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Overcoming multidrug-resistance in cancer: Statins offer a logical candidate
Medical Hypotheses 74 (2010) 237–239
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Overcoming multidrug-resistance in cancer: Statins offer a logical candidate Narendra G. Mehta a,*, Monica Mehta b,1 a b
3B/33 Takshila, Off, Mahakali Caves Road, Andheri (East), Mumbai, Maharashtra 400 093, India 1216 Carriage Drive, Longmont, CO 80501, USA
a r t i c l e
i n f o
Article history: Received 9 September 2009 Accepted 17 September 2009
s u m m a r y About seven million people die of cancer every year. This is largely due to development of drug resistance, particularly multidrug resistance, in the tumor cells. Multidrug resistance (MDR) arises due to overexpression of MDR proteins in the cancer cells, which cause efflux of anticancer drugs from the cells using ATP. MDR proteins are members of the family of ABC transporters that occur universally, and are structurally and functionally conserved during evolution. In Drosophila, the germ cell attractant peptide is secreted by an ABC transport protein, mdr49. Recently, the peptide has been shown to undergo conjugation with the lipid geranylgeranyl before secretion. If conjugation with the lipid is inhibited, mdr49 protein is unable to transport the peptide. Similarly, in the case of yeast mating factor pheromone, farnesylation is required to occur before the export of the pheromone by ste6 protein, an ABC transporter. In view of the homology of mdr49 and ste6 proteins with mammalian MDR proteins, we postulate that the drug transporters also require their ligands to be conjugated to a lipid. This view finds support from the studies with synthetic inhibitors of geranylgeranyl-/farnesyl-diphosphate synthetase or transferase: The inhibitors are reported to overcome multidrug resistance in cancer cell lines or xenografts in animals. Thus, the MDR transporters also appear to require their substrates to be conjugated with a lipid. Statins are the widely used inhibitors of HMG-CoA reductase. By depleting precursors of the mevalonate pathway, statins can prevent the formation of lipids like geranylgeranyl and farnesyl. Accordingly, they should also be able to overcome multidrug resistance in cancer. A few reports in the literature indicate that they appear to do so. Statins are in wide clinical use, and their pharmacology is well known. Besides, statins per se have mild beneficial effect on the outcome of the disease. We propose that statins should be seriously investigated for their ability to overcome multidrug resistance in cancer. This should be done after careful standardization of the protocol of simultaneous treatment with anticancer drugs and a statin. Ó 2009 Elsevier Ltd. All rights reserved.
Things growing to themselves are growth’s abuse. –Venus and Adonis, 166 ....and where’t will not extend, Thither he darts it. –King Henry VIII, I, 1, 111-112
Introduction What makes cancer such a perilous disease is the metastasis of its cells. Two of the three main-line therapies, viz. surgery and radiation, cannot reach every site where cancer spreads; while the third line of treatment, chemotherapy, often becomes ineffective due to development of resistance of the cells to the drugs [1]. What is most frustrating to the therapist and equally intriguing * Corresponding author. Tel.: +91 22 32511000. E-mail addresses: [email protected] (N.G. Mehta), monicanmehta@ gmail.com (M. Mehta). 1 Tel.: +1 720 530 4549. 0306-9877/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2009.09.039
to the researcher is that, frequently, the cancer cells become resistant not only to the drug administered, but also to a plethora of cancer chemotherapeutic agents that the patient has never been exposed to before. The multidrug resistance (MDR) in cancer arises from increased copy number of the mdr genes, and over-expression of the encoded membrane proteins [2]. The MDR proteins, of which P-glycoprotein [(P-pg), also called ABCB1/MDR1], is the most prominent member, cause efflux of the drugs from cancer cells [1]. Currently about seven million people die of cancer and 12 million new cases arise a year [3]. Over 90% of the deaths are attributable to failure of chemotherapy, mostly due to multidrug resistance [4]. Finding ways to overcome MDR in cancer is thus a problem as urgent as any in medical science. Logically, the solution will reside in finding a way to interfere with the efflux of the drugs by P-gp from the resistant cells. In order to do so, it is necessary to understand how the MDR protein recognizes drugs of diverse chemical nature and exports them. Since the drugs that P-gp pumps out come in a variety of structures, it is a priori unlikely that the protein–ligand interaction involves stereospecificity — the problem of substrate promiscuity.
