A Review on Apoptosis in Cancer: From Pathogenesis to Cancer

1.0 Introduction – Role of Apoptosis in Cancer

Apoptosis is a natural, tightly regulated process of programmed cell death vital for development and tissue maintenance. It acts as a defense mechanism by eliminating damaged or dangerous cells, and can be triggered by factors like infections, DNA damage, and cancer treatments. Central to apoptosis are caspase enzymes, activated through internal (mitochondrial) or external (receptor-mediated) pathways. In cancer, tumor cells often avoid apoptosis, helping them survive, multiply, and resist therapy. Some aggressive cancers even show high apoptosis levels, suggesting that dying tumor cells might aid tumor growth under certain conditions. The disruption of the balance between cell division and cell death is a key feature in cancer. For instance, mutations in the tumor-suppressor gene p53, which promotes apoptosis, are common. Additionally, treatments that kill cancer cells may unintentionally promote tumor recovery by altering the surrounding tumor microenvironment (TME). Therefore, effective cancer therapies must not only target tumor cells but also address their complex interactions within the TME to overcome resistance and improve treatment outcomes.[1]

2.0 Morphological Changes in Apoptosis

Apoptosis, or programmed cell death, involves distinct and orderly structural changes in both the nucleus and cytoplasm of the cell. These changes are consistent across various cell types and usually take several hours to complete, depending on the cell type and stimulus.

2.1 Nuclear Changes

• Chromatin Condensation: DNA and proteins (chromatin) condense near the nuclear membrane, forming crescent or ring shapes.
• Nuclear Fragmentation (Karyorrhexis): The condensed chromatin breaks apart, while the cell membrane remains intact—unlike necrosis.

2.2 Cytoplasmic and Cellular Changes

• Cell Shrinkage and Rounding: The cell becomes smaller and rounder (pyknosis).
• Retraction of Pseudopodes: The cell pulls in its projections used for movement.
• Membrane Blebbing: The outer membrane forms bubble-like blebs—a key sign of late-stage apoptosis.
• Ultrastructural Modifications: Internal organelles change in preparation for controlled breakdown.[2]

Importantly, during apoptosis, the cell’s outer membrane stays intact, preventing inflammation and distinguishing it from uncontrolled cell death processes like necrosis.

2.3 Final Stages and Clearance of Apoptosis

In the final phase of apoptosis, the cell breaks into small, membrane-enclosed fragments called apoptotic bodies, which safely contain parts of the nucleus and cytoplasm. These are quickly recognized and removed by nearby phagocytes (immune cells), ensuring no harmful substances leak out. This rapid and clean removal explains why apoptotic bodies are rarely observed under normal conditions.

2.4 Secondary Necrosis

If apoptotic cells are not cleared in time—such as in lab settings without phagocytes—they eventually undergo secondary necrosis, where the membrane breaks down. This process resembles necrosis and may lead to inflammation, unlike the controlled nature of normal apoptosis.

3.0 Biochemical Changes in Apoptosis

Apoptosis involves a series of organized biochemical events that dismantle the cell without causing inflammation. These changes are mainly grouped into three categories: caspase activation, DNA/protein breakdown, and membrane alterations aiding phagocytosis.

3.1 Activation of Caspases

Caspases are key enzymes that get activated during apoptosis. They cleave specific proteins and activate DNA-degrading enzymes, leading to controlled disassembly of the cell’s structure and function.

3.2 DNA and Protein Breakdown

Apoptotic cells show distinct DNA fragmentation. DNA is first broken into large pieces, then into smaller, 180–200 base-pair fragments forming a DNA ladder—a hallmark of apoptosis. However, this pattern is not exclusive to apoptosis and may appear in necrosis too.

3.3 Membrane Changes and Recognition by Phagocytes

A key membrane change is the flipping of phosphatidylserine (PS) from the inner to the outer membrane, signaling phagocytes to engulf the dying cell. This prevents inflammation and supports clean cell removal.[3]

3.4 Limitations and Considerations

While biochemical markers like caspase activity and DNA fragmentation help detect apoptosis, they have limitations. Not all apoptosis follows the same pathways—some may be caspase-independent. Hence, experts recommend emphasizing morphological features for accurate identification of apoptosis.

