- Email: [email protected]
Pharmacological modulators of DNA-interactive antitumor drugs
TiPS - September 1989 [Vof. 101 pyrimidinergic regulation of cellular functions is as yet poorly undersrood, owing to the lack of information on how the release of pyrimidine nucleotides is controlled. The development of potent and selective agonists and antagonists for pyrimidinoceptors is essential to characterize these receptors, and may provide a novel approach to intervene in various pathological states such as inflammatory processes and vascular diseases.
369
21 22 23 24
25 26
27 28
Acknowkdgemenb The authors are grateful to Mrs R. Kriiger for help in the preparation of the manuscript. Work of the authors cited herein was supported by grants of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
29
Naunyn-Sckmiedeberg’s Arch. Pharmacol. 337 (Suppl.), R44 UrquiIIa, P. R. (1978) Stroke 9,133-136 Hiiussinper, D., StebIe, T. and Gerok, W. (1987) Eur. 1. Biochem. 167,65-71 Needbam, L., Cusack, N. J., Pearson, J. D. and Gordon, J. L. (1987) Ear. j. Phanacal. 134,199-289 Goetz, U., Da Prada, M. and PIetscber, A. 11971) J. Pharmacaj. Exp. Ther. 178, 210-21s Keppler, D., Rudigr, J. and Decker, K. (1970) Anal. Biochem. 38,185-114 PauImicbI, M. and Lang, F. (1989) Bisthem. Biophys. Res. Crtmmun. I?. 1139-1143 Greenberg, S., Di VirSiiiz~ f., Steinberg, T. H. and Si!ve?&in, 5 1.11988) J. Bial. Chem. W, 1033%110363 Dubyak, G. R. and De Young, M. 3. (1985) f. Biot.Chem. 260,105t3--l_IOr;6~ Mustard, J. F. and Packbam. M. A (19&J) Phanacol. Rev. 22.97-187
38 Ford-Hutchinson, A. W. (1982) Br. J. Pharmacot. 76,36?-371 31 Okwon, I. L., B&man, T. R. and Gallo, R. C. (1982) Cancer Res. 42,392&3933 32 Limo& R., sdrvsrtz, I., Hasum, E., Ayalon, D. and Naor, Z. (1989) Biochem. Biopkys. Res. Commun. 159.209-215 33 Rehm. A. G., Wu, H. and Ha&da, S. P. (1988) FEES Lett. 234,316-320 34 Katsur&, T. and Furuksws. T. (1985) Trends Pharmacol. Sd. B337-339 35 Dusenbery, K. E., Mendiola, J. R. and’ Skubitz, K. M. (1988) Biochem. Biopkys. Res. Commun. 153,7-13 36 Keppens. S. snd de Wolf, H. (1986) ?“-frm?x. ;, 240.367-371 37 SwIzz C. L., Monir, A. j. and Harden, _ ..‘. (1989)j. BioJ. Ckem. 264,6202-6206 4 l+uber, I?., Ridrter, L and Re&er, W. !1982) F&BS J&t. 141,19&292 _ jessit~, A. J., ToIIey; J. 0. and AEen, IX. A. (1986; J. EiaZ. Char. 261, X3662-13669
References 1 Wii, mec41.
M. (1987) Annu. Rev. Phar27,315-345 J. L. (1986) Biockem. f. 233,
Toxicol.
2 Gordon, 309-319 3 Bumstock, G. and Kennedv, C. (1985) Gen. Pharauxol. 16,433-4404 Okaiima. F.. Tokumitsu. Y.. Kondo. Y. and’Ui,*M: (1987) J. B~oI.~Chem. 262, 13483-13498 5 Benbam, C. D. and Tsien, R. W. (1987) Nature 328,273-278 6 Dubyak, G. R. and De Young. M. 8. (1985) 1. BioJ. Ckem. 260.10653-10661 7 Forsbeg, E. J.,peuerstein, G..Shohemi, II. and PoIIard, H. II. (1987) Proc. Nal Acad. Sci. USA 84563iH634 8 Dubyak, G. R, Cowen, D. S. and Metier, L. M. (1988) J. Ihal. Chem. 263, 18108-18117 9 Kubns, D. B., Wright, D. G., Natb, J., KapIan, S. S. and Basford, R. E. (1988) Ial. Inuesf. 58,448-453 10 Seifert, R., Burde, R. and Schultz, G. (1989) Eiachem. J. 259,813-819 11 Seifert, R.. WeneeI, K., Eckstein, F. and ScI~uItz, G. (1989) Eur. 1. Biochem. 181, 277-285 12 Brown, C. M. and Burnstock, G. (1981) Eur. J. Pharmacol. 69,81-86 13 Hsrdebo,J.E.,Kabr&6m,J.,Gwmsn,C. and Salford, I, G. (1987) Blood Vessels 24, 150-l55 14 Sskai, K., Akima, M. and Matsushita, H. (1979) Jpn. J. Pkarmacai. 29,243-251 15 Martin, W., Cusack, N. J., Carleton, J. S. and Gordon, J. L. (1985) Eur. f. Pharmacof. 108,293-299 16 Mardonald, G., Asset, R. and Lo, E (1984) CJin. Exp. Pkarmacaol. Pkysiol. 11, 381-384 17 Hardebo, J. E., Hanko, J. and Owman, C. (1983) Gen. pharmacoI. 14,133-134 18 Shimsawa, Y., Wbite, R. P. and Robertson, J. T. (1983) Stroke 14‘347-355 19 von Kiigelen, I., Hliussinger, D. and Starke, K. (1987) Naunyn-Schmiedeberg’s Arch. Pharmncol. 336,556-560 20 von Kiigelen, I. and Starke, K. (1988)
John S. Laze and Robert R. Bahnson The poor therapeutic index and limited efficacy of current cancer chemotherap~~c agents represent an ~mpo~ant pha~acolo~ca~ prab~m. Athough there has been a significant increase in our understandjng of the nnechanisms by which anticancer drugs kill mammalian cells, identification of new, effective anticancer agents during the last decade has been exceeding slow. Thus, attention has focused on understanding the causes of drug resistance and on either sassing tumor cells to existing anticunc~ agents using what could be called ‘ckemoenhancers’, or protecting non-malignant tissues against serious untoward effects using ‘ckemoprotectors’. John Laze and Robert Bahnson review recent strategies attempting to modulate the activity of antineopiastic drugs. When demand exceeds supply, people look for ways to increase the usefulness of the existing sup ply. So it currently is in cancer research: there are many malignancies that do not respond to chemotherapy and too few exciting novel drugs to test. This, combined with an elevated understanding of the molecular basis of resistance to anticancer drugs, has kindled interest in identifying and developing pharmacological
f. S. Laze is Allegheny Foundation Professor and Ckairman of Pka~aco~gy and R. R. Bahnson is ~s~fant Professor of Urologic Surgey at tke University of Pittsburgh School of Medicine, Pittsbugh, PA 15261, USA. Both authors are also members of the Pittsburgh Cancer Institute.
