Báo cáo khoa học: Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells ppt

Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug,

microtubules, activates the mitotic checkpoint and

induces apoptosis in MCF-7 cells

Mithu Banerjee, Parminder Singh and Dulal Panda

Department of Biosciences & Bioengineering, Indian Institute of Technology Bombay, Mumbai, India

Introduction

Curcumin, a natural product found in the rhizome of

Curcuma longa, is emerging as an important anticancer

agent on account of its manifold clinical applications

[1–5] Although the phase I clinical trial of curcumin

for the prevention of colon cancer has already been

completed (clinicaltrials.gov Identifier: NCT00027495),

clinical trials to determine its efficacy in the

treat-ment of rectal cancer (clinicaltrials.gov Identifier:

NCT00745134), advanced pancreatic cancer

(clinicaltri-als.gov Identifier: NCT00094445), colorectal can-cer (clinicaltrials.gov Identifier: NCT00973869) and multiple myeloma (clinicaltrials.gov Identifier: NCT-00113841) are currently in progress In addition, the potential of curcumin to reduce the symptomatic side effects of chemoradiation in patients suffering from non-small cell lung cancer (clinicaltrials.gov Identifier: NCT01048983) is under clinical investigation Curcu-min has also entered into a phase II clinical trial for

Keywords

apoptosis; BubR1; combination study;

delayed mitosis; dynamic instability

Correspondence

D Panda, Department of Biosciences &

Bioengineering, Indian Institute of

Technology Bombay, Powai,

Mumbai-400076, India

Fax: +91 222 572 3480

Tel: +91 222 576 7838 ⁄ 7770

E-mail: panda@iitb.ac.in

(Received 14 April 2010, revised 21 May

2010, accepted 24 June 2010)

doi:10.1111/j.1742-4658.2010.07750.x

In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule–kinetochore attachment and disturbed the mitotic spindle structure Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indi-cating that it activated the mitotic checkpoint In addition, curcumin treat-ment increased the metaphase⁄ anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase We provide evidence suggesting that the affected cells underwent apoptosis via the p53-depen-dent apoptotic pathway The results support the idea that kinetic stabiliza-tion of microtubule dynamics assists in the nuclear translocastabiliza-tion of p53 Curcumin exerted additive effects when combined with vinblastine,

a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules

Abbreviations

CI, combination index; FITC, fluorescein isothiocyanate; PI, propidium iodide.

advanced pancreatic cancer [2] and a phase III clinical

trial in combination with gemcitabine and celebrex for

the treatment of metastatic colon cancer [2] Curcumin

inhibits tumor growth in animal models [3] Further,

the uptake of high doses of curcumin in both animals

and humans has been found to be nonhazardous and

relatively nontoxic [4,5] Curcumin has also been found

to be an effective stress reliever and neuroprotective

agent [6]

Curcumin has been shown to inhibit the

prolifera-tion of several types of cancer cells in culture,

includ-ing pancreatic, cervical, colon and breast cancer [7–15]

It arrests the cell-cycle progression of human

pancre-atic cancer cells (BxPC-3) and glioma cells (U251) at

the G2⁄ M phase of the cell cycle [7,8] and has been

shown to affect the progression of MCF-7 cells

through the G2⁄ M phase [9] Curcumin treatment

caused an increase in the G0⁄ G1 phase of the cell

pop-ulation implying apoptosis in MCF-7 cells [10]

Curcu-min is found to induce apoptosis in several cell lines

[1,7,10] It stimulated Bax-mediated p53-dependent

apoptosis in MCF-7 cells [10] Curcumin promotes the

action of certain drugs by overcoming chemoresistance

[11] It overcomes P-glycoprotein-mediated multidrug

resistance in multiple cell lines [1] The migration and

invasion of human lung cancer cells are also inhibited

by curcumin [1]