4.0 Mechanisms of Apoptosis

Fig 01:- The intrinsic and extrinsic pathways of apoptosis [12]

4.1 Extrinsic (Death Receptor) Pathway

This pathway is triggered by external signals, where death ligands (like TNF or FasL) bind to specific death receptors (TNFR1, Fas/CD95) on the cell surface. This binding forms a complex called DISC (Death-Inducing Signaling Complex), which activates caspase 8, an initiator enzyme. Caspase 8 then activates downstream executioner caspases, leading to controlled cell death.

4.2 Intrinsic (Mitochondrial) Pathway

This pathway starts from within the cell, usually due to internal stress like DNA damage, oxidative stress, or calcium imbalance. These stress signals increase mitochondrial membrane permeability, causing the release of cytochrome c and other apoptotic proteins. Cytochrome c helps form the apoptosome complex, which activates caspase 9. The pathway is tightly regulated by the Bcl-2 protein family, where pro-apoptotic members (e.g., Bax, Bak) promote cell death and anti-apoptotic members (e.g., Bcl-2, Bcl-XL) try to prevent it. Additional proteins like Smac/DIABLO and Omi/HtrA2 enhance apoptosis by blocking caspase inhibitors.[4]

4.3 The Common Execution Pathway

Both the extrinsic and intrinsic apoptotic pathways ultimately activate the common execution phase, centered around caspase 3, the key executioner enzyme. Caspase 9 (intrinsic) and caspase 8 (extrinsic) both activate caspase 3, which then cleaves the inhibitor of caspase-activated DNase (CAD), leading to DNA fragmentation—a hallmark of apoptosis. Caspase 3 also targets various other cellular components, including kinases, cytoskeletal proteins, DNA repair enzymes, and more. These cleavages cause structural and functional breakdowns in the cell, such as membrane blebbing, cytoskeleton collapse, and the formation of apoptotic bodies, ensuring an orderly dismantling process.

4.4 The Intrinsic Endoplasmic Reticulum (ER) Pathway

The ER stress-induced pathway is a third, less common apoptotic route, triggered by cellular stress like hypoxia, oxidative damage, or glucose deprivation, which leads to protein misfolding in the ER. This activates caspase 12, independent of mitochondria.[5]. Activation occurs when TRAF2 dissociates from procaspase 12, initiating a cascade that leads to apoptosis. This ER pathway provides an additional layer of regulation, allowing cells to respond to internal stress not directly involving mitochondria.

5.0 Apoptosis and Carcinogenesis

Apoptosis plays a crucial role in preventing cancer by eliminating damaged or abnormal cells. However, cancerous cells often evade apoptosis, enabling unchecked growth and survival. This evasion is considered a hallmark of cancer and occurs through several mechanisms:

Fig 02:-Mechanisms contributing to evasion of apoptosis and carcinogenesis.[13]

2.Reduced caspase activity

3.Defects in death receptor signalling

These changes allow malignant cells to resist death signals, persist, and accumulate further mutations.

5.1.1 Disrupted Balance of Pro-apoptotic and Anti-apoptotic Proteins

A key regulatory group in apoptosis is the Bcl-2 family, which includes both anti-apoptotic and pro-apoptotic members. The relative balance, rather than absolute levels, of these proteins determines a cell's fate.

• Anti-apoptotic proteins (e.g., Bcl-2, Bcl-xL, Mcl-1) prevent mitochondrial membrane permeabilization.
• Pro-apoptotic effector proteins (e.g., Bax, Bak) promote apoptosis by increasing membrane permeability.
• BH3-only proteins (e.g., Bid, Bim, Puma) sense stress and initiate apoptosis.

Cancer cells often overexpress anti-apoptotic proteins (e.g., Bcl-2 in prostate cancer, Bcl-xL in drug-resistant tumors) or downregulate pro-apoptotic ones (e.g., Bax mutations in colorectal cancer), leading to reduced apoptosis and therapy resistance.[6]

5.1.2 p53

p53, known as "the guardian of the genome," is a tumor suppressor protein that regulates the cell cycle and apoptosis in response to DNA damage. The TP53 gene is mutated in over 50% of cancers, impairing apoptosis and enabling tumor progression.

• Mutant p53 can lose its protective function or gain oncogenic activity.
• Aberrant p53 is linked to enhanced cell survival, reduced apoptosis, and increased inflammation in cancers like melanoma.
• Targeting mutant p53 in some cancers can restore apoptosis and reduce tumor growth.

