Week 6: Cancer Chemotherapy Flashcards
The two main categories of genes acted upon by mutation are recognised as:
Proto-oncogenes (inactive) transformed by mutation to oncogenes (active). These genes control cell division apoptosis and differentiation.
Tumour suppressor genes. The loss of function these suppressor genes can be critical in triggering carcinogenesis.
Transformation of a normal cell into a cancer cell requires a number of these control elements to be lost. Once this loss has taken place, the following characteristics of cancer cells define the magnitude of malignant threat.
Describe Loss of cell growth control as one of the hallmarks of cancer
The key feature of cancer cells is that the normal mechanism controlling proliferation and ordered cell death (apoptosis) is lost. This does not mean that the cancer cells are necessarily proliferating faster than normal cells, but that the ordered control of cell division balanced by apoptosis is lost in cancer cells.
Describe De-differentiation and loss of specific function as one of the hallmarks of cancer
Normal cells have a specific function to fulfil, which is lost when they turn cancerous. The degree of de-differentiation in cancers varies considerably, but the greater this loss of original identity, the worse the malignant challenge to the body generally is.
Describe blood supply as one of the hallmarks of cancer
As the cancer cells proliferate to reach the dimensions of a clinically discernible tumour, there is a natural limit to growth due to limited blood supply. This would restrict tumour growth to about 1-2 mm in diameter. To overcome this limitation, the tumour cells produce their own local angiogenesis factors to promote vessel growth into the tumour, so it can continue to develop.
Describe Metastasis and invasiveness as one of the hallmarks of cancer
Even with the additional vascular supply, if these de-differentiated cells in the tumour stayed in one location, then killing them would be easier. The threat they pose could be better contained through direct surgery and irradiation. However, malignant cells may also lose the very specific positional sense and identity within their parent tissue. This arises due to changes in cell surface proteins. These changes allow them to migrate or metastasise throughout the body.
This takes place via the lymphatic or vascular system or more locally by invasion of an adjacent body cavity. They can then proliferate further afield and produce new tumours. In addition as the cancer progresses over time, further mutations in cancer cells result in increased heterogeneity, with distinct cell lines arising that increase the malignant challenge to be met by chemotherapy
Given the physiological limitations on tumour growth, not all cells in a tumour are involved in active proliferation and normally belong to one of three compartments. These compartments tend also to relate to their spatial position in the body of the tumour. Outline the compartments.
Compartment A – Dividing cells receiving adequate nutrient/vascular supply
Compartment B – Resting cells remain in ‘G0’ phase but able to re- join Compartment
A if there are changes in cell signalling or the /local environment. – e.g. following surgery. More likely to situated in the middle of the body of the tumour.
Compartment C – Cells no longer able to divide, but act to contribute to the overall bulk of a tumour. These present no challenge.
Note: cancer cells can still die off on their own if minimal nutrient supply is not maintained.
Compartment A may represent between 5-20% of the actual tumour cell population and is the one most susceptible to chemotherapy targeting cells at one or more stages in the cell cycle.
Therapeutically, cells in Compartment B present a real problem. They may be affected to some degree by chemotherapy as some resting RNA and protein synthesis will be taking place. But the proportion of chromosomal DNA in ‘G0’ that is open to attack is much more limited. This makes the effective kill ratio attainable in this compartment much lower. They will thus be available to re-enter Compartment A, even following intense chemotherapy.
when is a tumor clinically detectable?
Typically, a tumour needs to consist of a cluster of about 109cells before it is clinically detectable or to reach the size of a small grape.
Describe The log kill ratio
Typically, a tumour needs to consist of a cluster of about 109cells before it is clinically detectable or to reach the size of a small grape. The underlying model utilized by clinicians is based on log kill ratios. This means that if a given treatment kills 104 or 99.99% of cancer cells then for a population of 109 cells, this would mean a reduction to 105 or to 0.01% of the original population of 109 cells. The 105 cancer cells remaining would resemble a sphere the size of a full stop.
This would be a four log kill ratio and further treatment would be warranted so another dose of the same four log kill therapy would then result in only 101 or just 10 cancer cells remaining. This proportionate killing is comparable with the ideas around first order pharmacokinetics covered in the first part of the Workbook.
In this theoretical model this sounds really good, but the factors outlined earlier mean that of the 109 cells perhaps only 107- 108 may be available for direct attack (Compartment A) with the more resistant resting Compartment B cells able to revert hydra like, to aggressive ‘A’ types post therapy.
In reality, more modest kill ratios than given in example above may be attained that require repeated chemotherapy. The kill rate for cancer cells has to be weighed up against the kill rate for healthy cells in susceptible tissues.
Cancer chemotherapy: Agents directly modifying DNA structure
DNA Intercalation and Topoisomerase II Inhibition – Anthracycline Antibiotics. Describe the mechanism of action.
The anthracycline antibiotics are used in synergistic combination with other drugs with different mechanisms to reduce the risk of additive ADRs. Members of this group include doxorubicin and daunorubicin. Their discrete molecular ring structure enables them to intercalate between the spaces between DNA base pairs. This intercalation would interfere with normal transcription and replication. The anthracyclines particularly affect the activity of Topoisomerase II.
