An essential relationship between ATP depletion and chemosensitizing activity of Pluronic® block copolymers

Abstract

Pluronic® block copolymers are known to sensitize multidrug resistant (MDR) tumors with respect to various anticancer agents, particularly, anthracycline antibiotics. After completion of the Phase I clinical trial, the formulation containing doxorubicin and Pluronic®, SP1049C, is undergoing Phase II clinical trials. Studies of the mechanism of the sensitization effect of Pluronic® suggested an essential role of ATP depletion in MDR tumors by the block copolymer. The ATP depletion phenomenon was further examined using a panel of cells with varying levels of expression of P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRPs). Cell responses were characterized in terms of EC₅₀ a concentration of Pluronic® P85 resulting in a 50% decrease in ATP intracellular levels. These studies suggested that the cells displaying high responses in ATP depletion with EC₅₀ < 0.01% were strongly sensitized by the block copolymer resulting in drastic increases of doxorubicin cytotoxic activity (over 100-fold). In contrast, the less responsive cells with EC₅₀ >ca. 0.02% were practically not sensitized by the block copolymer. The responses of the cells to P85 in ATP depletion studies correlated with the levels of expression of the drug efflux transport proteins, primarily Pgp. This provided initial evidence that Pgp may be useful as a gene expression marker for predicting potential responses to doxorubicin/Pluronic® formulation in chemotherapy of cancer.

1. Introduction

Chemotherapy treatment in many types of cancers is impeded by drug resistance [1,2]. One problem in treating drug resistance is that there are many mechanisms, through which the resistance is exhibited. In some cases several mechanisms act simultaneously and/or in concert, which may further complicate therapy. For example, tumors with the multiple drug resistance (MDR) phenotype over-express efflux transporters belonging to a superfamily of ATP binding cassette (ABC) proteins, such as P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRP) that pump drugs out of a cell [3,4]. The glutathione (GSH)/glutathione S-transferase (GST) detoxification system is frequently activated in MDR cells contributing to drug resistance [5,6]. MRP acts in concert with this system, providing for the efflux of glutathione conjugates of xenobiotics from the cells [5]. Another impediment to treatment, which is present in MDR cells, involves the sequestration of drugs within cytoplasmic vesicles, followed by drug extrusion out of the cell [7]. Drug sequestration in MDR cells is achieved through the maintenance of abnormally elevated pH gradients across organelle membranes—by the activity of H⁺ – ATPase, an ATP-dependent pump [8,9]. The combination of several mechanisms of drug resistance might complicate chemotherapy and reinforces the need for development of novel drugs and drug formulations effective against drug resistant cancers. One approach that has recently attracted attention uses Pluronic® block copolymers in formulations to treat drug resistant cancers [10–12]. Several review papers on this subject have been published during the last year [13–15]. This mini-review presents some new data reported at the 2nd International Symposium on Tumor Targeted Delivery Systems (National Cancer Institute, Rockville, MD, September 2002). It mainly focuses on the essential role that the ATP depletion plays in chemosensitizing activity of Pluronic® block copolymers.

2. Summary of previous studies on Pluronic® effects in MDR cancers

Pluronic® block copolymers (also known under their non-proprietary name ‘poloxamers’) consist of hydrophilic ethylene oxide (EO) and hydrophobic propylene oxide (PO) blocks arranged in a basic A–B–A structure: EO_n/2 –PO_m –EO_n/2. Copolymers with various numbers of hydrophilic EO (n) and hydrophobic PO (m) units are characterized by distinct hydrophilic–lipophilic balance (HLB).

Experimental studies demonstrated that Pluronic® block copolymers sensitize MDR cancer cells, resulting in an increase in the anticancer activity of anthracycline antibiotics by two to three orders of magnitude [10,12]. The most efficacious block copolymers are those with intermediate lengths of PO block and relatively hydrophobic (HLB<20), such as P85 or L61 [16]. Based on these studies one formulation of doxorubicin, SP1049 was developed, which contains a mixture of hydrophobic Pluronic® L61 and hydrophilic Pluronic® F127. An open labeled Phase I dose escalation and pharmacokinetics clinical trial of SP1049C has been completed under the sponsorship of the UK Cancer Research Campaign in two sites—Christie Hospital, Manchester (UK) and Queen Elizabeth Hospital, Birmingham (UK) [17]. Phase II clinical trials were begun in 2002.