Curcumin has been suggested to inhibit cell

prolifer-ation by diverse mechanisms [1,12–16]; however, the

primary mechanism by which inhibition occurs

remains obscure Recently, curcumin has been found

to bind to purified tubulin, to inhibit tubulin

polymeri-zation in vitro and to depolymerize microtubules in

HeLa and MCF-7 cells in culture [12] In addition,

curcumin has been shown to perturb the microtubule

spindle structure [12,13] and to stimulate

micronucle-ation in MCF-7 cells [13] In 32D cells, curcumin has

also been shown to affect the activity of the

chromo-somal passenger complex, resulting in multipolar

chro-mosome segregation promoting mitotic catastrophe

[14] Moreover, curcumin induced mitotic catastrophe

in Ishikawa and HepG2 cancer cells, indicating that it

might perturb microtubule assembly dynamics [15]

Dynamic microtubules are the key structural

ele-ments in mitotic spindle formation and they

orches-trate chromosome distribution during the cell division

[17,18] In this study, we found that curcumin strongly

suppressed the dynamic instability of individual

micro-tubules in live MCF-7 cells At low effective

prolifera-tion inhibitory concentrations, curcumin inhibited

microtubule dynamics in MCF-7 cells without causing

a significant depolymerization of microtubules

How-ever, high concentrations (‡ 2 · IC50) of curcumin

were found to depolymerize both the interphase and mitotic microtubules in MCF-7 cells Curcumin treat-ment perturbed the mitotic spindle network in MCF-7 cells, activated the mitotic checkpoint and delayed mitotic progression We present several lines of evi-dence indicating that curcumin inhibits cell prolifera-tion by inhibiting microtubule dynamics The results suggest that tubulin is one of the major targets for the antiproliferative activity of curcumin

Results

Curcumin inhibited the proliferation of MCF-7 cells and induced apoptosis

Consistent with previous studies [9,10,12], curcumin was found to inhibit the proliferation of MCF-7 cells

in a concentration-dependent manner (Fig 1A) For example, 20 and 40 lm curcumin inhibited the prolifer-ation of MCF-7 cells by 70% and 93%, respectively, and the half-maximal inhibition of proliferation (IC50) was determined to be 16 ± 0.3 lm MCF-7 cells were either treated with the vehicle or different concentra-tions of curcumin for 48 h Vehicle-treated MCF-7 cells did not display Annexin V and propidium iodide (PI) staining, although a population (46%) of cells treated with 12 lm curcumin stained positive for Ann-exin V alone, showing that the cells were at the early stage of apoptosis (Fig 1B) Cells treated with 24 lm curcumin showed greater numbers (71%) stained with Annexin V (Fig 1B) At a still higher curcumin con-centration (36 lm), cells were stained with both Ann-exin V and PI, indicating late apoptosis (Fig 1B) Further, curcumin treatment strongly increased the nuclear localization of p53 and p21 in MCF-7 cells (Fig 1C,D) For example, 4 and 24% of MCF-7 cells showed nuclear localization of p53 (Fig 1C), whereas

7 and 33% of cells showed nuclear localization of p21

in the absence and presence of 24 lm curcumin, respectively (Fig 1D)

Curcumin disrupted the mitotic spindle network inducing formation of monopolar spindles and depolymerized microtubules in MCF-7 cells MCF-7 cells were incubated without or with 24 and

36 lm curcumin for 6 h Curcumin treatment strongly depolymerized mitotic spindle microtubules (Fig 2A) However, it did not induce significant depolymeriza-tion of the interphase microtubules after 6 h of incuba-tion, suggesting that curcumin exerted a stronger depolymerizing effect on the microtubules of the mito-tic cells than those of the interphase cells (data not

B

Fig 1 Curcumin inhibited the proliferation of MCF-7 cells and induced cell death (A) MCF-7 cells were treated with different concentrations

of curcumin for one cell cycle and the inhibition of cell proliferation was determined by the sulforhodamine B assay (B) Curcumin induced apoptosis in MCF-7 cells MCF-7 cells were incubated with 0.1% dimethylsulfoxide (control) and different concentrations (12–36 l M ) of curc-umin for 48 h and then stained with Annexin V ⁄ PI Scale bar, 10 lm Curcumin (24 l M ) treatment increased the nuclear accumulation of p53 (C) and p21 (D) in MCF-7 cells Scale bar, 10 lm.

