ORIGINAL ARTICLE

Taxol Directly Induces EndoplasmicReticulum-Associated Calcium Changes That PromoteApoptosis in Breast Cancer Cells

Zhi Pan, PhD and Lauren Gollahon, PhD

Department of Biological Sciences, Texas Tech University, Lubbock, Texas

n Abstract: Calcium, a key regulator of cell survival, is also important in regulating apoptosis. Although the chemothera-peutic agent Taxol employs apoptosis to induce cell death, the exact mechanism of how it induces apoptosis and the roleof calcium in this process remains unclear. The main intracellular calcium storehouse, the endoplasmic reticulum, was iden-tified as a new important gateway in apoptosis, possibly providing a target for Taxol. The goal of this study was to investi-gate whether calcium changes associated with the endoplasmic reticulum, were directly or indirectly generated by Taxol atclinically relevant doses, and related to Taxol-induced apoptosis in breast cancer cells. Time-lapsed imaging techniquesfollowed by an endoplasmic reticulum-targeted construct, cameleon D1ER, were used to monitor cytosol––endoplasmicreticulum calcium dynamics in MDA-MB-468 (Bcl-2 negative) and MCF 7 (Bcl-2 positive) breast carcinoma cells. Apoptosislevels were measured with Annexin V and Propidium Iodide (PI) using flow cytometry. In both cell lines, Taxol at 2.5 lM(�10)6 M) was observed to induce significant internal calcium changes, first a rapid endoplasmic reticulum calcium releaseand a transient cytosolic calcium increase upon Taxol addition. After several hours of Taxol treatment, the endoplasmicreticulum calcium store was gradually depleted, and a sustained cytosolic calcium elevation was observed before significantinduction of apoptosis. Inhibition of these calcium changes decreased Taxol-induced apoptosis levels. In contrast, 0.2 lMTaxol (�10)7 M) induced only a slight cellular calcium change, not enough to regulate apoptosis. Our findings demonstratethat endoplasmic reticulum calcium stores provide a direct target for Taxol action and are important for induction of apopto-sis, independent of Bcl-2 status. Furthermore, our results show for the first time, that the role of calcium in Taxol-inducedendoplasmic reticulum-mediated apoptosis is dependent on Taxol dosage. n

Key Words: apoptosis, breast cancer, calcium (Ca2+), endoplasmic reticulum, Taxol

The main mechanism for most chemotherapeutic

agent-induced cell death is apoptosis, which is a

highly regulated, multi-pathway process (1–3). Since

the exact mechanism of apoptosis is dependent upon

both cell and inducer types, it needs to be studied in

those specific cell-inducer models (2–4). Taxol, an

important chemotherapeutic agent, is widely used in

breast cancer treatment. However, how Taxol induces

apoptosis in breast cancer cells remains unclear.

Although calcium (Ca2+) disturbances in regulating

apoptosis have been targeted by various inducers in

different cell types (5), the role of calcium in Taxol-

induced apoptosis of breast cancer cells is not widely

considered and as a result, is under-explored. There

are three possible reasons for this: (a) since Taxol was

initially found to be a microtubule stabilizer, previous

studies primarily focused on the relationship between

the microtubule and apoptosis (5–10). (b) The techno-

logical difficulty to measure dynamic calcium changes

in living cells (11,12) also limits our ability to study

the Taxol-Ca2+ relationship. (c) Calcium regulation in

nonexcitable cell type such as breast epithelium is

much more poorly elucidated than in excitable cell

types such as muscle and nervous cells (13–18). Given

its potency, Taxol may induce apoptosis through other

mechanisms in addition to stabilizing microtubules.

Calcium may act as an important cellular regulator

for Taxol-induced apoptosis since it has a dual role in

the fate of the cell. While stable calcium homeostasis

is necessary for normal cell survival, impaired calcium

homeostasis is toxic to cells and may induce cell death

(14–16). Normally, the cell must maintain a low cyto-

solic calcium level ([Ca2+]C�0.1 lM) due to calcium

cytotoxicity. Most internal calcium is stored in the

endoplasmic reticulum (ER) pool ([Ca2+]ER�0.1–

1 mM) (15–17). The high calcium content within the

Address correspondence and reprint requests to: Lauren Gollahon, PhD,

Department of Biological Sciences, Texas Tech University, Box 43131. Lub-

bock, TX 79409-3131, USA, or e-mail: lauren.gollahon@ttu.edu.

DOI: 10.1111/j.1524-4741.2010.00988.x

� 2010 Wiley Periodicals, Inc., 1075-122X/10The Breast Journal, Volume 17 Number 1, 2011 56–70

ER is maintained by the balance between passive

release through calcium channel inositol 1,4,5-tris-

phosphate receptor (IP3R) and active calcium pump-

ing by Sarco ⁄ Endoplasmic Reticulum Ca2+-ATPase

pumps, SERCA in epithelial cell type (13–18). In

regards to programmed cell death, the ER is important

in calcium-apoptosis regulation because its capability

to mobilize calcium determines the cell’s ability to

undergo apoptosis in response to specific inducers

(19–24). Therefore, the ER has been suggested as an

important upstream gateway in apoptosis. The higher

the basal ER calcium level, the more calcium may be

released from the ER through IP3R channel to the

cytosol, resulting in either sensitization of mitochon-

dria to apoptosis or activation of apoptosis-associated

enzymes (24,25).

Previous studies (26,27) showed that Taxol treat-

ment results in the production of intermediates such

as lipid ceramide through de novo pathways from the

lipid membranes and reactive oxygen species (ROS)

from the mitochondrial matrix after several hours. In

other studies, ceramide and ROS reportedly triggered

a massive calcium release from the ER, initiating a

downstream mitochondrial apoptotic pathway in a

strictly ER calcium-dependent manner (21,22,28).

Taken together these studies suggest that Taxol may

be engaged in the ER calcium pathway in an indirect

manner after long-term exposure. Another report also

showed that some ER calcium changes were observed

after long-term exposure Taxol treatment (29,30),

supporting the idea that Taxol may indirectly affect

the ER.

