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Pervilleines B and C, new tropane alkaloid aromatic esters that reverse the multidrug-resistance in the hollow fiber assay
Cancer Letters 184 (2002) 13–20 www.elsevier.com/locate/canlet
Pervilleines B and C, new tropane alkaloid aromatic esters that reverse the multidrug-resistance in the hollow fiber assay q Qiuwen Mi a,b, Baoliang Cui a,b,1, Gloria L. Silva a,b,2, Daniel Lantvit a,b, Eula Lim a,b, Heebyung Chai a,b, Melinda G. Hollingshead c, Joseph G. Mayo c, A. Douglas Kinghorn a,b, John M. Pezzuto a,b,* a
Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy (m/c 877), 833 South Wood Street, University of Illinois at Chicago, Chicago, IL 60612, USA b Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy (m/c 781), 833 South Wood Street, University of Illinois at Chicago, Chicago, IL 60612, USA c Biological Testing Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer InstituteFrederick Cancer Research and Development Center, Frederick, MD 21701, USA Received 4 March 2002; accepted 2 April 2002
Abstract P-Glycoprotein (Pgp)-mediated drug efflux can yield a multidrug-resistance phenotype that is associated with poor response to cancer chemotherapy. Pervilleines B and C (PB and PC), two new tropane alkaloid aromatic esters obtained from a chloroform extract of the roots of Erythroxylum pervillei as the result of bioactivity-guided fractionation, were found to restore the vinblastine (VLB) sensitivity of cultured multidrug-resistant KB-V1 cells, with 50% inhibitory concentration values of 0.17 mM in each case. To explore the potential relevance of this response, KB-V1 cells were placed in hollow fibers and implanted into NCr nu/nu mice. Cell growth was not significantly inhibited when VLB or PB or PC were administered as single agents, but when used in combination with vinblastine inhibition of up to 77.7% was observed. Equimolar doses of verapamil were less effective. These data suggest that PB and PC are effective inhibitors of Pgp and should be further evaluated for clinical utility. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pervilleines B and C; Multidrug-resistance; Hollow fiber test; Cancer chemotherapy; MDR1 gene; Verapamil
q Support for this work was provided by U19 CA52956 funded by the National Cancer Institute. * Corresponding author. Tel.: 11-312-996-5967; fax: 11-312-996-2815. E-mail address: [email protected] (J.M. Pezzuto). 1 Present address: PureWorld Botanicals, South Hackensack, NJ 07606, USA. 2 Present address: Departamento de Quı´mica Orga´nica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, 5000 Co´rdoba, Argentina. Abbreviations: DMEM, Dulbecco’s Modified Eagle medium; DMSO, dimethyl sulfoxide; IC50, 50% inhibitory concentration; MDR, multidrugresistance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PB, pervilleine B [3a-(3,4,5-trimethoxybenzoyloxy)-6b-(E)(3,4,5-trimethoxycinnamoyloxy) tropane]; PC, pervilleine C [3a,6b-di-(E)-(3,4,5-trimethoxycinnamoyloxy)tropane]; PBS, phosphate-buffered saline; Pgp, P-glycoprotein; PVP, polyvinylpyrrolidone; VLB, vinblastine; VP, verapamil
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00202-1
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Fig. 1. Structures of tropane alkaloid aromatic esters PA, PB and PC isolated from Erythroxylum pervillei, and VP.
1. Introduction One serious problem associated with cancer chemotherapy is the development of multidrug-resistant tumor cells during the course of treatment. An important mechanism of acquiring the multidrugresistance (MDR) phenotype in mammalian cells is the enhanced expression of a membrane glycoprotein, which has been termed P-glycoprotein (Pgp) [1]. With a molecular weight of 170 kDa, Pgp is coded by the MDR1 gene. Pgp functions as an energy-dependent multidrug membrane transporter that rapidly extrudes a variety of hydrophobic antitumor drugs from target cancer cells and thereby prevents the drugs from exerting cytotoxic effects. Several agents are known to overcome, or at least partially circumvent, MDR [2]. Examples are calcium
channel blockers such as verapamil (VP) [3,4], the immunosuppressive agent cyclosporin A [5], analogues of the antihypertensive reserpine and adrenergic blocking agent yohimbine [6], the neuroleptic, trifluroperazine [7], antiestrogens such as tamoxifen [8], a non-immunosuppressive analogue of cyclosporin D, SDZ PSC-833 [5], and others, such as MS-209 [9], S-9788 [10], GF120918 [11], and LY335979 [7]. However, while many of these pharmacological agents have been found to completely overcome drug resistance with in vitro models, reports showing efficacy with mammalian systems are more limited [12–15]. In general, it is difficult to maintain active doses of chemosensitizers without causing serious side effects [16]. Part of our natural product drug discovery program involves monitoring the potential of plant extracts to reverse multiple drug resistance. Standard cell survival assays are used to determine the dose of plant extracts or compounds required to inhibit cell growth by 50% inhibitory concentration (IC50) with drugsensitive human epidermoid carcinoma parental KB3 cells and Pgp-associated multidrug-resistant KB-V1 cells. To investigate the potential of plant extracts or compounds to reverse multidrug-resistance, KB-V1 cells are treated with different concentrations of plant extracts or compounds in the presence (1 mg/ ml) or absence of vinblastine (VLB). This concentration of VLB is lethal to KB-3 cells, but does not affect the growth of KB-V1 cells. Therefore, KB-3 cells serve as a control to differentiate between non-specific cytotoxicity and selective MDR antagonism. This assay employs 96-well microtiter plate technology and over 3000 different plant extracts have been tested. Using this model for bioassay-guided isolation of active principles, we have previously identified four moderate inhibitors of MDR: coronaridine, conoduramine, voacamine, and (2)-roemerine [17,18]. More recently, some extremely potent novel tropane alkaloid aromatic esters were obtained from a chloroform-soluble extract derived from the roots of Erythroxylum pervillei Baillon (Erythroxylaceae) and the stems of Erythroxylum rotundifolium Lanan (Erythroxylaceae) [19–22]. As shown in Fig. 1, these compounds have the general structure of tropane alkaloid 3a,6b-diesters, with the C-6 substituent bearing a trans-3,4,5-trimethoxycinnamoyl unit. Pervilleines A and B (PA and PB) have a 3,4,5-trimethoxybenzoy-
Q. Mi et al. / Cancer Letters 184 (2002) 13–20
loxy substituent at C-3, and a b-hydroxy and methylene substituents at C-7, respectively. Pervilleine C (PC) has a trans-3,4,5-trimethoxycinnamoyl substituent at C-3. Among the isolates, the mechanism of PA has been studied in some detail [19]. PA was found to restore the VLB sensitivity of cultured multidrugresistant KB-V1 and CEM/VLB100 cells, as well as the colchicine sensitivity of cultured KB-8-5 cells. PA competitively inhibited the binding the [ 3H]VLB with isolated KB-V1 cell membrane vesicles, increased the intracellular accumulation of [ 3H]VLB, and reversed the multidrug resistance of KB-V1 and KB-8-5 cells in the hollow fiber model. Starting with crude plant material, PA was obtained in 4.2% w/w yield. The additional isolates, PB and PC, were also obtained in good yield (3.5 and 4.3% w/w, respectively) [20]. Although these compounds bear a close structural resemblance, only PA has been evaluated for activity. Since small structural differences can have profound effects on biological responses, we currently report the activity of PB and PC with the in vivo hollow fiber model.