15 min

25 min

25 min

0 min

Fig 2 Curcumin-perturbed mitotic spindle structures of MCF-7 cells (A) MCF-7 cells were incubated without or with 24 and 36 l M of curcu-min for 6 h Microtubules are shown in red and the nucleus in blue Scale bar, 10 lm (B) Curcucurcu-min suppressed the reassembly of the cold-depolymerized mitotic spindle microtubules The upper and lower panels show growth kinetics of spindle microtubules in the absence or the presence of 36 l M curcumin Scale bar, 10 lm.

shown) Consistent with a previous study [12],

curcu-min was found to induce significant depolymerization

of both the interphase and mitotic microtubules of

MCF-7 cells after 24 h of incubation (Fig S1A,B) In

the control population, 70% and 30% of the mitotic

cells were found to be bipolar and monopolar,

respec-tively, whereas 85% of the mitotic cells were

mono-polar in the presence of 24 lm curcumin, suggesting

that curcumin induced the formation of monopolar

spindles

The effect of curcumin on the polymerized mass of

microtubules in MCF-7 cells was analysed by western

blotting The ratio of polymeric⁄ soluble tubulin was

found to be 2.44 ± 0.77 in the absence of curcumin,

and 1.97 ± 0.20, 1.56 ± 0.17 (P < 0.03) and

1.32 ± 0.15 (P < 0.01) in the presence of 12, 24 and

36 lm curcumin, respectively, indicating that curcumin

depolymerized microtubules in MCF-7 cells

(Fig S1C)

Curcumin inhibited the reassembly of mitotic

microtubules in MCF-7 cells

MCF-7 cells were synchronized in the M phase of the

cell cycle by treating with 1.3 lm nocodazole for 20 h

Nocodazole treatment completely depolymerized the

spindle microtubules Nocodazole was removed and

the cells were incubated with fresh media in the

absence or presence of 36 lm curcumin on ice for

30 min Subsequently, cells were incubated at 37C

Spindle microtubules in control cells reassembled

within 25 min to form normal mitotic spindle; the

spindle microtubules of curcumin-treated cells did not

reassemble (Fig 2B) The results showed that

cur-cumin inhibited reassembly of the mitotic spindle

microtubules

Curcumin suppressed the dynamic instability of

individual microtubules in live MCF-7 cells

Consistent with previous reports [19,20], microtubules

in control MCF-7 cells were found to be highly

dynamic (Fig 3A) Low concentrations of curcumin (5

and 12 lm) noticeably dampened the dynamic

instabil-ity of the individual microtubules in live MCF-7 cells

(Fig 3B,C) Curcumin treatment reduced the rate and

extent of both growing and shortening events

(Table 1) For example, 12 lm curcumin reduced the

rates of shortening and growing phases by 39% and

19%, respectively, and reduced the extent of the

grow-ing and shortengrow-ing phases by 60% and 65%,

respec-tively Like several other tubulin-targeted agents such

as benomyl, estramustine, epothilone B and paclitaxel

[19–22], curcumin also strongly increased the time that microtubules spent in the pause state, neither growing nor shortening detectably, and decreased the time microtubules spent in the growing or shortening phases Curcumin (12 lm) increased the time spent in the pause state from 28.9% (control) to 71.6% Fur-ther, curcumin (12 lm) altered both the time- and length-based transition frequencies of the interphase microtubules in MCF-7 cells The dynamicity (dimer exchange per unit time from the ends of microtubules) was reduced by 50% and 72% in the presence of

5 and 12 lm curcumin, respectively

A

B

C

Fig 3 Curcumin suppressed dynamic instability of individual micro-tubules in live MCF-7 cells Life-history traces of individual microtu-bules in MCF-7 cells in the absence (A) and presence of (B) 5 l M

curcumin and (C) 12 l M curcumin, respectively.

