulated in specific cells which showed a lower vacuolar pH than other mesophyll cells, as determined by neutral red staining. It is also interesting to notice that carrier mediated transport has been characterized by Deus Neumann and Zenk [145] mostly for vindoline (pKa=5.5), which has a pKa significantly lower than ajmalicine (6.3) and catharanthine (6.8), and thus would always be trapped less efficiently by the pH gradient alone. It was also for vindoline that McCaskill et al. [149] detected, apart fi:'om an ion-trapping, a specific energy-requiring uptake component. But the possible involvement of specific carriers, at least for vindoline transport, lacks confirmation, namely the final proof which is the purification of the carrier or carriers.
Although no fiirther work has been published on transport of indole alkaloids across the tonoplast, much work has since been published about tonoplast transporters, which may add some relevant clues. It was shown that in maize, the last step in anthocyanin biosynthesis involves its conjugation to glutathione and this conjugate is then recognized for transport into vacuoles by the glutathione pump [158]. This pump is
836
responsible for the vacuolar compartmentalization of glutathione conjugates of xenobiotics, thus enabling its detoxification. In view of their results, the authors fiirther suggested that many of the naturally synthesized plant secondary metabolites could also be recognized and transported by the glutathione pump. Hortensteiner et al [159] identified in vacuoles of Hordeum vulgare a transport system capable of actively transport the bile salt taurocholate across the tonoplast. The pysiological substrate(s) for this transporter of plants have not been identified, but vinblastine inhibited the taurocholate transport, and kinetic analysis of the inhibition revealed that vinblastine could be a substrate for the transporter [160].
Glutathione pumps and the bile salt transporter of Hordeum vulgare belong both to the ATP-binding-cassette (ABC) transporter superfamily that also includes the multidrug resistance protein, P-glycoprotein, responsible by resistance of cancer cells to drugs such as the Vinca alkaloids (see section "Uptake..." below). Knowledge about the fimction of ABC transporters in plants is still scarce but the vacuolar uptake of a number of compounds has been associated to these transporters, such as: glutathione conjugates of both exogenous (xenobiotic) and endogenous (secondary metabolites) compounds; clorophyll catabolites; glucuronides and glucosylated herbicides [161].
The Arabidopsis ABC transporter gene family has been shown to contain 131 members, exceeding the 48 reported for Homo sapiens [162], and Jasinski et al. [162] correlate the diversity and evolution of plant ABC transporters with the diversity of plant secondary metabolism, pointing out the requirement of adapted transporters to transfer those metabolites iato the vacuole or the extemal medium. In view of all this, we believe that TIA transport across the tonoplast may indeed be mediated by one of the several types of ABC transporters, and that this hypothesis should be fiirther investigated. Supporting this idea, an ABC protein has been shown to be responsible for the transport of the benzylisoquinoline alkaloid berberine across the plasma membrane of Coptis japonica [163]. Moreover, a gene encoding an ABC transporter has been cloned in C roseus, whose expression in cell cultures is enhanced by the addition of citokinins, methyl jasmonate and auxin suppression, all conditions that increase the production of terpenoid indole alkaloids by the same cell suspensions [164].
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MECHANISM OF ACTION OF THE CATHARANTHUS ALKALOIDS
Vinca alkaloids are cytotoxic to most cells (including plant cells) with a stronger effect in actively dividing cells like cancer cells. They have been shown to interfere with several distinct cellular processes like protein synthesis and degradation, lipid metabolism and calcium movements [13, 165, 166], but, until now, their main target in what concerns their cytotoxicity to cancer cells is still considered to be the microtubules, with consequent effects in mitosis.
After the discovery and clinical application of vinblastine and vincristine as anti cancer drugs, it was soon pointed out that the anticancer action of these compounds seemed to be due to microtubule depolymerization and mitosis arrest as a result of the absence of mitotic spindle [167-169]. The Vinca alkaloids were thus classified as mitotic blockers with their primary site of action being M phase of the cell cycle. However, more recently, it has been shown that although Vinca alkaloids depolymerize microtubules at |imolar concentrations, they actually have a more subtle effect at low concentrations (nanomolar range), stabilizing microtubules due to inhibition of normal microtubule dynamics [170, 171]. This effect, in actively dividing cells, seems to be able to induce a program of cell death [172-174], The antineoplastic activity of Vinca alkaloids in the clinical treatment of cancer may thus arise fi-om perturbation of a variety of microtubule-dependent processes, including the cell cycle, ultimately inducing programmed cell death.