2. Materials and methods 2.1. Cell lines and agents Human oral epidermoid carcinoma KB-3 were purchased from the American Type Culture Collection (ATCC, Rockville, MD), and KB-V1 cells were supplied by Dr I.B. Roninson (Department of Molecular Genetics, University of Illinois at Chicago, Chicago, IL). KB-3 cells were maintained in Dulbecco’s Modified Eagle medium supplemented with 10% heat-inactivated calf serum and 100 units/ml penicillin G, 100 mg/ml streptomycin sulfate, 250 ng/ml amphotericin B. KB-V1 cells were grown in the same medium, which was further supplemented with VLB (1 mg/ml). Cell culture media and supplements were obtained from Life Technologies, Inc. (Grand Island, NY). The tropane alkaloid aromatic esters PB and PC (Fig. 1) were obtained by bioassay-guided fractionation from extracts derived from the roots of E. pervillei Baillon (Erythroxylaceae) [20–22]. All general chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. Polyvinylidene fluoride hollow fibers (500 000
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Da molecular weight cut-off, 1.0 mm ID) were purchased from Spectrum Medical Industries (Luguan Hills, CA). 2.2. Cytotoxic potential The cytotoxic potential of test substances with KB3 and KB-V1 cells was determined as described previously [18]. Briefly, various concentrations of test compounds (dissolved in 10 ml of 10% dimethyl sulfoxide, DMSO) were transferred to 96-well plates, and 190 ml aliquots of cell suspensions (5 £ 10 4 cells/ ml) were added to each well. The plates were then incubated for 72 h at 378C (100% humidity with a 5% CO2 atmosphere in air), and 100 ml of cold 20% aqueous trichloroacetic acid were added to the growth medium in each well to fix the cells. The cultures were incubated at 48C for 30 min, washed, air-dried, stained with sulforhodamine B solution, and washed with 1% acetic acid. Finally, 200 ml of 10 mM Tris base were added to each well and the optical densities were determined at 515 nm utilizing an enzyme-linked immunosorbent assay plate reader. In each case, a zero-day control was performed by adding an equivalent number of cells to several wells and incubating at 378C for 30 min, and processing as described above. Optical density values obtained with the zero-day control were subtracted, and cell survival, relative to control (solvent-treated) cultures, was calculated. 2.3. Evaluation of PB and PC with in vivo hollow fiber tests The in vivo hollow fiber test was performed using a literature procedure with some modifications [23–25]. Confluent monolayers of KB-3, and KB-V1 cells were harvested, collected by centrifugation and resuspended in conditioned medium at a concentration of 5 £ 10 6 cells/ml. Fibers filled with cells were incubated in 6-well plates overnight at 378C in a 5% CO2 atmosphere. Female athymic NCr nu/nu mice at 5–6 weeks of age were obtained from Frederick Cancer Research Facility. Each mouse hosted up to six fibers, which were cultured in two physiological compartments. For intraperitoneal implants, a small incision was made through the skin and musculature of the dorsal abdominal wall, the fiber samples were inserted into the peritoneal cavity in a craniocaudal direction and the incision was closed with skin
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Table 1 MDR-reversing activity of pervilleines A–C and verapamil Compound
KB-3 a
KB-V (2) a,b
KB-V (1) a,b
IC50[KB-V (2)]/IC50 [KB-V (1)]
Pervilleine A Pervilleine B Pervilleine C Verapamil
24 . 35 3.7 37
. 34 15 15 44
0.36 0.17 0.17 0.79
. 95 88 88 56
a Results are expressed as IC50 values (concentration required to inhibit cell growth by 50%) in mM. Results are the means of the two independent experiments, with each concentration tested in triplicate. For additional experimental details, see Section 2. b Incubations were performed in the presence (1) (1 mg/ml) or absence (2) of VLB.
