15 Topoisomerase inhibitors: Doxorubicin and Etoposide

15.1 Mechanisms of antitumour activity

We will now look at topoisomerase II inhibitors, in particular doxorubicin and etoposide. These inhibitors are non-covalent inhibitors, in contrast to the covalent inhibitors, the alkylating agents and cisplatin, we discussed previously. These inhibitors have been shown to have several mechanisms of action:

  • They are known to inhibit topoisomerase II and are therefore classified as topoisomerase II inhibitors,
  • they have been shown to bind to double stranded DNA,
  • and as an additional mechanism, they contribute to the formation of reactive oxygen species (ROS) that damage DNA, proteins and cell membranes thus contributing to the anti-tumour activity of anthracyclines.

After a brief recapitulation into the field of DNA packing and topoisomerases we will look into these mechanisms in more detail.

15.2 The functions of topoisomerases: packaging of DNA

How is DNA packed into the nucleus? We first have to repeat some basics to understand how topoisomerase II inhibitors work. The human genome contains the complete set of genetic information of humans. This information is encoded in the DNA sequences of 23 chromosome pairs. Diploid genomes consist of twice 3 billion DNA base pairs. Does anybody know how long the length of the set of human chromosome is? Each human cell contains about 1.8 meters of DNA. The question is then how we can pack the complete set of chromosomes into a cell or to be more precise into the nucleus? Double stranded DNA is packaged and organised into structural units called nucleosomes (Figure 13.1). Although each human cell has about 1.8 meters of DNA, wound on the histones it fits into the nucleus, which has a diameter of about 10 μm. The DNA-nucleosome complexes are further organised into higher ordered structures leading to chromosomes. Very importantly, these higher ordered structures involve DNA supercoiling.

Higher ordered structures of DNA involve DNA supercoiling. DNA is packaged into the nucleus, an organelle found in eukaryotic cells. The basic unit of DNA packaging is the nucleosome, containing a short segment of DNA wound around a complex of eight histone proteins.

Figure 15.1: Higher ordered structures of DNA involve DNA supercoiling. DNA is packaged into the nucleus, an organelle found in eukaryotic cells. The basic unit of DNA packaging is the nucleosome, containing a short segment of DNA wound around a complex of eight histone proteins.

Supercoiling is important in a number of biological processes and topoisomerases are therefore required to fulfil multiple functions: - Compacting DNA, - supercoiled DNA can be savely stored in the nucleus, - DNA has to be even more compact during mitosis, when the chromosomes are segregated into the two daughter cells. - in contrast to this, parts of the DNA also have to be accessed which is also essential for DNA replication, without damaging the genetic material.

There are specialised enzymes that induce well-controlled DNA strand breaks in order to control the supercoiling of DNA. These enzymes are called topoisomerases.

There are two classes of topoisomerases:

  • Type I topoisomerases cut one strand of a DNA double helix, relaxation occurs and then the cut strand is resealed,
  • Type II topoisomerases cut both strands of one DNA double helix.

In this chapter we will concentrate on topoisomerase II.

15.3 Importance of topoisomerase II for cancer treatment and functional mechanism

Why is topoisomerase II to important? “Some drugs work by interfering with topoisomerase II in cancer cells. This interference leads to permanent breaks in the DNA that ultimately lead to programmed cell death.

To understand how cancer drugs interfere with topoisomerase II, we first have to appreciate the detailed molecular mechanism of the enzyme. In brief, Topoisomerase II cuts both strands of one DNA double helix. Once cut, the ends of the DNA are separated and a second DNA duplex is passed through the break. Following passage, the cut DNA is resealed (Nitiss (2009)).

Detailed mechanism of topoisomerase II function. a) Forms of DNA, which can be generated through the function of topoisomerase II. b) Blue: double-stranded DNA, G-segment or also called gate; red: double-stranded DNA, T-segment, also named transport; read: central DNA binding gate; yellow: N-terminal ATPase domain; dark green: C-terminal gate.Figure adopted from xxx

Figure 15.2: Detailed mechanism of topoisomerase II function. a) Forms of DNA, which can be generated through the function of topoisomerase II. b) Blue: double-stranded DNA, G-segment or also called gate; red: double-stranded DNA, T-segment, also named transport; read: central DNA binding gate; yellow: N-terminal ATPase domain; dark green: C-terminal gate.Figure adopted from xxx