However, the Taxol-ER Ca2+-apoptosis relationship

in breast cancer cells was not tested or confirmed, and

calcium regulation in cancer derived from epithelial

cell origin is not understood. No studies address

whether clinically relevant doses (0.1–10 lM) of

Taxol can directly affect calcium homeostasis and thus

regulate apoptosis in breast cancer cells. The purpose

of this study was to elucidate the role of calcium in

regulating Taxol-induced apoptosis in breast cancer

cells by addressing four questions: (a) Whether Taxol

directly or indirectly affects calcium homeostasis, espe-

cially the ER calcium store. (b) Whether Taxol-

induced calcium changes are related to apoptosis. (c)

Whether Taxol application conditions such as dosage

and exposure time are critical for Taxol-induced cal-

cium changes and apoptosis. (d) Whether other factors

such as antiapoptotic protein Bcl-2 status affect the

Taxol-ER Ca2+ calcium-apoptosis relationship.

MATERIALS AND METHODS

Cell Culture and Reagents

For this study, the breast cancer cell lines MDA-

MB-468 and MCF 7 were analyzed. M468 cells are

negative for the endogenous antiapoptotic Bcl-2 pro-

tein (31). Thus, the effects of Bcl-2 on calcium homeo-

stasis are eliminated. In contrast, MCF 7 breast cancer

cells show strong Bcl-2 expression (32). In addition,

M468 (estrogen receptor negative) and MCF 7 (estro-

gen receptor positive) representative two major sub-

types of breast cancer based on their estrogen receptor

status and hormone dependence (33,34). Both cell

lines were obtained from the American Tissue Culture

Center (ATCC, Manassas, VA), and cultured in

Dulbecco’s Modified Eagle’s Medium (DMEM),

(Sigma, St. Louis, MO), supplemented with 10% calf

serum (Hyclone, Logan, UT), 2 mM l-glutamine

(Sigma) and 1% penicillin ⁄ streptomycin (Sigma) at

37�C in 5% CO2. After reaching 70% confluence in

T25 flasks, cells were harvested with 1X 0.05% Tryp-

sin- 0.53 mM EDTA (Invitrogen-Gibco, Grand Island,

NY) and counted with a hemocytometer (Hausser

Scientific, Horsham, PA). Cells were plated at 50K

into each well of 24-well plates (BD Falcon, Franklin

Lakes, NJ) for 36 hours before commencing treat-

ments.

Taxol Treatment

Since the actions of Taxol on the cells depend on

the dose administered and the greatest difference was

observed between 10)6 and 10)7 M (7,35,36), Taxol

doses tested in this study needed to be considered. In

this study, two specific clinically relevant Taxol doses,

2.5 lM (high dose) and 0.2 lM (low dose) were used.

These are the estimated plasma levels in patients after

being administered a common dosage 135 mg ⁄ m2

for 3 hours and 24 infusion schedules, respectively

(37–39). Although the in vivo conditions are much

more complicated, we tried to more closely simulate a

clinically relevant phenomenon in studying the Taxol-

calcium relationship in breast cancer cells by recapitu-

lating these Taxol concentrations. In this study, Taxol

treatment within 10 minutes was defined as ‘‘short-

term’’ to capture rapid calcium changes directly

induced by Taxol addition. Taxol treatment for

greater than 1 hour was defined as ‘‘long-term’’ since

it may involve indirect and stable cellular changes.

Taxol was dissolved in dimethyl sulfoxide (DMSO)

solution and then diluted to the desired concentration

Calcium Regulates Taxol-Induced ER-Associated Apoptosis • 57

with medium. DMSO concentrations were kept below

0.5% in all experiments. Both Taxol and DMSO were

purchased from Sigma. Bovine serum albumin (BSA)

was purchased from Fisher Biotech Chemical (Fair

Lawn, NJ). The cytosolic calcium chelator 1,2-bis (o-

aminophenoxy) ethane-N,N,N¢,N¢-tetraacetic acid

with acetoxymethyl esters (BAPTA-AM), the ER cal-

cium pump inhibitor Thapsigargin (TG), the calcium

calibrating agents Ionomycin and EDTA were all

obtained from Invitrogen (Carlsbad, CA). The ER cal-

cium channel inhibitor 2-APB was purchased from

EMD Biosciences (San Diego, CA).

Apoptosis Measurements

Changes in apoptosis levels of cultured cells, under

different treatments, were measured by double label-

ing using an Annexin V-Fluorescein isothiocyanate

(FITC, green) and Propidium Iodide (PI, red) Apopto-

sis Kit (Biovision, Mountain View, CA). After trypsi-

nization, the suspended cells were stained with

Annexin V-FITC and PI according to the manufac-

turer’s instructions. The Annexin V-FITC green signal

(visualized at 526 nm) localized to the cell membrane,

signifying early apoptosis. PI signal (observed at

620 nm) bound to nuclear material with the accompa-

nying green fluorescence, indicated a later apoptotic

event. If PI alone was observed, this indicated a cell

necrotic event. An Epics XL-MCL flow cytometer

(Beckman Coulter, Miami, FL) was used to count the

populations of living, early apoptotic, late apoptotic

and necrotic cells based on their fluorescent proper-

ties. The Annexin V-FITC signal and the PI signal

were counted through flow cytometer channels FL1

and FL3, respectively. Between 5,000 and 10,000

cells were analyzed per run. A two-parameter dot plot

was constructed to show the log of FL1 on the X-axis

versus the log of FL3 on the Y-axis. The quadrant

region, based on FL1 ⁄ FL3 signal distribution, divided

the plot into four parts to calculate the percentages of

healthy, early apoptotic, late apoptotic and necrotic

cells.

