Steven Grant

Differentiation therapy of human cancer: basic science and clinical applications

Current cancer therapies are highly toxic and often nonspecific. A potentially less toxic approach to treating this prevalent disease employs agents that modify cancer cell differentiation, termed differentiation therapy. This approach is based on the tacit assumption that many neoplastic cell types exhibit reversible defects in differentiation, which upon appropriate treatment, results in tumor reprogramming and a concomitant loss in proliferative capacity and induction of terminal differentiation or apoptosis (programmed cell death). Laboratory studies that focus on elucidating mechanisms of action are demonstrating the effectiveness of differentiation therapy, which is now beginning to show translational promise in the clinical setting.

Ara-C: Cellular and Molecular Pharmacology

The antimetabolite cytosine arabinoside (ara-C) represents a prototype of the nucleoside analog class of antineoplastic agents and remains one of the most effective drugs used in the treatment of acute leukemia as well as other hematopoietic malignancies. The ability of ara-C to kill neoplastic cells is regulated at three distinct but interrelated levels. First, the activity of ara-C depends on conversion to its lethal triphosphate derivative, ara-CTP, a process that is influenced by multiple factors, including nucleoside transport, phosphorylation, deamination, and levels of competing metabolites, particularly dCTP. Second, the antiproliferative and lethal effects of ara-C are linked to the ability of ara-CTP to interfere with one or more DNA polymerases as well as the degree to which it is incorporated into elongating DNA strands, leading to DNA fragmentation and chain termination. Finally, the fate of the cell is ultimately determined by whether a threshold level of ara-C-mediated DNA damage is exceeded, thereby inducing apoptosis, or programmed cell death. The latter process is influenced by components of various signal transduction pathways (e.g., PKC) and expression of oncogenes (e.g., bcl-2, c-Jun), perturbations in which may significantly alter ara-C sensitivity. A better understanding of these factors could eventually lead to the development of novel therapeutic strategies capable of overcoming ara-C resistance and improving therapeutic efficacy.

Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)

Interactions between the histone deacetylase inhibitor SAHA (suberoylanilide hydroxamic acid) and the cyclin-dependent kinase (CDK) inhibitor flavopiridol (FP) were examined in human leukemia cells. Simultaneous exposure (24u2009h) of myelomonocytic leukemia cells (U937) to SAHA (1u2009μM) and FP (100u2009nM), which were minimally toxic alone (1.5u2009±u20090.5% and 16.3u2009±u20090.5% apoptosis respectively), produced a dramatic increase in cell death (ie 63.2u2009±u20091.9% apoptotic), reflected by morphology, procaspase-3 and -8 cleavage, Bid activation, diminished ΔΨm, and enhanced cytochrome c release. FP blocked SAHA-mediated up-regulation of p21CIP1 and CD11b expression, while inducing caspase-dependent Bcl-2 and pRb cleavage. Similar interactions were observed in HL-60 and Jurkat leukemic cells. Enhanced apoptosis in SAHA/FP-treated cells was accompanied by a marked reduction in clonogenic surivival. Ectopic expression of either dominant-negative caspase-8 (C8-DN) or CrmA partially attenuated SAHA/FP-mediated apoptosis (eg 45u2009±u20091.5% and 38.2u2009±u20092.0% apoptotic vs 78u2009±u20091.5% in controls) and Bid cleavage. SAHA/FP induced-apoptosis was unaffected by the free radical scavenger L-N-acetyl cysteine or the PKC inhibitor GFX. Finally, ectopic Bcl-2 expression marginally attenuated SAHA/FP-related apoptosis/cytochrome c release, and failed to restore clonogenicity in cells exposed to these agents. Together, these findings indicate that SAHA and FP interact synergistically to induce mitochondrial damage and apoptosis in human leukemia cells, and suggest that this process may also involve engagement of the caspase-8-dependent apoptotic cascade.

Histone deacetylase inhibitors in cancer therapy.