The complexity of microtubule behavior and the difficulty in determining exactly how occurs the interference of Vinca alkaloids with that complexity has made it very difficult to characterize the precise anticancer mechanism of action of these drugs, which is still not fiiUy understood. A thorough and clear review on what is known about microtubule behavior and the way Vinca alkaloids interact with microtubules and the ceU cycle has been published very recently by Mary Ann Jordan [172]. Here, a more general overview will be presented, trying to highlight adequately the main points of this difficult subject.
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Interaction of Vinca alkaloids with tubulin and microtubules
Microtubules are highly dynamic protein polymers present in all eucaryotic cells, with important roles in the determination of cell shape, in cell signaling, in cellular and intracellular movements, and in cell division. Microtubules are built from dimeric subunits composed of two similar globular proteins, a- and p-tubulin, that are stacked in 13 linear chains arranged in parallel to form hollow tubes with 25 nm in diameter and a variable length that may reach many |im. Cells contain a mixture of free tubulin dimers and microtubules that undergo continual remodeling by constant assembly and disassembly of tubulin subunits at the microtubule ends. During mitosis, the microtubules become organized in two arrays that form the mitotic spindle, responsible for the precise distribution of chromosomes between the two daughter cells - during this process the microtubules suffer constant and dramatic reshaping [175].
Vinblastine can bind with high aflSnity to the dimeric tubulin subunits and the microtubule ends (Kd = 1-2 |iM), and with lower aflBnity (Kd = 0.25-0.3 mM) to tubulin sites located along the sides of the microtubule cylinder [172, 176]. This binding of the Vinca alkaloids along the length of the microtubule must be the responsible for their ability to depolymerise microtubules at high concentrations (> 1 |iM). Among vinblastine, vincristine and vinorelbine, vincristine demonstrates the highest overall aflHnity for tubulin and vinorelbine the lowest [177]. Vinflunine has been shown to bind to tubulin with even lower affinity than vinorelbine [178]. Affinity is thus ranked vincristine > vinblastine > vinorelbine > vinflunine, parallel to drug toxicity, but not to therapeutic potential. These values correlate inversely with drug doses used in clinical treatments, since vincristine is used at the lowest dose and vinorelbine at the highest.
Binding of Vinca alkaloids to tubulin is reversible, and the precise location and number of binding sites in tubulin dimers and microtubule ends is not clear. Rai and Wolff [176], using a fluorescent vinblastine derivative, detected and characterized a single high affinity binding site in p-tubulin and also detected the presence of several low affinity sites that were not possible to characterize. More recently, nuclear magnetic resonance analyses revealed the presence of three binding sites in the a/p-tubulin dimer for vinorelbine and vinflunine [179]. At 30^ C, binding of
839
vinflimine to tubulin was hardly detected, in agreement with the significant lower aflSnity of this drug to tubulin.
In reconstituted microtubule systems and in cells, the interaction of Vinca alkaloids with microtubules results in different effects depending on drug concentration: i) at low concentrations (< 1 jiM = nanomolar range), the drugs diminish microtubule dynamics increasing the time spent in the "resting state"; ii) at intermediate concentrations (1-2 fiM), they depolymerize microtubules and inihibit assembly; and iii) at high concentrations (> 10 }iM), they induce self-aggregation of tubulin with formation of large paracrystals and other aggregates [172, 176]. In all cases, normal microtubule fimction is compromised.
In spite of all that is known about the interaction between Vinca alkaloids and tubulin, the precise molecular location and chemical bonds established during that interaction are not known what prevents a rationale design of new Vinca alkaloids.
The different therapeutic profiles and toxicities of the Vinca alkaloids are thought to be partially due to the different aflSnities they show towards tubulin [172, 177], namely towards different tubulin isotypes that may have tissue specific expression [180]. It is interesting to remark that, although vinorelbine and vinflunine show significant lower affinity to tubulin than vincristine and vinblastine, a fact correlated with their lower toxicity, they actually present a higher anticancer therapeutic action, a fact correlated with their higher intracellular accumulation (see section "Uptake..." below) [23, 25, 181]. This means that vinorelbine and vinflunine present a differential effect between normal cells, where lower aflSnity to tubulin prevents toxic effects in spite of high intracellular concentrations, and cancer cells, where the weak interaction of the high concentrated drugs is sufficient to induce a strong effect. There is no explanation for this fact but Ngan et al. [181] consider that nontumor cells, with normal checkpoint proteins, may tolerate better the relatively less powerfiil inhibitory effects of vinflunine and vinorelbine on microtubule dynamics, than cancer cells, with abnormal cell cycle regulation.