ER Calcium Measurements

Since calcium fluctuates among different cellular

compartments to maintain its intracellular homeosta-

sis, it is difficult to detect the calcium dynamics and

determine the sources of calcium changes. In this

study, a time-lapsed imaging technique was used to

monitor the calcium dynamics to detect calcium in the

endoplasmic reticular (ER) lumen of cultured breast

cancer cells. A novel ER-targeted cameleon (kind gift

of Dr. Roger Tsien, University of California San

Diego) was used to specifically detect ER calcium lev-

els and provide direct results (40,41). The cameleon

D1ER plasmid was transfected into the cells using

Lipofectamine (Invitrogen) according to the manufac-

turer’s instructions. M468 and MCF 7 cells were

imaged 48 hours after transfection using an Olympus

IX 71-based deconvolving fluorescence microscope

with a digital cooled charge-coupled (CCD) camera

(C4742-95-12ER, Hamatatsu Photonics). The analysis

software used was SimplePCI, (Compix Imaging Cor-

poration, Sewickley, PA). Dual-emission ratio imaging

for D1ER was performed with a D436 ⁄ 20x excitation

filter, DCLP455 dichroic mirror, and two emission

filters D480 ⁄ 40 m and D535 ⁄ 30 m (Chroma Techno-

logy, Rockingham, VT) controlled by a lambda 10-2

filter wheel (Sutter Instruments, Novato, CA). For

each D1ER expressed cell, the FRET (535) ⁄ CFP (480)

emission ratio was fixed arbitrarily as 1 at the start of

the acquisition. Calibration, background and crosstalk

were corrected according to the protocol developed by

Palmer and Tsien (42). Images were collected every

15 seconds for 5 minutes at room temperature (RT).

Cytosolic Calcium Measurements

To detect cytosolic calcium levels, the calcium-spe-

cific fluorescent dye Fluo4-AM (Molecular Probes,

Eugene, OR), was loaded into M468 and MCF 7 cells

using a modified procedure adapted from the manu-

facturer (Molecular Probes). Briefly, cells were incu-

bated for 30 minutes at 37�C with 4 lM Fluo4-AM in

Hank’s buffered salt solution (HBSS) (Invitrogen,

Grand island, NY) with 1.3 mM calcium and 0.5%

BSA. The 0.02% nonionic detergent Pluronic F-127

(Molecular Probes) was added to assist in solubilizing

the nonpolar AM ester of Fluo4 in the loading solu-

tion. Cells were then washed two times with fresh

HBSS and incubated in HBSS at RT prior to detection.

The Fluo4-AM green fluorescence, ex 488 nm ⁄ em

526 nm, was proportional to cytosolic calcium levels.

The changes of Fluo4 intensity over time in cell popu-

lations, in response to different agents, were moni-

tored using flow cytometry (FL1 channel). After

measuring the basal intensity level of the unstimulated

Fluo4 cells, Taxol or DMSO was added into the tube

and acquisition continued for 5–10 minutes. In order

to evaluate the calcium release from the ER into the

cytosol in Taxol-treated cells, the ER calcium pump

inhibitor Thapsigargin (TG) was added into the media

58 • pan and gollahon

and the changes were recorded using a similar time-

lapsed fluorescence imaging technique with the same

microscope system described in ER calcium measure-

ments. Fluo4 imaging was accomplished by using an

S484 ⁄ 15x excitation filter, a BP505 dichroic mirror,

and an emission filter HQ525 ⁄ 50 m (Chroma Tech-

nology). The time scan was started in order to moni-

tor resting levels of Fluo4 for 30 seconds. TG was

added just prior to the next capture and monitoring

was continued every 15 seconds for 5 minutes. Off-

line data analysis was used to measure mean green

intensity for 10–15 cells at each time point to generate

the intensity plot over time. All imaging was done at

20X at RT.

Immunoblotting

Total protein was extracted from the M468 and

MCF 7 cells using M-PER mammalian cell protein

extraction reagent (Pierce, Rockford, IL). Protein con-

centration was determined with the BCA Assay

(Pierce). Protein samples were denatured by heating at

95�C for 5 minutes and then analyzed by SDS-PAGE

gel. A prestained molecular weight marker (Bio-Rad,

Hercules, CA) was used to monitor protein gel migra-

tion during the run and the subsequent transfer. The

membrane was incubated with anti-IP3R antibody

(Rabbit anti-human polyclonal IgG) from Santa Cruz

Biotechnology (Santa Cruz, CA) at a dilution of 1:200

to detect ER calcium channel IP3R levels. Anti-IP3R

antibody was raised against amino acids 2402-2701

mapped at the C terminus of human IP3R. The result-

ing band is around 115 kD in size. The signal was

visualized with horseradish peroxidase (HRP) second-

ary antibody (goat anti-rabbit IgG) at a dilution of

1:5000 and was developed using Supersignal Ultra

Chemiluminescence Kit (Pierce). b-Tubulin expression

was used as an internal control (1:1000 dilution) for

equal protein loading. The developed films were

scanned and images were processed using Photoshop

software.

Statistical Analyses

Data from this study were generated from three

independent experiments. Apoptosis levels measured

were represented as mean ± SD (standard deviation)

in the figures. In order to show the calcium changes

over time clearly, calcium curves were represented as

mean values in the figures, but peak levels were shown

as mean ± SD in the additional figures for comparison.

Statistical analysis was performed with a two-sided

independent Student’s t-test to compare two means,

and one-way analysis of variance (ANOVA) to com-

pare more than two means of one variable. If one-way

ANOVA demonstrated unequal means, the Tukey’s

honestly significant difference (HSD) test was used to

find which mean was different by conducting multiple

pair-wise comparisons. In order to determine whether

an interaction between two independent variables

existed, the two-way ANOVA test performed. For all

analyses, differences with p < 0.05 were considered

statistically significant and indicated with *, p < 0.01

was indicated with **.

RESULTS

Taxol Induces Dose and Time-Dependent Apoptosis

in Breast Cancer Cells

For total apoptosis in both cell lines, results using

two-way ANOVA indicated that low (0.2 lM) and

high (2.5 lM) Taxol doses showed parallel time-

dependent tendencies (Fig. 1a,d) with no significant

interaction between each other. For apoptotic effects

within each dose, one-way ANOVA analysis showed

that both doses induced a significant increase in apop-

tosis from 12 hours on (p < 0.01, Fig. 1a,d). For

M468 cells, differences in apoptosis levels between the

two doses was significant at 12 hours (p < 0.01,

Fig. 1a) and 24 hours (p < 0.05, Fig. 1a). However,

there was no significant dose difference observed in

MCF 7 cells.

