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A novel Cu(II)–mal–picoline complex induces mitotic catastrophe mediated bydeacetylation of histones and a-tubulin leading to apoptosis in human cell lines

Biswarup Saha,†a Ananda Mukherjee,†a Saheli Samanta,†a Susmita Paul,†a Debalina Bhattacharya,†a

Chitta Ranjan Santra†b and Parimal Karmakar†*a

Received 14th November 2011, Accepted 2nd June 2012

DOI: 10.1039/c2md00285j

In this study, we investigated mitotic catastrophe followed by apoptosis induced in human cell lines

[HeLa, HepG2 and THP1] by a novel Cu(II) complex having malonate as the primary ligand and

protonated 2-amino-4-picoline as the counter ion; whose in vitro DNA binding ability was

demonstrated previously (B. Saha et al., J. Phys. Chem. B, 2010, 114(17), 5851–5861). Using the auto-

fluorescence property of the complex, it was observed that the complex entered into the cells within 15

min after the exposure and was able to kill cells as determined by clonogenic survivability and MTT

assay in a dose and time dependent manner. While dissecting the cell killing mechanisms, it was found

that initially the complex induced multinucleated cells by inhibiting acetylation of a histone acetyl

transferase (HAT) domain of CBP/p300, although histone deacetylase 6 (HDAC6) expression did not

change much. As a result, histone proteins, H3 andH2AX, along with a non-histone protein, a-tubulin,

were mostly deacetylated after 48 h of the treatment. This eventually led to mitotic catastrophe (MC),

as histone acetylation–deacetylation dynamics is essential for the successful mitosis. DNA damage-

induced gH2AX and 53BP1 foci in the treated cells were also observed after 72 h of treatment, as

abnormal mitosis with decondensed chromosomes are prone to nucleolytic attack. These molecular

phenomena ultimately rendered apoptosis. Taken together, our results provided evidence that the said

complex perturbed the signaling events associated with mitosis and consequently induced cell death.

1. Introduction

The transition element copper is vital for the healthy functioning

of higher organisms including respiration, angiogenesis, and

immune response.2–4 Recently, medical research has focused on

copper complexes for the development of different therapeutic

agents including cancer.5,6 The rationale for using copper

complexes as anti-cancer agents is to avoid severe side effects

induced by known anticancer agents like bleomycin, cis-platin,

etoposide, etc.7–11 Copper modulation has been suggested to be a

potential modality in therapy for several diseases. The ability to

cycle between +1 and +2 oxidation states of copper is one of the

main features that has been exploited in biological systems.12–14

Depending on the nature of the complex associated with copper,

diverse cellular response has been elucidated. Perturbation in

aDepartment of Life Science and Biotechnology, Jadavpur University, 188,Raja S.C. Mullick Road, Kolkata-700 032, West Bengal, India. E-mail:pkarmakar_28@yahoo.co.in; karmakarparimal@gmail.com; Fax: +91 332413 7121; Tel: +91 33 2414 6710bDepartment of Chemistry, Netaji Nagar Day College, NSC Bose Road,Regent Estate, Kolkata-700 092, West Bengal, India

† PK conceived and designed the experiments; BS, AM, SS, SP and DBperformed the experiments; PK, BS, AM and CRS analyzed the data; PKcontributed reagents/materials/analysis tools; PK and BS wrote thepaper.

This journal is ª The Royal Society of Chemistry 2012

distinct mode of cell survival signal or induction of specific

pathways associated with cell dismissal has been shown to induce

by the copper-complex in vitro.15–19 Many copper complexes are

demonstrated for their anticancer activity, which is mainly

mediated through the induction of oxidative stress.20–22 N-Sali-

cylidene-L-glutamato diaqua copper(II) complex (CuC) was

shown to induce cell death via production of ROS in mice

leukemia cells L1210.23 Recently, copper N-(2-hydrox-

yacetophenone)glycinate (CuNG) has been reported to increase

the ROS level in the liver of doxorubicin-resistant Ehrlich ascites

carcinoma (EAC/Dox) bearing swiss albino mice and can

modulate different anti-oxidant enzymes.24 Another copper(II)

complex of ethyl 2-[bis(2-pyridylmethyl)amino]propionate

ligand (ETDPA) has shown to be effective in killing HeLa cells

via the ROS-triggered autophagic pathway.25 Similarly, copper–

dopamine complex induces mitochondrial autophagy in the

cultured cells prior to caspase independent apoptotic death.26

Several organic ligands have been found to complex with copper

spontaneously or can be made to complex with copper easily.

For example, it has been reported that 8-hydroxylquinoline (8-

OHQ) is able to form a copper complex that inhibits proteasome

and induces apoptosis in cancer cells.27 Similarly, glutamine

Schiff base complexed with copper has been reported to selec-

tively inhibit the proteasomal activity and induce cell death in

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breast cancer cells.28 A detailed signaling mechanism of pyrroli-

