In Vivo Characterization of Combination Antitumor Chemotherapy with Calcium Channel Blockers and Ci5-Diamminedichloroplatinum(II)1
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(CANCER RESEARCH 49, 2844-2850. June 1, 1989] In Vivo Characterization of Combination Antitumor Chemotherapy with Calcium Channel Blockers and ci5-Diamminedichloroplatinum(II)1 James M. Onoda,2 Kevin K. Nelson, John D. Taylor, and Kenneth V. Honn Departments of Radiation Oncology [J. M. O., K. K. N., J. D. T., K. V. H.], Biological Sciences [J. D. T.], and Chemistry ¡K.V. H.], Wayne State University, Detroit, Michigan 48202; and the Cershenson Radiation Oncology Center [J. M. O., K. K. N., J. D. T., K. V. H.], Harper/Grace Hospitals, Detroit, Michigan 4820I ABSTRACT dulin antagonists to enhance the antitumor actions of the more We have examined nifedipine, a dihydropyridine class calcium channel commonly prescribed organic or natural product chemothera blocker, for ability to overcome m-diamminedichloroplatinum(II) (cis- peutic agents (3, 4). The ability of verapamil to reverse multi- platin) resistance in a murine tumor line variant, B16a-Pt, which we drug resistance or pleiotropic drug resistance correlates with developed for resistance to cisplatin. Nifedipine significantly enhanced the expression of a M, 170,000 glycoprotein in drug-resistant the antitumor actions of cisplatin against primary subcutaneous B16a-Pt tumor cell plasma membranes (5, 6). This glycoprotein is now tumors and their spontaneous pulmonary métastases.We have charac commonly referred to as the P-glycoprotein (7, 8) and is re terized, in vivo, the pharmacokinetics and dose-response interactions sponsible for the active efflux of many organic/natural product between nifedipine and cisplatin. We now report our studies designed to cytotoxic chemotherapeutic agents (9-11). The current hypoth compare, in vivo, the efficacy of nifedipine and other calcium active esis suggests that verapamil interacts with the P-glycoprotein compounds including: (a) structurally similar calcium channel blockers to block drug efflux (12, 13); and that its actions are independ (nimodipine, nicardipine) from the dihydropyridine class, (b) structurally ent of the classical slow-inward Ca2+ channel (14, 15). Calmo different calcium channel blockers from the benzothiazepine (diltiazem) and the phenylalkylamine (verapamil) classes, and (c) calmodulin antag dulin antagonists (16), CCBs of different chemical classes (13), onists (trifluoperazine and calmidazolium) for ability to enhance the and analogues of CCBs in a single class (17) often fail to work antitumor action of cisplatin. Nifedipine was included as the standard or as well as verapamil, suggesting that verapamil may interact reference compound. In these studies verapamil and diltiazem failed to with the P-glycoprotein at structurally definitive "binding sites" enhance the antitumor actions of cisplatin as did both calmodulin antag (18, 19). onists. Our findings suggest that nifedipine has a greater degree of In contrast to the abundant information available on MDR specificity for B16a-Pt cells than structurally different calcium channel less is known about tumor cell resistance to cisplatin, an inor blockers from other chemical classes (i.e., diltiazem and verapamil), or ganic cytotoxic agent. The P-glycoprotein may not be respon the two calmodulin antagonists u.c.. trifluoperazine and calmidazolium). sible for cisplatin-resistance, since cells exhibiting MDR resist We concluded that nifedipine interacts with specific target site(s) which are not accessible by verapamil, by diltiazem, or by the calmodulin ance usually remain sensitive to the cytotoxic effects of cisplatin antagonists. Surprisingly, the two dihydropyridine class calcium channel (20,21). blockers, nimodipine and nicardipine, also failed to enhance cisplatin's We recently reported (22, 23) that nifedipine, a dihydropyri antitumor actions despite the fact that their specificity and kinetics for dine class CCB, significantly enhances the cytotoxic antitumor binding to the dihydropyridine receptor component of the calcium channel and antimetastatic of effects of cisplatin against cisplatin-re favors them (nimodipine and nicardipine) over nifedipine. Therefore, we sistant primary tumors and their spontaneous pulmonary mé postulate that the synergism between cisplatin and nifedipine is inde pendent of the latter's effect on the voltage sensitive, slow inward calcium tastases. Our findings led us to test, in vivo, other calcium channel blockers of the dihydropyridine class, CCBs from dif channel. We suggest that cisplatin cytotoxicity is enhanced by nifedipine's ferent chemical classes (i.e., benzothiazepine, phenylalkyla interaction with an as yet unidentified specific "target site," as opposed mine) and