Arsenic trioxide enhances the inhibitory effect of lenvatinib on hepatocellular carcinoma through HMOX1-mediated ferroptosis

Currently, Lenvatinib, a tyrosine kinase inhibitor, is used as a first-line treatment for advanced hepatocellular carcinoma (HCC), but its efficacy remains unsatisfactory. Therefore, we investigated the effect of Lenvatinib in combination with Arsenic trioxide (ATO) on HCC and the underlying mechanisms. The antitumor activity of the combination was evaluated in vitro using MTT and colony formation assays, and in vivo using xenograft models in nude mice. mRNA-seq was performed to explore the potential mechanisms. Reactive oxygen species (ROS) and intracellular Fe²⁺ were measured to evaluate ferroptosis in HCC cells. Our findings showed that the addition of ATO significantly enhanced the anti-HCC effects of Lenvatinib both in vitro and in vivo. Moreover, RNA sequencing analysis indicated that ATO augmented the anti-HCC effect of Lenvatinib by inducing ferroptosis. Both ATO alone and the combination treatment markedly induced a significant elevation of ROS and ferroptosis, which was effectively blocked by the administration of ferrostatin-1 and deferoxamine mesylate. Furthermore, ATO upregulated the levels of heme oxygenase 1 (HMOX1) and Fe²⁺ in HCCLM3 and Huh7 cells. Knockdown of HMOX1 attenuated the effect of ATO on HCC cell viability, ROS and Fe²⁺ levels in HCC cells. Additionally, ATO and the combination of Lenvatinib and ATO also decreased the expression of GPX4 protein both in vitro and in vivo. In conclusion, ATO enhances the antitumor activity of Lenvatinib against HCC by inducing ferroptosis through upregulation of HMOX1 and downregulation GPX4.