Topoisomerase II works through a “two-gate mechanism,” a mechanism supported by biochemical as well as structural work” (Figure 15.2):

  • Double-stranded DNA (shown in blue, also named G-segment, or gate) is bound by a central DNA binding gate.
  • A second double-stranded DNA (coloured in green, also named T-segment, or transport) is captured by the dimerisation of the N-terminal ATPase domain (in yellow).
  • Energy gained from the hydrolysis of ATP leads to the cleavage of the G-segment, creating a double stranded break.
  • The T-segment is transferred through the G-segment.
  • The G-segment is sealed, leading to the C-terminal gate to open allowing the release of the T-segment.
  • ADP-release leads to the reset of the system.

15.4 Predominant mechanism of antitumour activity: topoisomerase II inhibition

Having understood how topoisomerase II works, we can finally talk about the mechanism of antitumour activity of Doxorubicin and related anthracyclines. It is believed that the main mechanism associated with the cytotoxicity of these drugs is the inhibition of topoisomerase II. This is also the reason these inhibitors are called topoisomerase inhibitors.

Anthracyclines are a class of drugs used in cancer chemotherapy derived from the Streptomyces bacterium. These compounds consist of a planar, hydrophobic tetracyclic ring system linked to an amino sugar (daunosamine) through a glycosidic linkage. These agents are positively charged at physiological pH, favouring intercalation with DNA. Doxorubicin and Daunorubicin, two anthracyclines, differ only by a single hydroxyl at the R1 position, yet they have distinct spectra of anti-tumour activity.
Chemical structures of Doxorubicin, Daunorubicin and Idarubicin. Differences in their chemical structures are highlighted in red colour.

Figure 15.3: Chemical structures of Doxorubicin, Daunorubicin and Idarubicin. Differences in their chemical structures are highlighted in red colour.

What is the mechanism of action of Doxorubicin? Please remember, one of the intermediates of topoisomerase II was the cleavage of the G-segment, creating a double stranded break (Figure 13.2; intermediate state in parenthesis). By forming a stable ternary complex of Doxorubicin-topoisomerase II and DNA, it prevents topoisomerase II from resealing the double strand DNA breaks, interfering with the process of replication. In principle, cells can deal with DNA strand breaks using special DNA repair systems. However, when too many double strand DNA breaks accumulate, the cell decides to initiate the apoptotic cell death program.

Add figure of anthracyclin-topo II complex here

Crystal structure oft the topoisomerase II – anthacycline complex (add ref and PFB ID).

Figure 15.4: Crystal structure oft the topoisomerase II – anthacycline complex (add ref and PFB ID).

15.5 Mechanism of antitumour activity: binding to double-stranded DNA

Having looked at the inhibition mechanism through topoisomerase II we now want to explore additional antitumour activities. A further mechanism is binding to double stranded DNA. Although the binding of anthracyclines to DNA is believed to be critical for drug action, this is not the main mechanism that leads to cell death.

We already discussed the chemical structure of Doxorubicin (Figure 13.3). The planar aromatic structure of anthracyclines allows them to intercalate into DNA. “Intercalation is the reversible inclusion or insertion of a molecule between the bases of DNA.” The amino sugar moiety can be positioned in the minor groove of the DNA. When intercalating agents, in this case anthracyclines, become associated with DNA, there is a partial unwinding that induces local structural changes to the DNA strand, such as lengthening of the DNA strand or twisting of the bases pairs. These structural changes can lead to functional changes, often in the inhibition of replication and transcription of rapidly growing cancer cells, which leads to subsequent cell death.

Schematic drawing of normal non-disturbed DNA -left panel- and structural changes in the DNA induced by intercalation of anthracyclines -right panel, coloured in red.

Figure 15.5: Schematic drawing of normal non-disturbed DNA -left panel- and structural changes in the DNA induced by intercalation of anthracyclines -right panel, coloured in red.