Histone deacetylase inhibitors (HDAC inhibitors) represent a novel class of antineoplastic agents that act by promoting acetylation of histones, leading in turn to uncoiling of chromatin and activation of a variety of genes implicated in the regulation of cell surivival, proliferation, differentiation, and apoptosis. The major classes of HDIs include short-chain fatty acids, hydroxamic acid derivatives, synthetic benzamide derivatives, and cyclic tetrapeptides. Members of each of these classes have now entered clinical trials in humans. Despite their shared capacity to trigger histone deacetylation, individual HDIs exert diverse actions on cell cycle regulatory, signal transduction, and survival-related proteins which in all probability accounts for their disparate actions. Major areas of investigation surrounding HDIs include elucidating the mechanisms by which they induce apoptosis in neoplastic cells, and characterizing the factors responsible for the decision of such cells to undergo maturation versus cell death in response to these agents. In this context, attention has recently focused on the ability of HDIs to induce perturbations in cell cycle regulatory proteins (e.g., p21CIP1), downregulation of survival signaling pathways (e.g., Raf/MEK/ERK), and disruption of cellular redox state (e.g., induction of reactive oxygen species; ROS). Aside from efforts to combine HDIs with established cytotoxic drugs, attempts are underway to establish a rational basis for combining HDIs with differentiation-inducing agents (e.g., ATRA, hypomethylating agents such as 5-deoxyazacytine) with the goal of triggering re-expression of tumor suppressor and/or differentiation-associated genes. Finally, the results of recent preclinical studies provide a strong rationale for combining HDIs with other novel, molecularly targeted agents, including inhibitors of survival signaling pathways or cell cycle progression. Collectively, these findings should provide a fertile environment for the development of novel HDI-containing regimens in the treatment of cancer for many years to come.

Histone deacetylase inhibitors: insights into mechanisms of lethality

Histone deacetylases (HDACs) have recently emerged as an important target for therapeutic intervention in cancer and potentially other human diseases. By modulating the acetylation status of histones, histone deacetylase inhibitors (HDACIs) alter the transcription of genes involved in cell growth, maturation, survival and apoptosis, among other processes. Early clinical results suggest a potentially useful role for HDACIs in the treatment of certain forms of lymphoma (e.g., cutaneous T cell lymphoma) and acute leukaemia. An unresolved question is how HDACIs induce cell death in tumour cells. Recent studies suggest that acetylation of nonhistone proteins may play an important role in the biological effects of this class of compounds, and may explain lack of correlation between histone acetylation and induction of cell death by HDACIs in some circumstances. Recently, attention has focussed on the effects of HDACIs on disruption of co-repressor complexes, induction of oxidative injury, upregulation of the expression of death receptors, generation of lipid second messengers such as ceramide, interference with the function of chaperone proteins and modulation of the activity of NF-κB as critical determinants of lethality. Aside from providing critical insights into the mechanism of action of HDACIs in neoplastic disease, these findings may provide a foundation for the rational development of combination studies, involving HDACIs in combination with either conventional cytotoxic drugs as well as more novel targeted agents.

Histone deacetylase inhibitors in clinical development

In addition to a variety of other novel agents, interest in histone deacetylase inhibitors (HDACIs) as antineoplastic drugs has recently accelerated and increasing numbers of these compounds have entered clinical trials in humans. HDACIs represent a prototype of molecularly targeted agents that perturb signal transduction, cell cycle-regulatory and survival-related pathways. Newer generation HDACIs have been introduced into the clinical arena that are considerably more potent on a molar basis than their predecessors and are beginning to show early evidence of activity, particularly in hematopoietic malignancies. In addition, there is an increasing appreciation of the fact that HDACIs may act through mechanisms other than induction of histone acetylation and, as in the case of other molecularly-targeted agents, it is conceivable that the ultimate role of HDACIs in cancer therapy will be as modulators of apoptosis induced by other cytotoxic agents. One particularly promising strategy involves attempts to combine HDACIs with other novel agents to promote tumour cell differentiation or apoptosis. The present review focuses on recent insights into the mechanisms by which HDACIs exert their anticancer effects, either alone or in combination with other compounds, as well as attempts to translate these findings into the clinic.

