Pgp-mediated Multidrug Resistance Research Articles

Abstract 4701: A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile

Abstract Initial evaluation of the prenylated hydroxy-stilbene isolated from bee propolis, AUS_001, in the NCI 60 human cell line anticancer screen showed significant growth inhibitory effects with the most robust being observed in leukemia, central nervous system and breast cancer cell lines. The aim of the current study was to further assess the potency of AUS_001 in vitro and investigate its safety profile and mechanism of action. A commercially available cell-based profiling screen (OncoPanel®) identified significant AUS_001-induced growth inhibition in 273 out of 280 cancer cell lines with a concentration causing 50% cell growth inhibition in the range of 0.021-0.94 µM, while non-neoplastic cells required an average of 20x higher concentration to generate similar efficacy. These findings indicate that AUS_001 exerts a high degree of potency across 30 cancer types with a selectivity for cancer versus normal cells. Maximum tolerated dose studies in mice illustrated that orally administered AUS_001 is well tolerated at doses 4 times greater than those that conferred antitumor efficacy, with no myelosuppression or other overt toxicities observed. Predictive toxicology and safety screening indicated that AUS_001 exerts a strong safety profile as evidenced by absence of mutagenic potential in the Ames test using up to 100 µM drug doses and low hERG-blocking liability with a half-maximal inhibitory concentration of 65 µM. Notably, AUS_001 is a poor substrate of the drug efflux transporter, P-glycoprotein 1 (Pgp), as it does not inhibit drug-stimulated Pgp ATPase activity and retains efficacy in high Pgp-expressing models in vitro. Subsequently, the effects of AUS_001 were explored in the ToxTracker® assay, consisting of a panel of mammalian stem cell lines that contain fluorescent reporters for the exploration of DNA damage, oxidative stress, and protein damage using flow cytometry. Robust activation of the Rtkn genotoxicity reporter under low cytotoxicity conditions, in combination with accumulation of cells in G2/M phases of the cell cycle within 4 h and an increase in aneuploid cells after 24 h of drug treatment, suggested an aneugenic mode of action, typically caused by tubulin-targeting agents or mitotic kinase inhibitors. Biochemical tubulin polymerization assays then revealed that AUS_001 acts as a direct tubulin destabilizing agent and monitoring microtubule dynamics in GFP-tubulin reporter cells provided evidence that AUS_001 blocks microtubule assembly. Our studies have elaborated the mechanism of AUS_001 as an inhibitor of tubulin polymerization. The favorable safety profile of AUS_001, along with its ability to circumvent Pgp-mediated multidrug resistance, provides potential for efficacy against multiple cancers where microtubule destabilization is proven to be an effective target. Citation Format: Herman Lelie, Inger Brandsma, Giel Hendriks, Lee R. Cavedine, Brogan A. Epkins, Steven M. Garner, Andrew J. Cook, Muthukrishnan Renganathan, Yong Zhao, Alastair J. King, Peter Y.W. Chan, April Risinger, Marina Koutsioumpa. A novel microtubule disruptor exerts broad anticancer efficacy with a tolerable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4701.

Pyxinol bearing amino acid residues: Easily achievable and promising modulators of P-glycoprotein-mediated multidrug resistance.

The P-glycoprotein (Pgp) is a major transporter involved in multidrug resistance (MDR) of cancer cells leading to chemotherapy failure. In our previous study, we demonstrated that the amide derivatives of pyxinol are promising modulators against Pgp-mediated MDR in cancer. In the present study, we designed and synthesized novel pyxinol derivatives linked to amino acid residues. We evaluated MDR (paclitaxel (Ptx) resistance) reversal potency of forty pyxinol derivatives in KBV cells and analyzed their structure-activity relationships. Half of our derivatives sensitized KBV cells to Ptx at non-toxic concentrations, among which the pyxinol compound bearing a methionine residue (3c) exhibited the best activity in MDR reversal. Compound 3c was found to possess high selectivity toward Pgp and sensitize the KBV cells to Pgp substrates by blocking the efflux function of Pgp. This manifestation may be attributed to its high binding affinity with Pgp, as suggested by docking studies. Overall, the biological profile and ease of synthesizing these pyxinol derivatives render them promising lead compounds for further development for Pgp-mediated MDR.

