Journal of Cancer Therapy, 2013, 4, 32-40
http://dx.doi.org/10.4236/jct.2013.47A006 Published Online August 2013 (http://www.scirp.org/journal/jct)
Exploiting MCF-7 Cells’ Calcium Dependence with
Interlaced Therapy
Jonathan Pottle1, Chengrong Sun1, Lloyd Gray2, Ming Li1*
1Department of Physiology, School of Medicine, Tulane University, New Orleans, USA; 2Tau Therapeutics, LLC, Charlottesville, USA.
Received May 3rd, 2013; revised June 4th, 2013; accepted June 12th, 2013
Copyright © 2013 Jonathan Pottle et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The purpose of this study is to demonstrate MCF-7 cells’ dependence on calcium for growth and to exploit that de-
pendence to improve chemotherapy efficacy. Fura-2 fluorescence imaging shows that MCF-7 cells maintain a higher
basal intracellular calcium concentration than non-tumorigenic MCF-10A cells. Blocking T-type calcium channels with
mibefradil reduced MCF-7 intracellular calcium concentration. Flow cytometry shows that knocking down T-type cal-
cium channel expression with siRNA caused an increase in MCF-7 cells in G1 phase and a decrease in cells in S phase.
Proliferation assays of MCF-7 cells treated with EGTA and thapsigargin reveal the dependence of MCF-7 cell growth
on extracellular and intracellular calcium sources, respectively. In vitro, interlaced treatment that alternated the T-type
calcium channel blocker NNC-55-0396 with paclitaxel more effectively reduced MCF-7 cell number than chemother-
apy alone. In a mouse in vivo model, interlaced mibefradil and paclitaxel more effectively reduced MCF-7 xenograft
size than chemotherapy alone. These findings indicate that MCF-7 cells are dependent on calcium for proliferation, par-
ticularly in passing the G1/S cell cycle checkpoint. Further, this dependence on calcium can be exploited by alternating
treatment with T-type calcium channel blockers with paclitaxel in an interlaced therapy scheme that increases the effi-
cacy of the chemotherapy.
Keywords: Breast Cancer; Calcium; Calcium Channel Blockers; Ion Channels; Interlaced Therapy
1. Introduction
Any given cell type expresses a unique cadre of calcium
signaling molecules that allows that cell to generate and
respond to various calcium signals [1]. The many roles of
calcium in eukaryote cell biology are so wide ranging in
type and scope that the complement of calcium signaling
modalities in a cell must be subject to exquisite regula-
tion in order to maintain normal growth and function.
Permanent alteration of a calcium signaling mechanism
can have a profound impact on the biology of a cell. The
abnormal growth characteristic to cancer is often associ-
ated with dysfunctional calcium signaling. Elevated cal-
cium levels normally cause keratinocytes to differentiate,
but transformed keratinocytes are far less likely to un-
dergo differentiation regardless of calcium concentration
[2]. The calcium binding protein S100-A8, which con-
tains two EF-hand motifs and normally participates in
cell cycle regulation and differentiation, has been shown
to influence the activity of telomerase [3], an enzyme
that lengthens telomeres and allows for unlimited chro-
mosomal replication in cancer cells. Expression and
function of the sarco/endoplasmic reticulum calcium
ATPase, a pump that stores calcium ions in the endo-
plasmic reticulum lumen, is important for proliferation in
LNCaP prostate cancer cells [4]. Calcium channels,
which conduct calcium ions from the extracellular envi-
ronment or intracellular stores into the cytosol, have been
found to play roles in cancer cell proliferation. A mem-
ber of the transient receptor potential superfamily of ion
channels, TRPV6, has been shown to be essential to pro-
liferation in some prostate cancer cells [5]. Voltage-gated
T-type calcium channels, characterized by a low thresh-
old for activation, have been found to be broadly ex-
pressed in cancer cells [6], and their role in proliferation
is well observed in cancer cells from a wide variety of
tissues of origin. MCF-7 breast cancer cells express
T-type calcium channels that conduct current and play a
functional role in proliferation [7]. In particular, blocking
T-type calcium channels with pharmacological agents
and knocking down channel expression with RNA inter-
*Corresponding author.

