Attenuation of cytotoxic natural product DNA intercalating agents by caffeine.
ABSTRACT Many anti-tumor drugs function by intercalating into DNA. The xanthine alkaloid caffeine can also intercalate into DNA as well as form π-π molecular complexes with other planar alkaloids and anti-tumor drugs. The presence of caffeine could interfere with the intercalating anti-tumor drug by forming π-π molecular complexes with the drug, thereby blocking the planar aromatic drugs from intercalating into the DNA and ultimately lowering the toxicity of the drug to the cancer cells. The cytotoxic activities of several known DNA intercalators (berberine, camptothecin, chelerythrine, doxorubicin, ellipticine, and sanguinarine) on MCF-7 breast cancer cells, both with and without caffeine present (200 μg/mL) were determined. Significant attenuation of the cytotoxicities by caffeine was found. Computational molecular modeling studies involving the intercalating anti-tumor drugs with caffeine were also carried out using density functional theory (DFT) and the recently developed M06 functional. Relatively strong π-π interaction energies between caffeine and the intercalators were found, suggesting an "interceptor" role of caffeine protecting the DNA from intercalation.
- SourceAvailable from: Joana P Miranda[show abstract] [hide abstract]
ABSTRACT: We report the development of a new microwave-based synthetic methodology mediated by Woollins' reagent that allowed an efficient conversion of caffeine into 6-selenocaffeine. A preliminary evaluation on the modulation of antioxidant activity upon selenation of caffeine, using the DPPH assay, indicated a mild antioxidant activity for 6-selenocaffeine, contrasting with caffeine, that exhibited no antioxidant activity under the same experimental conditions. Interestingly, whereas 6-selenocaffeine has revealed to have a low cytotoxic potential in both MCF10A and MCF-7 breast cells (24 h, up to 100 µM, MTT assay), a differential effect was observed when used in combination with the anticancer agents doxorubicin and oxaliplatin in MCF-7 breast cancer cells. The co-treatment of doxorubicin (1 µM) and 6-selenocaffeine (100 µM) resulted in a slight decrease in cellular viability when compared to doxorubicin (1 µM) alone. Conversely, the seleno-caffeine derivative at the same concentration markedly increased the viability of oxaliplatin (100 µM)-treated cells (p < 0.01). Overall, this work highlights an emerging methodology to synthesize organoselenium compounds and points out the differential roles of 6-selenocaffeine in the modulation of the cytotoxicity of anticancer agents.Molecules 01/2013; 18(5):5251-5264. · 2.43 Impact Factor
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ABSTRACT: Caffeine (CAF) and other methylxanthines (MTX) may interact directly with several aromatic, intercalating ligands through mixed stacking aggregation. Formation of such stacking hetero-complexes may decrease their free form concentration and, in consequence, diminish their biological activity, which is often related to their direct interaction with DNA. In this paper interactions of acridine mutagen (ICR191) with DNA in the presence of three MTX: caffeine (CAF), pentoxifylline (PTX) and theophylline (TH) are investigated. Several mathematical models are used to calculate all association constant values and every component concentration in each analyzed mixture. Model McGhee-von Hippel is used to analyze ligand-DNA interaction, and model Zdunek et al.-to analyze ligand-MTX interactions. Finally, two distinct mathematical models are employed to analyze three-component mixture containing ligand, MTX and DNA molecules. The first model describes possible interactions of ligand with DNA and MTX, and rejects direct MTX interactions with DNA. The second model describes all interactions mentioned above and, additionally, allows MTX to interact directly with DNA. Results obtained using these models are similar. However, correspondence of theoretical results to experimental data is better for the first model than the second one. In this paper possible interactions of ICR191 with eukaryotic cell chromatin are also analyzed, showing that CAF reduces acridine mutagen potential to interact directly with cell chromatin. Additionally, it is demonstrated that MTX inhibit mutagenic activity of ICR191 in a dose-dependent manner. Furthermore, biological activity of ICR191-MTX mixtures corresponds with concentration of free mutagen form calculated using appropriate mathematical model.Bioorganic & medicinal chemistry 04/2013; · 2.82 Impact Factor
S c i Pharm
Attenuation of Cytotoxic Natural Produc t DNA
Interc alating Agents by Caffeine
Open Ac c ess
Gabrielle M. HILL 1, Debra M. MORIARITY 2, William N. SETZER * 1
1 Department of Chemistry, University of Alabama in Huntsville, Huntsville, Alabama 35899, USA.
2 Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, Alabama 35899, USA.
