Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide, with limited long-term survival despite advances in surgical resection, locoregional therapies, and systemic treatments. While angiogenesis inhibitors such as sorafenib, lenvatinib, and cabozantinib have demonstrated clinical benefit, monotherapy outcomes remain suboptimal due to intrinsic or acquired resistance. Novel therapeutic strategies targeting multiple oncogenic pathways are urgently needed. Here, we investigate the synergistic antitumor potential of AL3810, a novel orally bioavailable multi-tyrosine kinase inhibitor targeting VEGFR, PDGFR, and FGFR, combined with CYH33, a highly selective PI3K inhibitor currently under clinical evaluation (NCT03544905). Our study reveals that co-targeting the PI3K-AKT and MAPK-ERK signaling axes via AL3810 and CYH33 induces potent synergistic inhibition of HCC proliferation, survival, and angiogenesis.
In vitro analyses using 22 human HCC cell lines and a patient-derived cell line (CLC2) demonstrated consistent synergy between AL3810 and CYH33, with combination index (CI) values predominantly below 0.8 across all tested models. This synergistic effect was further confirmed by dose-response curves, colony formation assays, and apoptosis detection via Annexin V/PI staining. Western blotting showed that AL3810 selectively suppressed p-ERK without affecting p-AKT, while CYH33 specifically inhibited p-AKT. Notably, their combination led to simultaneous blockade of both ERK and AKT phosphorylation within 30 minutes, persisting for up to 24 hours. Similar dual suppression was observed in xenograft tumor tissues derived from Bel-7402 and SMMC-7721 cells, confirming pathway inhibition in vivo.
Functional studies revealed that the combination significantly increased apoptosis, as evidenced by elevated cleaved PARP and reduced XIAP levels, and induced G1-phase cell cycle arrest associated with upregulation of p27 and downregulation of CyclinD1. These effects were validated in three-dimensional (3D) spheroid cultures incorporating fibroblasts (WI38/MRC9), which mimic the tumor microenvironment (TME).CHCHD3 Antibody medchemexpress The combination dramatically enhanced cytotoxicity and induced extensive cell death in co-cultured spheroids, indicating effective disruption of tumor-stroma interactions.PAFAH1B3 Antibody Data Sheet
Moreover, the antiangiogenic activity of AL3810 was significantly augmented by CYH33.PMID:35076560 Combined treatment synergistically inhibited HUVEC proliferation, tube formation, migration, and rat aortic ring sprouting. In 3D spheroids containing both HCC cells and HUVECs, the combination caused a marked increase in dead spheres compared to single agents. In vivo, CDX and PDX models exhibited profound tumor growth suppression with no significant body weight loss, indicating favorable tolerability. Immunohistochemistry revealed reduced CD31 expression and decreased α-SMA-positive fibroblasts in combination-treated tumors, underscoring suppression of both vascularization and stromal activation.
Mechanistically, depletion of either ERK or AKT partially reversed the antiproliferative effect of the combination, confirming the necessity of dual pathway inhibition. Furthermore, combining AL3810 with other AKT inhibitors (GDC0068, MK2206) also yielded strong synergy, reinforcing the central role of AKT signaling in this therapeutic strategy.
Collectively, these findings establish that AL3810 and PI3K inhibitor CYH33 act synergistically in HCC by simultaneously blocking the PI3K-AKT and MAPK-ERK pathways within tumor cells and the surrounding microenvironment. This dual inhibition leads to enhanced antiproliferative, pro-apoptotic, cell cycle-arresting, and antiangiogenic effects. Our data provide strong preclinical rationale for evaluating AL3810 in combination with PI3K inhibitors in future clinical trials for advanced HCC, offering a promising new approach to overcome resistance and improve patient outcomes.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