staples. For subcutaneous implants, a small skin incision was made at the nape of the neck to allow insertion of an 11-gauge tumor implant trocar. The trocar, containing the hollow fiber samples, was inserted caudally through the subcutaneous tissues and fibers were deposited during withdrawal of the trocar. The incision was closed with a skin staple. In preliminary studies, cell growth was assessed with fibers containing various cell densities. As a result, a cell density of 5 £ 10 6 cells/ml was found to be suitable for drug studies for KB-3, and KB-V1 cells. For treatment protocols, VLB and VP were dissolved in phosphate-buffered saline (PBS); PB and PC were coprecipitated with polyvinylpyrrolidone [26] to increase solubility, and then dissolved in PBS. Mice were randomized into eight groups (six mice per control group; three mice per treatment group): PBS vehicle control group; VLB treatment group; VP treatment group; PB or PC treatment group; VP plus VLB group; and PB or PC plus VLB group. Test compounds were administrated once daily by intraperitoneal injection from days 3 to 6 after implantation. Body weights were measured daily. On day 7, mice were sacrificed and fibers were retrieved. The fibers were placed into 6-well plates, with each well containing 2 ml of fresh, pre-warmed culture medium and allowed to equilibrate for 30 min at 378C. To define the viable cell mass contained within the intact hollow fibers, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye conversion assay was used. Briefly, 1 ml of pre-
Fig. 2. In vivo hollow fiber studies conducted with KB-3 (Panels A and C) and KB-V1 (Panels B and D) cells implanted at the i.p. compartment of NCr nu/nu mice. Confluent monolayers of KB-3 and KB-V1 cells were harvested, pelleted by centrifugation and resuspended in conditioned medium at a concentration of 5 £ 10 6 cells/ml. Fibers filled with the cells were incubated in 6-well plates overnight at 378C in a 5% CO2 atmosphere, and then inserted into the peritoneal cavity of NCr nu/nu mice in a craniocaudal direction. The incisions were closed with skin staples. The animals were treated with PBS (control), VLB (250 mg/kg), VP (0.136 mmol/kg), PB (0.136 mmol/kg), PC (0.136 mmol/kg), a combination of VLB and VP, a combination of VLB and PB, or a combination of VLB and PC (doses of individual agents in combination regimens were the same as given above). Drugs were administrated once daily by intraperitoneal injection from days 3 to 6 after implantation. On day 7, mice were sacrificed and fibers were retrieved. The effectiveness of the drugs was evaluated on the basis of net growth percentage of the cells determined by MTT assays (Panels A and B). Body weight was determined on days 1 and 7 of the study, and expressed as the difference observed during this time period (Panels C and D). Additional experiment details are provided in Section 2. *The calculated % inhibitions were significantly different from the observed % inhibitions (P , 0:0001) using Student’s t-test (n ¼ 6).
warmed culture medium containing 1 mg MTT/ml was added to each dish. After incubating at 378C for 4 h, the culture medium was aspirated and the samples were washed twice with normal saline containing 2.5% protamine sulfate solution by overnight incubation at 48C. To assess the optical density of the samples, the fibers were transferred to 24-well plates, cut in half and allowed to dry overnight. The formazan was extracted from each sample with DMSO (250 ml/ well) for 4 h at room temperature on a rotation plat-
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Table 2 Calculated and observed growth inhibition of KB-V1 cells implanted at i.p. and s.c. sites Cell line tested
Reversing Agent
Growth inhibition (%) a i.p.
KB-V1
Verapamil Pervilleine B Pervilleine C
s.c.
Calculated inhibition b
Observed inhibition c
Calculated inhibition b
Observed inhibition c
7.4 1 0 ¼ 7.4 7.4 1 2.0 ¼ 9.4 7.4 1 8.5 ¼ 15.9
60.3 (P , 0.0001) 66.8 (P , 0.0001) 77.7 (P , 0.0001)
3.8 1 0 ¼ 3.8 3.8 1 0 ¼ 3.8 3.8 1 0 ¼ 3.8
7.8 (P ¼ 0.73) 1.4 (P ¼ 0.87) 0.2 (P ¼ 0.96)
a Fibers filled with cells were implanted into the intraperitoneal and subcutaneous compartments of host mice. The test compounds were administered once daily by intraperitonal injection from days 3 to 6 after implantation. Fibers were retrieved on day 7. The efficacy of drugs (expressed as % growth inhibition) was determined by quantifying cells using the MTT assay (n ¼ 6). Significance (P) was evaluated using Student’s t-test, (calculated % inhibition versus observed % inhibition, n ¼ 6). For additional experimental details, see Section 2. b Calculated % inhibition is the summation of inhibition noted when VLB (first value) and the reversing agent (second value) were used as single agents. c Observed % inhibition resulting from co-administration of agents as described in Section 2.
form. Aliquots (150 ml) of extracted MTT formazan were transferred to individual wells of 96-well plates and assessed for optical density at a wavelength of 540 nm. The effect of the treatment regimen was determined by the net growth percentage of the cells relative to change in body weight.
3. Results An in vitro cell survival assay was employed as an initial method for monitoring the potential of test compounds to reverse multidrug-resistance. The KB-3 cell line is highly susceptible to VLB (IC50 ¼ 0.04 mM). As summarized in Table 1, neither PB nor VP demonstrated appreciable growth inhibitory potential with KB-3 cells in culture (.35 and 37 mM, respectively), but PC showed non-specific toxicity (IC50 ¼ 3.7 mM). Compared with PA (IC50 ¼ 24 mM), PB is less cytotoxic, but PC is more toxic with KB-3 cells. In the absence of VLB, none of the test substrates significantly inhibited the growth of KB-V1 cells (IC50 values 15, 15, or 44 mM for PB, PC, or VP, respectively). However, when VLB was added to the media in the presence of PB, PC, or VP, chemosensitivity was restored (IC50 ¼ 0.17, 0.17 or 0.79 mM, respectively). Even though PB or PC showed greater growth inhibitory activity (IC50 ¼ 15 mM) than VP (IC50 ¼ 44 mM) in the absence of VLB, the IC50 ratios of KB-V1 (2 VLB)/KB-V1 (1 VLB) were still favorable (88 for PB or PC, and 56 for VP), indicating a
relative lack of non-specific cytotoxicity. With an IC50 value .34 mM in the absence of VLB, 0.36 mM in the presence of VLB, and an IC50 ratio of KB-V1 (2 VLB)/KB-V1 (1 VLB) .95, PA appears somewhat superior to PB and PC, but the effectiveness appears to be of the same order of magnitude. In vivo evaluations were then performed with PB and PC. In preliminary growth assays, it was found that a dose 250 mg/kg of VLB inhibited the growth of KB-3 cells (Fig. 2A) without significantly influencing the growth of KB-V1 cells (less than 7.4% growth inhibition) (Fig. 2B). None of these cell types was sensitive to PB (77.6 mg/kg), PC (81.2 mg/kg), or VP (61.4 mg/kg), at a dose of 0.136 mmol/kg (less than 8.5% growth inhibition) (Fig. 2B). However, when VLB was co-administered with PB, or PC or VP, a significant growth inhibitory effect (P , 0:0001) was observed with KB-V1 (Fig. 2B) implanted at the i.p. site. As summarized in Table 2, when PB, PC or VLB was administered as single agents, growth inhibitory effects of 2.0, 8.5, or 0%, respectively, were observed, but when given together, inhibitory effects of 66.9, 77.7, or 60.3% resulted. In each case, relative to percent inhibition which was calculated as a summation of inhibition noted when the agents were administered singly, enhancements were observed when the agents were co-administered. Thus, since observed inhibitions were greater than those calculated, all agents were effective, and PB and PC showed a stronger effect than VP. In previous studies with PA, when administered as a single agent,
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growth inhibition of 1% was observed, but when given together with VLB, an inhibitory effect of 68.6% resulted [19]. Thus, relative to PA, PB and PC demonstrated similar levels of reversing activity. No significant response was observed with cells implanted at the s.c. site. In all of the cases, no significant loss in mouse body weight was observed (Figs. 2C,D), based on established criteria [25].