* Corresponding author. E-mail: firstname.lastname@example.org (W. N. Setzer)
Sci Pharm. 2011; 79: 729–747
September 17th 2011
Accepted: September 17th 2011
Received: July 14th 2011
This article is available from: http://dx.doi.org/10.3797/scipharm.1107-19
© Hill et al.; licensee Österreichische Apotheker-Verlagsgesellschaft m. b. H., Vienna, Austria.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Many anti-tumor drugs function by intercalating into DNA. The xanthine alkaloid
caffeine can also intercalate into DNA as well as form π-π molecular complexes
with other planar alkaloids and anti-tumor drugs. The presence of caffeine could
interfere with the intercalating anti-tumor drug by forming π-π molecular
complexes with the drug, thereby blocking the planar aromatic drugs from
intercalating into the DNA and ultimately lowering the toxicity of the drug to the
cancer cells. The cytotoxic activities of several known DNA intercalators
(berberine, camptothecin, chelerythrine, doxorubicin, ellipticine, and sanguinar-
ine) on MCF-7 breast cancer cells, both with and without caffeine present
(200 μg/mL) were determined. Significant attenuation of the cytotoxicities by
caffeine was found. Computational molecular modeling studies involving the
intercalating anti-tumor drugs with caffeine were also carried out using density
functional theory (DFT) and the recently developed M06 functional. Relatively
strong π–π interaction energies between caffeine and the intercalators were
found, suggesting an “interceptor” role of caffeine protecting the DNA from
Caffeine • DNA Intercalation • Cytotoxicity • π–π Complex • Density Functional Theory
Caffeine is ingested by millions of people on a daily basis in tea, coffee, soft drinks, and
various other foods. Because of its wide-spread usage, caffeine has been the focus of
730 G. M. Hill, D. M. Moriarity, and W. N. Setzer:
Sci Pharm. 2011; 79: 729–747
many studies for their effects on the body . Overuse of caffeine is associated with heart
problems, addiction due to its stimulant qualities and reproductive problems. However, it
acts as a vasodilator, and hence alleviates pain associated with migraines, and moderate
use of caffeine is linked to prevention of some autoimmune diseases, diabetes, and it can
act as an anti-oxidant . The actual interaction between caffeine and DNA is a
controversial subject; it is not fully understood, but a possible scenario is that it intercalates
Caffeine is a planar aromatic xanthine alkaloid which leads to the hypothesis that it could
very easily form π–π complexes with other planar aromatic molecules such as
nucleobases in DNA and several types of anticancer drugs known to intercalate DNA
based on their planar structures [2, 3]. Caffeine has been shown to associate with the
intercalators doxorubicin [4–6], mitoxantrone [5–7], topotecan , acridine orange , and
with itself . It has been previously confirmed that caffeine and theophylline can protect
DNA by affecting the binding of toxic compounds [1, 11, 12]. However, in cancer patients
undergoing chemotherapy, this property is not preferable. Several chemotherapy drugs
such as doxorubicin and daunorubicin work by intercalating into DNA, and caffeine has
been shown to reduce the toxicity of these drugs [1, 13]. Previous studies have shown that
caffeine forms “stacking complexes” with these anti-cancer drugs which affects the binding
of the drugs into the DNA and regulates the movement through cell membranes, and also
that caffeine actually displaces drugs that have already been bound to DNA .
Two mechanisms have been proposed for the modulation of DNA intercalating drugs
[14, 15], which involve equilibria of complexed drug and caffeine, caffeine and DNA, and
drug and DNA. The mechanism proposed works within a system that consists of two
ligands in the presence of DNA. X represents the first ligand, which is the anticancer drug,
and Y represents the other binding molecule such as caffeine. The caffeine molecule (Y)
can essentially do two things. It can either bind to the anticancer drug, acting as the
“interceptor” molecule or it can bind to the DNA, acting as the “protector” molecule as it is
“protecting” the DNA molecule from being bound by the anticancer drug (X) (Fig. 1).