Exploiting MCF-7 Cells’ Calcium Dependence with Interlaced Therapy 33
ference reduced the proliferation of MCF-7 cells. Similar
findings indicating a dependence of cancer cell prolifera-
tion on T-type calcium channels have been reported in
esophageal cancer cells [8], retinoblastoma cells [9], co-
lorectal and gastric cancer cells [10], and ovarian cancer
cells [11] that express T-type calcium channels. Overex-
pression of T-type calcium channels doubled the prolif-
eration rate of astrocytoma and neuroblastoma cells that
already expressed the channels, while antisense oligonu-
cleotides directed against mRNA coding for T-type cal-
cium channels reduced the proliferation rate in these cells
but had no effect in cancer cells that lack T-type calcium
channel expression [12]. Similar results were found the
prostate cancer cell line LNCaP, in which T-type calcium
channels play a role in intracellular calcium regulation [13,14].
In this study, we set out to investigate the physiology
of calcium in breast cancer cells by estimating the intra-
cellular calcium concentration in cancerous and non-
cancerous cell lines, and by assessing the importance of
extracellular and intracellular calcium sources to breast
cancer cell growth. We also examined how T-type cal-
cium channel inhibition can affect passage through the
cell cycle, and we employed both in vitro and in vivo
models to determine if these channels may be potential
targets in breast cancer therapy.
2. Materials and Methods
2.1. Cell Culture
MCF-7 cells were cultured in Dulbecco’s Modified Ea-
gle’s Medium (DMEM; ATCC, Manassas, VA) supple-
mented with 10% fetal bovine serum (FBS; Gibco by
Life Technologies, Carlsbad, CA), 1% pen/strep (Gibco),
and 0.01 mg/mL insulin (Gibco). Non-tumorignenic
MCF-10A cells were grown in serum-free Mammary
Epithelial Growth Medium (MEGM; Lonza Group Ltd.,
Basel, Switzerland) supplemented with 100 ng/mL chol-
era toxin. Culture flasks, plates and dishes were incu-
bated in a humidified, 37˚C incubator. For subculturing,
growth medium was removed by vacuum and the cell
monolayer was rinsed with DPBS (Gibco). The mono-
layer was detached by addition of trypsin/EDTA (ATCC)
followed by incubation for 5 minutes. For MCF-7 cells,
complete growth medium was added to neutralize the
trypsin, and the cell suspension was centrifuged at 1500
rpm. In the culture of MCF-10A cells, to neutralize tryp-
sin soybean trypsin inhibitor (0.5 mg/mL; Gibco) was
added in volume equal to that of the trypsin solution.
Cells were resuspended in complete growth medium and
split at 1:3 - 1:6.
2.2. Fura-2 Fluorescence
Intracellular calcium concentrations were estimated with
the calcium-binding fluorophore fura-2 AM (Molecular
Probes by Life Technologies). Cells were plated on
poly-D-lysine-coated glass coverslips and cultured over-
night. The blank measurement solution contained, in mM:
130 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 15 HEPES; solution
pH was adjusted to 7.4 with NaOH. Cells were loaded
with the fura-2 AM loading solution, consisting of meas-
urement solution plus 3 - 5 μM fura-2 AM (stock in
DMSO; 3 - 5 μL stock per mL of loading solution) and
an identical volume of a 10% pluronic acid solution
(Sigma-Aldrich), in a humidified 37˚C incubator for 1 h.
Cells were then washed thrice with measurement solution,
then incubated in measurement solution for 30 minutes to
allow for fura-2 AM deesterification. Coverslips bearing
fura-2 AM loaded cells were mounted in an Attofluor
cell chamber (Molecular Probes) for analysis. Excitation
wavelengths were 340 nm and 380 nm, and emission was
measured at 510 nm. The emission intensities measured
for 340 nm and 380 nm were used to calculate an emis-
sion intensity ratio, which can be converted to free cal-
cium concentration. Fluorescence measurements were
taken with an inverted microscope in conjunction with a
175 watt xenon arc lamp and Metafluor imaging system
(Nikon Instruments, Melville, NY). The fluorescent emis-
sion signal was captured by an ADC 20 MHz Photo-
metric Coolsnap FX monochrome camera (Nikon). Data
were analyzed with Metafluor software (Molecular De-
vices, LLC, Sunnyvale, CA).
