**2. Proposed action mechanisms**

#### **2.1 Anthracyclines as DNA intercalators**

The exact mechanism of the anthracycline in the body is not known and still under investigation. However, DNA is recognized as the prime target of well-known anthracycline like doxorubicin. The primary mechanism involves the intercalation of planar tetracyclic chromophore between the DNA base pairs subsequently affecting the transcription and translation of DNA. The binding affinity of the drug to DNA is not only the factor contributing to the cytotoxic activity of anthracyclines but other factors like binding mode and binding site of anthracycline also play important role in exerting cytotoxic effects.

**157**

**Figure 3.**

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

The specificity, binding affinity, and the binding mode of every anthracycline differ with the difference in the sequence of DNA bases (**Figures 3**–**5**). Structural, computational and solution studies on the daunomycin-DNA complex have provided the information that daunomycin has preferential binding where AT is present between two GC base pairs i.e. GCATGC [9, 10]. Equilibrium binding and DNase footprinting methods were utilized to study site and sequence specificity of daunorubicin to DNA. The results of these experiments demonstrate that daunomycin indeed recognizes specific DNA sequences and its binding affinity with DNA increases with an increase in GC content [11]. Moreover, the effect of daunorubicin in cleaving the linear pBR322 DNA by restriction endonuclease *Eco*RI and PvuI was investigated to assess the sequence-specific binding of daunorubicin [11]. The recognition sequence of *Eco*RI and PvuI are 5′-GAATTC-3′and 5′-CGATCG-3′ respectively. Chaires et al. observed that PvuI inhibits the rate of digestion of pBR322 DNA more than the *Eco*RI suggesting preferential sequence specificity of daunorubicin. Similar results were evident from the crystal structure analysis of daunomycin-DNA d (CpGpTpApCpG) complexes (**Figure 4**) [10]. The specificity for the GC base pair is due to the hydrogen bonds formed during the interaction. The hydroxyl group on C9 of daunorubicin interact with N2 and N3 of guanine with two hydrogen bonds. This preference to GC base pair also explains the increase in binding affinity of the drug with an increase in GC content of DNA [10]. Similarly, crystal structures of anthracyclines like doxorubicin, epirubicin, and idarubicin show sequence-specific intercalative binding mode between DNA

Moreover, several experiments were done to study the intercalation mechanism of anthracyclines with DNA. A spectrofluorometric method was also developed for the estimation of anthracycline intercalation in living cells and DNA solutions [12]. Belloc et al. have done measurement of anthracyclines (daunorubicin and idarubicin) intercalation in the DNA of living cells by flow cytometry [13]. Ashley et al. has demonstrated the intercalative property of anthracyclines in nuclear as well as in mitochondrial DNA using picogreen (fluorescent DNA binding dye) [14]. The intercalation of anthracycline into mitochondrial DNA has a significant impact

*Intercalation of doxorubicin between GC basepairs. Crystal structure of doxorubicin getting intercalated between GC bases. Structures are taken from protein data bank. Light green color depicts AT baseparing and dark green color shows GC bases. Red dots represent crystallized water molecules. Figure clearly depicts intercalation of doxorubicin in GC bases of DNA and preferential binding with nucleotide sequences.*

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

bases (**Figures 3** and **5**).

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

The specificity, binding affinity, and the binding mode of every anthracycline differ with the difference in the sequence of DNA bases (**Figures 3**–**5**). Structural, computational and solution studies on the daunomycin-DNA complex have provided the information that daunomycin has preferential binding where AT is present between two GC base pairs i.e. GCATGC [9, 10]. Equilibrium binding and DNase footprinting methods were utilized to study site and sequence specificity of daunorubicin to DNA. The results of these experiments demonstrate that daunomycin indeed recognizes specific DNA sequences and its binding affinity with DNA increases with an increase in GC content [11]. Moreover, the effect of daunorubicin in cleaving the linear pBR322 DNA by restriction endonuclease *Eco*RI and PvuI was investigated to assess the sequence-specific binding of daunorubicin [11]. The recognition sequence of *Eco*RI and PvuI are 5′-GAATTC-3′and 5′-CGATCG-3′ respectively. Chaires et al. observed that PvuI inhibits the rate of digestion of pBR322 DNA more than the *Eco*RI suggesting preferential sequence specificity of daunorubicin. Similar results were evident from the crystal structure analysis of daunomycin-DNA d (CpGpTpApCpG) complexes (**Figure 4**) [10]. The specificity for the GC base pair is due to the hydrogen bonds formed during the interaction. The hydroxyl group on C9 of daunorubicin interact with N2 and N3 of guanine with two hydrogen bonds. This preference to GC base pair also explains the increase in binding affinity of the drug with an increase in GC content of DNA [10]. Similarly, crystal structures of anthracyclines like doxorubicin, epirubicin, and idarubicin show sequence-specific intercalative binding mode between DNA bases (**Figures 3** and **5**).

