Lecture 12: Mechanism of Action of Chemotherapeutic Agents (I)

What is Chemotherapy?

• The use of cytotoxic drugs to kill cancer cells or inhibit their proliferation

• Primarily target rapidly dividing cells → interfere with DNA replication, mitosis or other cell division processes

  • Some can also affect non-dividing cell (cells in a resting state).

What are the 3 main approaches to treating cancer?

• The approach will depend on the type and stage of the tumour.

• This includes:

  • Surgical excision

  • Chemotherapy

  • Radiation

• These approaches are typically used in combination with each other

What are the clinical contexts in which chemotherapy is used?

• 1. Induction therapy: Initial intensive treatment to induce remission

  • Example: Used in AML

• 2. Adjuvant therapy: Aims to eradicate any microscopic residual disease, following primary treatment, to reduce the risk of recurrence

  • Example: Used following surgery (e.g. mastectomy) to reduce the risk of recurrence in breast cancer

• 3. Curative-Intent therapy: Use of chemotherapy with the intent of eradicating the disease completely

  • Example: R-CHOP for diffuse large B-cell lymphoma

• 4. Palliative Therapy: Aims to relieve symptoms/ slow progression, and improve quality of life

  • Used in advanced/ metastatic disease

What is the cell cycle and how is it altered in tumour cells?

• The process by which a somatic cell duplicates its content to divide

• Controlled by checkpoints that regulate progression

• Cells can exit the cell cycle, entering G0 (resting phase)

• Normal checkpoints ensure the cell proliferates as expected (regulated proliferation)

• In tumour cells: there is a loss of normal checkpoint control → disrupted cell cell cycle progression → dysregulated proliferation

What are the 4 Major Phases of the Cell Cycle?

• G1: Cell growth and preparation for DNA synthesis

• S phase: DNA synthesis/replication

• G2: Further (rapid) growth and preparation for mitosis

• M phase (Mitosis): Separation of replicated DNA and cell division

What are the major cell cycle checkpoint?

• G1/S checkpoint (Before S Phase): Commit to DNA replication

• G2/M checkpoint (After S Phase): Controls entry into mitosis

• Mitotic spindle checkpoint: Ensures correct chromosome separation

• Control points that regulate progression through the cell cycle

How Do Cancer Cells Grow?

• Cancer cells do not divide faster than normal cells

• Some tumours divide slowly (e.g. plasma tumour cells) compared to fast-dividing tissues (e.g. bone marrow, hair follicles or GIT epithelium)

• Primary characteristic of cancer cells: dysregulated proliferation due to defects in the cell cycle

• Tumour growth results from the imbalance between proliferation and cell loss

How Do Chemotherapeutic Agents Interact With The Cell Cycle?

• Some drugs only act in specific cell cycle phases, e.g.

  • anti-metabolites act only on S-phase

  • Taxanes act only on the M-phase

• Others (Phase Non-Specific Drugs) can act in any phase

How Do Non-Cell Cycle Specific Drugs Act?

• Damage cells in any phase of the cell cycle

  • Cytotoxicity is greater in proliferating cells than in resting (G₀) cells (as expected)

• Effect is dose/concentration dependent

  • Implications for the dosing and scheduling of treatment for the patient

• Includes:

  • Alkylating agents

  • Platinum complexes

  • Anthracycline antibiotics

How Do Cell-Cycle Specific Drug Act?

• Target cells at specific phases of the cell cycle

• Effect dependent on the duration of exposure and the fraction of cells entering the relevant phase

• Dose-dependent effect not important → once a threshold is reached, all the cells in the cycle are killed

  • Must not exceed the threshold → kills all cells and results in diminished return (increased side effects without tumour-specific effects)

What is Platinum Based Chemotherapy?

• A cell-cycle non-specific chemotherapeutic agent

• Informally referred to as ‘platins → characterised by the presence of platinum metal aton

• Examples:

  • Cisplatin (no alkyl group)

  • Carboplatin

  • Oxaliplatin:

Give Three Examples of Platinum-Based Chemotherapy

• Similar core mechanisms, but each differs in toxicity profile

• Cisplatin (no alkyl group)

  • Dose-limiting nephrotoxicity effects (kidney damage)

  • Moderate neurotoxicity

  • High otoxoticity

• Carboplatin

  • High risk of bone marrow suppression (myelosuppression)

• Oxaliplatin:

  • Severe peripheral neuropathy (neurotoxicity)

  • Moderate myelosuppression (e.g. neutropenia)

(MoA) How Does Cisplatin Enter Cells?

• In the bloodstream, cisplatin has two chloride ions attached to the platinum atom

  • Neutrally charged as the bloodstream has a high concentration of Cl- ions

• Entry into the cells is facilitated by transport-mediated uptake and passive diffusion.