modulators of existing cancer chemotherapeutic agents to improve their therapeutic indices. Two classes of modulator are being examined: chemoenhancers, which would sensitize tumor cells; and chemoprotectors, which would selectively protect non-ma~~ant tissue. To be a successful chemoenhancer or chemoprotector, an agent must exhibit little toxicity itself; many such agents have no anticancer activity at all and some have other useful pharmacological properties. The clinically successful combination of methotrexate with leucovorin (folinate} provides an important precedent for the chemoprotective approach; cisplatin, currently one of the most popular anticancer drugs,
3M would not be useful if pharmacological methods had not been identified to reduce its renal toxicity. A number of pharmacological agents have been identity as chemoenhancers or chemoprotectors. While many modulators of antimetabolites have been described, we wiII focus on pharmacological means to improve the therapeutic index of DNA-interactive anticancer drugs (see Ref. 1 for review of their molecular interactions), because they have become the most important class of drug for the treatment of solid tumors, and solid tumors are the most difficuh neoplastic diseases to cure. These anticancer drugs have some of the lowest therapeutic indices of aII available drugs and thus represent good candidates for improvement. A partial list of modulators of DNA-interactive drugs identified in preclinical studies with cultured cells or transplantable tumors in oioo is found in Table I. Most of the chemoenhancers or chemopmtectors are,. thought to have cellular targets that either affect celluIar responsiveness to the DNA-inpctive agents or emulate potential alternative cellular targets (Pig. 1). We will briefly highlight some of the recently acquired information on three classes of biochemical mediator of anticancer drug resistance and examine the pharmacological attempts to alter the levels or actions of these endogenous mediators of resistance. IIioehemicaImediators of resistance htrinsic drug resistance occurs in cds that have not previously been exposed to anticancer drugs; acquired resistance develops after exposure to anticancer drugs. It has become obvious that more than one mechanism of resistance can be identified even with a single DNA-~tera~ve agent or within a single tumor population-. The mechanisms of resistance seen within populations of cells having intrinsic or acquired drug resistance can be similar or different. The challenge for the Cancer p~acol~st is not only to establish which mechanisms are operative but also to devise Strategies to circumvent the resistance without increasing the
toxicity of the drugs to normal tissue. Both genetic and epigenetic processes have been implicated in anticancer drug resistance3; pharmacolo~c~ attempts either to increase the sensitivity of tumor cells or to decrease the sensitivity of non-malignant tissues have focused principally upon changing the steady-state levels of proteins or peptides, rather than on making any fundamental genetic aher-. ations. The preclinical studies to investigate whether modulators alter or emulate the hypothhized targets have been extensive for some of these modulators; clinical investigations with some of these agents are being initiated or considered. P-gIycoproteIn Resistance of cultured cells to a number of commonly used natural products that are antineoplastic including doxorubicin, dntgsdaunorubicin, etoposide, teniposide, plicamycin, vinblastine, vincristine and actinomycin D has been associated with the overexpression of a plasma membrane (P-glycoprotein). glycopmtein Most but not all of these drugs are believed to require interactions with DNA or DNA-associated proteins for their cytotoxic activity, although as a group they share little obvious chemical and mechanistic similarity. P-glycoprotein migrates in polyacrylamide gels with an apparent molecular mass of 150-180 kDa depending upon post-translational modificatio&. Our understanding of the molecular and bioTABLE I. Examples of chemoenhancers or ~~~~~rn Chemoanhancera
Chemeprotectore
Ca*+ channel blockers Phenothiazines
Amiistine
Ciclosporin Buthionine s~~rni~
(m)
TC%ElOXif@l
Ttfparanol lndole alkaloids Acridines
Thiosulfate Diethyldithiocarbamate Ilium&s Pw estem Bismuth salts Oxothiazoliiine+ carboxylate Razoxane
Amioderone
ICRF-187
Phorbol esters
~~~
streptozlcin SR-2508
!Semids
Pmgesterone Etacrynic Acid
esters
chemical properties of Pglycoproteins has expanded greatly in the past few years (see Ref. 4 for review). Briefly, a small gene family encodes P-glycoproteins: there are two genes in humans and three genes in rodents. The nucleotide sequences for these genes are now known, and the amino acid sequences of the proteins have been deduced. The proteins consist of highly homologous halves, each half containing six transmembrane segments and a nucleotide-binding site. DNA transfection studies suggest that only one human gene product (mdrl) produces resistance to antineoplastic drugs. The human m&l gene and its murine counterparts appear to encode a drug efflux pump glycoprotein that is responsible for the lower intracellular levels of cytotoxic drugs seen in the multiple-drugresistant phenotype. How antineoplastic drugs are removed from the cytosol by Pglycoprotein is currently being examined by several laboratories. In highIy resistant cells grown in culture, amplification of the mdrl gene is observed. The level of expression of m&l is also high in non-malignant adrenal gland, liver, kidney and colon. This has led to the speculation that the normal function of P-glycoprotein is to remove environmental toxins from the body*. cDNA probes for m&l, monocIonaI antibodies that recognize external epitopes of P-glycoproteins, and photoaffinity labeling reagents have been used to demonstrate overexpression of m&l mRNA and overproduction of P-glycoprotein in human tumor tissue. With hematol~i~ malignancies, such as multiple myeloma, initial results suggest that overexpression of P-glycoprotein is uncommon in previously untreated disease but is frequently found in eelIs from unresponsive patients who have been treated with drugs associated with the phenomultiple-drug-resistant type&s. Although overexpression of mdrl mRNA and overproduction of P-glycoprotein in solid tumors have been reported, its relationship to acquired or intrinsic drug resistance has not yet been well characterized. This may reflect the heterogeneity of tumor cells found in solid tumors or the
TiPS - September 1989 [Vol. 101 existence of multiple mechanisms of drug resistance. The implementation of immunocytochemical methods and in-situ hybridization techniques should provide additional information concerning the overexpression or overproduction of m&l gene and P-glycoprotein in human solid tumors. Numerous studies indicate that multiple drug resistance can be circumvented in cell culture by compounds such as verapamil, diltiazem, ciclosporin, reserpine, tamoxifen, mepacrine (quinacrine), triparanols, phenothiazine and amiodarone. In general, pretreatment of cells with nontoxic concentrations of circumventing agents can selectively reverse the resistant phenotype with little or no effect on the drugsensitive cell types. Many of these compounds can compete with a radiolabeled photoaffinity vinblastine analog for binding to the P-glycoprotein (see Ref. 6 for review of the stmclure-activity relationship of some of these compounds). Studies with drug-resistant transplantable murine tumors suggest that circumvention of the multiple-drug-resistant phenotype may have therapeutic application. For example, Slater et ~2.’ have reported that ciclosporln restores the sensitivity of daunorubicin-resistant Ehrlich ascites carcinoma in viva. Some investigators have attempted to apply the preclinical results to clinical protocoissfi*p. The severe cardiac toxicity seen with the initial phase I clinical studies with dtixorubicin and verapamil in women with ovarian carcinoma indicates the need for appropriate consideration of the pharmacokinetics and for rational design of chemoenhancers’. High levels of verapamil are needed in culture to overcome multiple drug resistance (- 2-10 PM) and it is difficult to achieve these levels clinically. Subsequent phase I trials with modified drug regimens or different modulators suggest chemoenhancement may be possible in patient@. Notably, Dalton et al. monitored levels of P-glycoprotein in the tumors of patients and concluded that administration of verapamil with vincristine, doxorubicin and dexamethasone may partially cirdrug resistance in cumvent
371 ---_
Fg. 1. Ptup0.d sites of infemcffon of some modulahnswithc&i/ar targets.
