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inhibitor CMZ [36,37] and lactate production was monitored another pathway that is regulated by the cellular redox
as described in Methods. Results shown in Figure 3C state is cellular energy metabolism. The interrelation
indicate that inhibition of CYP2E1 in MCF-7 cells resulted between ROS and aerobic glycolysis which is the main
in reduction of lactate production in these cells whereas pathway through which cancer cells produce energy has
inhibition of CYP2E1 in MDA-MB-231 cells did not have been extensively investigated. [31,32] Since cytochrome P450
any effect on lactate production [Figure 3D] reiterating the enzymes are one of the endogenous sources of ROS [46,47] it
concept that CYP2E1 effects are cell type dependent. was hypothesized that CYP450s might be involved in the
regulation of cellular energy metabolism.
It is known that oxidative stress can trigger the
mitochondrial permeability transition and Δψ collapse The role of the CYP2E1 mediated ROS generation in the
leading to defects in ATP production. Taking into energy metabolism of breast cancer cells was investigated
[38]
account these observations, we next assessed potential in the estrogen receptor positive MCF-7 and estrogen
changes in the mitochondrial membrane potential in breast receptor negative MDA-MB-231 cells. Given that
[48]
cancer cells treated with the CYP2E1 activator APAP CYP2E1 gene expression is under the transcriptional
or the CYP2E1 inhibitor CMZ. APAP treatment of both control of factors responsive to inflammation [19,29,49,50] it
MCF-7 and MDA-MB-231 cells led to decrease of Δψ was theorized that inhibition of glycolysis in breast cancer
compared to untreated cells [Figure 4B, compare APAP cells bearing diverse genetic background would lead to
white (0.652) and black (0.698) bars to UN white (1) alternative CYP2E1 cellular levels and hence dissimilar
and black (1) bars]. In contrast, CMZ treatment did not ROS. On the other side inhibition of glycolysis would
[42]
[31]
exert any effects on Δψ which remained the same as that lead to increased ROS generation that could be altered
observed in the untreated cells [Figure 4B, compare CMZ by CYP2E1 enzymatic activity. [42,51-53] In accord with
white and black bars to UN white and black bars]. These published results treatment of both MCF-7 and MDA-
results indicate a potential role of CYP2E1 mediated ROS MB-231 cells with the glycolytic inhibitors 3BP and
generation in the process of alterations of mitochondrial 2DG increased ROS levels in the two cell lines. [33,54,55]
membrane potential. Combination of either 3BP or 2DG with the CYP2E1
inhibitor CMZ reduced dramatically the oxygen radicals’
Alterations in Δψ might in some cases lead to cell death levels in the MCF-7 but not in the MDA-MB-231 cells
[39]
and in order to explore whether that was the case in breast [Figure 2] implying that the dissimilar genetic background
cancer cells treated with APAP or CMZ MCF-7 and in the two cell lines (wild type ER and p53 in MCF-7 and
MDA-MB-231 cell death was determined by PI staining defective ER and mutated p53 in MDA-MB-231 cells)
upon treatment with APAP or CMZ. APAP treatment of determines the differential response of these cells to the
MCF-7 and MDA-MB-231 cells led to increased cell glycolytic and CYP2E1 inhibitors. [56]
death in both cell lines (MCF-7 cells from 9% to 18%
and MDA-MB-231 cells from 16% to 20%) [Figure 5B, The observation that CMZ decreased ROS generation
compare APAP white and black bars to UN white and stimulated by 3BP and 2DG treatment in MCF-7 cells
black bars]. CMZ treatment of both MCF-7 and MDA- prompted our interest to explore the possibility that
MB-231 cells did not exert any effects on cell death as CYP2E1 is involved in the process of energy metabolism
it did not affect the percentage of PI positive compared in breast cancer cells. The potential link between CYP2E1
to untreated MCF-7 and MDA-MB-231 cells [Figure 5B, and energy metabolism was investigated in the MCF-7
compare CMZ white and black bars to UN white and black and MDA-MB-231 cells by estimating the ATP production
bars]. Taken together these results indicate that at least in after treating these cells with the CYP2E1 inhibitor CMZ.
part CYP2E1 mediated generation of ROS alters collapse Results shown in Figure 3 indicate that CMZ treatment
of mitochondrial membrane potential determining cell did not significantly affect ATP generation in the two cell
survival or death in a cell type dependent manner. lines implying that if CYP2E1 had inhibitory effect on
one of the pathways of ATP generation another pathway
DISCUSSION compensates for the loss facilitating cells to meet their
energy requirements, or CYP2E1 is not involved in
[57]
Accumulating evidence supports the view that ROS ATP production in these cells. To answer these questions
generated by CYP2E1 activity mediate cell signalling the lactate concentration was determined in CMZ treated
events that promote alterations in the cellular physiology MCF-7 and MDA-MB-231 cells. CMZ treatment
[58]
and disease development. [40-43] Studies in our and other reduced lactate efflux in MCF-7 but not in MDA-MB-231
laboratories have indicated diverse levels of CYP2E1 cells [Figure 3C and 3D] implying that CYP2E1 exerts
gene expression in a manner dependent on the genetic cell type dependent effects on energy metabolism. These
background and the migratory potential of these results are in line with those shown in Figure 2 indicating
cells. [16,17,29,34] CYP2E1 overexpression in breast cancer reversion of the effect of the glycolytic inhibitors 3BP
cells is involved in the alteration of numerous pathways and 2DG on ROS levels by CMZ in MCF-7 cells, and
linked to the disease such as cell cycle control, apoptosis, published studies reporting that high ROS levels induce
autophagy, ER stress and UPR. [29,44,45] In addition to these, hypoxia inducible factor 1 alpha (HIF-1α) thereby
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Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ July 29, 2016 ¦