Clodronate and trehalose are known to modulate metabolic and autophagy-related pathways, respectively, yet their combined effects on tumour cell proliferation remain poorly understood. This study investigated the individual and combined effects of these compounds on the canine mammary tumour cell line CMT-U27 and explored underlying mechanisms using network pharmacology. Cell proliferation was assessed following treatment with clodronate, trehalose, and their combination across a range of concentrations. Growth rate analysis and Bliss independence modelling were used to evaluate drug interactions. Target prediction was performed using SwissTargetPrediction, followed by homology mapping to canine proteins and protein-protein interaction and enrichment analysis in STRING. Hybrid compounds (CT compounds) predicted to be derived from clodronate and trehalose were also analysed. Target expression relevance was validated using gene expression data, and molecular docking was conducted using AutoDock Vina. Clodronate and trehalose individually promoted proliferation in CMT-U27 cells, whereas their combination produced a dose-dependent suppression of proliferation. Network analysis revealed that clodronate targets were enriched in IGFR and Ras signalling pathways, while Trehalose targets were associated with proteostasis and metabolic regulation pathways. These findings supported their individual proliferative effects but did not explain the suppressive combination effect. CT compounds demonstrated distinct target profiles enriched in extracellular matrix remodelling and lipid metabolism pathways, consistent with anti-proliferative activity. Integration with gene expression data identified key targets associated with proliferation (clodronate and trehalose) and inhibition (CT compounds). Molecular docking suggested favourable binding interactions with these targets. The paradoxical suppression of tumour cell proliferation by the combined use of clodronate and trehalose, may be due to metabolic-autophagic incompatibility and the potential formation of novel bioactive compounds. These findings highlight the importance of pathway integration and suggest exploring therapeutic strategy based on simultaneous targeting of metabolic and proteostatic mechanisms.
