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harmful effects ROS act as second messenger signalling the hypothesis that overexpression of CYP2E1 and the
molecules regulating numerous pathways including cell resultant elevated ROS production might regulate cellular
cycle, autophagy, [4,5] apoptosis, endoplasmic reticulum energy metabolism in cancer cells pointing out CYP2E1
[6]
[3]
(ER) stress and cellular energy metabolism. [8,9] as a potential cancer biomarker. The understanding of the
[7]
interplay between CYP2E1 -- ROS generation -- cellular
Sources of intracellular ROS generation include both energy metabolism can provide important conclusions
organelles such as mitochondria, ER and peroxisomes as towards establishing novel breast cancer biomarkers
well as enzymes such as the NADPH oxidases, xanthine and overcoming drug resistance. The estrogen receptor-
oxidase, lipoxygenases and cytochrome P450 enzymes, positive MCF-7 and the triple negative MDA-MB-231
which produce ROS through their enzymatic activities. [estrogen receptor-negative, progesterone receptor-
[10]
CYP450 enzymes are mainly involved in the phase I negative and human epidermal growth factor receptor 2
metabolism of a wide range of exogenous and endogenous (HER2)-negative] breast cancer cells were used in this
compounds oxidizing them to form more hydrophilic study to evaluate the impact of the CYP2E1 mediated ROS
molecules thereby facilitating easier clearance. In the generation on the energy metabolism of these cells.
[11]
case the monooxygenation reaction catalysed by CYP
enzymes is uncoupled from the NADPH reaction instead METHODS
of a monooxygenated substrate production of ROS
occurs. The CYP450 family member CYP2E1 is the Cell culture
[12]
most active enzyme of the family in terms of generating The human breast carcinoma cell lines MCF-7 and MDA-
ROS sometimes inducing production of oxygen radicals MB-231 [obtained from the European Collection of Cell
even in the absence of substrates. [13] Cultures (ECACC)] were maintained in Dulbecco’s
modified Eagle’s medium (Sigma-Aldrich, Gillingham,
Apart from the liver CYP2E1 gene expression has been UK), supplemented with 10% foetal bovine serum (Gibco,
detected in other tissues such as breast, lung, kidney Paisley, UK) and 1% penicillin/streptomycin (Lonza,
and hematopoietic tissues and has been reported to Allendale, NJ, USA) at 37°C in a humidified atmosphere
[13]
be over expressed in malignant compared to normal containing 5% CO . Cells were treated with 100 µM
2
tissues. [14-17] CYP2E1 overexpression in cancer is 3-bromopyruvate (3BP) (Sigma-Aldrich) for 3 h, 20
attributed to the inflammatory conditions present in the mmol/L 2-deoxyglucose (2DG) (Sigma-Aldrich) for 24 h,
tumor microenvironment characterised by increased 2.5 mmol/L acetaminophen (APAP) (Sigma-Aldrich) for
inflammatory cytokine production which affects CYP2E1 3 h and 20 μM chlormethiazole (CMZ) (Sigma-Aldrich)
gene expression. [18-20] Several CYP2E1 dependent for 16 h.
mechanisms contributing to tumorigenesis have been
suggested including formation of toxic intermediate Transient transfection
derivatives and activated carcinogens. [21-23] CYP2E1 Transient transfections were carried out using the polyfect
mediated ROS generation could also contribute to tumor transfection reagent (Qiagen, Crawley, UK), according to
development through pathways in which ROS play the manufacturer’s instructions. Constructs used for ectopic
vital role such as DNA damage, enhanced angiogenic expression included the pcDNA™3.1 (Invitrogen) and the
responses autophagy [4,25,26] ER stress and unfolded pCI-neo-CYP2E1 (kindly provided by Dr. Cederbaum,
[27]
[24]
protein response (UPR). Furthermore, research in our Mount Sinai School of Medicine, New York). [29]
[28]
laboratory has indicated that CYP2E1 is differentially
expressed in a manner dependent on the genetic background Measurement of ROS
and the stage of breast cancer, regulating oxidative stress Cells were grown until they reached 80% confluence
response and metastasis. [29] prior to transient transfection and different treatments.
ROS levels were measured using flow cytometry as
Cancer cells produce energy predominantly through described previously. Cells were transiently transfected
[29]
aerobic glycolysis -- a phenomenon also called Warburg with the indicated constructs and 16 h after transfection
effect -- rather than oxidative phosphorylation even in the they were harvested and incubated with 1 mL of APC-
presence of oxygen and functional mitochondria. The H7-conjugated CD20 antibody (BD Biosciences, Franklin
[30]
Warburg effect is induced in cancer cells by increased Lakes, NJ, USA) to detect only the cells ectopically
cellular glucose uptake stimulated by ROS mediated expressing CYP2E1. Cells were then incubated with
upregulation of gene expression of glucose transporters H2DCFDA (Invitrogen, Carlsbad CA, USA) in the dark
such as GLUT-1. On the other hand, experimental at 37°C for 30 min and subjected to flow cytometry using
[31]
evidence supports the view that increased glycolytic CYAN-ADP flow cytometer (Dako, Glostrup, Denmark)
conversion to pyruvate leads to ROS generation following the fluorescence profile of the H2DCFDA and
[32]
suggesting the existence of an interrelation between ROS APC-H7 probes.
generation with glycolysis and vice versa. [9,33]
Adenosine triphosphate (ATP) assay
Taken together, the above mentioned observations allow ATP levels were measured using the ViaLight plus kit
Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ July 29, 2016 ¦ 269