The term multiple drug resistance (MDR) ()refers to the phenomenon in which tumor cells which survive an initial round of chemotherapy subsequently demonstrate decreased sensitivity to both the original therapeutic agent and other seemingly unrelated drugs (reviewed in (1, 2, 3, 4, 5) ). This is commonly mediated by overexpression of Pgp, a transmembrane protein (M = 170,000) which acts as an energy-dependent drug efflux pump. This transporter actively removes a variety of structurally diverse compounds, including anthracyclines, Vinca alkaloids, epipodophyllotoxins, actinomycin D, and paclitaxel. Enhanced efflux of these compounds reduces their intracellular accumulation and so reduces their cytotoxicity. In contrast, Pgp does not export small hydrophilic drugs such as cisplatin, 5-fluorouracil and melphalan. Certain agents, such as verapamil, have been shown to reverse MDR by competing with the cytotoxic drugs for binding to Pgp, thereby promoting drug accumulation and cytotoxicity (reviewed in (6) ). Because of their potential usefulness as anticancer agents, the identification of compounds with this ability to reverse MDR is an area of research receiving high priority in both academic and pharmaceutical settings.

Another approach to the development of MDR reversing compounds involves characterization of the molecular mechanisms which regulate Pgp function and expression. Many cellular activities are regulated by the phosphorylation and dephosphorylation of specific proteins. Shortly after its discovery, Pgp was shown to be phosphorylated on serine residues in resting cells(7, 8, 9, 10, 11) , most likely at consensus recognition sites for PKC and PKA (12) present in the deduced amino acid sequence of mdr genes. Recently, convincing data that Pgp is a substrate for both PKC and PKA in vitro and in intact cells has been provided(13, 14, 15) . These kinases phosphorylate serine residues (669 and 681, respectively) in the linker region of Pgp(13, 16) . Additional studies demonstrated that treatment of MDR cells with PKC-activating phorbol esters enhances Pgp phosphorylation(9, 17, 18) , while the nonspecific protein kinase inhibitor staurosporine reduces Pgp labeling(19, 20) . However, the effects of phosphorylation on Pgp activity remain controversial since both stimulation (17, 21, 22, 23) and inhibition (9, 24) of drug transport have been reported. Additionally, phorbol esters have been found to decrease the drug sensitivities of cells which do not express Pgp(25, 26) , casting doubt on a specific role of Pgp in these responses

The combined information indicates that multiple protein kinases are able to phosphorylate Pgp; however, the significance of these reactions in regulating Pgp activity remains undefined. To address this ambiguity, we have characterized the effects of a battery of pharmacological inhibitors and activators of several protein kinases on Pgp activity in human breast carcinoma cells which overexpress Pgp (MCF-7/ADR). In these cells, reduction of Pgp activity is manifested as increased cytotoxicity of substrate drugs such as actinomycin D, daunomycin, and vinblastine(27, 28, 29) . If phosphorylation is important in regulating the function of Pgp, phosphorylation modulators would be expected to demonstrate patterns of reversal of MDR consistent with their effects on specific types of protein kinases

EXPERIMENTAL PROCEDURES

Materials

Cell Culture and Cytotoxicity Assay

[H]Drug Accumulation Assay

Phosphorylation of Pgp

Photoaffinity Labeling of Pgp

Other Methods

RESULTS

Cytotoxicities of Phosphorylation-modulating Compounds toward Drug-sensitive and -resistant Cells

()

Figure 1: Structures and properties of PKC modulators. Two-dimensional structures of the phorbol ester family (left), mezerein (center), and staurosporine (right) are indicated. Partition coefficients (water:octanol) were calculated using the HyperChem molecular modeling program with Chem Plus extensions as indicated under ``Experimental Procedures.'' The IC values for MCF-7/ADR cells were determined as indicated under ``Experimental Procedures'' and represent the mean ± S.E. for at least three experiments.

Effects of Phosphorylation-modulating Compounds on Drug Resistance

Several phorbol esters were tested to explore structure-function relationships among these compounds for reversal of MDR. Dose-response curves for the inhibition of cell proliferation by actinomycin D (Fig. 2A) indicate that the 80-fold resistance of the MCF-7/ADR cells can be fully eliminated by 10 µM verapamil. Similarly, resistance was completely reversed by 5 µM mezerein and slightly reversed by 0.5 µM mezerein. The resistance of MCF-7/ADR cells was reduced 30-fold by 10 µM of either 4- or 4-PMA (Fig. 2B), while a 100 nM concentration of either compound was ineffective. High concentrations (50 µM) of 4- and 4-PDBu reduced the resistance factor of MCF-7/ADR cells by 20- and 80-fold, respectively, whereas 500 nM doses had no effects on the IC for actinomycin D (Fig. 2C). Similar studies with vinblastine demonstrated that high, but not low, doses of PMA and PDBu sensitized MCF-7/ADR cells to this drug regardless of the stereochemistry at the 4-position of the phorbol ester (data not shown). None of the compounds tested, i.e. verapamil, mezerein, or the 4- and 4-isomers of PMA and PDBu, affected the sensitivity of parental MCF-7 cells to either actinomycin D or vinblastine (data not shown).