-, chemoenhanaws; +. chemqwt&om.
P-glycoprotein-positive tumors. Phase II studies as well as additional phase I trials are currently being conducted in which P-glycoprotein levels will be measured and newer modulators used. The recent observations that Pglycoprotein exists in non-malignant tissue and on endothelial cells4,10 emphasize the need for clinical trials to ensure that drugs selectively enhance malignant tissue destruction. The results of these clinical trials should help resolve questions concerning the importance of P-glycoprotein in clinical drug resistance’* and should provide information concerning the ultimate therapeutic utility of this modulation approach. Glutathione The tripeptide y-glutamylcysteinylglycine (glutathione, GSH) is the predominant intracellular non-protein thiol in mammalian cells. GSH has multiple cellular functions including protection of essential thiol groups on macromolecules from oxidation, detoxification of metabolites of physiological and xenobiotic origin via formation of mercapturic acids, and scavenging of cytotoxic free radicals”. It is primarily through nucleophilic
thioether formation or oxidationreduction reactions that GSH is thought to participate in the detoxification and repair of cellular injury of a variety of anticancer agents, including alkylating agents, quinone antibiotics and platinating agents=. Thus, it is not surprising that overproduction of GSH appears to be a mechanism of resistance to members of these clgsses of DNAinteractive anticancer agent. Intracellular levels of GSH are maintained by several interrelated processes including its synthesis, degradation and removal from the cell. Vistica and co-workersi have extensively studied the thiol status in model cell systems (murine L1210 cells with acquired resistance to melphalan) and found that in these drug-resistant cells the elevated levels of GSH result primarily from elevated y-glutamylcysteine synthetase activity. Although differences in GSHlevels or in the enzyme systems involved in GSH synthesis or degradation have been noted with some cells that overexpress P-glycoprotein and are resistant to doxorubicin and other classes of compound, it does not appear that these changes in GSH or GSH-related enzyme systems cause the multiple-drugresistant phenotype”.
TiPS - Se~~e~~er 1989 fVot. 101
372 The MS~of :hioi-depleting or repleting agents as chemoenhancers or chemopreventors for therapeutic use was limited in the past by their lack of selectivity and marked cellular toxicity. The development of buthionine sulfoximine (BSO), which is an irreversible inhibitor of y-glutamylcysteine synthetase15 and blocks the synthesis of GSH, has altered this. Treatment of cells with BSO can reduce GSH levels to less than 10% of the basal levels with littIe evidence of toxicity’“. In-viva studies have confirmed that BSO treatment can markedly reduce GSH in some tissues”. GSH can also be depleted with the diuretic etacrynic acid, which forms GSH conjugates through a reaction mediated by glutathione S-transferase’*. Experimentally it is useful that cellular GSH levels can be increased by tnratment with oxothiazolidine-&carboxylate, which elevates the intracelhrlar pool of cysteine, a precursor required for GSH synthesislg. Monoethyl GSH ester, which is more lipophihc than GSH, can also elevate intracellular levels of GSH (Ref. 20). A number of laboratories have used euhured malignant cells and BSO to examine the effects of lowered GSH levels on the cytotoxicity of antineoplastic agents13,“*17L1. In general, depletion of celldar GSH has lead to increased cytotoxicity with Certain antineoplastic agents, including melphalan, nitmsoureas, mitomycin and amsacrine (m~‘&)13.‘4,17,21; some controversy exists concerning other agents, such as doxorubicin, daunorubitin, bleomycin and cisplatin21. it appears that decreases in GSH to X&20% of control levels are required for drug sensitization. Treatment of cells with etacrynic acid increases the sensitivity of human and murine cells to chlorambucil”. Monoethyl GSH ester or oxothiazolidine-4carboxylate protect cells from some DNAinteractive drugs. Studies with animal models carrying transplantable tumors treated with anticancer agents such as melphalan and BSO appear promising For example, 0~01s and co-workers”, using an in-oivo model of human ovarian carcinoma, found that oral administration of BSO in the drinking water decreased tumor GSH
by 96%; subsequent treatment of tumor-bearing mice with melphalan resulted in an improved therapeutic response and several long-term survivors. Friedman et al.” have found increased melphalan activity in intracranial medulloblastoma and human glioma xenografts following BSO treatment. Treatment of animals with BSO can lead to reduction in GSH levels in many normal tissues, such as liver, muscle and kidney. The host toxicity of melphalan alone is primarily to bone marrow but through dietary restriction of L-cysteine and methionine selective sensitization of tumor tissue has been obtained; GSH in bone marrow decreased slightly but without an increase in sensitivity to melphalanss. Thus, the preclinical studies attempting to sensitize tumors selectively are promising. Phase I clinical trials are about to be initiated with BSO and melphalan. Preclinical studies with etacrynic acid and hying agents also are being conducted. MethaIIothioneIns Methallothioneins (MT) are a family of inducible proteins of low molecular weight, which are synthesized in mammalian tissues, are cysteine-rich, and have high affinity for heavy metals. A physiological role for MT in heavy metal detoxification has been postulatedz4. There is also considerable experimental evidence to support the h~othesis that intracellular levels of MT are an important factor in determining cellular responsiveness to some DNA-interactive anticancer agents Cells with acquired resistance to heavy metals overexpress MT and are cross-resistant to alkylating agents, such as chlorambucil or melaphalan, or to cisplat#. In addition, some but not all cells with acquired resistance to cisplatin overexpress MT. For example, we found that So% of human cell lines grown in culture that have acquired resistance to cisplatin have elevated levels of MT mRNA and proteir?. Thus increases in MT appear to be one biochemical alteration that occurs with acquired resistance to cisplatin ~though other mechanisms of resistance certainly exi&. Further evidence that MT is causally important in the resistant phenotype came from studies
