Combined Action of Hyper-Harmonized Hydroxylated Fullerene Water Complex and Hyperpolarized Light Leads to Melanoma Cell Reprogramming In Vitro

Abstract

(1) Background: Their unique structure and electron deficiency have brought fullerenes into the focus of research in many fields, including medicine. The hyper-harmonized hydroxylated fullerene water complex (3HFWC) formulation has solved the limitations of the poor solubility and bioavailability of fullerenes. To achieve better antitumor activity, 3HFWC was combined with short-term irradiation of cells with hyperpolarized light (HPL) generated by the application of a nanophotonic fullerene filter in a Bioptron^® device. The benefits of HPL were confirmed in the microcirculation, wound healing and immunological function. (2) Methods: B16, B16-F10 and A375 melanoma cells were exposed to a wide spectrum of 3HFWC doses and to a single short-term HPL irradiation. (3) Results: Apart from the differences in the redox status and level of invasiveness, the effects of the treatments were quite similar. Decreased viability, morphological alteration, signs of melanocytic differentiation and cellular senescence were observed upon the successful internalization of the nanoquantum substance. (4) Conclusions: Overall, 3HFWC/HPL promoted melanoma cell reprogramming toward a normal phenotype.

Keywords: differentiation; hyperpolarized light; melanoma; senescence; water-layered fullerol.

Figure 1

Inhibition of melanoma cell viability by hyper-harmonized hydroxylated fullerene water complex (3HFWC) and hyperpolarized light (HPL) in vitro. (A) B16, (B) B16-F10 and (C) A375 cells were treated with the indicated dose range of 3HFWC (measured as µg/mL of fullerol) and with HPL, for 15 min. The viability was evaluated by MTT (left panel) and crystal violet (CV) (right panel) assays. The results after 48 h (A,B) and after 72 h of incubation (C) are shown. * Significant if p < 0.05 in comparison to untreated cells.

Figure 2

Inhibition of melanoma cell proliferation by 3HFWC and/or HPL in vitro. (A) Cell abundance (cells/mm²) and propidium iodide staining (white squares—mitotic figures; magnification: ×630, scale bar: 50 µm); (B) carboxyfluorescein succinimidyl ester (CFSE) staining; *** significant if p < 0.001 in comparison to untreated (control) cells.

Figure 3

Morphometric and microscopic analysis of melanoma cells’ nuclei after the treatments with 3HFWC and/or HPL. (A) Average nuclear surface area (µm²); (B) distribution of nuclei based on their surface area; (C) representative micrographs of hematoxylin and eosin (HE)-stained melanoma cells; sl—senescence-like; dl—dendritic-like phenotype (magnification and scale bar: ×1000, 20 µm). Statistical significance, in comparison to untreated (control) cells: * if p < 0.05, ** p < 0.01, *** p < 0.001; in comparison to identical treatment regarding 3HFWC without HPL irradiation: ⁺⁺ if p < 0.01, ⁺⁺⁺ p < 0.001.

Figure 4

Cell senescence stimulation by 3HFWC and/or HPL. (A) Senescence-associated β-galactosidase detection; (B) microscopic analysis of senescent phenotype; TEM analysis of B16-F10 cells (Nu—nucleus; ld—lipid droplet; in rectangles—mitochondria) and Sudan black B staining of lipid droplets (inserts, black arrowheads); magnification and scale bar: TEM: ×13,000, 1 µm; LM: ×1000, 20 µm.

Figure 5

Melanocytic differentiation of B16 and B16-F10 melanoma cells treated with 3HFWC and/or HPL. (A) HE staining: occurrence of melanoma cells with brownish mature melanosomes (white triangles); (B) TEM: melanosomes in different stages of maturation (I–IV) in B16-F10 cells; small coated vesicles in the cytoplasm (white arrowheads) (Nu—nucleus; m—mitochondrion; ld—lipid droplet); (C) immunocytochemical detection of tubulin in B16 cells treated with 3HFWC and HPL; melanosomes along these cytoskeletal elements (black arrows) (magnification and scale bar: A—×400, 50 µm; B—×13,000, 1 µm; C—×1000, 20 µm).

Figure 6

Production of nitric oxide (NO) and reactive oxygen and nitrogen species (ROS/RNS) in melanoma cells after the treatments with 3HFWC and/or HPL. (A) DAF-FM diacetate staining; (B) dihydrorhodamine 123 (DHR123) staining.