Current Medicinal Chemistry - Anti-Cancer Agents - Volume 3, Issue 4, 2003

Anticancer agents are used to kill tumour cells with collateral damage as limited as possible. One of the cell death triggered by these agents is apoptosis, an orderly and synchronised process that involves a mitochondrial-dependent, Bcl-2 regulated pathway ending with caspase activation. In this issue of Current Medical Chemistry - Anticancer Agents, five different aspects of tumour cell apoptosis triggered by chemotherapeutic drugs are reviewed.Simone Fulda and Klaus-Michael Debatin explore the contribution of death receptor signalling in cancer therapy. A number of studies support the assertion that anticancer agents induce the formation of a plasma membrane-associated, death inducing signalling complex involving CD95, FADD and caspase-8. Activation of caspase-8 in this complex may contribute to druginduced cell death and negative regulators of this pathway, including caspase-8 down-regulation and overexpression of IAPs (Inhibitor of Apoptosis Proteins) or FLIP contribute to drug resistance at the cellular level. In addition, anticancer drugs sensitise tumour cells to death induced by engagement of death receptors such as CD95, DR4 and DR5, which suggests that death receptor agonists could be used in combination with common anticancer drugs to improve their efficacy. The DR4- and DR5-ligand known as TRAIL (Tumor necrosis factor-Related Apoptosis-Inducing Ligand), whose toxicity is limited in preclinical studies, may be a good candidate for such a combination.Christine Bezombes et al analyse the role of raft microdomains in death response to DNA damaging agents such as cytarabine and daunorubicin. A ceramide-mediated pathway is initiated by these agents through the activation of a sphingomyelinase that hydrolyses sphingomyelin to produce ceramide. This pathway leads to a protein kinase cascade that contributes to apoptosis. The spatio-temporal organisation of sphingomyelinase activation is discussed with a special attention to the role of lipid microdomains in the plasma membrane. Defects in rafts could contribute to cellular drug resistance and pharmacological manipulation of this lipid / kinase pathway could increase cancer cell chemosensitivity.Olivier Sordet et al focus on connections between topoisomerase inhibitor-induced DNA damage and apoptotic pathways. These drugs generate cleavage complexes that are converted into DNA lesions during DNA replication and transcription. Sensor protein kinases such as DNA-PK (DNA-dependent protein kinase), ATM (Ataxia Telangiectasia Modified) and ATR (Ataxia Telangiectasia and Red 3-modified) bind to DNA breaks, then phosphorylate downstream substrates such a c-Abl and Chk2. The authors provide nice molecular interaction maps, demonstrating that the next challenge will be to elucidate how manipulation of these connected pathways will improve the efficacy of these anticancer agents.Another target for anticancer drugs is tubulin. Taxanes are commonly used antineoplastic agents that promote microtubule polymerisation and inhibit tubulin depolymerisation. Valérie Ganansia-Leymarie et al summarise the protein kinase signalling pathways activated by these anticancer drugs, leading to caspase-dependent apoptotic cell death. Bcl-2 has been shown to be phosphorylated at serine residues between BH3 and BH4 regions by microtubule-targeting drugs and this phosphorylation was suggested to contribute either to cell death or cell cycle arrest.Proteins of the Bcl-2 family are key regulators of apoptosis. Whether they act at the mitochondrial level by preventing the release of soluble molecules or upstream of these mitochondrial events is currently a controversial issue. Whatever the exact answer to this question, anti-apoptotic proteins of the Bcl-2 family are attractive targets for a chemosensitising strategy. Current strategies to target Bcl-2 proteins are explored by Ali Bettaïeb et al who analyse the current clinical results obtained with an antisense oligonucleotide strategy and report that alternative approaches are currently developed. Altogether, these papers summarise a large piece of data demonstrating that understanding of cell death mechanisms has opened numerous opportunities to improve the clinical use of classical anticancer agents.

Apoptosis, the cell's intrinsic death program, is a key regulator of tissue homeostasis. An imbalance between cell death and proliferation may result in tumor formation. Also, killing of cancer cells by cytotoxic therapies, such as chemotherapy, γ-irradiation or ligation of death receptors is predominantly mediated by triggering apoptosis in target cells. Death receptor signaling pathways have been implied to contribute to the efficacy of cancer therapy. Failure to undergo apoptosis in response to anticancer therapy may lead to resistance. Understanding the molecular events that regulate apoptosis induced by anticancer therapy and how cancer cells evade apoptosis may provide new opportunities for drug development. Thus, novel strategies targeting tumor cell resistance will be based on insights into the molecular mechanisms of cell death.

DNA damaging agents such as 1-β-D-arabinofuranosylcytosine (Ara-C) and daunorubicin (DNR) are widely used in the treatment of acute nonlymphocytic leukemia. These drugs have, of course, been the objects of intense basic research, as well as preclinical and clinical study. Although specific biochemical lesions (DNA damage) have been associated with Ara-C- and DNR-mediated cytotoxicity, the pathways leading to the induction of apoptosis remain ill defined. This standpoint has forced investigators to explore a new concept in cell response to cytotoxic stress: apoptosis signaling. The recent identification of a ceramide (CER) mediated apoptotic signaling pathway triggered by antitumor agents offers a new perspective for the treatment of neoplastic cells. Indeed, these agents have been shown to induce apoptosis through the activation of a sphingomyelinase (SMase) responsible for the hydrolysis of sphingomyelin (SM) and the generation of CER. The latter acts as a potent apoptosis mediator, triggering several downstream signaling pathways among which the stress-activated protein kinase cascade (MEKK1-SEK1-SAP / JNK) plays a critical role in apoptosis induction. However, the spacio-temporal organization of the key early signaling events is unclear. The present review delineates what appears to be a critical factor in apoptosis signaling: sphingomyelin enriched plasma membrane rafts. The apparent topological partitioning between DNA damage and apoptosis signaling (integrated into specialized plasma membrane domains) is discussed.

