The workplan for the PhD thesis: “Bioenergetics of colorectal cancer: changes in the
mitochondrial respiratory chain supercomplex assembly”
“Jämesoolevähi bioenergeetika: muutused mitokondriaalse hingamisahela superkompleksides”
Majority of previous studies about the bioenergetics of malignant tumors were performed on different
cell models with the conclusion that cancer cells have increased glucose uptake and, due to
mitochondrial damage, it is not metabolized via oxidative phosphorylation (OXPHOS). The OXPHOS
system may be the principal ATP producer (<90%) for several malignant tumor cell types under
normoxic conditions. The experiments of the Laboratory of Bioenergetics (NICPB) showed that
human colorectal cancer (HCC) cannot be considered a hypoxic tumor, since the malignancy itself and
cells surrounding it exhibited even higher rates of OXPHOS than healthy large intestine. The
mitochondria of HCC are homogenous in terms of regulation of the mitochondrial respiration.
The main task of this PhD thesis is to define the architecture and composition of the RC
supercomplex assembly in HCC clinical material and cell cultures in comparison with healthy smooth
muscle and mucosa. Five multi-subunit enzyme complexes constitute the respiratory chain system:
NADH-coenzyme Q reductase (Complex I, CI), succinate-coenzyme Q reductase (Complex II, CII),
ubiquinol-cytochrome c reductase (Complex III, CIII), cytochrome c oxidase (Complex IV, CIV) and
ATP Synthase (Complex V, CV). Kinetic testing of metabolic fluxes by means of metabolic control
analysis can provide preliminary information about the supramolecular organization of the energy
transfer system and enables to quantify the flux exerted by different RC and the ATP synthasome
complexes. The molecular mechanisms and equilibrium between associations and dissociations of RC
supercomplexes determines electron flux from different substrates through the respiratory chain. These
dynamic rearrangements range from all-bound to all-free RC complexes and they open up the
possibility that different modes of RC organization are switched on/switched off to regulate diverse
physiological and pathophysiological processes. Four tissue samples are studied separately: tumor,
nearby (tissue on the junction between cancer and healthy tissue) and control tissue, which contain
healthy colon smooth muscle and colon mucosa, therefore two different tissues. Caco-2 (ATCC®
HTB-37™), HCT 116 (ATCC® CCL-247™) for HCC and cell culture witch is characterized as
normal epithelial tissue CCD 841 CoN (ATCC® CRL-1790™) will be used as reference material. The
complexes will be studied on the level of gene expression and also activities of respiratory chain
complexes will be studied. Genome and transcriptome data are important, but not sufficient to describe
the final in situ modifications and the final outcomes of a pathway or cellular processes, these are
defined by actual activities of their separate proteins – or their assemblies – together with the
respective regulatory mechanisms. To understand the roles of mitochondrial respiratory chain
supercomplexes, methods for consistently separating and preparing supercomplexes must be
established in this work. Quantitative reverse transcription PCR (qRT-PCR) was performed for the
analysis of mRNA. 2D BN/SDS PAGE followed by Western Blot method is used to visualize the
composition of the respiratory chain complexes healthy and cancer affected material. All this data give
the results necessary for the (quantitative)analysis of the changes in the content of ATP synthasome
and respiratory chain supercomplexes in pathological conditions and healthy colon. The main aim of
this PhD thesis is the evaluation of candidates for the mitochondrial checkpoints at the respiratory
chain supercomplexes involved into cellular energy metabolism for colorectal cancer development.
Part of this work is possible to carry out at our collaboration partners from COST MITOEAGLE
(CA15203).
Source of funding IUT 23-1 (NICPB, Laboratory of Bioenergetics, PI Tuuli Käämbre).