Moreover, several experiments were done to study the intercalation mechanism of anthracyclines with DNA. A spectrofluorometric method was also developed for the estimation of anthracycline intercalation in living cells and DNA solutions [12]. Belloc et al. have done measurement of anthracyclines (daunorubicin and idarubicin) intercalation in the DNA of living cells by flow cytometry [13]. Ashley et al. has demonstrated the intercalative property of anthracyclines in nuclear as well as in mitochondrial DNA using picogreen (fluorescent DNA binding dye) [14]. The intercalation of anthracycline into mitochondrial DNA has a significant impact

#### **Figure 3.**

*Advances in Precision Medicine Oncology*

in the model list of medicines [7]. But it has been discovered that repeated administration of these drugs can impart chemotherapy-resistance to the tumors and cardiotoxicity [4]. To reduce or subside these side effects, major efforts are being done to find better alternatives. Therefore, the study of more than 2000 analogs has

*Chemical structure of epirubicin and idarubicin. Epirubicin and doxorubicin (Figure 1) differs at position no. 3 (C4′ of sugar moiety, stereoisomer). Idarubicin differs from daunorubicin (Figure 1) at position 1 by* 

*absence of methoxy group. Chemical structures were rendered using ChemDraw software.*

*Chemical structure of doxorubicin and daunorubicin. Dotted circle and dotted line arrow represents probable substitution position in anthracyclines. Doxorubicin and daunorubicin differ at C14 position encircled with 2. Green dotted line is used to depict aglycone and sugar moieties of anthracycline drugs. Chemical structures were* 

Regarding the chemical structure of daunomycin C27H29NO10 and doxorubicin C27H29NO11, they share the same carbon skeleton (**Figure 1**). The difference in their chemical structure comes at the side chain at C-14 position; daunomycin has a hydrogen atom whereas doxorubicin has an alcohol group (**Figure 1**) [5, 6].

The exact mechanism of the anthracycline in the body is not known and still under investigation. However, DNA is recognized as the prime target of well-known anthracycline like doxorubicin. The primary mechanism involves the intercalation of planar tetracyclic chromophore between the DNA base pairs subsequently affecting the transcription and translation of DNA. The binding affinity of the drug to DNA is not only the factor contributing to the cytotoxic activity of anthracyclines but other factors like binding mode and binding site of anthracycline also play

**156**

been done so far [8].

**Figure 1.**

**Figure 2.**

*rendered using ChemDeaw software.*

**2. Proposed action mechanisms**

**2.1 Anthracyclines as DNA intercalators**

important role in exerting cytotoxic effects.

*Intercalation of doxorubicin between GC basepairs. Crystal structure of doxorubicin getting intercalated between GC bases. Structures are taken from protein data bank. Light green color depicts AT baseparing and dark green color shows GC bases. Red dots represent crystallized water molecules. Figure clearly depicts intercalation of doxorubicin in GC bases of DNA and preferential binding with nucleotide sequences.*

#### **Figure 4.**

*Intercalation of daunorubicin between GC basepairs. Crystal structure of daunorubicin getting intercalated between GC bases. Structures are taken from protein data bank. Light green color depicts AT baseparing and dark green color shows GC bases. Red dots represent crystallized water molecules. Figure clearly depicts intercalation of doxorubicin in GC bases of DNA and preferential binding with nucleotide sequences.*

#### **Figure 5.**

*Intercalation of epirubicin and idarubicin between GC basepairs. Crystal structure of epirubicin getting intercalated between AT and GC basepairs. Crystal structure of idarubicin getting intercalated between GC bases. Structures are taken from protein data bank. Red dots represent crystallized water molecules. Figure clearly depicts intercalation of epirubicin and idarubicin in GC bases of DNA and preferential binding with nucleotide sequences.*

on mitochondrial toxicity. The effect of doxorubicin binding on the morphology of the single stranded DNA was further quantitatively analyzed using Atomic Force Microscopy (AFM). AFM studies strengthen the probable mechanism of intercalative binding mode as consequences of doxorubicin interaction with DNA [15].

Other studies found that B-DNA is preferred over Z-DNA by the daunorubicin for binding. Allosteric conversion of the Z form into B form has also been observed in some cases. Ionic concentration in which usually Z form of DNA is present changes to B form on the binding of daunorubicin to poly dGdC or resist to change from B form to Z form [16]. There are several pieces of evidence suggesting drug binding to DNA results in the inhibition of specific DNA function contributing towards their therapeutic effects.

**159**

cleavage of DNA.

dividing cells [18, 19].

*2.2.2 Via oxidative stress*

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

Anthracyclines are known to damage DNA by several mechanisms which include topoisomerase-II poisoning, free radical formation, and DNA-anthracycline adduct formation. The semiquinone radical of anthracycline can intercalate between DNA base pair resulting in DNA damage by forming reactive oxygen species (ROS).

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

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

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

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

**2.2 How anthracyclines damage DNA?**

*2.2.1 Via topoisomerase II poisoning*
