Multiparametric analysis of the effectiveness of cisplatin on cutaneous squamous carcinoma cells using two different types of adjuvants

Abstract

The objective of this study was to regulate the cytotoxicity of cisplatin (cisPt) minimizing its adverse effects. For this purpose, the lowest cisPt concentration needed to obtain a significant positive response in cutaneous squamous cell carcinoma (cSCC) was explored. Two adjuvant agents as gold nanoparticles (AuNP) and chelating tricine were tested as enhancers in cisPt treatment. Effectiveness of all treatments was assessed by means of biochemical techniques, which offer quantitative data, as well as two microscopy-based techniques that provided qualitative cell imaging. The present work confirms the effectiveness of free cisplatin at very low concentrations. In order to enhance its effectiveness while the side effects were probably diminished, cisPt 3.5 μM was administered with AuNP 2.5 mM, showing an effectiveness practically equal to that observed with free cisPt. However, the second treatment investigated, based on cisPt 3.5 μM combined with tricine 50 mM, enhanced drug effectiveness, increasing the percentage of cells dying by apoptosis. This treatment was even better in terms of cell damage than free cisPt at 15 μM. Images obtained by TEM and cryo-SXT confirmed these results, since a notable number of apoptotic bodies were detected when cisPt was combined with tricine. Thus, tricine was clearly a better adjuvant for cisPt treatments.

Single-Cell Chemistry of Photoactivatable Platinum Anticancer Complexes

“…Endosomes were identified in two independent cells treated with Pt2 under dark conditions (0.14−0.99 μm 3 in volume, average: 0.29 ± 0.18 μm 3 ), and exhibited a variety of dense or lucent interiors, in agreement with literature cryo-SXT characterizations of endosomes. 35,43 Some of these endosomal-structures are dark in appearance, which may be indicative of internalized platinum due to the enhanced X-ray absorption of heavy metals compared to endogenous elements. Importantly, diazido-Pt(IV) complexes are relatively inert in the dark.…”

“…In cryo-SXT, cells are illuminated with soft X-rays in the “water” window, the region between the K-absorption edges of carbon (285 eV) and oxygen (543 eV), where carbon-rich biological matter absorbs photons more than the oxygen-rich medium that surrounds it, providing natural contrast for imaging. − This allows 3D imaging of vitrified cell populations under cryogenic conditions and the investigation of drug-induced changes in cancer cells at near physiological states, thus avoiding the need to use chemical or mechanical treatments. − Cryo-SXT has previously been used to probe drug-induced morphological changes to cancer cells treated with iridium complexes, , iron nanoparticles, and cisplatin (in combination with other chemotherapeutics), allowing the monitoring of cancer-related cellular events which cannot be achieved using conventional light or electron microscopy . Complementary to this, structured illumination microscopy (SIM) is a powerful 3D imaging technique that pushes the resolution of optical microscopy past the Abbe diffraction limit, − and under cryogenic conditions can provide high resolution fluorescence imaging of cells in their near-native state, and high sensitivity and contrast with minimal reconstruction artifacts .…”

Metallodrugs are unique: opportunities and challenges of discovery and development

“…304 Cryo-XRT has been used to probe the effectiveness of cisplatin in the presence of chelating tricine, which causes signicant cell damage and numerous apoptotic bodies. 305 Similarly, cryo-XRT revealed signicant mitochondrial damage in photoirradiated cancer cells treated with a cyclometallated Ir III photosensitiser Ir12. 306 Cryo-XRT can be coupled to other techniques such as structured illumination microscopy (cryo-SIM) 304 or hard-XRF, which enables the direct detection of metal complexes overlaid with the 3D subcellular morphology.…”

Soft X-ray Microscopy in Cell Biology: Current Status, Contributions and Prospects

“…The ability of cryo-SXT to allow one to visualize the entire cell volume, identify many subcellular systems, and measure organelle sizes without using contrast/labeling was employed to study the dynamics of the redistribution of mast cell secretory granules upon antigen stimulation [ 33 ] and the emptying of insulin vesicles in secretory pancreatic cells in response to glucose stimulation [ 34 ]. It was also used to visualize and quantify pre-apoptotic changes under the influence of the anticancer agent cisplatin, in combination with adjuvants to reduce its effective concentration [ 35 ], as well as to estimate the average volume of mitochondrial fragments in cancer cells after exposure to the free radicals generated by an iridium-based photosensitizer [ 36 ]. Finally, that capacity was used to measure the mitochondrial volume, the radius of lipid droplets and cytoplasmic vesicles when cells are infected with the SaRS-CoV-2 virus [ 37 ], to analyze the redistribution of cytoplasmic vesicles and changes in the mitochondrial morphology under the influence of the herpes simplex virus [ 22 ], and to collect quantitative parameters on the response of endothelial cells to glucose stimulation (the in vitro model of vascular damage processes in diabetes) [ 38 ].…”