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N.G. Mehta, M. Mehta / Medical Hypotheses 74 (2010) 237–239
One way of looking at the problem is to envisage that the potentially toxic compounds are modified by the detoxification machinery of the cell, and the modifying tag is the means of recognition by the MDR protein.
tance of the cells to drugs and antibiotics [12]. The protein was able to substitute for human MDR1 when expressed in lung fibroblasts. It is possible that ligand modification is a mechanism common to all ABC transporters.
ABC transporters
Multidrug transport
The multidrug resistance proteins are members of the universally distributed ATP-binding cassette (ABC) transporters [5]. The structure and function of these proteins are conserved in evolution. Their role is to export physiologically important molecules and cytotoxic substances from the cell using ATP. Bacteria use this mechanism to take up some nutrients from the outside by active transport [6].
Some indirect evidence indicates that the P-gp-ligands also undergo conjugation with lipid in cancer cells. SCH66336, a synthetic inhibitor of farnesyl/geranylgeranyl synthase /transferase, is a potent inhibitor of P-gp function, and enhances the efficacy of various cytotoxic agents in vivo and in vitro [13–15]. If an anticancer drug requires conjugation with lipid for export by the MDR protein, as postulated here, it cannot be complexed with lipid in presence of the inhibitor, and hence cannot be expelled. It would thus remain within the cell and build up a high enough concentration to kill the cell. Another compound, zoledronate, that inhibits geranylgeranyl transferase primarily, also is able to surmount multidrug resistance [16–20]. (Both these compounds are generally believed to bring about their effects by preventing prenylation of Rho and allied proteins. Since P-gp function is also shown to be affected [14], the transferase must obviously be non-specific.)
Mechanism of export by ABC Transporters: ligand modification prior to efflux A recent paper by Ricardo and Lehmann [7] (see also [8]) throws light on the nature of the efflux mechanism. In Drosophila melanogaster, as in most organisms, the primordial gonadal cells migrate towards the somatic cells in response to an attractant peptide secreted by the latter cell type. The report show that geranylgeranylation of the peptide is necessary before its secretion by the mdr49 gene product, an ABC transporter. ‘‘Reducing hmgcr, bGGT1, and ABC transporter (mdr49) expression fully blocked germ cell migration [7].” Thus the MDR49 protein-mediated transport of the attractant peptide requires: (i) HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (the hmgcr gene product) that supplies precursors for the synthesis of downstream lipids in the mevalonate pathway, including geranylgeranyl; (ii) the geranylgeranyl transferase (bGGT1 is the b-subunit of the enzyme) that puts the lipid moiety on the attractant peptide; and (iii) the MDR protein which pumps the lipid-conjugated attractant out of the cell. In the absence of any of these, the embryo is malformed. The above mechanism of ABC transport requiring prior conjugation of the ligand with lipid is supported by results from the yeast mating system, reported earlier [9]. The cells of Schizosaccharomyces pombe, signal those of the opposite mating type via a diffusible nonapeptide. The peptide pheromone is found to undergo farnesylation before secretion by Mam1 protein. The latter, an ABC transporter, is highly homologous to human MDR proteins [9]. In Saccharomyces cerevisiae, the a-factor pheromone is transported by ste6 gene product, a P-gp homologue [10]. The remarkable similarity of the mechanism of yeast peptide efflux with that of multidrug resistance was noted: ‘‘the STE6 protein functions to export the hydrophobic a-factor lipopeptide in a manner analogous to the efflux of hydrophobic cytotoxic drugs by the related mammalian P-glycoprotein [10].” Multidrug transporters How relevant are these results to transport of drugs by MDR proteins? It is remarkable that two very divergent organisms show a similar mechanism of ABC export, viz. the conjugation of ligand with lipid prior to efflux. As mentioned earlier, the ABC transporters are structurally and functionally conserved in evolution [5]. The functional conservation is strikingly demonstrated in two papers: The S. cerevisae cells lacking ste6 gene are unable to mate. The complementary DNA for the mouse mdr3 gene, when expressed in the deficient yeast cells, was able to restore the ability of the cells to mate [11]. Since the mating-peptide is farnesylation before export [9], mammalian P-glycoprotein (the mdr3 protein), expressed in yeast, must also recognize the peptide in complex with the lipid. In Lactobacillus lactis, the Lmr protein probably determines resis-
