Bioenergetics of Human Cancer Cells and Normal Cells

Bioenergetics of Human Cancer Cells and Normal
Cells During Proliferation and Differentiation

Nina A. Mikirova¹, Joseph J. Casciari¹, *Michael J. Gonzalez², Jorge R. Miranda-Massari³, Neil H. Riordan¹ and
Jorge Duconge³
Riordan Clinic, 3100 Hillside Ave, Wichita, Kansas 67219, University of Puerto Rico, Medical Sciences Campus, RECNAC
Schools of Public Health, San Juan P.R
School of Pharmacy, GPO Box 365067San Juan PR 00936-5067
Submission: February 24, 2017; Published: March 22, 2017
*Correspondence Address: Dr. Michael J Gonzalez, University of Puerto Rico, Medical Sciences Campus, School of Public Health, Department
of Human Development, Nutrition Program, GPO Box 365067, San Juan P.R 00936-5067, Tel: x 1405; 787-649-2737

Cancer cells are known to have different metabolic properties than normal cells, particularly their tendency to undergo glycolysis even
under aerobic favoring conditions. This has created interest in how mitochondrial function in tumor cells may differ from that in normal cells.
Using human malignant cells (SW-620, PC-3, HT-1080, SK-MEL, HL-60, K-562 and MOLT-3), human fibroblast (CCL-153) and human T Cells,
we investigated three key parameters that have been typically to describe mitochondrial function: cellular ATP production, mitochondrial
potential and cellular cardiolipin levels. On average, tumor cancer cells had more ATP production and greater mitochondrial potentials. For
example, ATP levels in malignant cells ranged from 20 to 69 µmole/106 cells, with a cancer cell average of 40 ± 18 µmole/106 cells. For
normal cells, the ATP level range went from 9 to 24 µmole/106 cells, for an average of 15 ± 11 µmole/106 cells. Mitochondrial potentials
tended to be three times higher in cancer cells, perhaps because overall mitochondrial mass (as measured by relative cardiolipin levels) were
twice as high in cancer cells. Higher mitochondrial masses are consistent with proliferation. Proliferating cells in general showed higher
mitochondrial function compared to quiescent cells (confluent monolayers), and HL-60 cells showed reductions in all three mitochondrial
parameters measured here when the cells were exposed to the differentiating agent TPA. The effects of ATP production inhibitors CCCP
and oligomycin on mitochondrial function in normal and cancer cells were also compared. In general, in these experiments, cancer cell
mitochondrial inhibition with these agents produced a decrease ATP levels by 30-40% while in normal cells ATP production was reduced by
60%. These results provideevidence of a mitochondrial dysfunction in cancer cells. Cancer cells appear to better withstand interference with
ATP synthesis in mitochondria since they rely mainly on glycolysis as an energy producing mechanism.