The Histone Deacetylase Inhibitor LAQ824 Induces Human Leukemia Cell Death through a Process Involving XIAP Down- Regulation, Oxidative Injury, and the Acid Sphingomyelinase- Dependent Generation of Ceramide

Determinants of differentiation and apoptosis induction by the novel histone deacetylase inhibitor (HDACI) LAQ824 were examined in human leukemia cells (U937 and Jurkat). Exposure of U937 cells to a low concentration of LAQ824 (30 nM) resulted in a delayed (2 h) increase in reactive oxygen species (ROS), induction of p21WAF1/CIP1, pRb dephosphorylation, growth arrest of cells in G0/G1 phase, and differentiation. On the other hand, exposure of cells to a higher concentration of LAQ824 (75 nM) resulted in the early (30 min) generation of ROS, arrest of cells in G2/M phase, down-regulation of XIAP (at the transcriptional level) and Mcl-1 (through a caspase-mediated process), the acid sphingomyelinase-dependent generation of ceramide, and profound mitochondrial injury, caspase activation, and apoptosis. LAQ824-induced lethality in U937 cells did not involve the extrinsic apoptotic pathway, nor was it associated with death receptor up-regulation; instead, it was markedly inhibited by ectopic expression of Bcl-2, Bcl-xL, XIAP, and Mcl-1. The free radical scavenger N-acetyl cysteine blocked LAQ824-mediated ROS generation, mitochondrial injury, Mcl-1 down-regulation, ceramide generation, and apoptosis, suggesting a primary role for oxidative injury in LAQ824 lethality. Together, these findings indicate that LAQ824-induced lethality represents a multifactorial process in which LAQ824-mediated ROS generation is necessary but not sufficient to induce apoptosis, and that the degree of XIAP and Mcl-1 down-regulation and ceramide generation determines whether this agent engages a maturation rather than an apoptotic program.

Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[β-d-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells

We have demonstrated previously that bryostatin 1, a macrocylic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[beta-D-arabinofuranosyl]cytosine (ara-C) in HL-60 human promyelocytic leukemia cells (Grant et al., Biochem Pharmacol 42: 853-867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grant et al., Cancer Res 52: 6270-6278, 1992). In the present studies, we have assessed the extent of this damage by quantitative spectrofluorophotometry of small molecular weight, double-stranded DNA fragments in order to provide: (a) a more complete characterization of the interaction between ara-C and bryostatin 1, and (b) a direct comparison of the relative effects of bryostatin 1 treatment with other pharmacological manipulations known to modulate protein kinase C activity. Exposure of cells to ara-C (10(-9) to 10(-4) M; 1-24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10(-11) to 10(-7) M; 1-24 hr) alone failed to induce DNA damage, but promoted substantial time- and concentration-related increases in the extent of fragmentation induced by a subsequent 6-hr exposure to ara-C. Maximal potentiation of fragmentation (e.g. 2- to 3-fold greater than that obtained with ara-C alone) was observed following a 24-hr pretreatment with 10(-8) M or 10(-7) M bryostatin 1, and correlated closely with enhanced inhibition of HL-60 cell clonogenicity. The stage-1 tumor-promoter phorbol dibutyrate potentiated the effects of ara-C in a biphasic manner, maximally augmenting the response at 2.5 x 10(-8) M, but exerting no effect at 10(-7) M, whereas the stage-2 tumor-promoter mezerein failed to augment ara-C-related DNA fragmentation at low concentrations, and antagonized ara-C action at high concentrations. In contrast, ara-C-related DNA fragmentation was attenuated or abolished either by continual preexposure to synthetic diglyceride or by pretreatment with exogenous phospholipase C at all concentrations tested. Increased DNA fragmentation was not specifically related to recruitment of cells into S-phase or enhancement of ara-C-related cellular differentiation. Finally, concentrations of bryostatin 1 that maximally potentiated ara-C-related DNA fragmentation were associated with virtually complete down-regulation of total cellular PKC activity, whereas diglyceride and phospholipase C, which suppressed the response to ara-C, moderately increased total PKC activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein Kinase C Targeting in Antineoplastic Treatment Strategies

