Modulation of NFκB Signaling by Natural Compounds in Sarcoma and Normal Muscle Models

Berberine, curcumin, biochanin A, cucurbitacin E, and caffeic acid phenethyl ester (CAPE) are plant-derived compounds with long histories of use in traditional medicine for inflammatory and proliferative conditions. Their known capacity to modulate NF-κB signaling makes them candidates for anticancer investigation, particularly in mesenchymal malignancies such as fibrosarcoma, which arise in muscle-rich environments shared with normal myogenic tissue. To evaluate the selective anticancer potential of these compounds in fibrosarcoma (WEHI-164) and normal muscle (L6) cells, with focus on mitochondrial function, mitophagy, cellular senescence, and NF-κB-related metabolic pathways, alongside preliminary in vivo toxicity assessment. IC50 values were determined using MTT and PrestoBlue® assays. Mitochondrial membrane potential was assessed using JC-1 and normalized to the matched untreated control for each cell line, and mitophagy by PINK1/PARKIN immunofluorescence colocalisation together with a mitophagy dye assay. Cellular senescence was measured using a β-galactosidase assay, and ATP levels by a luminescence-based method. Gene expression of NF-κB pathway components and PFKFB3 was analyzed by RT-qPCR. In vivo–like toxicity was assessed using the Galleria mellonella model, including PBS handling, DMSO vehicle, and 70% ethanol utility controls, with survival data analyzed by Kaplan–Meier curves and the log-rank test. The compounds differentially affected normal and cancer cells, indicating selectivity toward malignant phenotypes. Decreased ATP and mitochondrial depolarization suggest disruption of bioenergetic homeostasis, supported by modulation of mitophagy. Stronger effects in WEHI-164 cells indicate higher susceptibility to mitochondrial dysfunction. Increased cellular senescence suggests inhibition of tumor proliferation. These findings indicate that natural NF-κB modulators may exert anticancer effects by targeting mitochondrial and metabolic homeostasis. Differential sensitivity between normal and tumor cells highlights therapeutic potential. In the G. mellonella model, berberine and curcumin did not differ significantly from the PBS or DMSO controls, whereas CAPE, CurE, and particularly biochanin A produced significantly greater larval mortality. The G. mellonella assay should be regarded only as a preliminary acute toxicity screen, and further in vivo studies in mammalian models are required to clarify mechanisms and clinical relevance.