A cell-based high-throughput screen identifies inhibitors that overcome P-glycoprotein (Pgp)-mediated multidrug resistance

Multidrug resistance (MDR) to chemotherapeutic drugs remains one of the major impediments to the treatment of cancer. Discovery and development of drugs that can prevent and reverse the acquisition of multidrug resistance constitute a foremost challenge in cancer therapeutics. In this work, we screened a library of 1,127 compounds with known targets for their ability to overcome Pgp-mediated multidrug resistance in cancer cell lines. We identified four compounds (CHIR-124, Elesclomol, Tyrphostin-9 and Brefeldin A) that inhibited the growth of two pairs of parental and Pgp-overexpressing multidrug-resistant cell lines with similar potency irrespective of their Pgp status. Mechanistically, CHIR-124 (a potent inhibitor of Chk1 kinase) inhibited Pgp activity in both multidrug-resistant cell lines (KB-V1 and A2780-Pac-Res) as determined through cell-based Pgp-efflux assays. Other three inhibitors on the contrary, were effective in Pgp-overexpressing resistant cells without increasing the cellular accumulation of a Pgp substrate, indicating that they overcome resistance by avoiding efflux through Pgp. None of these compounds modulated the expression of Pgp in resistant cell lines. PIK-75, a PI3 Kinase inhibitor, was also determined to inhibit Pgp activity, despite being equally potent in only one of the two pairs of resistant and parental cell lines. Strong binding of both CHIR-124 and PIK-75 to Pgp was predicted through docking studies and both compounds inhibited Pgp in a biochemical assay. The inhibition of Pgp causes accumulation of these compounds in the cells where they can modulate the function of their target proteins and thereby inhibit cell proliferation. In conclusion, we have identified compounds with various cellular targets that overcome multidrug resistance in Pgp-overexpressing cell lines through mechanisms that include Pgp inhibition and efflux evasion. These compounds, therefore, can avoid challenges associated with the co-administration of Pgp inhibitors with chemotherapeutic or targeted drugs such as additive toxicities and differing pharmacokinetic properties.

Celecoxib Prevents Doxorubicin-Induced Multidrug Resistance in Canine and Mouse Lymphoma Cell Lines.

Background: Treatment of malignancies is still a major challenge in human and canine cancer, mostly due to the emergence of multidrug resistance (MDR). One of the main contributors of MDR is the overexpression P-glycoprotein (Pgp), which recognizes and extrudes various chemotherapeutics from cancer cells. Methods: To study mechanisms underlying the development of drug resistance, we established an in vitro treatment protocol to rapidly induce Pgp-mediated MDR in cancer cells. Based on a clinical observation showing that a 33-day-long, unplanned drug holiday can reverse the MDR phenotype of a canine diffuse large B-cell lymphoma patient, our aim was to use the established assay to prevent the emergence of drug resistance in the early stages of treatment. Results: We showed that an in vitro drug holiday results in the decrease of Pgp expression in MDR cell lines. Surprisingly, celecoxib, a known COX-2 inhibitor, prevented the emergence of drug-induced MDR in murine and canine lymphoma cell lines. Conclusions: Our findings suggest that celecoxib could significantly improve the efficiency of chemotherapy by preventing the development of MDR in B-cell lymphoma.

P-glycoprotein targeted and near-infrared light-guided depletion of chemoresistant tumors

Drug resistance remains a formidable challenge to cancer therapy. P-glycoprotein (Pgp) contributes to multidrug resistance in numerous cancers by preventing accumulation of anticancer drugs in cancer cells. Strategies to overcome this resistance have been vigorously sought for over 3 decades, yet clinical solutions do not exist. The main reason for the failure is lack of cancer specificity of small-molecule Pgp inhibitors, thus causing severe toxicity in normal tissues. In this study, Pgp-targeted photodynamic therapy (PDT) was developed to achieve superior cancer specificity through antibody targeting plus locoregional light activation. Thus, a Pgp monoclonal antibody was chemically modified with IR700, a porphyrin photosensitizer. In vitro studies showed that the antibody-photosensitizer conjugates specifically bind to Pgp-expressing drug resistant cancer cells, and caused dramatic cytotoxicity upon irradiation with a near infrared light. We then tested our Pgp-targeted approach in mouse xenograft models of chemoresistant ovarian cancer and head and neck cancer. In both models, targeted PDT produced rapid tumor shrinkage, and significantly prolonged survival of tumor-bearing mice. We conclude that our targeted PDT approach produces molecularly targeted and spatially selective ablation of chemoresistant tumors, and thereby provides an effective approach to overcome Pgp-mediated multidrug resistance in cancer, where conventional approaches have failed.