The active site of P-gp In another recent paper, Aller et al. [21] report an X-ray crystallographic study of the mouse P-gp, which is 87% identical to human P-gp. They find a 6000 Å3 cavity that is exposed to cytoplasm as well as to the inner leaflet of the lipid bilayer. The interpretation of the authors is that the latter, the part of the active site in contact with the inner leaflet, is involved in mopping up and pumping out the anticancer drugs sequestered in the membrane. While this mechanism could well operate, we believe it would not be the major one: (i) although many P-gp substrates are hydrophobic, many are amphiphilic [5]; (ii) a specificity of P-gp is transport of amphiphilic cations [5]; (iii) P-gp substrates are known to be acted upon by CYP1B1, a component of detoxifying cytochrome P450 system, that appears to have a cancer-specific expression [22], and may modify the drugs, for example, by hydroxylation; and (iv) the larger, cytoplasmically-exposed active site has 15 hydrophilic and two charged residues out of a total of 73 (Ref. [21]). It makes sense that the larger, relatively hydrophilic region of the active site plays the major role in efflux of (at least the amphiphilic) anticancer drugs. It is also possible that the smaller, lipid-embedded part of the active site is involved in the flippase activity that P-gp possesses [23]. The active site of P-gp is large and lipophilic, and has a few polar residues [21]. Yet, it is doubtful if it can stereoselectively bind to anticancer drugs of a variety of structures that P-gp is able to transport. If, however, the drug is modified by tagging a lipid, such as geranylgeranyl or farnesyl, as in Drosophila and yeast [7,9,10] and indirectly suggested for the mammalian MDR system [13– 20], P-gp could bind the complex via the lipid moiety, and expel the drug together with the lipid. If the drug is conjugated to a lipid, one has to look at the export of the complex rather than the drug per se. Most interestingly, the P-gp’s active site is found to be large enough to ‘‘simultaneously accommodate two different drugs [24,25].” The two subsites, binding to dissimilar substances, could in fact be accommodating the complex consisting of the drug and the lipid. P-gp is the most prominent of multidrug resistant proteins in humans, and transports uncharged or positively charged hydrophobic substances [5]. MRP1/ABCC1 is the next in importance to P-gp in matter of drug efflux. It favors the transport of organic anions, including anticancer drugs conjugated to glutathione, glucu-
N.G. Mehta, M. Mehta / Medical Hypotheses 74 (2010) 237–239
ronate or sulfate [26]. It is also able to transport free glutathione. This suggests that MRP1 has a binding site for glutathione, and very likely, the transport of the drug-glutathione conjugate occurs via the glutathione-binding site. This is exactly similar to what is proposed here for P-gp mediated transport. Based on the suggestion made here, the following outline emerges for the transport of anticancer drugs from the cancer cell. The drug is possibly acted upon by CYT1B1, and metabolized further by conjugation to lipid (such farnesyl or geranylgeranyl in the case of P-gp), or an anion (glutathione in the case of MRP1). (Additional modifications cannot be ruled out.) This is followed by the binding of the conjugate to the MDR protein via the lipid moiety (or glutathione), and transport using ATP. Such a mechanism would also provide a simple explanation for the enigma of ‘substrate promiscuity.’ Effect of statins If anticancer drugs are indeed modified by addition of lipid prior to efflux from resistant cells, it should be possible to overcome the resistance by blocking the synthesis (or transfer) of the lipids. The effects of SCH66336 and zoledrone are along this line [13–20]. Treatment with statins, the inhibitors of HMG-CoA reductase, can starve the cells of precursors of the mevalonate pathway. This can prevent addition of lipid to the anticancer drugs and, in turn, their efflux; as happens in the case of export of the attractant peptide in Drosophila embryos upon reduction in the level of HMG-CoA reductase [7]. Statins are widely prescribed for lowering blood