Research into the energy metabolism of cancer cells began
in the early 20th century with Otto Warburg, whoobserved
that tumor tissues appear defective in respiration and have
abnormally high rates of aerobic glycolysis [1]. This led Warburg
to propose that cancer arouse as a result of mitochondrial injury
[2]. Since then several cancer cell metabolism and mitochondrial
function has been subject to extensive study. Two of the most
well-known and acceptedfeatures of tumor cell metabolism are
the “Crabtree effect” [3] and the “Pasteur effect” [4]. The former
refers to inhibition of cancer cell respiration by elevated glucose
concentrations, while the latter refers to inhibition of glycolysis
by elevated oxygen concentration. Presumably, the Crabtree
effect arises due to competition between glycolysis and oxidative
phosphorylation for Pi and ADP [5]. It has also been observed
in some cancer cells that the consumption rate of one nutrient
(oxygen or glucose) increases when concentration of the other
nutrient is reduced, suggesting an ability of cancer cells to
adjust their metabolism based on micro-environment [6]. These
observations show that cancer has a very relevant metabolic
A variety of abnormalities in cancer cell mitochondrial
structure and function have been reported [7-27]. These
peculiarities in glucose metabolism may be linked to differences
between the mitochondria of cancer cells and those of normal
cells [15-27]. These include increases in, and alteration of,
mitochondrial DNA [8, 10-14,20-21], elevation ofhexokinase
production [15], lysis of cristae structures [23-26] and altered
mitochondrial protein and lipid content [24-26]. Interestingly,
genetic alterations in cancer cells, such as deregulation of the
PI3K/Akt pathway, or imbalances in activity of c-MYC, HIF, or
p53, can alter glucose and amino acid metabolism [7,28]. In addition, cancer cells have abnormal content and composition of
cardiolipin [29-33], a key mitochondrial lipid that is necessary
for proper cell respiration. Cardiolipin normally resides in
the inner membrane of mitochondria, where it plays a role in
chemiosmosis. In addition, cardiolipin can move to the outer
mitochondrial membrane to trigger apoptosis. The “defect” in
mitochondrial respiration reported by Warburg may be related
to cardiolipin defects in tumor cells [34]. A generalized increase
in anabolism characterizes nearly all cancer types [35-36],
perhaps indicating consumption of metabolic intermediates
toward anabolic reactions, and concomitantly less conversion
of pyruvate to oxaloacetate, leaning toward an augmented
formation of lactic acid thus indicating an increase dependency
on glycolysis as energy mechanism.
In the present study, we examine differences between
cancer cells and normal cells in three parameters related to
mitochondria: ATP production, cardiolipin concentration,
and mitochondrial potentials. Moreover, we examine how, in
cancer cells and normal cells, these parameters are affected by
ATP synthesis inhibitors and, in case of one cancer cell type,
chemically induced differentiation. Our results support the idea
that mitochondria in cancer cells rely more in glycolysis as their
main energy production mechanism.
Human cancer cell lines used in this study include
HL-60 cells (acute promyelocytic leukemia), K-562 cells
(chronic myelogenic leukemia, lymphoblast), MOLT-3 (acute
lymphoblastic leukemia, T cells, human), SK-MEL (skin
melanoma), PC-3 (prostate carcinoma), HT-1080 (connective
tissue, fibrosarcoma), CRL-1977 (uterine sarcoma), and SW-620
(colon adenocarsinoma). Normal human cell lines used in this
study include CCD18-lu (lung fibroblasts) and CCL-153 (lung
fibroblast). All cell lines were obtained from ATCC (Manassas,
VA). In addition, T-lymphocytes were obtained from peripheral
blood by incubation in RosetteSep™ antibody cocktail (StemCell
Technologies). RosetteSep™ crosslinks unwanted cells to red
blood cells, forming immune-rosettes. These immune-rosettes
pellet during centrifugation, leaving untouched, highly purified
target cells at the interface between the plasma and the density
gradient medium.
ATP Measurements
Cellular ATP levels were measured using the CellTiter-GLO
Luminescent Cell Viability Assay Kit (Promega, Madison WI).
This assay generates a luciferase reaction that is proportional
the amount of ATP present within the cells. ATP concentration
versus luminescence is linear between zero and 1.5 Million
cells (r = 0.90), allowing ATP levels (µmoles) per 106 cells to
be determined from luminescence signals using standard curves
generated with pure ATP (Sigma, St. Louis MO).
Mitochondrial Potential Measurements
Mitochondrial potential was measured based on fluorescence
intensity of JC-1 (Cayman Scientific, Ann Arbor MI), a dye that
exhibits potential-dependent accumulation in mitochondria.
The dye’s emission shifts from green (535 nm) to red (595
nm) in mitochondria, with the red-to-green ratio indicating
mitochondrial potential. To exclude the effect of membrane
emission during measurements, membrane potential was
dissipated by gentle buffers which do not affect mitochondria
(Promega lysis buffer, or by PBS with 0.2% Triton –X, 1mM
DTT) before fluorescent spectra were measured. Cells were
counted, and 5 x 105 cells were stained using 2.5 µg/mL JC-1.
After wash and membrane lysis, emission spectra in range 500
nm to 750 nm were obtained using a SPEX fluorometer (SPEX
Industries, Edison NJ). The total accumulation of dye at 595nm
is proportional to the mitochondrial potential and the number of
mitochondria, while the signal at 535 nm should be independent
of this. Thus, we used the ratio of fluorescence at 595 nm to that
at 535 nm as our measure of mitochondrial potential.
Cardiolipin Measurements
We used 10-N-nonyl acridine orange (NAO) dye as a
cardiolipin probe.NAO monomers have a green fluorescence
(flow cytometry channel FL-1) while dimers, formed on contact
with cardiolipin, emit a red fluorescence (flow cytometry channel
FL-3). NAO specifically binds to cardiolipin with a stoichiometry
of 2:1. To measure cardiolipin, 0.5M cells were incubated in
medium with NAO (dye concentration in range 1-12 µM). After
the 30 min of incubation at 37C, 50µg/ml propidium iodide (PI)
was added to stain the DNA of dead cells. Emission of NAO was
measured by flow-cytometer.
Cell Differentiation
The HL-60 leukemia model allows us to examine
the effects of differentiation on mitochondrial metrics.
12-O-tetradecanoylphorbol-13-acetate (TPA) induces cellular
differentiation of a number of leukemia cell lines including HL60. TPA was dissolved in DMSO. HL-60 cells were plated in 6-well
plates with concentration 106 cells in 5ml growth medium. Cells
were then treated with TPA (Sigma, St. Louis MO). Two TPA
concentrations were tested, 32 nM and 64 nM. After 24hours,
control and TPA treated cells were used in assays described
Three key variables were assessed in this study, mitochondrial
potential, mitochondrial mass and cellular ATP concentration.
Results for several tumor and normal cell types are given in
Table 1. There was some variation in values with cell type, but
there was a general pattern of the cancer cells having higher
ATP levels, greater mitochondrial mass levels (as determined
by cardiolipin levels) and higher mitochondrial potentials. This
confirms the hypothesis that cancer cell mitochondria have
different properties than normal cell mitochondria, consistent with the differences in cancer cell metabolism described in the
introduction. Some, but not all, of the variation in mitochondrial
potential and ATP production can be explained by differences in
mitochondrial mass, as shown in Figure 1(a). According to these
data, levels of ATP show a statistically significant correlation
with cardiolipin levels in normal cells (r=0.8) and relation with
ATP in cancer cells with lower measured ATP for higher levels
of cardiolipin. When mitochondrial potential or ATP levels are
normalized with mitochondrial mass (dividing by cardiolipin),
they are roughly thirty percent higher in cancer cells than
in normal cells. This suggests that the larger mitochondrial
mass in cancer cells may account in part for their increased
ATP production and mitochondrial potentials, although it
should be noted that cardiolipin levels may be an imperfect
corollary to mitochondrial mass if cardiolipin concentrations
vary significantly from one cell type to another. As expected,
ATP production rates are highly correlated with mitochondrial
potentials (Figure 1b) with higher measured ATP in normal
cells in comparison with cancer cells at the same value of
mitochondrial potential.