Discodermolide and Taxol: A Synergistic Drug Combination in Human

Chapter 5 Discodermolide

and

Taxol:

A Synergistic

Drug

Downloaded by UNIV OF GUELPH LIBRARY on September 11, 2012 | http://pubs.acs.org Publication Date: August 24, 2001 | doi: 10.1021/bk-2001-0796.ch005

Combination in Human Carcinoma Cell Lines

1

1

1

Susan Band Horwitz , Laura A. Martello , Chia-Ping H. Y a n g , Amos B. Smith, III , and Hayley M. McDaid 2

1

1

Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104 2

New natural products with Taxol-like activities have been identified during a search for compounds with the same mechanism of action as Taxol, but with better therapeutic properties. The epothilones, eleutherobin and discodermolide, like Taxol, all enhance the polymerization of stable microtubules. Careful analyses of these compounds have indicated that Taxol and discodermolide have differential effects in cells. The presence of low concentrations of Taxol significantly increased the cytotoxicity of discodermolide. Median effect analysis, using the combination index method, revealed a schedule-independent synergistic interaction between Taxol and discodermolide in human carcinoma cell lines, suggesting that these two drugs could represent an important drug combination in the treatment of cancer.

© 2001 American Chemical Society In Anticancer Agents; Ojima, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

81

82


Introduction Taxol is an effective antitumor drug that has been approved by the FDA for the treatment of ovarian, breast and lung carcinomas ( 1 ) and is under evaluation for the treatment of Kaposi's sarcoma (2). It is being used both as a single agent and in combination chemotherapy. The microtubule polymer is the cellular target for Taxol, specifically the β-tubulin subunit with which Taxol interacts (3). The drug binds specifically, but not covalently, to microtubules with a stoichiometry approaching one mole of Taxol per mole of tubulin heterodimer (4,5). Two hallmarks of this antimitotic agent are (i) the enhancement of assembly of microtubules, even in the absence of GTP that is normally required for in vitro tubulin assembly (6), and (ii) the formation of stable bundles or parallel arrays of microtubules that results from a reorganization of the microtubule cytoskeleton (7). The normal assembly/disassembly dynamics of microtubules are disrupted by Taxol and cells are arrested at metaphase. This perturbation of normal tubulin kinetics results in cell death. High Taxol concentrations result in sustained mitotic arrest, while low Taxol concentrations which inhibit microtubule dynamics also cause cell death in the absence of an apparent mitotic block. Low concentrations of Taxol have a major effect on microtubule dynamics, inhibiting this process even at nM concentrations of drug (8-10). Extensive photoaffinity labeling studies have been done with Taxol analogs bearing photoreactive groups at the C-2, C-3 or C-7 positions to identify the sites of interaction between Taxol and β-tubulin (11-13). In an early study (3), we demonstrated by direct photolabeling, using radiolabeled Taxol, that the drug interacted specifically with β-tubulin. Further studies with [ H]3'-(pazidobenzamido)Taxol (where the arylazide was incorporated into the C-13 side chain) resulted in the isolation of a photolabeled peptide containing amino acid residues 1-31 of β-tubulin (11). Studies with [ H]2-(m-azidobenzoyl)Taxol (where the photoreactive group is attached to the Β ring of the taxoid nucleus) have shown that a peptide containing amino acid residues 217-233 of β-tubulin also is involved in Taxol binding (12). More recently we have utilized a photoreactive Taxol analog containing a benzoyldihydrocinnamoyl (BzDC) substituent at the C-7 position of the drug (13) that allowed the determination of the single amino acid in tubulin which interacts with Taxol, a limitation with arylazide containing Taxol analogs. Studies done with the BzDC analog indicated that it photo-crosslinked to Arg of β-tubulin. The information gained from our photoaffinity labeling studies and from electron crystallographic studies done by Nogales and colleagues (14) has allowed the visualization of the binding pocket for Taxol in β-tubulin. f

3

3

282

The success of Taxol in the clinic plus the desire to improve on the properties of the drug, have prompted the search for new natural products with Taxol-like

In Anticancer Agents; Ojima, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.


83 activity. It would be advantageous to have Taxol-like compounds with a broad range of tumor efficacy, with improved aqueous solubility, and most importantly, the ability to retain potency in Taxol-resistant tumors. In the past five years, at least three new natural products, the epothilones, (15,16) eleutherobin (17,18), and discodermolide (19-21) have been isolated, each from distinct natural sources, a bacterium, marine coral and sponge, respectively. These drugs share the same mechanism of action as Taxol, although the structures of each of the four compounds is unique (Fig. 1). One focus of our laboratory is to delineate the mechanistic similarities and differences between these molecules, with particular emphasis on Taxol and discodermolide.

