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Difference between revisions of "Bastos Sant'Anna Silva 2018 TRANSMIT Bertinoro di Romagna IT"

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(Created page with "{{Abstract |title=Effect of cell-permeable succinate and malonate prodrugs on mitochondrial respiration in prostate cancer cells. |authors=Sant'Anna-Silva ACB, Elmer E, Mesza...")
 
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{{Abstract
{{Abstract
|title=Effect of cell-permeable succinate and malonate prodrugs on mitochondrial respiration in prostate cancer cells.
|title=Cellular succinate transport and mitochondrial respiratory function in prostate cancer.
|authors=Sant'Anna-Silva ACB, Elmer E,  Meszaros AT, Gnaiger E
|authors=Sant'Anna-Silva ACB, Klocker H, Weber A, Elmer E,  Meszaros AT, Gnaiger E
|year=2018
|year=2018
|event=Life Sciences Meeting 2018 Innsbruck AT
|event=TRANSMIT Course in Cancer and Metabolism
|abstract=Succinate is a substrate mainly metabolized to fumarate in mitochondria by succinate dehydrogenase (SDH) or Complex II. SDH is located at the inner mitochondrial membrane, coupling the oxidation of succinate to fumarate in the tricarboxylic acid cycle (TCA) with electron transfer to ubiquinone. Inhibition or downregulation of SDH leads to an impairment of TCA cycle and respiratory activity, and consequently to accumulation of succinate. This, in turn, transmits an oncogenic signal from mitochondria to the cytosol. Cytosolic succinate inhibits the hypoxia inducible factor 1α (HIF1α) prolyl hydroxylase (PHD) leading to HIF1α stabilization. In this “pseudohypoxic” state angiogenesis and anaerobic metabolism are enhanced, ultimately leading to tumour progression.  
|abstract=Succinate dehydrogenase (SDH, mitochondrial Complex II) links the oxidation of succinate and FAD to fumarate and FADH2 in the tricarboxylic acid (TCA) cycle to electron transfer (ET) from FADH2 to ubiquinone in the ET system. Changes in ET capacity through the succinate pathway affect TCA cycle function and cell respiration [1]. In addition, succinate transmits oncogenic signals from mitochondria to the cytosol by stabilization of hypoxia inducible factor 1α. This, in turn, stimulates the expression of genes involved in angiogenesis and anaerobic metabolism [2], finally enabling tumour progression and metastasis. Succinate uptake is enhanced in various cancer cells [3], and its mitochondrial utilisation is increased in permeabilized prostate cancer cells [4].
 
To decipher the pathophysiological role of succinate in prostate cancer, we measured the plasma membrane permeability for succinate and utilization of external succinate by mitochondria in terms of succinate pathway capacity and kinetic properties in prostate cancer (multiple metastatic origins) and control cell lines.
While succinate has essential implications on prostate cancer development, it is difficult to control intracellular succinate concentrations in intact cells due to the low permeability of plasma membranes to the compound. To overcome this limitation, we applied novel plasma membrane-permeable succinate (NV118) and malonate (inhibitor of SDH, NV161) prodrugs in high-resolution respirometry (Oroboros O2k-FluoRespirometer). Mitochondrial respiration was assessed in three cell lines: RWPE-1 (prostate; noncancerous), LNCaP (prostate; cancer), and HEK293T (embryonic kidney; control).  
Respiration in RWPE-1 (prostate; noncancerous), LNCaP (prostate; lymph node metastasis) and DU145 (prostate; brain metastasis) cells was measured using High-Resolution FluoRespirometry (O2k, Oroboros Instruments) and substrate-uncoupler-inhibitor titration (SUIT) protocols developed specifically for the study. To assess succinate utilization in intact cells independent of a plasma membrane succinate transporter, we applied novel plasma membrane-permeable succinate prodrugs (pS) [5].
 
In LNCaP cells, transport of external succinate is enhanced through the plasma membrane as compared to the other cell lines, while pS exerted similar effects in all cell lines, suggesting an important regulatory role of the transport mechanism. Furthermore, in LNCaP cells, mitochondria utilize succinate with higher affinity than control cells. Importantly, kinetic measurements demonstrated the most pronounced difference in the affinities in the physiological intracellular succinate concentration range (< 100 µM), underlining its pathophysiological role.
NV118 (250 µM) stimulated ROUTINE respiration in LNCaP cancer cells by 18% as compared to vehicle (DMSO), while respiration remained unchanged in RWPE-1 (4% increase) and HEK 293T cells, even at higher concentrations of the prodrug. NV161 (66 µM) had no effect on ROUTINE respiration of HEK 293T cells.
Our results indicate a “succinate phenotype” in LNCaP, with enhanced transport and utilization. As such, succinate is a potential mitochondrial metabolic biomarker in prostate cancer cells. We propose a model in which succinate does not only play a role in the signalling but has a central role in the maintenance of mitochondrial respiration as a fuel substrate.
 