4. Discussion Cell culture has proven to be an invaluable tool for the discovery and characterization of agents capable of altering the MDR phenotype. Relative to parental KB-3 cells, KB-V1 cells are .200-fold more resistant to the growth inhibitory effect of VLB [27], and we have employed this as a model for the discovery of various agents capable of reversing MDR [17,18]. As a result of this process, the most interesting natural product MDR reversing agents we have discovered are some new tropane alkaloid aromatic esters [19– 22]. Since PB and PC bear a structural resemblance to VP (Fig. 1), a well-known prototype-reversing agent, comparative studies were performed. In each case, the responses mediated by PB or PC were found to be equal to or greater than the response mediated by VP. Compared with PA, similar effectiveness was observed with PB and PC. Many studies have been performed to define the structural or physicochemical features of reversing agents that might account for their effectiveness [4,28–32]. As noted previously [19], VP, and PB or PC (Fig. 1), as well as PA, bear features shared by other MDR reversing agents, in having a tertiary nitrogen, two aromatic rings, and methoxyphenyl groups. ClogP values and molar refractivity weakly correlated with potential to reverse MDR [22]. Since PB and PC demonstrated promising activities with in vitro studies, we employed the hollow fiber model to further investigate in vivo potential. As currently described, in combination with VLB, the hollow fiber test was used to assess the capacity of VP or PB or PC to effect the growth of multidrugresistant tumor cells growing in the i.p. and s.c. compartments of mice. At pharmacologically relevant doses, the data in Table 2 indicate both VP and PB or PC were capable of reversing VLB sensitivity with
KB-V1 cells implanted at the i.p. site (P , 0:0001), which was consistent with in vitro data (Table 1). Poor responses were observed at the s.c. site (Table 2), possibly due to ineffective drug delivery, or the lack of blood vessel development, since the present hollow fiber assay protocol would not enable angiogeneisis [33]. However, these results indicate the utility of the hollow fiber model for combination drug studies, and the promising results with PB or PC would support more advanced testing with conventional in vivo animal models. A primary question addressed by the current study was the in vivo activity of PB and PC, relative to PA. The compounds bear different substituents at C-3 and C-7, and it is important to understand the potential ramifications of these differences prior to making decisions on future development. As now demonstrated, the potential of each of these agents to reverse multidrug-resistance was similar. Therefore, since each of the alkaloids bear the trans-3,4,5-trimethoxycinnamoyl unit at the C-6 position, this may be the fundamental structural requirement for activity within this compound class, and the substituents at C-3 and C-7 may play less important roles. In sum, since PA, PB and PC are effective inhibitors of Pgp, with comparable or greater activity than VP, it is reasonable to conduct more advanced in vivo tests. The resulting data will be pivotal in contemplating further development as clinically useful pharmaceutical agents.
Acknowledgements The authors are grateful to Dr I.B. Roninson for supplying KB-V1 cells.
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[18] M. You, D.B.M. Wickramaratne, G.L. Silva, H. Chai, T.E. Chagwedera, N.R. Farnsworth, G.A. Cordell, A.D. Kinghorn, J.M. Pezzuto, (2) Roemerine, an aporphine alkaloid from Annona senegalensis that reverses the multidrug-resistance phenotype with cultured cells, J. Nat. Prod. 58 (1995) 598–604. [19] Q. Mi, B. Cui, G.L. Silva, D. Lantvit, E. Lim, H. Chai, M. You, M.G. Hollingshead, J.G. Mayo, A.D. Kinghorn, J.M. Pezzuto, Pervilleine A, a novel tropane alkaloid that reverses the multidrug-resistance phenotype, Cancer Res. 61 (2001) 4030–4037. [20] G.L. Silva, B. Cui, D. Cha´ vez, M. You, H. Chai, P. Rasoanaivo, S.M. Lynn, M.J. O’Neill, J.A. Lewis, J.M. Besterman, A. Monks, N.R. Farnsworth, G.A. Cordell, J.M. Pezzuto, A.D. Kinghorn, Modulation of the multidrug-resistance phenotype by new tropane alkaloid aromatic esters from Erythroxylum pervillei, J. Nat. Prod. 64 (2001) 1514–1520. [21] D. Cha´ vez, B. Cui, H. Chai, R. Garcı´a, M. Mejı´a, N.R. Farnsworth, G.A. Cordell, J.M. Pezzuto, A.D. Kinghorn, Multidrug resistance inhibitory tropane alkaloids from the stems of Erythroxylum rotundifolium, J. Nat. Prod. 65 (2002) 606–610. [22] Q. Mi, B. Cui, D. Cha´ vez, H. Chai, G.A. Cordell, S. Hedayat, H. Zhu, A.D. Kinghorn, J.M. Pezzuto, Characterization of novel tropane alkaloid aromatic esters that reverse the multidrug-resistance phenotype, Anticancer Res. (2002) In press. [23] M. Hollingshead, B. Abbott, J. Mayo, M. Grever, Short-term in vivo