dine dithiocarbamate (PDTC)/copper induced apoptosis has also

been reported recently.29 Apart from these studies, a Cu(II)–thi-

oxotriazole complex has been reported to trigger a non-apoptotic

type 3B programmed cell death on HT1080 human fibrosarcoma

cells.30 Disulfiram (DS)–Cu complex significantly enhanced the

cytotoxicity of gemcitabine resistant cells by inhibiting the NFkB

activity.31 Copper–chitosan complex inhibits tumor cell prolif-

eration by arresting the cell cycle progression at the S phase.32

Tetraaza macrocyclic copper complex [Cu(TAAB)Cl2] has also

shown to trigger apoptosis in L1210 murine leukemia cells.33

Different mononuclear ligands of copper(II) complexes of the

type [Cu(L-tyr)(diimine)](ClO4) were tested for their ability to

induce cell death on lung cancer (H-460) cells. It was reported

that when diimines of a Cu(II)-complex were replaced by dipyr-

ido[3,2-d20,30-f]quinoxaline (dpq), the resulting complex induced

apoptosis but when diimines were replaced by 5,6-dimethyl-1,10-

phenanthroline (5,6-dmp), the resulting copper complex induced

mitotic catastrophe.34 Thus unlike other conventional chemo-

therapeutic agents, different copper complexes not only elicit

apoptosis but other forms of non-apoptotic cell death, such as

autophagy, mitotic catastrophe, etc. provide an opportunity to

test different copper complexes for their ability to induce various

kinds of signaling mechanisms associated with the cell-death.35

A water soluble Cu(II) complex, [Cu(mal)2](picH)2$2H2O, was

synthesized and characterized, drawn interest to the crystal

engineers for its remarkable supramolecular features in the solid-

state.36 Our previous in vitro study showed that the complex

interacts with DNA as a partial intercalator as well as a partial

minor groove binder.1 However, in the present work, we have

provided evidence that the complex can induce death in HeLa,

HepG2 and THP1 cells in a dose and time dependent manner.

The mechanism of cell death is coupled with the induction of

mitotic catastrophe (MC) followed by apoptosis. The induction

of MC after the treatment with the complex was associated with

deacetylation of histone acetylase (HAT) domain of CBP/p300

followed by deacetylation of the downstream histone proteins,

H3 and H2AX, and a non-histone protein, a-tubulin. A major

cytoskeleton protein, actin, was degraded in the presence of the

complex as observed by immunolabeling. Such mitotic catas-

trophe was also featured with DNA damage associated with

gH2AX and 53BP1 foci in the decondensed chromosomes.

However, after 72 h of incubation, the damaged cells entered into

apoptosis, which was confirmed by annexin V-FITC staining,

activation of caspase 3 and initiation of PARP cleavage. Thus

taken together, the novel Cu(II)-complex has a unique property

of inducing mitotic catastrophe followed by apoptosis, which

may have a potential therapeutic implication in the near future.

2. Materials and methods

2.1. Cell lines, culture conditions and treatments

HeLa (commercially available from National Centre for Cell

Science, NCCS, Pune, India), HepG2 (NCCS, Pune, India) and

THP1 (NCCS, Pune, India) cells were maintained at 37 �C, 5%CO2 and 95% relative humidity (RH) in DMEM (for HeLa and

HepG2) and RPMI 1640 (for THP1) medium, supplemented

with 10% heat-inactivated fetal bovine serum, 2 mM

1394 | Med. Chem. Commun., 2012, 3, 1393–1405

L-glutamine, penicillin (100 U ml�1) and streptomycin

(100 U ml�1). Cells were seeded for 24 h prior to treatment with

Cu(II)-complex or picoline (10–150 mM). In most of the experi-

ments, 60 mM of either Cu(II)-complex or picoline was used or

mentioned otherwise. All the treatments were performed at 37 �Cand at a cell density allowing exponential growth.

2.2. Reagents, antibodies and plasmid

The title Cu(II)-complex was synthesized in Prof. Subrata

Mukhopadhyay’s laboratory (Department of Chemistry,

Jadavpur University), using the method described previously.36

The Cu(II)-complex is readily soluble in water while purified

2-amino-4-picoline (henceforth, picoline, Sigma) requires slight

warming. These were separately dissolved at a concentration of

10 mM (stock) in double distilled water and all further dilutions

were made freshly in the respective medium.

Primary antibodies anti-PARP (1 : 250, rabbit polyclonal,

Santa Cruz Biotechnology), anti-caspase 3 (1 : 1000, rabbit

polyclonal, Cell Signaling Technology), anti-b-Actin (1 : 1000,

rabbit polyclonal, Cell Signaling Technology), anti-a-tubulin

(1 : 100 for Immunofluorescence, 1 : 1000 for WB, rabbit poly-

clonal, Cell Signaling Technology), anti-phospho-H2AX

(Ser139) (1 : 100 for Immunofluorescence, rabbit monoclonal),

anti-Acetyl-CBP(Lys1535)/p300(Lys1499) (1 : 1000, rabbit

polyclonal, Cell Signaling Technology), anti-Acetyl (Lys9/Lys14)

Histone H3 (1 : 1000, rabbit polyclonal, Cell Signaling Tech-

nology), anti-HDAC6 (1 : 1000, rabbit polyclonal, Abcam), anti-

a-tubulin (1 : 100 for Immunoprecipitation, mouse monoclonal,

Santa Cruz Biotechnology), anti-Acetylated Lysine (1 : 1000,

mouse monoclonal, Cell Signaling Technology) and anti-Histone

H2AX (1 : 100 for Immunoprecipitation, 1 : 2000 for W.B.,

rabbit polyclonal, Upstate, Millipore) were used. Anti-mouse or

anti-rabbit IgG conjugated with either alkaline phosphatase

(1 : 1000, Bangalore Genei, India) or HRP (1 : 1000, Bangalore

Genei, India), anti-rabbit IgG conjugated with Alexa Fluor 568

(1 : 400, Molecular Probes) and phalloidin conjugated with

Alexa Fluor 488 (1 : 200, Molecular Probes) were used as

secondary antibodies. Aprotinin and leupeptin (Roche) were

used as protease inhibitors.

pEGFP-C1 containing 53BP1 clone (kind gift from Dr VA

Bohr, NIH, Baltimore, USA) was used in the transfection

experiments.

