Actions of cytotoxic drugs

Question 1

What are the main classes of cytotoxic drugs?

Answer 1

Anti-metabolites
Alkylating drugs
Patinating drugs
Topoisomerase poisons
Mitotic poisons

Question 2

What are antimetabolites?

Answer 2

These are drugs that interfere with one or more enzymes or reactions necessary for nucleic acid synthesis and are often structurally similar to the metabolite
They restrict the flow of precursors for DNA synthesis or get incorporated into the DNA and produce stress signals

Question 3

What are cytosine arabinoside and gemcitabine?

Answer 3

These drugs are transported into tumour cells via specific nucleoside transporters and must be phosphorylated by deoxycytidine kinase to produce a triphosphate form which will be retained in the cell, inhibit DNA synthesis and activate the S phase checkpoint, they may also be incorporated into DNA causing chain termination and activate the S phase checkpoint
These are both drugs which are inactivated by cytidine deaminase to the corresponding uridine derivatives

Question 4

What treatment is cytosine arabinoside used for?

Answer 4

Generally used in the treatment of leukaemia

Question 5

What treatment is gemcitabine used for?

Answer 5

A variety of solid bulk tumours

Question 6

What is methotrexate?

Answer 6

This is a drug used in a wide variety of tumours such as breast cancer, it is taken up by cells and binds to dihydrofolate reductase this inhibition stops thymidine monophosphate synthesis and thus activates the S phase checkpoint

Question 7

What is 5-Fluorouracil?

Answer 7

This is a drug commonly used for the treatment of colorectal cancer
It is taken up by the same transporter as uracil and metabolised to ribonucleosides and deoxyribonucleosides its main action is inhibition of thymidylate synthase which activates the S phase point
While other actions include misincorporation into DNA and RNA
The drug is inactivated by conversion to dihydrofluoruracil by dihydropyrimidine dehydrogenase

Question 8

What is the principle behind DNA alkylating drugs?

Answer 8

These are drugs which rely on having a chemically reactive group (typically a substituted alkyl halide) that reacts with the most electronegative areas on DNA (mainly guanine-7-nitrogen)

Question 9

What are the different DNA alkylating drugs?

Answer 9

Nitrogen mustard was the first developed others include
Chlorambucil, melphan which are nitrogen mustard derivatives used in lymphoma, ovarian cancer, breast cancer and multiple myeloma
Nitrosoureas used in lymphoma and brain tumours
Cyclophosphamide and ifosfamide used in lymphoma ovarian cancer and breast cancer
Dacarbazine and temozolomide used in brain cancers and melanoma

Question 10

What is the action of nitrogen mustard?

Answer 10

This drug gets taken up into cells by the choline transport pathway which may provide it some selectivity
It is a bifunctional drug as it alkylates firstly at guanine N-7 position and then the second chlorethyl group alkylates a second guanine
This forms an inter strand crosslink which distorts DNA structure which will activate the S phase checkpoint during DNA replication

Question 11

What is cyclophosphamide?

Answer 11

This is the most commonly used of the clinical alkylating agents and can be given orally
It is a prodrug which is activated by liver P450 enzymes to 4-hydroxy cyclophosphamide
This exists in equilibrium with aldophosphamide which is taken up by tumour cells and converted to phosphormaide mustard which causes DNA crosslinks through the same mechanism as nitrogen mustard

Question 12

What are the actions of DNA crosslinking agents?

Answer 12

Interstrand crosslinks prevent translation and replication by inhibitind DNA strand separation
This causes a DNA repair foci to from

Question 13

What are dacarbazine and tomozolomide?

Answer 13

These are methylating agents used mainly for treating gliomas but do also have some action against carcinomas
Darcarbazine must be activated by the liver while temozolomide will spontaneously activate in plasma
The activated form of both of these drugs is called MTIC and will methylate DNA-O6-methylguanine (this may be reversed by MGMT)

Question 14

Why is methylated DNA harmful to the cell?

Answer 14

Methylated DNA itself is not cytotxic however O6-guanine which is methylated can mispair with thymine instead of cytosine during DNA replication which leads to activation of the mismatch repair complex which initially activates the S phase checkpoint and eventually results in G2/M phase arrest

Question 15

What are the platinum based anti-cancer drugs?

Answer 15

Cisplatin was discovered by accident during an electrophoresis experiment and has particular activity against testicular cancer though also acts against ovarian and otyher cancers
Carboplatin was developed due to this discovery, this is an analogue with reduced toxicity and is active against ovarian cancer

Question 16

What is the action of cisplatin?