Effects of curcumin on cell-cycle progression

It was previously reported that curcumin treatment

markedly increased the number of MCF-7 cells in

metaphase [13] Because curcumin suppressed

microtu-bule dynamic instability (Fig 3 and Table 1), we

examined whether it could inhibit mitosis The number

of cells in mitosis in the absence or presence of

differ-ent concdiffer-entrations of curcumin was determined by

Hoechst 33258 staining of the chromosomes Only

2.6 ± 1.6% of the control (vehicle-treated) cells were

found to be in mitosis, whereas 3.2 ± 0.2%,

5.0 ± 0.1% and 6.2 ± 1% (P < 0.0001) of the cells

were found to be in mitosis in the presence of 12, 24

and 36 lm curcumin, respectively If curcumin caused

a delay in mitosis, it might lead to an increase in the

metaphase⁄ anaphase ratio The metaphase ⁄ anaphase

ratio was calculated to be 0.43 ± 0.06 and

1.88 ± 0.40 (P < 0.0001) in the absence and presence

of 24 lm curcumin, supporting the idea that curcumin

could prolong the duration of metaphase

Further, MCF-7 cells were synchronized in the M

phase of the cell cycle by nocodazole treatment for

20 h Nocodazole-blocked cells were washed with fresh

medium and subsequently incubated in medium

with-out and with curcumin Flow cytometry analysis

dem-onstrated that nocodazole-induced mitotic arrest was

gradually released over time for control cells For

example, the percentage of cells in mitosis was 87%,

53% and 16% in nocodazole-treated control flask

immediately, and 4 and 8 h after release of the

noco-dazole block However, in the presence of curcumin,

the percentage of cells in the mitotic phase was 83%

and 81% after 4 and 8 h of block release Thus, treat-ment of cells with curcumin significantly delayed release of the mitotic block (Fig 4A) However, flow cytometric analysis of the cell cycle using PI staining showed that there was no significant cell-cycle block after 24 h of curcumin treatment (Fig S2)

Microtubule inhibitors are known to induce mitotic block by activating the spindle assembly checkpoint proteins [20,23,24] It has been suggested that a com-pound may drive the cells towards delayed mitosis through activation of spindle checkpoint proteins such

as BubR1 [23] and Mad2 [24] Nocodazole, a well-known inhibitor of mitosis, led to the accumulation of Mad2 and BubR1 at the kinetochores (Fig 4B,C) Similar to the action of nocodazole, curcumin treat-ment also activated Mad2 and BubR1 in MCF-7 cells (Fig 4B,C)

Curcumin exhibited antagonism with paclitaxel, but an additive effect with vinblastine for inhibition of MCF-7 cell proliferation Curcumin, paclitaxel and vinblastine inhibited MCF-7 cell proliferation with median inhibitory doses of

15 ± 4 lm, 40 ± 6 nm and 17 ± 10 nm (Fig S3A–C) Curcumin (8 lm) and paclitaxel (2 nm) inhibited prolif-eration of MCF-7 cells by 26% and 13%, respectively, when used alone, whereas their combination inhibited proliferation by 9% The combination index (CI) for the combination of 8 lm curcumin and 2 nm paclitaxel was found to be 3.1 ± 1.5 The proliferation of

MCF-7 cells was inhibited by 22% and 24% in the presence

of 2 and 3 nm vinblastine, respectively, whereas in

Table 1 Effects of curcumin on the dynamic instability parameters of the interphase microtubules in MCF-7 cells Twenty-five microtubules were measured for each condition Data are given as mean ± SD.

a

P < 0.0001;bP < 0.001.