cultivation of human tumor cell lines for assessing potential chemotherapeutic agents, Proc. Am. Assoc. Cancer Res. 34 (1993) 429. [24] J.J. Casciari, M.G. Hollingshead, M.C. Alley, J.G. Mayo, L. Malspeis, S. Miyauchi, M.R. Grever, J.N. Weinstein, Growth and chemotherapeutic response of cells in a hollow-fiber in vitro solid tumor model, J. Natl. Cancer Inst. 86 (1994) 1846– 1852. [25] M.G. Hollingshead, M.C. Alley, R.F. Camalier, B.J. Abbott, J.G. Mayo, L. Malspeis, M.R. Grever, In vivo cultivation of tumor cells in hollow fibers, Life Sci. 57 (1995) 131–141. [26] D.P. Waller, L.J.D. Zaneveld, H.H.S. Fong, In vitro spermicidal activity of gossypol, Contraception 22 (1980) 183–187. [27] T. Watanabe, H. Tsuge, T. Oh-Hara, M. Naito, T. Tsuruo, Comparative study on reversal efficacy of SDZ PSC 833, cyclosporin A and verapamil on multidrug resistance in vitro and in vivo, Acta Oncol. 34 (1995) 235–241. [28] W.D. Stein, Kinetics of the multidurg transporter (P-glycoprotein) and its reversal, Physiol. Rev. 77 (1997) 545–590. [29] G. Klopman, L.M. Shi, A. Ramu, Quantitative structure-activity relationship of multidrug resistance reversal agents, Mol. Pharmacol. 52 (1997) 323–334. [30] J.M. Zamora, H.L. Pearce, W.T. Beck, Physical-chemical properties shared by compounds that modulate multidrug resistance in human leukemic cells, Mol. Pharmacol. 33 (1988) 454–462. [31] J.L. Weaver, G. Szabo Jr, P.S. Pine, M.M. Gottesman, S. Goldenberg, A. Aszalos, The effect of ion channel blockers, immunosuppressive agents, and other drugs on the activity of the multi-drug transporter, Int. J. Cancer 54 (1993) 456–461. [32] G. Klopman, S. Srivastava, I. Kolossvary, R.F. Epand, N. Ahmed, R.M. Epand, Structure-activity study and design of
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Pervilleines B and C, new tropane alkaloid aromatic esters that reverse the multidrug-resistance in the hollow fiber assay q Qiuwen Mi a,b, Baoliang Cui a,b,1, Gloria L. Silva a,b,2, Daniel Lantvit a,b, Eula Lim a,b, Heebyung Chai a,b, Melinda G. Hollingshead c, Joseph G. Mayo c, A. Douglas Kinghorn a,b, John M. Pezzuto a,b,* a
Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy (m/c 877), 833 South Wood Street, University of Illinois at Chicago, Chicago, IL 60612, USA b Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy (m/c 781), 833 South Wood Street, University of Illinois at Chicago, Chicago, IL 60612, USA c Biological Testing Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer InstituteFrederick Cancer Research and Development Center, Frederick, MD 21701, USA Received 4 March 2002; accepted 2 April 2002
Abstract P-Glycoprotein (Pgp)-mediated drug efflux can yield a multidrug-resistance phenotype that is associated with poor response to cancer chemotherapy. Pervilleines B and C (PB and PC), two new tropane alkaloid aromatic esters obtained from a chloroform extract of the roots of Erythroxylum pervillei as the result of bioactivity-guided fractionation, were found to restore the vinblastine (VLB) sensitivity of cultured multidrug-resistant KB-V1 cells, with 50% inhibitory concentration values of 0.17 mM in each case. To explore the potential relevance of this response, KB-V1 cells were placed in hollow fibers and implanted into NCr nu/nu mice. Cell growth was not significantly inhibited when VLB or PB or PC were administered as single agents, but when used in combination with vinblastine inhibition of up to 77.7% was observed. Equimolar doses of verapamil were less effective. These data suggest that PB and PC are effective inhibitors of Pgp and should be further evaluated for clinical utility. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pervilleines B and C; Multidrug-resistance; Hollow fiber test; Cancer chemotherapy; MDR1 gene; Verapamil
q Support for this work was provided by U19 CA52956 funded by the National Cancer Institute. * Corresponding author. Tel.: 11-312-996-5967; fax: 11-312-996-2815. E-mail address: [email protected] (J.M. Pezzuto). 1 Present address: PureWorld Botanicals, South Hackensack, NJ 07606, USA. 2 Present address: Departamento de Quı´mica Orga´nica, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, 5000 Co´rdoba, Argentina. Abbreviations: DMEM, Dulbecco’s Modified Eagle medium; DMSO, dimethyl sulfoxide; IC50, 50% inhibitory concentration; MDR, multidrugresistance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PB, pervilleine B [3a-(3,4,5-trimethoxybenzoyloxy)-6b-(E)(3,4,5-trimethoxycinnamoyloxy) tropane]; PC, pervilleine C [3a,6b-di-(E)-(3,4,5-trimethoxycinnamoyloxy)tropane]; PBS, phosphate-buffered saline; Pgp, P-glycoprotein; PVP, polyvinylpyrrolidone; VLB, vinblastine; VP, verapamil
0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(02)00202-1
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Fig. 1. Structures of tropane alkaloid aromatic esters PA, PB and PC isolated from Erythroxylum pervillei, and VP.
