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Targeting Apoptotic Pathways to Develop Anticancer Therapy

Posted By Arun Kumar
Tags: Apoptosis, Cancer

Apoptosis is a highly ordered and orchestrated cellular pathway that occurs under normal physiological and pathological conditions. This evolutionarily conserved process has crucial roles that range from tissue sculpting during embryonic development to execution of immune effector functions. Apoptosis, also known as Type 1 Programmed Cell Death (PCD) plays a critical role for maintaining homeostatic balance between the rates of cell proliferation and cell death. Other major types of PCD that also serve to trigger cell death are type II (autophagy) and type III (necrosis). The past decade has witnessed a steady accumulation of findings suggesting that apoptosis, necrosis and autophagy are often regulated by similar pathways, engage common sub-cellular sites and organelles, and even share initiator and effector molecules. Defective or inefficient apoptosis is a factor in many human conditions including neurodegenerative diseases, ischemic damage, autoimmune disorders and many types of cancer. An understanding of the underlying mechanism of apoptosis is important as it plays a pivotal role in the pathogenesis of many diseases. In certain disease conditions, the problem is due to too much apoptosis, such as in the case of degenerative diseases. Whereas, there is too little apoptosis with cancer, resulting in malignant cells that are not targeted for cell death. Apoptotic cell death-based therapy has received attention for the development of anticancer drugs. The mechanism of apoptosis is complex and involves many pathways. Defects can occur at any point along these pathways, leading to malignant transformation of the affected cells, tumor metastasis and resistance to anticancer drugs. Thus, a thorough understanding of apoptotic signaling pathways and insights into apoptosis resistance mechanisms are imperative to unravel novel drug targets for effective selective therapeutic strategies. At Enzo, we offer a complete set of tools for advancing apoptosis research including antibodies, activators and inhibitors, enzyme assays, and recombinant proteins.

Mechanisms of Apoptosis

Activation of caspase is a hallmark of apoptosis. Caspases are a family of conserved cysteine proteases that play a central role in apoptosis as both the initiators and executioners. Activated caspases cleave many vital cellular proteins and break up the nuclear scaffold and cytoskeleton. They also activate DNAase, which further degrades nuclear DNA. The removal of apoptotic cells is the final step in the execution of apoptosis. Historically, this process has presented a significant hurdle in evaluating the amount of apoptosis, as the dying cells are rapidly cleared by phagocytes, making it very difficult to detect apoptotic cells in animal tissues. The engulfment process can be divided into the following stages: (1) sensing of the apoptotic cell, (2) recognition by the phagocyte, (3) internalization of target cell, (4) ingestion, and (5) post engulfment response of the phagocyte. Dying apoptotic cells secrete “find me” and “eat me” signals that attract and recruit phagocytes.
Once caspases are activated, there seems to be an irreversible commitment towards cell death. Mammalian caspases can be subdivided into three functional groups: initiator caspases (caspase-2, -8, -9 and -10), executioner caspases (caspase-3, -6 and -7), and inflammatory caspases (caspase-1, -4, -5, -11 and -12). There are three pathways by which caspases can be activated. The two commonly described initiation pathways are the intrinsic (or mitochondrial) and extrinsic (or death receptor) pathways of apoptosis. Both pathways eventually lead to a common pathway or the execution phase of apoptosis. Enzo provides a range of caspase assays, enzymes, inhibitors and antibodies for your caspase research needs.

Figure 1: Caspase-1 activity is stable for at least 50 min. under the conditions of the YVAD-pNA cleavage assay. [YVAD-pNA]=200 μM and 20 μM; 25°C.

The Extrinsic Death Receptor Pathway

Activation of the extrinsic cell death pathway occurs following the binding on the cell surface of “death receptors” (DRs) to their corresponding ligands such as Fas (CD95), TNF receptor (TNFR1) and TNFR, or TRAIL. The DRs have two distinct signaling motifs: death domains (DD) and death effector domains (DED) that allow them to interact and recruit other adaptor molecules, such as FAS-associated death domain protein (FADD) and caspase-8, which can then directly cleave and activate caspase-3 and caspase-7, leading to apoptosis. Death receptor mediated apoptosis can be inhibited by a protein called c-FLIP which bind to FADD and caspase-8, rendering them ineffective.