combination with 8 lm curcumin, these concentrations

of vinblastine inhibited proliferation by 44% and 51%,

respectively The CI values for the combination of

8 lm curcumin with 2 and 3 nm of vinblastine were

estimated to be 0.92 ± 0.23 and 0.97 ± 0.19,

respec-tively A CI value < 1 indicates a synergistic effect,

1 indicates an additive effect and > 1 indicates an

antagonistic effect [25,26] The results suggested that

curcumin was antagonistic to paclitaxel, whereas it

dis-played an additive effect with vinblastine in inhibiting

MCF-7 cell proliferation

Curcumin affected the localization of the kinesin

protein Eg5

Because curcumin produced monopolar spindles in

MCF-7 cells, we examined the effect of curcumin on

the localization of Eg5, a motor protein that plays an

essential role in bipolar spindle formation [27,28] In

control cells, Eg5 was localized throughout the bipolar

spindle and remained concentrated at the spindle poles

(Fig 5A) Consistent with a previous study [27],

mon-astrol (50 lm) was found to induce monopolar spindle

formation (Fig 5B) In monastrol-treated cells, Eg5

mainly localized to the pole of the monoastral spindle

and also diffused all along the monoastral microtu-bules (Fig 5B) In the presence of 24 lm curcumin, Eg5 primarily remained confined to the pole of the monopolar spindles Some Eg5 also delocalized along the microtubules of the monopolar spindles (Fig 5C)

Discussion

In this study, we have provided several lines of evi-dence indicating that the antiproliferative mechanism

of action of curcumin involves the perturbation of microtubule dynamics Brief incubation of curcumin with MCF-7 cells produced a noticeable depolymeriz-ing effect on the mitotic microtubules of MCF-7 cells and also inhibited the assembly of cold-depolymerized spindle microtubules indicating that curcumin perturbs microtubule assembly in cells Further, similar to the effects of several other microtubule-targeted drugs such

as benomyl [19], estramustine [20], epothilone [21] and paclitaxel [22] on microtubule dynamics, curcumin was also found to reduce the dynamic instability of individ-ual microtubules in live MCF-7 cells Curcumin treatment caused defective chromosome alignment in the mitotic spindles and the cells eventually died via the p53-dependent apoptotic pathway Curcumin was

Fig 4 Curcumin treatment delayed mitotic progression in MCF-7 cells (A) MCF-7 cells were incubated with 1.3 l M nocodazole Nocodazole was washed off with fresh medium Cells were incubated in the absence or presence of 35 l M curcumin for 4 and 8 h and then stained with PI DNA content of the cells was quantified by flow cytometry Nocodazole and curcumin treatment activated Mad2 (B) and BubR1 (C)

in MCF-7 cells MCF-7 cells were incubated with nocodazole (500 n M ) and curcumin (36 l M ) for 24 h and cells were then stained with Mad2 and BubR1 antibodies Scale bar, 10 lm.

found to bind to purified tubulin and to perturb

microtubule assembly in vitro [12] The results together

indicated that curcumin inhibits MCF-7 cell

prolifera-tion by targeting microtubules

The plus-end-directed motor Eg5 (kinesin spindle

protein) plays an important role in proper

chromo-some separation and the formation of a proper bipolar

spindle [27,28] Similar to the action of monastrol [27],

curcumin also induced monopolar spindle formation in

association with the perturbation of Eg5 localization

in MCF-7 cells, indicating that curcumin may inhibit

Eg5 function and thereby induce monopolar spindle

formation Curcumin might inhibit the binding of Eg5

to microtubules and perturb the movement of Eg5

over the microtubules leading to abnormal spindle

for-mation Alternatively, curcumin might directly interact

with Eg5 and inhibit its function

Effects of curcumin on the progression of the

cell cycle

Curcumin increased the metaphase⁄ anaphase ratio and

slowed the release of mitotic block in

nocodazole-synchronized MCF-7 cells, indicating that it can delay

cell-cycle progression at mitosis However, it failed to

induce substantial mitotic block in MCF-7 cells In several cases, higher concentrations of microtubule-targeted agents are required to inhibit cell-cycle progression at mitosis than are required to inhibit the proliferation [29–32] In a KB⁄ HeLa (human cervical epitheloid carcinoma) cell line, a derivative of benzylid-ene-9(10H)-anthracenone gave an IC50 value of 0.09 lm for the inhibition of cell proliferation, whereas 50% arrest in the G2⁄ M phase occurred in the pres-ence of 0.205 lm of compound [29] The anthracenone derivative caused cell-cycle arrest in a K-562 cell line