1. Introduction One serious problem associated with cancer chemotherapy is the development of multidrug-resistant tumor cells during the course of treatment. An important mechanism of acquiring the multidrugresistance (MDR) phenotype in mammalian cells is the enhanced expression of a membrane glycoprotein, which has been termed P-glycoprotein (Pgp) [1]. With a molecular weight of 170 kDa, Pgp is coded by the MDR1 gene. Pgp functions as an energy-dependent multidrug membrane transporter that rapidly extrudes a variety of hydrophobic antitumor drugs from target cancer cells and thereby prevents the drugs from exerting cytotoxic effects. Several agents are known to overcome, or at least partially circumvent, MDR [2]. Examples are calcium
channel blockers such as verapamil (VP) [3,4], the immunosuppressive agent cyclosporin A [5], analogues of the antihypertensive reserpine and adrenergic blocking agent yohimbine [6], the neuroleptic, trifluroperazine [7], antiestrogens such as tamoxifen [8], a non-immunosuppressive analogue of cyclosporin D, SDZ PSC-833 [5], and others, such as MS-209 [9], S-9788 [10], GF120918 [11], and LY335979 [7]. However, while many of these pharmacological agents have been found to completely overcome drug resistance with in vitro models, reports showing efficacy with mammalian systems are more limited [12–15]. In general, it is difficult to maintain active doses of chemosensitizers without causing serious side effects [16]. Part of our natural product drug discovery program involves monitoring the potential of plant extracts to reverse multiple drug resistance. Standard cell survival assays are used to determine the dose of plant extracts or compounds required to inhibit cell growth by 50% inhibitory concentration (IC50) with drugsensitive human epidermoid carcinoma parental KB3 cells and Pgp-associated multidrug-resistant KB-V1 cells. To investigate the potential of plant extracts or compounds to reverse multidrug-resistance, KB-V1 cells are treated with different concentrations of plant extracts or compounds in the presence (1 mg/ ml) or absence of vinblastine (VLB). This concentration of VLB is lethal to KB-3 cells, but does not affect the growth of KB-V1 cells. Therefore, KB-3 cells serve as a control to differentiate between non-specific cytotoxicity and selective MDR antagonism. This assay employs 96-well microtiter plate technology and over 3000 different plant extracts have been tested. Using this model for bioassay-guided isolation of active principles, we have previously identified four moderate inhibitors of MDR: coronaridine, conoduramine, voacamine, and (2)-roemerine [17,18]. More recently, some extremely potent novel tropane alkaloid aromatic esters were obtained from a chloroform-soluble extract derived from the roots of Erythroxylum pervillei Baillon (Erythroxylaceae) and the stems of Erythroxylum rotundifolium Lanan (Erythroxylaceae) [19–22]. As shown in Fig. 1, these compounds have the general structure of tropane alkaloid 3a,6b-diesters, with the C-6 substituent bearing a trans-3,4,5-trimethoxycinnamoyl unit. Pervilleines A and B (PA and PB) have a 3,4,5-trimethoxybenzoy-
Q. Mi et al. / Cancer Letters 184 (2002) 13–20
loxy substituent at C-3, and a b-hydroxy and methylene substituents at C-7, respectively. Pervilleine C (PC) has a trans-3,4,5-trimethoxycinnamoyl substituent at C-3. Among the isolates, the mechanism of PA has been studied in some detail [19]. PA was found to restore the VLB sensitivity of cultured multidrugresistant KB-V1 and CEM/VLB100 cells, as well as the colchicine sensitivity of cultured KB-8-5 cells. PA competitively inhibited the binding the [ 3H]VLB with isolated KB-V1 cell membrane vesicles, increased the intracellular accumulation of [ 3H]VLB, and reversed the multidrug resistance of KB-V1 and KB-8-5 cells in the hollow fiber model. Starting with crude plant material, PA was obtained in 4.2% w/w yield. The additional isolates, PB and PC, were also obtained in good yield (3.5 and 4.3% w/w, respectively) [20]. Although these compounds bear a close structural resemblance, only PA has been evaluated for activity. Since small structural differences can have profound effects on biological responses, we currently report the activity of PB and PC with the in vivo hollow fiber model.
2. Materials and methods 2.1. Cell lines and agents Human oral epidermoid carcinoma KB-3 were purchased from the American Type Culture Collection (ATCC, Rockville, MD), and KB-V1 cells were supplied by Dr I.B. Roninson (Department of Molecular Genetics, University of Illinois at Chicago, Chicago, IL). KB-3 cells were maintained in Dulbecco’s Modified Eagle medium supplemented with 10% heat-inactivated calf serum and 100 units/ml penicillin G, 100 mg/ml streptomycin sulfate, 250 ng/ml amphotericin B. KB-V1 cells were grown in the same medium, which was further supplemented with VLB (1 mg/ml). Cell culture media and supplements were obtained from Life Technologies, Inc. (Grand Island, NY). The tropane alkaloid aromatic esters PB and PC (Fig. 1) were obtained by bioassay-guided fractionation from extracts derived from the roots of E. pervillei Baillon (Erythroxylaceae) [20–22]. All general chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. Polyvinylidene fluoride hollow fibers (500 000
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Da molecular weight cut-off, 1.0 mm ID) were purchased from Spectrum Medical Industries (Luguan Hills, CA). 2.2. Cytotoxic potential The cytotoxic potential of test substances with KB3 and KB-V1 cells was determined as described previously [18]. Briefly, various concentrations of test compounds (dissolved in 10 ml of 10% dimethyl sulfoxide, DMSO) were transferred to 96-well plates, and 190 ml aliquots of cell suspensions (5 £ 10 4 cells/ ml) were added to each well. The plates were then incubated for 72 h at 378C (100% humidity with a 5% CO2 atmosphere in air), and 100 ml of cold 20% aqueous trichloroacetic acid were added to the growth medium in each well to fix the cells. The cultures were incubated at 48C for 30 min, washed, air-dried, stained with sulforhodamine B solution, and washed with 1% acetic acid. Finally, 200 ml of 10 mM Tris base were added to each well and the optical densities were determined at 515 nm utilizing an enzyme-linked immunosorbent assay plate reader. In each case, a zero-day control was performed by adding an equivalent number of cells to several wells and incubating at 378C for 30 min, and processing as described above. Optical density values obtained with the zero-day control were subtracted, and cell survival, relative to control (solvent-treated) cultures, was calculated. 2.3. Evaluation of PB and PC with in vivo hollow fiber tests The in vivo hollow fiber test was performed using a literature procedure with some modifications [23–25]. Confluent monolayers of KB-3, and KB-V1 cells were harvested, collected by centrifugation and resuspended in conditioned medium at a concentration of 5 £ 10 6 cells/ml. Fibers filled with cells were incubated in 6-well plates overnight at 378C in a 5% CO2 atmosphere. Female athymic NCr nu/nu mice at 5–6 weeks of age were obtained from Frederick Cancer Research Facility. Each mouse hosted up to six fibers, which were cultured in two physiological compartments. For intraperitoneal implants, a small incision was made through the skin and musculature of the dorsal abdominal wall, the fiber samples were inserted into the peritoneal cavity in a craniocaudal direction and the incision was closed with skin
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Table 1 MDR-reversing activity of pervilleines A–C and verapamil Compound
KB-3 a
KB-V (2) a,b
KB-V (1) a,b
IC50[KB-V (2)]/IC50 [KB-V (1)]
Pervilleine A Pervilleine B Pervilleine C Verapamil
24 . 35 3.7 37
. 34 15 15 44
0.36 0.17 0.17 0.79
. 95 88 88 56
a Results are expressed as IC50 values (concentration required to inhibit cell growth by 50%) in mM. Results are the means of the two independent experiments, with each concentration tested in triplicate. For additional experimental details, see Section 2. b Incubations were performed in the presence (1) (1 mg/ml) or absence (2) of VLB.
