Protocol for analyzing energy metabolic pathway dependency in human liver cancer cell lines.

Cellular energy metabolism analysis is complex, expensive, and indirect. We present a protocol to analyze relative contribution of metabolic pathways to ATP production by directly measuring ATP levels. We describe steps for cell counting and seeding in 96-well plate, treating with metformin, and systematic inhibition with metabolic inhibitors. We then detail procedures for a viability and ATP assay and calculating energy metabolism dependency. This high-throughput and accessible protocol works with any cell line and allows for flexible perturbation studies.

Identification of glucose-independent and reversible metabolic pathways associated with anti-proliferative effect of metformin in liver cancer cells.

INTRODUCTION: Despite the ability of cancer cells to survive glucose deprivation, most studies on anti-cancer effect of metformin explored its impact on glucose metabolism. No study ever examined whether its anti-cancer effect is reversible. Existing evidences warrant understanding of glucose-independent non-cytotoxic anti-proliferative effect of metformin to rationalize its role in liver cancer. OBJECTIVES: Characterization of glucose-independent anti-proliferative metabolic effects of metformin as well as analysis of their reversibility in liver cancer cells. METHODOLOGY: The dose-dependent effects of metformin on HepG2 cells were examined in presence and absence of glucose. The longitudinal evolution of metabolome was analyzed along with gene and protein expression as well as their correlations with and reversibility of cellular phenotype and metabolic signatures. RESULTS: Metformin concentrations up to 2.5 mM were found to be anti-proliferative irrespective of presence of glucose without significant increase in cytotoxicity. Apart from mitochondrial impairment, derangement of fatty acid desaturation, one-carbon, glutathione, and polyamine metabolism were associated with metformin treatment irrespective of glucose supplementation. Depletion of pantothenic acid, downregulation of essential amino acid uptake and metabolism alongside purine salvage were identified as novel glucose-independent effects of metformin. These were significantly correlated with cMyc expression and reduction in proliferation. Rescue experiments established reversibility upon metformin withdrawal and tight association between proliferation, metabotype, and cMyc expression. CONCLUSIONS: The derangement of multiple glucose-independent metabolic pathways, which are often upregulated in therapy-resistant cancer, and concomitant cMyc downregulation coordinately contribute to the anti-proliferative effect of metformin in liver cancer cells. These are reversible and may influence its therapeutic utility.

Effect of short-term use of FFP2 (N95) masks on the salivary metabolome of young healthy volunteers: a pilot study.

The use of face masks has become an integral part of public life in the post-pandemic era. However, the understanding of the effect of wearing masks on physiology remains incomplete and is required for informing public health policies. For the first time, we report the effects of wearing FFP2 masks on the metabolic composition of saliva, a proximal matrix to breath, along with cardiopulmonary parameters. Un-induced saliva was collected from young (31.2 ± 6.3 years) healthy volunteers (n = 10) before and after wearing FFP2 (N95) masks for 30 minutes and analyzed using GCMS. The results showed that such short-term mask use did not cause any significant change in heart rate, pulse rate or SpO2. Three independent data normalization approaches were used to analyze the changes in metabolomic signature. The individuality of the overall salivary metabotype was found to be unaffected by mask use. However, a trend of an increase in the salivary abundance of L-fucose, 5-aminovaleric acid, putrescine and phloretic acid was indicated irrespective of the method of data normalization. Quantitative analysis confirmed increases in concentrations of these metabolites in saliva of paired samples amid high inter-individual variability. The results showed that while there was no significant change in measured physiological parameters and individual salivary metabotypes, mask use was associated with correlated changes in these metabolites plausibly originating from altered microbial metabolic activity. These results might also explain the change in odour perception reported to be associated with mask use. Potential implications of these changes on mucosal health and immunity warrants further investigation to evolve more prudent mask use policies.

Thiourea mediated ROS-metabolites reprogramming restores root system architecture under arsenic stress in rice.

Arsenic (As) is a ubiquitous carcinogenic metalloid that enters into human food chain, through rice consumption. To unravel the conundrum of oxidative vs. reductive stress, the differential root-system architecture (RSA) was studied under As (a ROS producer) and thiourea (TU; a ROS scavenger) alone treatments, which indicated 0.80- and 0.74-fold reduction in the number of lateral roots (NLR), respectively compared with those of control. In case of As+TU treatment, NLR was increased by 4.35-fold compared with those of As-stress, which coincided with partial restoration of redox-status and auxin transport towards the root-tip. The expression levels of 16 ROS related genes, including RBOHC, UPB-1 C, SHR1, PUCHI, were quantified which provided the molecular fingerprint, in accordance with endogenous ROS signature. LC-MS based untargeted and targeted metabolomics data revealed that As-induced oxidative stress was metabolically more challenging than TU alone-induced reductive stress. Cis/trans-ferruloyl putrescine and γ-glutamyl leucine were identified as novel As-responsive metabolites whose levels were decreased and increased, respectively under As+TU than As-treated roots. In addition, the overall amino acid accumulation was increased in As+TU than As-treated roots, indicating the improved nutritional availability. Thus, the study revealed dynamic interplay between "ROS-metabolites-RSA", to the broader context of TU-mediated amelioration of As-stress in rice.

Lipidomic Analysis of Cancer Cell and Tumor Tissues.

Due to their role in cellular structure, energetics, and signaling, characterization of changes in cellular and extracellular lipid composition is of key importance to understand cancer biology. In addition, several mass spectrometry-based profiling as well as imaging studies have indicated that lipid molecules may be useful to augment existing biochemical and histopathological methods for diagnosis, staging, and prognosis of cancer. Therefore, analysis of lipidomic changes associated with cancer cells and tumor tissues can be useful for both fundamental and translational studies. Here, we provide a high-throughput single-extraction-based method that can be used for simultaneous lipidomic and metabolomic analysis of cancer cells or healthy or tumor tissue samples. In this chapter, a modified Bligh-Dyer method is described for extraction of lipids followed by analysis of fatty acid composition by gas chromatography-mass spectrometry (GC-MS) or untargeted lipidomics using electrospray ionization mass spectrometry (ESIMS) coupled with reverse-phase (RP) ultraperformance liquid chromatography (UPLC) followed by multivariate data analysis to identify features of interest.