In order to provide additional insight into the molecular interactions of caffeine with
intercalating natural products and with DNA, we have examined: (a) the cytotoxic activities
of the antitumor agents chelerythrine, camptothecin, ellipticine, doxorubicin, berberine, and
sanguinarine, both alone and in the presence of caffeine, on the MCF-7 human breast
adenocarcinoma cell line; (b) molecular docking of caffeine and the antitumor agents with
DNA; and (c) molecular modeling of π–π interactions between caffeine and the antitumor
agents and between these compounds and the guanine-cytosine base pair using density
Results and Discussion
In-vitro cytotoxicity assays on MCF-7 cells were carried out for six compounds (berberine,
camptothecin, chelerythrine, doxorubicin, ellipticine, and sanguinarine) at various
concentrations in order to determine their IC50 values. In a separate assay, the medium
was supplemented with caffeine at a concentration of 200 μg/mL (previously determined to
have little effect on the cells), and IC50 values of the compounds re-determined in the
π–π Interactions Between Caffeine and Intercalators 731
Sci Pharm. 2011; 79: 729–747
presence of caffeine. The effects of caffeine on the cytotoxic activity of the intercalating
antitumor agents are shown in Table 1.
Cytotoxicity attenuation of intercalating antitumor agents by caffeine.
a Standard deviations in parentheses. b Caffeine concentration = 1030 μM.
Caffeine resulted in attenuation on the cytotoxic activities of all the intercalating drugs in
the present study. Some of the intercalating drugs were more affected than others as
shown in their IC50 calculation in Table 1. Significant attenuation (P < 0.001) was observed
for all intercalating drugs in this study; large attenuations were observed for berberine,
camptothecin, doxorubicin and ellipticine, while chelerythrine and sanguinarine were
marginally attenuated. The increased in IC50 due to caffeine may be attributed to either
caffeine competing for intercalation sites in DNA (the “protector” scheme), or that the
caffeine has formed a π–π complex with the intercalating drug (the “interceptor” scheme,
Fig. 1) .
Modulation of DNA intercalation by caffeine via “interceptor” (left) or “protector”
(right) interactions .
732 G. M. Hill, D. M. Moriarity, and W. N. Setzer:
Sci Pharm. 2011; 79: 729–747
The slight attenuation in the IC50 values of sanguinarine and chelerythrine from the system
with no caffeine to the system with caffeine could indicate there is another mechanism of
cytotoxic activity in addition to DNA intercalation. It has been reported that sanguinarine
induces oxidation within the cell which causes double-stranded DNA breaks .
Additionally, in some cell lines, sanguinarine was found to induce caspase activation 
or severe glutathione depletion [18, 19], leading to apoptosis. It has also been suggested
that both sanguinarine and chelerythrine produce H2O2 and other reactive oxygen species,
which cause oxidation and subsequent apoptosis .
The Molegro Virtual Docker (MVD) [21, 22] was used to carry out a molecular docking
analysis of the intercalating antitumor agents as well as caffeine with DNA in order to
compare docking energies. The molecular docking studies were based on structures of
various intercalators with DNA that are available in the Protein Data Bank (PDB). A total of
nine different DNA structures with two intercalation sites each were modeled in this
docking study. The docking energies are summarized in Table 2.
Molegro Virtual Docker (MVD) docking energies (kcal/mol) of caffeine and
intercalating antitumor agents with DNA.
−14.9 −15.0 −16.5 −16.4 −16.3 −16.5 −16.0 −16.0 −16.4 −16.5
−18.5 −18.8 −22.9 −22.3 −23.2 −22.7 −17.7 −18.1 −20.0 −21.6
Camptothecin −18.5 −18.6 −22.0 −22.0 −22.2 −22.6 −19.4 −19.3 −22.1 −22.4
−19.7 −20.4 −21.1 −21.4 −22.8 −22.2 −20.2 −20.2 −21.6 −21.9
−26.7 −26.0 −25.3 −25.3 −26.1 −26.1 −25.7 −25.3 −23.7 −23.5
−18.1 −17.8 −20.8 −20.8 −20.8 −20.8 −18.8 −18.6 −21.3 −21.4
−20.1 −20.1 −21.6 −21.5 −22.7 −23.2 −20.3 −20.1 −22.1 −22.5
1 2 1 2
−16.4 −16.5 −16.4 −16.7 −16.5 −16.7 −16.5 −16.7
−23.1 −23.3 −22.9 −20.1 −23.2 −23.4 −19.1 −20.1
Camptothecin −22.2 −22.6 −22.4 −22.8 −22.1 −23.3 −21.6 −22.9
−22.6 −21.6 −22.9 −22.4 −23.1 −23.1 −21.9 −22.6
−25.7 −25.3 −25.4 −27.3 −26.5 −26.7 −23.9 −26.4
−20.9 −21.0 −21.0 −21.0 −20.7 −20.9 −20.5 −20.5
−22.4 −22.9 −22.7 −23.3 −23.0 −23.8 −21.8 −23.3
a Protein Data Bank identification number. b Intercalation site number in the DNA structure. c Average
intercalation docking energy for all structures.
