The mechanism of CPT1A involved in hepatocellular carcinoma growth and Bufalin regulates malignant behavior of hepatocellular carcinoma via CPT1A

Objective Cinobufacini exhibits significant anti-cancer effects on various malignant tumors, particularly demonstrating outstanding efficacy against hepatocarcinoma. The anti-tumor effects of Cinobufacini primarily manifest as inhibition of tumor cell proliferation, cell cycle arrest, and modulation of immune responses. Bufalin, the most potent active component in Cinobufacini, requires further exploration of its anti-tumor mechanisms. We aim to elucidate the potential mechanisms of Bufalin in treating hepatocarcinoma through experimental research guided by proteomic clues. Materials and Methods In this study, Bufalin was employed to target human hepatocellular carcinoma cell line HepG2. Quantitative proteomic analysis using tandem mass tag (TMT) was conducted to explore differentially expressed proteins (DEPs) before and after Bufalin treatment. The bioinformatics analysis of DEPs was performed using hierarchical clustering, volcano plots, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG). The PPARα/CPT1A pathway was selected for further analysis. Immunohistochemistry was performed on postoperative liver cancer tissues collected from 91 liver cancer patients to analyze the correlation between relevant DEPs, differentially expressed protein CPT1A, and hepatocellular carcinoma prognosis, as well as the expression differences of CPT1A in cancer tissue and adjacent tissue. Western blot, qRT-PCR, scratch assay, transwell invasion assay, Oil Red O staining, ATP analysis, and other in vitro experiments were conducted to further identify the mechanism of Bufalin in treating hepatocarcinoma. Furthermore, in vivo experiments in nude mice were carried out to validate the reversal of Sorafenib resistance in hepatocarcinoma by Bufalin through CPT1A. Results TMT labeling quantitative proteomic analysis revealed significant differences in protein expression before and after Bufalin treatment in the HepG2 cells. A total of 835 proteins showed significant differences between the comparison groups, with 373 proteins upregulated and 462 proteins downregulated. GO analysis indicated that the DEPs were mainly associated with cellular processes, metabolic processes, and biological regulation. KEGG pathway analysis showed that DEPs were primarily related to lysosomes, complement and coagulation cascades, extracellular matrix (ECM)-receptor interaction, cholesterol metabolism, and the PPAR signaling pathway. Among these, the PPARα/CPT1A pathway may be a crucial pathway for Bufalin in hepatocellular carcinoma. Clinical significance of CPT1A was elucidated in postoperative tissues from hepatocarcinoma patients, with high CPT1A expression affecting tumor prognosis. Further analysis and validation of the PPARα/CPT1A fatty acid oxidation pathway revealed that Bufalin could downregulate the expression of the PPARα/CPT1A pathway, inhibit the proliferation of liver cancer cells, reduce their migration and invasion capabilities, and attenuate their fatty acid oxidation. Moreover, it demonstrated that Bufalin could reverse Sorafenib resistance in hepatocarcinoma by modulating CPT1A in vivo. Conclusion 1. CPT1A is an adverse prognostic factor for hepatocarcinoma. 2. Downregulation of CPT1A can inhibit the growth of hepatocellular carcinoma cells. 3. Bufalin can intervene in tumor growth and suppress fatty acid oxidation in hepatocarcinoma by regulating CPT1A expression, which may be one of the mechanisms by which Bufalin inhibits liver cancer growth. 4. Bufalin can reverse Sorafenib resistance by modulating CPT1A in hepatocellular carcinoma.