Garipi 2018 MiPschool Tromso E2

Garipi Enis Phenotyping mitochondrial metabolism in Barrett’s metaplasia-dysplasia-adenocarcinoma sequence: respiratory capacity, extracelular proton flux and ROS production

Link: MitoEAGLE

Garipi E, Iglesias-Gonzalez J, Gnaiger E (2018)

Event: MiPschool Tromso-Bergen 2018

With an increasing global incidence of cancer, prevention and therapy is one of the major public health challenges of the 21st century. According to the data of the Global Cancer Observatory, approximately 18.1 million of new cases of cancer are expected in 2018. Esophageal cancer (EC) accounts for 3.2% of the total number of new diagnosed cases. Its severity is reflected in the fact that it is responsible for more than half a million deaths per year and has a 5-year survival rate of only 18% [1]. The main causes for the devastating influence of esophageal cancer are the lack of convenient biological markers and effective treatment options. Metabolism of cancer cells is highly adaptable and expresses great plasticity when it comes to shifting the metabolic pathways with one ultimate goal: to survive and to spread. Numerous advantages are ascribed to such changes, the generation of the energy for life and building blocks for growth being the main one [2,3]. Recently, dysregulated pH emerged as a key player in the survival mechanism of mammalian cancer cells and it also confers resistance to chemotherapy and invasiveness to them [4,5]. Whether it is a cause or consequence, this hallmark of most cancer cells contributes to the metabolic shift and to changes in their microenvironment. Also, the alteration in ROS production has been described as beneficial for cancer cells genesis and survivability. Reactive oxygen species (ROS) are vital for various cellular processes in both healthy and cancer cells but can also cause oxidative stress in excessive amounts, leading to genetic and functional damage. It is supposed that limiting the oxidative phosphorylation (OXPHOS) in cancer cells have a protective role in the sense of limiting the amount of ROS generated [3,6]. Our plan is to establish Substrate-Uncoupler-Inhibitor Titration (SUIT) protocols for functional analysis of respiratory capacities, extracellular proton flux and ROS production in cell lines and human tissue samples of esophageal mucosa and to investigate the influence of different extracellular pH values regarding the above-mentioned parameters. Following human cell lines will be included: radioresistant SCC-25, radiosensitive SCC-090 and HGF cell lines. Experiments are going to be performed on the Oroboros Oxygraph-2k (O2k-FluoRespirometer) that provides real-time measurement of mitochondrial respiration, ROS production rate and extracellular proton flux in one trial. Esophageal cell culture models will be used and compared with fresh human esophageal tissue representing non-cancerous cells/tissue and three succeeding stages in Barrett’s metaplasia-dysplasia-adenocarcinoma-sequence. This will give clinical significance to our study. Before starting experiments with cell lines and human biopsies, training with HEK293 cells and proficiency test must be accomplished by every researcher in our lab. It is a proof of competence and skills to carry out complete protocol from cell culturing to performing the experiment and only then is the researcher allowed to work with human biopsies. This study is directed towards discovery of new possibilities for diagnosis and potential targets for a new therapeutic approach in the treatment of esophageal cancer.

• Keywords: cancer, mitochondrial physiology, ROS, pH, esophagus • Bioblast editor: Garcia-Souza LF • O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E

Labels: MiParea: Respiration, Patients  Pathology: Cancer  Stress:Hypoxia  Organism: Human  Tissue;cell: Endothelial;epithelial;mesothelial cell, Other cell lines  Preparation: Permeabilized cells, Permeabilized tissue  Enzyme: Complex I, Complex II;succinate dehydrogenase, Complex III, Complex IV;cytochrome c oxidase, Complex V;ATP synthase, TCA cycle and matrix dehydrogenases, Uncoupling protein 

Coupling state: LEAK, ROUTINE, OXPHOS, ET  Pathway: CIV, NS, ROX  HRR: O2k-Protocol, O2k-FluoRespirometer"O2k-FluoRespirometer" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

Affiliations

Garipi E(1,2), Iglesias-Gonzalez J(1,2), Gnaiger E(1,2)

1. Oroboros Instruments, Innsbruck
2. Daniel Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck

Acknowledgements

Supported by the Marie Skłodowska-Curie PhD Fellowship TRACT.

References

1. Globocan. Estimated number of new cases in 2018, South Africa, all cancers, both sexes, all ages.(2018)
2. Lunt SY, Vander Heiden MG (2011) Aerobic Glycolysis: Meeting the Metabolic Requirements of Cell Proliferation. Annu Rev Cell Dev Biol 27:441–64.
3. Vander Heiden, M, Cantley L, Thompson C (2009) Understanding the Warburg effect: The metabolic Requirements of cell proliferation. Science 324:1029–1033.
4. Webb BA, Chimenti M, Jacobson MP, Barber DL (2011) Dysregulated pH: A perfect storm for cancer progression. Nat Rev Cancer 11:671–7.
5. Walsh M et al (2015) Proton pump inhibitors for the treatment of cancer in companion animals. J Exp Clin Cancer Res 34:93.
6. Hervouet E et al (2008) HIF and reactive oxygen species regulate oxidative phosphorylation in cancer. Carcinogenesis 29:1528–37.