Figure 2: Chemosensitization of MCF-7/ADR cells by verapamil, mezerein, and phorbol esters. MCF-7/ADR cells were treated with the indicated concentrations of actinomycin D with the following additions. A, none, ; 10 µM verapamil, ; 0.5 µM mezerein, ; or 5 µM mezerein, . The effects of actinomycin alone on MCF-7 cells are also indicated (). B, none, ; 0.1 µM 4-PMA, ; 10 µM 4-PMA, ; 0.1 µM 4-PMA, ; or 10 µM 4-PMA, . C, none, ; 0.5 µM 4-PDBu, ; 50 µM 4-PDBu, ; 0.5 µM 4-PDBu, ; or 50 µM 4-PDBu, . The inhibition of cell proliferation was quantitated as indicated under ``Experimental Procedures.'' Values represent the mean ± S.D. of triplicate samples in one of three virtually identical experiments.

The effects of multiple concentrations of mezerein and 11 phorbol compounds on the cytotoxicities of daunomycin, actinomycin D, and cisplatin toward MCF-7/ADR cells were examined. As indicated in Fig. 3, 5 µM mezerein strongly enhanced the abilities of daunomycin and actinomycin D to kill these cells, but did not modulate the cytotoxicity of cisplatin. Similar studies indicated that neither the 4- nor the 4-isomers of phorbol or PDD, at doses up to 100 µM, modulated the toxicities of actinomycin D (Fig. 4A). 4-PDBu was slightly more potent than 4-PDBu in enhancing the cytotoxicities of actinomycin D (Fig. 4B), while the monobutyrated phorbol was ineffective. 4-PMA increased the cytotoxicities of actinomycin D at 10 µM (Fig. 4C); however, its IC of 20 µM precluded analysis of its effects on MDR at higher doses. The 4-isomer of PMA and 4-phorbol 12-myristate were substantially less effective at reversing MDR, while 4-phorbol 13-acetate was essentially inactive even at 100 µM. Virtually identical results were obtained when these phorbol compounds were tested in combination with daunomycin, whereas none of these compounds modulated the cytotoxicity of cisplatin (data not shown).

Figure 3: Chemosensitization of MCF-7/ADR cells by mezerein. MCF-7/ADR cells were treated with the indicated concentrations of mezerein in the presence of 5 µM daunomycin (), 50 nM actinomycin D (), or 2 µM cisplatin (). Cell survival was assayed as indicated under ``Experimental Procedures.'' Values represent the mean ± S.D. of triplicate samples in one of three virtually identical experiments.

Figure 4: Chemosensitization of MCF-7/ADR cells by phorbol esters. MCF-7/ADR cells were treated with 50 nM actinomycin D in the presence of the indicated concentrations. A, 4-phorbol, ; 4-phorbol, ; 4-PDD, ; or 4-PDD, . B, 4-PDBu, ; 4-PDBu, ; or 4-phorbol 13-butyrate, . C, 4-PMA, ; 4-PMA, ; 4-phorbol 13-monoacetate, ; or 4-phorbol 12-monomyristate, . Cell survival was assayed as indicated under ``Experimental Procedures.'' Values represent the mean ± S.D. of triplicate samples in one of two virtually identical experiments.

Staurosporine, which inhibits PKC with a K of approximately 1 nM(33) , was tested for its effects on reversal of MDR by mezerein and phorbol esters. As demonstrated in Fig. 5A, pretreatment of MCF-7/ADR cells with 50 nM staurosporine did not reduce cell killing by combinations of daunomycin and mezerein, 4-PDBu, 4-PMA, or 4-PMA. Similarly, staurosporine did not antagonize the effects of these compounds on the cytotoxicity of actinomycin D (Fig. 5B).

