The histone deacetylase inhibitor suberic bishydroxamate: a potential sensitizer of melanoma to TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis

Abstract

TRAIL appears to be a promising anticancer agent in that it induces apoptosis in a wide range of cancer cells but not normal tissues. Sensitivity of melanoma cells to TRAIL-induced apoptosis varied considerably because of their development of various resistance mechanisms against apoptosis. We discuss in this report the potential effect of a histone deacetylase inhibitor SBHA on TRAIL-induced apoptosis. Histone deacetylase (HDAC) inhibitors regulate histone acetylation and thereby modulate the transcriptional activity of certain genes leading to cell growth arrest, cellular differentiation, and apoptosis. Suberic bishydroxamate (SBHA) is a relatively new HDAC inhibitor that induced apoptosis in the majority of melanoma cell lines through a mitochondrial and caspase-dependent pathway. This was due to its regulation of the expression of multiple proteins that are involved in either the mitochondrial apoptotic pathway (Bcl-2 family members) or the final phase of apoptosis (caspase-3 and XIAP). Co-treatment with SBHA at nontoxic doses and TRAIL resulted in a marked increase in TRAIL-induced apoptosis of melanoma, but showed no toxicity to melanocytes. SBHA appeared to sensitize melanoma to TRAIL-induced apoptosis by up-regulation of pro-apoptotic proteins in the TRAIL-induced apoptotic pathway such as caspase-8, caspase-3, Bid, Bak, and Bax, and up-regulation of the BH3 domain only protein, Bim. This, together with activated Bid, may have acted synergistically to cause changes in mitochondria. Treatment with SBHA also resulted in down-regulation of antiapoptotic members of the Bcl-2 family, Bcl-X_L and Mcl-1, and the IAP member, XIAP. These changes would further facilitate apoptotic signaling. SBHA appeared therefore to be a potent agent in overcoming resistance of melanoma to TRAIL-induced apoptosis.

Introduction

Histones are basic proteins that, by complexing with DNA, form nucleosomes leading to the compact structure of chromatin. Basic amino acids of the histones can be post-translationally modified with methyl, acetyl or phosphate groups. The balance between histone acetylation and deacetylation is regulated by histone acetyltransferase (HAT) and HDACs, and plays an important role in transcriptional regulation of genes [1], [2], [3], [4], [5]. Acetylation of lysine residues of histones results in more open chromatin structure and therefore activation of transcription, whereas hypoacetylation of histones is associated with a condensed chromatin structure resulting in the repression of gene transcription [4], [5]. Deregulation of HAT and HDACs has been implicated in the formation and development of certain human cancers by changing the expression pattern of various genes [6], [7], [8], [9].

HDACs are a family of enzymes that regulate histone acetylation by catalyzing the removal of acetyl groups on lysine residues of the nucleosomal histones [4], [5]. HDAC inhibitors are members of a class of agents that modulate the expression of genes by inhibiting the HDAC activity, resulting in an increase in histone acetylation [4], [5]. DNA microarray studies using malignant cell lines cultured in the presence of a HDAC inhibitor showed that only a small number (1–2%) of genes were regulated [10]. The altered gene expression after exposure to a HDAC inhibitor has been demonstrated to arrest cell growth [11], [12], [13] and to reverse neoplastic characteristics by inducing differentiation [14], [15], [16], [17]. In addition, HDAC inhibitors can induce apoptotic cell death in a variety of tumor cell lines [14], [18], [19], [20]. HDAC inhibitors are therefore considered to be promising chemotherapeutic agents for treatment of malignant tumors. The potential significance of HDAC inhibitors as anticancer agents has been supported by studies in animal models and clinical trials showing antitumor activity with minor toxicity to normal tissues in vivo[21], [22].

Although a number of HDAC inhibitors have been shown to induce apoptosis of cultured tumor cells, the mechanism(s) underlying this appear to vary. For example, explanations for the induction of apoptosis by the nonspecific HDAC inhibitor sodium butyrate include alterations in Bcl-2 family protein expression [23], [24], [25], [26], increased caspase activity [20], increased sensitivity to Fas–Fas ligand interaction [27], [28] and changes in the expression of genes such as c-myc and k-ras[29]. Moreover, caspase-independent mechanisms have also been suggested as causative [25]. It appeared that sodium butyrate-induced apoptosis was mediated through the mitochondrial pathway in that apoptosis was inhabitable by overexpression of Bcl-X_L[25]. Studies with another HDAC inhibitor, apicidin, showed that this was associated with translocation of Bax to mitochondria and subsequent release of cytochrome c[30]. The HDAC inhibitor suberoylanilide (SAHA) appeared to induce apoptosis by direct noncaspase-dependent activation of Bid [31].