calmodulin antagonists, to determine whether the to nonspecific interactions with the tumor cell plasma membrane or observed synergism between nifedipine and cisplatin was spe specific interactions with calmodulin or the P-glycoprotein (which is cific for nifedipine or was a general phenomenon exhibited by responsible for pleiotropic resistance). all classes of CCBs, by all dihydropyridine class CCBs or by calmodulin antagonists. INTRODUCTION Recently, novel chemotherapeutic agents and combination MATERIALS AND METHODS chemotherapies have resulted in promising new protocols for the treatment of recurrent and drug-resistant malignancies (1, Tumors 2). These regimens allow for an improved therapeutic efficacy but are often limited to a specific tumor type or to specific The B16 amelanotic melanoma (B16a) was originally obtained from cytotoxic agent(s). A significant advance in experimental ther the Mason Research Institute (Worchester, MA), Routine tumor trans apeutics involves the use of CCB3 (e.g., verapamil) and calmo- plantation was performed every 2 weeks into male, syngeneic host mice (C56BL/6 J; The Jackson Laboratory, Bar Harbor, ME). Mice used Received 9/20/88; revised 1/30/89: accepted 3/3/89. for transplantation were between 18 and 22 g (approximately 49 days The costs of publication of this article were defrayed in part by the payment old) and were housed under identical conditions of photoperiod, feeding of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. regime, temperature, etc. All animals were quarantined and acclimated 1This investigation was partially supported by a grant from the Meyer L. for a minimum of 10 days after receipt. Tumor brei (implanted into the Prentis Comprehensive Cancer Center of Metropolitan Detroit. Detroit. MI left axillary region) were used for routine transplantation. In order to 48201 [85-SC11: J. M. O.). and a grant from Harper Hospital |J. M. O., maintain metastatic phenotype, the B16a line was routinely restarted K. V. H.]. Detroit. Michigan, 48201. 2To whom requests for reprints should be addressed, at Department of from liquid N2 frozen stocks after eight to ten isotransplant generations Radiation Oncology, 431 Chemistry Building, Wayne State University, Detroit. in vivo. In addition, each line was recharacterized before use. Passages Michigan 48202. one and two after restarting each line from liquid N2 stocks were used ' The abbreviations and trivial name used are: CCB, calcium channel blockers; for characterization. Characterization included histology, transplanta- cisplatin. fM-diamminedichloroplatinum(ll): B16a. B16 amelanotic melanoma tumor line: B16a-Pt. cisplatin-resistant variant of the B16 amelanotic melanoma: bility, screen for murine tumor viruses, growth rate, metastatic pattern, 3LL, Lewis lung carcinoma tumor line; MEM, minimal essential media; MDR. number of lung colonies formed in the experimental metastasis and multidrug resistance. spontaneous metastasis assay and doubling time in tissue culture. 2844 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1989 American Association for Cancer Research. COMBINATION CHEMOTHERAPY WITH CALCIUM ANTAGONISTS AND CISPLATIN Development of the Cisplatin-resistant Tumor Line B16a-Pt the antitumor effects of cisplatin with combination nifedipine therapy, (c) to avoid death due to cisplatin, nifedipine and/or cisplatin/nifedipine The cisplatin-resistant B16a variant (B16a-Pt) line, was developed toxicity, and (d) to administer nifedipine and cisplatin to mice via the by a modification of the protocol of Schmid et al. (24). This was route most often used for human therapy, i.e., oral (nifedipine) and achieved by sequential treatment of primary subcutaneous tumors (ap intravenous (cisplatin). proximately 2 g) with two courses of cisplatin (4 mg/kg) on Days 14 Drug Dosage and Injection Schedule. We used drug dosage and and 21 posttumor brei implantation. On Day 33, primary tumors were injection protocols previously reported for combination nifedipine/ excised and adapted for growth in culture as described previously (22). cisplatin therapy against the B16a melanoma (22) and the Lewis lung The resulting primary cultures were treated with incremental (0.5-2.0 carcinoma (23). Nifedipine was administered orally (10 mg/kg; solubi- MM)exposure to cisplatin. Typically, exponentially growing cells were lized in 0.1 ml polyethylene glycol 400) 20 min prior to the i.v. (tail treated for one day with cisplatin, followed by removal of cisplatin, and vein) injection of cisplatin (4 mg/kg). Drugs were administered on Days readdition at the next highest dose. The cells which survived the in 14, 17, and 25 posttumor cell injection. Nifedipine, because it is vitro cisplatin treatment were reinjected s.c. into syngeneic mice. Mice absorbed from the gut, was administered