with a mouse cell line infected with a bovine papilloma virus containing a human MT gene. This cell line overexpresses human MT and is n dstant to cisplatin and alkylating agents25. It is also slightly resistant to doxorubicin and Meomycin but not to 54luorouracil or vincristine. How cisplatin increases MT levels is not known but there is no evidence for gene amplification or for MT induction in a manner similar to that seen with heavy metals; perhaps the overexpression reflects the selection of cells that overexpress MT. This is an important area for future studies. In humans there are at least five forms of MT (Ref. 24) and it is not certain which form(s) is increased within cells with acquired resistance to anticancer agents. With Cd’+-resistant cells overexpression of MT&, is seen=. Understanding which forms are overexpressed may provide important isolation ~n~eming the different functions of the isoforms and the mechanisms that regulate the synthesis of MT. One contentious issue is precisely how MT might protect cells against the toxicities of DNAinteractive anticancer agents. More than 25% of intracellular cisplatin is bound to MT in cells that overexpress MT (Refs 2628). By contrast, less than 5% of cisplatin is associated with MT in cells with normal levels of MT. Similar results were seen with radiolabeled ch1orambuci1** Collectively, these results have led to the suggestion that elevated levels of MT can sequester electrophilic anticancer agents and reduce their DNA-damaging properties. A reduction in DNA adducts has not yet been demonstrated in cells with elevated MT, however, and it must be recognized that a large number of enzymes and macromolecules require Zn*+ or other metal ions that bind to MT; interactions between anticancer drugs and MT could alter the intracellular levels of these ions and thus the function of the proteins. Furthermore, MT could influence intracellular redox status or scavenge radical species%. Because MT is a highly inducible family of proteins, several groups have begun to examine whether changes in in-z& MT levels can alter the toxicity of
TiPS - September 1989 Wol. 101 anticancer agents to normal or malignant tissues. Webber et ~2.~ claimed that treatment of cells in culture with doxorubiti decreased the synthesis of MT; in these studies, however, only the ratio of [%]cysteine incorporation into macromolecules was determined, rather than actual synthesis of MT. Naganuma and co-workers31 have observed that pretreatment of mice with bismuth compounds decreased the lethality and renal, hematopoietic and gastrointestinal toxicity caused by cisplatin. Furthermore, pretreatment with bismuth salts protected mice against the cardiac toxicity of doxorubicin3*. This protection from the serious untoward effects of the anticancer agent occurred without a detectable reduction in antitumor activity. These observations with bismuth, which is a potent inducer of MT (Refs 31 and 32), could be of great therapeutic significance since bismuth compounds have been used as antidiarrheal drugs for many years and since the doses used in the studies closely approximate those used clinically. It is unclear, however, why the induction of MT is selective for non-malignant tissues and whether protection can be extended to other DNA-interactive drugs. 0
0
cl
In addition to the three classes of biochemical mediator of resistance to DNA-interactive anticancer drugs discussed here other mediators, such as glutathione Stransferasela and topoisomerase II (Ref. 33), are being studied and could be the focus of pharmacological modulation. The therapeutic advances obtained when leucovorin was added to methotrexate and when diuretics were administered with cisplatin provide important precedents for current preclinical and clinical studies with chemoenhancers and chemoprotectors. Not only may these therapeutic studies allow us to answer questions concerning the nature of intrinsic and acquired resistance to currently used antineoplastic drugs, but they could result in improved treatments for solid tumors.
373 Acknowledgements John S. Laze is the recipient of a US Public Health Service Research Career Development Award (CA01012). Robert R. Bahnson is the recipient of an American Cancer Society Clinical Oncology Career Development Award (86-48). References 1 Hurley, L. H. and Boyd, F. L. (1988)
Trends Phannacol Sci. 9,402-407 2 T&her, 8. A. et al. (1987) Cancer Res.47, 38R-393 ---_3 RIordan, J. R. and Ling, V. (1985) Pharmacol. Ther. 28.51-75
4 Gottesman, M. M. &d Pastan, I. (1988) Trends Pharmacol Sci. 9.54-58 5 Dalton, W. S. et al. (19eSj J. Clin. Oncol. 7.415-424 6 Ramu, A. (1989) in Resistance to Antineoplastic Drags (Kessel, D., ea.), pp. 63-80, CRC Press 7 Slater, L. M., Sweet, P., Stupecky, M., Wetzel, M. W. and Gupta, S. (1986) Br. J. Cancer 54‘235-238 8 0~01s. R. F. et al. (1987) J. Clin. Oncol. 5, 641-647 9 Miller, R. L. et al. (1988) 1. Clin. Oncol. 6, 880-888 10 Cordon-Cardo, C. et al. (1989) Pmt. Natl Acad. Sci. USA 86.695-698 11 Cass, C. E. and Beck, W. T. (1989) in Resistance to Antineapbstic Drugs (Kessel, D., ed.), pp. 141-165, CRC Press 12 Arrick, B. A. and Nathan, C. F. (1984) Cancer Res. 44.4224-4232 13 Ahmad, S., Okine, L., Wood, R., Fijian, J. and Vistica, D. T. (1987) J. Biol. Chetn. 262,15048-15053 14 Bellamy, W. T., Dalton, W. S., Mel-r, P. and Dorr, R. T. (1989) Biochem. Pharmacol. 38.787-793 15 Meister, A. and Anderson, M. A. (1983) Annu. Reu. Biochem. 52,711-760 16 Somfai-Relle, S., Suzukake, K., Vistica, 8. P. and Vistica, D. T. (1984) Biochem. Pharmacol. 33,485-490