P-glycoprotein-targeted photodynamic therapy boosts cancer nanomedicine by priming tumor microenvironment

Cancer nanomedicines only modestly improve the overall survival of patients because their anticancer activity is limited by biological barriers posed by the tumor microenvironment. Currently, all the drugs in FDA-approved cancer nanomedicines are substrates for P-glycoprotein (Pgp), and thus, Pgp-mediated multidrug resistance (MDR) remains a hurdle for cancer nanomedicines.Methods: In this study, Pgp-targeted photodynamic therapy (PDT) was developed to enhance the anticancer efficacy of nanomedicines by depleting MDR cancer cells as well as enhancing tumor penetration of nanomedicines. We first examined the Pgp specificity of our targeted PDT approach, and then tested combination therapy of PDT with Doxil in mixed tumor models of MDR cancer cells and stromal cells, mimicking human heterogeneous tumors.Results: In vitro studies showed that the antibody-photosensitizer conjugates produced Pgp-specific cytotoxicity towards MDR cancer cells upon irradiation with a near-infrared light. The studies with a co-culture model of MDR cancer cells and stromal cells revealed synergistic effects in the combination therapy of PDT with Doxil. Using a mouse model of mixed tumors containing MDR cancer cells and stroma cells, we observed markedly enhanced tumor delivery of Doxil after PDT in vivo. We further examined the effects of the two modalities on individual cell populations and their synergism using an in vivo dual substrate bioluminescence assay. The results indicated that Pgp-targeted PDT specifically depleted MDR cancer cells and further enhanced Doxil's actions on both MDR cancer cells and stromal cells.Conclusion: We conclude that our targeted PDT approach markedly enhances anticancer actions of nanomedicines by depleting MDR cancer cells and increasing their tumor penetration, and thereby, may provide an effective approach to facilitate translation of cancer nanomedicines.

Lawsone derivatives target the Wnt/β-catenin signaling pathway in multidrug-resistant acute lymphoblastic leukemia cells

Multidrug resistance (MDR) represents a serious problem in cancer treatment. One strategy to overcome this obstacle is to identify agents that are selectively lethal to MDR cells. The aim of this study was to discover novel compounds against MDR leukemia and to determine the molecular mechanisms behind collateral sensitivity. A library of 1162 compounds was tested against parental, drug-sensitive CCRF-CEM cells using the resazurin assay. A total of 302 compounds showed reasonable activity (less than 50% cell viability). Eleven out of 30 lawsone derivatives revealed considerable collateral sensitivity in MDR P-glycoprotein (Pgp)-overexpressing CEM/ADR5000 cells. They reduced β-catenin activity in a Wnt/β-catenin reporter cell line. Their activities significantly correlated with apolar desolvation (R = 0.819). Compound (1) (3-hydroxy-1,4-dioxo-N-phenyl-naphthalene-2-carboxamide) was the most active compound and dose-dependently down-regulated protein expression of β-catenin, c-MYC, Pgp and Frizzled 7. By molecular docking, we predicted that compound (1) bound to the palmitoyl-binding groove of the cysteine-rich domain of Frizzled-7 and Frizzled-8. Compound (1) neither stimulated ATPase activity of Pgp nor reactive oxygen species generation, both of which have been previously described as possible mechanisms of collateral sensitivity. Instead, we found that Wnt/β-catenin signaling was selectively inhibited in CEM/ADR5000 cells, but not in CCRF-CEM cells. In conclusion, we found for the first time that the inhibition of Wnt/β-catenin signaling may represent a novel molecular mechanism of collateral sensitivity in MDR cells. Wnt/β-catenin signaling, therefore, represents a potential therapeutic target for the selective killing of Pgp-mediated MDR.

Physical and biological characteristics of multi drug resistance (MDR): An integral approach considering pH and drug resistance in cancer.

The role of the Warburg effect in cancer remains to be elucidated with a resurgence in research efforts over the past decade. Why a cancer cell would prefer to use energy inefficient glycolysis, leading to an alteration of pH both inside and outside of the cell, remains to be uncovered. The development of MDR represents a major challenge in the treatment of cancer and it is explained, so far, by the over expression of drug transporters such as the well-known and archetypal P-glycoprotein (Pgp). However, controversies exist regarding the function of Pgp in multi-drug resistance. We suggest here that Pgp-mediated MDR relies fundamentally on pH alterations mediated by the Warburg effect. Furthermore, we propose that the use of proton pump and/or transporters inhibitors (PPIs/PTIs) in cancer are key to controlling both MDR, i.e. sensitize tumors to antineoplastic agents, and drug-related adverse effects.