2.3. Clonogenic survival assay

500 cells were seeded into six-well plates. 24 h after seeding, cells

were treated with designated concentrations of either Cu(II)-

complex or picoline. The cells were fed at an interval of 3 days

with fresh medium and two weeks after that, the cells were fixed,

stained with 2%methylene blue (Sigma) in 50% of methanol for 5

min. Colonies consisting of 50 or more cells were counted under a

bright field microscope.37

2.4. MTT assay

After the treatment for designated dose or time, cells were

washed thrice with phosphate buffered saline (PBS) and incu-

bated in phenol red free medium with 3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyl tetrazolium bromide (MTT) (450 mg ml�1) for

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3 h at 37 �C. The resulting formazan crystals were dissolved in an

MTT solubilization buffer and the absorbances were taken with

a UV-visible spectrophotometer (Hitachi) at a wavelength of

650 nm.38

2.5. Annexin V-FITC staining

After the treatment, cells were washed in ice-cold PBS and

incubated with 100 ml of a buffer (10 mM HEPES pH 7.4,

140 mM NaCl and 2.5 mM CaCl2) containing annexin V-FITC

solution (10 : 1 (v/v), Sigma). After 15 min of incubation in the

dark at room temperature, coverslips were mounted with Vec-

tashield containing DAPI (0.2 mg ml�1, Vector Laboratories Inc.)

on glass slides and immediately analyzed under a fluorescence

microscope.39

2.6. Imaging of live cells exploiting autofluorescence of

complex

After the incubation for indicated times, the treated cells in

coverslips were washed twice, mounted with PBS in grease-free

glass slides and observed under a fluorescence microscope (Leica)

using the DAPI filter.

2.7. Lactate dehydrogenase (LDH) assay

After the incubation for 72 h with treatments, culture media were

collected separately and then cells were lysed for 10 min in 0.5%

(v/v) Triton X-100 in 0.1 M potassium phosphate buffer, pH 7.4.

The supernatant was separated after centrifugation at 10 000g

for 5 min. The percentage release of LDH was determined by the

spectrophotometric method. The absorbances of both the culture

medium and the supernatant after cell lysis at 340 nm were taken.

The percentage of LDH released was calculated using a standard

relation described elsewhere.40

2.8. Reactive oxygen species (ROS) assay

A fluorescent probe, 20,70-dichlorofluorescein diacetate (DCFH-

DA, Sigma), was used to estimate the cellular level of reactive

oxygen species (ROS), as previously described.41 Briefly, after the

treatment for 72 h, cultured cells were harvested and incubated

with DCFH-DA (20 mM) in PBS for 1 h at 37 �C in the dark. The

chemical diffuses through the cell membrane, enzymatically

hydrolyzed by intracellular esterases and oxidized to produce a

fluorescent product 20,70-dichlorofluorescein (DCF) in the pres-

ence of ROS. The intensity of fluorescence is proportional to the

level of intracellular reactive oxygen species.

2.9. Cell cycle analysis in HepG2 cells

After the treatment for the indicated times, cells were harvested

and fixed with 70% ethanol for 2 h at 4 �C. Prior to staining with

50 mg ml�1 propidium iodide (PI, Sigma), cells were incubated for

1 h with 100 mg ml�1 of DNAse free RNAse A (SRL, India) at

37 �C. The cell cycle was analyzed with a Becton Dickinson

(FACSCalibur) flow cytometer, equipped with an air-cooled

20 mW argon laser. 25 000 events were counted at each data

point.40

This journal is ª The Royal Society of Chemistry 2012

2.10. Hematoxylin–eosin staining for multinucleus

After the treatment for the designated times, cultured cells in

coverslips were washed with PBS twice, fixed with ice-cold

methanol and then stained with 4% hematoxylin (Himedia)

solution (dissolved in warm PBS) for 2 h at room temperature.

Cells were then washed vigorously with PBS until the redness of

the coverslips disappeared completely. Standard eosin solution

(Himedia) was used as a counter stain prior to mounting the cells

with PBS in stain-free glass slides. Enlarged cells with multi-

nucleus were observed under a light microscope (Leica).42

2.11. Immunolabeling for a-tubulin and gH2AX

After the treatment for 48 h, cells in coverslips were washed twice

with ice-cold PBS and fixed with freshly prepared 4% para-

formaldehyde (HiMedia, India) in PBS for 15 min at room

temperature. After the fixation, cells were washed again with PBS

and then permeabilized with 0.2% Triton X-100 in PBS. Subse-

quently, the cells were blocked with 1% BSA for 30 min at room

temperature and then incubated with appropriate primary anti-

body diluted in wash buffer (1 : 100) containing 0.1% BSA and

0.05% Tween 20 in PBS overnight at 4 �C. The cells were then

washed and labeled with the appropriate secondary antibody

conjugated with Alexa Fluor 568.40 After washing with wash

buffer, the labeled cells were finally observed under either a

fluorescence microscope (Leica) or a laser scanned confocal

microscope (Zeiss LSM 510 META).