In vitro Polymerization of Microtubules and Bundle Formation An in vitro assay has been used extensively to monitor the polymerization of soluble tubulin dimers to form the microtubule polymer. This assay requires the presence of purified bovine brain tubulin, GTP and MES buffer and is carried out at 37°C. The turbidity or change in absorbance (light scattering) at 350 nm correlates with the initiation and elongation of microtubules (22) and is routinely confirmed by electron microscopy. Normal microtubules assembled in the presence of GTP depolymerize in the presence of mM concentrations of Ca"\ Since all of the small molecules used in this assay are hydrophobic and are solubilized in DMSO, the control also contains DMSO. The four drugs that were compared all share the same ability as Taxol to stabilize microtubules. Each was assayed at 10 μΜ and shown to assemble tubulin, in the absence of GTP, into stable microtubules that are not depolymerized by Ca or cold temperatures (Fig. 2). The inset depicts the first two minutes of polymerization, from which it is clear that epothilone B, and particularly discodermolide, have an initial slope that is extremely steep, exceeding that of Taxol. This indicates that these two drugs have a major effect on the initiation of tubulin polymerization. The microtubules formed in the presence of discodermolide are distinctly shorter than normal microtubules or even Taxol-induced microtubules (23), emphasizing the effect that this drug has on initiation of microtubule polymerization. ++

Although both Taxol and discodermolide enhance the assembly of stable microtubules, there are distinct differences in the way the two drugs reorganize the microtubule cytoskeleton. As depicted in Figure 3, both drugs enhance the formation of microtubule bundles, but with unique morphologic characteristics. Taxol-treated cells have microtubule bundles, often centered around the nucleus but also localized throughout the cells, whereas discodermolide treatment induces short bundles, predominantly at the periphery of the cells. The latter are



Figuré 1.

Epothilone A (R=H) Epothilone Β (R=Me)

Chemical structures of Taxol, epothilone A and B, eleutherobin, and discodermolide. (Reproduced with permission from reference 21).

Ph


85

0.145 ε


c ο

ΙΟ CO

φ ο c

0.095 Η

CB .Ω λΟ W

5000 >609.8

Eleu

50

Ratio of IC for S K V L B resistant cell line to that for S K O V 3 sensitive cell line.

50>

IC drug concentration that inhibits cell division by 5 0 % after 6 days. V B L , vinblastine; EpoA, epothilone A ; EpoB, epothilone B; Disco, discodermolide; Eleu, eleutherobin. Mean ± S E .

1.1 ±0.1 1880.3 ±347.5 1709.4

VBL

50

American Association for Cancer Research.

SOURCE: Reproduced with permissionfromreference 2 1 . Copyright 2000

d

c

b

a

d

2.0 ±0.1 13333 ±1154 6666.5

SKOV3 SKVLB Fold resistant c

Taxol

Cell lines

"C

cell line that overexpresses P-glycoprotein

Table I: Cytotoxicity of antimitotic agents in a drug-resistant



d

50

e

c

9.4

2±0.14 18.7 ± 0 . 9

Taxol

5.3

6.4 ± 2.0 34 ± 3 . 1

EpoA

b

Eleu

a

4.7

4.9

14 ± 2 . 8 0.7 ±0.1 3.3 ± 0.72 69 ± 11.3

EpoB

(nM)

0.8

8.1 ± 0 . 1 4 6.5 ± 2.4

Disco

1.0

1.8 ± 0 . 2 1 1.8 ± 0 . 1 8

VBL

1.0

33 ± 10.6 33 ± 12.7

CLC

American Association for Cancer Research.

e

d

c

b

I C , drug concentration that inhibits cell division by 5 0 % after 72 h. E p o A , epothilone A ; E p o B , epothilone B; Eleu, eleutherobin; Disco, discodermolide; V B L , vinblastine; C L C , colchicine. Mean ± SE. Cells were maintained in 2 nM Taxol during cross resistance experiments. Ratio of IC 5 0 for A549-T12 resistant cell line to that for A 5 4 9 sensitive cell line. SOURCE: Reproduced with permission from reference 2 1 . Copyright 2000

a

Fold resistant

A549 A549-T12

Cell lines

50

"c

Table II: Cytotoxicity of antimitotic agents in a Taxol-resistant cell line that does not express P-glycoprotein


