Apoptosis: The Deadly Process Manipulated by Novel Therapeutics

What is Apoptosis?

Apoptosis is a biological mechanism that ultimately results in programmed cell death. With around 10 billion adult cells undergoing cell death every day, apoptosis is a natural part of the body’s homeostasis, allowing for new cells to arise. It is also a vital part of an organism’s development, aging process, and defense mechanism to eliminate diseased or damaged cells from the body. Although this process is ‘deadly’ to cells it is an essential and useful process in our body. So much so, if this highly regulated process is dysregulated, diseases may arise. For example, increased apoptosis has been found in AIDs and neurodegenerative diseases such as Alzheimer’s, whereas reduced apoptosis has been associated with autoimmune disorders and cancer. Research into how apoptosis contributes to specific diseases is paramount and may allow scientists to control a cell’s fate. This scientific manipulation offers significant therapeutic potential, with even natural products offering their therapeutic services. Find out more about the process of apoptosis and how it can be manipulated by disease and therapeutics below.

Figure 1: The extrinsic and intrinsic pathways of apoptosis, linked by the activation of Bid by caspase 8 in the extrinsic pathway.

Two Main Mechanisms of Apoptosis

The intrinsic and extrinsic pathways are two mechanisms by which apoptosis can occur. While the extrinsic pathway depends on receptor activation by extracellular signals, the intrinsic pathway can be described as being mediated by the mitochondria. Despite this, both pathways share a commonality, the use of caspases. Caspase enzymes must first be activated by either the intrinsic or extrinsic pathway. Only then can they go onto cleave the C-terminal side of aspartate residues located on their substrates. Caspases can even cleave and activate other caspases to amplify the apoptotic signal. It is largely recognized that once caspases have been activated, the process of apoptosis is irreversible, and cell death is inevitable.

Intrinsic Pathway

Figure 2: Schematic of apoptosis activation by the intrinsic pathway

The mammalian intrinsic pathway, also known as the mitochondrial-mediated apoptotic pathway, is activated upon extracellular and intracellular stress, such as irradiation, cytotoxic drugs, and oxidative stress. In response to this signal, there is a p53-dependent activation of the BCL-2 family proteins Bax and Bak, which are inserted into the mitochondrial membrane, allowing for the release of cytochrome c from the mitochondria. At the same time, the anti-apoptotic Bcl-2 family proteins, BCL-2 and BCL-xL, are inhibited. The release of cytochrome c is a critical event in forming a structure called the apoptosome, which comprises Apaf-1, procaspase-9, and cytochrome c. Cytochrome c promotes the heptamerization of the Apaf-1 protein so that it binds to procaspase-9 to form the apoptosome. Only once procaspase-9 has been activated can downstream caspases become functional, such as caspase 3. For this reason, procaspase-9 is termed an initiator caspase, whereas those downstream are termed effectors caspases. These effector caspases carry out the degradation of the cell. In mammals, the activation of caspases in the intrinsic pathway can be inhibited by Inhibitor of Apoptosis Proteins (IAPs), which are produced when IAP antagonists such as Smac/Diablo are expressed. Both BCL-2 and IAPs regulate the intrinsic pathway in mammals.

Extrinsic Pathway

The extrinsic pathway is activated in response to extracellular signals, which activate death receptors such as Fas/CD95 and the Tumour Necrosis Factor α (TNFα). These death receptors are located on the cell’s surface and activate caspases to stimulate apoptosis.

Figure 3: Schematic of apoptosis activation by the extrinsic pathway

Extracellular Fas ligands (FasL) bind to a death receptor domain (DR) located extracellularly on the Fas receptors. This initiates the oligomerization of the death receptors. Using a death domain (DD), the now-activated death receptors bind to an adaptor molecule known as the Fas-associated death domain (FADD). FADD has a death effector domain (DED), which it uses to recruit the initiator procaspases -8 and -10, which also have a DED. This FAS, FADD, procaspase – 8, and -10 complex is termed the death-inducing signaling complex (DISC).

Within this DISC complex, procaspase-8 and procaspase-10 become activated by autoproteolytic cleavage, meaning they can activate downstream effector caspases. Specifically, Caspase 8 goes on to cleave effector caspases 3, 6, and 7 and Bid (a BCL-2 protein family member that is proapoptotic). Bid then activates Bax and Bak, which oligomerize to activate mitochondrial outer membrane permeabilization (MOMP) and apoptosis (Figure 1). At the same time, activated Bid also prevents BCL-2 and BCL-xL from inhibiting the oligomerization of Bax and Bak. MOMP releases cytochrome c, Smac/Diablo, and Htra2/Omi from the mitochondria, all of which are proapoptotic.