Our group has focused on determining the mechanisms through which Pluronic® block copolymers sensitize drug resistant cancer cells. It has been demonstrated that exposure of MDR cells to Pluronic® results in inhibition of several distinct mechanisms of drug resistance exhibited by these cells, including inhibition of drug efflux transporters, such as P-glycoprotein (Pgp), and abolishment of sequestration of drugs within intracellular vesicles [12]. Furthermore, recent studies have demonstrated that these block copolymers induce drastic decreases in ATP levels in MDR cells [18]. For example, P85 at quite low concentrations (10⁻³ to 10⁻² wt.%) induces energy depletion in cells displaying MDR phenotype but does not decrease ATP levels in non-MDR cells at these concentrations.

It has been known that Pluronic® block copolymers can affect metabolic processes in the cells. For example, decreased ATP levels were observed, following exposure to P85, in the Jurkat T-cell lymphoma [19]. A study by Kirillova et al. [20] suggested that Pluronic® inhibits respiration both in isolated mitochondria and in whole cells. Our studies demonstrated that Pluronic® molecules are transported into the MDR cells and can reach mitochondria membranes [21]. Being membrane active agents, these block copolymers possibly affect the structure of mitochondrial membranes and inhibit function of the respiratory chain [14]. This hypothesis is reinforced by a study by Rapoport et al. [22] suggesting that Pluronic® inhibits the activity of electron transport chains in the mitochondria of HL-60 cells. However, the reasons for a remarkable ‘selectivity’ of the block copolymers with respect to the MDR phenotype cells are presently unknown. One hypothesis relating the ATP depletion with the high energy consumption in MDR cells is discussed in the fourth section of this manuscript.

The energy depletion induced by Pluronic® in MDR cells is transient as ATP levels restore once the block copolymer is removed from the cells. There-fore exposure of MDR cells to Pluronic® for a limited period of time does not induce cytotoxic effects in the cells [18]. However, once a drug, such as doxorubicin, is co-administered with the block copolymer, the cytotoxic effect of the drug is drastically increased. The increased cytotoxicity of the drug appears to be related to the ATP depletion by Pluronic®, since restoration of energy levels using an ATP supplementation system abolishes the sensitization effect of the block copolymer in MDR cells [18].

Since the various mechanisms of drug resistance require energy to sustain their activity we hypothesize that ATP depletion in resistant cells, induced by Pluronic®, can affect various drug resistance systems and result in sensitization of the resistant cells. This hypothesis has been validated using an example of Pgp drug efflux pump. Pluronic® displays a pronounced inhibitory effect on Pgp resulting in decreased drug efflux and increased drug uptake in Pgp expressing cells [10,12,23]. Supplementation of ATP abolishes the inhibitory effect of the block copolymer resulting in increased efflux of the Pgp substrates in the cell [18,21]. Recent studies also suggested that in addition to energy depletion in MDR cells, Pluronic® induces membrane fluidization [21,24]. Membrane fluidization by various agents, including non-ionic surfactants such as Tween 20, Nonidet P-40 and Triton X-100, is known to contribute to inhibition of Pgp efflux function [25]. Indeed our recent study using membranes displaying human Pgp demonstrated that Pluronic® P85 drastically inhibits Pgp ATPase activity [18]. Therefore, it is likely that Pluronic® block copolymers have a ‘double-punch’ effect in MDR cells: through ATP depletion and membrane fluidization, which both have a combined result of potent inhibition of Pgp [21].

It has long been suggested that a broadly successful strategy for killing drug-resistant cancer cells could be based on selective energy depletion in these cells, since many mechanisms of drug resistance are energy-dependent [26]. However, no other agent was known so far that induces such a significant decrease in ATP levels, observed selectively in drug resistant cells, as Pluronic® block copolymers. Therefore, the finding of energy-depleting effects of Pluronic® block copolymers, in combination with their very high sensitization effects and ability to inhibit multiple mechanisms of drug resistance in MDR cells is of considerable theoretical and practical significance.