Although the results for total apoptosis were com-

pelling, they did not allow delineation of cell death as

a result of early apoptosis or late apoptotic events.

Therefore the results were separated into early

(Fig. 1b,e) and late (Fig. 1c,f) stage apoptosis. Early

apoptosis was demonstrated to be the major form of

cell death induced by Taxol after a 12 hour exposure

(Fig. 1b,e, p < 0.01). However after 48 hours, most

cell death was attributed to late apoptosis and necrosis

(Fig. 1c,f). There is a dose-dependent difference in

early apoptosis similar to total apoptosis in M468

cells: significant at 12 hours (p < 0.01, Fig. 1b) and

24 hours (p < 0.05, Fig. 1b). In contrast, MCF 7 does

not demonstrate the same dose-dependence (Fig. 1e).

As with the reports from previous studies (43,44), our

results showed that MCF 7 cells are more resistant to

Taxol treatment. After a 12 hours exposure, the per-

centage of early apoptotic cells increased only 2 fold

and 2.5 fold for low and high dose Taxol respectively,

Calcium Regulates Taxol-Induced ER-Associated Apoptosis • 59

compared to 3.8 fold and 5.2 fold for the same Taxol

treatment in M468 cells (p < 0.01, Fig. 1b,e).

As expected, control medium and 0.5% DMSO did

not show a significant change in the apoptosis level.

From these experimental data, Taxol 12 hours treat-

ment, which resulted in significant apoptosis in both

cells lines (p < 0.01), was chosen as the optimal time

for the subsequent calcium interfering agent applica-

tions. After 12 hours exposure, calcium changes could

not be differentiated as a cause or consequence of

apoptosis.

Taxol Induces Dose and Time-Dependent Cytosolic

Calcium Changes in Breast Cancer Cells

To test the hypothesis that Taxol-induced apoptosis

is regulated by calcium, the question of whether Taxol

had an effect on cellular calcium homeostasis was

investigated first. Changes in [Ca2+]C were determined

by Fluo4 calcium indicator intensity measurements.

Both short-term and long-term responses of Taxol

treatment were evaluated. As shown in the curves

(Fig. 2a,b), exposure to high dose Taxol treatment

caused a relatively rapid increase in Fluo4 fluores-

cence, indicating an elevation in [Ca2+]C. The increase

in M468 cells peaked to 1.5 fold (p < 0.01, Fig. 2a) at

�4 minutes and then decreased gradually, but it was

still above the basal levels after 10 minutes of expo-

sure. The increase in MCF 7 cells peaked to 1.4 fold

(p < 0.01, Fig. 2b) faster (�2 minutes) and then

decreased rapidly, but remained above the basal levels

after 10 minutes of exposure. In contrast, low dose

Taxol only induced a slight and slow elevation that

0

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Figure 1. Percentage of apoptotic breast cancer cells depends on Taxol dosage and exposure time. Apoptosis levels were analyzed by

flow cytometry using Annexin V-FITC and PI in M468 and MCF 7 breast cancer cells. Cells were treated in four groups: Control normal

medium (C), 0.5% DMSO medium (D), low dose (0.2 lM) Taxol containing medium (L), and high dose (2.5 lM) Taxol containing medium

(H) with 0.5% DMSO as the vehicle. Floating cells were collected with the medium and attached cells were collected using trypsin at

different time points (3, 6, 12, 24, 48, 72 hours). All cells in this solution were analyzed. Data are representative of mean ± SD of three

experiments at each time point. One-way ANOVA plus Tukey’s HSD test were used to evaluate significance and two-way ANOVA was

used to test the interaction between dose and time effects (**p < 0.01 and *p < 0.05). M468 cells––a: Total Apoptosis; b: Early Apoptosis;

c: Late Apoptosis. MCF 7 cells––d: Total Apoptosis; e: Early Apoptosis; f: Late Apoptosis.

60 • pan and gollahon

was not significant when compared to vehicle control.

The results indicate that Taxol had a direct transient

effect on [Ca2+]C that was dose-dependent. After this

direct transient change, long-term treatment with high

dose Taxol continued to induce a stable calcium

change. High dose Taxol showed a slow but sustained

effect on [Ca2+]C that was significant after 6 hours

exposure in both cell lines (M468 cells, p < 0.01,

Fig. 3a; MCF-7 cells, p < 0.05, Fig. 3c). However, low

dose Taxol did not induce such a significant change

(Fig. 3b,d) before 12 hours. In addition to the

observed the time-dependent changes, a significant dif-

ference in cytosolic calcium levels, induced by Taxol

was also observed between the two doses (p < 0.01).

Taken together, the effect of Taxol on cellular cal-

cium homeostasis in breast cancer cells is dependent

both on dose and exposure time. Taxol showed a

two-phase cytosolic calcium increase. A transient

response was observed after the addition of Taxol

followed by a much slower but sustained elevation

after long-term treatment. The results showed at high

dose, Taxol directly induced a significant cytosolic

calcium increase, before significant apoptosis occurred

(12 hours treatment). These results indicate that these

calcium changes may play a role in the induction of

apoptosis induced by Taxol. Whether the calcium

increase was derived from the endoplasmic reticulum

calcium store was addressed in the subsequent experi-

ments.

Taxol Induces Dose and Time-Dependent ER Calcium

Changes in Breast Cancer Cells

To evaluate the short-term and long-term ER cal-

cium release in response to Taxol treatment, a direct

D1ER calcium indicator and an indirect Thapsigargin

(TG) simulation method were used respectively. As

illustrated in Figure 4a,b, addition of high dose Taxol

immediately induced a significant decrease in ER free

calcium levels in both cell lines (p < 0.01), directly

detected by the D1ER cameleon. In contrast, low dose

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Figure 2. Cytosolic calcium changes were induced by short-term Taxol treatment. Time-response curve of Fluo4 fluorescence intensity

changes, which are proportional with the cytosolic calcium levels upon Taxol addition. The curve was drawn from the mean results gener-

ated in three independent tests, peak levels of calcium changes after treatment, from the curve, was represented as mean ± SD and statis-

tical analysis was performed. After Fluo4 loading and incubation, cells were washed and kept in Ca-HBSS, which contains the normal

extracellular calcium level (1.3 mM). The arrow shows the time point at which agents Taxol, DMSO or normal medium was added to the

cells M468 cells (a) and MCF 7 (b). Final concentrations of Taxol are 2.5 lM (H), or 0.2 lM (L); DMSO is 0.5% (D); normal medium is the

control (C). The basal Fluo4 fluorescence levels of all groups before treatment were similar and were expressed as 1.

