Norcantharidin induces apoptosis in hepatocellular carcinoma MHCC97-H cells in association with altered NF-κB/IκBα signaling dynamics and JNK activation.

Abstract

Norcantharidin (NCTD), a demethylated derivative of cantharidin, has antitumor activity against multiple cancer types; however, its effects on the NF-κB/JNK signaling axis in hepatocellular carcinoma (HCC) remain incompletely defined. MHCC97-H HCC cells were treated with NCTD at 5, 20, and 40 μg/mL (approximately 29.7, 118.9, and 237.9 μM), alone or in combination with pyrrolidine dithiocarbamate (PDTC, 50 μM), a compound with reported NF-κB inhibitory and antioxidant properties. NCTD suppressed cell viability in a dose- and time-dependent manner, with 48-h inhibition rates of 24.3%, 51.9%, and 72.1% at the three concentrations. Based on the available MTS data, the estimated NCTD IC50 was > 40 μg/mL at 24 h and approximately 19.0 μg/mL at 48 h. Flow cytometry showed corresponding apoptosis rates of 13.3%, 36.8%, and 43.2%, which increased to 50.2% after PDTC co-treatment. Western blotting showed reduced nuclear NF-κB p65 signal and decreased cytoplasmic IκBα levels, together with increased phosphorylated JNK (p-JNK), while total JNK remained unchanged. RT-qPCR showed upregulation of pro-apoptotic Bax, downregulation of anti-apoptotic Bcl-2, and increased c-Jun, TNF-α, and IL-6 transcripts. NCTD further impaired colony formation, induced G0/G1 arrest, depolarized mitochondrial membrane potential, and increased reactive oxygen species. TCGA-LIHC bulk RNA-seq data were analyzed to provide clinical context. NF-κB pathway genes were differentially expressed in HCC, and enrichment analyses implicated NF-κB, apoptosis, TNF, and MAPK-related pathways. The NF-κB Core activity score showed no significant tumor-normal difference, but immune infiltration analysis revealed associations between NF-κB activity and the HCC immune microenvironment. These findings indicate that NCTD triggers mitochondrial apoptosis in MHCC97-H cells in association with reduced nuclear p65 signal, altered IκBα turnover, stress/inflammatory gene induction, and enhanced JNK activation, rather than demonstrating uniform blockade of the entire NF-κB pathway.