β-Hydroxybutyrate Inhibits Angiogenesis, Suppresses Non-Small Cell Lung Cancer Growth, and Enhances Gemcitabine Antitumor Activity

Lung cancer remains the most prevalent malignancy and the leading cause of cancer-related mortality worldwide, highlighting the need for novel therapeutic strategies. β-Hydroxybutyrate (BHB), the primary circulating ketone body, has shown antitumor activity in other cancers, but its role in non-small cell lung cancer (NSCLC) is not well defined. This study investigated the anticancer and anti-angiogenic effects of BHB alone and in combination with Gemcitabine using in vitro and in vivo approaches. In A549 (adenocarcinoma) and LNM35 (large-cell carcinoma) NSCLC cells, BHB significantly reduced cell number and colony growth in a concentration-dependent manner, with LNM35 cells exhibiting greater sensitivity. Migration of both A549 and LNM35 cells was also markedly inhibited. In endothelial Telo-HAEC cells, BHB exhibited no cytotoxicity but significantly inhibited migration, tube formation, and VEGF-induced spheroid sprouting, indicating anti-angiogenic activity. Combination studies showed that BHB enhanced Gemcitabine-mediated suppression of NSCLC cell number and colony growth, consistent with an additive to synergistic effect. In the chick embryo chorioallantoic membrane model, BHB and Gemcitabine reduced tumor growth individually, while their combination further decreased tumor weight, particularly in LNM35 xenografts, without observable toxicity. These findings provide preclinical evidence that BHB enhances Gemcitabine antitumor activity against NSCLC and reveals its in vitro anti-angiogenic effects.