cholesterol levels in normal individuals and in cardiovascular disease and stroke patients. Statins themselves have been tried as cancer chemotherapeutic agents [27–29], and found to ‘‘exert several beneficial antineoplastic properties, including decreased tumor growth, angiogenesis, and metastasis. [29].” There are several reports of statins potentiating or even synergizing the effect of anticancer drugs in tumor cell lines or animal models (e.g. [30–32]), including reversing multidrug resistance [32–34]. However, there is also a report of halting a phase II clinical trial because of the lack of effect of high dose simvastatin in multiple myeloma refractory to chemotherapy [35]. Statins are in wide clinical use and their pharmacology is well known. Therefore, their application in multidrug resistant cancer should be possible with minimal further effort and time. However, the protocol of statin treatment with respect to the type [hydrophobic/hydrophilic], concentration, time of administration relative to drug therapy, duration, etc., need to be standardized for proper evaluation of statin effect in overcoming MDR in cancer. Conflicts of interest statement None declared. References [1] Szakács G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Nat Rev Drug Discov 2006;5:219–34. [2] Riordan JR, Deuchars K, Kartner N, Alon N, Trent J, Ling V. Amplification of Pglycoprotein genes in multidrug-resistant mammalian cell lines. Nature 1985;316:817–9. [3] Garcia M, Jemal A, Ward EM, et al. Global cancer facts and figures 2007. Atlanta, GA: American Cancer Society; 2007. [4] Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol 2005;205:275–92. [5] Glavinas H, Krajcsi P, Cserepes J, Sarkadi B. The role of ABC transporters in drug resistance, metabolism and toxicity. Curr Drug Deliv 2004;1:27–42.
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[6] Dawson R, Kaspar JP, Locher P. Structure of a bacterial multidrug ABC transporter. Nature 2006;443:180–5. [7] Ricardo S, Lehmann R. An ABC transporter controls export of a Drosophila germ cell attractant. Science 2009;323:943–6. [8] Hla T, Im DS. The ABCs of lipophile transport. Science 2009;323:883–4. [9] Christensen PU, Davey J, Nielsen O. The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor. Mol Gen Genet 1997;255:226–36. [10] McGrath JP, Varshavsky A. The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature 1989;340:400–4. [11] Raymond M, Gros P, Whiteway M, Thomas DY. Functional complementation of yeast ste6 by a mammalian multidrug resistance mdr gene. Science 1992;256:232–4. [12] van Veen HW, Callaghan R, Soceneantu L, Sardini A, Konings WN, Higgins CF. A bacterial antibiotic-resistance gene that complements the human multidrugresistance P-glycoprotein gene. Nature 1998;391:291–5. [13] Liu M, Bryant MS, Chen J, et al. Antitumor activity of SCH 66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res 1998;58: 4947–56. [14] Wang E, Casciano CN, Clement RP, Johnson WW. The farnesyl protein transferase inhibitor SCH66336 is a potent inhibitor of MDR1 product Pglycoprotein. Cancer Res 2001;61:7525–9. [15] Wang EJ, Johnson WW. The farnesyl protein transferase inhibitor lonafarnib (SCH66336) is an inhibitor of multidrug resistance proteins 1 and 2. Chemotherapy 2003;49:303–8. [16] Morgan MA, Sebil T, Aydilek E, Peest D, Ganser A, Reuter CW. Combining prenylation inhibitors causes synergistic cytotoxicity, apoptosis and disruption of RAS-to-MAP kinase signalling in multiple myeloma cells. Br J Haematol 2005;130:912–25. [17] Horie N, Murata H, Nishigaki Y, et al. The third-generation bisphosphonates inhibit proliferation of murine osteosarcoma cells with induction of apoptosis. Cancer Lett 2006;238:111–8. [18] Kuroda J, Kimura S, Andreef M, et al. ABT-737 is a useful component of combinatory chemotherapies for chronic myeloid leukemias with diverse drug-resistance mechanisms. Brit J Haematol 2008;140:181–90. [19] Zhang Y, Cao R, Yin F, et al. Lipophilic bisphosphonates as dual farnesyl/ geranylgeranyl diphosphate synthase inhibitors: an X-ray and NMR investigation. J Am Chem Soc 2009;131:5153–62. [20] Gnant M, Mlineritsch B, Schippinger W, et al. Endocrine therapy plus zoledronic acid in premenopausal breast cancer. N Engl J Med 2009;360:679–91. [21] Aller SG, Yu J, Ward A, et al. Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding. Science 2009;323:1718–22. [22] McFadyen MC, McLeod HL, Jackson FC, Melvin WT, Doehmer J, Murray GI. Cytochrome P450 CYP1B1 protein expression: a novel mechanism of anticancer drug resistance. Biochem Pharmacol 2001;62:207–12. [23] Bosch I, Dunussi-Joannopoulos K, Wu RL, Furlong ST, Croop J. Phosphatidylcholine and phosphatidylethanolamine behave as substrates of the human MDR1 P-glycoprotein. Biochemistry 1997;36:5685–94. [24] Loo TW, Bartlett MC, Clarke DM. Simultaneous binding of two different drugs in the binding pocket of the human multidrug resistance P-glycoprotein. J Biol Chem 2003;278:39706–10. [25] Lugo MR, Sharom FJ. Interaction of LDS-751 and rhodamine 123 with Pglycoprotein: evidence for simultaneous binding of both drugs. Biochemistry 2005;44:14020–9. [26] Tew KD. Glutathione-associated enzymes in anticancer drug resistance. Cancer Res 1994;54(16):4313–20. [27] Fritz G. HMG-CoA reductase inhibitors (statins) as anticancer drugs (review). Int J Oncol 2005;27:1401–9. [28] Demierre MF, Higgins PD, Gruber SB, Hawk E, Lippman SM. Statins and cancer prevention. Nat Rev Cancer 2005;5:930–42. [29] Hindler K, Cleeland CS, Rivera E, Collard CD. The role of statins in cancer therapy. Oncologist 2006;11:306–15. [30] Agarwal B, Bhendwal S, Halmos B, Moss SF, Ramey WG, Holt PR. Lovastatin augments apoptosis induced by chemotherapeutic agents in colon cancer cells. Clin Cancer Res 1999;5:2223–9. [31] Holstein SA, Hohl RJ. Synergistic interaction of lovastatin and paclitaxel in human cancer cells. Mol Cancer Ther 2001;1:141–9. [32] Feleszko W, Mlynarczuk I, Olszewska D, et al. Lovastatin potentiates antitumor activity of doxorubicin in murine melanoma via an apoptosis-dependent mechanism. Int J Cancer 2002;100:111–8. [33] Schmidmaier R, Baumann P, Simsek M, Dayyani F, Emmerich B, Meinhardt G. The HMG-CoA reductase inhibitor simvastatin overcomes cell adhesionmediated drug resistance in multiple myeloma by geranylgeranylation of Rho protein and activation of Rho kinase. Blood 2004;104:1825–32. 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Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Overcoming multidrug-resistance in cancer: Statins offer a logical candidate Narendra G. Mehta a,*, Monica Mehta b,1 a b
3B/33 Takshila, Off, Mahakali Caves Road, Andheri (East), Mumbai, Maharashtra 400 093, India 1216 Carriage Drive, Longmont, CO 80501, USA
a r t i c l e
i n f o
Article history: Received 9 September 2009 Accepted 17 September 2009
s u m m a r y About seven million people die of cancer every year. This is largely due to development of drug resistance, particularly multidrug resistance, in the tumor cells. Multidrug resistance (MDR) arises due to overexpression of MDR proteins in the cancer cells, which cause efflux of anticancer drugs from the cells using ATP. MDR proteins are members of the family of ABC transporters that occur universally, and are structurally and functionally conserved during evolution. In Drosophila, the germ cell attractant peptide is secreted by an ABC transport protein, mdr49. Recently, the peptide has been shown to undergo conjugation with the lipid geranylgeranyl before secretion. If conjugation with the lipid is inhibited, mdr49 protein is unable to transport the peptide. Similarly, in the case of yeast mating factor pheromone, farnesylation is required to occur before the export of the pheromone by ste6 protein, an ABC transporter. In view of the homology of mdr49 and ste6 proteins with mammalian MDR proteins, we postulate that the drug transporters also require their ligands to be conjugated to a lipid. This view finds support from the studies with synthetic inhibitors of geranylgeranyl-/farnesyl-diphosphate synthetase or transferase: The inhibitors are reported to overcome multidrug resistance in cancer cell lines or xenografts in animals. Thus, the MDR transporters also appear to require their substrates to be conjugated with a lipid. Statins are the widely used inhibitors of HMG-CoA reductase. By depleting precursors of the mevalonate pathway, statins can prevent the formation of lipids like geranylgeranyl and farnesyl. Accordingly, they should also be able to overcome multidrug resistance in cancer. A few reports in the literature indicate that they appear to do so. Statins are in wide clinical use, and their pharmacology is well known. Besides, statins per se have mild beneficial effect on the outcome of the disease. We propose that statins should be seriously investigated for their ability to overcome multidrug resistance in cancer. This should be done after careful standardization of the protocol of simultaneous treatment with anticancer drugs and a statin. Ó 2009 Elsevier Ltd. All rights reserved.