3. Relationship between ATP depletion and sensitization phenomena

Modulation of intracellular energy can have complex effects in cancer cells. It is well known that intracellular ATP pools play an essential role in the regulation of processes involved in cell death, although the critical ATP-dependent steps have not yet been characterized in detail [27]. Exposure of cancer cells to conditions inhibiting both glycolysis and respiration can abolish drug-induced apoptosis and result in necrotic death [28–30]. However, more selective metabolic modulators that can inhibit energy conservation at various levels (i.e. glucose uptake, glycolysis, citric acid cycle, oxidative phosphorylation) have been shown to induce apoptotic cell death in cancer cells [31]. Similarly, the multiple drug combination, PMA ((N-phosphonacetyl)-aspartate, 6-methylmercaptopurine, 6-aminonicotinamide), which contains inhibitors of pyrimidine and purine pools, can amplify drug activity and enhance drug-induced apoptosis in cancer tumors [32–35]. ATP depletion has been shown to restore drug accumulation in resistant cells [36]. Furthermore, both inhibitors of respiration [37] as well as H⁺ –ATPase [9,38] were shown to amplify drug activity in resistant cells. Taken together, these data provided a basis for further examination of the relationship between ATP depletion and sensitization of drug resistant cancers by Pluronic®.

There are several indications that the ATP depletion and sensitization affects of Pluronic® are related. The most compelling evidence in support of this relationship is that ATP supplementation that results in restoration of the intracellular ATP pools also abolishes the sensitization effect of Pluronic® in MDR cells [18]. This relationship is further reinforced by the structural–functional relationship observed in Pluronic® block copolymers [18]. The block copolymers that are the most active in ATP depletion, such as P85 and L61, also display the highest efficacy in amplifying the cytotoxic activity of the drug in resistant cells. On the contrary, the copolymers that are less active in ATP depletion, such as F127, are also the least efficacious in amplifying the drug cytotoxic activity.

Furthermore, a clear correlation has been observed between the extent of the ATP depletion induced by a given block copolymer, such as P85, and the extent of amplification of the drug activity in the MDR cells [18]. This correlation is illustrated in Fig. 1 that presents the data on the doxorubicin cytotoxicity and ATP levels determined in MDR cell line, KBv. The curve on this figure represents the relationship between (i) the ATP levels determined after exposure of the cells to various concentrations (% wt.) of P85 and (ii) the IC₅₀ of doxorubicin determined in the presence of the same concentrations of P85. This data suggest that the effect of P85 on ATP levels in MDR cells correlated well with the sensitization effect of this block copolymer. The lower the ATP level is, the more cytotoxic the drug becomes. Very similar results supporting the correlation between ATP depletion and drug cytotoxicity with Pluronic® were obtained using another MDR cell line, MCF-7/ADR [18].

Fig. 1.

Relationship between the P85 induced changes in IC₅₀ of doxorubicin and ATP levels in KBv cells. Cells were exposed to treatment solutions containing various concentrations of P85 (% wt. as indicated in the plot) for 2 h followed by measurements of ATP levels by luciferin–luciferase assay [18]. In the cytotoxicity studies, cells were exposed to the drug solutions containing the same concentrations of the block copolymer for 2 h and the standard MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) cytotoxicity assay was performed [18]. From Ref. [18] with permission.

The fact that the relationships between the ATP depletion and sensitization affects of Pluronic® were displayed in certain drug resistant cell lines has prompted us to ask whether such a relationship will also be observed when several different cell lines are compared? To address this question we have evaluated the energy status and cytotoxicity changes induced by Pluronic® in several cell lines that either display or do not display drug resistance mechanisms. First, we determined the responsiveness of these cell lines to P85 in ATP depletion experiments. The effective concentration of P85 that induced a 50% decrease in ATP levels in the cells (EC₅₀) determined from the dose–response curves, was used as an indicator of the cell responsiveness the block copolymer [18]. Second, using doxorubicin as a drug we determined the extent of the sensitizing affect of P85 in the same cell lines. These effects were expressed in the form of a ‘resistance reversion index’ (RRI), i.e. ratio of IC₅₀ of the drug in the 50 assay buffer and P85 solution (IC_50,0/IC₅₀). This study utilized 0.1% wt. P85 as a representative dose of the block copolymer, which usually sensitizes the MDR cells [16]. The results are summarized in Fig. 2 that presents the P85 sensitization effect, expressed as RRI, vs. the ATP depletion, expressed as EC₅₀. There appeared to be a simple relationship between the RRI and EC₅₀ values. Specifically, the cells that were poorly responsive to P85 in ATP depletion studies (EC₅₀ >ca. 0.02%) were also not responsive to the block copolymer in the drug sensitization studies. The cells that were relatively responsive in ATP depletion studies (EC₅₀ <0.01%) also displayed significant sensitization in response to exposure to the block copolymer.