at 0.3 lm, whereas its IC50 in the same cell line was 0.02 lm In smooth muscle cells, 68.6% of the cells were arrested in the G2⁄ M phase at 100 nm concentra-tion of LY290181 (IC50of inhibition of cell prolifera-tion being 20 nm) [30] In human non-small cell lung carcinoma cells A549, low concentrations of paclitaxel (3-6 nm) inhibited cell proliferation without causing mitotic arrest [31] Moreover, treatment with a low concentration of paclitaxel induced abnormal cell for-mation without the G2⁄ M block [32] A 50% inhibi-tion of cell growth after 72 h incubainhibi-tion required 3.4 nm paclitaxel and 9.5 nm discodermolide [32] These concentrations were closer to that required for aneuploidy induction rather than mitotic arrest [32]

Tubulin Eg5 DNA Tubulin + Eg5 Tubulin + Eg5 + DNA

A

B

C

Fig 5 Localization of Eg5 in control and curcumin-treated MCF-7 cells Cells were treated without and with curcumin for 24 h, fixed, and co-immunostained with a-tubulin (green), Eg5 antibody (red) and DNA was stained with Hoechst 33258 (A) In control mitotic cells, Eg5 remained mainly concentrated at the poles of the bipolar spindle and to some extent delocalized along the spindle microtubules (B) In the presence of 50 l M monastrol, monopolar spindles were formed Eg5 localized mainly at the pole of the monopolar spindle and remained dif-fused along the microtubules in the overlayed image (C) Curcumin at a concentration of 24 l M induced monopolar spindle formation In the overlain image the Eg5 localized to the centre of the monopolar spindle and also remained dispersed over the microtubules Scale bar,

10 lm.

Thus, the induction of abnormal mitosis and

aneu-ploidy is dependent on the drug mechanism and the

concentration of the drug used [31,32]

Several microtubule-targeted agents are known to

activate checkpoint proteins and to arrest cells in

mito-sis [20,33–35] The checkpoint proteins accumulate in

the kinetochoric region after detecting a flaw in

kineto-chore–microtubule attachment or reduced tension at the

kinetochores [24] For example, nocodazole enhances

the accumulation of Mad2 and BubR1 to the

kinetoch-ores and induces mitotic arrest (Fig 4B,C) [36]

Several inhibitors of microtubule dynamics were

found to delay G2⁄ M transition [37] The ability of a

compound to activate spindle checkpoint proteins may

sometimes lead to delayed mitosis [38] Conditions that

perturb proper kinetochore–microtubule attachment

may cause checkpoint protein translocation and the

affected cells may be held back from progressing

fur-ther in the cell cycle, leading to a delay in mitosis [38]

Curcumin was found to perturb

microtubule–kineto-chore attachment and also activated the mitotic

check-point, resulting in delayed mitosis A delay in mitosis

has been shown to induce apoptosis in cancer cells

[39]

Curcumin treatment enhanced the nuclear

accumulation of p53 in MCF-7 cells

An alteration in expression of the tumor suppressor

gene p53 is known to induce apoptosis in several types

of cells [40–42] It has been suggested that p53 is

trans-ported into the nucleus through the microtubule

net-work [40,41] Compounds that stabilize microtubule

dynamics have been suggested to promote p53

translo-cation to the nucleus [19,41] Several antimitotic drugs

have been found to induce apoptosis by inhibiting

microtubule assembly dynamics [43] Curcumin

suppresses the dynamic instability of microtubules,

therefore, it may enhance nuclear translocation of p53

through the stabilized microtubule track

Curcumin in combination with vinblastine, a

micro-tubule depolymerizing agent, inhibited cell

prolifera-tion in an additive fashion However, it antagonized

the action of paclitaxel, a compound that promotes

microtubule assembly; supporting the idea that

curcu-min inhibits cell proliferation by targeting

micro-tubules The results also indicated that curcumin may be

used in combination with microtubule depolymerizing

agents such as vinblastine to improve the efficacy and

reduce the toxic dose of the drug It has been found

that an oral intake of curcumin is not toxic to humans

up to 8000 mgÆday)1 for 3 months [44] Moreover,

curcumin (C3 Complex, Sabinsa Corp., East

Wind-sor, NJ, USA) in single oral doses up to 12 000 mg was found to be well tolerated in healthy volunteers [45] Therefore, the concentrations of curcumin used in this study are expected to be within tolerable doses It has been suggested that less potent dietary compounds can enhance the effect of a more potent and toxic drug

