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Cite this: Dalton Trans., 2014, 43,6070

Received 28th October 2013,Accepted 3rd December 2013

DOI: 10.1039/c3dt53030b

www.rsc.org/dalton

The inhibitory effects of a new cobalt-basedpolyoxometalate on the growth of human cancercells†

Lu Wang,a,b Kai Yu,a Bai-Bin Zhou,*a Zhan-Hua Su,a Song Gao,a Li-Li Chua andJia-Ren Liu*c

A new cobalt-based polyoxometalate, (Himi)2[Bi2W20O66(OH)4Co2(H2O)6Na4 (H2O)14]·17H2O (imi = imi-

nazole) (BWCN) has been synthesized and structurally characterized. The inhibitory activities against

selected human cancer lines were also determined in this study. The cell viability and chemoresistance of

BWCN on human colon carcinoma HT-29 cells were assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyl tetrazoliumbromide), cell morphology changes, a comet assay and western blot analysis. The

typical morphologic changes of apoptosis and DNA damage indicated that BWCN could have a distinct

proliferation inhibitory effect on cancer cells. BWCN as a chemotherapeutic agent also induced apoptosis

on HT-29 cells and showed a significant expression of cleaved-caspase-3. These results suggested that

the active site of BWCN is the polymeric anion based on the basic tectonic block {BiW9}, and the possible

mechanism is related to the interference of DNA synthesis in cancer cells.

Introduction

Colorectal cancer is the third most common visceral malignancyand remains one of the leading causes of cancer- related deathsdue to therapy resistance.1 Worldwide colorectal1 cancer affectsover 1 million people every year and is responsible for over5 million cancer-related annual deaths.2 These patients are typi-cally offered palliative surgery and chemotherapy. Chemotherapyhas become an important method in tumor treatment. Most ofthe chemotherapeutic agents investigated in clinical applicationsand experimental exploration mainly consist of synthetical com-pounds and adjuvant chemotherapeutic natural substances orphotochemicals. However, the synthetical compounds are thepriority due to their effectiveness. In 2000, fluorouracil (5-FU) inconjunction with leucovorin (LV) remained the mainstay first-

line treatment for metastatic colon cancer.3,4 In 2001, the DNAtopoisomerase inhibitor, irinothecan (CPT-11), was added to thecombination of 5-FU and LV. In 2002, the addition of oxaliplatinresulted in a further increase in the survival of colon carcino-genesis and metastasis.5,6 Other chemotherapeutic agents suchas bevacizumab,7 isoreserpine,8 cisplatin,9 digalloylresveratrol,10

AEE788 and celecoxib11 have also been reported to potentiatethe effects in colon cancer.

As is known, chemotherapy is an important treatment for avariety of clinical malignancies. However, chemotherapy isalso well known for its visible side-effects. Globally, severalresearch groups are working towards the effective treatment ofcolon cancer.12–15 The novel therapeutic intervention isneeded to increase the survival rate in patients with advancedcolorectal cancer and to improve the efficacy of current chemo-therapeutic treatments while minimizing drug-induced toxici-ties. In recent years, many researchers have raised widespreadconcerns about the significant pharmaceutical properties ofthe metal complexes. The considerable novel efficiency andlow toxicity metallic compounds with antitumor activity weresynthesized constantly, such as bismuth complexes16–19 andpolyoxometalates,20 etc. In particular, polyoxometalates inten-sively attracted the attention coming from scholars in the med-icinal realm due to their great molecular diversity of elementand structure, and biological activities manifested in antivirusand antitumor properties. A myriad of polyoxometalate(POM) analogs have been designed to overcome tumor cellresistance. In 1965, Mukherjee reported that a combination of

†Electronic supplementary information (ESI) available: summary of crystal data,selected bond lengths and angles, W-183NMR, XPS, IR, TG, cyclic voltammetricbehaviors, stability studies in aqueous media, and cif files for BWCN. CCDCnumber 730764. For ESI and crystallographic data in CIF or other electronicformat see DOI: 10.1039/c3dt53030b

aKey Laboratory of Synthesis of Functional Materials and Green Catalysis Colleges of

Heilongjiang Province, School of Chemistry and Chemical Engineering, Harbin

Normal University, Harbin, Heilongjiang 150025, the People’s Republic of China.