To visualize actin filaments, the treated cells were directly labeled

with phalloidin conjugated with Alexa Fluor 488 and analyzed

under a fluorescence microscope after counter stained with DAPI.

2.12. Western blot analysis

After the treatment, whole cell lysates were extracted with a lysis

buffer containing 1% Triton X-100, 50 mM NaCl, 50 mM NaF,

20 mM Tris–Cl (pH 6.8), 1 mM EDTA, 1 mM EGTA, 1 mM

sodium vanadate, 0.2 mM PMSF, 0.5% NP-40, 20 mg ml�1

aprotinin, 10 mg ml�1 leupeptin, 10 U ml�1 DNAse and phos-

phatase inhibitors (1 : 1000). Equal quantities of cell lysates

(50 mg) were solubilized in loading buffer, boiled for 5 min and

electrophoresed on a 7–12% polyacrylamide (ICN) gel in Tris–

glycine buffer (pH 8.3). Proteins were then transferred to poly-

vinylidine difluoride (PVDF) membranes. Nonspecific binding

was blocked with 5% non-fat dry milk and 0.05% Tween-20 in

20 mM Tris–Cl, pH 7.6 (TBS–T). After incubation with the

appropriate primary antibodies overnight at 4 �C, membranes

were washed with TBS–T and were then reincubated with

appropriate secondary antibodies conjugated with either alkaline

phosphatase or HRP. Immunoreactivity was visualized by

incubating the blots either in a BCIP/NBT (SRL, India)

substrate buffer40 or the enhanced chemiluminescence signals

were developed in films using an ECL-plus kit (GEHealthcare).43

2.13. Immunoprecipitation analysis

After 48 h of treatment, whole-cell lysates were prepared by using

non-denaturing RIPA lysis buffer (150 mM NaCl, 1% Triton

X-100, 0.5% NP-40, 10 mM Tris–Cl (pH 6.5), 0.5% sodium

deoxycholate, 0.2 mM PMSF, 1 mM sodium orthovanadate,

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10 U ml�1 DNAse, 20 mg ml�1 aprotinin, 10 mg ml�1 leupeptin) at

4 �C. 1 mg of the whole cell lysates were incubated for 1 h at 4 �Cwith protein A/G agarose beads and then centrifuged to discard the

pellet. Pre-cleared supernatants were then incubated with

appropriate antibodies at room temperature for 4 h. To this

antigen–antibody complex, protein A/G agarose (Santa Cruz

Biotechnology) beads were added and kept at 4 �C overnight in a

shaker (Rotospin, Tarsons). The beads were washed three times

with ice-cold RIPA buffer without protease inhibitors. Finally,

40 ml of denaturing lysis buffer containing 0.5% SDS and gel

loading dye were added to the beads.44 The samples were boiled for

3 min before analysis by the standard western blot technique,

described in the previous section.

2.14. Live cell imaging after transfection

The expression plasmid vector pEGFP-C1 containing 53BP1

clone was transfected into HeLa cells using a transfection

reagent, TransPass� D1 (New England BioLabs Inc., Hercules,

Fig. 1 Structure of [Cu(mal)2](picH)2$2H2O.

Fig. 2 Clonogenic survivability assay of HeLa, THP1 and HepG2 cells in the

mean � S.E. of three independent experiments. The concentration profile (C)

picoline (figure in the inset) byMTT assay. Temporal kinetics of HeLa cells (D

mock treated cells in each day. Values represent the mean � S.E. of three ind

1396 | Med. Chem. Commun., 2012, 3, 1393–1405

CA), according to the instructions provided by the manufac-

turer. 24 h after the transfection, Cu(II)-complex was treated for

48 h and finally the cells were observed under a laser scanned

confocal microscope (Zeiss LSM 510 META).

3. Results

Copper complex [Cu(mal)2](picH)2$2H2O (Fig. 1) having

established supramolecular structure in the solid state36 has been

shown to bind with DNA.1 In the present study, the possible

cytotoxicity of the complex and its mechanism of action have

been explored on the human cell lines. Clonogenic survival as

well as MTT assay gave us initial impression about the cytotoxic

effect induced by the complex. Three human cancerous cell lines,

HeLa, THP1 and HepG2, were treated individually with the

increasing concentrations of the complex and then subjected to

clonogenic survival and MTT assay. As seen in Fig. 2A and C,

the percentages of cell viability were decreased with the

increasing concentrations of the complex. In solution, the

complex may generate picH+. So the purified ligand, picoline,

was also used as a control in all the experiments, where no

significant effects on cell viability were observed (Fig. 2B and the

inset figure in Fig. 2C) in the concentration ranges tested under

the same experimental conditions. From the clonogenic surviv-

ability assay, the lethal dose for 50% cell-viability (LD50) value

for the Cu(II)-complex in each cell line was estimated and these

are approximately 20 mM for HeLa, 110 mM for THP1 and

17 mM for HepG2, respectively. The concentration profiles of the

presence of Cu(II)-complex (A) and picoline (B). Each value represents the

of HeLa, THP1 and HepG2 cells in the presence of Cu(II)-complex and

) viability inMTT assay. Absorbances were normalized with respect to the

ependent experiments.

