Nanodiamonds coupled with 5,7-dimethoxycoumarin, a plant bioactive metabolite, interfere with the mitotic process in B16F10 cells altering the actin organization

Abstract

For the first time, we coupled reduced detonation nanodiamonds (NDs) with a plant secondary metabolite, citropten (5,7-dimethoxycoumarin), and demonstrated how this complex was able to reduce B16F10 tumor cell growth more effectively than treatment with the pure molecule. These results encouraged us to find out the specific mechanism underlying this phenomenon. Internalization kinetics and quantification of citropten in cells after treatment with its pure or ND-conjugated form were measured, and it was revealed that the coupling between NDs and citropten was essential for the biological properties of the complex. We showed that the adduct was not able to induce apoptosis, senescence, or differentiation, but it determined cell cycle arrest, morphological changes, and alteration of mRNA levels of the cytoskeletal-related genes. The identification of metaphasic nuclei and irregular disposition of β-actin in the cell cytoplasm supported the hypothesis that citropten conjugated with NDs showed antimitotic properties in B16F10 cells. This work can be considered a pioneering piece of research that could promote and support the biomedical use of plant drug-functionalized NDs in cancer therapy.

Keywords:

• citropten
• cytoskeletal structure
• plant secondary metabolite
• melanoma
• internalization kinetics

Supplementary materials

Figure S1 The names of the primers used in qPCR assay, their nucleotide sequences and corresponding references, were reported.

Note: The melting temperature for all qPCR amplifications was 58.5°C.

Abbreviation: qPCR, quantitative polymerase chain reaction.

Figure S2 Senescence investigation.

Notes: Microscopic images of B16F10 cells after treatment, for 72 hours, with PBS (A), DMSO (B), ND (200 μg/mL) (C), ND + C (200 μg/mL) (D), C (640 μM) (E), and DOX (F). In green were evidenced, by a specific kit, the senescent cells. The arrows indicate single cells showing the senescent phenotype. The black bars indicate 45 μm.

Abbreviations: PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; ND, nanodiamond; C, citropten; DOX, doxorubicin.

Figure S3 Hypothetic model of the molecular mechanism supposed in the present study.

Notes: B16F10 cells treated with PBS or pure ND (A) and ND + C (B) are shown. The images represent mitotic cells. Hexagons, triangles, and half circles symbolize ND, citropten, and β-actin monomers, respectively. The chains of these last elements are the F-actin. In (A), the cell can complete the anaphase and the levels of G and F-actin are balanced (as indicated by the two arrows of similar thickness), while in presence of ND + C (B) the nuclei remain in prometaphase, as indicated by the overlapping chromosomes present in the nuclear region, and actin equilibrium is moved toward the monomeric form. In this last condition, indeed, the incapacity to build filamentous actin structures, probably due to the capture of G-actin by ND + C adducts, inhibits the mitotic process and the separation of the duplicated genome.

Abbreviations: ND, nanodiamond; C, citropten; Cyt, cytoplasm; Nuc, nuclear region; Ev, endocytic vesicles; ExC, extracellular compartment.

Acknowledgments

The authors thank the Center of Advanced Microscopy “PB Albertano” of the Department of Biology of the University of Rome “Tor Vergata” for the microscopic analysis and Dr Valentina Iannizzotto for her assistance in fluorescence microscopic analysis. This research was supported by the grant “Uncovering Excellence 2014” of the University of Rome ‘Tor Vergata’ for the project entitled “NDDrugCell”.

Disclosure

The authors report no conflicts of interests in this work.