Calcium Regulates Taxol-Induced ER-Associated Apoptosis • 61

Taxol only induced a slight decrease. No significant

changes were observed in control and 0.5% DMSO

vehicle control groups. This result showed Taxol

alone, especially at high dose, directly induced a sig-

nificant ER calcium release upon addition.

The ER calcium release after long-term Taxol treat-

ment (3, 6, and 12 hours) was analyzed using the TG

method (45,46) (see time-lapsed images in Fig. 5). As

shown in Figure 6a, after 3 hours high dose Taxol

exposure, M468 cells only showed a 55% increase in

[Ca2+]C in response to TG treatment, compared to the

control group (p < 0.05). Additionally, 6 hours and

12 hours exposure only showed a 35% and 30%

increase respectively (p < 0.01, Fig. 6a). Interestingly,

upon TG treatment, the basal ER calcium release in

MCF 7 cells is 35% higher than M468 cells, indicat-

ing that MCF cells has more releasable ER calcium at

the resting level (Fig. 6a,b, p < 0.01). The releasable

ER calcium level did not show a significant change in

MCF 7 cells until 6 hours (62.5% increase, p < 0.01),

and further at 12 hours (40% increase, p < 0.01).

These results indicated that the releasable calcium in

the ER was gradually decreased in both cell lines, but

MCF 7 ER calcium depletion occurred more gradually

and at a slower rate than M468 cells. Therefore, long-

term exposure to high dose Taxol induced a gradual

ER calcium depletion dependent on exposure time. In

contrast, the low dose Taxol did not induce a deple-

tion of ER calcium stores for the 3, 6, or 12 hours

exposure groups (Fig. 7). Since Taxol, especially at

high dose, demonstrated a direct effect on cytosolic

and ER calcium homeostasis, we investigated whether

these calcium changes were related to apoptosis using

calcium interfering agents.

Cytosolic and ER Calcium Interfering Agents Inhibit

Taxol-Induced Apoptosis

To evaluate whether Taxol-induced calcium

changes were related to this apoptotic event, both the

cytosolic calcium chelator BAPTA-AM and the ER

calcium release inhibitor 2-APB were applied. As

shown in Figure 8a,b, BAPTA-AM showed a 40%

Treatment

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(d)(c)

Treatment

Figure 3. Cytosolic calcium changes were induced by long-term Taxol treatment. Changes in free cytosolic calcium levels were deter-

mined by changes in Fluo4 fluorescence intensity. High dose Taxol (2.5 lM) treatment for 3, 6 and 12 hours in M468 cells (a) and MCF 7

cells (c). Low dose Taxol (0.2 lM) treatment for 3, 6 and 12 hours in M468 cells (b) and MCF 7 cells (d). The means ± SD were calculated

from three independent experiments and statistical analysis was performed. After Fluo4 loading and incubation, cells were washed and kept

in Ca-HBSS containing the normal extracellular calcium level (1.3 mM). The basal Fluo4 fluorescence levels of control groups before treat-

ment were expressed as 1.

62 • pan and gollahon

and 28% inhibition on high dose Taxol-induced total

apoptosis in M468 and MCF 7 cells, respectively

(p < 0.01). In contrast, low dose Taxol-induced apop-

tosis did not show significant inhibition. Furthermore,

the effect of BAPTA-AM was shown to negatively

impact Taxol-induced early apoptosis (p < 0.01), sup-

porting the role of cytosolic calcium in the induction

of apoptosis. To further determine whether calcium

release from the ER was required for the induction of

apoptosis by Taxol, the ER calcium release channel

(IP3R) inhibitor 2-APB was also tested. IP3R expres-

sion was confirmed in both M468 and MCF-7 cells

(Fig. 9). The results showed 2-APB pretreatment had a

similar inhibitory effect on high dose Taxol-induced

apoptosis in M468 and MCF-7 cells (Fig. 8c,d,

p < 0.01), whereas no significant inhibitory effect was

observed for low dose Taxol-induced apoptosis.

Therefore, interfering with cytosolic and ER calcium

changes caused a significant decrease of the Taxol-

induced apoptosis levels after 12 hours. These results

indicate that calcium promotes Taxol-induced apopto-

sis and the internal ER calcium store plays an impor-

tant role during this process.

DISCUSSION

High Dose Taxol (·10)6 M) had a Direct Effect on

Calcium Homeostasis Whereas Low Dose (·10)7 M)

did not, Indicating Taxol-Induced Calcium Change is

Dose-Dependent

In the introduction, the possible effect of Taxol on

calcium homeostasis was presented under the premise

that Taxol indirectly affects calcium through the pro-

duction of intermediates ROS or ceramide. However,

our results demonstrated definitively that Taxol

(·10)6 M) has a direct rapid effect on both cytosolic

and ER calcium levels in breast cancer cells. We

0.8

0.85

0.9

0.95

1

1.05

0 60 120 180 240 300 360 420

D1E

R F

RE

T r

atio

Time (s)

CDLH

0.8

0.85

0.9

0.95

1

1.05

C D L H

M468

**

(a)

Treatment

0.8

0.85

0.9

0.95

1

1.05

0 60 120 180 240 300 360 420

D1E

R F

RE

T r

atio

Time (s)

CDHL

0.8

0.85

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0.95

1

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C D L H**

(b)

Treatment

MCF-7

Figure 4. Endoplasmic reticulum calcium changes induced by short-term Taxol treatment directly measured using the D1ER cameleon.