To measure basal calcium, MCF-7 or MCF-10A cells
were subjected to ratiometric fluorescent analysis with
blank measurement solution in the cell chamber. To test
the effects of inhibiting T-type calcium channel window
current on the basal calcium concentration in MCF-7
cells, measurement solution with 3 mΜ mibefradil (Sig-
ma-Aldrich) was perfused in the cell chamber. Blank
measurement solution was perfused through the chamber
to serve as a perfusion control. To mitigate calcium buff-
ering from intracellular stores in the mibefradil experi-
ments, measurement solution with 1 μM thapsigargin (an
irreversible inhibitor of the sarcoplasmic/endoplasmic
reticulum calcium ATPase; Sigma-Aldrich) was perfused
through the chambde hydrostatic pressure driving force.
Solution flow rates were approximer prior to addition of
mibefradil. Solution perfusion was controlled by a War-
ner Instruments valve controller (Hamden, CT), with
solution reservoirs elevated to proviately 5 mL/min. Sin-
gle-cell epifluorescence measurement images were cap-
tured throughout the experiments. Exposure times were
initially adjusted to maximize usage of the camera’s dy-
namic range, but kept constant throughout a given ex-
periment.
2.3. Proliferation Assay
To assess the effects of a particular agent on the prolif-

Exploiting MCF-7 Cells’ Calcium Dependence with Interlaced Therapy 34
eration/survival of MCF-7 cells in vitro, proliferation
assays were performed. Cells were plated in 96 well
plates and cultured overnight in a humidified, 37˚C in-
cubator. The next day, experimental medium was pre-
pared by mixing drug stocks into complete growth me-
dium to achieve the desired concentration, with stock
solvent added to make vehicle concentrations equal
across all concentrations of the agent of interest. EGTA
was dissolved in distilled water to 10 mM for working
stock, then diluted in complete medium to concentrations
of (in μM): 1.0, 1.5, 2.0, and 3.0. Thapsigargin was dis-
solved in DMSO to 1 mM for working stock and diluted
in complete medium to experimental concentrations (in
nM): 10, 30, 100, 300, and 1000. Mibefradil and NNC-
55-0396 were dissolved at 30 mM in distilled water, then
diluted to 3 mM for working stock; experimental con-
centration was 10 μM in complete medium. Paclitaxel
was dissolved in DMSO to 1 mM, and diluted in DMSO
to 100 μM working stock; experimental concentration
was 10 nM. For vehicle control medium, only stock sol-
vent was added to complete growth medium. The culture
medium was removed from plate wells and 0.1 mL of the
appropriate experimental medium was added to each well
for the appropriate experimental time period. Cell num-
ber was WST-8 assay kit (Cell Counting Kit-8; Dojindo
Molecular Technologies, Kumamoto, Japan) according to
the manufacturer’s instructions. Absorbance at 540 nm
was measured with a Multiskan Multisoft multi-well rea-
der.
2.4. RNA Interference
T-type calcium channel expression levels were knocked
down with short interfering RNA (siRNAs) designed to
target both the α1G and α1H isoforms of T-type calcium
channels, as described earlier [7]. The sequence targeted
by the siRNAs used were: scrambled control
5’-UAGUGAAGGGAGUCGGAUCUC-3’, T-type
5’-GCCAUCUUCCAGGUCAUCACA-3’. MCF-7 cells
were plated in 96 well plates and cultured overnight in a
humidified 37˚C incubator. Cells were transfected with
100 nM siRNA, aided by Lipofectamine 2000 (Invitro-
gen) according to the manufacturer’s instructions. Re-
duced mRNA levels were verified with quantitative RT-
PCR (data not shown).
2.5. Flow Cytometry
To assess the effects of inhibiting T-type calcium chan-
nels on passage of MCF-7 cells through the phases of the
cell cycle, flow cytometry experiments were performed.
