*2.2.1 Via topoisomerase II poisoning*

Along with DNA intercalation, topoisomerase II is also considered as the primary target of anthracyclines [17]. Topoisomerases help in solving the topological problems like supercoiling, knotting, and catenation of DNA during replication, transcription, and recombination by creating single and double stranded breaks and subsequently rejoining the breaks. Based on structure and function, mammalian cells have two types of topoisomerases which are topoisomerase I and II. Topoisomerase I is monomeric and forms single strand breaks in DNA whereas topoisomerase II is dimeric and introduces double stranded breaks in DNA. Anthracyclines interfere with the normal functioning of breaking and rejoining of DNA strands by topoisomerases, particularly topoisomerase II consequence in the formation of an abortive anthracycline- DNA-topoisomerase ternary complex, hence poisoning the enzyme action. This ternary complex impends the religation of breaks in the dsDNA. Hence, anthracyclines act on topoisomerase II and stabilize the DNA-topoisomerase II complex. Due to this topoisomerase II which otherwise is essential for the normal functioning of the cell now acts as a lethal toxin to the cell and leads the cell to apoptosis. During intercalation, the planar ring of the aglycone and sugar moiety remains in contact with DNA bases while the A ring and the substituents present the C9 which are present in the minor groove of DNA interact with the enzyme (**Figures 1** and **2**). Perhaps that's why modifying the C9 substituent changes the activity of the drug. Cleavage does not occur on all the sites recognized by the Topoisomerase II and depends on the specific base sequence where the drug interacts with the enzyme. An increase in drug activity is seen when the 4-methoxy group is removed and the sugar moiety is substituted on 3′. 3′ substitution also has a significant role in the determination of specific sites for anthracycline associated cleavage of DNA.

Several lines of evidence have shown that these anthracyclines induce irreversible DNA damage by forming a ternary complex with DNA topoisomerase which introduces permanent double stranded breaks which ultimately lead to apoptosis in rapidly dividing cells [18, 19].

#### *2.2.2 Via oxidative stress*

Anthracyclines also causes the production of free radicals inside the cell which are responsible for the cytotoxic effect of these drugs. Though the mechanism of this process is still unclear increased number of reactive oxygen species (ROS) and the presence of deoxyaglycone in the urine after the administration of drugs indicates the possibility of this mechanism [5]. Oxidative stress is the imbalance between the reactive nitrogen species and reactive oxygen species in the cell. Mitochondria are believed to take part in this process. Quinone ring of the anthracyline aglycone act as electron acceptor (**Figures 1** and **2**). In the electron transport chain (ETC), one electron is transferred from NADPH to flavoprotein and then to the aglycone due to which quinone gets reduced to form semiquinone free radical. This reaction is catalyzed by NADPH cytochrome P-450 reductase [20]. From

the semiquinone, this electron gets transferred to oxygen and semiquinone gets converted into stable hydroquinone in this redox cycle. Due to this electron transfer oxygen gets reduced to superoxide which readily gets converted to other reactive oxygen species like hydroxyl radical which is harmful to cells. These reactive oxygen species can lead to DNA damage and lipid peroxidation which finally result in cell apoptosis.

When oxygen becomes limiting, semiquinone free radical transfer the electron to other electron acceptors, and rearrangement in anthracyclines takes place. The aglycone ring remains connected to sugar moiety by a glycosidic bond which is an ether containing linkage with oxygen in it which accepts the unpaired electron and due to this cleavage of glycosidic bond takes place. As a result, the formation of 7-deoxyaglycone takes place which either directly passes through urine or as conjugates in the bile [5]. There are several hypotheses explaining why superoxide dismutase (SOD), catalase, and antioxidants do not work effectively after the administration of anthracyclines. Studies have shown that anthracyclines can alter the glutathione (GSH) level and the enzymes involved in its redox pathway. According to another hypothesis free radicals are formed on the cell surface or internally in the cell and then get transferred to the cell surface due to which SOD and catalase fail to act upon these ROS [17].

