William T. Beck

Photoaffinity substrates for P-glycoprotein

A variety of compounds can inhibit the function of P-glycoprotein (Pgp) by binding to it and preventing the efflux of anticancer drug substrates. While the molecular architecture of the drug binding site(s) in Pgp is not known, it is clear that modulators in general appear to conform to some general physical-chemical rules. In this paper, we discuss the basic concepts of drug recognition by Pgp as currently understood. We also examine the compounds used to photoaffinity label this protein and discuss their utility in identifying drug binding sites. Finally, we show that a photoaffinity analog of daunorubicin, [3H]azidobenzoyl-daunorubicin ([3H]AB-DNR), is a good affinity labeling reagent for Pgp. A finding of interest is that vinblastine and verapamil compete more effectively than daunorubicin for [3H]AB-DNR binding to Pgp, suggesting that vinblastine and verapamil have similar structural features not shared by daunorubicin.

Effects of indole alkaloids on multidrug resistance and labeling of P-glycoprotein by a photoaffinity analog of vinblastine.

Multidrug resistant cells are characterized by decreased drug accumulation and retention, thought to be mediated by a high molecular weight glycoprotein, P-glycoprotein (P-gp). Agents such as verapamil have been shown to increase anticancer drug cytotoxicity and increase the amount of drug accumulated and retained by such cells. We show here that in addition to verapamil, reserpine, chloroquine, quinine, quinacrine, yohimbine, vindoline, and catharanthine also enhance the cytotoxicity of vinblastine (VLB) in a multidrug resistant, human leukemic cell line, CEM/VLB1K, described here for the first time. These cells express P-gp as a doublet that is photoaffinity labeled by the analog of VLB, N(p-azido-[3-125I]salicyl)-N-beta-aminoethylvindesine ([125I]NASV). Both reserpine and, to a lesser extent, verapamil, compete with [125I]NASV for binding to P-gp. We also found that chloroquine, quinacrine, vindoline, and catharanthine, each of which enhanced VLB cytotoxicity in CEM/VLB1K cells by 10- to 15-fold, similarly inhibited [125I]NASV labeling of P-gp. However, neither quinine nor yohimbine inhibited this labeling, and the inhibition produced by catharanthine and vindoline was the greatest or exclusively on the lower band of the P-gp doublet. Our results suggest a complex relationship between the ability of a compound to modulate MDR and its ability to compete for binding to P-gp.

Chloroquine enhancement of anticancer drug cytotoxicity in multiple drug resistant human leukemic cells

Vinblastine-sensitive (CCRF-CEM) and -resistant (CEM/VLB100) human T-cell lymphoblasts were treated with the lysosomotropic agent chloroquine. As measured by growth inhibition, this drug enhanced the cytotoxicity of vinblastine in the CEM/VLB100 cells but was less effective in the CCRF-CEM cells. Chloroquine also enhanced the cytotoxic activity of vincristine, daunorubicin and doxorubicin and, to a lesser extent, teniposide (VM-26) in the CEM/VLB100 cells. Histological examination revealed that the vinblastine-resistant cells contained more cytoplasmic vacuoles than their drug-sensitive counter-parts. When the CEM/VLB100 cells were treated with chloroquine, vinblatine, or a combination of the two, the cells displayed many more cytoplasmic vacuoles than the controls. Coincident with the increased number of vacuoles, these treated cells stained more intensely than controls for the lysosomal enzyme, acid phosphatase, but not for lipid. The vacuolization did not increase as much in the CCRF-CEM cell line when these cells were exposed to the chloroquine + vinblastine combination. Vacuolization was also associated with vincristine, doxorubicin, and daunorubicin treatments, but not with VM-26. We conclude that chloroquine is a modulator of anticancer drug action in the CEM/VLB100 cell line.

Structural characteristics of compounds that modulate P-glycoprotein-associated multidrug resistance

Multidrug resistance is mediated by a membrane-bound protein, P-gp, that functions as an energy dependent efflux system to reduce the intracellular concentration of anticancer drugs by binding to these drugs and actively exporting them from the cell. Compounds that interact with P-gp and compete with anticancer drug binding modulate the degree of drug resistance and therefore enhance the cytotoxicity of anticancer drugs against the resistant cell. Effective modulators share certain physical and chemical properties including octanol/water partitioning and molecular size, but the physical properties of size and shape seem to correlate best with modulator effectiveness. Using a photoactivatable analog of vinblastine as a probe, together with a semi-synthetic series of structurally homologous reserpine and yohimbine analogs, the need for two planar aromatic domains and a basic nitrogen atom was established within the structural context of these compounds. The use of three-dimensional comparisons was extended to examine important structural features in other modulator types such as the condensed-ring aromatics. This approach indicates that structural similarities between different classes of compounds are present in compounds recognized by the MDR phenotype. These studies emphasize the importance of a ligand-receptor relationship for modulators of MDR, and begin to define the P-gp-binding pharmacophore. It is likely that this approach will be useful in directing the de novo synthesis of compounds that modulate MDR and help to further define the requirements for molecular recognition by this system.