Topoisomerase IIenables the breaking, rotation and re-ligation of DNA strands during DNA replication and repair. The intercalated antibiotic molecule physically interferes with this. This action is further augmented by the anthracycline binding to the Topoisomerase II thus forming a tripartite DNA – anthracycline – Topoisomerase II complex. This hinders its availability for deployment elsewhere. The resulting non-ligated free strands of DNA act as a trigger to apoptosis.
As a secondary action, the anthracyclines are able to generate free radicals by binding free Fe2+. The locally produced free radicals then go onto further damage the DNA. Overall, the damage caused by anthracyclines is detected by DNA damage sensing mechanisms in the cell which then trigger apoptosis. In contrast to bleomycin, (see below) their action is non-cell cycle specific
Cancer chemotherapy: Agents directly modifying DNA structure
DNA Scission –Bleomycin.
Describe the mechanism of action
Bleomycin is a glycopeptide antibiotic derived from Streptomyces fungi. Bleomycin can both bind with DNA and chelate with free Fe2+ ions. Its structure allows it to closely align itself within DNA by both intercalation but also by binding via its terminal NH2 group to DNA. When it chelates with free cytoplasmic Fe2+ ions, this reaction site then catalyses production of both superoxide and hydroxide free radical species These highly reactive free radical species then attack phosphodiester bonds in the DNA strand resulting in a cutting or scission of DNA strands. This scission is considered to be the primary mechanism underlying its cytotoxicity and is comparable with the anthracyclines in this respect. The production of free radicals is also considered to underlie its pulmonary toxicity. Bleomycin is most effective during G2, but also has some effect in non-replicating G0 cells.
Cancer chemotherapy: Agents directly modifying DNA structure.
Alkylating Agents Related Compounds and The Platins
Describe the mechanism of action
Clinically, the name is often used to refer to compounds used in chemotherapy that covalently bind to DNA. The primary underlying mechanism for these related agents is that they typically possess two highly labile or ‘leaving’ groups such as – Cl, that are necessary for instigating the covalent reaction with the DNA.
Nucleophilic target sites for these agents are spread along the whole length of DNA
Along the length of DNA strands the specific molecular target sites are nucleophilic, or positive charge attracting, groups. These are very common and their locations are very well defined. These include:
N7 and O6 atoms in guanine; N1 and N3 in adenine; N3 in cytosine. These all have spare electron pairs to donate to a vacant electron orbital. They can therefore make a covalent bond with those agents the alkylating group. The reactive group on alkylating agents are therefore electrophilic or ‘electron attracting’
Antimetabolites – interference with precursors to purine and pyrimidine synthesis
Describe the mechanism of action
Antimetabolites used in cancer chemotherapy are structurally related to precursors involved in DNA synthesis. They act to interfere with the production of the purine or pyrimidine nucleotides. This is by acting as a direct competitive analogue in DNA or RNA synthesis such as 6- Mercaptopurine (a purine analogue), 5-Fluorouracil or 5-FU (a pyrimidine analogue).
In addition, inhibition of key enzymes involved in precursor synthesis is a common therapeutic option, with methotrexate used as a highly potent inhibitor of Dihydrofolate Reductase or DHFR. Brief consideration is given here to the mechanism of methotrexate and 5-fluorouracil from this large group of therapeutic agents.
Methotrexate acts by inhibiting DHFR to interfere with folate metabolism. Describe this process
Folates are vital for production of purine nucleotides and thymidilate. These feed into the pathway that enables DNA and RNA synthesis. After being actively taken up into the cell, folate is converted in to a polyglutamate. Then, the polyglutamated folate is reduced in two steps by the enzyme DHFR to produce a tetradyhdrofolate, or FH4.
Polyglutamated FH4acts as a co-factor acting as a methyl group carrier. This methyl group is then used in the transformation of 2 - deoxyuridylate or DUMP into 2- deoxythymidylate or DTMP. This transformation is carried out by the Thymidylate Synthase enzyme.
Along with other related pathways, the production of DTMP contributes to the production of the purines and pyrimidines that act as molecular building blocks for both DNA and RNA. Methotrexate is structurally very similar to folic acid and is actively transported into the cell and also polyglutamated. Methotrexate on its own has a much higher (x1000) affinity for DHFR
As a result, both Methotrexate alone and its polyglutamated form act to vastly reduce the metabolism of folate to reduce nucleotide production. The decrease in thymidine levels being the most marked. The controlled dosages used in cancer chemotherapy are much higher than used in RA and the aim is to reduce cancer cell growth as rapidly as possible by effectively removing nucleotide precursors from the cell metabolite pool.
why is chemotherapy most commonly prescribed IV?
With many of these drugs, their toxicity rules out oral delivery as they would severely damage the GI tract. In addition bioavailability is often variable and only a few agents are given orally. In addition, the patient is highly likely to experience nausea and vomiting throughout treatment further limiting practicability of the oral route.