staples. For subcutaneous implants, a small skin incision was made at the nape of the neck to allow insertion of an 11-gauge tumor implant trocar. The trocar, containing the hollow fiber samples, was inserted caudally through the subcutaneous tissues and fibers were deposited during withdrawal of the trocar. The incision was closed with a skin staple. In preliminary studies, cell growth was assessed with fibers containing various cell densities. As a result, a cell density of 5 £ 10 6 cells/ml was found to be suitable for drug studies for KB-3, and KB-V1 cells. For treatment protocols, VLB and VP were dissolved in phosphate-buffered saline (PBS); PB and PC were coprecipitated with polyvinylpyrrolidone [26] to increase solubility, and then dissolved in PBS. Mice were randomized into eight groups (six mice per control group; three mice per treatment group): PBS vehicle control group; VLB treatment group; VP treatment group; PB or PC treatment group; VP plus VLB group; and PB or PC plus VLB group. Test compounds were administrated once daily by intraperitoneal injection from days 3 to 6 after implantation. Body weights were measured daily. On day 7, mice were sacrificed and fibers were retrieved. The fibers were placed into 6-well plates, with each well containing 2 ml of fresh, pre-warmed culture medium and allowed to equilibrate for 30 min at 378C. To define the viable cell mass contained within the intact hollow fibers, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye conversion assay was used. Briefly, 1 ml of pre-
Fig. 2. In vivo hollow fiber studies conducted with KB-3 (Panels A and C) and KB-V1 (Panels B and D) cells implanted at the i.p. compartment of NCr nu/nu mice. Confluent monolayers of KB-3 and KB-V1 cells were harvested, pelleted by centrifugation and resuspended in conditioned medium at a concentration of 5 £ 10 6 cells/ml. Fibers filled with the cells were incubated in 6-well plates overnight at 378C in a 5% CO2 atmosphere, and then inserted into the peritoneal cavity of NCr nu/nu mice in a craniocaudal direction. The incisions were closed with skin staples. The animals were treated with PBS (control), VLB (250 mg/kg), VP (0.136 mmol/kg), PB (0.136 mmol/kg), PC (0.136 mmol/kg), a combination of VLB and VP, a combination of VLB and PB, or a combination of VLB and PC (doses of individual agents in combination regimens were the same as given above). Drugs were administrated once daily by intraperitoneal injection from days 3 to 6 after implantation. On day 7, mice were sacrificed and fibers were retrieved. The effectiveness of the drugs was evaluated on the basis of net growth percentage of the cells determined by MTT assays (Panels A and B). Body weight was determined on days 1 and 7 of the study, and expressed as the difference observed during this time period (Panels C and D). Additional experiment details are provided in Section 2. *The calculated % inhibitions were significantly different from the observed % inhibitions (P , 0:0001) using Student’s t-test (n ¼ 6).
warmed culture medium containing 1 mg MTT/ml was added to each dish. After incubating at 378C for 4 h, the culture medium was aspirated and the samples were washed twice with normal saline containing 2.5% protamine sulfate solution by overnight incubation at 48C. To assess the optical density of the samples, the fibers were transferred to 24-well plates, cut in half and allowed to dry overnight. The formazan was extracted from each sample with DMSO (250 ml/ well) for 4 h at room temperature on a rotation plat-
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Table 2 Calculated and observed growth inhibition of KB-V1 cells implanted at i.p. and s.c. sites Cell line tested
Reversing Agent
Growth inhibition (%) a i.p.
KB-V1
Verapamil Pervilleine B Pervilleine C
s.c.
Calculated inhibition b
Observed inhibition c
Calculated inhibition b
Observed inhibition c
7.4 1 0 ¼ 7.4 7.4 1 2.0 ¼ 9.4 7.4 1 8.5 ¼ 15.9
60.3 (P , 0.0001) 66.8 (P , 0.0001) 77.7 (P , 0.0001)
3.8 1 0 ¼ 3.8 3.8 1 0 ¼ 3.8 3.8 1 0 ¼ 3.8
7.8 (P ¼ 0.73) 1.4 (P ¼ 0.87) 0.2 (P ¼ 0.96)
a Fibers filled with cells were implanted into the intraperitoneal and subcutaneous compartments of host mice. The test compounds were administered once daily by intraperitonal injection from days 3 to 6 after implantation. Fibers were retrieved on day 7. The efficacy of drugs (expressed as % growth inhibition) was determined by quantifying cells using the MTT assay (n ¼ 6). Significance (P) was evaluated using Student’s t-test, (calculated % inhibition versus observed % inhibition, n ¼ 6). For additional experimental details, see Section 2. b Calculated % inhibition is the summation of inhibition noted when VLB (first value) and the reversing agent (second value) were used as single agents. c Observed % inhibition resulting from co-administration of agents as described in Section 2.
form. Aliquots (150 ml) of extracted MTT formazan were transferred to individual wells of 96-well plates and assessed for optical density at a wavelength of 540 nm. The effect of the treatment regimen was determined by the net growth percentage of the cells relative to change in body weight.