Non-canonical functions of the cellular transporter P-glycoprotein

P-glycoprotein/ABCB1 (Pgp) is a well known protein of cell defense system. It is localized in cell membrane and pumps different drugs out of various cells using ATP energy. Its overexpression is associated with the development of multidrug resistance (MDR) in cancer cells. The data showing that Pgp also has other functions appeared recently, and this review surveys these data. In particular, (1) Pgp can protect cells from apoptosis; it suppresses the expression of endogenous protein TRAIL and decreases the activity of caspases 8 and 3; (2) Pgp is able to act as an outwardly directed flippase; (3) Pgp participates in a proper development of the innate immune response to intracellular pathogens and in the development of inflammation; (4) functionally active Pgp can be transferred from drug-resistant to drug-sensitive cells by microvesicles (MV). This is a new way of the Pgp-mediated MDR emergence in populations of tumor cells. Thus, Pgp functions as a regulator of some cellular processes. Molecular mechanisms of the Pgp influence on tumor cell viability are related not only with the drug efflux but also with some other functions.

Predictive value of microparticle-associated tissue factor activity for permeability glycoprotein-mediated multidrug resistance in cancer.

Multidrug resistance (MDR) protein 1, which is also known as permeability glycoprotein (Pgp), and tissue factor (TF) are recurrently overexpressed on the surface of cancer cells, likely in response to stimuli such as chemotherapy. Microparticles (MPs) released from cancer cells into the bloodstream express tumour markers on their surface that may be useful as predictive biomarkers for evaluating disease progression. The present study measured the level of TF/factor VII (FVII)-dependent coagulation of MPs isolated from the plasma of cancer patients with various tumours, who were undergoing chemotherapy. Furthermore, Pgp expression on the surface of MPs was evaluated by immunohistochemistry. A total of 50 cancer patients, as well as 10 healthy volunteers, were enrolled in the present study. MP-associated TF/FVII-dependent coagulation pathways were evaluated as the effect of an anti-FVII antibody on the time to thrombin generation, as compared with controls treated with saline. The significantly lengthened times of coagulation [obtained in 20/50 samples (36.5 ± 16%) after treatment with anti-FVIIa when compared with controls] suggest the presence of TF activity is associated with circulating MPs. Furthermore, the 20 MP/TF-positive samples were associated with Pgp overexpression on their surface. Conversely, in the remaining samples (n=30), treatment with the anti-FVIIa antibody did not significantly lengthen the time to clotting (<10%), and Pgp overexpression was not detected. In addition, in the control samples from healthy individuals, Pgp expression at the plasma membrane and clotting in the presence of the anti-FVII antibody were not observed, indicating the absence of MPs. The present study demonstrated that MPs in the blood of cancer patients promoted fibrin generation via TF/FVII-dependent pathways, thus suggesting that the evaluation of MP-TF activity may have a predictive value for Pgp-mediated MDR in various cancer types. Although further studies are required, the measurement of plasma MP-associated TF activity as a predictive biomarker may provide novel therapeutic perspectives to improve the prognosis and effectiveness of anti-cancer drugs in patients who are at a high-risk of Pgp-mediated MDR.

A pH-Responsive Carrier System that Generates NO Bubbles to Trigger Drug Release and Reverse P-Glycoprotein-Mediated Multidrug Resistance.

Multidrug resistance (MDR) resulting from the overexpression of drug transporters such as P-glycoprotein (Pgp) increases the efflux of drugs and thereby limits the effectiveness of chemotherapy. To address this issue, this work develops an injectable hollow microsphere (HM) system that carries the anticancer agent irinotecan (CPT-11) and a NO-releasing donor (NONOate). Upon injection of this system into acidic tumor tissue, environmental protons infiltrate the shell of the HMs and react with their encapsulated NONOate to form NO bubbles that trigger localized drug release and serve as a Pgp-mediated MDR reversal agent. The site-specific drug release and the NO-reduced Pgp-mediated transport can cause the intracellular accumulation of the drug at a concentration that exceeds the cell-killing threshold, eventually inducing its antitumor activity. These results reveal that this pH-responsive HM carrier system provides a potentially effective method for treating cancers that develop MDR.

Carbonic anhydrase XII is a new therapeutic target to overcome chemoresistance in cancer cells.