MCF-7 cells were plated in culture dishes and cultured
overnight. Experimental treatments were applied the fol-
lowing day and maintained for the appropriate lengths of
time. At the end of the experimental period, cells were
collected and suspended in 5 mL PBS, then centrifuged.
After resuspension in 0.5 mL PBS, the cells were added
to 4.5 mL 70% ethanol for fixing overnight. The next day,
the fixed cells were centrifuged and the ethanol solution
decanted. The cell pellet was resuspended in 10 mL PBS
with 0.1% v/v Triton X-100 (Sigma-Aldrich), 2 mg
DNAse-free RNAse (Sigma-Aldrich) and 200 μL of 1
mg/mL propidium iodide (PI), and the suspension was
incubated at room temperature for 30 min. A BD LSR II
(BD Biosciences, San Jose, CA) flow cytometer was
used to measure cell fluorescence in flow cytometry,
with excitiation with blue light and detection of PI
emission with red light. The DNA content frequency
histogram was analyzed for cell-cycle progression us-
ing ModFit LT v3.0 software (Verity Software House,
Maine).
2.6. In Vivo Xenografts
Ovariectomized 25 - 35 days old nu/nu athymic female
mice (Charles River Laboratories, Wilmington, MA)
were obtained and allowed to acclimate to the vivarium
environment for 1 week. Mice then had slow-release
17-beta-estradiol pellets (0.72 mg/60 days relase) im-
planted subcutaneously with a trochar. MCF-7 cells were
cultured in standard conditions in 225 cm2 cell culture
flasks, harvested via trypsinization, and 5 * 106 cells (in
80 μL PBS) were mixed with cold Matrigel and kept on
ice to maintain the fluidity of the gel. Aliquots of the
Matrigel-cell suspension (80 μL, BD Biosciences) were
implanted in the abdominal mammary fat pads of the
mice with surgical needle-syringes to form tumor xeno-
grafts. All implantations followed anesthetization with
ketamine/xylazine (0.88 ml/kg, intraperitoneal). After 5
days for tumor establishment, mice were randomized into
four treatment groups: control (estradiol pellets only),
mibefradil only, paclitaxel only, and alternating mibe-
fradil/paclitaxel. Mibefradil (Sigma-Aldrich) was sus-
pended in distilled water (6 mg/mL) and administered by
oral gavage at 30 mg/kg for 3 days every week. Pacli-
taxel (Sigma-Aldrich) stock, 32 mg/mL, was made in
ethanol/Cremphor EL (polyethoxylated castor oil, glyc-
erol polyethelene glycol ricinoleate) solution. For ad-
ministration, paclitaxel stock was diluted in PBS to 2.4
mg/mL and administered via IP injection once per week
(12 mg/kg in 0.1 mL). The control group received no
treatment, the mibefradil group received mibefradil
treatment via oral gavage only, the paclitaxel group re-
ceived paclitaxel treatment via IP injection only, and the
alternating group received both treatments.
Tumor dimensions were monitored with digital cali-
pers thrice weekly, with volumes calculated by the for-
mula Volume = 0.5 * width2 * length. Mice body weights
were also measured thrice weekly, as a general measure
of animal health. At completion of the experiment (35
days), the mice were sacrificed and tumors, hearts, livers,

Exploiting MCF-7 Cells’ Calcium Dependence with Interlaced Therapy 35
kidneys, spleens, and bladders were weighed, and then
snap frozen in liquid nitrogen and stored at −80˚C. All
protocols involving animals were approved by the Tulane
University Institutional Animal Care and Use Commit-
tee.
3. Results
3.1. Basal Calcium
The comprehensive details of breast cancer cell calcium
signaling remain elusive. As calcium is one of the most
ubiquitous and promiscuous messengers in biology, a
greater understanding of the fine temporal and spatial
balance of calcium signaling stands to shed light on
many aspects of breast cancer cell proliferation. To this
end, basal intracellular calcium concentration was esti-
mated in the tumorigenic MCF-7 breast cancer cell line
and the non-tumorigenic MCF-10A cell line. MCF-7
cells were found to have a significantly higher (p < 0.05,
t-test) average basal intracellular calcium concentration,
102.3 ± 5.0 nM (mean ± SE, data from 100 s in 17 cells),
than that found in MCF-10A cells, 84.6 ± 5.9 nM (data
from 100 s in 19 cells; Figure 1).