Fig. 2.

Relationship between the responsiveness of the cancer cells to P85 in ATP depletion and cytotoxicity studies (paper submitted). The studies included the following cell lines: human breast carcinoma cells, MCF-7, and their MDR subline, MCF-7/ADR; human oral epidermoid carcinoma cells, KB and their MDR subline, KBv; human pancreatic adenocarcinoma cells, Panc-1; human lung carcinoma epithelial cells, COR-L23 and their MRP1 subline, COR-L23/R; wild type porcine kidney epithelial cells, LLC-PK1 and human MDR1-transfected cells, LLC-MDR1; wild type canine kidney epithelial cells, MDCKIIwt and human MRP2-transfected cells, MDCKII-MRP2. EC₅₀ values for each of the cell lines were determined from the dose response curves as the concentration of Pluronic® vP85 inducing a 50% decrease in intracellular ATP following 2 h exposure of the cells to the block copolymer. Resistance reversion index (RRI) was determined as previously described [16] as the ratio of IC₅₀ of doxorubicin in the assay buffer and in 0.1% wt. P85 solution. Vertical arrow separates cell lines with relatively high and low response to P85.

In this respect it is remarkable that Pluronic® induced no changes in the drug cytotoxicity in doxorubicin-selected resistant cell line, COR-L23/R that overexpresses MRP1. At the same time, accumulation studies suggested several fold increases in the transport of doxorubicin in these cells in the presence of the block copolymer (paper in preparation). These accumulation increases were comparable to those observed in MDCKII-MRP2 cells, which displayed very significant sensitization effects. One major difference between these cell lines is in their response to Pluronic® in ATP depletion studies. COR-L23/R cells appeared to be much less responsive than MDCKII-MRP2 cells. Therefore, it is possible, that the lack of sensitization of COR-L23/R cells with respect to Pluronic® is due to a small extent of the block copolymer effect on the metabolic systems in these cells.

The current phenomenological characterization of the relationship between ATP depletion and chemosensitizing activity of Pluronic® does not yet provide a mechanistic explanation of this dependence. However, the ATP depletion phenomenon could be essential for elucidating the reasons for the elevated anticancer activity of Pluronic® -based formulations in drug resistant tumors. It is quite possible that chemosensitization phenomenon is a direct result of the decrease in the ATP pools available within the drug resistant cells. The studies suggesting that chemosensitizing activity of Pluronic® is reversed by ATP supplementation reinforce this point of view. Clearly, the ATP-dependent drug resistance systems including drug efflux transporters and drug sequestration in cytoplasmic vesicles can be inhibited as a result of the ATP depletion. This has been directly demonstrated for Pgp by correlating efflux activity of this drug efflux transporter with the ATP levels in the MDR cells affected by Pluronic [18,21]. For example, as is shown in Table 1 there is a clear correlation between the levels of uptake of Pgp substrate rhodamine 123 and ATP levels in the Pgp overexpressing MCF-7/ADR cells. By increasing the intracellular ATP levels by adding the ATP supplementation system in the extracellular media this study demonstrates abolishment of the inhibitory effect of Pluronic® P85 on the Pgp efflux system and suggest direct relationship between Pgp activity and ATP levels. In the non-MDR MCF-7 cells Pluronic® P85 has little effect on both rhodamine 123 and ATP levels. Furthermore, an increase in intracellular ATP by adding the ATP supplementation system does not affect rhodamine 123 levels. More recently, a similar result suggesting a direct relationship between MRP efflux function and ATP levels modulated by Pluronic® P85 was obtained in the studies of accumulation of MRP substrate, vinblastine, in MRP1 overexpressing in COR-L23/R cell line (Batrakova et al., submitted).

Table 1.

