Cell lines used in the experiments

Introduction

genomes, and indirectly regulating the three mitochondrial-encoded COX subunit (Whelan and Zuckerbraun 2013). If oxygen species overwhelm the protective capacity of antioxidants, enhanced lipid peroxidation leads to increased permeability and progression to programmed cell death.

Introduction

through altered cell differentiation and cell growth (Escandell et al. 2008). A colorectal cancer cell line which differed only by the presence of an activated Ki-ras allele was called HCT-116, whereas Hke-3 was derived from HCT-116 by deletion of the activated mutant Ki-ras allele. Compared with parental cells, Hke-3 was described as morphologically altered. It had lost the capacity for anchorage-independent growth, grew more slowly both in vitro and in nude mice, and showed reduced expression of c-Myc (Shirasawa et al., 1993).

It was observed that oncogenic Ki-ras mutations correlated in particular with alterations in an energy metabolism, including increase of glucose and glutamine consumption, lactic acid accumulation, changed expression of mitochondrial genes, and reduction of mitochondrial activity (Gaglio et al. 2011).

Mitotane appears to require transformation into active metabolites for therapeutic action, which is connected with mitochondrial P450-mediated hydroxylation (described in Section 1.1.5). The observed susceptibility of this cell line in response to mitotane might thus be connected with Ki-ras disruption. Since a well-known side effect of mitotane is gastroenterological disturbance, what might reflect its greater sensitivity of the Hke-3 cell line to mitotane.

H1975 is a cell line derived from a lung carcinoma, popularly called non-small-cell lung cancer. It is an adherent cell line with a mutation in the epidermal growth factor receptor (EGFR) (Zhao et al. 2015).The process of tumourigenesis often involves a shift in key regulatory steps of energy metabolism, in which cancer cells become highly dependent on glycolysis rather than oxidative phosphorylation. As described above (in section 1.1.2). This may result from different oncogenic signaling pathways. Aberrant signaling from the EGFR though the PI3K/AKT pathway, in particular, was found to change gene expression connected with glycolytic transporters and enzymes. In non- small cell lung cancer, a mutated EGFR is a crucial oncogenesis driver. Mutation in the kinase domain of EGFR enhances EGFR tyrosine kinase activity, with downstream signaling causing tumour progression. H1975 cells bear an activation point mutation in exon 21(L858R) of EGFR and a T790M mutation in tyrosine kinase of EGFR. The T790M mutation makes this cell line resistant to erlotnib – an inhibitor of the EGFR receptor via binding to the intracellular portion, in contrast to responsive HCC827 cells, another non-small lung cell line.(De Rosa et al. 2015). Their findings indicate that normal

Introduction

Figure 7. Demonstration of the reverse Warburg effect using MCF-7 cells (taken from Sotgia et al. (2011)).

MCF-7 — is a cell line that was first isolated in 1970 from the breast adenocarcinoma of a 69-year old Caucasian woman. It has ideal characteristics particular to the mammary epithelium, possessing oestrogen receptors (ER) and the ability to covert oestrone to oestradiol (Horwitz, Costlow, and McGuire 1975). These cells has been used to demonstrate that oestrogenic organochlorine pesticides, including o,p'-DDT, mimic the endogenous oestrogen, estradiol-17β and suppress apoptosis (Burow et al. 1999).

MCF-7 has been used to study the reverse Warburg effect (Figure 7). Co-culture with fibroblasts, which more closely mirrors the microenvironment of a naturally occurring

(a) Via oxida tive s tre s s , ca nce r ce lls a ctiva te two ma jor tra ns cription fa ctors in adjacent stromal fibroblasts (HIF1α and NFκB). This leads to the onset of both a utopha gy a nd mitopha gy, a s we ll a s a e robic glycolys is , which the n produce s re cycle d nutrie nts (s uch a s la cta te , ke tone s , a nd gluta mine ). The s e high- e ne rgy che mica l building blocks ca n the n be tra ns fe rre d a nd us e d a s fue l in the trica rboxylic a cid cycle (TCA) in a dja ce nt ca nce r ce lls . The outcome is high ATP production in ca nce r ce lls , a nd prote ction a ga ins t ce ll de a th.

(b) Homotypic culture s (uppe r pa ne ls ) of MCF-7 ce lls (right) a nd hTERT-fibrobla s ts (le ft) we re

immunos ta ine d with a mitochondria l me mbra ne a ntibody (re d). Note tha t mitochondria l ma s s is lowe r in monoculture s of MCF-7 ce lls compa re d to fibrobla s ts . Howe ve r, co-culture of MCF-7 ce lls with fibrobla s ts (lowe r pa ne l) induce s a dra ma tic incre a s e in mitochondria l ma s s in the 'ce ntra l MCF-7 ce ll colony.

The “Reverse Warburg Effect”

(a) (b)

(la cta te , ke tone s , glutim ine & othe rs )

Canc e r c e lls S tro mal fibro blas ts

ROS HIF1α NFκB

Ae robic gyc olys is

&

Re c yc le d nutrie nts Auto fag y/

Mito fag y Oxidativ e

mito c ho ndrial me tabo lis m Pro te c tio n ag ains t apo pto s is

(ce ll de ath)

Image redrawn

Introduction

cultured under homotypic conditions, the cells have a very low mitochondrial mass, which characterises the conventional Warburg effect (Sotgia et al. 2011).

Since homotopic culture was used in the current study, it could be that the strongest relative unresponsiveness after mitotane treatment, on energy metabolisms level, is partly caused by the described lack of mitochondria

1.4 He te ro g e ne ity in c o nte xt o f ACC s tudy and g e ne e xpre s s io n