The most practical route for systemic administration of these agents is intravenous. This allows fine control of delivery by injected bolus an infusion bag or pump infusion. If an emergency arises due to appearance of adverse effects, the infusion can be stopped immediately.
If a tumour is localised, then direct intraregional delivery may be possible. If it is within a defined space for example the bladder or lung effusion may be performed. Intrathecal and intraventricular delivery is used for treating tumours in the CNS.
Drug resistance in cancer chemotherapy - Primary and acquired resistance
Traditional agents used in cancer chemotherapy are, by definition, cytotoxic. Neither healthy or cancer ‘know’ these drugs are being used to defeat a systemic threat to the whole body. Consequently they will treat many of these agents in the way they would deal with any other potentially toxic xenobiotic molecule.
The terms primary and acquired resistance are respectively defined as resistance being present prior to drug exposure and subsequent to drug exposure. These resistances in tumour cells can be due to their adapting and upregulating responses to repeated ‘xenobiotic’ chemotherapeutic challenge, or they can arise as a result of mutations in the cancer cell genome driving increased MDRP expression/activity.
Main factors contributing to chemotherapy drug resistance
Multidrug Resistance Protein – MDRP. This P-Glycoprotein (P is for ‘permeability’) is generally expressed throughout most healthy cells at low levels. High levels are notably expressed in the kidney, liver and GI tract. MDRP functions to generically remove hydrophobic (ie charged) large xenobiotics
If cancer cells repeatedly encounters one or more of the chemotherapeutic drugs described here then expression of MDRP may increase as a result. Importantly, as MDRP activity is non-specific, it can then act can remove structurally dissimilar molecules used in combination chemotherapy. For example prior exposure to doxorubicin can then also increase efflux and resistance to vinblastine or cis-platin to limit efficacy of future dosing.
Other inducible mechanisms – Drug exposure can also act to decrease the rate of active uptake of drugs such as methotrexate or cis-platin by downregulation of the active carrier.
Conversely, drug target enzymes expression can be upregulated to offset the decrease in production of metabolites e.g. DHFR ↑ with methotrexate. Further examples include increased rate of repair of drug induced lesions in DNA or membrane following increased expression of repair enzymes in cancer cells. For example, this is seen with use of the alkylating agents or bleomycin which act to damage DNA.
Cancer chemotherapeutic ADRs
As they are primarily targeted at rapidly dividing cells, they also affect normal cells with a higher rate of proliferation. This includes most of the GI tract, especially the buccal mucosa, and hair follicles. There are however other ADRs, which are manifest with specific groups or drugs.
Common ADRs
These include severe nausea, vomiting and diarrhoea. Mucositis, alopecia and myelosuppresion, impaired wound healing skin toxicity are also common. More specific within group ADRs, include neurotoxicity, cardio, bladder, lung, and renal toxicity. The latter three are irreversible.
Of the ADRs, the most frequent dose limiting is haematological toxicity and is the most frequent cause of death during chemotherapy. Different agents have varying effects in both degree and lineage e.g. neutrophils and platelets.
In addition, acute renal failure is also an expected ADR. Hyperuricaemia caused by rapid tumour lysis during treatment can result in a large increases in purines being released into the circulation. The subsequent increase in purine metabolism generates urates. This can lead to precipitation of urate crystals in renal tubules and in extreme cases subsequent kidney failure and death.
Some side effects associated with main cytotoxic groups
Antimetabolites – The example drugs given here largely exhibit all the general ones listed. With high dose methotrexate treatment there is higher risk of renal failure.
Cytotoxic antibiotics 1 – Cardiotoxicity: The anthracyclines doxorubicin and daunorubicin are especially cardiotoxic due to free radical generation.
Cytotoxic antibiotics 2 – Pulmonary Toxicity: Risk of pulmonary fibrosis with bleomycin is high at about 10% with a 1% fatality rate. These effects of both are cumulative and irreversible.
Alkylating agents – Cis-platin at high dose, peripheral, sensory and motor neuropathy. High frequency ototoxicity is common.
Mitotic spindle inhibitors – High dose neurotoxicity as ‘stocking and glove’ peripheral neuropathy is often reported.
describe the basic components of DNA
- Nucleotide = sugarphosphate- base
- DNA = double helix of nucleotides
- Purines = Adenine & Guanine
- Pyridimines = Cytosine & Thymine (Uracil in RNA)
Classification of Tumours According to Chemo-sensitivity
Highly Sensitive
Lymphomas
Germ cell tumours Small cell lung Neuroblastoma Wilm’s tumour
Modest Sensitivity
Breast Colorectal Bladder Ovary Cervix
Low Sensitivity
Prostate Renal cell Brain tumours Endometrial
site of Action of antimetabolites
DNA synthesis
site of Action of alkylating agents
DNA
site of Action of intercalating agents
dna transcription and duplication
site of Action of spindle poisons
mitosis
• Once chromosomes are aligned at metaphase plate, spindle microtubules depolymerize, moving sister chromatids toward opposite poles
• Nuclear membrane re-forms and cytoplasm divides