Execution Pathway

The intrinsic and extrinsic pathways feature the execution pathway, the final stage of apoptosis. In this phase, effector caspases, also called executioner caspases, activate cytoplasmic endonuclease, which inflicts chromatic condensation and forms apoptotic bodies and cytoplasmic blebs. Simultaneously the nuclear and cytoskeletal proteins are degraded. Caspase 3 is one of the most important executioner caspases. Any of the initiator caspases can activate Caspase 3, and it exclusively activates endonuclease Caspase-activated DNAse for chromosomal DNA degradation within nuclei. The condensation of chromatin results in cell shrinkage and pyknosis, key morphological features of apoptosis that can be visibly detected using light and electron microscopy. Plasma membrane blebbing occurs, and karyorrhexis
and cell fragments are separated into apoptotic bodies. These apoptotic bodies comprise an intact plasma membrane in a cytoplasm with tightly packed organelles. Apoptotic bodies are ultimately phagocytosed and degraded by macrophages, neoplastic cells, or parenchymal cells. 

What are the Consequences of Apoptotic Dysregulation?
Apoptosis is crucial in preventing diseases by balancing cell death and survival. When apoptosis is dysregulated, it can result in autoimmune disease and cancer. Within the central nervous system, apoptosis dysregulation is also associated with lateral sclerosis, Alzheimer’s, Parkinson’s, and Huntington’s diseases. Generally, the apoptosis rate increases in immunodeficiency, infertility, and acute and chronic degenerative disease, whereas reduced apoptosis is found in autoimmune diseases and cancer. It has also been found that reducing the fas-mediated apoptosis pathway can lead to increased cell proliferation, as seen in hepatocellular carcinoma. On the other hand, increased fas-mediated apoptosis has been linked to Wilson’s disease, hepatic fibrosis, plus viral and alcoholic hepatitis.

Apoptosis and Neurodegenerative Disease

In neuronal cell death, the balance of pro-apoptotic and anti-apoptotic Bcl-2 family proteins has been shown to play a role. Pro-apoptotic being those such as BCL and BCL-w, and anti-apoptotic being those like Bad, Bim, and Bax. Furthermore, these proteins may alter vulnerable neurons in Alzheimer’s disease. In Huntington’s disease, caspase 3 is involved in the cleavage of huntingtin, and its activity is important for oligomeric Aβ-mediated facilitation of Alzheimer’s disease.

Apoptosis and Cancer

Cancerous cells have developed mechanisms to evade apoptosis, including upregulation of IAPs, decreased caspase expression, p53 mutations, and a misbalance of BCL-2 family proteins and receptor signaling pathways. One noticeable feature seen in over half of cancers is the overexpression of the BCL-2 protein. This provides cancer cells with resistance to intrinsic apoptotic stimuli and some cancer therapeutics. In turn, cancer cells survive, mutate and fulfill the hallmarks of cancer: uncontrolled tumor growth and angiogenesis.

It is possible to target both the intrinsic and extrinsic apoptosis pathways to stop the uncontrolled growth of cancer cells. Therefore, therapeutics are being developed that either stimulate proapoptotic proteins or inhibit antiapoptotic proteins. Researchers have found potential targets such as BCL-2 inhibitors, ligands for death receptors, and IAP inhibition, but it is yet to be determined which is most effective. Many traditional cancer therapeutics rely on BCL-2/BAX-dependent mechanisms. However, chemoresistance occurs if this mechanism is disrupted. Below are some of the ways the process of apoptosis can be exploited to treat cancer.

Targeting the BCL-2 family:

• Oblimersen sodium was one of the first drugs to target the BCL-2 pathway and was found to increase sensitivity to chemotherapy in chronic lymphocytic leukemia patients.
• ABT-263, also known as navitoclax, was found to target BCL-2 expression, and when used alongside doxorubicin or dinaciclib, apoptosis was induced in small cell lung cancer cell lines.
• In 2016, the FDA approved a BH3 mimetic anti-cancer drug for treating chronic lymphocytic leukemia called venetoclax

Targeting the tumor suppressor gene p53 (a tumor inhibitor and proapoptotic protein):

• Many cancer treatments aim to activate p53 or restore p53 function in cases where it has become mutated. Tumor progression often occurs in the event p53 has become mutated.
• One study used the CRISPER/Cas technology to replace, within the human genome, a mutated p53 gene with a functional p53 gene. This showed great potential in inducing apoptosis

The use of death receptor agonists:

• Another example of death receptors are DR4 and DR5 whose extracellular ligand is Apo2L/TRAIL (TNF-Related Apoptosis-Inducing Ligand). DR4 and DR5 agonists induce apoptosis in cancer cells without inflicting damage on normal cells.