#### *2.2.3 Via forming DNA-anthracycline adduct*

Formation of the anthracycline-DNA adduct in the presence of formaldehyde is another proposed mechanism of anthracycline action. There are studies that show the formation of doxorubicin-DNA at clinically relevant concentrations of drugs [21]. Anthracycline forms covalent bond with one DNA strand and hydrogen bond with other DNA strand resulting in DNA damage. The reaction between anthracycline and cellular formaldehyde is the foremost step to form activated Schiff base which then forms aminal linkage with the exocyclic amino group of guanosine. Formaldehyde in the cells is derived from the various carbon sources like lipids and spermine by iron mediated free radical reactions. In addition, Kato et al. have found increased formaldehyde levels in the cancer cells as compared to normal cells [22].

Phillips et al. using the bidirectional transcription footprinting technique demonstrate that adriamycin-DNA adduct formation induces transcription inhibition with alkylation at specific DNA sites [23]. However, Bilardi et al. have demonstrated that anthracycline-DNA adduct induces breaks in DNA via a mechanism independent of topoisomerase-II [24]. Moreover, DNA breaks formation occurs mainly due to the stalled or collapsed replication fork and these DNA breaks are repaired by homologous recombination dependent process. Forrest et al. investigated the activation of DNA damage response pathways after doxorubicin-DNA adduct formation and found that the downstream processing is dependent on various stages like recognition of adduct during replication, transcription, or any other stage of the cell cycle. Ataxia telangiectasia mutated (ATM) and ATM and Rad3 related protein kinase (ATR) are the DNA damage recognition proteins that get activated in response to double and single DNA strand breaks respectively. Forrest et al. demonstrate that both ATM and ATR proteins have the capability to react with intermediates produced as a result of doxorubicin-DNA adduct formation [25].

DNA-anthracycline adduct formation probably follows a similar pattern as followed by cisplatin-DNA adduct (**Figure 6**). The same process also takes place in presence of xanthine oxidase which is present in the living cells [23, 26]. Other studies revealed that formaldehyde is also an important factor for the DNA adduct formation which is formed in the reaction media due to oxidation of different components in media by hydrogen peroxide. Formaldehyde has the carbon through

**161**

*Molecular-Level Understanding of the Anticancer Action Mechanism of Anthracyclines*

which N-C-N i.e. covalent bond forms between 3′ NH2 of amino sugar of doxorubicin/daunorubicin and 2-NH2 of guanosine residue of DNA backbone (**Figure 6**) [25]. Quinone methide 1 is present as a transient form in this reaction. Further, it is stabilized by the hydrogen bond formed between two strands of double helical DNA. Though the structure of the adduct is known but the mechanism by which it

*DNA-drug adduct formation. DNA cisplatin structure was visualized and rendered using Chimera software. Chemical structures were rendered using ChemDraw software. (a) DNA-cisplatin adduct (PDB ID: 2NPW).* 

Doxorubicin forms DNA adduct with the assistance of formaldehyde and the concentration of formaldehyde in the cell is low. So, to overcome this, some prodrugs are administered which gets converted to formaldehyde inside the cell for e.g. pivaloyloxymethyl butyrate give rise to formaldehyde inside the cell when cleaved by esterase. Due to DNA-anthracycline adduct formation cytotoxicity of anthracy-

Cardiotoxicity is one of the main side effects caused by anthracyclines. Types of cardiotoxicity are Type I and Type II, Type I cardiotoxicity results irreversible cell death, while Type II cause reversible damage. At the beginning, cardiotoxicity thought to directly correlate with the cumulative drug dose, but later on, it was discovered that cardiotoxicity can occur even during the treatment [28, 29].