Effect of ATP supplementation on the uptake of a Pgp substrate rhodamine 123 in MCF-7/ADR and MCF-7 cells. Based on the data presented in Ref. [18]

Cells and treatment solutionsa	Intracellular ATP	Rhodamine 123 uptake
MCF-7/ADR
Control	300±21	0.45±0.02
Pluronic® P85	16±2.3	1.44±0.05
Pluronic® P85 and ATP supplementation system	560±34	0.26±0.05
MCF-7
Control	28±1.5	2.92±0.1
Pluronic® P85	28.6±1.5	2.9±0.1
Pluronic® P85 and ATP supplementation system	150±15	3.1±0.26

^aCells were treated for 1.5 h with the following treatment solutions: (i) copolymer free media, (ii) 0.1% Pluronic® P85, (iii) 0.1% Pluronic® P85 with ATP supplementation system containing 50 µM extracellular ATP and 10 µM dodecylamine. For rhodamine 123 accumulation studies the same treatment solutions also contained 3.2 µM of the probe. ATP levels (nmol/mg protein) were determined by luciferin–luciferase assay [18]. Rhodamine 123 accumulation levels (mmol/mg protein) were measured as described in Ref. [18].

Inhibition of drug efflux systems and abolishment of drug sequestration in cytoplasmic vesicles should result in enhanced delivery of the drug to its target site of action, nucleus DNA. However, it is possible that Pluronic® also affects some other biochemical pathways resulting in amplification of the cytotoxic response following the interaction of the drug with the DNA. It has been long suggested that MDR cells exposed to Pluronic® are much more responsive to anthracycline antibiotics than the parental non-MDR cells [10]. Specifically once the block copolymer was present, the MDR cells displayed greater cytotoxic responses than the parental non-MDR cells, with much lower amounts of daunorubicin delivered to the DNA. Based on these studies there appears to be some major component of Pluronic® activity beyond the inhibition of the drug efflux and sequestration pathways. Recently evidence was obtained that ATP depletion induced by Pluronic® can trigger GSH depletion in drug resistant cells (paper submitted). It is also possible that the block copolymer amplifies drug-induced apoptosis in the resistant cells. Increase in the DNA strand breaks induced by anthracyclines by adding Pluronic® reported by Alakhov et al. [10] reinforces this possibility. Furthermore, ATP depletion by Pluronic® may hinder the DNA repair systems in the MDR cells. This may explain why Pluronic® block copolymers are particularly efficacious in the case of anthracyclines, which are known to function through the DNA damage. Elucidation of these possible pathways of Pluronic® effect is an important task of the future studies. It has to be also emphasized that so far the studies were mainly limited to cells expressing Pgp and some MRP drug efflux transporters. It is important that the future studies also validate the relationship between ATP depletion and chemosensitizing activity of Pluronic® including cases of other drug resistance mechanisms and drugs other than anthracyclines.

4. Correlation between ATP depletion and Pgp expression

Earlier reports suggested that ATP responses of the cells to Pluronic® correlate with the expression of the drug efflux transporters in these cells [18]. Specifically, drug resistant cells that overexpress Pgp are much more responsive to Pluronic® than the parental cells that do not overexpress Pgp [18]. In the present work we attempted to quantitatively correlate drug transporter expression and ATP responses to Pluronic®. Cell models used in this study displayed varying expression levels of Pgp, MRP1 and MRP2. First, the expression levels of these proteins were determined by the Western blot analysis of the cell lysates and normalized to constitutively expressed β-actin. Second, the EC₅₀ values were determined in ATP depletion experiments as described above. Finally, EC₅₀ values were plotted as a function of normalized Pgp expression. The results are presented in Fig. 3. Analysis of these data suggested a correlation between ATP depletion and Pgp expression. In particular cells displaying higher levels of Pgp expression (ca. 0.4 Pgp/β-actin) were also generally more responsive to the block copolymer treatment in the ATP depletion study. Conversely, cells displaying lower levels of Pgp expression as a rule were much less responsive to the block copolymer. Although there were certain variations in EC₅₀ and Pgp expression values between the cells of different origin, for all the related pairs of MDR and non-MDR cells the overexpression of Pgp was accompanied by a significant increase of the responsiveness to Pluronic® (compare EC₅₀ for each of KBv and Kb; MCF-7/ADR and MCF-7, LLC-MDR1 and LLC-PK).

Fig. 3.

Relationship between ATP depletion and Pgp expression levels in various cells. In addition to the cell lines presented in Fig. 2 this study included canine MRP1-transfected cells, MDCKII-MRP1; human umbilical vein endothelial cells, HUVEC; bovine brain microvessel endothelial cells, BBMEC; and murine myoblast cells, C2C12. Identification of the drug efflux transport proteins was done using the immunoblot technique [18]. The levels of Pgp expression were quantified by densitometry and normalized to constitutively expressed β-actin. EC₅₀ values for ATP depletion following 2 h exposure of the cells to P85 were determined as discussed above. Vertical arrow separates cells with relatively high and low Pgp expression levels.