Mechanism of inhibition of ceU proUferation
The mechanism of action of the Vinca alkaloids was initially thought to involve the depolymerization of spindle microtubules and induction of
840
paracrystalline tubulin-Fmca alkaloid aggregates, the eflFects observed at intermediate and high concentrations of the drugs [167-169]. However, the more recent observation that, at low concentrations, the Vinca alkaloids inhibit the microtubule dynamic behavior raised two questions: i) whether this effect could also block mitosis and ii) which of the two situations is actually involved in anticancer therapy with this group of drugs.
Investigating this question, Jordan and collaborators [182] observed that, at the lowest effective concentrations of five Vinca alkaloids, inhibition of cell proliferation and blockage of HeLa cells at metaphase occurred with little or no microtubule depolymerization and no spindle disorganization. With increasing drug concentrations, the authors observed that the organization of microtubules and chromosomes started to deteriorate. Dhamodharan et al. [183] also observed that low vinblastine concentrations (nM levels) block mitosis in BS-C-1 cells, in association with suppression of microtubule dynamics but in the absence of appreciable changes in microtubule mass. Recently, it has been shown that the precise parameters of microtubule dynamics that are inhibited at low concentrations by vinorelbine and vinflunine differ significantly fi-om vinblastine [181]. In spite of those differences, fiirther investigations showed that all the three drugs produced remarkably similar effects on spindle organization. In all cases, proliferation inhibition seemed to be induced my mitotic block at the metaphase/anaphase transition with formation of aberrant spindles, consistent with induction of block by suppression of microtubule dynamics [23].
As a whole, the results presented above indicate that low concentrations of Vinca alkaloids, probably similar to therapeutic concentrations, have an antiproliferative activity that is due to inhibition of mitotic spindle fimction by changing the dynamics of microtubules rather than by depolymerizing them. A growing body of evidence seems to indicate that, specially in cancer cells, where mitosis regulation is already disrupted, the suppression of microtubule dynamics with mitosis arrest induces a signaling cascade leading to cell death by apoptosis (a type of programmed cell death) [172-174].
Other cellular targets of Vinca alkaloids
Vinca alkaloids are toxic molecules that easily cross cellular membranes due to their lipophilicity and interfere with a multitude of cell targets.
841
Although tubulin and microtubules are undoubtedly the main target responsible by their anticancer action, other targets may also contribute to this action, while some others may be associated with their toxic side eifects.
Recently, it has been shown that proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent proteolysis pathway, can also be considered a target of vinblastine. Proteasomes have a crucial role in the regulation of the cell cycle, and proteasome inhibitors can block cell cycle progression and induce apoptosis in certain cell lines. Vinblastine seems to have an inhibitory effect on proteasomes and could thus interfere with mitosis also through this path [165].
Another target of Vinca alkaloids seems to be DNA. Tiburi et al. [184] showed that vinblastine, vincristine and vinorelbine all had significant genotoxicity, as assayed by the vraig Somatic Mutation and Recombination Test (SMART) of Drosophila, All the three drugs caused increments in the incidence of mutational events and somatic recombination.
An effect of Vinca alkaloids that may also be important in their anti tumour activity is their antivascular action. It has been shown that vincristine and vindesine are able to reduce the capillary network formation by HUVEC cells cultured on Matrigel at non-cytotoxic concentrations, while vinblastine and vinorelbine produce anti-angiogenic effects by direct cytotoxicity [185]. Vinflunine seems to have an antivascular activity consistently superior to that of vinorelbine [25].