90


epothilones, eleutherobin and discodermolide to substitute for Taxol in sustaining the growth of A549-T12 cells was evaluated and it was determined that only discodermolide could not substitute (21). This result further substantiated our other findings, suggesting a distinct difference between Taxol and discodermolide. Due to their requirement for Taxol, the experiments to determine cytotoxicity and cross resistant profiles in A549-T12 cells were carried out in the presence of 2 nM Taxol. In the absence of 2nM Taxol, it was found that the A549-T12 cells were approximately 20-fold less sensitive to discodermolide. Over a range of discodermolide concentrations, maximum cytotoxicity was observed in the presence of 2 nM Taxol (Fig. 4). In contrast, the presence of Taxol did not significantly potentiate the cytotoxicities of the epothilones or eleutherobin (21). These observations led us to the idea that there may be an interaction between Taxol and discodermolide.

Taxol and Discodermolide are a Synergistic Drug Combination Median effect analysis using the combination index (CI) method of Chou and Talalay (27) was used to analyze the nature of the interaction, if any, between Taxol and discodermolide. This method resolves the degree of synergy, additivity or antagonism at various levels of cytotoxicity. The type of interaction is expressed as a combination index where l=additivity, >l=antagonism and + CO

κ-

Figure 4.

> + CO h-

=0.0005)***

0.25

A549

0.00

0.50 FA

0.75 Mean ON0.273 (P=0.0007)*** Median CI=0.252 (PO.0001)***

0.25

Taxol and discodermolide are a synergistic drug combination in human carcinoma cell lines. Cells were incubated with different drug concentrations at their equipotent ratios for 72 h after which cell counts were determined. Data points represent the mean CI values, based on the mutually nonexclusive assumption, ± SE from at least three independent experiments. Probabilities (P) indicate the level of significance of the mean and median CI values compared to a CI=1. (Reproduced with permission from reference 21).

1.00

MCF-7


1.00


0.00

1

2

3

FA

0.50

— ι —

Mean CI=1.21 (PO.0001)*** Median CI=1.12 (P=0.0005)***

0.25 0.75

Figure 6. Continued.

1.00

0.00

5-

5-

: m j IIII111II11 imj

6-

6-

4

87-

7-

9-

9-

8-

10-,

10-

FA

0.50

Mean CI=2.37 (P=0.045)* Median CI=1.62 (PO.0001)*

0.25


95

Taxol U

1 10 15 20

Discodermolide Combination 0/

5 50 75 100 1/5 10/5015/75 2 l00 Drug Concentration (nM) Parp

24 kDa


Taxol & ο

1

10 20

Discodermolide Combination 5

50 100

1/5 10/50 20/100 Drug Concentration (nM) Bcl-Xy

26kDa Figure 7.

Potentiation of apoptosis by Taxol and discodermolide. A549 cells were incubated with Taxol, discodermolide, or both drugs at their equipotent ratios for 18 h and cell lysates prepared. Proteins were electrophoresed, blotted onto nitrocellulose, and subjected to immunodetection by specific antibodies.

Classically one would not expect a synergistic interaction between two drugs with a similar mechanism of action. Most cancer chemotherapeutic regimens include drugs with distinct mechanisms of action and toxicities. The results reported in this paper indicate that one must be open to new strategies when developing combination therapies. This potentially interesting drug combination must now be explored in animals.

References 1. 2.

Rowinsky,E.KAnnu Rev Med. 1997, 48, 353-74. Gill, P.S.; Tulpule, Α.; Espina, B.M.; Cabriales,S.;Bresnahan,J.;Ilaw,M.; Louie, S.; Gustafson, N.F.; Brown, M.A.; Orcutt, C.; Winograd, B.; Scadden, D.T. J Clin Oncol. 1999,17,1876-83. 3. Rao, S.; Horwitz,S.B.;Ringel, I. J. Natl. Cancer Inst. 1992, 84, 785-788. 4. Parness,J.;Horwitz, S.B. J. Cell Biol. 1981, 91, 479-487. 5. Diaz,J.F.;Andreu, J.M. Biochemistry 1993, 32, 2747-2755. 6. Schiff, P.B.; Fant,J.;Horwitz,S.B.Nature(Lond.), 1979, 277, 665-667. 7. Schiff, P.B.; Horwitz, S.B. Proc. Natl. Acad. Sci. USA, 1980, 77, 15611565. 8. Jordan, M.A.; Toso, R.J.; Thrower, D; Wilson, L. Proc. Natl. Acad. Sci. USA, 1993, 90, 9552-9556. 9. Jordan,M.A.;Wendell,K.;Gardiner, S.; Derry, W.B.; Copp, H.; Wilson, L. Cancer Res., 1996, 56, 816-825. 10. Torres, K; Horwitz, S.B. Cancer Res., 1998, 58, 3620-2626. In Anticancer Agents; Ojima, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.