  • Cisplatin is small and neutrally charged so enters cells via passive diffusion across the lipid bilayer

  • Transport-mediated uptake facilitated by the CTR1 copper influx transporter

(MoA) How is Cisplatin Activated?

• Intracellular compartment has a lower [Cl^-] compared to the blood → Cl- ions lost

• Intracellular displacement of the leaving group (Cl) attached to Pt by water molecules

• Cisplatin undergoes aquation, generating a reactive (positively charged) Pt species that binds to the negatively charged DNA

(MoA): How Does Cisplatin Kill Cells?

• Preferentially binds to the N7 position of guanine residues of DNA

• Predominantly forms intrastrand (DNA) crosslinks (bonds between bases on the same strand (adducts)) → distorts DNA helix and blocks replication

  • Interstrand links are also possible, but less common

• DNA damage triggers apoptosis via p53-dependent and independent pathways

How Does Cisplatin Differ to Oxaliplatin and Carboplatin?

• Cisplatin’s leaving group undergoes aqueation

• Oxaliplatin and carboplatin leaving groups undergo hydrolysis

Why are some tumours resistant to cisplatin?

• Resistance can occur either intrinsically (before treatment) or acquired during therapy

• There are two main reasons for this resistance

  • Failure to achieve a sufficient amount of platinum reaching the DNA

  • Failure to achieve cell death after platinum DNA cross-link formation

How can failure to achieve sufficient platinum at DNA lead to Cisplatin resistance?

• This can be due to 3 main reasons (never just a standalone reason, usually a mix)

• 1. Decreased uptake of the drug

  • Reduced activity/expression of membrane transport systems needed for Pt entry by the tumour

• 2. Increased intracellular detoxification

  • Elevated levels of sulphur-rich molecules, e.g. glutathione, bind to the reactive Pt species and form inactive complexes

• 3. Enhanced efflux/sequesteration mechanisms, e.g. upregulation of copper-exporting ATPases → lowers the effective intracellular Pt concentration

How can failure to achieve cell death after platinum-DNA crosslink/adduct formation lead to Cisplatin resistance?

• This can be due to 3 main reasons:

  • Increased repair of Pt-induced DNA lesions:

    • Upregulation of nucleotide excision repair system (e.g. ERCC1)

    • Increased removal of intrastand adducts (before they become lethal) and reduced cytotoxicity

  • Increased tolerance to DNA damage

    • Defects in mismatch repair

    • DNA damage is not properly recognised or responded to

    • Pt adducts/lesions bypassed by specialised DNA polymerases

  • Altered apoptosis signalling

    • Loss of p53 function

    • Increased anti-apoptotic proteins, e.g. BCL-2

Why was Oxaliplatin developed as an alternative to Cisplatin?

• To address two major problems with cisplatin:

  • Resistance

  • Severe nephrotoxicity

• Structural changes induced by chemists aimed to:

  • Overcome some resistance mechanisms

  • Reduce certain toxicities

• Toxicity was not eliminated — it was changed rather than removed

What structural changes distinguish Oxaliplatin from Cisplatin, and why do they matter?

• Two key differences:

  • Oxalate leaving group (vs chloride)

  • Bulk Diaminocyclohexane (DACH) ligand (vs amine groups)

• Effects:

  • Alter activation profile

  • Alters the toxicity profile

  • Helps overcome some cisplatin resistance

• Clinical use:

  • IV infusion for colorectal cancer (part of FOLFOX regimen)

• When used in combination therapies and regimens, can increase myelosuppression → additive toxicity

What are the Side Effects Associated with Oxaliplatin?

• Tingling, numbness, nausea

• Neurotoxicity is the most frequent dose-limiting toxicity

• Distinctive feature: acute cold-induced neuropathy → exposure to cold stimuli, e.g. cold drinks/ air can trigger or worsen sensory symptoms

What is the Structural Basis for Oxaliplatin Activity?

• Two key structural features:

  1. Oxalate leaving group

  • Bidentate ligand (2 bonds to Pt) → more stable

  • Hydrolyses more slowly than the chloride ligands of cisplatin (easily displaced by water)

  • Slower activation rate

  • Alters the toxicity profile

  2. Bulky DACH ligand

  • Remains attached when binding DNA → protrudes from the DNA helix

  • Distorts DNA helix conformation differently from cisplatin

  • Produces distinct DNA adducts → less efficiently recognises and is removed by nucleotide excision repair

• DNA adducts are processed differently by DNA repair pathways (including mismatch repair) → overcomes some of the cisplatin resistance mechanisms

• → Different activation, toxicity profile and pattern of DNA distortion

How Does Oxaliplatin Activate Cellular DNA Damage Responses

• Oxaliplatin cross-links with DNA and distorts the DNA helix (via its bulky groups)

• Results in DNA damage response activation

  • Activates ATM / ATR kinases

  • Kinases phosphorylate TP53 (p53),

  • This leads to p53 stabilisation and prevents MDM2-mediated ubiquitination and proteasomal degradation

• p53 accumulates and triggers two outcomes:

  1. G2/M cell cycle arrest

  • Allows/attempts DNA repair

  2. Apoptosis

  • If DNA damage is too extensive

  • p53 wild-type (proficient) cells are often more sensitive to oxaliplatin

  • p53-deficient/mutant cells may show resistance to oxaliplatin

What is the Acute Peripheral Neurotoxicity Associated with Oxaliplatin?

