Silkuniene 2017 MiPschool Obergurgl
Event: MiPschool Obergurgl 2017
Since the suggestion of the so called Warburg effect in 1956 it was believed that glycolytic energy production predominates in most cancer cells due to inactivation of mitochondria (Mt) or lack of oxygen in the cancer cells . The shift in understanding metabolic changes in cancer cells occurred recently regarding the role and activity of Mt [2,3]. It was established that Mt are not inactivated in cancer cells but change the main function: instead of being sites for oxidative phosphorylation (as in normal cell), Mt become efficient suppliers of precursors for biosynthetic pathways in rapidly growing tumor cells. In cancer cells Mt produce non-essential amino acids from TCA intermediates and export excess of citrate to cytosol to support fatty acid synthesis and NADPH production (reviewed by ). To keep TCA running in the absence of pyruvate, cancer cells use glutaminolysis, which involves deamination of glutamine, yielding glutamate and ammonia. Glutamate dehydrogenase (GDH) then converts glutamate to intermediate of TCA cycle – alpha-ketoglutarate (α-KG). Therefore GDH is a critical enzyme for the growth and redox regulation in cancer cells . The aim of this study was to estimate the effect of hyperthermia on GDH activity in healthy rat brain and liver mitochondria as well as in different cancer and normal cell lines.
The effect of hyperthermia (40, 42, 45 and 47°C) was determined on GDH activity in Mt isolated from rat brain and liver, and in lysates of cancer and normal cells of different origin. Brain and liver mitochondria were isolated from 3-4 month old Wistar rats, using deferential centrifugation. Not tumorigenic (Chinese hamster ovary - CHO, hamster kidney fibroblast - BHK-21, murine fibroblast - McCoy) and tumorigenic cells (murine liver cancer - MH-22A, human pancreatic carcinoma - PANC1, human primary pancreatic adenocarcinoma - BxPC-3, murine fibroblast - WEHI 164 and L929) before experimental procedures were cultivated for five days at normal conditions: 37°C, 5% CO2 and humidity. Activity of GDH was evaluated spectrophotometrically at 340 nm, measuring NADH oxidation rate in isolated mitochondria or crude cell lysates (0.2 mg protein in reaction) at 37, 40, 42, 45ºC in the measurement medium containing 20 mM Tris, 110 mM KCl, 2 mM EGTA, 50 mM creatine, 50 mM (NH4)2SO4, pH 7.4. Reaction is started by adding 0.1 mM NADH, 50 mM 2-oxoglutarate and measurement duration is 3 min at the indicated temperature.
Hyperthermia had a strong inhibitory effect on GDH activity in healthy rat brain and liver mitochondria – GDH activity in isolated mitochondria of both tissues decreased more than 50% at 42°C as compared to control (37°C). These findings seemed to be relevant for the explanation of the successful elimination of some cancer cells or tumors by hyperthermic treatment. Therefore we also compared the effect of hyperthermia on GDH activity in cancer and normal cells. However, we found that the response of GDH activity to hyperthermia in investigated cells is highly heterogeneous. The effect of hyperthermia on GDH activity in some cell lines (all three not tumorigenic CHO, BHK 21, McCoy and two tumorigenic MH-22A, WEHI164 cell lines) was similar to that in mitochondria isolated from healthy tissues – the extent of inhibition varied between 50-60%, as compared with control. However, GDH activity was stimulated by hyperthermia (over 20%, as compared with control) in three out of five tumorigenic cell lines – PANC-1, BxPC-3 and L929. Thus, in Mt from the normal tissues and non-tumorigenic cells GDH is inhibited by hyperthermia, but the response is heterogeneous in different cancer types – GDH activity can be stimulated or inhibited under the same conditions. We suppose that these differences are related to cell line dependent variations in the expression of GDH isoforms with different thermal sensitivity. Different pattern of GDH isoform expression at least partially might responsible for the observed large differences in efficiency of hyperthermic oncotherapy in treatment of various cancers.
Labels: Pathology: Cancer Stress:Temperature Organism: Human, Other mammals, Rat Tissue;cell: Nervous system, Liver, Kidney, Islet cell;pancreas;thymus, Genital, Fibroblast Preparation: Isolated mitochondria
Event: B1, Oral
- Vytautas Magnus Univ, Fac Natural Sciences, Kaunas, Lithuania.- [email protected]
References and support
- Warburg O (1956) On the origin of cancer cells. Science 23:309-14.
- Frezza C, Gottlieb E (2009) Mitochondria in cancer: Not just innocent bystanders. Seminars Cancer Biol 19:4-11.
- DeBerardinis RJ, Mancuso A, Daikhin E, Nissim I, Yudkoff M, Wehrli S, Thompson CB (2007) Beyond aerobic glycolysis: Transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc Natl Acad Sci U S A 104:19345-50.
- Jin L, Li D, Alesi GN, Fan J, Kang HB, Lu Z, Boggon TJ, Jin P, Yi H, Wright ER, Duong D, Seyfried NT, Egnatchik R, DeBerardinis RJ, Magliocca KR, He C, Arellano ML, Khoury HJ, Shin DM, Khuri FR, Kang S (2015) Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. Cancer Cell 27:257-70.
- Selected mentor: Vilmante Borutaite