Figure 2: Inhibition of Caspase-3 by Ac-DEVD-CHO. The enzyme was incubated with the inhibitor for 10 minutes prior to addition of substrate. 30 U/well; [DEVD-CHO]=0.1 μM; [DEVD-pNA]=200 μM; 25°C

The Intrinsic Mitochondrial Pathway

The intrinsic pathway is initiated by intracellular signals that act directly on targets within the cell and are mitochondria-initiated events. Internal stimuli such as irreparable genetic damage, hypoxia, extremely high concentrations of cytosolic Ca2+ and severe oxidative stress are some triggers of the initiation of the intrinsic mitochondrial pathway. The key step in the intrinsic cell death pathway is permeabilization of the mitochondrial outer membrane and the release of pro-apoptotic molecules such as cytochrome- c into the cytoplasm. This has been identified as a ‘point of no return’ after which cells are committed to cell death. This pathway is closely regulated by a group of proteins belonging to the Bcl-2 family.

There are two main groups of the Bcl-2 proteins, namely the pro-apoptotic proteins (e.g. Bax, Bak, Bad, Bcl-Xs, Bid, Bik, Bim and Hrk) and the anti-apoptotic proteins (e.g. Bcl-2, Bcl-XL). While the anti-apoptotic proteins regulate apoptosis by blocking the mitochondrial release of cytochrome-c, the pro-apoptotic proteins act by promoting such release. It is not the absolute quantity but rather the balance between the pro- and anti-apoptotic proteins that determines whether apoptosis will be initiated.

Figure 3: A typical standard curve for Human Total Bcl-2 Concentration (pg/ml)

Apoptosis and Carcinogenesis

Cancer is viewed as the result of a succession of genetic changes during which a normal cell is transformed into a malignant one while evasion of cell death is one of the essential changes in a cell that cause this malignant transformation. Generally, the mechanisms by which evasion of apoptosis occurs can be broadly divided into: 1) disrupted balance of pro-apoptotic and anti-apoptotic proteins, 2) reduced caspase function and 3) impaired death receptor signaling. Many proteins have been reported to exert pro- or anti apoptotic activity in the cell. The Bcl-2 family of proteins is comprised of pro-apoptotic and anti-apoptotic proteins that play a pivotal role in the regulation of apoptosis, especially via the intrinsic pathway as they reside upstream of irreversible cellular damage and act mainly at the mitochondria level. When there is disruption in the balance of anti-apoptotic and pro-apoptotic members of the Bcl-2 family, the result is dysregulated apoptosis in the affected cells. This can be due to an overexpression of one or more anti-apoptotic proteins or an under expression of one or more pro-apoptotic proteins or a combination of both. Overexpression of Bcl-xL has also been reported to confer a multi-drug resistance phenotype in tumor cells and prevents them from undergoing apoptosis.
Other abnormalities include downregulation of the receptor or impairment of receptor function as well as a reduced level in the death signals, all of which contribute to impaired signaling and hence a reduction of apoptosis. For instance, down regulation of receptor surface expression has been indicated in some studies as a mechanism of acquired drug resistance. A reduced expression of CD95 was found to play a role in treatment-resistant leukemia or neuroblastoma cells.

Targeting Apoptosis in Cancer Treatment

Drugs or treatment strategies that can restore the apoptotic signaling pathways towards normality have the potential to eliminate cancer cells, which depend on these defects to stay alive. One good example of these agents is the drug oblimersensodium, which is a Bcl-2 antisense oligomer, the first agent targeting Bcl-2 to enter clinical trial. Several drugs have been designed to synthetically activate caspases. Small molecules, caspase activators, have also been designed and found to have lower the activation threshold thereby activating caspase, contributing to an increase in drug sensitivity of cancer cells. In addition to caspase-based drug therapy, caspase-based gene therapy has been attempted in several studies. For instance, human caspase-3 gene therapy was used in addition to etoposide treatment in liver tumor model studies to induce extensive apoptosis and reduce tumor volume. Apoptosis-targeted cancer therapy has been an indispensable approach in combating cancer. As research further defines the intricate nuances underlying apoptotic pathways and the ways in which they are dysregulated in cancer, our ability to design effective therapies targeting such mechanisms grows.

Enzo Life Sciences provides a wide variety of products for monitoring cell death, drug discovery and cancer research. Our decades of experience in the design and manufacture of active enzymes, small molecules and their substrates supports development of an ever-expanding portfolio of biochemical assays. Please check out our Successful Research Tips or feel free to contact our Technical Support Service for further assistance.

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