Neoplastic cell survival is governed by a balance between pro-apoptotic and anti-apoptotic signals. Noteworthy among several anti-apoptotic signaling elements is the protein kinase C (PKC) isoenzyme family, which mediates a central cytoprotective effect in the regulation of cell survival. Activation of PKC, and subsequent recruitment of numerous downstream elements such as the mitogen-activated protein kinase (MAPK) cascade, opposes initiation of the apoptotic cell death program by diverse cytotoxic stimuli. The understanding that the lethal actions of numerous antineoplastic agents are, in many instances, antagonized by cytoprotective signaling systems has been an important stimulus for the development of novel antineoplastic strategies. In this regard, inhibition of PKC, which has been shown to initiate apoptosis in a variety of malignant cell types, has recently been the focus of intense interest. Furthermore, there is accumulating evidence that selective targeting of PKC may prove useful in improving the therapeutic efficacy of established antineoplastic agents. Such chemosensitizing strategies can involve either (a) direct inhibition of PKC (e.g., following acute treatment with relatively specific inhibitors such as the synthetic sphingoid base analog safingol, or the novel staurosporine derivatives UCN-01 and CGP-41251) or (b) down-regulation (e.g., following chronic treatment with the non-tumor-promoting PKC activator bryostatin 1). In preclinical model systems, suppression of the cytoprotective function(s) of PKC potentiates the activity of cytotoxic agents (e.g., cytarabine) as well as ionizing radiation, and efforts to translate these findings into the clinical arena in humans are currently underway. Although the PKC-driven cytoprotective signaling systems affected by these treatments have not been definitively characterized, interference with PKC activity has been associated with loss of the mitogen-activated protein kinase (MAPK) response. Accordingly, recent pre-clinical studies have demonstrated that pharmacological disruption of the primary MEK-ERK module can mimic the chemopotentiating and radiopotentiating actions of PKC inhibition and/or down-regulation.

Effect of bryostatin 1 on taxol-induced apoptosis and cytotoxicity in human leukemia cells (U937)

We have examined the effects of the macrocyclic lactone protein kinase C (PKC) activator bryostatin 1 on taxol-induced apoptosis and inhibition of clonogenicity in the human monocytic leukemia cell line U937. Exposure of cells to bryostatin 1 (10 nM; 15 hr) after (but not before) a 6-hr incubation with 0.5 microM taxol significantly increased apoptosis and resulted in an approximately 3 log reduction in clonogenicity. Cell cycle analysis revealed that the increase in apoptotic cells following bryostatin 1 treatment occurred primarily in the population undergoing taxol-mediated G2M arrest. The actions of bryostatin 1 were not attributable to potentiation of taxol-induced tubulin stabilization or to a reduction in the intracellular retention of taxol. Following exposure of cells to taxol, the Bcl-2 protein displayed an alteration in mobility that was not modified appreciably by bryostatin 1 treatment. The mobility shift in Bcl-2 protein from cells exposed to taxol followed by bryostatin 1 was eliminated by treatment of lysates with the protein phosphatase 2A (PP2A); the latter effect was blocked by okadaic acid. Treatment of cells with taxol followed by bryostatin 1 did not increase the amount of total Bax (compared with treatment with taxol alone), but did increase the amount of free Bax in the supernatant fraction. Finally, the ability of bryostatin 1 to potentiate taxol-induced apoptosis in U937 cells was mimicked closely by 2-amino-3-methoxyflavone (PD98059), a specific inhibitor of the mitogen-activated protein kinase (MAPK) kinase (MEK). Collectively, these findings indicate that bryostatin 1 increases the susceptibility of U937 cells to taxol-induced apoptosis and inhibition of clonogenicity. They also raise the possibility that this phenomenon may involve functional alterations in Bcl-2 and/or other proteins involved in regulation of the cell death pathway.