Forty-eight hours after transient transfection with the D1ER vector, breast cancer cells were examined using FRET time-lapsed imaging to

monitor the changes of free ER calcium levels. These were represented by the FRET ratio (535 ⁄ 480 fluorescence emission) in D1ER ca-

meleon expressed cells. DMSO and Taxol were added to observe whether these agents could affect the ER calcium levels in M468 cells

(a) and MCF 7 cells (b). Time-response curve (mean) was determined from data generated in three independent tests. For the curve, the

lowest decreased ER calcium levels upon treatment were represented by the mean ± SD and statistical analysis were performed. Arrow

indicates the time point at which agents or normal medium were added into these groups. Final concentrations of Taxol were 2.5 lM (H) or

0.2 lM (L); DMSO is 0.5% (D); Normal medium is the control (C).

Calcium Regulates Taxol-Induced ER-Associated Apoptosis • 63

showed that addition of Taxol alone at a dose compa-

rable to clinical plasma levels (2.5 lM) induced a

rapid ER calcium release (Fig. 4) as well as a transient

increase in cytosolic calcium levels (Fig. 2) in both

M468 and MCF-7 breast cancer cell lines. After this

transient, rapid increase, a gradual ER calcium deple-

tion (Fig. 6) and sustained cytosolic calcium increase

(Fig. 3) occurred after long Taxol exposure. For both

short-term and long-term Taxol exposure, ER calcium

release occurred before the cytosolic calcium increases,

suggesting the ER provides a direct target for Taxol.

In contrast, the effects of Taxol at 0.2 lM

(·10)7 M) on calcium were much slower and less dra-

matic. Cytosolic calcium levels only slightly increased

in response to Taxol addition (Fig. 2). Nor was a sta-

ble cytosolic calcium increase observed until 12 hours

exposure to Taxol in M468 cells (Fig. 3b). By this

time, significant apoptosis had already occurred and

thus it became difficult to exclude whether calcium

release was a cause or an effect of apoptosis. In MCF

7 cells, low dose Taxol did not induce a significant

cytosolic calcium increase after long-term treatment.

The ER calcium responses were consistent with cyto-

solic calcium responses. Only a slow and slight

calcium release from the ER calcium store in M468

cells, and a transient calcium release in MCF 7 cells

were observed (Fig. 4a,b). Neither was significant. The

ER depletion did not occur during long-term exposure

of low dose Taxol treatment for both cell lines

(Fig. 7). Therefore, there is an obvious difference

between the dose effects of 10)6 M and 10)7 M Taxol

on calcium homeostasis with high dose directly and

rapidly changing calcium levels by triggering calcium

release from the ER, and low dose demonstrating a

nonsignificant change.

High Dose Taxol (·10)6 M) -Induced Calcium

Changes Promote Apoptosis, Whereas Low Dose

Taxol (·10)7 M) did not, Indicating an Induction of

Apoptosis Through Different Mechanisms

High dose (·10)6 M) Taxol induced significant ER

and cytosolic calcium changes in a biphasic manner. A

Figure 5. Time-lapsed images of Thapsigargin (TG)-induced Fluo4 changes in untreated M468 and MCF 7 cells. In order to evaluate the

calcium release from the ER into the cytosol in Taxol-treated cells, the ER calcium pump SERCA inhibitor Thapsigargin (TG) was added

into the media at the indicated ‘‘0 seconds’’ time point and the changes were recorded using a time-lapsed fluorescence imaging technique.

The Fluo4-AM fluorescence intensity changes were proportional to cytosolic calcium levels. Images were collected every 15 seconds for

5 minutes at room temperature (RT) at 20X. The ineffective SERCA pump failed to compensate for the ER passive release and resulted in

an increase in cytosolic calcium, visualized as an increase in signal intensity. Therefore, in the absence of extracellular calcium, upon TG

addition, all the observed cytosolic calcium increase originated from the ER store. This provided an indirect method to evaluate the ER cal-

cium release.

64 • pan and gollahon

rapid transient response was observed after its addi-

tion, followed by a slower but sustained change after

long-term treatment. All of these calcium changes

induced by high dose Taxol occurred before significant

apoptosis appeared (12 hours, Fig. 1). Since early

apoptosis was the major form of cell death at that

time, it is possible that calcium changes induced by

Taxol before 12 hours exposure may trigger or regu-

late induction of early apoptosis. The relationship

between the high dose Taxol-induced calcium changes

and consequent apoptosis was confirmed in the cal-

cium targeting agent experiments. Both cytosolic cal-

cium chelator BAPTA-AM (Fig. 8a) and 2-APB

(inhibitor of ER calcium channel release through the

IP3 receptor, Fig. 8b) significantly inhibits high dose

Taxol-induced apoptosis.

In contrast, low dose (·10)7 M) Taxol did not

induce a significant calcium change. In addition, cal-

cium interfering agents did not show significant inhibi-

tion to low dose Taxol-induced apoptosis. This

suggested that low dose Taxol-induced apoptosis is

not significantly regulated by calcium changes. How-

ever, at 12 hours exposure, low dose Taxol still

induced significant apoptosis, (Fig. 1). These results

suggest that high dose Taxol induces apoptosis

through an internal calcium-dependent mechanism,

whereas low dose Taxol induces apoptosis through an

internal calcium-independent mechanism.

Taxol was first identified as a potent inhibitor of cell

replication through dose-dependent stabilization of

microtubules. Low doses [0.01–0.2 lM] stop mitosis

and induce mitotic arrest-dependent apoptosis. High

doses [>1 lM] form a static microtubule environment,

effectively stopping daughter cell formation and induc-

ing mitotic arrest-independent apoptosis (7). This raises

the intriguing possibility that a relationship could exist

between Taxol-induced calcium changes and Taxol-

induced microtubule changes. High dose Taxol-

induced ER calcium release may provide an important

signal to bypass the mitotic arrest. Therefore, calcium

changes, mitotic arrest, and apoptosis may become

directly linked depending on Taxol dosage. Therefore,

Taxol action is much more complex than originally

expected and needs to be further elucidated.