• Acute, transient neurotoxicity → Symptoms are usually transient and reversible between cycles (early in treatment).

• Occurs in the majority of patients.

• Occurs early on in treatment

• Appears during infusion or within hours.

• Symptoms often induced/worsened by exposure to cold stimulus

• Sensory symptoms: distal paraesthesia and/or dysesthesias(tingling or numbness in finger or toes); perioral numbness (around mouth)

• ~1-2% of patients report cold-induced pharyngolaryngeal dysesthesia (intense discomfort in throat/ non-lethal choking-like feeling.

• Motor symptoms (less common): muscle cramps or tetanic spasms, transient neuromuscular hyperexcitability

• May reflect hyperexcitability and misfiring of peripheral neurons rather than structural nerve damage.

  • Most symptoms are sensory

What is the Chronic Peripheral Neurotoxicity Associated with Oxaliplatin?

• Cumulative sensory neuropathy

• Seen in 10%–15% of patients after repeated exposure (dose-dependent).

  • Occurs as doses of oxaliplatin accumulate

• Primarily non-cold-related dysesthesias and paraesthesia of extremities.

• Clinical features: Impaired sensation, sensory ataxia (loss of balance and coordination – unique to neuropathy), distal dysesthesias and paraesthesia, deficit in fine sensory-motor coordination.

• Symptoms generally persist between cycles and worsen with cumulative doses.

  • A key problem with the FOLFOX protocol and explains the reason patients stop treatment due to this chronic neuropathy

• At higher grades, symptoms can interfere with daily activities.

• Often partially reversible:

  • Most with grade 3 neurotoxicity improve to grade 1 or less within 6-12 months after treatment discontinuation.

  • Not completely reversible

How Does Oxaliplatin Cause Acute Neuropathy?

• Often triggered/worsened by cold exposure

• Beings in dorsal root ganglion (DRG) sensory neurons, which are vulnerable to drug accumulation (lack BBB → permeable blood supply)

• Hallmark feature: ion channel dysfunction (functional, reversible) involving:

  • Voltage-gated Na⁺ channels

  • K⁺ channels

  • Ca²⁺ channels

• Oxalate byproduct chelates Ca²⁺ and Mg²⁺ → disrupts ion channel gating

• Oxaliplatin directly alters Na⁺/K⁺ channels

• Leads to disrupted ion fluxes, neuronal hyperexcitability and abnormal sensory signal transmission → paraesthesia/dysaesthesia

• Functional, not structural nerve damage

What is the Mechanism Behind Oxaliplatin Induced Acute Neuropathy?

• Cold sensitivity: Due to TRPA1 sensitisation (cold-sensitive ion channel)

  • Oxaliplatin enhances TRPA1 activity through oxidative stress and altered calcium homeostasis (area of active research)

  • Lower cold activation threshold → Cold stimuli activate the channels more easily

• Copper transporters (CTRs):

  • CTR1 contributes to oxaliplatin uptake (Uptake is less CTR1-dependent than cisplatin).

What are the Two Reasons for Oxaliplatin Induced Chronic Neuropathy Development?

• Neuroinflammation

  • Increased production of pro-inflammatory cytokines & chemokines (e.g. TNF-α, IL-8).

  • This sustained activation of inflammatory signalling contributes to progressive neuronal sensitisation.

  • There is also abnormal communication between neurons & glia (which should support the neurons), which sustains neuropathic pain.

  • Glial activation and inflammatory signalling can occur early on in treatment; it becomes chronic when sustained.

  • Sustained neuroinflammation characteristic of chronic neuropathy.

• DNA damage in dorsal root ganglia

  • Limited DNA repair capacity of neurons, as neurons are post-mitotic (do not divide)

  • Thus, increased vulnerability to Pt-induced DNA damage

Why are Oxidative Stress and Mitochondrial Dysfunction Also Observed in Oxaliplatin-Induced Chronic Neuropathy?

