ABSTRACT
High attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity. In drug development for cancer, metabolic, and neurological disorders and for antibiotics, cytotoxicity is increasingly being assessed using extracellular flux (XF) analysis, which measures cellular bioenergetic metabolism in real time. Here, we adopt the XF platform to investigate the cytotoxicity of drugs currently used in TB treatment on the bioenergetic metabolism of HepG2 cells, THP-1 macrophages, and human monocyte-derived macrophages (hMDMs). We found that the XF analysis reveals earlier drug-induced effects on the cells' bioenergetic metabolism prior to cell death, measured by conventional viability assays. Furthermore, each cell type has a distinct response to drug treatment, suggesting that more than one cell type should be considered to examine cytotoxicity in TB drug development. Interestingly, chemically unrelated drugs with different modes of action on Mycobacterium tuberculosis have similar effects on the bioenergetic parameters of the cells, thus discouraging the prediction of potential cytotoxicity based on chemical structure and mode of action of new chemical entities. The clustering of the drug-induced effects on the hMDM bioenergetic parameters are reflected in the clustering of the effects of the drugs on cytokine production in hMDMs, demonstrating concurrence between the effects of the drugs on the metabolism and functioning of the macrophages. These findings can be used as a benchmark to establish XF analysis as a new tool to assay cytotoxicity in TB drug development.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Antitubercular Agents/toxicity , Energy Metabolism , Humans , MacrophagesABSTRACT
BACKGROUND: Administration of amplitude modulated 27·12â¯MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2â¯T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.