UPTAKE AND EXTRUSION OF VINCA ALKALOIDS IN ANIMAL CELLS
Vinca alkaloids are lipophilic molecules that can readily cross membranes by simple diffusion [186]. Experiments performed with several human cancer and tissue cell lines have shown in all cases rapid uptake of every one of Vinca alkaloids [23, 187-189]. Uptake is thought to occur by diffusion although energy dependence or independence of the uptake is seldom mentioned in reports. However, for instance in cultured human promyelocytic leukemia HL-60/C1 cells it has been shown that rates of uptake of vinblastine were unaffected by depletion of cellular adenosine triphosphate, reinforcing that uptake is not mediated by an energy-dependent system [189].
842
When incubated with human hepatocytes, vinorelbine was the most rapidly and intensely accumulated Vinca alkaloid followed by vinblastine, vindesine and vincristine, as would be suggested by the lipophilicities of the molecules [187]. Vinflunine is even more lipophilic than vinorelbine and accumulates faster inside cells [23].
Although uptake is considered to occur by difiusion, the Vinca alkaloids accumulate inside animal cells to concentrations many times higher than extracellular concentrations. Vinblastine and vincristine have been shown to accumulate more than 100 fold in cultured human promyelocytic leukemia HL-60/C1 cells [189]. Addition of 10 nM vinblastine to the culture medium of HeLa cells resulted in a 40 fold accumulation, while addition of 100 nM vinblastine resulted in a 31 fold accumulation [182], In BS-C-1 cells, 32 nM vinblastine accumulated 284 fold [183]. This intracellular accumulation is thought to result, at least in part, from the binding of the drugs to tubulin and microtubules [172]. For example, the maximum vinblastine intracellular levels observed in HeLa cells are similar to the intracellular levels of tubulin [182]. However, the existence of other intracellular reservoirs for drug accumulation is not discarded and, recently, some evidence has been obtained that suggest that vinorelbine and vinflunine may be sequestered inside the cell by other mechanism than binding to tubulin [23]. The authors characterized uptake of 1 nM vinblastine, 3 nM vinorelbine and 30 nM vinflunine by HeLa cells. The concentrations used for each drug were the ones inducing the same effect in cells, i.e., a 50% inhibition of mitosis. As predicted from their lipophilicity, uptake rate was much higher for vinflunine, followed by vinorelbine, and the peak concentration for the three drugs was 4.2 jxM for vinflimine (140 fold), 1.3 |LIM for vinorelbine (430 fold) and 130 nM for vinblastine (130 fold). In these conditions, mitosis was blocked but microtubules were not disassembled. Since micromolar concentrations of vinorelbine and vinflunine significantly reduce microtubule polymerization in vitro [181], the authors suggest that not all intracellular vinflunine and vinorelbine is available to bind to tubulin and must be sequestered in other cellular reservoirs such as membrane compartments [23].
In animal models, concentrations in tissues can also exceed those in plasma, and it has been observed that, in certain tissues, the drugs are retained for prolonged periods, sometimes related to the specific therapeutic indications of each drug [190]. In mice, vinblastine is
843
selectively retained in genetic tract and lymphatic tissues, a fact that may be the basis of the activity of this alkaloid against malignant transformations with an origin in these tissues [191]. Likewise, in rats, the lungs were among the organs with higher accumulation of vinorelbine, which is used in the treatment of small cell lung cancer [190], This differential retention may result in some cases from different relaxation times of the binding of the drug to different tissue isotypes of tubulin, but, in other cases, it seems that the tissues not retaining the alkaloids possess effective means of extruding the drug when plasma levels decrease [191]. Meaning that extrusion of the Vinca alkaloids from animal cells may occur not only by difiusion but also as a result of a more efficient transport mechanism.
In many cases, the Vinca alkaloids are just released by difiusion after exposure to the drug ends [189], with a rate dependent on the strength of their binding to tubulin and/or on the release rate from other intracellular sequestration mechanisms. However, as stated above, some tissues possess a more effective way of extruding the drugs, namely cancer cells that have become resistant to chemotherapy. In fact, the best characterized mechanism of resistance of cancer cells to chemotherapy drugs, like the Vinca alkaloids, is the phenomenom known as multidrug resistance (MDR), which is due to the overexpression of the mdrl gene coding for the membrane localized P-glycoprotein, that actively pumps the drugs out ofthecell[15, 192].