17 Ozols, R. F. et al. (i%7) Biochem. Pharmacol. 36.147-153 18 Tew. K. D.. Bomber, A. M. and Hoffman, S. 1. (1988) Cancer Res. 48, 3622-3625 19 Williamson, J. M., Boettcher, B. and Meister, A. (1982) Proc. IVat! Acad. Sci. USA 79,6246-6249 20 Anderson, M. E.. Powrie. F.. Puri. R. N. and Meister, A. (196) Arch. Biochem. Biophys. 239,538-548 21 Andrews. I’. A., Murphy, M. P. and Howell, S. B. (1986) Mol. Pharmacol. 30, 643x% 22 Friedman, H. S. et al. (1989) 1. Nat1 Cancer ht. 81.524-527 23 Somfai-Relle, S., Suzukake., K., Vistica, B. P. and Vistica, D. T. (1984) Cancer Treat. Rev. 11 t%m~L A). 43-54 24 Hamer, D. (l&j Xnnuy Rev. Biochem. 55,9l3-951 25 Kelley, S. L. et al. (1989) Science 241, 181%1815 26 Bakka, A., Endersen, L., Johnsen, A. 6. S., Edminson, P. D. and Rugstad, H. E. (1981) Toxicol. Appl. Pharmac~l. 61, 21%226 27 Zelazowski, A., Garvey, J. and Hoeschele, J. (1984) Arch. Biochem. Biophys. 229,246-252 28 Kraker, A., Schmidt, J., Krezoski, S. and Petering, D. H. (1985) Biochem. Biophys. Res. Commun. 130,78&792 29 Endresen, L., B&&a, A. and Rugstaad, H. (1963) Cancer Res. 43,2916-2926 30 Webber, M. M., Maseehur Rehman, S. M. and James, G. T. (1988) Cancer Res. 48,1503-1508 31 Naganuma, A., Satoh, M. and &ura, N. (1987) Cancer Res. 47,983-987 32 Nagaruuna, A., Satoh, M. and lmura, N. (1988) Ipn. 1. Cancer Res. 79,406-411 33 Sullivan, D. M., Chow, K-C. and Ross, W. E. (1989) in Resishnce to Antineoplastic Drugs (Kessel, D., ed.), pp. 281-292, CRC Press SR-2508: N-(2-hy~xyethyl)-2-nitro_1Himidazole-l-Ycetamide ICRF-I8~(+)-1,2-bis(3,5-dioxopiperazinI-yl)propane
ducts Fbllcolour
reprints of the following ceakefolds
publishedearlierinWSare available:
o&idreceptortypE?s pharmacology of lipoprotein
me Tmnsmembranesi~g Dopandae receptor s&types Eicosanoids (updated May 198?I . Flrmcma receptors for 5 Peptides, receptors, cytokines
Available in multiples of six (any combination). Price per six copies is E8.00or us$15.00. Larger orders (lOO+) available at 15% discount. ordensErom: Fkevier Science publishers Ltd Crown House, Linton Road, Barking, Essex lG118JU UK
369
21 22 23 24
25 26
27 28
Acknowkdgemenb The authors are grateful to Mrs R. Kriiger for help in the preparation of the manuscript. Work of the authors cited herein was supported by grants of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
29
Naunyn-Sckmiedeberg’s Arch. Pharmacol. 337 (Suppl.), R44 UrquiIIa, P. R. (1978) Stroke 9,133-136 Hiiussinper, D., StebIe, T. and Gerok, W. (1987) Eur. 1. Biochem. 167,65-71 Needbam, L., Cusack, N. J., Pearson, J. D. and Gordon, J. L. (1987) Ear. j. Phanacal. 134,199-289 Goetz, U., Da Prada, M. and PIetscber, A. 11971) J. Pharmacaj. Exp. Ther. 178, 210-21s Keppler, D., Rudigr, J. and Decker, K. (1970) Anal. Biochem. 38,185-114 PauImicbI, M. and Lang, F. (1989) Bisthem. Biophys. Res. Crtmmun. I?. 1139-1143 Greenberg, S., Di VirSiiiz~ f., Steinberg, T. H. and Si!ve?&in, 5 1.11988) J. Bial. Chem. W, 1033%110363 Dubyak, G. R. and De Young, M. 3. (1985) f. Biot.Chem. 260,105t3--l_IOr;6~ Mustard, J. F. and Packbam. M. A (19&J) Phanacol. Rev. 22.97-187
38 Ford-Hutchinson, A. W. (1982) Br. J. Pharmacot. 76,36?-371 31 Okwon, I. L., B&man, T. R. and Gallo, R. C. (1982) Cancer Res. 42,392&3933 32 Limo& R., sdrvsrtz, I., Hasum, E., Ayalon, D. and Naor, Z. (1989) Biochem. Biopkys. Res. Commun. 159.209-215 33 Rehm. A. G., Wu, H. and Ha&da, S. P. (1988) FEES Lett. 234,316-320 34 Katsur&, T. and Furuksws. T. (1985) Trends Pharmacol. Sd. B337-339 35 Dusenbery, K. E., Mendiola, J. R. and’ Skubitz, K. M. (1988) Biochem. Biopkys. Res. Commun. 153,7-13 36 Keppens. S. snd de Wolf, H. (1986) ?“-frm?x. ;, 240.367-371 37 SwIzz C. L., Monir, A. j. and Harden, _ ..‘. (1989)j. BioJ. Ckem. 264,6202-6206 4 l+uber, I?., Ridrter, L and Re&er, W. !1982) F&BS J&t. 141,19&292 _ jessit~, A. J., ToIIey; J. 0. and AEen, IX. A. (1986; J. EiaZ. Char. 261, X3662-13669
References 1 Wii, mec41.
M. (1987) Annu. Rev. Phar27,315-345 J. L. (1986) Biockem. f. 233,
Toxicol.
2 Gordon, 309-319 3 Bumstock, G. and Kennedv, C. (1985) Gen. Pharauxol. 16,433-4404 Okaiima. F.. Tokumitsu. Y.. Kondo. Y. and’Ui,*M: (1987) J. B~oI.~Chem. 262, 13483-13498 5 Benbam, C. D. and Tsien, R. W. (1987) Nature 328,273-278 6 Dubyak, G. R. and De Young. M. 8. (1985) 1. BioJ. Ckem. 260.10653-10661 7 Forsbeg, E. J.,peuerstein, G..Shohemi, II. and PoIIard, H. II. (1987) Proc. Nal Acad. Sci. USA 84563iH634 8 Dubyak, G. R, Cowen, D. S. and Metier, L. M. (1988) J. Ihal. Chem. 263, 18108-18117 9 Kubns, D. B., Wright, D. G., Natb, J., KapIan, S. S. and Basford, R. E. (1988) Ial. Inuesf. 58,448-453 10 Seifert, R., Burde, R. and Schultz, G. (1989) Eiachem. J. 259,813-819 11 Seifert, R.. WeneeI, K., Eckstein, F. and ScI~uItz, G. (1989) Eur. 1. Biochem. 181, 277-285 12 Brown, C. M. and Burnstock, G. (1981) Eur. J. Pharmacol. 69,81-86 13 Hsrdebo,J.E.,Kabr&6m,J.,Gwmsn,C. and Salford, I, G. (1987) Blood Vessels 24, 150-l55 14 Sskai, K., Akima, M. and Matsushita, H. (1979) Jpn. J. Pkarmacai. 29,243-251 15 Martin, W., Cusack, N. J., Carleton, J. S. and Gordon, J. L. (1985) Eur. f. Pharmacof. 108,293-299 16 Mardonald, G., Asset, R. and Lo, E (1984) CJin. Exp. Pkarmacaol. Pkysiol. 11, 381-384 17 Hardebo, J. E., Hanko, J. and Owman, C. (1983) Gen. pharmacoI. 14,133-134 18 Shimsawa, Y., Wbite, R. P. and Robertson, J. T. (1983) Stroke 14‘347-355 19 von Kiigelen, I., Hliussinger, D. and Starke, K. (1987) Naunyn-Schmiedeberg’s Arch. Pharmncol. 336,556-560 20 von Kiigelen, I. and Starke, K. (1988)
John S. Laze and Robert R. Bahnson The poor therapeutic index and limited efficacy of current cancer chemotherap~~c agents represent an ~mpo~ant pha~acolo~ca~ prab~m. Athough there has been a significant increase in our understandjng of the nnechanisms by which anticancer drugs kill mammalian cells, identification of new, effective anticancer agents during the last decade has been exceeding slow. Thus, attention has focused on understanding the causes of drug resistance and on either sassing tumor cells to existing anticunc~ agents using what could be called ‘ckemoenhancers’, or protecting non-malignant tissues against serious untoward effects using ‘ckemoprotectors’. John Laze and Robert Bahnson review recent strategies attempting to modulate the activity of antineopiastic drugs. When demand exceeds supply, people look for ways to increase the usefulness of the existing sup ply. So it currently is in cancer research: there are many malignancies that do not respond to chemotherapy and too few exciting novel drugs to test. This, combined with an elevated understanding of the molecular basis of resistance to anticancer drugs, has kindled interest in identifying and developing pharmacological
f. S. Laze is Allegheny Foundation Professor and Ckairman of Pka~aco~gy and R. R. Bahnson is ~s~fant Professor of Urologic Surgey at tke University of Pittsburgh School of Medicine, Pittsbugh, PA 15261, USA. Both authors are also members of the Pittsburgh Cancer Institute.
