Docetaxel loaded oleic acid-coated hydroxyapatite nanoparticles enhance the docetaxel-induced apoptosis through activation of caspase-2 in androgen independent prostate cancer cells

Abstract

Docetaxel (Dtxl) remains the preferred choice of improving the survival of patients with hormone refractory prostate cancer (HRPC), but many patients suffer from modest drug response and significant toxicity. In the present study, we investigated the efficiency of novel Dtxl loaded-[1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-carboxy(polyethylene glycol)]2000 (DSPE-PEG-COOH) stabilized-oleic acid (OA) coated hydroxyapatite (HA) nanoparticles (Dtxl-NPs) and gained insights into the molecular mechanism of the apoptosis induced by these novel Dtxl-loaded nanoparticles. The drug encapsulation efficiency of Dtxl was 83.6% and the sustained drug release was observed over 30 days. The Dtxl-NPs exhibited significantly more cytotoxicity in both prostate cancer cell lines (PC3 and DU145) compared with Dtxl in vitro and increased the Dtxl-induced apoptosis in the PC3 cells. Cell cycle analysis showed that the PC3 cells treated with Dtxl-NPs exhibited significant arrest in the G2-M phase but a higher sub-G₀/G₁ population when compared with Dtxl. The enhanced apoptosis induced by Dtxl-NPs in the PC3 cells was associated with the changes in mitochondrial membrane potential (MMP) and seemed to involve the activation of caspase-2. The kinetic studies of caspases demonstrated an early activation of caspase-2 in Dtxl-NPs-induced apoptosis in PC3 cells, which differs from Dtxl-induced apoptosis. The inhibition of caspase-2 activation by small interfering RNA (siRNA) knockdown resulted in the significant inhibition of Dtxl-NPs-induced disruption of MMP and Dtxl-NPs-induced apoptosis, indicating that the activation of caspase-2 was the critical event before the mitochondrial depolarization in the PC3 cells. Our findings showed that nanoparticles, more than simple drug carriers, may play an active role in mediating the biological effects.

Introduction

Prostate cancer (PCa) is the most common solid organ malignancy affecting men and the second leading cause of cancer death in the United States [1]. Although most PCa patients initially respond to androgen deprivation, a large proportion of patients eventually develop androgen independent, hormone refractory prostate cancer (HRPC) and suffer skeletal metastasis. The treatment of HRPC, which is resistant to conventional therapies, mostly relies on chemotherapeutic drugs [2]. Especially, patients with skeletal metastasis require the systemic administration of chemotherapeutic drugs for the treatment of disseminated tumors.

Docetaxel (Dtxl), which is a semisynthetic product of the European yew Taxus baccata [3], similar to paclitaxel, binds tubulin to promote the polymerization and prevent the depolymerization of microtubules causing mitotic arrest, which leads to apoptosis [4]. Previous studies have shown that Dtxl has a broad spectrum of activity against a variety of tumors, including HRPC [5]. Clinical trials have demonstrated that Dtxl (Taxotere; Aventis Pharmaceuticals, Inc, Bridgewater, NJ) could effectively reduce prostate specific antigen (PSA) levels and improve symptoms [6], and even the survival rate in HRPC patients [5], [7].

However, its clinical application is limited by its intrinsic or acquired multidrug resistance (MDR), its poor water solubility and severe toxic side effects [8]. In daily clinical practice, systematic administration of Dtxl requires the use of Taxotere formulation (containing ploysorbate 80) due to its lipophilic nature, which can cause hypersensitivity reaction, reduced uptake by tumor tissue and increased exposure to other body compartments. Therefore, it is desirable to develop a novel and effective delivery system that would deliver Dtxl to the tumor site. The advantages of preparing such a system would be improved efficiency of drug delivery to the HRPC, reduction in the dosage required to achieve effective antiproliferation for less toxicity, enhanced solubility, controlled drug release to maintain the drug level within a desired range, and suppression of the drug resistance [9], [10].

Owing to their small size, prolonged circulation time, and sustained drug release profile, nanoparticles bearing chemotherapeutic drugs have received an increasing amount of attention for their ability to improve the efficacy of anticancer drugs and will bring an alternative to patients with HRPC [11]. The nanoparticles can spontaneously extravasate and accumulate into the tumor interstitium, resulting from a unique physiology of fenestrated vascular architecture and poor lymphatic drainage, which is referred to as the enhanced permeability and retention (EPR) effect [12]. Moreover, using nanoparticles might overcome, or at least reduce drug resistance, which attenuates the efficacy of conventional chemotherapeutic agents.

Hydroxyapatite (HA), with the structural formula of Ca₁₀(PO₄)₆(OH)₂, is the principal inorganic constituent of human bones and teeth, which has been proposed as a bone substitute material [13]. The nano-HAs have special characteristics such as improved biocompatibility, non-immunogenicity, non-inflammatory behavior, high osteoconductivity and/or osteoinductivity and good bioactivity [14], which are important for the potential applications as the filling material for bone defect. Previous studies have demonstrated that drugs are either chemically conjugated or ionically adsorbed to the surface of HA nanoparticles, but result in limited drug loading capacity and rapid drug release [15]. Herein we devised a novel, biocompatible, efficient and more controllable release system using a well-defined formulation strategy for the treatment of HRPC and its skeletal metastasis. The nanoparticles were constituted of three biomaterials, (1) HA was selected for the core due to its biocompatibility and osteoconductivity; (2) OA was chosen for the shell surrounding the HA core; and (3) DSPE-PEG-COOH interspersed on the OA layer to form a PEG shell. We hypothesized that hydrophobic drugs would partition into the OA shell, and DSPE-PEG-COOH would anchor at the interface of the OA shell to confer an aqueous dispersity to the formulation. In addition, the nanoparticles, which were accumulated in the tumor site via EPR effect, would act as both the osteoinductive materials and the drug reservoirs for the bone defect caused by the metastasis of HRPC.

It is widely believed that Dtxl retards cell cycle progression in the G2-M phase [16]. During the G2-M phase arrest of cancer cells, Bax is released, translocates, and inserts into the mitochondrial membrane releasing cytochrome C and apoptosis-inducing factor (AIF), leading to apoptosis, which is referred to as the intrinsic mitochondrial apoptotic pathway [17]. Recently, nano-HA was also found to be able to inhibit tumor cell proliferation and induce apoptosis [18], [19], but the biomedical pathway is poorly understood. In this study, we determined whether Dtxl-loaded OA-coated HA nanoparticles could increase the Dtxl-induced apoptosis in HRPC cancer cells and explored the biochemical pathways involved.