Figure 5: Effects of staurosporine on the chemosensitization of MCF-7/ADR cells by phorbol esters. MCF-7/ADR cells were incubated with 0 (cross-hatched bars) or 50 nM staurosporine (solid bars) for 15 min before the addition of 5 µM daunomycin (A) or 50 nM actinomycin D (B) in the presence of ethanol (solvent control), 5 µM mezerein, 100 µM 4-PDBu, 10 µM 4-PMA, or 10 µM 4-PMA. Cell survival was assayed as indicated under ``Experimental Procedures.'' Values represent the mean ± S.D. of triplicate samples in one of two virtually identical experiments.

Effects of Phosphorylation-modulating Compounds on Pgp Activity

The dose dependences for modulation of the intracellular accumulation of [H]vinblastine by mezerein and 5 phorbol esters were tested. None of these compounds significantly altered the accumulation of [H]vinblastine by MCF-7 cells at doses up to at least 100 µM (50 µM for mezerein) (data not shown). In contrast, mezerein caused dose-dependent and very marked (up to 12-fold) increases in [H]vinblastine accumulation in MCF-7/ADR cells (Fig. 6), with doses as low as 50 nM being effective. 4-PMA modestly increased [H]vinblastine accumulation at very low doses (10 nM), followed by much more substantial accumulations at 100 µM. 4-PMA, 4-PDBu, 4-PDBu, and 4-phorbol 12-myristate all enhanced [H]vinblastine accumulation only at 100 µM.

Figure 6: Effects of mezerein and phorbol esters on [H]vinblastine accumulation in MCF-7/ADR cells. MCF-7/ADR cells were incubated with the indicated concentrations of mezerein (), 4-PMA (), 4-PMA (), 4-PDBu (), 4-PDBu (), or 4-phorbol 12-monomyristate () for 30 min as indicated under ``Experimental Procedures.'' [H]Vinblastine was then added, and its intracellular accumulation after 60 min was determined. Values represent the mean ± S.D. accumulation of [H]vinblastine (solvent control = 100%) in one of three similar experiments.

Pretreatment of the cells with 50 nM staurosporine did not reduce the abilities of mezerein or the phorbol esters to increase intracellular accumulation of [H]vinblastine (Fig. 7). Staurosporine, K-252a, and calphostin C promoted modest dose-dependent increases in [H]vinblastine accumulation (Fig. 8); however, responses were seen only at concentrations of these kinase inhibitors which were very close to their IC values. In contrast, the PKA inhibitor H-89 caused very marked accumulation of [H]vinblastine at doses much lower than its IC for either MCF-7 or MCF-7/ADR cells.

Figure 7: Effects of staurosporine on [H]vinblastine accumulation in MCF-7/ADR cells. MCF-7/ADR cells were incubated with 0 (cross-hatched bars) or 50 nM staurosporine (solid bars) for 15 min before the addition of 5 µM mezerein, 10 µM 4-PDBu, 10 µM 4-PDBu, 10 µM 4-PMA, or 10 µM 4-PMA. After an additional 30 min, [H]vinblastine was added and its intracellular accumulation after 60 min was determined as indicated under ``Experimental Procedures.'' Values represent the mean ± S.D. accumulation of [H]vinblastine (solvent control = 100%) in one of three similar experiments.

Figure 8: Effects of phosphorylation inhibitors on [H]vinblastine accumulation in MCF-7/ADR cells. MCF-7/ADR cells were incubated with the indicated concentrations of staurosporine (), K-252a (), calphostin C (), or H-89 (]) for 30 min as indicated under ``Experimental Procedures.'' [H]Vinblastine was then added and its intracellular accumulation after 60 min was determined. Values represent the mean ± S.D. accumulation of [H]vinblastine (solvent control = 100%) in one of two similar experiments.

Effects of Phosphorylation-modulating Compounds on Pgp Phosphorylation and Photolabeling

Figure 9: Phosphorylation of Pgp. PO-labeled MCF-7/ADR cells were incubated for 5 min with 0 (even-numbered lanes) or 200 nM staurosporine (odd-numbered lanes), followed by incubation for 30 min with ethanol (lane 1), 0.1 µM 4-PMA (lanes 2 and 3), 10 µM 4-PMA (lanes 4 and 5), 0.1 µM 4-PMA (lanes 6 and 7), or 10 µM 4-PMA (lanes 8 and 9). Pgp was then immunoprecipitated using the Oncogene Science antibody, subjected to SDS-PAGE, and dried gels were analyzed by autoradiography and scintillation counting as indicated under ``Experimental Procedures.'' The positions of the following prestained molecular mass markers are indicated: myosin, 217 kDa, and -galactosidase, 130 kDa.