modulators of existing cancer chemotherapeutic agents to improve their therapeutic indices. Two classes of modulator are being examined: chemoenhancers, which would sensitize tumor cells; and chemoprotectors, which would selectively protect non-ma~~ant tissue. To be a successful chemoenhancer or chemoprotector, an agent must exhibit little toxicity itself; many such agents have no anticancer activity at all and some have other useful pharmacological properties. The clinically successful combination of methotrexate with leucovorin (folinate} provides an important precedent for the chemoprotective approach; cisplatin, currently one of the most popular anticancer drugs,
3M would not be useful if pharmacological methods had not been identified to reduce its renal toxicity. A number of pharmacological agents have been identity as chemoenhancers or chemoprotectors. While many modulators of antimetabolites have been described, we wiII focus on pharmacological means to improve the therapeutic index of DNA-interactive anticancer drugs (see Ref. 1 for review of their molecular interactions), because they have become the most important class of drug for the treatment of solid tumors, and solid tumors are the most difficuh neoplastic diseases to cure. These anticancer drugs have some of the lowest therapeutic indices of aII available drugs and thus represent good candidates for improvement. A partial list of modulators of DNA-interactive drugs identified in preclinical studies with cultured cells or transplantable tumors in oioo is found in Table I. Most of the chemoenhancers or chemopmtectors are,. thought to have cellular targets that either affect celluIar responsiveness to the DNA-inpctive agents or emulate potential alternative cellular targets (Pig. 1). We will briefly highlight some of the recently acquired information on three classes of biochemical mediator of anticancer drug resistance and examine the pharmacological attempts to alter the levels or actions of these endogenous mediators of resistance. IIioehemicaImediators of resistance htrinsic drug resistance occurs in cds that have not previously been exposed to anticancer drugs; acquired resistance develops after exposure to anticancer drugs. It has become obvious that more than one mechanism of resistance can be identified even with a single DNA-~tera~ve agent or within a single tumor population-. The mechanisms of resistance seen within populations of cells having intrinsic or acquired drug resistance can be similar or different. The challenge for the Cancer p~acol~st is not only to establish which mechanisms are operative but also to devise Strategies to circumvent the resistance without increasing the
toxicity of the drugs to normal tissue. Both genetic and epigenetic processes have been implicated in anticancer drug resistance3; pharmacolo~c~ attempts either to increase the sensitivity of tumor cells or to decrease the sensitivity of non-malignant tissues have focused principally upon changing the steady-state levels of proteins or peptides, rather than on making any fundamental genetic aher-. ations. The preclinical studies to investigate whether modulators alter or emulate the hypothhized targets have been extensive for some of these modulators; clinical investigations with some of these agents are being initiated or considered. P-gIycoproteIn Resistance of cultured cells to a number of commonly used natural products that are antineoplastic including doxorubicin, dntgsdaunorubicin, etoposide, teniposide, plicamycin, vinblastine, vincristine and actinomycin D has been associated with the overexpression of a plasma membrane (P-glycoprotein). glycopmtein Most but not all of these drugs are believed to require interactions with DNA or DNA-associated proteins for their cytotoxic activity, although as a group they share little obvious chemical and mechanistic similarity. P-glycoprotein migrates in polyacrylamide gels with an apparent molecular mass of 150-180 kDa depending upon post-translational modificatio&. Our understanding of the molecular and bioTABLE I. Examples of chemoenhancers or ~~~~~rn Chemoanhancera
Chemeprotectore
Ca*+ channel blockers Phenothiazines
Amiistine
Ciclosporin Buthionine s~~rni~
(m)
TC%ElOXif@l
Ttfparanol lndole alkaloids Acridines
Thiosulfate Diethyldithiocarbamate Ilium&s Pw estem Bismuth salts Oxothiazoliiine+ carboxylate Razoxane
Amioderone
ICRF-187
Phorbol esters
~~~
streptozlcin SR-2508
!Semids
Pmgesterone Etacrynic Acid
esters
chemical properties of Pglycoproteins has expanded greatly in the past few years (see Ref. 4 for review). Briefly, a small gene family encodes P-glycoproteins: there are two genes in humans and three genes in rodents. The nucleotide sequences for these genes are now known, and the amino acid sequences of the proteins have been deduced. The proteins consist of highly homologous halves, each half containing six transmembrane segments and a nucleotide-binding site. DNA transfection studies suggest that only one human gene product (mdrl) produces resistance to antineoplastic drugs. The human m&l gene and its murine counterparts appear to encode a drug efflux pump glycoprotein that is responsible for the lower intracellular levels of cytotoxic drugs seen in the multiple-drugresistant phenotype. How antineoplastic drugs are removed from the cytosol by Pglycoprotein is currently being examined by several laboratories. In highIy resistant cells grown in culture, amplification of the mdrl gene is observed. The level of expression of m&l is also high in non-malignant adrenal gland, liver, kidney and colon. This has led to the speculation that the normal function of P-glycoprotein is to remove environmental toxins from the body*. cDNA probes for m&l, monocIonaI antibodies that recognize external epitopes of P-glycoproteins, and photoaffinity labeling reagents have been used to demonstrate overexpression of m&l mRNA and overproduction of P-glycoprotein in human tumor tissue. With hematol~i~ malignancies, such as multiple myeloma, initial results suggest that overexpression of P-glycoprotein is uncommon in previously untreated disease but is frequently found in eelIs from unresponsive patients who have been treated with drugs associated with the phenomultiple-drug-resistant type&s. Although overexpression of mdrl mRNA and overproduction of P-glycoprotein in solid tumors have been reported, its relationship to acquired or intrinsic drug resistance has not yet been well characterized. This may reflect the heterogeneity of tumor cells found in solid tumors or the
TiPS - September 1989 [Vol. 101 existence of multiple mechanisms of drug resistance. The implementation of immunocytochemical methods and in-situ hybridization techniques should provide additional information concerning the overexpression or overproduction of m&l gene and P-glycoprotein in human solid tumors. Numerous studies indicate that multiple drug resistance can be circumvented in cell culture by compounds such as verapamil, diltiazem, ciclosporin, reserpine, tamoxifen, mepacrine (quinacrine), triparanols, phenothiazine and amiodarone. In general, pretreatment of cells with nontoxic concentrations of circumventing agents can selectively reverse the resistant phenotype with little or no effect on the drugsensitive cell types. Many of these compounds can compete with a radiolabeled photoaffinity vinblastine analog for binding to the P-glycoprotein (see Ref. 6 for review of the stmclure-activity relationship of some of these compounds). Studies with drug-resistant transplantable murine tumors suggest that circumvention of the multiple-drug-resistant phenotype may have therapeutic application. For example, Slater et ~2.’ have reported that ciclosporln restores the sensitivity of daunorubicin-resistant Ehrlich ascites carcinoma in viva. Some investigators have attempted to apply the preclinical results to clinical protocoissfi*p. The severe cardiac toxicity seen with the initial phase I clinical studies with dtixorubicin and verapamil in women with ovarian carcinoma indicates the need for appropriate consideration of the pharmacokinetics and for rational design of chemoenhancers’. High levels of verapamil are needed in culture to overcome multiple drug resistance (- 2-10 PM) and it is difficult to achieve these levels clinically. Subsequent phase I trials with modified drug regimens or different modulators suggest chemoenhancement may be possible in patient@. Notably, Dalton et al. monitored levels of P-glycoprotein in the tumors of patients and concluded that administration of verapamil with vincristine, doxorubicin and dexamethasone may partially cirdrug resistance in cumvent
371 ---_
Fg. 1. Ptup0.d sites of infemcffon of some modulahnswithc&i/ar targets.