3.2. Extracellular Calcium Concentration and
Intracellular Calcium Stores
Much of calcium signaling is accomplished through an
elevation in cytosolic calcium concentration via flow of
calcium ions into the cytosol from a comparatively cal-
cium-rich environment. These calcium sources include
the extracellular fluid and intracellular stores contained
Figure 1. Basal calcium concentration in MCF-7 and MCF-
10A cells. The tumorigenic MCF-7 cell line was found to
have significantly higher basal intracellular calcium con-
centration (102.3 ± 5.0 nM, data from 17 cells) than that
found in the non-tumorigenic MCF-10A cell line (84.6 ± 5.9
nM, data from 19 cells). Data are mean ± SE. *p < 0.05,
t-test.
within organelles such as the endoplasmic reticulum.
Manipulation of these sources can reveal their impor-
tance to breast cancer cell proliferation. Lowering growth
medium calcium concentrations to 34 μM and below
caused a significant reduction in MCF-7 cell number
compared to control for all time points (p < 0.05,
ANOVA followed by Bonferroni comparison; see Fig-
ure 2). When extracellular calcium was 500 μM or above,
there was no difference between experimental groups and
controls.
Depleting ER calcium stores by inhibiting the SERCA
pump with 1 μM thapsigargin significantly inhibited
MCF-7 cell proliferation (p < 0.05, ANOVA followed by
Bonferroni comparison; see Figure 3). At 48 h, signifi-
cant growth inhibition was observed in cells treated with
30 nM, 100 nM, 300 nM and 1 μM. At 72 h, all thapsi-
gargin concentrations significantly reduced proliferation
in MCF-7 cells. By contrast, loading vehicle control
(DMSO) did not inhibit MCF-7 cell growth at any time
point measured.
3.3. T-Type Calcium Channel Effect on Average
Intracellular Calcium Concentration
Knowing that MCF-7 cells maintain higher basal calcium
concentration than their non-tumorigenic counterpart
MCF-10A and that MCF-7 cells express T-type calcium
channels [7], the effects of blocking T-type calcium
channels on MCF-7 intracellular calcium concentration
was examined. After thapsigargin treatment to remove
ER-derived calcium buffering and emission intensity
stabilization, MCF-7 cells were treated with 3 μM mibe-
fradil. Emission intensity values over 100 s pooled from
17 cells were analyzed in both baseline and experimental
(treated with mibefradil or blank solution) conditions.
The MCF-7 cells were observed to have a small but sta-
tistically significant decrease in their intracellular cal-
cium concentration within 5 min of mibefradil applica-
tion, while cells given control measurement solution saw
no decrease in intracellular calcium concentration in the
same time period after application (see Figure 4). These
results indicate that pharamacological blockade of T-type
calcium channels can significantly affect the overall in-
tracellular calcium concentration in MCF-7 cells.
3.4. Cell Cycle
Passage through the cell cycle is known to be dependent
on calcium [15], e.g. the dependence of passing the G1/S
checkpoint on high intracellular calcium concentration.
Given that T-type calcium channels influence calcium
concentration (described above) and proliferation of
MCF-7 cells [7], cell cycle analysis was performed to
clarify the effects of inhibition of T-type calcium chan-
nels on MCF-7 cells’ passage through the cell cycle.

Exploiting MCF-7 Cells’ Calcium Dependence with Interlaced Therapy 36
Figure 2. Effects of EGTA on MCF-7 cell proliferation. EGTA, a calcium chelator, has a significant inhibitory effect on
MCF-7 cell proliferation compared to control at concentrations equal to or greater than 2 mM (corresponds to free calcium
concentration of 34 μM or lower), p < 0.05 for 2 mM and 3 mM EGTA. A higher concentration of EGTA in the growth me-
dium results in a lower extracellular calcium concentration. Data normalized to that of cells in untreated control medium. n =
6. Data analyzed with ANOVA for overall difference, then columns compared to medium control with Dunnett’s multiple
comparison test.
