A systematic node-specific autophagy inhibition approach unveils U0126 as a potent enhancer of sorafenib efficacy in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a complex malignancy with limited therapeutic options. Although the reduced efficacy of first-line treatment option sorafenib therapy has been largely attributed to autophagy in HCC cells, the specific pharmacological targets involved in this phenomenon remain unclear. We systematically evaluated seven small molecule autophagy inhibitors— SBP-7455, U0126, wortmannin, hydroxychloroquine, S130, SAR405, SP600125—each targeting distinct regulatory nodes within the autophagy pathway. The inhibitory concentrations of these compounds were determined, followed by combination treatments with sorafenib to assess additive or synergistic effects in three HCC cell lines (Huh7, HepG2, and Mahlavu) representing distinct HCC characteristics. Cell viability and migration were assessed in 2D cultures, while spheroid size was analyzed in 3D hanging drop models. Most inhibitors exhibited additive effects in 2D cultures; however, only U0126—a dual MEK1 and autophagy inhibitor—markedly enhanced sorafenib efficacy in 3D cultures across all cell lines. Combination treatments were also tested on sorafenib-resistant Huh7 cells, again revealing U0126 as the most effective synergistic inhibitor in this pre-established resistance model. Notably, the levels of HCC stemness and sorafenib resistance marker CD24 decreased when sorafenib was combined with U0126. These results highlight the potential importance of targeting autophagy-associated pathways and suggest that U0126 may enhance the efficacy of sorafenib in HCC. Further investigation of U0126’s dual action mechanism is warranted to optimize treatment strategies and develop newer drugs.