0.8

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2

0 60 120 180 240 300

Time (s)

H3hH6hH12hC

1

1.2

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1.6

1.8

2

C H3h H6h H12h

TG

*** **

(a) M468

Peak level

Treatment

Flu

o4

inte

nsi

ty

chan

ges

Flu

o4

inte

nsi

ty

chan

ges

0.8

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H6h

H12h

C

Time (s)

1

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C H3h H6h H12h

****

Peak level

TG

Treatment

Flu

o4

inte

nsi

ty

chan

ges

MCF-7(b)

Flu

o4

inte

nsi

ty

chan

ges

Figure 6. Long-term effects of high dose Taxol (2.5 lM) on the ER calcium release measured using TG. Time-response curves (mean)

were determined from data generated in three independent tests for M468 cells (a) and MCF 7 cells (b). After exposure to Taxol for 3, 6

and 12 hours treatments, cells were loaded and incubated with Fluo 4. After incubation, the control and Taxol-treated cell samples were

washed and kept in HBSS without calcium to remove the effect of extracellular calcium influx. For the curve, peak levels of ER calcium

release upon TG treatment were represented by the mean ± SD, and statistical analysis was performed. Arrows show the addition of 3 lM

TG.

Calcium Regulates Taxol-Induced ER-Associated Apoptosis • 65

There are several possibilities as to how high dose

Taxol induces this rapid ER calcium release. First,

high dose Taxol may act as a modulator for ER

release channels such as IP3R. As reported in neuron

cell lines, Taxol may directly bind to neuronal calcium

sensor 1 (NCS-1) which interacts with IP3R, and thus

stimulates ER calcium release (47). However, direct

targets for epithelial cells are unknown and need to be

further investigated. Alternatively, Taxol may directly

damage the ER membrane, inducing a calcium flux

based on the calcium gradient. In response to ER cal-

cium release, the cell may increase calcium efflux to

the extracelluar medium in order to maintain normal

cytosolic calcium levels. Recall that the cytosolic cal-

cium levels did not return back to the baseline sug-

gesting that the ER calcium release continued upon

Taxol addition. This rapid calcium change may serve

as an upstream signal to sensitize mitochondria or

activate calcium-dependent caspase, thus regulating

the initiation of apoptosis. Furthermore, the expres-

sion of some apoptosis-related genes or protein

modifications may be regulated through activated

calcium-dependent enzymes, indirectly affecting apop-

tosis. For example, in breast cells, Bid was reported to

be cleaved by calcium-activated proteases Calpain into

tBid, which is an active pro-apoptotic protein (48). It

is interesting to note that Taxol causes activation of

calpain in neuron cell lines and inhibition of calpain

has a protective effect against Taxol-induced neuropa-

thy (49). Therefore, side effects in nervous tissue

induced by Taxol treatment can be also associated

with calcium regulation. High dose Taxol (·10)6 M)

may therefore be involved in more side effects due to

its significant effects on ER calcium regulation.

How does high dose Taxol induce the gradual ER

calcium depletion after long-term exposure? In addi-

tion to the direct repetitive stimulation of high dose

Taxol on the ER (47), other possible causes of the

time dependent ER calcium depletion may involve reg-

ulation of ER calcium release channels or other ER

calcium buffer proteins. Alternatively, intermediate

production of ROS and ceramide, which could be pro-

duced after several hours, may affect ER calcium

depletion (26,27). The ceramide and ROS could

0.8

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Time (s)

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Peak level(b)

TreatmentF

luo

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ten

sity

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ang

es

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Time (s)

L3hL6hL12hC

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TG

M468(a)

Peak level

Treatment

Flu

o4

inte

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ty

chan

ges

Flu

o4

inte

nsi

ty

chan

ges

MCF-7

Figure 7. Long-term effects of low dose Taxol (0.2 lM) on the ER calcium release measured using TG. Time-response curves (mean)

were determined in M468 cells (a) and MCF 7 cells (b) from data generated in three independent tests. After exposure to Taxol for 3, 6

and 12 hours treatments, cells were loaded and incubated with Fluo 4. After incubation, the control and Taxol-treated cell samples were

washed and kept in HBSS without calcium to remove the effect of extracellular calcium influx. For the curve, peak levels of ER calcium

release upon TG treatment were represented by mean ± SD, and statistical analyses were performed. Arrows show the addition of 3 lM

TG.

66 • pan and gollahon

induce the calcium transfer from the ER to the nearby

mitochondria and result in mitochondrial calcium

overload (21). Recent studies in lymphoma cell lines

showed that long-term Taxol (100 lM) exposure may

regulate some ER stress proteins and induce an ER

stress pathway (50), providing other possible effectors

for Taxol. Regardingless of the possible causes, the

key finding from our study is that Taxol, depending

on dosage, can directly induce ER calcium immediate

release and gradual depletion in breast cancer cells.

This is a direct ER stress response that signals the

induction of the ER apoptotic pathway.

Taxol Induces Calcium Changes in Both Bcl-2

Negative M468 Cells and Bcl-2 Positive MCF-7 Cells,

Indicating Taxol can Directly Attack ER Calcium

Stores Independent of Bcl-2 Status

B cell lymphoma 2 protein is an antiapoptotic pro-

tein, which is important for tumor resistance to che-

motherapy. Although its exact mechanism of action is

not fully understood, Bcl-2 regulation on calcium

homeostasis is suggested to play an important role

(51–53). Recent studies show that there is an interac-

tion between Bcl-2 and the ER calcium release channel

IP3R, which most likely inhibits the ER calcium

release and subsequent apoptosis (54,55). Another

(b)

0

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40

L12h LBA H12h HBA

*

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**

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ptos

is%

Apo

ptos

is%

0

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40

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ptos

is%

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ptos

is%

****

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(d)

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ptos

is%

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ptos

is%

(c) MCF-7

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ptos

is%

Apo

ptos

is%

****

ER Calcium Channel Inhibitor 2-APB

M468(a)