5.1.3 Inhibitor of Apoptosis Proteins (IAPs)

IAPs are a family of proteins that block apoptosis by inhibiting caspases directly or promoting their degradation. They contain BIR domains that bind caspases and prevent cell death.

• Overexpression of IAPs in cancer cells helps them resist apoptosis.
• Targeting IAPs is an emerging strategy for sensitizing cancer cells to therapy.

5.2 Reduced Caspase Activity

Caspases are critical enzymes involved in apoptosis and inflammation. In cancer, reduced expression or loss of caspase activity impairs apoptosis, allowing abnormal cells to survive, proliferate, and resist treatment—contributing to carcinogenesis.

Types of Caspases:

• Inflammation-related caspases: Caspase-1, -4, -5, -13, -14 (cytokine processing)
• Apoptosis-related caspases:
  • Initiator caspases: Caspase-2, -8, -9, -10 (trigger apoptosis)
  • Effector caspases: Caspase-3, -6, -7 (execute apoptosis)

Clinical Evidence of Caspase Downregulation in Cancer:

• Caspase-9 is frequently downregulated in stage II colorectal cancer, correlating with poor outcomes.
• Caspase-3 is absent or significantly reduced in breast, cervical, and ovarian cancers (e.g., MCF-7 breast cancer cells). Restoring caspase-3 reactivates apoptosis and increases treatment sensitivity.
• Caspase-8 and -10 are simultaneously downregulated in choriocarcinoma, severely disrupting apoptotic signaling.[7]

5.3 Impaired Death Receptor Signaling

The extrinsic pathway of apoptosis is initiated by death receptors located on the cell surface, which respond to external death signals (ligands) like FasL, TNF, and TRAIL. These receptors include TNFR1, Fas (CD95), DR3, DR4, DR5, and others, each containing a death domain that activates downstream caspases to induce apoptosis.

Mechanism of Normal Death Receptor Function:

• Binding of death ligands triggers the formation of Death-Inducing Signalling Complex (DISC).
• This activates initiator caspases, leading to cell death.

Mechanisms of Impairment in Cancer:

1. Downregulation or Loss of Receptor Expression:
   • Lower levels of receptors like CD95 reduce apoptosis sensitivity.
   • Found in treatment-resistant leukemia and neuroblastoma.
2. Overexpression of Decoy Receptors:
   • Decoy receptors mimic real receptors but lack death domains, blocking the apoptotic signal.
3. Mutations or Defects in Death Receptors:
   • Even if receptors are present, mutations in their structure can prevent DISC formation and signal transmission.
4. Reduced Death Ligands:
   • Decreased availability of FasL or TRAIL weakens the extrinsic apoptotic pathway.[8]

6.0 Targeting the Bcl-2 Family in Cancer Therapy

1. Antisense Inhibitors

• Oblimersen sodium (G3139):
  A synthetic antisense oligonucleotide that binds to Bcl-2 mRNA, preventing its translation and leading to decreased Bcl-2 protein levels.
• Effect: Sensitizes cancer cells to chemotherapy by promoting apoptosis.

2. Small Molecule Inhibitors

• Examples: Gossypol, TW-37, ABT-737, ABT-263 (Navitoclax)
• Mechanism: These compounds directly bind and inhibit anti-apoptotic Bcl-2 family proteins, allowing pro-apoptotic proteins to initiate cell death.

3. BH3 Mimetics

• Function: Imitate BH3-only proteins, displacing pro-apoptotic proteins from Bcl-2 complexes.
• Result: Facilitates mitochondrial outer membrane permeabilization (MOMP), leading to apoptosis.

4. Gene Silencing

• Approach: Use of small interfering RNAs (siRNAs) to knock down expression of Bcl-2 and related genes (e.g., Bmi-1).

Impact: Increases cancer cell susceptibility to apoptosis and reduces therapy resistance.

6.1Targeting Apoptosis in Cancer Therapy

Apoptotic pathways, when disrupted, not only contribute to cancer development but also offer promising targets for treatment. Defects or abnormalities within these pathways can be exploited therapeutically—by designing drugs or interventions that restore normal apoptotic signaling, it becomes possible to selectively eliminate cancer cells that rely on such defects for survival. Recent advances in research have led to the discovery of novel anticancer agents that specifically target these apoptotic dysfunctions[14]. This section focuses on emerging treatment strategies aimed at correcting the apoptotic defects discussed earlier in Section 3. For a detailed overview of these drugs and approaches, please refer to Table 1.