E-mail: zhou_bai_bin@163.com; Tel: (+86) 0451-88060770bDepartment of Biochemical Engineering, Harbin Institute of Technology, Harbin,

Heilongjiang 150090, the People’s Republic of ChinacBoston Children’s Hospital and Harvard Medical School, 300 Longwood Ave,

Boston, MA 02115-5737, USA. E-mail: Jia-Ren.Liu@childrens.harvard.edu

6070 | Dalton Trans., 2014, 43, 6070–6078 This journal is © The Royal Society of Chemistry 2014

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phosphotungstic acid, H3[PW12O40], phosphomolybdic acid,H3[PMo12O40], and caffeine was used on patients sufferingfrom carcinoma of the intestinal tract.21 The biological activi-ties of the polymeric anion [SiW12O40]

4− against mousesarcoma virus were first reported about 40 years ago, sincethen, the researchers successively discovered that a great classof polymeric anions such as [BW12O40]

4−, [P2W18O62]6− and

[As2W18O62]6− exhibited high inhibitory activities against

various viruses and tumors in vitro and in vivo.22 In 1992, Na[IMo6O24], as an anti-colon cancer drug, was applied clinicallyin Japan.23 [NH3Pri]6[Mo7O24]·3H2O (PM-8) exhibited tumorgrowth inhibition against CO-4 human colon cancer xeno-grafted on CD-1 mice.24 In 2002, Wang25 synthesizedK4H3[(CH3CO2CH2CH2Sn)3(a-GeW9O37)]·10H2O, the isomers oforganotin substituted Keggin polyoxotungstates, and reportedthe antitumor activity of isomers. In 2010, the potential anti-proliferative effects of studied molybdenum(VI) complexes,[MoO2L(CH3OH)]·CH3OH(L = 2-hydroxy-3-methoxybenzalde-hyde), were tested by using an MTT test in human colon carci-noma SW620 cell lines.26 The antitumor mechanism ofpolyoxometalates is mainly based on the redox reaction.27

Studies have also shown that the anticancer activities of com-pounds with the same polyanion and organic metal groupwere positively correlated with the oxidizing ability of the poly-anions, whether organic metal monosubstituted, polysubsti-tuted or forming sandwich structure compounds.28 Cobalt isan excellent transition metal element to replace polyoxometa-lates, and it has been reported that considerable polyoxometa-lates substituted by cobalt with a unique structure andalterable molecular properties have been synthesized.29–34

However, chemical inhibitors of cobalt-based polyoxometalatesof trilacunary Keggin-type tungstobismuthates modified withiminazole (imi) with preclinical anti-colon tumor activity as asingle agent with chemotherapeutics have not been reported.

Based on our previous research works,35 using Na2WO4, Bi-(NO3)3, imi and CoCl2 as raw materials, a cobalt-based polyoxo-metalate, (Himi)2[Bi2W20O66(OH)4Co2(H2O)6Na4(H2O)14]·17H2O (BWCN), is synthesized by a conventional aqueous solu-tion, structurally characterized by elemental analysis, IR, TGand single-crystal X-ray diffraction, and practiced in coloncancer HT-29 cells as a chemotherapeutic agent. In this study,BWCN was synthesized and structurally characterized, and itwas shown to inhibit cell proliferation and induce apoptosis incancer cells.

ExperimentalMaterials and physical measurements

Fluorescent DNA dye Hoechst 33342, MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide), Ethy-lene diamine tetra acetic acid disodium salt (EDTA), anddimethylsulfoxide were purchased from Sigma Chemicals Co.(St. Louis, MO). Rabbit polyclonal antibodies for β-actin (sc-1616-R) and cleaved-caspase-3 (sc-7148) were bought fromSanta Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-

rabbit (w3960) secondary antibodies was purchased fromPromega.