|keywords=mitochondrial respiration, intact cells, cell-permeable succinate, Prostate cancer, Succinate,
Our results indicate enhanced utilization of external, plasma membrane-permeable succinate in mitochondrial respiration in LNCaP prostate cancer cells but not in control cell lines. The cell-permeable prodrugs offer promising research tools to elucidate the roles of succinate and inhibition of SDH in metabolic reprograming towards a malignant phenotype.
|editor=[[Sant'Anna-Silva ACB]],
|keywords=mitochondrial respiration, intact cells, cell-permeable succinate,
|editor=[[Sant'Anna-Silva ACB]], [[Kandolf G]],
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck Oroboros, SE Lund Elmer E
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck Oroboros, SE Lund Elmer E
}}
}}
Line 18: Line 16:
|area=Respiration, Pharmacology;toxicology
|area=Respiration, Pharmacology;toxicology
|diseases=Cancer
|diseases=Cancer
|tissues=Kidney, Other cell lines, HEK
|organism=Human
|tissues=Genital, HEK
|preparations=Intact cells, Permeabilized cells
|preparations=Intact cells, Permeabilized cells
|enzymes=Complex II;succinate dehydrogenase
|enzymes=Complex II;succinate dehydrogenase
|topics=Calcium, Substrate
|topics=Ion;substrate transport, Substrate
|couplingstates=ROUTINE
|couplingstates=LEAK, ROUTINE, OXPHOS, ET
|instruments=Oxygraph-2k
|pathways=S, ROX
|instruments=Oxygraph-2k, O2k-Protocol
|event=Oral
|event=Oral
}}
}}
== Affiliations ==
== Affiliations ==


:::Sant´Anna-Silva ACB(1,2), Elmer E(3), Meszaros AT(1,4), Gnaiger E(1,2)
:::Sant´Anna-Silva ACB(1,2), Klocker H (3), Weber A (3), Elmer E(4), Meszaros AT(1,2), Gnaiger E(1,2)


::::# Oroboros Instruments, Innsbruck, Austria. - [email protected]
::::# Oroboros Instruments, Innsbruck, Austria. - [email protected]
::::# Daniel Swarovsky Inst, Medical Univ Innsbruck, Austria
::::# Daniel Swarovsky Inst, Medical Univ Innsbruck, Austria
::::# Lund Univ, Sweden
::::# Dept of Urology, Medical Univ Innsbruck, Austria
::::# Inst Surgical Research, Univ Szeged, Hungary
::::# Dept of Clinical Sciences, Lund Univ, Sweden

Revision as of 12:53, 21 August 2018

Cellular succinate transport and mitochondrial respiratory function in prostate cancer.

Link:

Sant'Anna-Silva ACB, Klocker H, Weber A, Elmer E, Meszaros AT, Gnaiger E (2018)

Event: TRANSMIT Course in Cancer and Metabolism

Succinate dehydrogenase (SDH, mitochondrial Complex II) links the oxidation of succinate and FAD to fumarate and FADH2 in the tricarboxylic acid (TCA) cycle to electron transfer (ET) from FADH2 to ubiquinone in the ET system. Changes in ET capacity through the succinate pathway affect TCA cycle function and cell respiration [1]. In addition, succinate transmits oncogenic signals from mitochondria to the cytosol by stabilization of hypoxia inducible factor 1α. This, in turn, stimulates the expression of genes involved in angiogenesis and anaerobic metabolism [2], finally enabling tumour progression and metastasis. Succinate uptake is enhanced in various cancer cells [3], and its mitochondrial utilisation is increased in permeabilized prostate cancer cells [4]. To decipher the pathophysiological role of succinate in prostate cancer, we measured the plasma membrane permeability for succinate and utilization of external succinate by mitochondria in terms of succinate pathway capacity and kinetic properties in prostate cancer (multiple metastatic origins) and control cell lines. Respiration in RWPE-1 (prostate; noncancerous), LNCaP (prostate; lymph node metastasis) and DU145 (prostate; brain metastasis) cells was measured using High-Resolution FluoRespirometry (O2k, Oroboros Instruments) and substrate-uncoupler-inhibitor titration (SUIT) protocols developed specifically for the study. To assess succinate utilization in intact cells independent of a plasma membrane succinate transporter, we applied novel plasma membrane-permeable succinate prodrugs (pS) [5]. In LNCaP cells, transport of external succinate is enhanced through the plasma membrane as compared to the other cell lines, while pS exerted similar effects in all cell lines, suggesting an important regulatory role of the transport mechanism. Furthermore, in LNCaP cells, mitochondria utilize succinate with higher affinity than control cells. Importantly, kinetic measurements demonstrated the most pronounced difference in the affinities in the physiological intracellular succinate concentration range (< 100 µM), underlining its pathophysiological role. Our results indicate a “succinate phenotype” in LNCaP, with enhanced transport and utilization. As such, succinate is a potential mitochondrial metabolic biomarker in prostate cancer cells. We propose a model in which succinate does not only play a role in the signalling but has a central role in the maintenance of mitochondrial respiration as a fuel substrate.

Keywords: mitochondrial respiration, intact cells, cell-permeable succinate, Prostate cancer, Succinate Bioblast editor: Sant'Anna-Silva ACB O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck Oroboros, SE Lund Elmer E


Labels: MiParea: Respiration, Pharmacology;toxicology  Pathology: Cancer 

Organism: Human  Tissue;cell: Genital, HEK  Preparation: Intact cells, Permeabilized cells  Enzyme: Complex II;succinate dehydrogenase  Regulation: Ion;substrate transport, Substrate  Coupling state: LEAK, ROUTINE, OXPHOS, ET  Pathway: S, ROX  HRR: Oxygraph-2k, O2k-Protocol  Event: Oral 


Affiliations

Sant´Anna-Silva ACB(1,2), Klocker H (3), Weber A (3), Elmer E(4), Meszaros AT(1,2), Gnaiger E(1,2)
  1. Oroboros Instruments, Innsbruck, Austria. - [email protected]
  2. Daniel Swarovsky Inst, Medical Univ Innsbruck, Austria
  3. Dept of Urology, Medical Univ Innsbruck, Austria
  4. Dept of Clinical Sciences, Lund Univ, Sweden