Skin cancer, strongly associated with UV exposure, is the most common malignancy worldwide, including squamous cell carcinoma (SCC), and cutaneous melanoma (CM). Although cisplatin and 5-FU are standard treatments for SCC and CM, the development of new therapeutic alternatives is crucial. Silver complexes have shown promising anticancer potential, while the monoterpene limonene has demonstrated efficacy in enhancing skin permeation, supporting its application in topical drug delivery.

Our study aimed to evaluate the in vitro antiproliferative effects of silver complexes and limonene, isolated and in association.

The silver complexes identified as I and II were synthesized at the Institute of Chemistry of University of Campinas. The pure substances R-(+)-limonene and S-(-)-limonene were acquired from Merck. Pharyngeal SCC (FaDU) and melanoma (A-375, SK-MEL-28) cells (4 × 10³ cells/mL) were treated with complexes I e II (0.4–400 µM) or their combination with R-(+) and S-(-) limonene (4 µM). Cisplatin and 5-FU (100 μL/well, 0.4 to 400 µg/mL, in triplicate) were used as positive controls. Before (T0) and after (T1) sample addition, cells were fixed with 50% trichloroacetic acid (TCA, 50 μL/well), and were then resuspended in Tris base for subsequent absorbance at 540 nm with a microplate reader spectrophotometer (VersaMax, Molecular Devices). The difference between T0 and T1 absorbance values represented 100% cell growth. Effective concentration representing the sample concentration required to promote 50% growth inhibition (IC50) for each cell line was calculated by sigmoidal regression using Origin 8.0 software. The Combination Reduction Index (CRI) was calculated as IC 50 of the metal complex + monoterpene / IC 50 of the metal complex alone.

Both silver complexes exhibited potent antiproliferative activity. The antiproliferative effect of silver complex I ranged from 4 µM to 10 µM in SCC and CM cell lines, whereas the antiproliferative effect of silver complex II ranged from 1 µM to 6 µM in the same cell lines. The association of silver complex I in combination with S-(-)-limonene resulted in synergism effect in the FaDu cell line with IC50 < 2 µM, CRI = 0.4. The association of silver complex II with both enantiomers demonstrated a partial synergistic effect in the FaDu and SK-MEL-28 cells, with IC50 <1.5 µM and CRI = 0.5.

Silver complexes are promising candidates for in vivo studies as potential alternatives for the treatment of patients with SCC and CM. Additional experiments are necessary to evaluate their mechanism of action and toxicity. The study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Apoio ao Ensino e à Pesquisa do Estado de São Paulo (FAPESP #2016/07729-4; #2023/09738-4 and Cancer Theranostics Innovation Center, (CancerThera), CEPID FAPESP #2021/10265-8).