by lowering its toxicity level [46,47] Therefore, combi-nation between two such drugs can provide superior clinical efficacy than a single drug alone [46]

Materials and methods

Reagents

Curcumin, sulforhodamine B, fetal bovine serum, BSA and G418 were purchased from Sigma (St Louis, MO, USA) Annexin V and PI were purchased from Santa Cruz Bio-technology (Santa Cruz, CA, USA) All other reagents were

of analytical grade

Cell culture

MCF-7 cells, human breast carcinoma cells, were grown in minimum essential medium (HiMedia, Mumbai, India)

bicarbonate, along with 1% antibacterial and antimycotic solution containing streptomycin, amphotericin B and

solution was prepared in 100% dimethylsulfoxide and dif-ferent concentrations of curcumin were added to the culture

seeding Dimethylsulfoxide (0.1%) was used as a vehicle control

Cell proliferation assay and mitotic index calculation

The effect of curcumin on the proliferation of MCF-7 cells was determined by sulforhodamine B assay [49] For mito-tic index calculation, MCF-7 cells were seeded at a density

cover-slips followed by treatment with curcumin for 24 h [20] The coverslips were centrifuged in a Labofuge 400R cyto-spin (Heraeus, Hanau, Germany) for 10 min (1200 g at

stained with Hoechst 33258 The number of cells in mitosis and interphase were counted using the Eclipse TE2000-U microscope (Nikon, Tokyo, Japan) At least 800 cells were counted for each set and the experiment was repeated three times The numbers of cells at the metaphase and anaphase stages of the cell cycle were calculated for both the control and curcumin-treated cells

Immunofluorescence microscopy and

transfection

cover-slips in 24-well plates for 24 h and incubated with different

concentrations of curcumin for another 24 h The cells were

immuno-stained as reported earlier [48] Cells were immuno-stained with the

following primary antibodies: mouse monoclonal

anti-(a-tubulin IgG) (1 : 300) from Sigma, rabbit polyclonal

anti-(a-tubulin IgG) (1 : 300) from Abcam (Cambridge,

MA, USA), mouse monoclonal anti-p53 IgG (1 : 300),

mouse monoclonal anti-p21 IgG (1 : 300) purchased from

Santa Cruz (Santa Cruz, CA, USA), mouse anti-BubR1

IgG (1 : 500) from BD Biosciences (San Jose, CA, USA)

rabbit Mad2 IgG (1 : 300), mouse monoclonal

anti-Eg5 IgG (1 : 800) from Abcam (Cambridge, MA, USA)

The secondary antibodies used were Alexa 568-conjugated

sheep anti-(mouse IgG) (1 : 400) purchased from Molecular

Probes (Eugene, OR, USA), fluorescein isothiocyanate

(FITC)-conjugated anti-(mouse IgG) (1 : 400) and

FITC-conjugated anti-(rabbit IgG) (1 : 400) from Sigma The

(Sigma) The slides were observed under an Eclipse

objective The images were captured using CoolSNAP-Pro

camera image-pro plus software 4.0 (Media Cybernetics,

Bethesda, MD, USA) was used for image acquisition and

processing MCF-7 cells were transfected with EGFP–

a-tubulin plasmid, as described previously [20] and the

stably transfected MCF-7 cells were maintained in the

pres-ence of the antibiotic G418

Annexin V⁄ propidium iodide staining

MCF-7 cells were grown in the absence and presence of

dif-ferent concentrations of curcumin for 48 h and were stained

with Annexin V⁄ PI, as reported previously [20,48] The

manufacturer’s protocol was used for staining the cells

using an Annexin V apoptosis detection kit (Santa Cruz

Biotechnology) and processed for microscopy [20,48] The

cells exhibiting positive Annexin V and PI staining were

seen under microscope using the FITC and PI fluorescence,

differential interference contrast microscopy was used for

visualizing total number of cells

Cell-cycle analysis

MCF-7 cells were grown in the absence and presence of 25

and 35 lm curcumin for 24 h The cells were first fixed in

flow cytometer (FACS Aria; Becton Dickinson, San Jose,

CA, USA)

