Background: The inhibition of ubiquitin-specific proteases (USPs) is a novel and promising direction in the development of molecularly targeted therapies in oncology. The aim of the present study was to examine whether Degrasyn could be a potential therapeutic agent against bladder cancer (BC). Also, we aimed to determine whether Degrasyn is more effective in terms of anti-cancer activity compared to the non-selective DUB inhibitor PR-619. To facilitate the translational value of the obtained results, our experiments were performed using both human and canine in vitro models of BC. Methods: Human T24 (urothelial grade III BC) and SV-HUC-1 (non-tumorigenic urothelial cell line), as well as canine K9TCC-PU-NK and RDSVS-TCC1 (both derived from invasive grade III urothelial bladder tumors) cell lines, were used in the present study. Cell proliferation was determined using the MTT assay and Ki-67 proliferation assay, and the level of apoptosis induced by Degrasyn and PR-619 was evaluated by Annexin V-FITC staining and caspase 3/7 activation assay. Western blot was used to assess DNA damage and key proteins involved in apoptosis. Results: Degrasyn inhibited the proliferation of all BC cell lines in a concentration- and time-dependent manner. Lower concentrations of Degrasyn were more potent against human and canine BC cell lines compared to PR-619. Degrasyn induced caspase-dependent apoptosis and triggered DNA damage. PR-619 did not show a significant pro-apoptotic effect. Conclusions: Our results demonstrate that Degrasyn significantly impairs the growth of in vitro models of human and canine BC. Selective USP inhibition with Degrasyn seems to be more effective in reducing BC cell proliferation and inducing apoptosis and DNA damage than non-selective USP inhibition with PR-619.

Within the “One Health, One Medicine” and comparative oncology paradigms, algal extracts have attracted attention, containing natural compounds (NCs) with biological activities, including anti-cancer properties. To characterize the biological effects of a Sphaerococcus coronopifolius extract (SCE), two canine mastocytoma and two normal cell lines were used. After a preliminary screening of three algal extracts, SCE cytotoxicity was measured using Alamar Blue, Sulforhodamine B, and Neutral Red Uptake assays. After assessing the selectivity versus tumor cells and its chemical characterization, SCE mechanisms of action were investigated using RNA-seq, quantitative PCR, flow cytometry and immunoblotting approaches. SCE showed an IC₅₀ comprised between 25 and 35 μg/mL in tumor cell lines, but it also affected normal ones (selectivity index < 2.0). RNA-seq and flow cytometry revealed that SCE negatively affected cell cycle and mevalonate pathway in tumor cells. Additional flow cytometry and immunoblotting investigations suggested a concentration- and time-dependent pro-apoptotic effect of SCE and DNA damage events. In conclusion, SCE demonstrated promising anti-cancer activity in mastocytoma cell lines by targeting the mevalonate pathway, arresting the cell cycle, and inducing apoptosis and DNA damage. Furthermore, the results presented here reinforce the idea that NCs may be promising candidates in comparative anti-cancer chemotherapy.

A series of novel γ-halo-δ-lactones and δ-halo-γ-lactones bearing a phenolic ring at the β-position were synthesized from vanillin. The divergent seven-step synthetic route commenced with the benzyl protection of the hydroxy group of the starting material, followed by a four-step transformation that led to the corresponding β-aryl-γ,δ-unsaturated carboxylic acids. Subsequent halolactonization (iodo-, bromo-, and chlorolactonization), followed by selective benzyl deprotection, gave the target halolactones. The structures of all intermediates and final lactones were confirmed by comprehensive spectroscopic analyses, including NMR and HRMS. The resulting halolactones were evaluated for antiproliferative activity against two canine (CLBL-1, CLB70) and two human (T-24, CaCo-2) cancer cell lines, as well as non-cancerous mouse embryonic fibroblasts (NIH/3T3). Hemolytic assays were performed to assess toxicity against human red blood cells (RBCs). Among the tested lactones, the transδ-iodo-γ-lactone was the most active, particularly against CLBL-1 and T-24 cells. All compounds demonstrated no inhibitory effects on normal fibroblasts and no hemolytic toxicity. This favorable selectivity profile positions this group of lactones, particularly trans-δ-iodo-γ-lactone, as a promising candidate for further development as potential anticancer agents.

