Fenretinide inhibits myeloma cell growth, osteoclastogenesis and osteoclast viability

Abstract

Fenretinide (4HPR), a nontoxic analog of ATRA, has been investigated in various malignancies but not in multiple myeloma (MM), a plasma cell malignancy associated with induction of osteolytic bone disease. Here we show that 4HPR induces apoptosis through increased level of ROS and activation of caspase-8, 9 and 3, and inhibits growth of several MM cell lines in a dose-dependent manner. Serum or co-culture with the supportive osteoclasts partially protects MM cells from 4HPR-induced growth inhibition. Sphingosine-1 phosphate (S1P) significantly protects MM cells from 4HPR-induced apoptosis suggesting that as in other malignancies, this drug up-regulates ceramide in MM cells. 4HPR has no toxic effects on non-malignant cells such as blood mononucleated cells, mesenchymal stem cells and osteoblasts, but markedly reduces viability of endothelial cells and mature osteoclasts and inhibits differentiation of osteoclasts and MM-induced tube formation. 4HPR is a potential anti-MM agent, affecting MM cells and MM-induced bone disease and angiogenesis.

Introduction

Multiple myeloma (MM) is characterized by the clonal expansion of monoclonal immunoglobulin-secreting plasma cells within the bone marrow (BM). Myeloma cells are often dependent on the BM microenvironment for growth and their dissemination within the hematopoietic BM are typically associated with induction of severe osteolytic bone disease [1] and angiogenesis [2]. Treatment with high dose therapy and novel agents substantially improved clinical outcome but treatment with these agents often associated with severe adverse effects and development of drug resistance [3]. These clinical observations emphasize the need of additional potent but yet relatively safe, anti-myeloma agents.

Fenretinide (N-(4-hydroxyphenyl) retinamide, or 4HPR), a neoclassical analog of the retinoids all-trans retinoid acid (ATRA), has been successfully tested as a chemopreventive and chemotherapeutic agent on various malignancies, such as prostate, breast and colorectal cancer [4]. It is less toxic and teratogenic than other retinoids [5], thereby making it one of the most promising retinoid anti-tumor compounds. Compared with ATRA, 4HPR exhibits reduced hepatotoxicity and increased efficacy in inhibiting mammary carcinogenesis in animal models [6]. Furthermore, 4HPR lacks the ability to induce point mutations or chromosomal aberrations, and is therefore not genotoxic [7]. 4HPR is currently under clinical evaluation in different cancers but the biological effect and therapeutic value in MM have not been investigated.

The cellular and molecular mechanisms by which 4HPR elicit its cytotoxic action are still not clearly understood. Compared with retinoic acid, a key distinction is that 4HPR induces cell apoptosis rather than differentiation and shows synergistic responses with chemotherapeutic drugs. Induction of apoptosis by 4HPR is mediated through a receptor-independent mechanism and is accompanied by increased cellular levels of ceramide [8], [9] and ROS [10], and activation of caspases [11]. We reasoned that if ceramide is a major mediator of 4HPR-induced apoptosis, the effect of the drug could be counteracted by sphingosine-1-phosphate (S1P), which is another sphingolipid metabolite known to regulate cell growth and survival [12]. Whereas increased ceramide and sphingosine are associated with growth arrest and apoptosis, increased intracellular level of S1P is associated with suppression of apoptosis. Intracellularly, the balance between S1P and ceramide/sphingosine levels, which is regulated by a network of key enzymes, determines the fate of normal and malignant cells, and chemoresistance [12], [13].

In this study, we have tested the effect of 4HPR on growth and survival of MM cells in the absence and presence the protective osteoclasts [14]. We also tested the effect of 4HPR on angiogenesis and osteoclastogenesis, two physiological processes typically stimulated in myelomatous bones.