Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.

Most cancer cells rely on aerobic glycolysis to support the mitochondrial oxidative phosphorylation system (OXPHOS). The persistent oxic-anoxic cycle exerts selection pressures which lead to constitutive activation of glycolysis even in the presence of abundant oxygen. Expression of hypoxia-inducible factor 1 (HIF1) increases following hypoxia in neoplastic cells. This leads to the induction of pyruvate dehydrogenase kinase 1 (PDK1). The latter inactivates pyruvate dehydrogenase (PDH) that converts pyruvate to acetyl-coenzyme A for delivery to the tricarboxylic acid cycle (TAC). Dichloroacetic acid (DCA) is an inhibitor of PDK that forces cells into oxidative phosphorylation thereby suppressing cancer growth. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-3-ol-17-one (C9), along with a few other 17β-estradiol analogs, are a novel class of in silico-designed inhibitors of microtubule dynamics. These newly designed and synthesized antimitotic compounds induce G2/M arrest and apoptosis by docking to colchicine binding site between α- and β-tubulin. These compounds are 5 to 20 times more potent than their source molecule, 2-methoxyestradiol (2ME). To improve bioavailability C9 has been in silico-modified at carbon positions C2, C3 and C17 compared to 2ME. The approach to investigate the anticancer potential of the in silico-designed antimitotic C9 in combination with the glycolytic inhibitor DCA in vitro is novel. Human breast carcinoma cell line MCF-7 and non-tumorigenic breast cells MCF-12A were used as an experimental model system. The present study demonstrated that DCA (7.5 mM) in combination with C9 (130 nM) selectively inhibited half of MCF-7 cells‘ population (50.8%). Under the same treatment conditions, MCF-12A cells displayed high number of cell survival (70% cell growth). Qualitative morphological studies revealed decreased cell density in both cell lines, as well as hallmarks of apoptosis and autophagic processes including formation of apoptotic bodies, DNA fragmentation and autophagic vacuoles. Cell cycle- and apoptosis quantification analyses revealed C9+DCA treatment induced apoptosis in both cell lines and exhibited selectivity towards tumorigenic cells. Presence of autophagosome was observed and microtubule-associated protein 1 light chain 3 (II) (LC3-II) expression was elevated. Reduction of mitochondrial membrane potential depolarization in tumorigenic MCF-7 cells was demonstrated, but not in MCF-12A cells. Oxidative stress tests suggested the combination treatment C9+DCA is able to induce lysosomal rupture and/or mitochondrial damage in tumorigenic MCF-7 cells. Kinase inhibition studies revealed that transient activation of c-Jun N-terminal kinase (JNK) plays an important role in cell proliferation. However, C9+DCA stimulated prolonged JNK activation and, in turn, promoted Bcl-2 phosphorylation, thereby facilitating autophagic and apoptotic cell death. C9+DCA induced expression of a number of genes related to stress in MCF-7 treated cells including TP53BP1, MDM2 and BBC3/PUMA. Genes related to cell motility and maintenance of the cytoskeleton such as ACTG1, MAP7, TUBA1, TUBA6, TUBA8 and TUBB2A genes were down-regulated. In MCF-12A cells, treatment of C9+DCA induced expression of multidrug resistance gene ABCB1. Moreover, genes involved in reactive oxygen species metabolism FTH1, GSTA2, NOS2A, SMOX, SOD1 and SOD2 were also up-regulated. In conclusion, the novel 17β-estradiol derivative, C9, in combination with DCA is a potent antiproliferative treatment. This study addressed the mechanisms of combination treatment at the basis of molecular and cellular level, warranting further research projects to develop viable and functional combination treatment as clinically useable anticancer agents. / Thesis (PhD)--University of Pretoria, 2014. / lk2014 / Physiology / PhD / Unrestricted

Yes / Organometallic complexes offer the prospect of targeting multiple pathways that are important in cancer biology. Here, the preclinical activity and mechanism(s) of action of a silver-bis(N-heterocyclic carbine) complex (Ag8) were evaluated. Ag8 induced DNA damage via several mechanisms including topoisomerase I/II and thioredoxin reductase inhibition and induction of reactive oxygen species. DNA damage induction was consistent with cytotoxicity observed against proliferating cells and Ag8 induced cell death by apoptosis. Ag8 also inhibited DNA repair enzyme PARP1, showed preferential activity against cisplatin resistant A2780 cells and potentiated the activity of temozolomide. Ag8 was substantially less active against non-proliferating non-cancer cells and selectively inhibited glycolysis in cancer cells. Ag8 also induced significant anti-tumour effects against cells implanted intraperitoneally in hollow fibres but lacked activity against hollow fibres implanted subcutaneously. Thus, Ag8 targets multiple pathways of importance in cancer biology, is less active against non-cancer cells and shows activity in vivo in a loco-regional setting. / RMP and MS funded by Yorkshire Cancer Research (pump priming grant BPP 046). IJS and AL funded by NIHR Research & Innovation Division, Strategic Project Funding 2013 and Manchester Pharmacy School Fellowship.