Things growing to themselves are growth’s abuse. –Venus and Adonis, 166 ....and where’t will not extend, Thither he darts it. –King Henry VIII, I, 1, 111-112
Introduction What makes cancer such a perilous disease is the metastasis of its cells. Two of the three main-line therapies, viz. surgery and radiation, cannot reach every site where cancer spreads; while the third line of treatment, chemotherapy, often becomes ineffective due to development of resistance of the cells to the drugs [1]. What is most frustrating to the therapist and equally intriguing * Corresponding author. Tel.: +91 22 32511000. E-mail addresses: [email protected] (N.G. Mehta), monicanmehta@ gmail.com (M. Mehta). 1 Tel.: +1 720 530 4549. 0306-9877/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2009.09.039
to the researcher is that, frequently, the cancer cells become resistant not only to the drug administered, but also to a plethora of cancer chemotherapeutic agents that the patient has never been exposed to before. The multidrug resistance (MDR) in cancer arises from increased copy number of the mdr genes, and over-expression of the encoded membrane proteins [2]. The MDR proteins, of which P-glycoprotein [(P-pg), also called ABCB1/MDR1], is the most prominent member, cause efflux of the drugs from cancer cells [1]. Currently about seven million people die of cancer and 12 million new cases arise a year [3]. Over 90% of the deaths are attributable to failure of chemotherapy, mostly due to multidrug resistance [4]. Finding ways to overcome MDR in cancer is thus a problem as urgent as any in medical science. Logically, the solution will reside in finding a way to interfere with the efflux of the drugs by P-gp from the resistant cells. In order to do so, it is necessary to understand how the MDR protein recognizes drugs of diverse chemical nature and exports them. Since the drugs that P-gp pumps out come in a variety of structures, it is a priori unlikely that the protein–ligand interaction involves stereospecificity — the problem of substrate promiscuity.
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N.G. Mehta, M. Mehta / Medical Hypotheses 74 (2010) 237–239
One way of looking at the problem is to envisage that the potentially toxic compounds are modified by the detoxification machinery of the cell, and the modifying tag is the means of recognition by the MDR protein.
tance of the cells to drugs and antibiotics [12]. The protein was able to substitute for human MDR1 when expressed in lung fibroblasts. It is possible that ligand modification is a mechanism common to all ABC transporters.
ABC transporters
Multidrug transport
The multidrug resistance proteins are members of the universally distributed ATP-binding cassette (ABC) transporters [5]. The structure and function of these proteins are conserved in evolution. Their role is to export physiologically important molecules and cytotoxic substances from the cell using ATP. Bacteria use this mechanism to take up some nutrients from the outside by active transport [6].
Some indirect evidence indicates that the P-gp-ligands also undergo conjugation with lipid in cancer cells. SCH66336, a synthetic inhibitor of farnesyl/geranylgeranyl synthase /transferase, is a potent inhibitor of P-gp function, and enhances the efficacy of various cytotoxic agents in vivo and in vitro [13–15]. If an anticancer drug requires conjugation with lipid for export by the MDR protein, as postulated here, it cannot be complexed with lipid in presence of the inhibitor, and hence cannot be expelled. It would thus remain within the cell and build up a high enough concentration to kill the cell. Another compound, zoledronate, that inhibits geranylgeranyl transferase primarily, also is able to surmount multidrug resistance [16–20]. (Both these compounds are generally believed to bring about their effects by preventing prenylation of Rho and allied proteins. Since P-gp function is also shown to be affected [14], the transferase must obviously be non-specific.)