Mechanisms of multidrug resistance in human tumor cells. The roles of P-glycoprotein, DNA topoisomerase II, and other factors

Multidrug resistance (MDR) associated with overexpression of P-glycoprotein (Pgp) is a well-described experimental phenomenon that appears to have clinical correlates. However, recent descriptions of non-P-glycoprotein forms of MDR have complicated efforts to detect and circumvent MDR in the tumors of patients. One major form of natural product MDR appears to be due to alterations in the amount of activity of DNA topoisomerase II. Compared to Pgp-MDR cells, cells expressing this form of MDR (at-MDR) do not overexpress the mdr1 gene or its product, Pgp, are unaltered in drug accumulation and retention, are unaffected by such modulators of Pgp-MDR as verapamil, and express this phenotype recessively. Recently, other MDR cell lines have been described with some characteristics of Pgp-MDR (decreased drug accumulation and retention, increased drug cytotoxicity by modulators of MDR), but not others (no expression of the mdr1 gene or Pgp). Whether any non-Pgp forms of MDR occur in patients tumors remains to be determined.

Drug resistance associated with altered DNA topoisomerase II

Abstract OLX-209 has readily measurable activity, is safe in experimental animals, and is efficacious in model systems. These results support the concept of OLX-209 and provide groundwork for further development of this oncoprotein targeted agent.

Cellular pharmacology of vinca alkaloid resistance and its circumvention

Vinca alkaloid-resistant human leukemic cells express the multiple drug-resistant phenotype, characterized by cross resistance to natural product compounds of unrelated structure and action. While there are also distinct biochemical lesions associated with this phenotype, their role(s) in the expressions of resistance is not known at this time. More clear, however, is our understanding of the pharmacologic determinants of this resistance--apparent decreased drug uptake and decreased drug retention. This latter phenomenon has been attributed to the workings of an active efflux pump, but data presented here and elsewhere permit an alternative explanation--that of altered drug binding to an as yet unidentified target(s). Elucidation of the mechanism of multiple drug resistance is important in the design of new chemotherapeutic strategies to overcome it. In this regard, calcium channel blocking agents and calmodulin inhibitors can cause an apparent reversal of resistance by enhancing the cytotoxic effectiveness of the anticancer drugs, possibly by increasing the amount of drug retained by the tumor cells. The basis for this enhanced retention and cytotoxicity is not presently known, but it may be related to cellular calcium fluxes, calmodulin content or membrane fluidity and permeability. The meaning of these findings is unclear at the present time, but they may provide new insights into the mechanism of action and ultimate cellular target(s) for Vinca alkaloids. Whether these modifying drugs sensitize the cells to the action of the alkaloids or potentiate the oncolytic drug effect in the cell remains to be determined. Regardless of the mechanism, calcium channel blocking agents may have a role in the combination chemotherapy of the leukemias with Vinca alkaloids.

Effects of 1,2-naphthoquinones on human tumor cell growth and lack of cross-resistance with other anticancer agents