Answer 16

This reacts with water in the cytoplasm to give an aquated form (where the chlorine groups are replaced by H2O groups) which is more reactive
This form will react with DNA mainly at the N7 position of purines
The most important adduct is the 1,2-intrastrand crosslinks where the platinum is covalently bound to adjacent purine bases
The DNA is unwound and bent towards the major groove, high mobility group proteins and other proteins bind to form a damage-induced nuclear focus activating the S phase checkpoint

Question 17

What are DNA topoisomerases?

Answer 17

Theses are enzymes which change the topology of tethered DNA causing the loops to have a number of topological forms which differ in linking number
There are two types
Type 1 which makes single strand DNA breaks and changes linking number of topologically constrained DNA in steps of 1
Type 2 which make double stranded breaks changing the linkage number in steps of two
Both of these types are associated with DNA replication forks

Question 18

What are the topoisomerases in mammalian cells and how do topoisomerase poisons act on them?

Answer 18

Topo 1A, Topo1B and two Topo 2 enzymes these catalyse a transesterification reaction between an enzyme tyrosol group and a DNA phosphate group which leads to breakage of the DNA backbone and the formation of a transient covalent link between the tyrosol group and the DNA 5’phosphoryl group
Topoisomerase poinsc can be used to prevent the reversal of this leading to a DNA double stranded break which activates multiple checkpoints

Question 19

What is topoisomerase 1 and how do cytotoxic drugs interact with it?

Answer 19

Topoisomerase 1 binds to DNA and has several domains with the C terminal domain containing the active site
Topoisomerase 1 breaks one DNA strand joining the 3’ phosphate end of the broken DNA to the tyrosine of the active site
The DNA rotates and religates, freeing the enzyme and the DNA with the DNA linking number being changed by 1
Topoisomerase poisons bind to the enzyme and prevent the relegation step

Question 20

What is camptothecin?

Answer 20

This is a type 1 topoisomerase poison which was originally isolated as a natural product but the toxicity of a metabolite masked its anti-tumour effects
Chemical modification overcame this problem
The most common clinical forms are topotecan, irinotecan and 9-aminocamptothecin

Question 21

What is the action of topoisomerase 1 poisons?

Answer 21

Camptothecin and related compounds inhibit the action of topoisomerase I, this alone causes no toxicity however in S phase cells the jammed topoisomerase I-CPT complex interacts with the DNA replication complex to activate the S phase checkpoint

Question 22

How is topoisomerase 2 used as a target?

Answer 22

Topoisomerase is a dimer of two identical proteins which can bind two DNA strands (the G and T segments)
During its normal action the G strand is broken the T segment is translocated downwards and the G segment is religated
Poisons of this complex like doxorubicin and etoposide interferes with the relegation step leading to the formation of a double stranded DNA break

Question 23

What is doxorubicin?

Answer 23

This is a member of the anthracyclines (antibiotics) which is active against breast cancer and lymphoma as well as some other tumours
Daunorubicin is a derivative of this which is active against non-lymphocytic leukaemia and actinomycin D

Question 24

What is etoposide?

Answer 24

This is a semi-synthetic drug which is derived from a plant product, podophyllotoxin
It is active against lymphoma, germ cell cancers, small cell lung cancer

Question 25

What are mitotic poisons?

Answer 25

These are all complex molecules derived from plant toxins and includes vinblastine and paclitaxel these bind to the beta subunit of tubulin
Vinblastine binds to the polymerising end of microtubules preventing its elongation while paclitaxel stabilises the microtubule preventing shortening or depolymerisation

Question 26

How are mitotic poisons cytotoxic?

Answer 26

Separation of chromatids during mitosis requires changes in the polymerisation of the protein tubule
These microtubules are formed by the self-assembly of tubulin which will link to the kinetochores of the chromatids improper attachment can activate the mitotic checkpoint
This can result in mitotic cells being unable to separate due to a lack of activity of APC/seperase
Chromosome condensation reverses and cells convert to a multinucleated form but remain intact until over time they die due to post mitotic catastrophe

Question 27

What is the basis for selectivity of cytotoxic drugs?

Answer 27

This may be based on uptake like the choline transporter in nitrogen mustard, and the copper transporter in cisplatin
It may also be based on the trapping of an active form inside the cell with phosphorylation of many of the anti-metabolites like polyglutamate with methotrexate
High target protein concentration when this directly related to its cytotoxic action like in topoisomerase poisons
Lack of an enzyme like MGMT which leads to sensitivity to temozolomide and darcarbazone
Short cell cycle gives a higher proportion of cells entering S-phase or mitosis which is the general principle of mitotic inhibitors

Question 28

What are the features of treatment with cytotoxic drugs?

Answer 28

Often 2 or more drugs are used in combination
Metastatic melanomas and gliomas are highly resistant to treatment but a lack of the MGMT enzyme in sme cases can allow for temozolidine and dacarbazine use
Topoisomerase 2 poisons have a small spectrum of action but etoposide is often used for lymphoma and doxorubicin for breast cancer