Increased ROS level in cardiac cells is considered as one of the factors contributing to anthracycline mediated cardiotoxicity. Administration of anthracyclines consequences in increased ROS involving the transfer of an electron to oxygen during the reduction of quinone to semiquinone which further gets converted to hydroquinone to complete the redox cycle. This process takes place in mitochondria and the number of mitochondria is mostly higher in cardiomyocytes as compared to other cells [30]. Moreover, enzymes such as SOD, catalase, glutathione transferase, and cytochrome P450 which can reduce the level of ROS in cells are comparatively less in cardiomyocytes resulting in an increased cardiotoxic effect [31]. According to some reports, doxorubicin also increases NADPH oxidase level and enzymes containing flavin like nitric oxide synthase, p450 reductase in cardiomyocytes. These enzymes increase the level of reactive oxygen species in the cell which leads to oxidative stress [31, 32].

*DOI: http://dx.doi.org/10.5772/intechopen.94180*

promotes cell death is not clear [27].

*(b) Schematic representation of doxorubicin-DNA adduct.*

clines readily increases [21].

**3. Side effects**

**Figure 6.**

*Molecular-Level Understanding of the Anticancer Action Mechanism of Anthracyclines DOI: http://dx.doi.org/10.5772/intechopen.94180*

#### **Figure 6.**

*Advances in Precision Medicine Oncology*

and catalase fail to act upon these ROS [17].

*2.2.3 Via forming DNA-anthracycline adduct*

apoptosis.

the semiquinone, this electron gets transferred to oxygen and semiquinone gets converted into stable hydroquinone in this redox cycle. Due to this electron transfer oxygen gets reduced to superoxide which readily gets converted to other reactive oxygen species like hydroxyl radical which is harmful to cells. These reactive oxygen species can lead to DNA damage and lipid peroxidation which finally result in cell

When oxygen becomes limiting, semiquinone free radical transfer the electron to other electron acceptors, and rearrangement in anthracyclines takes place. The aglycone ring remains connected to sugar moiety by a glycosidic bond which is an ether containing linkage with oxygen in it which accepts the unpaired electron and due to this cleavage of glycosidic bond takes place. As a result, the formation of 7-deoxyaglycone takes place which either directly passes through urine or as conjugates in the bile [5]. There are several hypotheses explaining why superoxide dismutase (SOD), catalase, and antioxidants do not work effectively after the administration of anthracyclines. Studies have shown that anthracyclines can alter the glutathione (GSH) level and the enzymes involved in its redox pathway. According to another hypothesis free radicals are formed on the cell surface or internally in the cell and then get transferred to the cell surface due to which SOD

Formation of the anthracycline-DNA adduct in the presence of formaldehyde is another proposed mechanism of anthracycline action. There are studies that show the formation of doxorubicin-DNA at clinically relevant concentrations of drugs [21]. Anthracycline forms covalent bond with one DNA strand and hydrogen bond with other DNA strand resulting in DNA damage. The reaction between anthracycline and cellular formaldehyde is the foremost step to form activated Schiff base which then forms aminal linkage with the exocyclic amino group of guanosine. Formaldehyde in the cells is derived from the various carbon sources like lipids and spermine by iron mediated free radical reactions. In addition, Kato et al. have found increased formaldehyde levels in the cancer cells as compared to normal cells [22]. Phillips et al. using the bidirectional transcription footprinting technique demonstrate that adriamycin-DNA adduct formation induces transcription inhibition with alkylation at specific DNA sites [23]. However, Bilardi et al. have demonstrated that anthracycline-DNA adduct induces breaks in DNA via a mechanism independent of topoisomerase-II [24]. Moreover, DNA breaks formation occurs mainly due to the stalled or collapsed replication fork and these DNA breaks are repaired by homologous recombination dependent process. Forrest et al. investigated the activation of DNA damage response pathways after doxorubicin-DNA adduct formation and found that the downstream processing is dependent on various stages like recognition of adduct during replication, transcription, or any other stage of the cell cycle. Ataxia telangiectasia mutated (ATM) and ATM and Rad3 related protein kinase (ATR) are the DNA damage recognition proteins that get activated in response to double and single DNA strand breaks respectively. Forrest et al. demonstrate that both ATM and ATR proteins have the capability to react with intermediates produced as a result of doxorubicin-DNA adduct formation [25]. DNA-anthracycline adduct formation probably follows a similar pattern as followed by cisplatin-DNA adduct (**Figure 6**). The same process also takes place in presence of xanthine oxidase which is present in the living cells [23, 26]. Other studies revealed that formaldehyde is also an important factor for the DNA adduct formation which is formed in the reaction media due to oxidation of different components in media by hydrogen peroxide. Formaldehyde has the carbon through