Figure 3. Effects of thapsigargin on MCF-7 cell proliferation. Thapsigargin, an inhibitor of the sarcoplasmic/endoplasmic
calcium ATPase, exerts time-dependent and dose-dependent inhibitory effect on MCF-7 cell proliferation. Data normalized to
that of cells in untreated control medium. n = 6. Data analyzed with ANOVA for overall difference, then columns compared
to medium control with Dunnett’s multiple comparison test.

Exploiting MCF-7 Cells’ Calcium Dependence with Interlaced Therapy 37
Figure 4. MCF-7 intracellular calcium concentration in
response to treatment with mibefradil. Fura-2 fluorescence
shows that blockade of T-type calcium channels with 3 μM
mibefradil results in a significant decrease compared to
basal level in MCF-7 intracellular calcium concentration
within 120 s of mibefradil application. The intracellular
calcium concentration of MCF-7 cells treated with control
solution (Blank) maintained basal level with no significant
change. Data from 100 s for each condition, pooled from 17
cells. Data analyzed with t-test, *p < 0.05 vs. baseline.
MCF-7 cells were treated with siRNA targeted against
T-type calcium channel mRNA (siRNA-2, see above) or
scrambled siRNA, and then subjected to flow cytome-
tryto assess their DNA content and thus infer their cell
cycle stage. Treatment with siRNA targeted against T-
type calcium channel mRNA resulted in a large increase
in the percentage of MCF-7 cells in G1 phase and a
smaller increase in cells in G2, along with a decrease in
cells in S phase, compared to cells treated with scram-
bled siRNA (Figure 5 and Table 1). This indicates that
functional inhibition of T-type calcium channels reduces
proliferation largely through halting cells in the G1 phase,
preventing them from passing through the checkpoint
into S phase.
3.5. Interlaced: In Vitro
Since T-type calcium channels play a functional role in
MCF-7 cell growth [7], T-type calcium channels may be a
viable target in breast cancer treatment. By virtue of their
role in the cell cycle, blocking T-type calcium channels
may serve as a means of reducing tumor cell growth in
between applications of cytotoxic chemotherapy. To as-
sess this possibility, MCF-7 cells were cultured in
96-well plates and subjected to a scheme that interlaced 3
μM NNC-55-0396 with 10 nM paclitaxel, each drug ap-
plied for 24 h. While treatment with NNC-55-0396 ini-
tially was not as effective as treatment with paclitaxel, by
the end of the experiment at 72 h, the cells receiving in-
terlaced NNC-55-0396 and paclitaxel were significantly
reduced in number compared to cells receiving either
Figure 5. Flow cytometry of MCF-7 cells treated with siRNA
directed against T-type calcium channel mRNA. RNA in-
terference directed against T-type calcium channel mRNA
causes a marked decrease in the proportion of MCF-7 cells
in S-phase, with most cells shifted into G1, compared to
cells treated with scrambled siRNA.
Table 1. Cell cycle analysis of MCF-7 cells treated with
siRNA-T.
Total G1 % Total S % Total G2 %
siRNA-T 70.33 11.85 17.82
Scrambled 61.07 25.13 13.80
Cell cycle analysis of MCF-7 cells treated with siRNA targeting T-type
calcium channel mRNA (siRNA-T) compared with cells treated with control
(scrambled) siRNA.
treatment alone. These results can be seen in Figure 6.
Of note is the overall slope of the interlaced treatment
group plot, which is steeper than that of either single-
agent treatment group, especially from 24 h to 48 h.
These results indicate that interlacing the chemothera-
peutic drug paclitaxel with T-type calcium channel
blocker NNC-55-0396 is effective at impeding the growth
of MCF-7 breast cancer cells in a manner that may be
synergistic.