Interaction of a compound with the drug binding site(s) of Pgp can be inferred if the compound antagonizes the ability of Pgp to bind and become photolabeled by [H]azidopine(27, 28, 35) . As demonstrated in Fig. 10, lanes 1, [H]azidopine can be cross-linked to Pgp in membranes isolated from MCF-7/ADR cells, whereas no such protein in membranes from MCF-7 cells is photolabeled by [H]azidopine (data not shown). As expected, photolabeling of Pgp by [H]azidopine was substantially reduced by including verapamil in the binding buffer (A, lane 2). Photolabeling was very strongly suppressed by H-89 and A23187 (lanes 6 and 7), while calyculin A, dibutyryl-cAMP, dibutyryl-cGMP, W-7, and genestein caused more modest decreases in [H]azidopine binding. The effects of PKC modulators were also tested (Fig. 10B). Photolabeling was inhibited by 4-PMA, 4-PMA, 4-PDBu, or 4-PDBu, indicating that these compounds which reversed MDR can directly interact with Pgp. K252-a and H-7 were more effective than staurosporine in reducing photolabeling by [H]azidopine. Calphostin C also strongly inhibited Pgp labeling by [H]azidopine, while both the 4- and 4-isomers of phorbol and PDD were ineffective (data not shown).

Figure 10: Photoaffinity labeling of Pgp. Membranes from MCF-7/ADR cells were incubated with the indicated compounds (all at 50 µM unless otherwise indicated) before Pgp was photolabeled with [H]azidopine as described under ``Experimental Procedures.'' A, samples contained ethanol (solvent control), 20 µM verapamil, 200 nM calyculin A, dibutyryl-cAMP, dibutyryl-cGMP, H-89, A23187, W-7, or genestein (lanes 1-9, respectively). B, samples contained ethanol, mezerein, -PMA, -PMA, -PDBu, -PDBu, staurosporine, K252-a, or H-7 (lanes 1-9, respectively). The positions of prestained molecular mass markers are indicated.

Role of Pgp in Transporting Phorbol Esters

DISCUSSION

Despite several years of effort, there have been no definitive demonstrations of reversal of MDR due to phosphorylation or dephosphorylation of Pgp. We sought to assess the roles of protein kinases in regulating Pgp function using a pharmacological approach. To this end, a panel of compounds which activate or inhibit protein kinases were tested for their effects on Pgp activity in MCF-7/ADR cells. These compounds included both general protein kinase inhibitors, such as staurosporine, and quite selective protein kinase inhibitors and activators, e.g. calphostin C, H-89, and phorbol esters.

Consideration of the effects of this large panel of phosphorylation modulators indicated that there were no patterns of differential cytotoxicity of inhibitors or activators of any particular class of protein kinase or phosphatase toward the two cell lines. Only calyculin A and A23187 demonstrated significantly different toxicities for the two cell lines. Therefore, PKC, PKA, PKG, and Ca/calmodulin-dependent protein kinases appear to be similarly regulated and functional in the two cell types, even though their actual levels may be different(15, 25) . Cell cycle times and cell phase distributions were similar in wild-type MCF-7 and drug-resistant MCF-7/ADR cells (data not shown), indicating that pathways involved in regulating cell proliferation have not been grossly altered in the selected cells.

Recently, we have identified several novel natural products which overcome MDR by acting as antagonists for Pgp (e.g. 27, 28). Using the same methods, we have assessed the ability of the phosphorylation modulators to overcome Pgp-mediated MDR in MCF-7/ADR cells. While certain of these compounds inhibited Pgp activity, there are no correlations with the activation or inhibition of any class of protein kinase. Similarly, phosphoprotein phosphatase inhibitors, which have more global effects on protein phosphorylation, did not modulate MDR at subcytotoxic doses.

Because of the interest in the potential role of PKC in regulating MDR, we conducted an extensive characterization of the effects of activators and inhibitors of this family of kinases. Overall, our data argues against a substantial role of PKC in regulating Pgp activity for the following reasons.

1. Quite specific activators and inhibitors of PKC do not consistently affect MDR or drug accumulation by MCF-7/ADR cells. For example, chelerythrine chloride and calphostin C are both potent and selective inhibitors of PKC; however, the latter compound demonstrated much greater ability to reverse MDR and promote the intracellular accumulation of [H]vinblastine. Previous studies have shown that calphostin C increases drug accumulation in MDR cells(36, 37) , but this effect is not mediated by inhibition of PKC (37) .

2. Drug resistance was substantially reversed by micromolar concentrations of several phorbol esters, including both the - and -isomers of PMA and PDBu. The high doses required and the lack of specificity for the -isomers suggest that these effects do not involve protein kinase C. Furthermore, 1,2-dioctanoylglycerol, another PKC activator, did not enhance the cytotoxicities of the drugs. 4-PDBu was particularly interesting since it demonstrated MDR reversing activity at doses at least 12-fold lower than its IC. This efficacy index of 12 is at least as good as the index for well-characterized Pgp antagonists, e.g. verapamil.