Cytosolic Calcium Chelator BAPTA-AM

MCF-7

M468

Figure 8. Calcium interfering agents inhibited Taxol-induced apoptosis. M468 cells (a) and MCF 7 cells (b) were pretreated with 5 lM of

the cytosolic calcium chelator BAPTA-AM for 1 hour before Taxol addition. The percentage of apoptosis was measured by flow cytometry

using Annexin V-FITC & PI as described in the Methods section. Results (mean ± SD) were determined from data generated in three inde-

pendent tests. L12h and LBA were the treatment groups of low dose Taxol (0.2 lM) for 12 hours and combined low dose Taxol + BAPTA-

AM test, respectively. H12h and HBA are the treatment groups of high dose Taxol (2.5 lM) and combined high dose Taxol + BAPTA-AM

test, respectively. Effects of the IP3R calcium channel inhibitor, 2-APB at 25 lM, were determined in M468 cells (c) and MCF 7 cells (d). 2-

APB was pretreated for 1 hour before Taxol addition. L12h and LP were the treatment groups of low dose Taxol (0.2 lM) for 12 hours and

combined low dose Taxol + 2-APB, respectively. H12h and HP were the treatment groups of high dose Taxol (2.5 lM) and combined high

dose Taxol + 2-APB, respectively.

Figure 9. Western results for IP3R protein expression levels in

M468 and MCF 7 cells. Cellular protein lysates of M468 and MCF

7 cells (25 lg) were separated on a 7.5% SDS-PAGE, blotted,

detected with an IP3R antibody at 1: 200 and then visualized with

goat anti-rabbit HRP at 1:5000. Anti-IP3R antibody was raised

against amino acids 2402-2701 mapped at the C terminus of

human IP3R, thus the resulting band is around 115 kD in size.

Tubulin (1:1000) was used as the internal control and visualized

under the same conditions.

Calcium Regulates Taxol-Induced ER-Associated Apoptosis • 67

recent study indicated that Taxol can directly interact

and attack Bcl-2 protein as it polymerizes tubulins

(56). Therefore, it is difficult to clarify whether the

calcium responses are directly induced by Taxol or by

a Taxol-Bcl-2 interaction in Bcl-2 positive cell lines.

To address this question, both the Bcl-2 negative cell

line MDA-MB-468 and the Bcl-2 positive cell line

MCF-7 were investigated in our study. We observed

similar dose-dependent Taxol-induced calcium changes.

High dose Taxol treatment induced ER calcium release

and then cytosolic calcium increase, which promoted

apoptosis, whereas low dose Taxol did not. Therefore,

this result is not limited to one cell line or breast

cancer type, but more likely indicates a general

phenomenon.

Although the patterns are similar, there are some

differences between Bcl-2 negative and positive cell

lines. First, our results showed that MCF-7 is more

resistant to Taxol treatment, which indicates Bcl-2

expression had an inhibitory effect on apoptosis.

Additionally, the releasable ER calcium levels were

much higher in MCF 7 cells at rest, which supports

Bcl-2 protein inhibits ER calcium release and conse-

quently increases the basal level of releasable ER cal-

cium. Upon Taxol treatment, the transient cytosolic

increase in MCF 7 cells is faster, consistent with the

faster ER calcium release observed. However, the

amplitude of the calcium changes is similar to M468

cells, indicating that Taxol make attack multiple tar-

gets associated with the ER, and its direct inhibition

on Bcl-2 action may accelerate its attack on the ER

calcium store. Although the transient phase is faster,

the ER calcium depletion and sustained cytosolic cal-

cium increase occurring in MCF 7 cells is slower and

smaller. Since no significant difference in expression

levels of IP3R was observed between M468 and

MCF-7 cells by Western blot (Fig. 9), this indicates

that the slower ER calcium depletion observed in

MCF-7 cells is most likely related to its higher releas-

able ER calcium levels, making the ER calcium store

more difficult to deplete. Thus, Taxol can directly

attack ER calcium independent of Bcl-2 status, the

Bcl-2 regulation of basal ER calcium release, and the

interaction of Taxol on Bcl-2 together, forming a

complex picture. The direct assault of Taxol of Bcl-2

may speed up the ER calcium release, but it does not

increase the amplitude. The final outcome is depen-

dent upon the competition between Bcl-2 and Taxol

for the ER calcium release, the mechanism of which

provides a new field need to be further investigated.

In summary, several interesting new observations

were revealed regarding the role of calcium in Taxol-

induced apoptosis in breast cancer cells. Within the

scope of this study, two major subtypes of breast can-

cer cell lines, hormone-dependent, Bcl-2 positive

MCF-7 and hormone-indpendent, Bcl-2 negative

MDA-MB-468, were investigated. The major finding

is that the addition of Taxol (10)6 M) can directly

induce a significant ER calcium release and a subse-

quent transient cytosolic calcium increase. Further-

more, ER calcium release induced by Taxol appeared

before the cytosolic calcium increase––after either

immediate addition or long-term exposure of Taxol

treatment, suggesting that the ER calcium store

provides Taxol a direct upstream target. These Taxol-

induced calcium changes significantly promoted

apoptosis, confirming calcium (especially ER calcium)

plays an important role in Taxol-induced apoptosis in

breast cancer cells. Interestingly, the same results were

not observed for low dose Taxol (10)7 M), although

low dose Taxol can also induce significant apoptosis.

Therefore, Taxol dosage is a key element in determin-

ing its involvement in the ER apoptotic pathway,

independent of Bcl-2 status. Bcl-2 acts more as a mod-

ulator on the ER calcium release and is not the only

target on the ER that Taxol can attack directly. These

findings provide new insights into the mechanism of

Taxol action and may aid in the development of more

effective breast cancer therapies. In addition, our

results indicate that calcium regulation of breast can-

cer cells is important for both tumor resistance and

side effects to chemotherapy. This information may

aid clinicians in devising more effective strategies for

the management of cancer treatment.

Acknowledgments

We thank Prof. R.Y. Tsien, University of California

San Diego for providing the D1ER cameleon plasmid.

We also thank the Imaging Center of Texas Tech

University for providing the necessary microscope

system and training for this research. Financial

support was provided by the Department of Biological

Sciences, Texas Tech University. This work was also

supported by research grants from the Texas Tech

University Association of Biologists.

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