Elemental analyses (C, H and N) were performed on aPerkin-Elmer 2400 CHN elemental analyzer. Na, Bi, W and Coanalyses were analyzed on a PLASMA-SPEC (I) ICP atomicemission spectrometer. The 183W-NMR spectra were recordedon a Bruker DRX 500 instrument at 16.67 MHz using D2O as asolvent in 10 mm tubes. As references (external standards), weused aqueous solutions of 1 mol L−1 Na2WO4. X-ray photo-electron spectrum (XPS) analyses were performed on a VGESCALAB MK II spectrometer with a Mg Kα (1253.6 eV) achro-matic X-ray source. The vacuum chamber was maintained at6.2 × 10−6 Pa during analysis. The IR spectrum was recorded inthe range 400–4000 cm−1 on an Alpha Centaurt FT/IR Spectro-photometer using KBr pellets. Diffuse reflectance UV-visspectra (BaSO4 pellets) were obtained with a Varian Cary 500UV-vis NIR spectrometer. TG analyses were performed on aPerkin–Elmer TGA7 instrument in flowing N2 with a heatingrate of 10 °C min−1. Electrochemical measurements were per-formed with a CHI660 electrochemical workstation. A conven-tional three-electrode system was used. The working electrodewas a glassy carbon electrode. Platinum gauze was used as acounter electrode and Ag/AgCl was used as reference electrode.The morphologies and sizes of the products were characterizedby using Hitachi S-4800 field emission scanning electronmicroscopy (FE-SEM) and TECNAI G2 F20 S-TWIN high-resolu-tion transmission electron microscope (TEM). Single cell gelelectrophoresis (SCGE) was assessed by fluorescencemicroscopy (Olympus IX70, Japan).

Synthesis BWCN

1.55 g (4.7 mmol) Na2WO4·2H2O was dissolved in 40 mL de-ionized water, then stirred and heated to 50 °C. 0.2425 g(0.5 mmol) Bi(NO3)3·5H2O dissolved in 1 mL 6 mol L−1 HCl,0.1162 g (0.7 mmol) CoCl2·2H2O, and 0.0681 g (1 mmol) solidimidazole were added to the above solution at the same time,and adjusted to pH = 6.8 with a 0.25 mol L−1 NaOH solution.The mixed solution was heated to 80 °C. The reaction was con-tinued for 2 h. The solution was then cooled to room tempera-ture and filtered. The pink block crystal were obtained a fewdays later. The yield was 41.6% (based on Bi; mg%). (H2imi)2-[Bi2W20O70Co2(H2O)6Na4 (H2O)14]·2H3O·15H2O (BWCN)(6152.74) was calculated: C 1.17, H 1.37, N 0.91, Co 1.92, W59.76, Bi 6.79, Na 1.49; found C 1.15, H 1.39, N 0.92, Co 1.89,W 59.95, Bi 6.67, Na 1.44. IR (KBr pellet, cm−1): 3386(br),2967(br), 1621(s), 1434(s), 1061(m), 944(s), 8191(s), 632(s).

X-ray crystallography

The crystal data were collected on a Bruker Smart CCDdiffractometer with Mo Kα radiation (λ = 0.71073 Å) at 273 K.Semi-empirical absorption correction was applied. The struc-tures of BWCN were solved by direct methods and refined onF2 by the full-matrix least squares using the SHELXTL-97 crys-tallographic software.36,37 Anisotropic thermal parameterswere refined to all of the non-hydrogen atoms. The hydrogenatoms were held in calculated positions on the carbon atoms

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and nitrogen atoms, and the hydrogen atoms on the oxygenatoms were directly included in the final molecular formula onthe water molecules and protonated oxygen atoms. The crystal-lographic data and structure refinement were provided in theESI, Table S1.† Selected bond lengths and angles were listed inthe ESI, Table S2.† CCDC number 730764 contains the sup-plementary crystallographic data for this paper.

Cell culture

Human gastric adenocarcinoma SGC-7901 cells, human colonadenocarcinoma HT-29 cells, and human liver adeno-carcinoma HepG-2 cells were obtained from the Cancer Insti-tute of Chinese Academy of Medical Science (Beijing, China).The BWCN solution was adjusted by dissolution in 10% fetalbovine serum (FBS) RPMI 1640 medium (GIBCO) or DMEMmedium (GIBCO). SGC-7901 cells and HT-29 cells were main-tained in 10% FBS RPMI 1640 medium, and HepG-2 cells weremaintained in 10% FBS DMEM medium supplemented with100 IU mL−1 penicillin, 100 μg mL−1 streptomycin and 2 mmolL−1 glutamine, at 37 °C in a 5% CO2 atmosphere. The mediumwas changed every three days, and cells were passaged with0.25% trypsin. The cells were plated at a density of 1 × 106

cells per 100 mm culture dish and allowed to grow to approxi-mately 70% confluence before experimentation.