Mechanism of export by ABC Transporters: ligand modification prior to efflux A recent paper by Ricardo and Lehmann [7] (see also [8]) throws light on the nature of the efflux mechanism. In Drosophila melanogaster, as in most organisms, the primordial gonadal cells migrate towards the somatic cells in response to an attractant peptide secreted by the latter cell type. The report show that geranylgeranylation of the peptide is necessary before its secretion by the mdr49 gene product, an ABC transporter. ‘‘Reducing hmgcr, bGGT1, and ABC transporter (mdr49) expression fully blocked germ cell migration [7].” Thus the MDR49 protein-mediated transport of the attractant peptide requires: (i) HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase (the hmgcr gene product) that supplies precursors for the synthesis of downstream lipids in the mevalonate pathway, including geranylgeranyl; (ii) the geranylgeranyl transferase (bGGT1 is the b-subunit of the enzyme) that puts the lipid moiety on the attractant peptide; and (iii) the MDR protein which pumps the lipid-conjugated attractant out of the cell. In the absence of any of these, the embryo is malformed. The above mechanism of ABC transport requiring prior conjugation of the ligand with lipid is supported by results from the yeast mating system, reported earlier [9]. The cells of Schizosaccharomyces pombe, signal those of the opposite mating type via a diffusible nonapeptide. The peptide pheromone is found to undergo farnesylation before secretion by Mam1 protein. The latter, an ABC transporter, is highly homologous to human MDR proteins [9]. In Saccharomyces cerevisiae, the a-factor pheromone is transported by ste6 gene product, a P-gp homologue [10]. The remarkable similarity of the mechanism of yeast peptide efflux with that of multidrug resistance was noted: ‘‘the STE6 protein functions to export the hydrophobic a-factor lipopeptide in a manner analogous to the efflux of hydrophobic cytotoxic drugs by the related mammalian P-glycoprotein [10].” Multidrug transporters How relevant are these results to transport of drugs by MDR proteins? It is remarkable that two very divergent organisms show a similar mechanism of ABC export, viz. the conjugation of ligand with lipid prior to efflux. As mentioned earlier, the ABC transporters are structurally and functionally conserved in evolution [5]. The functional conservation is strikingly demonstrated in two papers: The S. cerevisae cells lacking ste6 gene are unable to mate. The complementary DNA for the mouse mdr3 gene, when expressed in the deficient yeast cells, was able to restore the ability of the cells to mate [11]. Since the mating-peptide is farnesylation before export [9], mammalian P-glycoprotein (the mdr3 protein), expressed in yeast, must also recognize the peptide in complex with the lipid. In Lactobacillus lactis, the Lmr protein probably determines resis-
The active site of P-gp In another recent paper, Aller et al. [21] report an X-ray crystallographic study of the mouse P-gp, which is 87% identical to human P-gp. They find a 6000 Å3 cavity that is exposed to cytoplasm as well as to the inner leaflet of the lipid bilayer. The interpretation of the authors is that the latter, the part of the active site in contact with the inner leaflet, is involved in mopping up and pumping out the anticancer drugs sequestered in the membrane. While this mechanism could well operate, we believe it would not be the major one: (i) although many P-gp substrates are hydrophobic, many are amphiphilic [5]; (ii) a specificity of P-gp is transport of amphiphilic cations [5]; (iii) P-gp substrates are known to be acted upon by CYP1B1, a component of detoxifying cytochrome P450 system, that appears to have a cancer-specific expression [22], and may modify the drugs, for example, by hydroxylation; and (iv) the larger, cytoplasmically-exposed active site has 15 hydrophilic and two charged residues out of a total of 73 (Ref. [21]). It makes sense that the larger, relatively hydrophilic region of the active site plays the major role in efflux of (at least the amphiphilic) anticancer drugs. It is also possible that the smaller, lipid-embedded part of the active site is involved in the flippase activity that P-gp possesses [23]. The active site of P-gp is large and lipophilic, and has a few polar residues [21]. Yet, it is doubtful if it can stereoselectively bind to anticancer drugs of a variety of structures that P-gp is able to transport. If, however, the