metabolic signatures in apoptotic human cancer cell lines anna halama 1, gabriele möller 1, and...
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Metabolic Signatures in Apoptotic Human Cancer Cell LinesAnna Halama1, Gabriele Möller1, and Jerzy Adamski1,2
Vincent Torrecampo and Tammy Tran
Cell Fate: Proliferation, differentiation, cell death
Cell death: The end result of homeostasis decay1. 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 studyGoals: • 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 stimulationCell 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 DMSONecrosis 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 TreatmentBlack: decreased levelsGray: increased levelsWhite: 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): 1026-1035.• Application of targeted metabolomics in different cancer tissues to
observe markers
Questions?