3. The phosphorylation state of Pgp was significantly increased by 4-PMA, but was unaffected by 4-PMA, even at 10 µM, indicating that the effects of the latter compound are not mediated by phosphorylation.

4. The abilities of mezerein and phorbol esters to reverse MDR and to potentiate intracellular drug accumulation were not altered by pretreatment of the cells with staurosporine, even though this compound very effectively blocked PKC-mediated phosphorylation of Pgp.

5. All of the ``PKC modulators'' which reversed MDR inhibited the photolabeling of Pgp by [H]azidopine. Therefore, it is very likely that the reversing effects of these compounds reflect their abilities to interact directly with Pgp rather than alteration of Pgp phosphorylation by PKC.

Similar conclusions can be drawn from data on PKA modulators. For example, H-89, which is 650-fold more active toward PKA than PKC (38) , effectively inhibited drug transport by Pgp and reversed MDR. However, this was associated with high efficiency of inhibiting Pgp photolabeling by [H]azidopine, indicating that the activity of H-89 is due to direct interaction with Pgp rather than through alteration of transport activity via the phosphorylation state of Pgp. Notably, the efficacy index of H-89 (i.e. 10-20) is superior to many previously described Pgp antagonists, suggesting that this compound may be therapeutically useful for reversal of MDR. A structurally related PKA inhibitor, H-87, has been shown to partially reverse resistance in MDR cells(39) .

Antagonism of Pgp by staurosporine(40, 41, 42) , H-7(43) , chlorpromazine (44) , and trifluoperazine (45) have been described previously, and these compounds demonstrated moderate-to-good inhibition of Pgp in our model system. However, these agents simultaneously inhibit more than one protein kinase, making it difficult to assess the involvement of individual kinases. With the exception of staurosporine, all of these agents markedly inhibited Pgp photolabeling by [H]azidopine (Fig. 10), again suggesting that they act by direct antagonism of the drug binding site of Pgp rather than by altering its phosphorylation state.

The phorbol ester family represents a new class of Pgp antagonists which demonstrate interaction at low micromolar concentrations. These compounds are of moderate size and are composed of multiple ring systems, similar to several other Pgp antagonists(6) ; however, their lack of ionizable functional groups is somewhat unusual. The hydrophobicity of these compounds appears to determine interaction with Pgp such that log P values of >2, but <7, effectively block drug transport. While PMA and PDBu are able to bind to Pgp, our results indicate that they are not transported out of the cells by this protein. In this regard, they resemble estramustine(32, 34) .

In certain model systems, reversal of MDR is correlated with down-regulation of the expression of Pgp(24, 46, 47, 48) . Additional studies have suggested that PKA activity may be necessary for the expression of the mdr1 gene(39, 46, 49) . However, the relevance of study of the regulation of expression of highly amplified genes, e.g. mdr1 in MCF-7/ADR cells, is open to debate. Therefore, we have not shown the effects of phosphorylation modulators on levels of Pgp in these cells. We have recently found that A-498 kidney adenocarcinoma cells have significant levels of Pgp and are moderately resistant to Pgp-substrate drugs. ()Thus, it would seem appropriate to analyze mdr1 gene regulation in this nonamplified system as well.

In conclusion, it is apparent that several compounds which are commonly used as phosphorylation modulators, i.e. to study the roles of specific protein kinases in cellular regulation, are able to interact directly with Pgp. This results in inhibition of drug transport and reversal of the MDR phenotype independent of the involvement of protein kinases. There is little doubt that several protein kinases, including PKC and PKA, are able to phosphorylate Pgp, but this does not appear to significantly alter its activity in MCF-7/ADR cells. Protein phosphorylation may play a role in regulating Pgp expression; however, additional studies are needed to further explore such possibilities.

FOOTNOTES

*

This work was supported by Grant DHP-52 from the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Pharmacology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-3141; Fax: 215-728-4333; CD_Smith@FCCC.EDU.

()

The abbreviations used are: MDR, multiple drug resistance; IC, concentration which inhibits cell proliferation by 50%; Pgp, P-glycoprotein; PDBu, phorbol 12,13-dibutyrate; PDD, phorbol 12,13-didecanoate; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis.

()

Resistance factor = IC of resistant cell line/IC of parental cell line.

()

C. D. Smith, X. Zhang, and J. T. Zilfou, unpublished observations.

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