MCF-7 cells were treated without and with 1.3 lm noco-dazole for 20 h Noconoco-dazole was washed off with fresh media The cells were incubated without or with curcumin for 4 and 8 h, and then stained with PI The effect of curc-umin on the kinetics of the release of the mitotic block was examined in a flow cytometer and the data were analysed using the modfit lt program (Verity Software, Topsham,

ME, USA)

Effect of curcumin on the reassembly of cold-depolymerized mitotic microtubules

cover-slips for 24 h and then incubated with 1.3 lm nocodazole for 20 h Nocodazole was removed by washing with fresh medium Cells were then incubated without or with 36 lm curcumin on ice for 30 min Subsequently, cells were

microtubule network was visualized by staining the fixed cells with anti-a-tubulin Ig The DNA was stained with Hoechst 33258

Western blot analysis

The effect of curcumin on the polymeric mass of microtu-bules in the cells was analysed by western blot, as described previously [20] The protein concentrations of the polymeric and the soluble fraction were determined by the Bradford method [50] The polymeric and the soluble tubulin

poly(vinylidene difluoride) membranes The membranes were probed with mouse monoclonal anti-(a-tubulin IgG) (1 : 1000) and alkaline phosphatase-conjugated secondary anti-(mouse IgG) (1 : 5000) (Sigma) The band intensities were calculated using image j software

Effects of curcumin on the dynamic instability of individual microtubules in MCF-7 cells

The effects of curcumin on the dynamic instability of the interphase microtubules in MCF-7 cells were determined as described previously [20,51] Briefly, MCF-7 cells having stably transfected green fluorescent protein–a-tubulin were grown on glass coverslips for 24 h Cells were then incu-bated in the absence or presence of 5 and 12 lm curcumin for an additional 24 h The coverslips were transferred to glass-bottomed dishes (Prime BioScience, Pandan Loop, Singapore) containing media without phenol red and were

of microtubules was carried out using an FV-500 laser scanning confocal microscope (Olympus, Tokyo, Japan)

acquired at 4 s intervals for a maximum duration of 3 min using fluoview software (Olympus, Tokyo, Japan) The

plus end of microtubules was tracked using image j

soft-ware Life-history traces were obtained by plotting the

length of individual microtubules against time Length

were considered as growth or shortening excursions and a

change < 0.5 lm in length was considered as a pause state

A transition from a shortening to a growth or pause state

is called a rescue, whereas the transition from a growth or

pause state to a shortening state is defined as a catastrophe

[51] Twenty-five microtubules were analysed for each

experimental condition Statistical significance was

calcu-lated using the Student’s t-test

CI determination

MCF-7 cells were incubated either separately with

curcu-min, paclitaxel and vinblastine or in combination with

curc-umin and vinblastine or paclitaxel for one cell cycle The

combination index was calculated using the Chou and

Tala-lay method [26,52], with the help of the following equation

where (D)1 and (D)2 are the concentrations of drug 1 and

drug 2 used in combination, which produce a particular

effect, (Dx)1 and (Dx)2 are the concentrations of the drugs

that produce similar effect when used alone The

concentra-tion of curcumin which produced a particular effect was

calculated from the median effect equation

affected and fraction unaffected, respectively [27] Dm was

estimated from the antilog of the X-intercept of the median

the median effect plot

Acknowledgement

The work was partly supported by Swarnajayanti

Fel-lowship (to DP) from the Department of Science and

Technology and partly by a grant from the Council of

Scientific and Industrial Research, Government of

India

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