This journal is ª The Royal Society of Chemistry 2012

Fig. 3 Bar diagram (A) represents the day profile of percent apoptotic HeLa cells in annexin V-FITC staining. Values are represented in mean� S.E. of

three independent experiments. Western blot analysis (B) from the whole cell extract after treating HeLa cells with the complex for different times. The

relative intensity ratio below the first panel indicates the proportional band intensity with respect to the mock treated cells in each day.

Fig. 4 Cell cycle analysis (gated data) for HepG2 cells in increasing time

of incubation with the complex. Percentage distributions of cells in

different phases of the cell cycle are shown in the top of each figure.

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complex in MTT assay (Fig. 2C), in contrast, showed the tran-

sient effect of the complex on cell lines after 3 days of incubation.

So the cytotoxicity of the complex, in terms of its LD50 values,

was different than that of the clonogenic survival assay. Here the

estimated doses (LD50 for MTT assay) are around 60 mM for

HeLa, 43 mM for THP1 and 60 mM for HepG2 (Fig. 2C). As the

growth rate of THP1 is much higher than the other two cell lines

tested, the amount of complex required to kill 50% of THP1 cells

in the clonogenic survival assay was more than that in the MTT

assay. Thus, the extent of response induced by the complex in

terms of its cytotoxicity depends on the type of cell lines. Here

also picoline did not have any significant effect on any of the

three cell lines used (inset figure of Fig. 2C). In another MTT

assay, HeLa cells were treated with 60 mM of complex and the

cell viability was evaluated with respect to the mock treated cells

in each day. It was observed that more than 50% cells were killed

after 3 days of incubation (Fig. 2D) with the complex; although

picoline did not show any cytotoxicity within that same period of

time. Thus, the toxicity imposed by the complex is also

depending upon the time of exposure.

In order to explore the possible mode of cell killing by the

complex, we measured the level of LDH, catalase and also esti-

mated the ROS level in both the treated and mock-treated cells.

None of these parameters changed significantly compared to that

of the mock treated cells even with the highest dose or extended

time of exposure with the complex (data not shown). An elevated

level of LDH is a marker of necrosis, so the cell death in our case

was not due to necrosis.40,45

We next tried to estimate the apoptotic cell death induced by

the complex. HeLa cells were treated with 60 mM of the complex

for different times and subsequently stained with Annexin V-

FITC for the determination of apoptotic cells.40 As seen in

Fig. 3A, the percentage of apoptotic cell death sharply increased

after the third day; whereas such death was not significant for the

first two days. The induction of apoptosis is generally associated

with caspase 3 activation and PARP cleavage.46 We immuno-

blotted the proteins from the HeLa cell extract with antibodies

against PARP and caspase 3. From Fig. 3B, it is evident that the

procaspase 3 level was significantly reduced after 48 h of incu-

bation with the complex and subsequently at 72 h, most of the

caspase 3 was activated and PARP cleavage was also visible.

Usually, apoptotic cell death is associated with cell cycle arrest.47

To explore such possibility, we analyzed the cell cycle after

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treating the HepG2 cells with the complex. As seen in Fig. 4,

from 72 h onwards, a substantial amount of cells were arrested at

both S (18.59% to 25.86%) andG2/M (22.96% to 40.18%) phases.

It may be quite possible that the complex might take a long time

to enter into the cells but as soon as it enters, it induced apoptosis.

Consequently, this phenomenon might be responsible for the

delayed apoptosis observed in our earlier experiments. So we tried

to find out the intra-cellular localization of the complex with

increasing time of incubation, exploiting the intrinsic fluorescence

property of the complex, described earlier.1 As seen in Fig. 5, the

complex was clearly visible inside the HeLa cells after 15 min of

incubation and further with the increasing time, the complex

appeared to localize within the entire cells in a diffuse pattern. It is

therefore quite evident that the delayed apoptosis induced by the

complex was not due to the late entry of the complex within the

cells.

Fig. 5 Imaging of live cells exploiting the autofluorescence property of the c

fluorescence microscope through the DAPI filter.

1398 | Med. Chem. Commun., 2012, 3, 1393–1405

As the complex mainly resides within the cytoplasm (Fig. 5),

we were then interested to see the effect of the complex on the

major cytoplasmic proteins like b-actin and a-tubulin. The

labeling of b-actin (Fig. 6) by phalloidin conjugated with Alexa

Fluor 488 revealed that most of the actin filaments were desta-

bilized or damaged after 48 h of incubation in the presence of

complex (60 mM). Whereas, indirect immunolabeling of a-

tubulin (Fig. 7) showed a more intense localization of the

proteins and above all, around 50% of cells remarkably appeared

to be multi-nucleated.

Any abnormality in microtubules and/or actin filaments may

induce mitotic irregularity, where cells with more than one nucleus

can be generated.48,49 To ensure these mitotic abnormalities, the

complex-exposed HeLa cells were subjected to hematoxylin–eosin

staining and observed under a light microscope. As seen in Fig. 8A,

the complex induced multinucleated cells, which persisted even

omplex with increasing time of incubation. Cells were observed under a

This journal is ª The Royal Society of Chemistry 2012

Fig. 6 Staining of actin filaments in HeLa cells with phalloidin tagged with Alexa Fluor 488 (panel in the mid column) after 48 h of incubation with

different treatments. Panel in the right column represents their corresponding merge images with DAPI. Arrow-heads (in white) indicate damaged actin

filaments.