15.6 Mechanism of antitumour activity: formation of reactive oxygen species

A third mechanism of anti-tumour activity of anthracyclines is the formation of reactive oxygen species, which damage DNA, proteins and cell membranes. A few of you may have noticed that Doxorubicin and the other anthracyclines contain the quinone scaffold, (Figure 13.7). The quinone moieties allow these molecules to participate in electron transfer reactions and generate oxygen free radicals. Doxorubicin is oxidized to semiquinone, an unstable metabolite, which is converted back to Doxorubicin in a process that releases reactive oxygen species. Reactive oxygen species can lead to membrane and DNA damage, oxidative stress, and triggers apoptotic cell death. Semiquinone, superoxide and hydrogen peroxide can also reduce iron III to iron II to produce hydroxyl radicals. Unfortunately, this also leads to cardiotoxicity (discussed in the next section), because the heart contains low levels of detoxifying enzymes.

Reduction of quinone to semiquinone. Semiquinone is a free radical that is highly reactive, for example with oxygen, to generate the ROS superoxide (O2-) and hydrogen peroxide (H2O2).

Figure 15.6: Reduction of quinone to semiquinone. Semiquinone is a free radical that is highly reactive, for example with oxygen, to generate the ROS superoxide (O2-) and hydrogen peroxide (H2O2).

15.7 Administration, clinical uses and toxicities of anthracyclines

Doxorubicin is given intravenously and particular care has to be taken by a carefully trained nurse as it is a vesicant/blistering agents, that may cause serious damage if it leaks out of the vein.

Anthracyclines are used to treat solid and haematological malignancies. Doxorubicin shows broad-spectrum activity and is used to treat:

  • Breast cancer (one of the most active agents),
  • Bladder, endometrium, lung, ovarian, thyroid,
  • Sarcoma of the bone and soft tissue,
  • Hodgkin and non-Hodkin lymphomas,
  • Leukemias In contrast, Daunorubicin and Idarubicin are used to treat myelogenous leukaemia.

Common toxicities of anthracyclines include nausea and vomiting, alopecia and myelosuppression, in particular leukopenia. Cardiotoxicity, which is dose-limiting, has been intensively studied. Although there are short term and transient effects, congestive heart failure is of significantly greater clinical significance.

Mortality rates of 30% have been reported. Heart failure sometimes occurs many years after treatment and may be a long-term effect. Cardiotoxicity is complex but may result from reactive oxygen species in the heart, or the accumulation of anthracycline metabolites in the heart. This may be a result of the low catalase activity in cardiac tissue.

The incidence of congestive heart failure as a function of the cumulative dose is shown in Figure 13.8. There is an incidence of about 5% when the dose of Doxorubicin is 500–550 mg/m², 15% when the dose is 551–600 mg/m² and up to 30% when the dose exceeds 700 mg/m². This signifies that cardiotoxicity is related to a patient’s cumulative lifetime dose. A patient’s lifetime dose is calculated during drug administration, and anthracycline treatment is usually stopped upon reaching the maximum cumulative dose. Combination of Herceptin with Doxorubicin produces an increase in congestive heart failure at lower than the cumulative dose. Due to these complications, cardiac monitoring is recommended at three, six, and nine months.

The figure shows that the incidence of congestive heart failure is related to the cumulative lifetime dose. Anthracycline treatment is stopped upon reaching the cumulative dose, which is for example 450 mg/m2 for Doxorubicine (add ref for graph)

Figure 15.7: The figure shows that the incidence of congestive heart failure is related to the cumulative lifetime dose. Anthracycline treatment is stopped upon reaching the cumulative dose, which is for example 450 mg/m2 for Doxorubicine (add ref for graph)

15.8 Etoposide, a new topoisomerase II inhibitor

Another important topoisomerase II inhibitor is Etoposide. It is chemically related to Podophylotoxin, a plant toxin. As Doxorubicin, Etoposide forms a ternary complex with DNA and topoisomerase II.

Chemical structures of Etoposide and Podophulotoxin, a plant toxin

Figure 15.8: Chemical structures of Etoposide and Podophulotoxin, a plant toxin

Etoposide is given orally or intravenously.

Clinical uses include small cell bronchial carcinoma and testicular cancer. Etoposide is often given in combination with other drugs to treat:

  • Kaposi’s sarcoma,
  • Ewing’s sarcoma,
  • Lung cancer,
  • Lymphoma,
  • Nonlymphocytic leukemia,
  • Glioblastoma multiforme

Toxicities include nausea and vomiting, alopecia and myelosuppression, to name a few.

15.9 Bibliogrpahy

References to be added