Topoisomerase inhibitors are among the most efficient inducers of apoptosis. The main pathways leading from topoisomerase-mediated DNA damage to cell death involve activation of caspases in the cytoplasm by proapoptotic molecules released from mitochondria. In some cells, apoptotic response also involves the death receptor Fas (APO-1 / CD95). The engagement of these apoptotic effector pathways is tightly controlled by upstream regulatory pathways that respond to DNA lesions-induced by topoisomerase inhibitors in cells undergoing apoptosis. These include the proapoptotic Chk2, c-Abl and SAPK / JNK pathways, the survival PI(3)kinase-Akt-dependent pathway and the transcription factors p53 and NF-κB. Initiation of cellular responses to DNA lesions-induced by topoisomerase inhibitors is ensured by the protein kinases DNA-PK, ATM and ATR, which bind to DNA breaks. These kinases commonly called “DNA sensors” mediate their effects (DNA repair, cell cycle arrest and / or apoptosis) by phosphorylating a large number of substrates, including several downstream kinases such as c-Abl and the checkpoint protein Chk2. c-Abl induces apoptosis by activating cell death pathways (e.g., SAPK, p53 and p73) and inhibiting cell survival pathways [e.g., PI(3)kinase]. The DNA-damage regulating kinase Chk2, in addition to its role in cell cycle arrest and / or DNA repair, can induce apoptosis by phosphorylation / activation of the promyelocytic leukemia (PML) protein and p53. Finally, we will review the recent observations that support a role for topoisomerases in chromatin fragmentation during the execution phase of apoptosis.

Docetaxel (Taxotere) is a member of the taxane class of anticancer agents to reach clinical use. This semisynthetic analog of paclitaxel (Taxol) is one of the newer potent anti-neoplastic agents now undergoing extensive laboratory and clinical investigations. Several studies indicate that antimicrotubule agents are potent promoters of apoptosis in cancer cells. Cytotoxic mechanisms of antimitotic taxoids are not yet fully understood, but it has been demonstrated that docetaxel increases tubulin polymerisation, promotes microtubule assembly and also inhibits tubulin depolymerisation. Disruption of microtubules results also in the induction of tumor suppressor gene p53 and inhibitor of cyclin-dependent kinases and activation / inactivation of several protein kinases. As a consequence cells are arrested in the G2-M phase of the cell cycle, after which they may either undergo cell death by apoptosis or necrosis or overcome the G2-M stop and continue in the division cycle (often toward a post-mitotic cell death) depending on the tumor cell type. Nevertheless, how docetaxel induces apoptotic cell death or caspases activation is not yet defined. One may assume that taxanes are able to induce the phosphorylation of Bcl-XL / Bcl-2 members and thus inactivate their anti-apoptotic capacities. The down-regulation of Bcl-2 and / or the upregulation of p53 and p21 / WAF-1 are certainly one of the important modes of apoptosis induction by taxanes. The aim of this framework is to summarize the effects of microtubuline targeting agents on apoptotic signal transduction and new molecular pathways. Finally, we will also discuss the potential therapeutic interest in the association of docetaxel and ionizing radiation.

Proteins of the Bcl-2 family share one or several Bcl-2 homology (BH) regions and behave as pro- or antiapoptotic proteins. Prosurvival members such as Bcl-2 and Bcl-XL are supposed to preserve mitochondrial outer membrane integrity, thus preventing the release of soluble apoptogenic molecules. Pro-apoptotic members include BH3- only proteins that act as sensors of cellular damage and initiate the death process and Bax-like proteins that act downstream of BH3-only proteins to permeabilise the mitochondrial outer membrane. Whether BH3-only proteins directly activate Bax-like proteins or prevent prosurvival members of the family from inhibiting Bax-like proteins or both remains a matter of controversy. Expression of these proteins is altered in various human tumours and this abnormal expression may contribute to oncogenesis and tumour cell resistance to anticancer drug-induced cell death. Based on these observations, prosurvival proteins are attractive intracellular targets for inducing tumour cell death or sensitising tumour cells to death induced by chemotherapeutic drugs. The use of 18-mer antisense oligonucleotides (G3139 or Genasense) targeting the first six codons of bcl-2 mRNA is currently developed in clinics with phase I studies demonstrating that thrombocytopenia may be the main dose-limiting side effect. This strategy, that efficiently decreases Bcl-2 protein expression in some tumour cells, is currently tested in phase II and phase III trials. Alternative approaches to achieve the functional knock-out of Bcl-2 include the use of either peptides mimicking the BH3 domain of Bcl-2-related proteins or more stable, non peptidic BH3 mimetics and the pharmacological modulation of the post-translational modifications of the protein.