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after the withdrawal of the complex from the culture medium (data

not shown). The percentage of multinucleated cells produced with

increasing time of incubations with the complex is represented in

Fig. 8B.

Moreover, cells in mitotic catastrophe with decondensed

chromosomes are vulnerable for any in vivo nucleolytic attack in

their naked DNA.50 So we next tried to observe whether gH2AX

and 53BP1 were accumulated in the nuclei, as DNA breaks

rapidly accumulating these proteins in the damaged sites. We

immunolabeled HeLa cells with anti-gH2AX after 48 h of

incubation with the complex and subsequently strong gH2AX

foci were observed (Fig. 9A). We performed live cell

imaging after transfecting HeLa cells with pEGFP-C1 plasmid

expressing GFP tagged 53BP1 as well. After the transfection,

cells were treated with the complex for 48 h and then

visualized under a confocal microscope. As seen in Fig. 9B, DNA

damage-accumulated 53BP1 proteins gave distinct green fluo-

rescence foci in the nucleus of the treated cells. In both the

experiments, we used etoposide (25 mM) as a positive control,

which is known to inhibit topoisomerase II and induced

DNA damage (data not shown).51,52 Thus our results indicate

that the complex induced mitotic abnormality leading to DNA

damage.

To understand the mechanism of mitotic catastrophe induced

by the complex, we immunoblotted HeLa cell lysate to analyze

the expression of acetylated CBP/p300, acetylated H3 and

HDAC6. CBP/p300 is familiar as one of the histone acetyl

This journal is ª The Royal Society of Chemistry 2012

transferases (HATs), which plays a key role in mitosis. Any

mitotic abnormality is closely associated with the catalytic

activity of HATs, which mediates acetylation of different

downstream proteins including histones.50 Simultaneously, the

catalytic activity of CBP/p300 is also modulated by acetylation

apart from its phosphorylation in several cases.53–55 As seen in

Fig. 10, the level of acetylation at K1499 of the CBP/p300

protein was gradually reduced with the time of incubation in

the presence of complex compared to the mock treated cells in

each day. The effect was more pronounced at the third day,

where almost 40% protein was in deacetylated form compared

to the control (relative intensity ratio below the first immuno-

blot panel in Fig. 10). Being a direct substrate of CBP/p300

proteins, the acetylation status of histone H3 was also drasti-

cally reduced in the treated cells. As illustrated in the relative

intensity ratio below the second WB panel, around 66% of H3

protein was deacetylated in the third day compared to that of

the mock treated samples (Fig. 10). Conversely, differential

expression level of HDACs has been shown to modulate the

acetylation of histones in the literature.56–58 But in our case, the

HDAC6 level remained unchanged in the treated cells even on

the third day (Fig. 10). Here b-actin was blotted as a loading

control. Hence from our observations, it is clear that the

complex directly induced hypo-acetylation of CBP/p300

proteins, which in turn decreases the acetylation level of

histone H3, without hindering the overall expression level of

HDAC6.

Med. Chem. Commun., 2012, 3, 1393–1405 | 1399

Fig. 7 Immunolabeling of a-tubulin in HeLa cells after 48 h of incubation with different treatments. Panel in the left column represents the distribution

of a-tubulin (red) and the panel in the right column represents their corresponding merge images with the phase micrograph.

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It has also been reported recently that some other modifica-

tions of H2AX along with phosphorylation play a crucial role in

chromatin reorganization, which is essential for the DNA

metabolism.59 So, the acetylation status of H2AX was

Fig. 8 Hematoxylin–eosin staining (A) of HeLa cells after 48 h of incubation

cells. Percent multinucleated HeLa cells with days of incubation in the prese

represented in mean � S.E. of three independent experiments.

1400 | Med. Chem. Commun., 2012, 3, 1393–1405

investigated in our study. We immunoprecipitated H2AX from

the whole cell-extract and immunoblotted the precipitated

proteins with anti-acetylated lysine. As seen in Fig. 11A, the

acetylation of H2AX was completely absent in the cells treated

with different treatments. Arrow-heads (in black) indicate multinucleated

nce of Cu(II)-complex are represented in the bar diagram (B). Values are

This journal is ª The Royal Society of Chemistry 2012

Fig. 9 Immunolabeling of HeLa cells for gH2AX after 48 h of incubation with different treatments (A). Panel in the middle column represents the

distribution of gH2AX foci (red) and the panel in the right column represents their corresponding merge images with DAPI. Expression and distri-

butions of 53BP1 protein in live transfected HeLa cells after 48 h of incubation with different treatments (B). Panel in the left column represents the

distribution of GFP tagged 53BP1 protein (green) and the panel in the right column represents their corresponding merge images with the phase

micrograph.

Fig. 10 Immunoblot analysis from the whole cell extract after treating

HeLa cells for different time points. Relative intensity ratios below each

WB panel indicate the proportional band intensity with respect to the

mock treated cells in each day.

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with the complex for 48 h compared to that of either mock

treated or even picoline treated cells. Additionally, we used

Trichostatin A (TSA, 132 nM), a known HDAC6 inhibitor,60 as

a positive control of histone acetylation.