3.6. Interlaced: In Vivo
The success of the interlaced treatment scheme in vitro
justified a similar approach in an in vivo model. Athy-
micovarectemized female nude mice were implanted

Exploiting MCF-7 Cells’ Calcium Dependence with Interlaced Therapy 38
Figure 6. WST-8 assay of MCF-7 cells treated with NNC-
55-0396, paclitaxel, or both in alternation. MCF-7 cells
treated in vitro with an alternating schedule of NNC-55-
0396, paclitaxel, and NNC-55-0396 (24 h each) were signifi-
cantly inhibited in growth, compared to cells treated with
either NNC-55-0396 alone or paclitaxel alone for 72 h. Data
normalized to that of untreated control cells at the same
time point. ANOVA with Bonferonni multiple comparison
post-tests, #p < 0.05 vs. NNC-55-0396, *p < 0.05 vs. pacli-
taxel.
with subcutaneous slow-release estradiol pellets, fol-
lowed by being implanted with MCF-7 cell xenografts in
the mammary fat pads. After tumors were established,
animals were randomly segregated into groups and treat-
ed with mibefradil, paclitaxel, or interlaced mibefradil/
paclitaxel in a weekly treatment regimen. Mibefradil was
administered orally for 3 consecutive days in a given
week; paclitaxel was administered via intraperitoneal
injection once in a given week. Single-agent treatment
groups received one of these; the interlaced group re-
ceived both. The tumors started out very similar in size.
Over the course of the 35 days of the experiment, the
mice given interlaced therapy were the only animals
whose tumors became significantly smaller than those in
mice that received no treatment. The plot of tumor size
vs. time can be seen in Figure 7. While the control and
single-agent treatment groups’ tumors are steadily grow-
ing, the interlaced treatment groups’ tumors are held in
check. For days 30, 32 and 35 the interlaced group’s tu-
mors are significantly smaller than those of the control
group. Figure 8 shows the close correlation of column
plots of final tumor volume (Day 35) and tumor weight
as determined immediately following animal sacrifice
and tumor excision.
4. Discussion
This study demonstrates the importance of T-type cal-
cium channels in breast cancer cell calcium signaling and
growth. Both extracellular calcium and intracellular cal-
cium stores are crucial for MCF-7 cell growth. T-type
calcium channels play a significant role in the manage-
ment of MCF-7 intracellular calcium concentration. In-
hibition of T-type calcium channels prevents these breast
cancer cells from passing through the phases of the cell
Figure 7. In vivo MCF-7 xenograft growth inhibition by
interlaced mibefradil and paclitaxel. MCF-7 tumor xeno-
grafts in nude mice experienced significant growth inhibi-
tion from a treatment schedule that alternated mibefradil
and paclitaxel when compared to control or either mibe-
fradil alone or paclitaxel alone.
Figure 8. Final tumor volume and weight comparison.
MCF-7 tumors were removed from mice after completion of
the in vivo interlaced treatment study and weighed. The
tumor weight data closely resembles the final tumor volume
measurement data.
cycle. This dependence makes T-type calcium channels a
potential target in breast cancer therapy, through the ef-
fects of combining T-type calcium channel blockers with
chemotherapy.
The importance of calcium to MCF-7 cell proliferation
is demonstrated here. MCF-7 breast cancer cells maintain
a basal average intracellular calcium concentration high-
er than that maintained in non-tumorigenic MCF-10A
cells, perhaps allowing for enhanced calcium signaling.
Reduction in calcium available for signaling, through
either chelating extracellular calcium or depleting intra-
cellular stores, significantly reduces the growth of MCF-

Exploiting MCF-7 Cells’ Calcium Dependence with Interlaced Therapy 39
7 cells. A cell expressing T-type calcium channels has an
extra pathway for calcium ion entry into the cytosol from
the extracellular fluid, and so may be expected to have an
elevated intracellular calcium concentration at rest due to
the properties of these channels. T-type calcium channels
have a low threshold for activation, which would allow
for channel conductance at transmembrane voltages near
normal resting potential [16]. Blocking T-type calcium
channels with mibefradil reduces MCF-7 intracellular
calcium concentration, confirming the role that these
channels play in calcium concentration maintenance.
