Mechanism of DNA Intercalation by Chloroquine Provides Insights into Toxicity.

Chloroquine has been used as a potent antimalarial, anticancer drug, and prophylactic. While chloroquine is known to interact with DNA, the details of DNA-ligand interactions have remained unclear. Here we characterize chloroquine-double-stranded DNA binding with four complementary approaches, including optical tweezers, atomic force microscopy, duplex DNA melting measurements, and isothermal titration calorimetry. We show that chloroquine intercalates into double stranded DNA (dsDNA) with a KD ~ 200 µM, and this binding is entropically driven. We propose that chloroquine-induced dsDNA intercalation, which happens in the same concentration range as its observed toxic effects on cells, is responsible for the drug's cytotoxicity.

New Insights into the Mechanism of Action of the Drug Chloroquine: Direct Interaction with DNA and Cytotoxicity.

Chloroquine (CLQ) and hydroxychloroquine (HCLQ) are compounds largely employed in the treatment of various human diseases for decades. Nevertheless, a number of intrinsic details concerning their mechanisms of action, especially at the molecular level, are still unknown or have presented controversial results in the literature. Using optical tweezers, here, we investigate at the single-molecule level the molecular mechanism of action of the drug CLQ in its intrinsic interaction with the double-stranded (ds)DNA molecule, one of its targets inside cells, determining the binding modes and the physicochemical (binding) parameters of the interaction. In particular, we show that the ionic strength of the surrounding medium strongly influences such interaction, changing even the main binding mode. In addition, the cytotoxicity of CLQ against three different cell lines was also investigated here, allowing one to evaluate and compare the effect of the drug on the cell viability. In particular, we show that CLQ is highly cytotoxic at a very low (a few micromolar) concentration range for all cell lines tested. These results were rigorously compared to the equivalent ones obtained for the closely related compound hydroxychloroquine (HCLQ), allowing a critical comparison between the action of these drugs at the molecular and cellular levels.

Caffeine Enhances the Toxicity of Platinum-Based Drugs at the Molecular Level Even Outside of the Intracellular Environment: A Single-Molecule Force Spectroscopy Study.

It is well reported in the literature that caffeine, the most consumed alkaloid around the world, enhances the anticancer effects of the drug cisplatin by inhibiting DNA repair by the cellular machinery. Here, we perform single-molecule force spectroscopy assays with optical tweezers to show that caffeine enhances the toxicity not only of cisplatin but also of various different platinum-based drugs already at the molecular level, using samples containing only double-stranded (ds)DNA, platinum drugs, and the alkaloid in a simple phosphate buffer, that is, completely out of the complex environment found inside real living cells. In fact, our results show that caffeine acts as an allosteric catalyst which increases the effective equilibrium binding constant between DNA and the platinum drugs, also interfering in the cooperativity of the binding reactions. To the best of our knowledge, this is the first time that such a property of caffeine was demonstrated and characterized from a pure physicochemical perspective, outside the cellular environment. Thus, the present work provides new insights into the use of this alkaloid for current chemotherapeutic applications.

How light absorption modifies the radiative force on a microparticle in optical tweezers.

Reflection and refraction of light can be used to trap small dielectric particles in the geometrical optics regime. Absorption of light is usually neglected in theoretical calculations, but it is known that it occurs in the optical trapping of semi-transparent particles. Here, we propose a generalization of Ashkin's model for the radiative force exerted on a spherical bead, including the contribution due to attenuation/absorption of light in the bulk of the particle. We discuss in detail the balance between refraction, reflection, and absorption for different optical parameters and particle sizes. Our findings contribute to the understanding of optical trapping of light-absorbing particles and may be used to predict whenever absorption is important in real experiments.

New antineoplastic agent based on a dibenzoylmethane derivative: Cytotoxic effect and direct interaction with DNA.

Melanoma accounts for only 4% of all skin cancers but is among the most lethal cutaneous neoplasms. Dacarbazine is the drug of choice for the treatment of melanoma in Brazil through the public health system mainly because of its low cost. However, it is an alkylating agent of low specificity and elicits a therapeutic response in only 20% of cases. Other drugs available for the treatment of melanoma are expensive, and tumor cells commonly develop resistance to these drugs. The fight against melanoma demands novel, more specific drugs that are effective in killing drug-resistant tumor cells. Dibenzoylmethane (1,3-diphenylpropane-1,3-dione) derivatives are promising antitumor agents. In this study, we investigated the cytotoxic effect of 1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP) on B16F10 melanoma cells as well as its direct interaction with the DNA molecule using optical tweezers. DPBP showed promising results against tumor cells and had a selectivity index of 41.94. Also, we demonstrated the ability of DPBP to interact directly with the DNA molecule. The fact that DPBP can interact with DNA in vitro allows us to hypothesize that such an interaction may also occur in vivo and, therefore, that DPBP may be an alternative to treat patients with drug-resistant melanomas. These findings can guide the development of new and more effective drugs.