The overall correlation exhibited one notable exception—the human umbilical vein endothelial cells, HUVEC, having one of the lowest levels of Pgp expression, were almost as responsive to P85 as some of the Pgp-overexpressing cells (Fig. 3). Although the HUVEC line was characterized as Pgp-negative [39], we suggest that it might express some other proteins that could render these cells responsive to Pluronic®. Furthermore, it appeared that overexpression of MRP was also accompanied by an increase in the responsiveness to Pluronic®. This was evident from comparing the related series of MRP and non-MRP cells, such as MDCKII-MRP1, MDCKII-MRP2 and MDCKwt or COR-L23/R and COR-L23, which have comparable levels of Pgp expression, but very different levels of expression of the corresponding MRPs. (The data for the MRP expression is not shown). However, although MRP expression was obviously important, elevated levels of MRP alone without parallel expression of Pgp were not sufficient to render the cells highly responsive to Pluronic®. For example, doxorubicin-selected resistant cell line COR-L23/R displaying high levels of MRP1 (ca. 3.1 MRP1/β-actin), but very little expression of Pgp was poorly responsive to the block copolymer.

One hypothesis relating ATP depletion to expression of the drug efflux transporters is that the transporter’s function imposes high rates of the energy consumption MDR cells. Under conditions in which the cellular respiration necessary for ATP synthesis is inhibited by Pluronic®, the high rates of ATPase activity by the drug efflux pumps could result in a rapid exhaustion of the intracellular ATP pools. Alternatively, cells, which do not exhibit these resistance mechanisms, would appear to be less responsive to inhibition of respiration and would not exhibit energy depletion, at least to the extent observed in the resistant cells. Such a hypothesis is in line with the earlier observation that resistant cells have an increased glucose utilization rate compared to sensitive cells [40,41]. Furthermore, Batrakova et al. [18] demonstrated that metabolic inhibitors, such as rotenone, deplete ATP in the resistant cells more effectively than in the sensitive cells. Since Pgp is one of the major ATPases overexpressed in MDR cells it makes the high energy consumption in MDR cells a likely cause for the block copolymer selectivity in these cells. An alternative hypothesis would be that, for some reason, the metabolic processes in MDR cells are more sensitive to inhibition with Pluronic® than the metabolic processes in non-MDR cells. This could result in the more pronounced ATP depletion observed following exposure of MDR cells to the block copolymer. In addition, it has to be emphasized also that overexpression of Pgp is not the only factor implicated in the appearance of the MDR phenotype. The MDR cells frequently express other efflux proteins, such as MRP [5,6], have high levels of H⁺ –ATPase [8] and exhibit activated GSH/GST detoxification system [7]. These and possibly some other factors could impose high energy requirements in MDR cells and collectively contribute to the elevated responsiveness of these cells to Pluronic. The high responsiveness to P85 of the cell lines, which have relatively low levels of Pgp expression may be one indication of the possible involvement of alternative mechanisms in the energy depletion.

Overall, evaluation of the relationship between the ATP depletion and expression of drug efflux transport proteins is essential for understanding the mechanisms of Pluronic® affects in drug resistant cells. Furthermore, based on such studies the drug transporters, such as Pgp (and/or MRPs) may be used as gene expression markers for predicting potential responses in cancers displaying various drug resistant phenotypes. The latter is particularly important in view of the current clinical development of the doxorubicin/Pluronic® formulation.

5. Conclusions

In conclusion, ATP depletion plays an essential role in the sensitization activity of Pluronic® block copolymers in MDR tumors. A relationship has been established between the extent of amplification of the cytotoxic activity of doxorubicin in drug resistant cells and the extent of ATP depletion in these cells by the block copolymer. Furthermore, ATP depletion responses of the cells correlated with the levels of expression of the drug efflux transport proteins. This provides initial evidence that these proteins, in particular Pgp, may be useful as gene expression markers for predicting potential responses to doxorubicin/Pluronic® formulation during cancer chemotherapy. Both the mechanistic studies and clinical evaluations of Pluronic® -based formulations of antineoplastic agents are in progress and one should expect new developments in this area in the nearest future.