Fig. 11 HeLa cells were treated for 48 h with different agents and then immun

Level of acetylation in both the proteins was observed by anti-acetylated lysi

This journal is ª The Royal Society of Chemistry 2012

Further, multinucleation is closely associated with the post-

translational modification of cytoskeleton proteins like

a-tubulin.61,62 After immunoprecipitation, we observed that

(Fig. 11B) the acetylation status of a-tubulin was also completely

absent in cells treated with the complex compared to that of

either mock treated or even picoline treated cells. Moreover, TSA

co-treated with Cu(II)-complex did not affect or alter the deace-

tylation status of a-tubulin.

4. Discussion

The complex, [Cu(mal)2](picH)2$2H2O, has been shown previ-

ously to bind with DNA as a partial intercalator as well as a

partial minor groove binder.1 In the present study, the cytotoxic

effect of the complex has been explored; in which the possible

mechanism of cytotoxicity has been evaluated further. We have

seen that the complex induced mitotic abnormality followed by

apoptosis in human cell lines. Induction of cytotoxicity was not

mediated by the production of ROS or necrosis; rather the

complex interferes with cellular signaling associated with the cell

proliferation.

oprecipitated with either anti-a-tubulin (B) or anti-H2AX (A) antibodies.

ne antibody in standard WB analysis.

Med. Chem. Commun., 2012, 3, 1393–1405 | 1401

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Being a supramolecule in the solid state, we thought that the

complex might take a long time to incorporate into the cells. But

by using the intrinsic fluorescence property of the complex, our

observation on live cells indicated that the complex quickly

(within 15 min) entered into the cells (Fig. 5). Thus the solution

structure of the complex must be a simpler one, which can readily

incorporate into the cells. The complex mainly resides in the

cytoplasm even after 48 h of incubation (Fig. 5) and blocks cell-

cycle progression at both S and G2/M (Fig. 4). Thus, unlike most

of the other apoptosis inducing agents, the complex may interfere

with the activity of some important cytosolic proteins, whose

dynamics and/or activity contribute largely to the completion of

any successful mitosis. Though at higher concentrations, the

interaction of the complex with chromosomal DNA may not be

excluded, as our earlier observation suggests strongly that the

complex in vitro has modest affinity towards DNA1 and this

might be the cause of cell cycle arrest at the S phase. However,

the observed gH2AX and 53BP1 foci in the nucleus (Fig. 9)

might not be a consequence of direct DNA damage by the

complex; rather due to the accumulation of the proteins at the

sites of decondensed and vulnerable chromosomes, as reported

earlier.50 Moreover, the gH2AX and 53BP1 foci were only

observed after 48 h of incubation in the treated cells with the

complex. The signaling process, which might be the target of the

complex, is closely linked with the mitotic check point; failure of

which usually accompanied by morphological alterations in

cytoskeleton proteins, including formation of micronucleus and/

or multinucleus.63,64 Thus, cell cycle arrests (at G2/M and S in

Fig. 4) and multinucleation (Fig. 8) induced by the complex were

due to the failure and deregulation in the mitotic check point,

similar to mitotic catastrophe.65,66

However, induction of apoptosis by aberrant mitosis and/or

subsequent multinucleated state related to mitotic slippage is the

key consequence of cells treated with the complex. It has already

been reported that the acetylation–deacetylation dynamics of

histone protein, H3, plays a major role in the cell division cycle

and consequently, the role of its modulatory proteins like

HDAC6 and/or HAT activity, endowed by CBP/p300 cannot be

ignored.55,56,67 In our case, we have seen that the complex deac-

tivates histone acetyl transferase, CBP/p300, by inhibiting its

acetylation (Fig. 10), which in turn reduced the acetylation status

of its downstream protein, histone H3 (Fig. 10). Thus the

signaling events associated with the acetylation status of HAT

activity of CBP/p300 are the main target of the complex inside

the cells.

On the other hand, microtubules and actin filaments, which

play important roles in mitosis, cell signaling and cell-motility,

became the target of several anti-cancer drugs.68,69 In our study,

when the cells were treated with the complex, actin filaments

became destabilized (Fig. 6), whereas a-tubulin became highly

stabilized around the boundaries of the multinucleated cells

(Fig. 7). Evidence suggests that such differential fates await each

of the cell’s cytoskeleton components with the progression of

apoptosis.70–72 Nevertheless, the dynamics of such cytoskeleton

proteins are also regulated by post-translational modifications

and among them, acetylation of a-tubulin is mediated by

CBP/p300 protein. Here in our study, we have also observed that

the acetylation of a-tubulin was greatly reduced when the cells

were treated with the complex for 48 h (Fig. 11B). The

1402 | Med. Chem. Commun., 2012, 3, 1393–1405

acetylation of a-tubulin is indicative of microtubule stabilization

similar to the fact when cells are treated with taxol.61,62 Tubulin

heterodimers, the subunits of microtubules, are dynamic cyto-

skeletal polymers that have many important cellular functions

including the segregation of chromosomes to the daughter cells

during the process of mitosis and cell division.69,73,74 In several

reports, it has also been observed that subtle suppression of

microtubule dynamics by microtubule-targeted anti-mitotic

drugs, such as taxanes, Vinca alkaloids and estramustine,

prevents cell cycle progression at G2/M by inhibiting mitosis.75–77

Thus taken together, our complex seems to interfere with the

activity of CBP/p300 proteins, which in turn affects the acety-

lation status of histones and a-tubulin rendering G2/M arrest in

the cell cycle prior to multinucleation.