Targeting the Apoptosis-inhibitor proteins (IAPs):

• In some cancers, IAPs are overexpressed. Scientists have therefore been investigating the use of IAP antagonists that are naturally available such as SMAC/Diablo and HtrA2/Omi, to bind and inhibit IAPs. Synthetic IAP protein inhibitors have been developed, and these include SM164, AZD5582 and YM155.

Manipulation of Apoptosis Using Natural Products

Due to their non-toxic nature, researchers are turning to natural products to treat cancer. Products like Black Cohosh from Actaea racemose and genistein can induce apoptosis in cancer cells without harming healthy cells. For example, Graviola, a fruit tree, can inhibit BCL-2 proteins and increase BAX and hence apoptosis. Quercetin and Aloe-emodin, from Rheum palmatum, have been found to stimulate caspases. Finally, Curcumin has shown the ability to suppress tumor cells by acting on various pathways such as caspase activation, cell proliferation, and pathways such as death receptor, a tumor suppressor, and mitochondrial pathways. Curcumin inhibits BCL-2 and IAP, increasing BAX and BAK expression. Furthermore, it enhances mitochondrial membrane permeability. Further research needs to be undertaken to understand the anticancer properties of these natural products.
Figure 4: Rheum palmatum from which the natural products Quercetin and Aloe-emodin are derived

Biosynth Products for Research and Detection of Apoptosis

At Biosynth, we take pride in supplying critical raw materials to projects researching, diagnosing, and developing therapeutics for apoptosis-associated diseases. We recognize the importance of further understanding the process of apoptosis; that is why we offer a wide range of custom and catalog products to research, detect, and manipulate the apoptotic process. For the detection of key apoptosis markers, we have our Annexin V Apoptosis Detection Kit and the following ELISA kits:

• BCL2
• Annexin V
• Human Bax
• Human FAS
• Human FASL
• Human p53
• Human TNF alpha
• Human TRAIL
• Mouse FAS

We also have a selection of caspase, BCL-2, Bax, Bid, Apaf-1, p53, cytochrome C, COX4, Smac/Diablo, TNF alpha, Fas, phosphatidylserine, peptide, and antibody products for many research applications. In addition, we have dyes, inhibitors of IAPs, molecules targeting BCL-2, and natural products to induce apoptosis. Use our research products search bar on our website to search for apoptosis-related products for your project. We offer custom peptides and antibodies if you cannot find what you want in our research catalog.

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References

Abd-Elhamid, R. A., Nazmy, M. H., Fathy, M. (2020). Targeting Apoptosis as a Therapeutic Approach in Cancer. Minia Journal of Medical Research, 31(2): 321-334.

Cairrão, F., Domingos, P. M. (2010). Apoptosis: Molecular Mechanisms. Encyclopedia of Life Sciences.

Carneiro, B. A., El-Deiry, W. S. (2021). Targeting apoptosis in cancer therapy. Nature Reviews Clinical Oncology. 17(7), 395-417

Elmore, S. (2007) A Review of Programmed Cell Death. Toxicologic Pathology, 35(4), 495-516.

Guo, M., Lu, B. Gan, J., Wang, S., Jiang, X., Li, H. (2021) Apoptosis detection: a purpose-dependent approach selection. Cell Cycle, 20(11), 1033-1040.

Jan, R., Chaudhry, G. (2019). Understanding Apoptosis and Apoptotic Pathways Targeted Cancer Therapeutics. Advanced Pharmaceutical Bulletin, 9(2), 205-218.

Pfeffer, C. M., Singh, A. T. K. (2018) Apoptosis: A Target for Anticancer Therapy. International Journal of Molecular Sciences, 19(2), 448.

Renehan, A. G., Booth, C., Potten, C. S. (2001). What is apoptosis, and why is it important? British Medical Journal, 322(7301), 1536-1538.