Original article
Laboratory—bladder
Paclitaxel-hyaluronan hydrosoluble bioconjugate: Mechanism of action in human bladder cancer cell lines

https://doi.org/10.1016/j.urolonc.2012.01.005Get rights and content

Abstract

Objectives

A previously described hydrosoluble paclitaxel-hyaluronan bioconjugate appears particularly well suited for treatment of superficial bladder cancer because of its in vitro cytotoxic profile against urothelial carcinoma (UC) cell lines and in vivo biocompatibility. The aim of this work was to assess the mechanism of action of the bioconjugate in UC cells.

Materials and methods

Expression of CD44 and RHAMM hyaluronan-binding receptors in RT-4 and RT-112/84 UC cell lines, interaction of fluorochrome-labeled bioconjugate with tumor cells, CD44 modulation upon incubation with the compound or free hyaluronan, and caspase activation were assessed by flow cytometry. Cytotoxicity was studied by the MTT assay. Analysis of bioconjugate intracellular localization and effects on β-tubulin organization was carried out by confocal microscopy.

Results

The paclitaxel-hyaluronan bioconjugate bound to UC tumor cells entered intracellular compartments through a saturable and energy-dependent mechanism that involved CD44, as assessed by blocking with specific antibody. Upon internalization, the bioconjugate accumulated into lysosomes where the esteric bond between paclitaxel and the hyaluronan moiety was cleaved, leading to cytoplasmic diffusion of the free drug, caspase activation, and disruption of the β-tubulin microtubular mesh with subsequent cell death.

Conclusions

Conjugation of paclitaxel to hyaluronan results in a new chemical entity, characterized by selective targeting to polymer receptors on plasma membrane and cell entry through receptor-mediated endocytosis, followed by lysosomal accumulation. Ultimately, the active molecule is released, fully preserving the cytotoxic potential and profile of clinically used free paclitaxel.

Introduction

Bladder cancer represents a leading cause of malignancy in the world, with urothelial carcinoma (UC) being the most common histotype, as it represents more than 90% of cases [1]. Among UC, about 70% are diagnosed as superficial tumors with a high likelihood of recurrence (50% to 70%); thus, the real prevalence of UC exceeds its primary incidence significantly [2], [3].

Management of superficial bladder cancer usually relies on transurethral resection (TUR) of neoplastic foci, followed by adjuvant intravesical immunotherapy or chemotherapy to reduce recurrence and/or progression [2], [3], [4]. Bladder instillation of chemotherapeutics has a potential targeting advantage due to a selective delivery of high concentration of drugs to the urothelial cancer tissues, while minimizing systemic exposure.

Paclitaxel is a well known and widely used diterpenoid, currently adopted against a variety of different tumor histotypes [5]. As a primary mechanism of action in cells, it leads to a mitotic block by stabilizing microtubules, thereby decreasing the dynamic nature of these cytoskeletal structures [6]. With regard to bladder neoplasia, paclitaxel exhibits a higher activity than other antimicrotubule compounds, such as vinblastine, against human bladder cancer cells [7]. In addition, paclitaxel has physiochemical properties differing from agents commonly employed for intravesical therapy, such as the high molecular weight (853 Da) and the elevated lipophilicity [8]. Although these properties might improve the transport of the molecule across the urothelium, which is the major barrier to drug absorption, the partitioning and/or adhesion of lipophilic drugs into micelles can, on the other hand, reduce their interaction with biological membranes by decreasing the fraction of free drug [9].

Owing to its low solubility, paclitaxel is administered in a castor oil (Cremophor)/ethanol solution that requires hospitalization since side-effects, such as hypersensitivity, may occur [10]. Moreover, previous studies have shown a direct role of concentrated Cremophor in reducing the paclitaxel free fraction because of its entrapment in Cremophor micelles [9]. Because of these drawbacks, extensive research has been carried out to produce paclitaxel derivatives devoid of such properties, in particular through conjugation to a number of different hydrosoluble biopolymers [11], [12], including hyaluronic acid (HA) [13], [14], [15].

HA interacts with cell surfaces binding to specific receptors, among which CD44 and the receptor for hyaluronic acid mediated motility (RHAMM, CD168) are established signal-transducing receptors that are known to be relevant to cancer [16], [17]. CD44 is a cell-surface glycoprotein that mediates the cellular uptake and degradation of HA [17], [18]. RHAMM is alternatively spliced, and the different forms of the resulting protein can be found both on the cell surface and intracellularly [16], [19]. The interactions of HA with these receptors, particularly CD44, regulate specific cellular functions that influence cell proliferation, survival, and motility; indeed, a consistent CD44 over-expression can be frequently found on cancer cell surface [18], [20], [21].

HA is not degraded extracellularly but, rather, undergoes turnover by another distinct mechanism such as receptor-mediated endocytosis. Indeed, endocytosis and degradation of HA are inhibited by the anti-CD44 antibody, suggesting that CD44 has critical role in HA turnover [14]. The high molecular weight HA is tethered to the cell surface by the combined efforts of CD44 and hyaluronidase-2 (Hyal-2) [22]. HA is cleaved by Hyal-2 to the 20-kDa limited sized products corresponding to ∼50 disaccharide units. The Hyal-2-generated HA fragments are internalized, delivered to endosomes, and ultimately to lysosomes, where hyaluronidase-1 (Hyal-1) degradates the 20 kDa-fragments to small tetrasaccharides [22].

We previously reported the characterization of a novel HA-paclitaxel bioconjugate (ONCOFID-P) and showed that it appears to particularly indicate intravesical therapy of UC, and is endowed with a high biotolerability profile [13]. The present work aimed at providing further insight into the mechanism of action of the bioconjugate by assessing its interaction, biological fate, and activity in bladder UC cancer cells.

Section snippets

Drugs

The paclitaxel-hyaluronan bioconjugate (Fidia Farmaceutici, Abano Terme, Italy) with ∼20% of paclitaxel loading has been previously described [13]. The compound, formerly indicated as HYTAD1-p20 [13], was thereafter indicated as ONCOFID-P [23]. Paclitaxel (Taxol) was from Bristol-Myers Squibb Italia (Rome, Italy).

Tumor cell lines

RT-4, a slow growing, well-differentiated, moderately aggressive human bladder carcinoma cell line, was cultured in McCoy's 5A (Cambrex, Verviers, Belgium) supplemented with 2 mM

Evaluation of HA receptor expression

We previously reported that CD44 was highly expressed on both RT-4 and RT-112/84 cancer cell lines [13]. RHAMM is another important HA receptor that can be found at membrane and/or cytoplasm level, where it is involved in cell motility and proliferation. For this reason, we investigated CD168 expression in RT-4 and RT-112/84 bladder cancer cell lines to gain information about its potential interaction with ONCOFID-P. Cytofluorimetric analysis disclosed that RHAMM was heavily present

Discussion

The high recurrence of UC tumors after TUR has led to the development of adjuvant approaches based on the intravesical instillation of immunostimulators, such as bacillus Calmette-Guerin (BCG) [4], or chemotherapeutics, mainly mitomycin C and doxorubicin [2], [3], to control relapse and progression of the disease. In this regard in the last years, taxanes, and in particular paclitaxel, have emerged as potential useful drugs as they exhibit strong efficacy against bladder cancer cells [8].

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  • Cited by (0)

    This work was partly supported by the Italian Association for Cancer Research (AIRC) and the Italian Ministry of Health.

    1

    These authors contributed equally to this work.

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