Kandashvili 2014 Abstract MiP2014
|Regulation of energy metabolism in human colorectal cancer cells in situ.|
Human colorectal cancer (HCC) is an important cause of cancer-associated mortality worldwide. For this study we used saponin-treated, post-operational tissue samples of HCC in combination with high-resolution respirometry to estimate and characterize altered metabolism of mitochondria in HCC. Mitochondrial respiration of tissue samples was measured at 25 °C using the Oroboros Oxygraph-2k. For immunocytochemistry we used standard Abcam protocol for formalin fixed paraffin-embedded tissue sections. Confocal images were collected using Olympus FV10i-W microscope, RNA isolation and RT-PCR were performed using Kits from QIAGEN sciences and Applied Biosystems and enzymatic activities were assessed spectrophotometrically at 25 °C .
The apparent Michaelis-Menten constant (K’m) for ADP and maximal tissue-mass specific respiratory flux (Jmax) were calculated for the characterization of the affinity of mitochondria for exogenous ADP: normal colon tissue displays low affinity (K’m=260±55 µM) whereas in tumor tissue, affinity was significantly higher (K’m=126±17 µM). Despite the increased permeability of the outer mitochondrial membrane, OXPHOS capacity (Jmax) of the tumor samples was 60-80% higher than that in control tissue. These results show that HCC is a non-hypoxic oxidative tumor with significantly increased mitochondrial respiration and is sensitive to its inhibitors.
We found that the localization and function of β-tubulin isotypes varies in different malignant cells. The absence of βII-tubulin in cancer cells allows binding of HK-2 to VDAC, mediating thus the initiation of the Warburg effect. According to Pedersen’s model , the voltage dependent anion channel (VDAC), located within the mitochondrial outer membrane (mtOM) and linked with HK-2, usually shows increased activity in tumor cells and is one of the main pathways mediating the “Warburg effect” in cancer. Our RT-PCR and immunocytochemistry (ICH) studies have revealed that HCC cells express genes encoding HK-1 and HK-2. Studies using confocal microscopy and immunostaining showed the colocalization of the HK-2 isoenzyme with VDAC and a possible binding of HK-2 to mitochondria in these malignant cells. Consequently, in HCC cells the expression of HK-2 and HK-1 is associated with binding either one or both isoforms with VDAC in the mtOM.
Using spectrophotometric assessment of enzymatic activities in HCC showed that during carcinogenesis the total activity of hexokinase (HK) does not change in comparison with normal tissue. Furthermore, only minor alterations in the expression of HK-1 and HK-2 isoforms were observed, and similar results were obtained measuring the total activity of CK. In contrast, total adenylate kinase (AK) activity is upregulated in HCC cells, and there could be a functional coupling between oxidative phosphorylation and the AK system in contrast to normal colon tissue. The structure-function relationship in bioenergetic regulation in comparison to tumor and normal tissue of HCC requires further investigation.
• O2k-Network Lab: EE Tallinn Kaambre T
Labels: MiParea: Respiration, mt-Membrane Pathology: Cancer
Organism: Human Tissue;cell: Endothelial;epithelial;mesothelial cell Preparation: Permeabilized tissue
Regulation: ADP Coupling state: OXPHOS
HRR: Oxygraph-2k Event: B3, Poster MiP2014
1-Lab Bioenergetics, National Inst Chem Physics Biophysics; 2-Tallinn Univ; 3-Cancer Res Competence Center; 4-North Estonian Reg Hospital, Tallinn; 5-Tallinn Univ Technology; Tallinn Estonia. - [email protected]
- Klepinin A, Chekulayev V, Timohhina N, Shevchuk I, Tepp K, Kaldma A, Koit A, Saks V, Kaambre T (2014) Comparative analysis of some aspects of mitochondrial metabolism in differentiated and undifferentiated neuroblastoma cells. J Bioenerg Biomembr 46: 17-31.
- Pedersen PL (2008) Voltage dependent anion channels (VDACs): a brief introduction with a focus on the outer mitochondrial compartment's roles together with hexokinase-2 in the "Warburg effect" in cancer. J Bioenerg Biomembr 40: 123-6.