Modulation of Intracellular Ceramide Using Polymeric Nanoparticles to Overcome Multidrug Resistance in Cancer Lilian E. van Vlerken, 1 Zhenfeng Duan, 2 Michael V. Seiden, 2 and Mansoor M. Amiji 1 1 Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University and 2 Department of Hematology and Oncology, Massachusetts General Hospital, Boston, Massachusetts Abstract Although multidrug resistance (MDR) is known to develop through a variety of molecular mechanisms within the tumor cell, many tend to converge toward the alteration of apoptotic signaling. The enzyme glucosylceramide synthase (GCS), responsible for bioactivation of the proapoptotic mediator ceramide to a nonfunctional moiety glucosylceramide, is overexpressed in many MDR tumor types and has been implicated in cell survival in the presence of chemotherapy. The purpose of this study was to investigate the therapeutic strategy of coadministering ceramide with paclitaxel, a commonly used chemotherapeutic agent, in an attempt to restore apoptotic signaling and overcome MDR in the human ovarian cancer cell line SKOV3. Poly(ethylene oxide)-modified poly(epsilon-caprolactone) (PEO-PCL) nanoparticles were used to encapsulate and deliver the therapeutic agents for enhanced efficacy. Results show that indeed the cotherapy eradicates the complete population of MDR cancer cells when they are treated at their IC 50 dose of paclitaxel. More interestingly, when the cotherapy was combined with the properties of nanoparticle drug delivery, the MDR cells can be resensitized to a dose of paclitaxel near the IC 50 of non-MDR (drug sensitive) cells, indicating a 100-fold increase in chemosensitization via this approach. Molecular analysis of activity verified the hypothesis that the efficacy of this therapeutic approach is indeed due to a restoration in apoptotic signaling, although the beneficial properties of PEO-PCL nanoparticle delivery seemed to enhance the therapeutic success even further, showing the promising potential for the clinical use of this therapeutic strategy to overcome MDR. [Cancer Res 2007;67(10):4843–50] Introduction A major clinical obstacle in cancer therapy is the development of resistance to a multitude of chemotherapeutic agents, a phenom- enon termed multidrug resistance (MDR). The development of drug resistance in a small subset of tumor cells is believed to be the cause for tumor survival despite invasive chemotherapy (1), a burden particularly in the treatment of ovarian cancer, in which MDR occurs in at least 50% of patients upon relapse (2). Cancer cells can acquire MDR through several molecular mechanisms, in which often more than one mechanism may be responsible for the MDR phenotype (1, 3). These common causes for MDR include overexpression of membrane spanning ATP-dependent drug efflux pumps from the ABC transporter family (most notably P-glycoprotein/MDR-1), modifications in drug metabolism through glutathione-S -transferase or cytochrome P450 activity, alterations in DNA repair mechanisms, and modifications of apoptotic signaling (1, 3). Another major barrier to successful anticancer therapy is the challenge of delivering the required therapeutic concentration to the tumor site while minimizing undesirable side effects resulting from systemic administration. Site-specific drug delivery systems increase the therapeutic benefit by delivering a greater fraction of the dose at the target site which minimizes the amount of therapeutic that accumulates at nonspecific targets. Biodegradable polymeric nanoparticles, such as poly(epsilon-caprolactone) (PCL), are useful drug delivery carriers for such tumor targeted delivery (4, 5). Biocompatibility and degradation methods of this polymer have been widely studied (6–8) and found to be nontoxic, leading to the U.S. Food and Drug Administration approval and acceptance of PCL for medical applications. Additionally, PCL offers an advantage for drug delivery whereby its alkyl structure efficiently encapsulates hydrophobic compounds, whereas slow degradation of the particle allows for extended release of the drug (9). Surface modification of the nanoparticles with a poly(ethylene oxide)-poly(propylene oxide) triblock copolymer (PEO-PPO-PEO, Pluronic) improves the stability of the nanoparticle in the aqueous environment of the body, while decreasing immune activation, repelling plasma proteins, and decreasing reticuloendothelial uptake leading to an increase in circulation time (10). PEO- modified nanoparticles also preferentially localize in the tumor mass by the enhanced permeability and retention effect (11), whereby the fenestrated tumor interstitium and poor lymphatic drainage cause the nanoparticles to extravasate and deliver their drug load. Previous studies from our group have shown that paclitaxel-containing poly(ethylene oxide)-modified poly(epsilon- caprolactone) (PEO-PCL) nanoparticles remain stable in vivo and retain their Pluronic surface layer to increase the circulating half- life and plasma residence time of paclitaxel from a fraction of an hour to 25.3 and 24.0 h, respectively, alongside a nearly 8-fold decrease in total body clearance of the drug (10, 12). The concentration of paclitaxel inside the tumor mass of mice-bearing human ovarian carcinoma (SKOV3) xenografts, as a result, was 8.7-fold higher at 5 h postinjection compared with mice treated with paclitaxel solution (12). Paclitaxel, an antitumor chemotherapeutic agent originally derived from the bark of the Pacific yew tree (Taxus brevifolia ; ref. 13), is widely used in the treatment of solid tumors, particularly of the breast and ovaries (14). Paclitaxel exerts its cytotoxicity by inducing tubulin polymerization resulting in unstable microtubules which interferes with mitotic spindle function and ultimately arrests cells in the G 2 -M phase of mitosis (15, 16). Although it is understood that cell cycle arrest results in activation of the apoptotic signaling cascade, recent studies suggest that paclitaxel Requests for reprints: Mansoor M. Amiji, 110 Mugar Life Sciences Building, 360 Huntington Avenue, Boston, MA 02115. Phone: 617-373-3137; Fax: 617-373-8886; E-mail: m.amiji@neu.edu. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1648 www.aacrjournals.org 4843 Cancer Res 2007; 67: (10). 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