Cell viability

The effects of BWCN on the cell viability were determined inSGC-7901, HT-29 and HepG-2 cells as described a previousstudy.38–40 Briefly, cells were seeded in a 96-well microtiterplate (Nunc, Wiesbaden, Germany) at 1.0 × 104 cells per well.After 24 h of incubation, the cells were treated with 200 μL ofmedium containing various concentrations (40, 80, 160, 240and 320 μmol L−1) of BWCN for 24 h. For each concentration,five replicates were employed. Twenty microliters of a 5 mgmL−1 MTT solution was added to each well and the cells wereincubated for 4 h at 37 °C. After careful removal of themedium, 200 μL of dimethyl sulfoxide (DMSO) was added toeach well, and the plate was then shaken for about 10 minuntil the crystals were solubilized. The absorbance was thenmeasured at 490 nm in a microplate reader (Elx800 UniversalMicroplate Reader, Bio-Tek Instruments, USA). The inhibitoryrates of BWCN were calculated in SGC-7901, HepG2 or TH-29cells. The effects of the clinical anti-colon cancer drugs,5-fluorouracil (5-Fu) and oxaliplatin (OXA) on the cell viabilityof HT-29 cells were also determined. The inhibitory rate wascalculated using the following equation: inhibitory rate (%) =(ODcontrol − ODtreatment)/ODcontrol × 100%.

Morphological observation

Apoptosis was assessed on the changes of nuclear morphologyby staining with Hoechst 33342 dye (Sigma). The cells wereexposed to BWCN for 24 h, washed with PBS and then incu-bated with 1 μg mL−1 Hoechst 33342 for 30 min at 37 °C. Thecellular morphology was evaluated by fluorescence microscopy(Olympus IX70, Japan).41

SEM and TEM

For scanning electron microscopy (SEM), HT-29 cells weregrown on 20 mm2 coverslips in a 6-well microtiter plate (Nunc,Wiesbaden, Germany) at 1.0 × 104 cells per well until con-fluency, and scanning electron microscopy was performed onHT-29 cells treated with 160 μmmol L−1 of BWCN for 24 h. Thesamples were fixed with 2.5% glutaraldehyde dissolved in0.1 mol L−1 sodium cacodylate for 2 h, post-fixed in osmiumtetroxide, dehydrated with graded ethyl alcohol series,immersed in isoamyl acetate, dried under CO2 critical point,and finally pasted on the stage for specimen. The plating filmswere examined in a scanning electron microscope. For trans-mission electron microscopy (TEM), HT-29 cancer cells weretreated with 160 μmol L−1 of BWCN for 24 h. The cells wereharvested, fixed, post-fixed, dehydrated as described above,and finally embedded in Epon. Ultrathin sections were stainedand observed in a transmission electron microscope.42

SCGE assay

100 μL of 1% normal agarose was spread on cleansed anddried slides, and cooled at 4 °C for solidification. Cells wereharvested and mixed with 1% low melting agarose at 37 °C,and then 100 μL of a cell suspension was covered on the top ofthe first gel, fixed and split in lysate (10 mmol L−1 Tris, 1%Triton X-100) for 1 h at 4 °C away from light, electrophoresedin an buffer, and finally stained with 50 μL of ethidiumbromide (20 μg mL−1).43

Western blotting

After the treatment with BWCN for 24 h, the total protein ofthe HT-29 cells were extracted in the lysis buffer following themethod of a previous study.44 The total protein (50–80 µg) wasseparated in 10% polyacrylamide gels (SDS-PAGE). The gelswere transferred to a nitrocellulose membrane. The membranewas blocked in blocking buffer (1% BSA and 0.1% Tween-20 in20 mmol L−1 TBS, pH 7.6) for 1 h at 37 °C in a hybridizationoven (Amersham, Life Science), then incubated with cleaved-caspase-3 or β-actin primary antibodies in blocking buffer over-night at 4 °C. After washing with TBST 3 times, the mem-branes were incubated with the corresponding IgG HRPconjugate secondary antibody at 37 °C for 1 h. The membranewas washed two times with TBST, and then washed with TBS.The membranes were then incubated with alkaline phospha-tase-conjugated IgG. The protein bands were detected with theWestern Blue Stabilized Substrate for alkaline phosphatase(Promega).

Statistical analysis

The data were expressed as means ± S.D. Three independentexperiments were performed in this study. The difference wasanalyzed using the One-Way ANOVA with the Bonferroni posthoc multiple comparisons to assess the difference betweenindependent groups. The data analyses were generated usingSPSS for Windows Version 19.0 (SPSS Inc., Chicago, IL).

