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.

Background Breast cancer is the most common cause of cancer related deaths in women. Treatment of breast cancer has many limitations including a lack of accurate biomarkers to predict success of chemotherapy and intrinsic resistance of a significant group of patients to the gold standard of therapy. Therefore, new tools are needed to provide doctors with guidance in choosing the most effective treatment plan for a particular patient and thus to increase the survival rate for breast cancer patients. Methods Here, we present a successful method to grow in vitro spheroids from primary breast cancer tissue. Samples were received in accordance with relevant ethical guidelines and regulations. After tissue dissociation, in vitro spheroids were generated in a scaffold-free 96-well plate format. Spheroid composition was investigated by immunohistochemistry (IHC) of epithelial [pan cytokeratin (panCK)], stromal (vimentin) and breast cancer-specific markers (ER, PR, HER2, GATA). Growth and cell viability of the spheroids were assessed upon treatment with multiple anti-cancer compounds. Student’s t -test and two-way ANOVA test were used to determine statistical significance. Results We were able to successfully grow spheroids from 27 out of 31 samples from surgical resections of breast cancer tissue from previously untreated patients. Recapitulation of the histopathology of the tissue of origin was confirmed. Furthermore, a drug panel of standard first-line chemotherapy drugs used to treat breast cancer was applied to assess the viability of the patient-derived spheroids and revealed variation between samples in the response of the spheroids to different drug treatments. Conclusions We investigated the feasibility and the utility of an in vitro , patient-derived spheroid model for breast cancer therapy, and we conclude that spheroids serve as a highly effective platform to explore cancer therapeutics and personalized treatment efficacy. These results have significant implications for the application of this model in clinical personalized medicine.

Introduction Recently we have discovered a promising anticancer agent Anthrafuran (LCTA-2034, (S)À3-(3-aminopyrrolidine-1-carbonyl)À4,11-dihydroxy-2-methylanthra [2,3-b]furan-5,10-dione methanesulfonate) which targeted topoisomerases (Top) 1, 2 and protein kinases Aur B, Aur C, Pim-1 and Pim-3 important for tumour cell biology. LCTA-2034 demonstrated an outstanding antitumor efficacy in vivo that makes this class attractive for anticancer drug development [Treshalina E.M. et al., . Eur. J. Pharm. Sci., 2017] To evaluate the role of the key functional groups and investigate structure-activity relationships within this class, we next have synthesised a series of new analogues of LCTA-2034 using anthra[2,3-b]furan scaffold. Material and methods To prepare new derivatives of anthra [2,3-b]furan several synthetic schemes were developed and used for pointed modifications of LCTA-2034. Comparison of antiproliferative activity (IC 50 ) of LCTA-2034 and its new analogues was performed on a murine leukaemia L1210 cell line. Cells were incubated for 48 hour at 37°C in a humidified CO 2 -controlled atmosphere, counted in a Coulter counter, and IC 50 values were calculated. Results and discussions Replacement of the methyl group at the position 2 of LCTA-2034 by hydrogen led to a slight decrease of antiproliferative potency of new analogues. The congeners bearing different diamines in the carboxamide moiety potently inhibited tumour cell growth at low micromolar to submicromolar concentrations. Introduction of the trifluoromethyl group at the position 2 decreased the potency of the corresponded analogue. Substitution of hydroxy groups in the core structure for amino, methoxy groups or chlorine atoms also significantly reduced cytotoxicity compared to the parent LCTA-2034. Transformation of the carboxamide group in LCTA-2034 into the aminomethyl spacer linked to 3-aminopyrrolidine residue also dramatically increased IC 50 value. These data were in agreement with results of molecular modelling of binding of LCTA-2034 and its analogues with intracellular targets Aur B and DNA duplex. Conclusion Our results strongly suggest that the carboxamide group, as well as 4,11-hydroxy groups, are critical for the cytotoxic efficacy of LCTA-2034 and shown perspective direction for further drug development studies in this class of anticancer compounds. The study was partially supported by RFBR (17-53-45105) and DST (INT/RUS/RFBR/P-291).

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.

Interferon (IFN) signaling resulting from external or internal inflammatory processes initiates the rapid release of cytokines and chemokines to target viral or bacterial invasion, as well as cancer and other diseases. Prolonged exposure to IFNs, or the overexpression of other cytokines, leads to immune exhaustion, enhancing inflammation and leading to the persistence of infection and promotion of disease. Hence, to control and stabilize an excessive immune response, approaches for the management of inflammation are required. The potential use of peptides as anti-inflammatory agents has been previously demonstrated. Our team discovered, and previously published, a 9-amino-acid cyclic peptide named ALOS4 which exhibits anti-cancer properties in vivo and in vitro. We suggested that the anti-cancer effect of ALOS4 arises from interaction with the immune system, possibly through the modulation of inflammatory processes. Here, we show that treatment with ALOS4 decreases basal cytokine levels in mice with chronic inflammation and prolongs the lifespan of mice with acute systemic inflammation induced by irradiation. We also show that pretreatment with ALOS4 reduces the expression of IFN alpha, IFN lambda, and selected interferon-response genes triggered by polyinosinic-polycytidylic acid (Poly I:C), a synthetic analog of viral double-stranded RNA, while upregulating the expression of other genes with antiviral activity. Hence, we conclude that ALOS4 does not prevent IFN signaling, but rather supports the antiviral response by upregulating the expression of interferon-response genes in an interferon-independent manner.