-, chemoenhanaws; +. chemqwt&om.
P-glycoprotein-positive tumors. Phase II studies as well as additional phase I trials are currently being conducted in which P-glycoprotein levels will be measured and newer modulators used. The recent observations that Pglycoprotein exists in non-malignant tissue and on endothelial cells4,10 emphasize the need for clinical trials to ensure that drugs selectively enhance malignant tissue destruction. The results of these clinical trials should help resolve questions concerning the importance of P-glycoprotein in clinical drug resistance’* and should provide information concerning the ultimate therapeutic utility of this modulation approach. Glutathione The tripeptide y-glutamylcysteinylglycine (glutathione, GSH) is the predominant intracellular non-protein thiol in mammalian cells. GSH has multiple cellular functions including protection of essential thiol groups on macromolecules from oxidation, detoxification of metabolites of physiological and xenobiotic origin via formation of mercapturic acids, and scavenging of cytotoxic free radicals”. It is primarily through nucleophilic
thioether formation or oxidationreduction reactions that GSH is thought to participate in the detoxification and repair of cellular injury of a variety of anticancer agents, including alkylating agents, quinone antibiotics and platinating agents=. Thus, it is not surprising that overproduction of GSH appears to be a mechanism of resistance to members of these clgsses of DNAinteractive anticancer agent. Intracellular levels of GSH are maintained by several interrelated processes including its synthesis, degradation and removal from the cell. Vistica and co-workersi have extensively studied the thiol status in model cell systems (murine L1210 cells with acquired resistance to melphalan) and found that in these drug-resistant cells the elevated levels of GSH result primarily from elevated y-glutamylcysteine synthetase activity. Although differences in GSHlevels or in the enzyme systems involved in GSH synthesis or degradation have been noted with some cells that overexpress P-glycoprotein and are resistant to doxorubicin and other classes of compound, it does not appear that these changes in GSH or GSH-related enzyme systems cause the multiple-drugresistant phenotype”.
TiPS - Se~~e~~er 1989 fVot. 101
372 The MS~of :hioi-depleting or repleting agents as chemoenhancers or chemopreventors for therapeutic use was limited in the past by their lack of selectivity and marked cellular toxicity. The development of buthionine sulfoximine (BSO), which is an irreversible inhibitor of y-glutamylcysteine synthetase15 and blocks the synthesis of GSH, has altered this. Treatment of cells with BSO can reduce GSH levels to less than 10% of the basal levels with littIe evidence of toxicity’“. In-viva studies have confirmed that BSO treatment can markedly reduce GSH in some tissues”. GSH can also be depleted with the diuretic etacrynic acid, which forms GSH conjugates through a reaction mediated by glutathione S-transferase’*. Experimentally it is useful that cellular GSH levels can be increased by tnratment with oxothiazolidine-&carboxylate, which elevates the intracelhrlar pool of cysteine, a precursor required for GSH synthesislg. Monoethyl GSH ester, which is more lipophihc than GSH, can also elevate intracellular levels of GSH (Ref. 20). A number of laboratories have used euhured malignant cells and BSO to examine the effects of lowered GSH levels on the cytotoxicity of antineoplastic agents13,“*17L1. In general, depletion of celldar GSH has lead to increased cytotoxicity with Certain antineoplastic agents, including melphalan, nitmsoureas, mitomycin and amsacrine (m~‘&)13.‘4,17,21; some controversy exists concerning other agents, such as doxorubicin, daunorubitin, bleomycin and cisplatin21. it appears that decreases in GSH to X&20% of control levels are required for drug sensitization. Treatment of cells with etacrynic acid increases the sensitivity of human and murine cells to chlorambucil”. Monoethyl GSH ester or oxothiazolidine-4carboxylate protect cells from some DNAinteractive drugs. Studies with animal models carrying transplantable tumors treated with anticancer agents such as melphalan and BSO appear promising For example, 0~01s and co-workers”, using an in-oivo model of human ovarian carcinoma, found that oral administration of BSO in the drinking water decreased tumor GSH
by 96%; subsequent treatment of tumor-bearing mice with melphalan resulted in an improved therapeutic response and several long-term survivors. Friedman et al.” have found increased melphalan activity in intracranial medulloblastoma and human glioma xenografts following BSO treatment. Treatment of animals with BSO can lead to reduction in GSH levels in many normal tissues, such as liver, muscle and kidney. The host toxicity of melphalan alone is primarily to bone marrow but through dietary restriction of L-cysteine and methionine selective sensitization of tumor tissue has been obtained; GSH in bone marrow decreased slightly but without an increase in sensitivity to melphalanss. Thus, the preclinical studies attempting to sensitize tumors selectively are promising. Phase I clinical trials are about to be initiated with BSO and melphalan. Preclinical studies with etacrynic acid and hying agents also are being conducted. MethaIIothioneIns Methallothioneins (MT) are a family of inducible proteins of low molecular weight, which are synthesized in mammalian tissues, are cysteine-rich, and have high affinity for heavy metals. A physiological role for MT in heavy metal detoxification has been postulatedz4. There is also considerable experimental evidence to support the h~othesis that intracellular levels of MT are an important factor in determining cellular responsiveness to some DNA-interactive anticancer agents Cells with acquired resistance to heavy metals overexpress MT and are cross-resistant to alkylating agents, such as chlorambucil or melaphalan, or to cisplat#. In addition, some but not all cells with acquired resistance to cisplatin overexpress MT. For example, we found that So% of human cell lines grown in culture that have acquired resistance to cisplatin have elevated levels of MT mRNA and proteir?. Thus increases in MT appear to be one biochemical alteration that occurs with acquired resistance to cisplatin ~though other mechanisms of resistance certainly exi&. Further evidence that MT is causally important in the resistant phenotype came from studies
