Background/Aim: The ketogenic diet has recently gained interest as potential adjuvant therapy for cancer. Many researchers have endeavored to support this claim in vitro. One common model utilizes treatment with exogenous acetoacetate in lithium salt form (LiAcAc). We aimed to determine whether the effects of treatment with LiAcAc on cell viability, as reported in the literature, accurately reflect the influence of acetoacetate. Materials and Methods: Breast cancer and normal cell lines were treated with acetoacetate, in lithium and sodium salt forms, and cell viability was assessed. Results: The effect of LiAcAc on cells was mediated by Li ions. Our results showed that the cytotoxic effects of LiAcAc treatment were significantly similar to those caused by LiCl, and also treatment with NaAcAc did not cause any significant cytotoxic effect. Conclusion: Treatment of cells with LiAcAc is not a convincing in vitro model for studying ketogenic diet. These findings are highly important for interpreting previously published results, and for designing new experiments to study the ketogenic diet in vitro.

The ketogenic diet, based on high fat (over 70% of daily calories), low carbohydrate, and adequate protein intake, has become popular due to its potential therapeutic benefits for several diseases including cancer. Recent evidence suggests ketogenic metabolic therapy as a complementary or alternative approach to breast cancer treatment. Under ketogenic conditions, as in starvation, the levels of glucose, insulin, and insulin-like growth factors in the blood decrease and stabilize, and the liver produces ketone bodies, acetone, acetoacetate (AcA) and β-hydroxybutyrate (ΒHb) by beta-oxidation of fatty acids, as an alternative energy source. Due to the volatile nature of acetone and its instability in the blood, acetone has no effect on cancer cells under physiological conditions. AcA alone is unstable as well, and is used in research as a sodium or lithium salt. Despite the widely accepted use of lithium AcA as a ketone body source, we and others recently demonstrated that its cytotoxic effects result from the lithium and not from acetoacetate.In this study, we aimed to elucidate the effect of the third ketone body, ΒHb, on breast cancer cells in vitro. Using two cancer (MCF7 and MDA-MB-231) and one non-cancer (HB2) breast cell lines, we evaluated the effect of β-hydroxybutyrate treatment on cell growth parameters and assessed the effect of BHb on metabolism and the gene expression profile. We found that ΒHb increases viability and proliferation of MCF7, but has no affect MDA-MB-231 and non-cancer HB2 cell viability. We observed no changes in glucose intake or lactate output following BHb treatment, but an increase in ROS level was detected. Gene expression analysis revealed a differential effect of BHb treatment on changes in genes involved in lipid metabolism and oxidative phosphorylation, among the tested cell lines. While genes involved in lipid metabolism were downregulated in MCF7 cells, ΒHb mitochondrial monocarboxylate transporter-4 (MCT4) expression was found to be significantly upregulated in both cancer cell lines with 10mM BHb but not affected in non-cancer breast cells. 3-hydroxybutyrate dehydrogenase 1 (BDH1) and 3-oxoacid CoA-transferase 1 (OXCT1), enzymes involved in BHb oxidation in the mitochondria, were significantly increased in MCF7 with a non-significant trend toward upregulation in MDA-MB-231 cells. Treatment of MCF7 cells with 10mM of BHb also significantly downregulated AMP-activated protein kinase (AMPK) and upregulated HDAC1, both known to be involved in BHb metabolism. Based on our results, we conclude that differential response of breast cell lines to BHb treatment, as alternative energy source or signal to altered lipid metabolism and oncogenicity, supports the need for a personalized approach to breast cancer patient treatment. Citation Format: Elimelech Nesher, Hadas Fulman-Levy, Igor Koman, Raichel Cohen-Harazi. Beta-hydroxybutyrate alters lipid metabolism affecting oncogenicity of MCF7 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3016.