In vivo study of the effect of anlotinib on the stemness of the lenvatinib-resistant hepatocellular carcinoma cells and the underlying mechanisms

Abstract

Abstract Background In vivo experiments were conducted to examine the effects of the targeted drug anlotinib on the stemness of hepatocellular carcinoma (HCC) cells and lenvatinib-resistant liver cancer cells and to explore the underlying molecular mechanisms. Methods A subcutaneous xenograft model of Hep3B-derived HCC was established in nude mice, which were randomly divided into 2 groups (n = 5 males per group): (1) intragastric administration of anlotinib (0.4 mg/kg) and (2) intragastric administration of normal saline. We constructed lenvatinib-resistant cell lines and randomly divided the mice into 3 groups (n = 5 males per group): (1) intragastric administration of anlotinib, (2) intragastric administration of lenvatinib, and (3) intragastric administration of normal saline. After 2 weeks of treatment, tumor tissues were harvested, and mRNA and proteins were isolated from the tissues. Changes in the expression of cancer stemness markers (epithelial cell adhesion molecule [EpCAM], CD13, CD90, aldehyde dehydrogenase 1 [ALDH1], CD44, and CD45), totipotency factors (sex-determining region Y-box 2 [Sox2], Nanog, octamer-binding transcription factor 4 [Oct4]), and genes related to the Notch signaling pathway were examined. Results Compared with that in the control group, tumor size and weight were reduced in nude mice treated with anlotinib. These differences were statistically significant in both the types of nude mice. Anlotinib affected stemness markers and totipotency factors by downregulating the expression of CD133, CD90, and G-protein–coupled receptor 5 (LGR5) and upregulating the expression of intercellular adhesion molecule 1 (ICAM-1) and Sox2. In addition, lenvatinib-resistant cell lines increased Notch signaling pathway, whereas anlotinib inhibited Notch signaling pathway. Conclusions The antitumor effect of anlotinib on HCC and lenvatinib-resistant HCC cells may occur through inhibition of the Notch signaling pathway. Anlotinib may be the drug of choice for sequential therapy in lenvatinib-resistant liver cancer.