3. Results An in vitro cell survival assay was employed as an initial method for monitoring the potential of test compounds to reverse multidrug-resistance. The KB-3 cell line is highly susceptible to VLB (IC50 ¼ 0.04 mM). As summarized in Table 1, neither PB nor VP demonstrated appreciable growth inhibitory potential with KB-3 cells in culture (.35 and 37 mM, respectively), but PC showed non-specific toxicity (IC50 ¼ 3.7 mM). Compared with PA (IC50 ¼ 24 mM), PB is less cytotoxic, but PC is more toxic with KB-3 cells. In the absence of VLB, none of the test substrates significantly inhibited the growth of KB-V1 cells (IC50 values 15, 15, or 44 mM for PB, PC, or VP, respectively). However, when VLB was added to the media in the presence of PB, PC, or VP, chemosensitivity was restored (IC50 ¼ 0.17, 0.17 or 0.79 mM, respectively). Even though PB or PC showed greater growth inhibitory activity (IC50 ¼ 15 mM) than VP (IC50 ¼ 44 mM) in the absence of VLB, the IC50 ratios of KB-V1 (2 VLB)/KB-V1 (1 VLB) were still favorable (88 for PB or PC, and 56 for VP), indicating a
relative lack of non-specific cytotoxicity. With an IC50 value .34 mM in the absence of VLB, 0.36 mM in the presence of VLB, and an IC50 ratio of KB-V1 (2 VLB)/KB-V1 (1 VLB) .95, PA appears somewhat superior to PB and PC, but the effectiveness appears to be of the same order of magnitude. In vivo evaluations were then performed with PB and PC. In preliminary growth assays, it was found that a dose 250 mg/kg of VLB inhibited the growth of KB-3 cells (Fig. 2A) without significantly influencing the growth of KB-V1 cells (less than 7.4% growth inhibition) (Fig. 2B). None of these cell types was sensitive to PB (77.6 mg/kg), PC (81.2 mg/kg), or VP (61.4 mg/kg), at a dose of 0.136 mmol/kg (less than 8.5% growth inhibition) (Fig. 2B). However, when VLB was co-administered with PB, or PC or VP, a significant growth inhibitory effect (P , 0:0001) was observed with KB-V1 (Fig. 2B) implanted at the i.p. site. As summarized in Table 2, when PB, PC or VLB was administered as single agents, growth inhibitory effects of 2.0, 8.5, or 0%, respectively, were observed, but when given together, inhibitory effects of 66.9, 77.7, or 60.3% resulted. In each case, relative to percent inhibition which was calculated as a summation of inhibition noted when the agents were administered singly, enhancements were observed when the agents were co-administered. Thus, since observed inhibitions were greater than those calculated, all agents were effective, and PB and PC showed a stronger effect than VP. In previous studies with PA, when administered as a single agent,
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growth inhibition of 1% was observed, but when given together with VLB, an inhibitory effect of 68.6% resulted [19]. Thus, relative to PA, PB and PC demonstrated similar levels of reversing activity. No significant response was observed with cells implanted at the s.c. site. In all of the cases, no significant loss in mouse body weight was observed (Figs. 2C,D), based on established criteria [25].
4. Discussion Cell culture has proven to be an invaluable tool for the discovery and characterization of agents capable of altering the MDR phenotype. Relative to parental KB-3 cells, KB-V1 cells are .200-fold more resistant to the growth inhibitory effect of VLB [27], and we have employed this as a model for the discovery of various agents capable of reversing MDR [17,18]. As a result of this process, the most interesting natural product MDR reversing agents we have discovered are some new tropane alkaloid aromatic esters [19– 22]. Since PB and PC bear a structural resemblance to VP (Fig. 1), a well-known prototype-reversing agent, comparative studies were performed. In each case, the responses mediated by PB or PC were found to be equal to or greater than the response mediated by VP. Compared with PA, similar effectiveness was observed with PB and PC. Many studies have been performed to define the structural or physicochemical features of reversing agents that might account for their effectiveness [4,28–32]. As noted previously [19], VP, and PB or PC (Fig. 1), as well as PA, bear features shared by other MDR reversing agents, in having a tertiary nitrogen, two aromatic rings, and methoxyphenyl groups. ClogP values and molar refractivity weakly correlated with potential to reverse MDR [22]. Since PB and PC demonstrated promising activities with in vitro studies, we employed the hollow fiber model to further investigate in vivo potential. As currently described, in combination with VLB, the hollow fiber test was used to assess the capacity of VP or PB or PC to effect the growth of multidrugresistant tumor cells growing in the i.p. and s.c. compartments of mice. At pharmacologically relevant doses, the data in Table 2 indicate both VP and PB or PC were capable of reversing VLB sensitivity with
KB-V1 cells implanted at the i.p. site (P , 0:0001), which was consistent with in vitro data (Table 1). Poor responses were observed at the s.c. site (Table 2), possibly due to ineffective drug delivery, or the lack of blood vessel development, since the present hollow fiber assay protocol would not enable angiogeneisis [33]. However, these results indicate the utility of the hollow fiber model for combination drug studies, and the promising results with PB or PC would support more advanced testing with conventional in vivo animal models. A primary question addressed by the current study was the in vivo activity of PB and PC, relative to PA. The compounds bear different substituents at C-3 and C-7, and it is important to understand the potential ramifications of these differences prior to making decisions on future development. As now demonstrated, the potential of each of these agents to reverse multidrug-resistance was similar. Therefore, since each of the alkaloids bear the trans-3,4,5-trimethoxycinnamoyl unit at the C-6 position, this may be the fundamental structural requirement for activity within this compound class, and the substituents at C-3 and C-7 may play less important roles. In sum, since PA, PB and PC are effective inhibitors of Pgp, with comparable or greater activity than VP, it is reasonable to conduct more advanced in vivo tests. The resulting data will be pivotal in contemplating further development as clinically useful pharmaceutical agents.
Acknowledgements The authors are grateful to Dr I.B. Roninson for supplying KB-V1 cells.
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