The sensitivity of human tumor and rat prostate tumor cells to a series of naphthoquinones, including tricyclic compounds of the beta-lapachone and dunnione families as well as 4-alkoxy-1,2-naphthoquinones, was evaluated. To better understand the mechanism of cytotoxicity of 1,2-naphthoquinones, the roles of various resistance mechanisms including P-glycoprotein, multidrug resistant associated protein, glutathione (GSH) and related enzymes, altered topoisomerase activity, and overexpression of genes that control apoptosis (bcl-2 and bc-xL) were studied. MCF7 cells were most sensitive to the naphthoquinones with IC50 values ranging from 1.1 to 10.8 microM, as compared to 2.5 to >32 microM for HT29 human colon, A549 human lung, CEM leukemia and AT3.1 rat prostate cancer cells. MCF7 ADR cells, selected for resistance to adriamycin (ADR), displayed cross-resistance to the tricyclic 1,2-naphthoquinones. Drug efflux via a P-glycoprotein mechanism was ruled out as a mechanism of resistance to 1,2-naphthoquinones, since KB-V1 cells expressing high levels of P-glycoprotein and the KB-3.1 parent line were equally sensitive to these compounds. Any resistance of the tricyclic naphthoquinones noted in ADR-resistant cells appeared to relate to the GSH redox cycle and could be circumvented by exposure to buthionine sulfoximine or by changing the structure from a tricyclic derivative to a 4-alkoxy-1,2-naphthoquinone. The 1,2-naphthoquinones were found to be cytotoxic against CEM/VM-1 and CEM/M70-B1 cells that were selected for resistance to teniposide or merbarone, respectively. In addition, cells overexpressing bcl-2 or bcl-xL proteins were as sensitive to 1,2-naphthoquinones as were control cells. Because of their effectiveness in drug-resistant cells, these agents appear to hold promise as effective chemotherapeutic agents.

Resistance of Mammalian Tumor Cells to Inhibitors of DNA Topoisomerase II

Publisher Summary Clinically useful agents such as the epipodophyllotoxins (etoposide and teniposide), anthracyclines (doxorubicin and daunorubicin), and aminoacridines (arnsacrine) appear to exert their oncolytic effects at least in part through interaction with and inhibition of DNA topoisomerase II (topo II). Alteration in the cytotoxic mechanism(s) plays a role in the resistance of cells to topoisomerase inhibitors. Some forms of clinical drug resistance may be associated with alterations in topo II because of the drugs used and the relative ease in selecting such resistant cell lines in vitro . Accordingly, studies of cell culture models can provide insights and direction to studies of drug resistance associated with alterations in topo II in patients tumor cells. The chapter presents the current understanding of the cellular and molecular expressions of resistance of mammalian tumor cells to the inhibitors of DNA topo II, focus on current results from laboratories, and suggestions for future study. The chapter discusses resistance of tumor cells to DNA topo II inhibitors: cellular pharmacology of the at-MDR phenotype, biochemical features associated with the at-MDR Phenotype—isozymes of DNA topo II in mammalian cells, Increased ATP requirement for catalytic activity and phosphorylation of topo II in at-MDR cells, and the role of the nuclear matrix in at-MDR. The chapter discusses mutations in the topo IIα genes and their relationship to at-MDR, pleiotropic consequences to the cell of an altered topo II, and possible mechanisms of at-MDR—decreased topo II gene expression, increased strand religation or decreased drug binding to the topo II-DNA complex, resistance to “programmed cell death”, progression through G 2 block, resistance to induction of sister chromatid exchanges, and decreased poly (ADP-ribose) polymerase. Studies have demonstrated a correlation between the cytotoxicity produced by topo II inhibitors and the ability of these drugs to induce sister chromatid exchanges (SCEs).

Characteristics of Multidrug Resistance in Human Tumor Cells

MDR is now a well-characterized experimental phenomenon (see recent reviews by Beck, 1987; Pastan and Gottesman, 1987, 1988; Moscow and Cowan, 1988; Bradley et al., 1988; Croop et al., 1988; van der Bliek and Borst, 1989; and Endicott and Ling, 1989). The early observations of Kessel et al. (1968), Biedler and colleagues (Biedler and Riehm, 1970; Biedler et al., 1975), Dano (1973), and Ling and colleagues (Ling and Thompson, 1974; Juliano and Ling, 1976; Bech-Hansen et al., 1976) were made in rodent cell lines and revealed the essential features of the phenotype: broad cross-resistance to a variety of apparently dissimilar “natural-product” drugs, decreased drug accumulation and retention, and overexpression of a high-molecular-mass glycoprotein of ca. 170 kDa, now known as P-glycoprotein or P170. Prior to 1979, this phenomenon had been demonstrated only in rodent cells. Because of the implications of these early studies for the treatment of neoplastic diseases, we believed that it was important to determine whether this type of resistance and its associated biochemical and pharmacologic “markers” could in fact occur in human tumor cells as well. We (Beck et al., 1979) selected a human T-cell leukemia line, CCRF-CEM, for different degrees of resistance to the Vinca alkaloid VLB and showed in a series of studies (summarized by Beck, 1983, 1984) that these cells did indeed display what was then known as the “pleiotropic” drug resistance phenotype. We (Conter and Beck, 1984) subsequently developed a series of VCR resistant CEM sublines that also expressed what is now called MDR.