96

K.

S.B.

11. Rao,S.;Krauss,N.E.;Heerding,J.M.;Swindell,C.S.;Ringel, I.; Orr, G.A.; Horwitz,S.B.J.Biol.Chem.,1994, 69, 3132-3134. 12. Rao,S.;Orr,G.A.;Chaudhary, A.G.; Kingston, D.G.I.; Horwitz, S.B. J. Biol.Chem.,1995, 270, 20235-20238. 13. Rao,S.;He,L.;Chakravarty,S.;Ojima, I.; Orr,G.A.;Horwitz,S.B.J.Biol. Chem., 1999, 274, 37990-37994. 14. Nogales,E.;Wolf,S.G.;Downing,K.H.Nature, 1998, 391, 199-203. 15. Bollag,D.M.;McQueney,P.A.;Zhu,J.;Hensens, O.; Koupal, L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods,C.M.Cancer Res., 1995, 55, 23252333. 16. Su,D-S.;Balog, Α.; Meng, D.; Bertinato, P.; Danishefsky,S.J.;Zheng, Y -H.; Chou,T-C.;He,L.;Horwitz, S.B. Chem. Int. Ed. Engl. 1997, 36, 20932096, 17. Lindel, T.; Jensen,P.R.;Fenical,W.;Long,B.H.;Casazza,A.M.;Carboni, J.; Fairchild,C.R.J.AM. Chem.Soc.,1997, 119, 8744-8745. 18. McDaid, H.M.; Bhattacharya, S.K.; Chen, X-T.; He, L.; Shen, H-J.; Gutteridge, C.E.; Horwitz, S.B.; Danishefsky, S.J. Cancer Chemother. Pharmacol., 1999, 44, 131-137. 19. Ter Haar, E.; Kowalski, R.J.; Hamel, E.; Lin,C.M.;Longley, R.E.; Gunasekera, S.P.; Rosenkranz, H.S.; Day,B.W.Biochemistry, 1996, 35, 243-250. 20. Hung,D.T.;Chen,J.;Schreiber,S.L.Chem. Biol., 1996, 3, 287-293. 21. Martello,L.A.;McDaid,H.M.;Regl,D.L.;Yang, C-P.H.; Meng,D.;Pettus, T.R.R.; Kaufman, M.D.; Arimoto, H.; Danishefsky, S.J.; Smith, A.B.; Horwitz,S.B.Clin. Cancer Res. 2000, 6, 1978-1987. 22. Gaskin,F.;Cantor,C.R.;Shelanski,M.L.J.Mol.Biol., 1974, 89, 737-758. 23. Kowalsiki, R.J.; Giannakakou, P.; Gunasekera, S.P.; Longley,R.E.;Day, B.W.; Hamel, E. Mol. Pharm., 1997, 52, 612-622. 24. Lee, F.Y.F.; Vite,G.D.;Borzilleri,R.M.;Arico,M.A.;Clark, J.L.;, Fager, L.; Kan, D.; Kennedy,K.A.;Kim, A.S-H.; Smykla,R.A.;Wen, M.-L., Kramer, R.A. Proceedings of the 11th NCI-EORTC-AACR 2000, Symposium on New Drugs in Cancer Therapy section 573, 2000. 25. Giannakakou, P.; Gussio,R.;Nogales, E.; Downing,K.H.;Zaharevitz, D.; Bollbuck, B.; Poy,G.;Sackett, D.; Nicolaou,K.C.;& Fojo, T. Proc. Natl. Acad. Sci. USA 2000, 97, 2904 - 2909. 26. Reversal of multidrug-resistance in tumor cells. In: Chemotherapeutic Synergism and Antagonism. Yang, C.-P,H; Greenberger,L.M;and Horwitz, D. Rideout andT.-C.Chou, Eds.; Academic Press, N.Y. 1991; p. 311338. 27. Chou, T.-C.; Talalay,P.;Adv. EnzymeRegul.1984, 22, 27-55.


Discodermolide and Taxol: A Synergistic Drug Combination in Human

Recommend Documents