P-glycoprotein is constitutively overexpressed in various normal tissues including the renal tubular epithelium, the adrenal medulla, the liver, and the blood brain barrier, where it is thought to protect cells from toxic agents/xenobitotics [193, 194]. P-glycoprotein belongs to the family of ABC transporters (see section "Accumulation ..." above) and it is localized in the plasma membrane, being able to extrude from the cell a variety of structurally diverse drugs, drug conjugates and metabolites. Extrusion of these compounds by P-glycoprotein is ATP-dependent and can take place against considerable concentration gradients [192].
P-glycoprotein expression may occur in tumour types derived from tissues that normally express the protein, like renal cell cancer, but its overexpression may also be induced by the treatment with anticancer drugs. All Vinca alkaloids used in cancer therapy can induce the expression of P-glycoprotein and the associated multidrug resistance phenotype, due to the capacity of P-glycoprotein to pump out of the cell a
844
number of unrelated anticancer drugs, preventing therapeutic intracellular concentrations to be achieved [15].
METABOLISM OF VINCA ALKALOIDS IN ANIMAL CELLS
Vinca alkaloids are metabolized primarily by the liver, and metabolites are eliminated by biliary excretion [15]. When incubated with human hepatocytes in suspension, vinblastine, vincristine, vindesine and vinorelbine are rapidly taken up and intensely metabolized by the cells in a number of xmidentified products [187]. On the other hand, the capacity of cancer cells to metabolize these drugs is usually limited. For example, HPLC analysis of extracts of human promyelocytic leukaemia HL-60/CI cells incubated with growth-inhibitory concentrations of labelled vinblastine and vincristine indicated little or no metabolism of either drug by cells or culture fluids [189].
In the liver, the only and possibly main enzymatic system that has been shown to be involved in metabolism of Vinca alkaloids is the cytochrome P450 monooxygenase CYP3A4 [195-197]. The large interpatient variability in the sensitivity to the Vinca alkaloids has been frequently associated to individual disparities in the levels of CYP3A4. Recently, it has been shown that tumour CYP3A4 may also contribute to the development of drug resistance during chemotherapy [196].
Another enzyme that has been implicated in the metabolism of vincristine in acute myeloblastic leukaemia (AML) cells is myeloperoxidase. The fact that AML is resistant to vincristine has been related to the presence of mieloperoxidase, which is able to catalize the vincristine's oxidative breakdown [198, 199].
The compounds resulting from metabolism of the Vinca alkaloids are little characterized. The main hepatic metabolite of vinblastine, vincristine, vinorelbine and vinflunine seems to be, for each of the compounds, the respective 4-O-deacetyl alkaloid. Other metabolites have also been detected but only very seldom were structurally characterized [15,191,200-203].
845
THE VJNCA ALKALOIDS FROM PLANTS TO ANIMALS - THE EVOLUTIONARY LINK
The Vinca alkaloids metabolism and transport in the producing plant cells and in the treated animal cells illustrate some interesting aspects of how evolution can be winding and parsimonious in the solutions it creates.
Plants possess an incredibly diverse biosynthetic capacity leading to the production of a myriad of compounds that, although not having an apparent function for fiindamental life processes (growth, development and reproduction), seem to have vital roles as mediators of ecological interactions, being very important for the survival of plants. This chemical wealth is the basis of the use of plants in medicine, and is still largely unexplored. One example of application of the so called plant secondary metabolites are the terpenoid indole alkaloids of Catharanthus roseus, used in cancer therapy, and known as the Vinca alkaloids.
In the plant, the biosynthesis of the Vinca alkaloids involves more than 20 enzymatic steps including several cytochrome P450 monooxygenases and one class III plant peroxidase. Removal of the toxic alkaloids from the cytoplasm to the vacuole of plant cells is made by an uncharacterised transport mechanism that we suggest may be an ABC transporter, as already shown for several other plant secondary metabolites.
In human and model animal cells, metabolism of the exogenously applied Vinca alkaloids is carried out by a cytochrome P450 monooxygenase and in some cases by myeloperoxidase, and removal of the toxic alkaloids from the cytoplasm to the extracellular compartment is made by an ABC transporter. The mechanisms involved in metabolism and transport of the Vinca alkaloids in animals are thought to have developed, at least in part, as a defence against the toxic compounds present in the plants that animals eat [204].
The similarities between mechanisms involved in production and accumulation of toxic defence compounds in plants, and metabolism and transport of the same compounds as xenobiotics in animals, mean that essential building blocks of such complex and divergent organisms as plants and animals have a very ancient common origin, and that, through evolution, they were sometimes recruited to ftmctions that oppose to each other in nature.