Multidrug resistance (MDR) in cancer cells is a challenging phenomenon often associated with P-glycoprotein (Pgp) surface expression. Finding new ways to bypass Pgp-mediated MDR still remains a daunting challenge towards the successful treatment of malignant neoplasms such as colorectal cancer.We applied the Cell Surface Capture technology to chemosensitive and chemoresistant human colon cancer to explore the cell surface proteome of Pgp-expressing cells in a discovery-driven fashion. Comparative quantitative analysis of identified cell surface glycoproteins revealed carbonic anhydrase type XII (CAXII) to be up-regulated on the surface of chemoresistant cells, similarly to Pgp. In cellular models showing an acquired MDR phenotype due to the selective pressure of chemotherapy, the progressive increase of the transcription factor hypoxia-inducible factor-1 alpha was paralleled by the simultaneous up-regulation of Pgp and CAXII. CAXII and Pgp physically interacted at the cell surface. CAXII silencing or pharmacological inhibition with acetazolamide decreased the ATPase activity of Pgp by altering the optimal pH at which Pgp operated and promoted chemosensitization to Pgp substrates in MDR cells.We propose CAXII as a new secondary marker of the MDR phenotype that influences Pgp activity directly and can be used as a pharmacological target for MDR research and potential treatment.

P-glycoprotein Mediates Drug Resistance via a Novel Mechanism Involving Lysosomal Sequestration

Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). However, very little work has focused on the contribution of Pgp expressed in intracellular organelles to drug resistance. This investigation describes an additional mechanism for understanding how lysosomal Pgp contributes to MDR. These studies were performed using Pgp-expressing MDR cells and their non-resistant counterparts. Using confocal microscopy and lysosomal fractionation, we demonstrated that intracellular Pgp was localized to LAMP2-stained lysosomes. In Pgp-expressing cells, the Pgp substrate doxorubicin (DOX) became sequestered in LAMP2-stained lysosomes, but this was not observed in non-Pgp-expressing cells. Moreover, lysosomal Pgp was demonstrated to be functional because DOX accumulation in this organelle was prevented upon incubation with the established Pgp inhibitors valspodar or elacridar or by silencing Pgp expression with siRNA. Importantly, to elicit drug resistance via lysosomes, the cytotoxic chemotherapeutics (e.g. DOX, daunorubicin, or vinblastine) were required to be Pgp substrates and also ionized at lysosomal pH (pH 5), resulting in them being sequestered and trapped in lysosomes. This property was demonstrated using lysosomotropic weak bases (NH4Cl, chloroquine, or methylamine) that increased lysosomal pH and sensitized only Pgp-expressing cells to such cytotoxic drugs. Consequently, a lysosomal Pgp-mediated mechanism of MDR was not found for non-ionizable Pgp substrates (e.g. colchicine or paclitaxel) or ionizable non-Pgp substrates (e.g. cisplatin or carboplatin). Together, these studies reveal a new mechanism where Pgp-mediated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.

Discovery of Novel 2-Aryl-4-benzoyl-imidazole (ABI-III) Analogues Targeting Tubulin Polymerization As Antiproliferative Agents

Novel ABI-III compounds were designed and synthesized based on our previously reported ABI-I and ABI-II analogues. ABI-III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best compound having an average IC(50) value of 3.8 nM. They are not substrate of Pgp and thus may effectively overcome Pgp-mediated multidrug resistance. ABI-III analogues maintain their mechanisms of action by inhibition of tubulin polymerization.

Synthesis and Biological Evaluation of Phenoxy-N-phenylacetamide Derivatives as Novel P-glycoprotein Inhibitors

Overexpression of P-glycoprotein (Pgp) is associated with multidrug resistance (MDR) of tumor cells to a number of chemotherapeutic drugs. Pgp inhibitors have been shown to effectively reverse Pgp-mediated MDR. We prepared a series of phenoxy-N-phenylacetamide derivatives and tested for their ability to inhibit Pgp as potential MDR reversing agents, using a Pgp over-expressing MCF-7/ADR cell line. Some of the synthesized compounds exhibited moderate to potent reversal activity. Of note, compound 4o showed a 3.0-fold increased inhibition compared with verapamil, a well-known Pgp inhibitor. In addition, co-treatment of the representative compound 4o and a substrate anticancer agent doxorubicin resulted in a remarkable increase in doxorubicin's antitumor effect and inhibition of DNA synthesis in the MCF-7/ADR cell line. Taken together, these findings suggest that compound 4o could be a useful lead for development of a novel Pgp inhibitor for treatment of MDR.