with a mouse cell line infected with a bovine papilloma virus containing a human MT gene. This cell line overexpresses human MT and is n dstant to cisplatin and alkylating agents25. It is also slightly resistant to doxorubicin and Meomycin but not to 54luorouracil or vincristine. How cisplatin increases MT levels is not known but there is no evidence for gene amplification or for MT induction in a manner similar to that seen with heavy metals; perhaps the overexpression reflects the selection of cells that overexpress MT. This is an important area for future studies. In humans there are at least five forms of MT (Ref. 24) and it is not certain which form(s) is increased within cells with acquired resistance to anticancer agents. With Cd’+-resistant cells overexpression of MT&, is seen=. Understanding which forms are overexpressed may provide important isolation ~n~eming the different functions of the isoforms and the mechanisms that regulate the synthesis of MT. One contentious issue is precisely how MT might protect cells against the toxicities of DNAinteractive anticancer agents. More than 25% of intracellular cisplatin is bound to MT in cells that overexpress MT (Refs 2628). By contrast, less than 5% of cisplatin is associated with MT in cells with normal levels of MT. Similar results were seen with radiolabeled ch1orambuci1** Collectively, these results have led to the suggestion that elevated levels of MT can sequester electrophilic anticancer agents and reduce their DNA-damaging properties. A reduction in DNA adducts has not yet been demonstrated in cells with elevated MT, however, and it must be recognized that a large number of enzymes and macromolecules require Zn*+ or other metal ions that bind to MT; interactions between anticancer drugs and MT could alter the intracellular levels of these ions and thus the function of the proteins. Furthermore, MT could influence intracellular redox status or scavenge radical species%. Because MT is a highly inducible family of proteins, several groups have begun to examine whether changes in in-z& MT levels can alter the toxicity of
TiPS - September 1989 Wol. 101 anticancer agents to normal or malignant tissues. Webber et ~2.~ claimed that treatment of cells in culture with doxorubiti decreased the synthesis of MT; in these studies, however, only the ratio of [%]cysteine incorporation into macromolecules was determined, rather than actual synthesis of MT. Naganuma and co-workers31 have observed that pretreatment of mice with bismuth compounds decreased the lethality and renal, hematopoietic and gastrointestinal toxicity caused by cisplatin. Furthermore, pretreatment with bismuth salts protected mice against the cardiac toxicity of doxorubicin3*. This protection from the serious untoward effects of the anticancer agent occurred without a detectable reduction in antitumor activity. These observations with bismuth, which is a potent inducer of MT (Refs 31 and 32), could be of great therapeutic significance since bismuth compounds have been used as antidiarrheal drugs for many years and since the doses used in the studies closely approximate those used clinically. It is unclear, however, why the induction of MT is selective for non-malignant tissues and whether protection can be extended to other DNA-interactive drugs. 0
0
cl
In addition to the three classes of biochemical mediator of resistance to DNA-interactive anticancer drugs discussed here other mediators, such as glutathione Stransferasela and topoisomerase II (Ref. 33), are being studied and could be the focus of pharmacological modulation. The therapeutic advances obtained when leucovorin was added to methotrexate and when diuretics were administered with cisplatin provide important precedents for current preclinical and clinical studies with chemoenhancers and chemoprotectors. Not only may these therapeutic studies allow us to answer questions concerning the nature of intrinsic and acquired resistance to currently used antineoplastic drugs, but they could result in improved treatments for solid tumors.
373 Acknowledgements John S. Laze is the recipient of a US Public Health Service Research Career Development Award (CA01012). Robert R. Bahnson is the recipient of an American Cancer Society Clinical Oncology Career Development Award (86-48). References 1 Hurley, L. H. and Boyd, F. L. (1988)
Trends Phannacol Sci. 9,402-407 2 T&her, 8. A. et al. (1987) Cancer Res.47, 38R-393 ---_3 RIordan, J. R. and Ling, V. (1985) Pharmacol. Ther. 28.51-75
4 Gottesman, M. M. &d Pastan, I. (1988) Trends Pharmacol Sci. 9.54-58 5 Dalton, W. S. et al. (19eSj J. Clin. Oncol. 7.415-424 6 Ramu, A. (1989) in Resistance to Antineoplastic Drags (Kessel, D., ea.), pp. 63-80, CRC Press 7 Slater, L. M., Sweet, P., Stupecky, M., Wetzel, M. W. and Gupta, S. (1986) Br. J. Cancer 54‘235-238 8 0~01s. R. F. et al. (1987) J. Clin. Oncol. 5, 641-647 9 Miller, R. L. et al. (1988) 1. Clin. Oncol. 6, 880-888 10 Cordon-Cardo, C. et al. (1989) Pmt. Natl Acad. Sci. USA 86.695-698 11 Cass, C. E. and Beck, W. T. (1989) in Resistance to Antineapbstic Drugs (Kessel, D., ed.), pp. 141-165, CRC Press 12 Arrick, B. A. and Nathan, C. F. (1984) Cancer Res. 44.4224-4232 13 Ahmad, S., Okine, L., Wood, R., Fijian, J. and Vistica, D. T. (1987) J. Biol. Chetn. 262,15048-15053 14 Bellamy, W. T., Dalton, W. S., Mel-r, P. and Dorr, R. T. (1989) Biochem. Pharmacol. 38.787-793 15 Meister, A. and Anderson, M. A. (1983) Annu. Reu. Biochem. 52,711-760 16 Somfai-Relle, S., Suzukake, K., Vistica, 8. P. and Vistica, D. T. (1984) Biochem. Pharmacol. 33,485-490
17 Ozols, R. F. et al. (i%7) Biochem. Pharmacol. 36.147-153 18 Tew. K. D.. Bomber, A. M. and Hoffman, S. 1. (1988) Cancer Res. 48, 3622-3625 19 Williamson, J. M., Boettcher, B. and Meister, A. (1982) Proc. IVat! Acad. Sci. USA 79,6246-6249 20 Anderson, M. E.. Powrie. F.. Puri. R. N. and Meister, A. (196) Arch. Biochem. Biophys. 239,538-548 21 Andrews. I’. A., Murphy, M. P. and Howell, S. B. (1986) Mol. Pharmacol. 30, 643x% 22 Friedman, H. S. et al. (1989) 1. Nat1 Cancer ht. 81.524-527 23 Somfai-Relle, S., Suzukake., K., Vistica, B. P. and Vistica, D. T. (1984) Cancer Treat. Rev. 11 t%m~L A). 43-54 24 Hamer, D. (l&j Xnnuy Rev. Biochem. 55,9l3-951 25 Kelley, S. L. et al. (1989) Science 241, 181%1815 26 Bakka, A., Endersen, L., Johnsen, A. 6. S., Edminson, P. D. and Rugstad, H. E. (1981) Toxicol. Appl. Pharmac~l. 61, 21%226 27 Zelazowski, A., Garvey, J. and Hoeschele, J. (1984) Arch. Biochem. Biophys. 229,246-252 28 Kraker, A., Schmidt, J., Krezoski, S. and Petering, D. H. (1985) Biochem. Biophys. Res. Commun. 130,78&792 29 Endresen, L., B&&a, A. and Rugstaad, H. (1963) Cancer Res. 43,2916-2926 30 Webber, M. M., Maseehur Rehman, S. M. and James, G. T. (1988) Cancer Res. 48,1503-1508 31 Naganuma, A., Satoh, M. and &ura, N. (1987) Cancer Res. 47,983-987 32 Nagaruuna, A., Satoh, M. and lmura, N. (1988) Ipn. 1. Cancer Res. 79,406-411 33 Sullivan, D. M., Chow, K-C. and Ross, W. E. (1989) in Resishnce to Antineoplastic Drugs (Kessel, D., ed.), pp. 281-292, CRC Press SR-2508: N-(2-hy~xyethyl)-2-nitro_1Himidazole-l-Ycetamide ICRF-I8~(+)-1,2-bis(3,5-dioxopiperazinI-yl)propane
ducts Fbllcolour
reprints of the following ceakefolds
publishedearlierinWSare available:
o&idreceptortypE?s pharmacology of lipoprotein
me Tmnsmembranesi~g Dopandae receptor s&types Eicosanoids (updated May 198?I . Flrmcma receptors for 5 Peptides, receptors, cytokines
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