**160**

*DNA-drug adduct formation. DNA cisplatin structure was visualized and rendered using Chimera software. Chemical structures were rendered using ChemDraw software. (a) DNA-cisplatin adduct (PDB ID: 2NPW). (b) Schematic representation of doxorubicin-DNA adduct.*

which N-C-N i.e. covalent bond forms between 3′ NH2 of amino sugar of doxorubicin/daunorubicin and 2-NH2 of guanosine residue of DNA backbone (**Figure 6**) [25]. Quinone methide 1 is present as a transient form in this reaction. Further, it is stabilized by the hydrogen bond formed between two strands of double helical DNA. Though the structure of the adduct is known but the mechanism by which it promotes cell death is not clear [27].

Doxorubicin forms DNA adduct with the assistance of formaldehyde and the concentration of formaldehyde in the cell is low. So, to overcome this, some prodrugs are administered which gets converted to formaldehyde inside the cell for e.g. pivaloyloxymethyl butyrate give rise to formaldehyde inside the cell when cleaved by esterase. Due to DNA-anthracycline adduct formation cytotoxicity of anthracyclines readily increases [21].

### **3. Side effects**

Cardiotoxicity is one of the main side effects caused by anthracyclines. Types of cardiotoxicity are Type I and Type II, Type I cardiotoxicity results irreversible cell death, while Type II cause reversible damage. At the beginning, cardiotoxicity thought to directly correlate with the cumulative drug dose, but later on, it was discovered that cardiotoxicity can occur even during the treatment [28, 29].

Increased ROS level in cardiac cells is considered as one of the factors contributing to anthracycline mediated cardiotoxicity. Administration of anthracyclines consequences in increased ROS involving the transfer of an electron to oxygen during the reduction of quinone to semiquinone which further gets converted to hydroquinone to complete the redox cycle. This process takes place in mitochondria and the number of mitochondria is mostly higher in cardiomyocytes as compared to other cells [30]. Moreover, enzymes such as SOD, catalase, glutathione transferase, and cytochrome P450 which can reduce the level of ROS in cells are comparatively less in cardiomyocytes resulting in an increased cardiotoxic effect [31]. According to some reports, doxorubicin also increases NADPH oxidase level and enzymes containing flavin like nitric oxide synthase, p450 reductase in cardiomyocytes. These enzymes increase the level of reactive oxygen species in the cell which leads to oxidative stress [31, 32].

Doxorubicin has a strong affinity for Fe+3 ions and the reaction of ferric ions with hydroxyl and ketone groups of doxorubicin results in a free radical complex of doxorubicin-Fe+2 [33]. The interaction between the negatively charged cell membranes and as this positively charged doxorubicin-Fe complex consequences in lipid peroxidation [33, 34]. Normally the level of iron is quite low in cells and usually, at this low level, doxorubicin cannot interact with iron [29]. The balance of iron is maintained by some transport proteins in the cell and according to some recent studies, doxorubicin disturbs this balance and as a result accumulation of iron occurs in mitochondria [34, 35]. Doxorubicin also interacts with the Fe/S group of iron regulatory protein IRP1 and affects its post-translational modification due to which function of IRP1 is affected and its binding affinity to IRE (iron response element) becomes low resulting in an increased amount of iron in the cell [33]. Mitoferrin, a mitochondrial carrier protein that helps in the entry of iron into the mitochondria and another transfer protein mABC1 export out the iron from mitochondria. Doxorubicin affects the functioning of mABC1 protein due to which it stops the export of iron out of the mitochondria while mitoferrin functions normally and as a result amount of free iron increases inside mitochondria [36].
