Metabolic Signatures in Apoptotic Human Cancer Cell Lines Anna Halama 1, Gabriele Möller 1, and Jerzy Adamski 1,2 Vincent Torrecampo and Tammy Tran

Cell Fate: Proliferation, differentiation, cell death Cell death: The end result of homeostasis decay 1.Necrosis – characterized by vacuolization of cytoplasm, breakdown of plasma membrane, and unorganized chromatin condensation and fragmentation, induces inflammatory response 2.Apoptosis– characterized by nuclear condensation and fragmentation, plasma membrane blebbing, and cell shrinkage Why are these processes important? Cells undergoing cell death exhibit different metabolic response to provide specific biomarkers for detection

Reasons to study apoptosis in cancer Significance of apoptosis/cancer studies? Disruption of apoptotic machinery: Induces tumorigenesis Creates cancer therapy resistance Chemicals that promote apoptosis: powerful tools for cancer therapeutic patient tolerable therapy Distinct metabolic biomarkers: Useful for drug therapy screening Monitoring early apoptotic induction in clinical trials Current challenges: No reliable noninvasive way to specifically detect and monitor apoptosis in cancer therapies

Metabolomics: Application and approach Useful way to quantitatively measure metabolic response to stimuli Monitors concentrations of amino acids, lipids, steroids, sugars in cells, tissue, body fluid Able to provide: Metabolic signatures of a specific biological process Kinetics of metabolite flux Profiling metabolomics- discovery analyses used for identifying rather than quantification GC-MS, NMR, LC-FT-ICR, UPLC-MS Targeted metabolomics- metabolite quantification of chosen metabolites LC-MS, GC-MS, FIA-MS Untargeted metabolomics- quantification of large number of metabolites LC-MS and GC-MS used simultaneously

Aims and approach of study Goals: to design a fast, robust, reliable, and affordable system for metabolite measurement in cells To create a metabolic profile for apoptosis to be able to discriminate it from necrosis Approach: Adaptation of NBS assay for metabolite measurements in cell cultures to study early metabolomics signature of apoptosis

Cell cultures and apoptosis/necrosis stimulation Cell lines used: HepG2: hepatocellular carcinoma HEK 293: human embryonic kidney PC3: Human prostate adenocarcinoma MCF 7: Human breast adenocarcinoma Apoptosis induction: cells were treated with medium containing staurosporine and vehicle DMSO Necrosis induction: cells were grown in fresh medium and heated on a hotplate

MTT-assay: Cell viability MTT assay was used to test cell viability during apoptosis, necrosis, and normal control Measures the metabolic activity of cells Colorimetric assay: darker purple reflects higher activity

Caspase 3/7 assay: Apoptosis detection Mitochondrial Caspase 3 and 7 are activated in staurosporine- induced apoptosis Measures the enzymatic activity of caspase 3 and 7 using Caspase-Glo reagent

Adaption of NBS assay to measure metabolite in cell culture Original use: provide quantification of 42 amino acids and acylcarnitines in blood plasma of newborns Screened infants to manage/treat genetic and endocrine diseases Chosen for: efficiency and robustness metabolite panel covers amino acids and acyl carnitines altered in cancers Adaptation: Instead of using dried blood plasma, metabolites from cell cultures were extracted, placed on filter paper, and dried before detection and quantification.

Mass Spectrometry and Statistical analysis Metabolites were detected and quantified via FIA-MS/MS Kruskal-Wallis test: used to test association of metabolite concentrations with the multiclass categorical phenotypes exhibited by each cell line. Tests nonparametric data and compares >2 independent samples

Main Question Which metabolites change in expression during apoptosis and by how much?

Main Question: Which metabolites change in expression during apoptosis and by how much? First need to determine if we can successfully induce apoptosis

Main Question: Which metabolites change in expression during apoptosis and by how much? First need to determine if we can successfully induce apoptosis Also need to distinguish apoptosis from necrosis

Main Question: Which metabolites change in expression during apoptosis and by how much? First need to determine if we can successfully induce apoptosis Also need to distinguish apoptosis from necrosis How can we do this?

Main Question: Which metabolites change in expression during apoptosis and by how much? First need to determine if we can successfully induce apoptosis Also need to distinguish apoptosis from necrosis How can we do this? Induce apoptosis or necrosis in each cell line and evaluate effects

Assessment of Viability Viability is related to metabolic activity Dependent on both time and dosage (of staurosporine). 4µM SS more effective than 2µM in all cell lines Heat drastically decreased viability, especially in HEK 293 and HepG2

Caspase 3/7 Activity Assess apoptotic activity, also a check for viability assay Again, 4µM SS most effective Heat causes decreased caspase activity

INITIAL SCREENING: Goal was to create a baseline measurement for metabolites in each cell line prior to treatment.

INITIAL SCREENING: Goal was to create a baseline measurement for metabolites in each cell line prior to treatment.

INITIAL SCREENING: Goal was to create a baseline measurement for metabolites in each cell line prior to treatment.

Results After Treatment Black: decreased levels Gray: increased levels White: no change

Results Overview Changes in metabolite concentrations observable as early as 4 hours. Generally, apoptosis caused increase in metabolite concentrations. Necrosis caused decrease. Exception is MCF7 cells. Both treatments caused decrease in most metabolites - Glycine concentration did not change.

Conclusions/Take Home Message Overall objective was to test a cheap, reliable, and efficient system for measuring metabolites in cancer cells for use in monitoring treatment via apoptosis induction and they succeeded MS used instead of HNMR (metabonomics) because of higher sensitivity High sensitivity allowed very early detection of changes Sensitivity of each cell line to staurosporine was time and dosage dependent Each cell line has a different metabolic signature

Critiques Very clear progression and methods. Method of choice not perfect: Targeted metabolomics cannot detect unknown metabolites. Recycled assay protocol. Metabolites tested were not chosen based on previous association to the cell lines used.

Future Directions Repeat experiment after performing non-targeted metabolomics Future applications to animal and human studies Further Reading: D’Apolito, Oceania, et al. "Basic amino acids and dimethylarginines targeted metabolomics discriminates primary hepatocarcinoma from hepatic colorectal metastases." Metabolomics 10.5 (2014): Application of targeted metabolomics in different cancer tissues to observe markers

Questions?