The P450 superfamily, for example, is found in all groups of organisms, including Archae, and is believed to have originated in an
846
ancestral gene that existed over 3 billion years ago [205]. The ABC transporter superfamily is also referred as one of the biggest multigene families and it has been shown to exist in bacteria, fimgi, plants and animals [161, 194, 206]. Single ancestor genes have thus suffered major duplication events and evolved to result in a panoply of functions.
To construct the amazing diversity of life, evolution has sometimes played with a small number of pieces to construct the biochemical survival strategies inherent to that diversity.
[10] Sottomayor, M.; DiCosmo, F.; Ros Barcelo, A. In Advances in Plant Physiology; A. Hemantarajan, Ed.; Scientific Publishers (India): Jodhpur, 1999; Vol. 2, pp. 1-41.
[28] Van der Heijden, R.; Verpoorte, R.; Ten Hoopen, H.J.G.; Plant Cell Tiss. Org. Cult., 1989,18,231-280.
[29] Groger, D. In Biochemistry of Alkaloids; K. Mothes, H.R. Schiitte, M. Luckner, Eds.; VEB Deutscher Verlag der Wissenschaften: Berlin, 1985; pp. 272-313.
[30] Verpoorte, R.; Van der Heijden, R.; Van Gulik, W.M.; Ten Hoopen H.J.G. In The Alkaloids; A. Brossi, Ed.; Academic Press: San Diego, 1991; Vol. XL, pp. 1-187.
[31] Schutte, H.R.; Progress Bot., 1986, 48,151-166. [32] Wink, M. In Alkaloids. Biochemistry, Ecology, and Medicinal
Applications; M.F. Roberts, M. Wink, Eds.; Plenum Press: New York, 1998; pp. 265-300.
[33] Bennett, R.N.; Wallsgrove, R.M.; New Phytol., 1994, 127, 617-633.
[34] Frischknecht, P.M.; Battig, M.; Baumann, T.W.; Phytochemistry, 19S7,26,107-7\0.
[35] Kulkami, R.N.; Ravindra, N.S.; Planta Med, 1988,54, 356-359. [36] Chandravadana, M.V.; Nidiry, E.S.J.; Khan, R. M.; Rao, M.S.;
Fundam. Appl. Nematol, 1994,17, 185-192. [37] Patel, H.R.; Makwana, M.G.; Patel, B.N.; Ind J. Myc Plant
[64] Meijer, A.H.; Pennings, E.J.M.; DeWaal, A.; Verpoorte, R. In Progress in Plant Cellular and Molecular Biology; H.J.J. Nijkamp, L.H.W. Van der Plas, J. Van Aartrijk, Eds.; Kluwer Academic Publishers: Dordrecht, 1990; pp. 769-774.
[65] Meijer, A.H.; De Waal, A.; Verpoorte, R.; J. Chrom., 1993, 635, 237-249.
[130] Hilliou, F.; Costa, M.; Almeida, I.; Lopes Cardoso, I.; Leech, M.; Ros Barcelo, A.; Sottomayor, M. In Plant Peroxidases. Biochemistry and Physiology. VI International Plant Peroxidase Symposium; M. Acosta, J.N. Rodriguez-Lopez, M.A. Pedreno, Eds.; University of Murcia and University of A Coruna: Murcia, 2002.
[148] Guem, J.; Renaudin, J.P.; Brown, S.C. In Cell culture and somatic cell genetics of plants; F. Constabel, I.K. Vasil, Eds.; Academic Press: San Diego, 1987; Vol. 4, pp. 43-76.
[184] Tiburi, M.; Reguly, M.L.; Schwartsmann, G.; Cunha, K.S.; Lehmaim, M.; Andrade, H.H.R.; Mutation Res., 2002, 519, 141-149.
[185] Hayot, C ; Farinelle, S.; De Decker, R.; Decaestecker, C ; Darro, F.; Kiss, R.; Van Damme, M.; Int. J. Oncol., 2002,21,417-425.
[186] Wink, M.; Roberts, M. F. In Alkaloids. Biochemistry, Ecology, and Medicinal Applications; M.F. Roberts, M. Wink, Eds.; Plenum Press: New York, 1998; pp. 239-264.
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