Gas Chromatography-Triple Quadrupole Mass Spectrometry Analysis and Vasorelaxant Effect of Essential Oil from Protium heptaphyllum (Aubl.) March.

The Protium heptaphyllum species, also known as Almécega, produces an oily resin, used in folk medicine as an analgesic and anti-inflammatory agent, in healing, and as an expectorant, which is rich in pentacyclic triterpenes and essential oils. In this study, the essential oil obtained by hydrodistillation of Almécega's resin was analyzed by gas chromatography-triple quadrupole mass spectrometry and evaluated for chemical composition and vasorelaxant activity in rat superior mesenteric artery. The main constituents determined by gas chromatography-triple quadrupole mass spectrometry were limonene, p-cineole, and o-cymene. In intact rings precontracted with phenylephrine (Phe 1 µM), EOPh (3-750 µg/mL) induced relaxation, and the essential oil had a concentration-dependent vasorelaxant effect, without involvement of endothelial mediators.

Isolation, characterization and evaluation of antimicrobial and cytotoxic activity of estragole, obtained from the essential oil of Croton zehntneri (Euphorbiaceae).

Croton zehntneri (Euphorbiaceae) is a native aromatic plant from Northeast region of Brazil. The monoterpenoid estragole (ESL) has been isolated by classical chromatographic methods from the essential oil (EO) of C. zehnteneri leaves and characterized by GC-FID and GC-MS, its antimicrobial and cytotoxic potentials being assessed. The analysis of the EO enabled the identification of 100% of the integrated constituents, of which yield was about 1.8%. The main components identified were: eucalyptol, estragole (84.7%) and spathulenol. The dosage of 50 µg/disk of ESL presented fairly significant zones of inhibition against Gram-positive bacteria and fungi. The ESL presented toxicity against Artemia salina with LC50 and LC90 of 4,54 and 8,47 µg mL-1. However, in tumor inhibition assays (human cells), there were no rewarding inhibition in any of the human cancer cell lines (MCF-7, HEP-2 and NCI-H292).

Normal and tumoral melanocytes exhibit q-Gaussian random search patterns.

In multicellular organisms, cell motility is central in all morphogenetic processes, tissue maintenance, wound healing and immune surveillance. Hence, failures in its regulation potentiates numerous diseases. Here, cell migration assays on plastic 2D surfaces were performed using normal (Melan A) and tumoral (B16F10) murine melanocytes in random motility conditions. The trajectories of the centroids of the cell perimeters were tracked through time-lapse microscopy. The statistics of these trajectories was analyzed by building velocity and turn angle distributions, as well as velocity autocorrelations and the scaling of mean-squared displacements. We find that these cells exhibit a crossover from a normal to a super-diffusive motion without angular persistence at long time scales. Moreover, these melanocytes move with non-Gaussian velocity distributions. This major finding indicates that amongst those animal cells supposedly migrating through Lévy walks, some of them can instead perform q-Gaussian walks. Furthermore, our results reveal that B16F10 cells infected by mycoplasmas exhibit essentially the same diffusivity than their healthy counterparts. Finally, a q-Gaussian random walk model was proposed to account for these melanocytic migratory traits. Simulations based on this model correctly describe the crossover to super-diffusivity in the cell migration tracks.

Revisiting the neighbor exclusion model and its applications.

We review the neighbor exclusion model and some of its applications to analyze the binding data of DNA-ligand complexes. We revisit the closed form of the model developed by McGhee and von Hippel in 1974, showing that this classic model can be used to help studying the behavior of DNA contour and persistence lengths when interacting with intercalating ligands. We present methods to quantitatively analyze the variation of these two quantities, allowing one to determine important parameters of the interaction such as the intrinsic binding constant and the exclusion number of the ligand.

New tools to study biophysical properties of single molecules and single cells.

We present a review on two new tools to study biophysical properties of single molecules and single cells. A laser incident through a high numerical aperture microscope objective can trap small dielectric particles near the focus. This arrangement is named optical tweezers. This technique has the advantage to permit manipulation of a single individual object. We use optical tweezers to measure the entropic elasticity of a single DNA molecule and its interaction with the drug Psoralen. Optical tweezers are also used to hold a kidney cell MDCK away from the substrate to allow precise volume measurements of this single cell during an osmotic shock. This procedure allows us to obtain information about membrane water permeability and regulatory volume increase. Defocusing microscopy is a recent technique invented in our laboratory, which allows the observation of transparent objects, by simply defocusing the microscope in a controlled way. Our physical model of a defocused microscope shows that the image contrast observed in this case is proportional to the defocus distance and to the curvature of the transparent object. Defocusing microscopy is very useful to study motility and mechanical properties of cells. We show here the application of defocusing microscopy to measurements of macrophage surface fluctuations and their influence on phagocytosis.