G-quadruplexes have been identified as a promising anti-cancer target because of their ability to modulate the stability of mRNAs encoding oncogenes, tumor suppressor genes, and other potential therapeutic targets. Deregulation of DNA damage and Unfolded Protein Response pathways in cancer cells may create vulnerabilities that can be exploited therapeutically. Previous studies have shown variations in the relative expression of DDR and UPR components in canine lymphoma and leukemia cell lines CLBL-1, CLB70, and GL-1. In the present study, we report the presence of G-quadruplex structures in these canine cell lines. Downregulation of the expression of DDR and UPR components at the mRNA level was observed in the CLBL-1 and CLB70 cell lines after stabilization of G4 structures using the ligand PhenDC3. In contrast, in GL-1 cells, important components of the DDR pathway, such as PARP1, GADD45A, and PIK3CB were upregulated in response to PhenDC3 treatment. Downregulation of DDIT4 mRNA expression, which encodes an important UPR component, was detected in the CLBL-1 and GL-1 cell lines after PhenDC3 exposure. These results suggest that G4 structures can be used to manipulate the expression of potential targets to treat lymphoma in dogs. A substantial enrichment of DNA replication and pyrimidine metabolism pathways was found in the GL-1 cell line after G4 stabilization. This finding suggests that PhenDC3 may induce DNA replication stress in this cell line. Collectively, these results support the feasibility of employing canine cancer cells as a model system to investigate the role of G-quadruplex structures in cancer.

A series of novel γ-halo-δ-lactones and δ-halo-γ-lactones bearing a phenolic ring at the β-position was synthesized from the naturally occurring aromatic aldehyde vanillin. The seven-step synthetic route commenced with benzyl protection of the hydroxy group of the starting material, followed by the synthesis of a γ,δ-unsaturated carbox-ylic acid, subsequent halolactonization (iodo-, bromo-, and chlorolactonization), and concluded with selective benzyl deprotection. The structures of all synthesized com-pounds were confirmed by comprehensive spectroscopic analyses, including NMR and HRMS. The resulting halolactones were evaluated for antiproliferative activity against two canine (CLBL-1, CLB70) and two human (T-24, CaCo-2) cancer cell lines, with non-cancerous mouse embryonic fibroblasts (NIH/3T3) serving as controls. Hemolytic assays were performed to assess toxicity toward human red blood cells (RBCs). All compounds demonstrated selective inhibition of cancer cell proliferation, exhibiting no inhibitory effects on normal fibroblasts and no hemolytic toxicity. Among the tested lactones, the trans δ-iodo-γ-lactone was the most potent, particularly against CLBL-1 and T-24 cells. These findings underscore the potential of vanillin-derived halolactones as selective anticancer agents with an advantageous safety profile, owing to their lack of toxicity toward non-malignant cells.

Chalcones are tunable electrophiles whose bioactivity depends on aryl substitution and membrane interactions. We profiled eight 4'-hydroxychalcones with differing in methoxy topology on ring B in primary human PBMCs (24 h, XTT) and canine lymphoma/leukemia cell lines (CLBL-1, CLB70; 72 h, MTT), integrating RBC compatibility assays (hemolysis, morphology, osmotic fragility C50, transmembrane potential, and reduced glutathione), hemoglobin tryptophan-fluorescence quenching, and cell-free antioxidant assays. In canine tumor models, chalcones were more cytotoxic to neoplastic than to normal cells, indicating selectivity. Among them, 3-methoxy-4'-hydroxychalcone showed the most favorable profile, combining strong anticancer activity in CLBL-1 and CLB70 with lower toxicity toward normal cells, whereas 2,5-dimethoxy-4'-hydroxychalcone, although potent, showed a less favorable safety profile. RBC studies revealed marked echinocytosis after 1 h at 100 µM without acute hemolysis and no changes in osmotic resistance, transmembrane potential, or GSH levels. After 24 h, hemolysis was observed only for two compounds at concentrations ≥ 200 µM. Hb quenching was static (Kq above the diffusion limit), with the highest Ksv values for 3-methoxy-4'-hydroxychalcone and 4'-hydroxychalcone, but the highest Kb for 3,4,5-trimethoxy-4'-hydroxychalcone, followed by 4'-hydroxychalcone, indicating that fluorophore proximity and complex stability are not directly coupled. DPPH and CUPRAC assays showed no detectable direct antioxidant activity, whereas ABTS•+ revealed weak and comparable radical-scavenging activity across the series. A concise SAR emerged: meta- and ortho/meta-methoxy substitution enhanced biological activity, whereas para-methoxy and 3,4,5-trimethoxy substitution attenuated it; the unsubstituted parent compound retained substantial activity. Overall, methoxy topology shapes both cytotoxicity and interactions with blood components, with 3-methoxy-4'-hydroxychalcone emerging as the most promising lead for early preclinical evaluation.