1 2 2 2
Caffeine is the worst binding ligand according to the docking studies and averaged
4 kcal/mol weaker binding than the worst intercalating drug (ellipticine). The ligand with the
strongest docking energy was doxorubicin, which docked, on average, 9.3 kcal/mol more
strongly than caffeine. These docking data would suggest that displacement of
intercalating antitumor agents is not a thermodynamically favorable process. Previous
spectroscopic [23, 24] and theoretical  studies have suggested that intercalation is not
π–π Interactions Between Caffeine and Intercalators 733
Sci Pharm. 2011; 79: 729–747
the predominant mechanism for interaction of caffeine with DNA. Likewise, de-intercalation
of ethidium bromide from DNA by caffeine has been attributed to caffeine–ethidium
bromide stacking aggregation and not intercalation of caffeine into DNA .
Density Functional Theory Calculations
In order to probe π–π complexation between caffeine and the intercalators, DFT modeling
studies were carried out using Spartan ’08 for Windows  with the M06 functional 
and the 6-31G* basis set. The recently developed M06 suite of density functionals  has
been shown to give generally superior performance for non-covalent interactions such as
hydrogen bonding, dipole-dipole, and π–π stacking interactions, unlike widely used B3LYP
and BLYP methods [29–32]. In this work we have chosen to use the M06 hybrid functional
because of its previously reported performance with π–π stacking interactions . A
number of different orientations were constructed and full geometry optimization was
carried out. The π–π interaction energies for the lowest-energy orientations of the six
intercalating compounds with caffeine are summarized in Table 3.
The M06 calculations all indicate relatively strong π–π interactions between caffeine and
the antitumor drugs with gas-phase exothermic interaction energies ranging from −16.8 to
−21.6 kcal/mol and aqueous energies of −11.0 to −16.5 kcal/mol. Interactions that likely
contribute to these favorable π–π complexes include dipole-dipole interactions [33, 34],
electrostatic interactions [35, 36] and van der Waals interactions , as well as frontier
molecular orbital interactions [38, 39]. Frontier molecular orbital theory  suggests that
the important interactions of caffeine with the intercalating drugs will be the HOMO of
caffeine and the LUMO of the antitumor agents.
π–π Interaction energies of intercalating antitumor agents and caffeine.
Caffeine + Berberine
Caffeine + Camptothecin
Caffeine + Chelerythrine
Caffeine + Doxorubicin
Caffeine + Ellipticine
Caffeine + Sanguinarine
a Calculated interaction energies in the gas phase.
b Calculated interaction energies using an aqueous solvation model (SM5.4 ).
Of the different caffeine-berberine orientations, the lowest energy orientation (Fig. 2) is
such that there are favorable dipole-dipole and electrostatic interactions. Frontier
molecular orbital overlap, however, is not favorable. The lowest-energy π–π orientation for
caffeine with camptothecin (Fig. 3) has the molecular dipoles of the caffeine and
camptothecin aligned rather than opposed and the electrostatic interactions are also
unfavorable. They do, however, have favorable frontier molecular orbital alignments. In the
lowest-energy π–π complex between chelerythrine and caffeine the molecular dipoles are
perpendicular, frontier molecular orbital overlap is not evident, but electrostatic interactions
are generally favorable (Fig. 4). The molecular dipoles for caffeine and doxorubicin are
nearly perpendicular in the lowest energy orientation (Fig. 5), but there do seem to be