drug is modified by tagging a lipid, such as geranylgeranyl or farnesyl, as in Drosophila and yeast [7,9,10] and indirectly suggested for the mammalian MDR system [13– 20], P-gp could bind the complex via the lipid moiety, and expel the drug together with the lipid. If the drug is conjugated to a lipid, one has to look at the export of the complex rather than the drug per se. Most interestingly, the P-gp’s active site is found to be large enough to ‘‘simultaneously accommodate two different drugs [24,25].” The two subsites, binding to dissimilar substances, could in fact be accommodating the complex consisting of the drug and the lipid. P-gp is the most prominent of multidrug resistant proteins in humans, and transports uncharged or positively charged hydrophobic substances [5]. MRP1/ABCC1 is the next in importance to P-gp in matter of drug efflux. It favors the transport of organic anions, including anticancer drugs conjugated to glutathione, glucu-
N.G. Mehta, M. Mehta / Medical Hypotheses 74 (2010) 237–239
ronate or sulfate [26]. It is also able to transport free glutathione. This suggests that MRP1 has a binding site for glutathione, and very likely, the transport of the drug-glutathione conjugate occurs via the glutathione-binding site. This is exactly similar to what is proposed here for P-gp mediated transport. Based on the suggestion made here, the following outline emerges for the transport of anticancer drugs from the cancer cell. The drug is possibly acted upon by CYT1B1, and metabolized further by conjugation to lipid (such farnesyl or geranylgeranyl in the case of P-gp), or an anion (glutathione in the case of MRP1). (Additional modifications cannot be ruled out.) This is followed by the binding of the conjugate to the MDR protein via the lipid moiety (or glutathione), and transport using ATP. Such a mechanism would also provide a simple explanation for the enigma of ‘substrate promiscuity.’ Effect of statins If anticancer drugs are indeed modified by addition of lipid prior to efflux from resistant cells, it should be possible to overcome the resistance by blocking the synthesis (or transfer) of the lipids. The effects of SCH66336 and zoledrone are along this line [13–20]. Treatment with statins, the inhibitors of HMG-CoA reductase, can starve the cells of precursors of the mevalonate pathway. This can prevent addition of lipid to the anticancer drugs and, in turn, their efflux; as happens in the case of export of the attractant peptide in Drosophila embryos upon reduction in the level of HMG-CoA reductase [7]. Statins are widely prescribed for lowering blood cholesterol levels in normal individuals and in cardiovascular disease and stroke patients. Statins themselves have been tried as cancer chemotherapeutic agents [27–29], and found to ‘‘exert several beneficial antineoplastic properties, including decreased tumor growth, angiogenesis, and metastasis. [29].” There are several reports of statins potentiating or even synergizing the effect of anticancer drugs in tumor cell lines or animal models (e.g. [30–32]), including reversing multidrug resistance [32–34]. However, there is also a report of halting a phase II clinical trial because of the lack of effect of high dose simvastatin in multiple myeloma refractory to chemotherapy [35]. Statins are in wide clinical use and their pharmacology is well known. Therefore, their application in multidrug resistant cancer should be possible with minimal further effort and time. However, the protocol of statin treatment with respect to the type [hydrophobic/hydrophilic], concentration, time of administration relative to drug therapy, duration, etc., need to be standardized for proper evaluation of statin effect in overcoming MDR in cancer. Conflicts of interest statement None declared. References [1] Szakács G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Nat Rev Drug Discov 2006;5:219–34. [2] Riordan JR, Deuchars K, Kartner N, Alon N, Trent J, Ling V. Amplification of Pglycoprotein genes in multidrug-resistant mammalian cell lines. Nature 1985;316:817–9. [3] Garcia M, Jemal A, Ward EM, et al. Global cancer facts and figures 2007. Atlanta, GA: American Cancer Society; 2007. [4] Longley DB, Johnston PG. Molecular mechanisms of drug resistance. J Pathol 2005;205:275–92. [5] Glavinas H, Krajcsi P, Cserepes J, Sarkadi B. The role of ABC transporters in drug resistance, metabolism and toxicity. Curr Drug Deliv 2004;1:27–42.
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