Drug Delivery: Bypassing Multidrug Resistance in Cancer Cells with Biodegradable Polymer Capsules (Adv. Mater. 47/2010)

The inside cover illustrates a pathway to bypass Pgp-mediated multidrug resistance in cancer cells through endocytosis of microcapsules (right), compared with drug effluxing (left), as reported in work by Frank Caruso and co-workers on p. 5398. Biodegradable polymer capsules conjugated with drug were prepared through click chemistry via layer-by-layer assembly. The incorporated drug is subsequently released after the internalization of microcapsules by intracellular enzymatic environment. This approach opens a generic way to challenge Pgp-mediated multidrug resistance.

Bypassing Multidrug Resistance in Cancer Cells with Biodegradable Polymer Capsules

Biodegradable polymer capsules with drug-loaded multilayers were prepared through click chemistry via layer-by-layer assembly. The capsules can efficiently release incorporated drug by intracellular enzymatic degradation in both sensitive and resistant colorectal cancer cells in vitro. Endocytosis of drug-loaded capsules leads to bypass of Pgp-mediated multidrug resistance in cancer cells.

On a biophysical and mathematical model of Pgp-mediated multidrug resistance: understanding the “space–time” dimension of MDR

Multidrug resistance (MDR) is explained by drug transporters with a drug-handling activity. Despite much work, MDR remains multifaceted, and several conditions are required to generate drug resistance. The drug pumping was conceptually described using a kinetic, i.e., temporal, approach. The re-emergence of physical biology has allowed us to take into account new parameters focusing on the notion of space. This, in turn, has given us important clues regarding the process whereby drug and transporter interact. We will demonstrate that the likelihood of drug-transporter meeting (i.e., the affinity) and thus interaction are also driven by the mechanical interaction between drug molecular weight (MW) and the membrane mechanical properties. This should allow us to mechanically control drug delivery.

Identification of Compounds Selectively Killing Multidrug-Resistant Cancer Cells

There is a great need for the development of novel chemotherapeutic agents that overcome the emergence of multidrug resistance (MDR) in cancer. We catalogued the National Cancer Institute's DTP drug repository in search of compounds showing increased toxicity in MDR cells. By comparing the sensitivity of parental cell lines with MDR derivatives, we identified 22 compounds possessing MDR-selective activity. Analysis of structural congeners led to the identification of 15 additional drugs showing increased toxicity in Pgp-expressing cells. Analysis of MDR-selective compounds led to the formulation of structure activity relationships and pharmacophore models. This data mining coupled with experimental data points to a possible mechanism of action linked to metal chelation. Taken together, the discovery of the MDR-selective compound set shows the robustness of the developing field of MDR-targeting therapy as a new strategy for resolving Pgp-mediated MDR.

Liposome composition is important for retention of liposomal rhodamine in P-glycoprotein-overexpressing cancer cells

Multidrug resistance (MDR) caused by high expression of P-glycoprotein (Pgp) in cancer patients remains one of the major obstacles to successful therapy of cancer. Earlier studies have shown that the incorporation of Pgp-substrate drugs in liposomes may provide a strategy to circumvent Pgp-mediated drug efflux. The present study investigated the impact of liposome composition on the efflux of Pgp-substrate incorporated in liposomes. Liposomes with varying compositions were loaded with rhodamine 123, a fluorescent probe frequently used as a Pgp-substrate, and the retention of rhodamine was compared in two breast cancer cell lines: wild-type cells with no detectable Pgp expression (MCF-7/WT) and Pgp-expressing cells resulting from stable transfection of the MDR1 gene (MCF-7/Pgp). Pgp-expression decreased the rhodamine retention in MCF-7 cells, suggesting that Pgp is functional. Liposome loading increased rhodamine retention in MCF-7/Pgp cells, but not in MCF-7/WT cells. Surface charge of liposomes did not affect the rhodamine retention, whereas the incorporation of cholesterol and polyethyleneglycol-attached lipids was effective in further increasing the rhodamine retention in MCF-7/Pgp cells. Since further study demonstrated that the rate of rhodamine release from liposomes tended to be inversely correlated with rhodamine retention by cells, it seems likely that more rigid liposomes are able to sequester rhodamine more efficiently, thereby inhibiting direct interactions of rhodamine with Pgp proteins. Taken together, these findings suggest that Pgp-mediated MDR in cancer cells may be more effectively modulated by optimizing the composition of liposomes for loading Pgp-substrate anti-cancer drugs.