Cancer cells have been reported to be relatively insensi-
tive to reductions in extracellular calcium concentration
[17]. The results reported here indicate that extracellular
calcium concentration can affect MCF-7 cell prolifera-
tion, but only when reduced by nearly two orders of
magnitude. Calcium signaling may be altered in MCF-7
cells as demonstrated by elevated basal concentration and
aberrant channel expression, but it certainly is still nec-
essary. Depletion of endoplasmic reticulum calcium
stores by thapsigargin is known to exacerbate the endo-
plasmic reticulum stress response in MCF-7 cells [18],
and prolonged exposure to thapsigargin is known to in-
duce chromosomal fragmentation in MCF-7 cells [19].
Activating the endoplasmic reticulum stress response via
thapsigargin mediated endoplasmic reticulum calcium
store depletion may help explain the time- and dose-de-
pendent reduction in MCF-7 cell growth seen in this
study. Perhaps T-type calcium channels serve as an aux-
iliary conduit in MCF-7 cells for calcium ions to be
moved from the extracellular fluid through the cytosol
and into intracellular stores, to serve as insurance against
induction of endoplasmic reticulum stress responses.
The inhibitory effects of T-type calcium channel bloc-
kade on the proliferation of MCF-7 cells has been dem-
onstrated previously [7]. The reduction in intracellular
calcium concentration, an immediate consequence of T-
type calcium channel blockers, is demonstrated here.
Mibefradil significantly reduces MCF-7 cell average
intracellular calcium concentration. However, exactly
how that change in free calcium ions results in a growth
reduction is unclear. Mibefradil is known to upregulate
p21 expression in esophageal cancer cells, a finding con-
firmed with RNA interference [8]. Presumably, a similar
“end result” of growth regulating signaling initiated by
reduced T-type calcium channel activity may be detect-
able in MCF-7 cells, but there remain many unknown
steps between T-type calcium current and cell cycle
regulation. Further investigation of the mechanisms in-
volved in these phenomena is needed.
Application of NNC-55-0396 in conjunction with pa-
clitaxel in an interlaced treatment scheme is more effec-
tive at reducing MCF-7 cell number than either NNC-
55-0396 or paclitaxel alone. These results can be viewed
in light of our other findings. By halting cells at the G1/S
checkpoint, the first 24 h of NNC-55-0396 application
lowers MCF-7 cell number relative to control and re-
duces the population’s cell cycle heterogeneity. This re-
duction in heterogeneity makes the cells more susceptible
to paclitaxel, which kills them as they pass through the
remainder of the cell cycle. This illustrates the potential
of T-type calcium channels as a target in cancer therapy.
Our in vitro results led us to apply interlaced therapy
in an in vivo model. MCF-7 tumor xenografts were es-
tablished in nude athymic female mice and tumor size
was monitored via caliper measurements. While all tu-
mors initially are of very similar size, separation is seen
at the termination of the experiment. In the last week, the
tumors of the mice receiving interlaced therapy are sig-
nificantly smaller than tumors in control mice to a statis-
tically significant degree; the sizes of tumors in animals
receiving either mibefradil alone or paclitaxel alone were
not found to be significantly different from those of con-
trol animal tumors. That the interlaced treatment suc-
ceeded in vivo to a degree larger than traditional chemo-
therapy alone is in accord with our in vitro results and
our hypothesis. Mibefradil is an effective T-type calcium
channel blocker but it also blocks L-type calcium chan-
nels via an intracellular metabolite [20]. While this is not
a problem in vitro, as MCF-7 cells do not express L-type
calcium channels, this is a matter of concern in terms of
organismal physiology. L-type calcium channels are cri-
tical in the function of tissues such as cardiac muscle and
vascular smooth muscle. We did not observe any signifi-
cant effects on animal body weight, pulse or blood pres-
sure as a result of mibefradil administration (data not
shown), but side effects are a relevant concern. Further
investigation of highly specific channel blockers such as
NNC-55-0396 is important to realizing the potential of
targeting T-type calcium channels in clinical cancer the-
rapy.
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