• Develops with cumulative oxaliplatin dosing

• Repeated treatments increase the accumulation of ROS

• Leads to Mitochondrial DNA damage → poor repair capacity (less able to repair itself compared to nuclear DNA)

• This leads to dysfunction of respiratory complexes, reducing ATP production and subsequently ATP depletion

• Neurons are highly energy-dependent; ATP depletion contributes to energy failure and leads to axonal degeneration

• Results in persistent, often irreversible neuropathy, even after treatment stops

What are Alkylating Agents?

• Cell-cycle non-specific agents → Cause cytotoxicity by attaching alkyl groups to DNA

• Form DNA adducts and cross-links (intra- and inter-strand)

• This leads to

  • Abnormal base pairing

  • DNA strand breaks

  • Disrupts replication/translation

• Activates DNA damage response → apoptosis (p53 dependent or independent)

How Do Alkylating Agents Differ to Pt-based Agents?

• Have similar effects → shared biological mechanisms (DNA damage)

• Differences in chemistries result in different toxicity profiles and different activation rates

  • Platins attach to the DNA via platinum coordination bonds

  • The reactive group in alkylating agents is the alkylating group

How Do Alkylating Agents Kill Cells?

• Cell cycle phase non-specific agents, but cells in the S Phase are more vulnerable, as replication forks stall at unrepaired adducts

  • In DNA replication, the helix is unzipped, with DNA synthesis following

  • When an adduct is encountered, it physically blocks this replication

  • Machinery is no longer able to bypass the adduct/lesion, blocking further replication

• Alkylating agents are more sensitive in the S-Phase → more likely to encounter a DNA adduct and block DNA replication

Give 3 Examples of Alkylating Agents

• Nitrogen mustards: Cyclophosphamide Chlorambucil, Ifosfamide

• Nitrosoureas: e.g. Lomustine (lipid soluble; able to pass the BBB)

  • Drug of choice for gliomas

• Triazines: e.g. Temozolomide

What is the Mechanism By With Alkylating Agents Cause DNA Damage?

• 1. Generate reactive electrophilic intermediates that covalently bind to nucleophilic sites on DNA (most commonly the N7 position of Guanine)

• 2. Alkylation of the N7 guanine base results in

  • Abnormal base pairing (e.g. G pairing with T)

  • Accumulation of secondary strand breaks, following base exicison repair attempts (DNA fragmentation)

    • An alkylating agent overwhelms the DNA repair system

  • DNA intrastrand/interstrand cross-links

• Collectively, this interferes with DNA replication and transcription

• Interference with DNA replication leads to Bax and Bak activation, forming pores in the outer mitochondrial membrane

• Leads to MOMP and Cytochrome C release from the intermembrane space → triggers apoptosis or necrosis

How Are Nitrogen Mustards Used As Chemotherapeutic Agents?

• Used in a range of cancers (e.g. lymphoma, leukaemia, breast, ovarian) and as immunosuppressants for various autoimmune conditions (e.g., cyclophosphamide lupus)

• Prodrugs → not administered as reactive drugs

• Require metabolic activation in the liver by CYP450 enzymes

  • Cyclophosphamide: primarily CYP2B6;

  • Ifosfamide: greater CYP3A4 contribution

• Compounds converted into two very different metabolites:

  • phosphoramide mustard (active metabolite).

    • Therapeutically active drug metabolite

    • Anti-tumour activity; cytotoxic species

  • Acrolein

    • Highly reactive & cytotoxic byproduct, primarily responsible for urotoxicity

What is the Haematolological Toxicity Associated with Alkylating Agents?

• Most common dose-limiting toxicity:

• Alkylation damages rapidly dividing marrow progenitor cells.

• This results in

  • Leukopenia (most common);

  • Neutropenia;

    • Neutrophils are most affected cell type – lifespan ~8 hours

    • Body continuously makes neutrophils  

  • Thrombocytopenia;

    • Moderate lifespan

  • Anaemia- develops with treatment

    • RBC Lifespan ~120 days

• Neutrophils most affected, with platelets being moderately affected, and RBCs being least affected

What is the Urological Toxicity Associated with Alkylating Agents?

• Haemorrhagic cystitis (most common toxicity): Bladder mucosal damage – severe inflammation and bleeding of the bladder lining

  • Most characteristic of cyclophosphamide and ifosfamide (due to their metabolism to acrolein).

  • Ifosfamide is associated with a higher risk of haemorrhagic cystitis and requires routine mesna (sodium 2-mercaptoethane sulfonate) co-administration.

    • Mesna aims to bind to and detoxify the acrolein metabolite

How Does Acrolein Contribute to Urological Toxicity and How Can It Be Prevented?

• It does not contribute to the anti-tumour activity → byproduct of the drug causes toxicity

• Concentrates in the bladder and has direct contact with the urothelium, causing oxidative stress and epithelial injury

• Toxicity can be prevented by:

  • Aggressive hydration (flushes away toxic metabolites)

  • Use of mensa (binds and detoxifies acrolein).