Acetylation of histone H3 and a-tubulin is also reported to be

regulated by the expression level of HDAC6 in an opposite

manner compared to the HAT activity of CBP/p300.56,57,61

Although in our study, the HDAC6 expression did not change

much in the cells treated with the complex for 72 h (Fig. 10).

Interestingly, we have also seen that the microtubules were

concentrated around the nucleus of the cells (Fig. 7) after the

treatment with the complex. So it can also be speculated that the

complex directly inhibits the catalytic activity of HDAC6 and/or

interaction of HDAC6 with microtubules tip-binding proteins,

which may affect the microtubule dynamics within the cells.78

H2AX is an evolutionarily conserved variant of histone that

differs from H2A at various amino acid residues along the entire

protein, especially at the C-terminal extensions and is one of the

key components in the chromatin structure.79 Recent studies

have shown that H2AX and other components of histone

proteins in the damaged chromatin are modified by acetylation

and ubiquitylation.80 The exact role of acetylation in H2AX is

not yet known, though some findings suggest that acetylation of

H2AX is also related to DNA repair associated with chromatin

remodeling.59 However in the present study, the said complex

induced deacetylation of H2AX (Fig. 11A), indicating a change

in histone dynamics. We used the whole cell lysate in our

immunoprecipitation experiments and showed that the acetyla-

tion status of H2AX was significantly reduced after 48 h of

treatment (Fig. 11A). Thus the complex might induce stresses

within the cells in such a manner, which led to an increase in the

soluble H2AX portion and those non-chromatin-associated

H2AX sensitized cells to undergo apoptosis.81 Moreover, exces-

sive soluble H2AX causes chromatin aggregation, which can

inhibit transcriptional co-activators like CBP/p300.81 These are

well supported by our observation, where it was clearly observed

that CBP/p300 was gradually inactivated with time by means of

deacetylation after the treatment with Cu(II)-complex (Fig. 10).

Thus, alternations in cellular signaling associated with post-

translational modifications of histone proteins and a-tubulin are

responsible for the mitotic abnormality leading to generation of

multinucleated cells, where such abnormality (MC) may be a

process (pre-stage) preceding the cell death via apoptosis (our

data and ref. 82). After entering quickly into the cells, the

complex inhibits histone acetyl transferase, CBP/p300 and

subsequently the down-stream proteins like a-tubulin, H2AX

and H3 become largely deacetylated. Such circumstances within

the cells may lead to abnormal mitosis resulting in multi-

nucleation. The induction of apoptosis has subsequently become

This journal is ª The Royal Society of Chemistry 2012

Fig. 12 Proposed mechanism of action induced by the Cu(II)-complex in

human cell lines. / indicates stimulation, Cindicates inhibition.

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apparent, as the signaling events associated with cell survival and

chromatin remodeling are perturbed completely. The proposed

mechanism of action by the complex on human cell-lines is

depicted in Fig. 12.

The solid state structure of Cu(II)-complex reveals that the

Cu(II)-malonate units are hydrogen bonded with the protonated

picoline moieties and crystal waters through the oxygen

atoms.36,83 In the culture condition (aqueous solution), under

physiological pH, the speciation would thus be [Cu(mal)2]2� and

picH+ (both hydrated).84 So our data effectively for the first time

demonstrated the detailed mechanistic study of a Cu(II)–malo-

nate complex on human cell-lines, having a cumulative charge of

(�2). The modulation of cellular signaling induced by the said

Cu(II)-complex may be deserved for considering it as a potential

chemotherapeutic agent.

5. Conclusions

The manuscript disclosed a novel observation about the bio-

logical effects of a Cu(II)-complex, [Cu(mal)2](picH)2$2H2O.

Though, in vitro, the complex binds with DNA but when applied

on human cell lines, it not only induced DNA damage but also

This journal is ª The Royal Society of Chemistry 2012

induced multinucleation and mitotic catastrophe. The complex

also interferes with other cellular processes which are necessary

for the cell survival. The molecular analysis revealed that the

complex can mainly modulate acetylation dynamics of several

proteins, like CBP/p300, histone proteins, H3 and H2AX, along

with a non-histone protein, a-tubulin.

List of abbreviations

HAT

M

Histone acetyl transferase;

HDAC6

Histone deacetylase 6;

MC

Mitotic catastrophe;

ROS

Reactive oxygen species;

53BP1

P53 binding protein 1;

GFP

Green fluorescent protein;

PARP

Poly(ADP-ribose) polymerase;

PBS

Phosphate buffered saline;

WB

Western blot;

TSA

Trichostatin A.

Acknowledgements

Ananda Mukherjee is a Senior Research Fellow of CSIR, 9/

96(0585) 2K9-EMR-I and Saheli Samanta is a Senior Research

Fellow of Indian Council of Medical Research (grant no. 3/1/

JRF/45/MPD/2004 (41414). We acknowledge Prof. Subrata

Mukhopadhyay, Department of Chemistry, Jadavpur University

for providing the Cu(II)-complex. We also acknowledge Struc-

tural Genomics Section, SINP, Kolkata, W.B., India for the flow

cytometry facility.

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