Much interest has been given to the role of HDAC inhibitors in regulation of p21^WAF1/CIP1[19], [32], [33], which is one of the key regulators of the cell cycle. The transcription of p21^WAF1/CIP1 is up-regulated in response to HDAC inhibitors as a consequence of increased acetylation of the chromatin at the Sp1 binding sites in the promoter region of p21^WAF1/CIP1[33], [34]. Up-regulation of p21^WAF1/CIP1 by HDAC inhibitors plays a critical role in cell cycle arrest by inhibiting cyclin-dependent kinase (cdk) activity [13], [32]. It may also participate in the induction of apoptosis in that overexpression of p21^WAF1/CIP1 leads to induction of cytochrome c release, activation of caspases, and apoptosis [33], [34]. However, cell lines that did not have increased p21^WAF1/CIP1 protein levels in response to the HDAC inhibitor acelaic bishydroxamic (ABHA) were more sensitive to apoptosis than cell lines in which p21^WAF1/CIP1 protein levels were increased following treatment with ABHA [19].

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family that can induce apoptosis in a wide range of cultured malignant cells including melanoma [35], [36], [37], [38]. The potential significance of TRAIL as an anticancer agent has been supported by studies in animal models showing selective toxicity to human tumor xenografts but not normal tissues. Traditionally, two principal pathways to apoptosis have been recognized, the transmembrane “extrinsic” pathway and the mitochondrial “intrinsic” pathway. Both depend on activation of cysteine proteases that cleave at aspartate residues (caspases). These enzymes are synthesized as proenzymes that become activated by adaptor proteins. Initiator caspases once activated can activate so-called effector caspases, which act on a wide range of substrates to cause apoptosis. The initiator caspases for the extrinsic pathway are caspase-8 and -10, whereas caspase-9 and -2 are initiator caspases for the intrinsic pathway. The effector caspases are believed to be similar for both pathways, i.e. 3, 6 and 7 [39], [40]. TRAIL-induced apoptotic signaling is believed to be initiated by ligand-induced aggregation of death domains that reside on the cytoplasmic sides of the death receptors, TRAIL-R1 and -R2. This in turn orchestrates the assembly of adaptor proteins such as Fas-associated death domain (FADD) that activate initiator caspases, caspase-8 and -10 leading eventually to activation of effector caspases such as caspase-3 [37], [41], [42].

TRAIL-induced apoptosis of melanoma is largely dependent on induction of changes in mitochondrial membrane permeability [43], [44]. Release of Smac/DIABLO from mitochondria to the cytosol mediates TRAIL-induced apoptosis by binding to members of the inhibitors of apoptosis protein (IAP) family. This prevents the IAP proteins binding to the effector caspase-3 and -4, and to caspase-9, so allowing apoptosis to proceed [39], [40], [43]. TRAIL induces low levels of Smac/DIABLO release into the cytosol in TRAIL resistant melanoma cell lines but high levels in TRAIL sensitive cell lines [43]. The basis for this variation in Smac/DIABLO release remains largely unknown but did not appear to be due to differences in activation of caspase-8 and Bid in that they appeared to be cleaved to the same extent in both TRAIL sensitive and resistant melanoma cell lines [43]. It was however suggested that Bcl-2 family members play a pivotal role in regulating mitochondrion-mediated apoptosis. The antiapoptotic members of the Bcl-2 family, such as Bcl-2, Bcl-X_L and Mcl-1, appear to preserve the integrity of the mitochondrial outer membrane by binding to BH3 domains of the pro-apoptotic BH3-only proteins such as Bid, Bim, and Noxa [45], [46]. In the absence or deficiency of the antiapoptotic Bcl-2 family members, the pro-apoptotic BH3-only proteins cause the oligomerization of the multi-BH3 domain proteins, Bax and Bak, in the mitochondrial outer membrane and formation of pores which allow release of mitochondrial proteins such as Smac/DIABLO and cytochrome c[47]. A schematic illustration of TRAIL-induced apoptosis pathway in melanoma is shown in Fig. 1.

SBHA is a relatively new HDAC inhibitor that can induce apoptosis in the majority of melanoma cell lines through a mitochondrial and caspase-dependent pathway [48]. Several key antiapoptotic proteins are down-regulated in SBHA-treated cells [49]. These include X-linked IAP (XIAP) and the Bcl-2 family proteins, Mcl-1 and Bcl-X_L. In contrast, SBHA induces up-regulation of the Bcl-2 family pro-apoptotic proteins, Bid, Bim, Bax, and Bak. Moreover, the expression levels of pro-caspase-8 and pro-caspase-3 are also up-regulated by SBHA treatment. This combination of events strongly suggests that SBHA may be a valuable agent in sensitizing melanoma to other apoptosis inducers. In this report, we discuss the possible mechanisms by which SBHA induces apoptosis and the potential effect of SBHA on TRAIL-induced apoptosis of melanoma.