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A statistical significance was set at P < 0.05, and all P valueswere unadjusted for multiple comparisons.

Results and discussionStructure of BWCN

The crystal structure of (Himi)2[Bi2W20O66(OH)4Co2(H2O)6Na4-(H2O)14]·17H2O is shown in Fig. 1. A 1-D chain built up of thesandwich anions {Co2(H2O)6[W(OH)2]2(β-B-BiW9O33)2}

6− andthe two Na+ ions is shown in the ESI, Fig. S1 and S2.† In thechain, the coordination geometries of the two Na+ ions areoctahedral: the bridging one is coordinated by four waterligands and two μ2-O atoms from two neighboring sandwich-type {Co2(H2O)6[W(OH)2]2(β-B-BiW9O33)2}

6− anions, andfurther linked a Na(H2O)

6+ complex group by three bridgingwater ligands, resulting in a 1-D chain by double W–O–[Na2]–O–W bridges. Additionally, the independent organic imi justlocates in the window formed by two sandwich polyanions andtwo [Na2] linkers (ESI, Fig. S1†). In a sandwich-type unit, twoidentical [B-β-BiW9O33]

9− moieties are linked by two corner-sharing WO6 octahedra and two octahedral CoO3(H2O)3groups to form the Krebs-type structure, {Co2(H2O)6[W(OH)2]2-(β-B-BiW9O33)2}

6− (ESI, Fig. S2†).Bond valence sum (BVS) calculations45 confirm that all of

the Bi and W centers are in the oxidation states of +3 and +6,respectively. The Co and Na cations in the BWCN compoundsare in the +2 and +1 oxidation states, respectively. In addition,the BVS calculations were used for the water molecules linkedto the Co and Na centers (0.22 to 0.34). BVS also shows valuesof 1.21 and 1.06 for O6 and O8 (only linked to W10), respect-ively, which indicate that they are hydroxyl groups. Moreover,two extra protons should be added to the two isolated imimolecules of the BWCN compounds for charge balance. Thuscompound 1 can be formulated as (Himi)2 [Bi2W20O66(OH)4-Co2(H2O)6Na4 (H2O)14]·17H2O.

The polyanion BWCN used in 183W-NMR studies is dia-magnetic in solution. The room-temperature 183W-NMRspectra of BWCN exhibit six line patterns at −55.25, −91.97,

−116.21, −128.03, −164.02, and −326.87 ppm with the relativeintensity ratios 2 : 2 : 2 : 2 : 1 : 1 (see Fig. S3†). This result indi-cates that in solution a species with C2h symmetry is present,46

which is in agreement with the solid state structure of BWCN.Furthermore, the NMR spectrum does not change even aftertwo weeks, indicating the high stability of BWCN in theaqueous solution.

XPS spectroscopy

The oxidation states of W, Bi and Co are further confirmed byXPS spectra, which were carried out in the energy regions ofW4f7/2, W4f5/2, Bi4f7/2, Bi4f5/2, Co2p1/2, and Co2p3/2 respectively(Fig. S4†). The XPS spectra gave two peaks at 34.9 eV and37.3 eV which are attributed to the W6+ ions. The peaks at782.2 eV and 800.0 eV are assigned to the Co2+ ions, while thepeaks at 158.0 eV and 164.2 eV are ascribed to the Bi3+ ions.47

The oxidation states of them are in accordance with thevalence bond calculations.

IR spectroscopy

The IR spectra exhibited prominent characteristic peaks forthe sandwich-type structure at 944, 819, and 632 cm−1, whichcan be ascribed to the (WvOd), (W–Ob–W), (W–Oc–W)vibrations of the polyoxoanion cluster. The characteristicpeaks concerned with coordinated imi ligands are at 1434(w)and 1061(m) cm−1. The strong peak at 1621 cm−1 can beassigned to isolated solvent water molecules. Peaks at2967–3386 cm−1 were assigned to the N–H and O–H vibrations(Fig. S5†).48

TG analysis

The TG curve presented a two-step weight-loss process(Fig. S6†). The first step corresponded to the loss of isolatedand coordinated water molecules (calculated weight loss10.82%; found 10.64%) in the temperature range 39–295 °C.The second step corresponded to the loss of the organic imiligands (calculated weight loss 2.32%; found 2.21%) between434 and 556 °C.

Cyclic voltammogram analysis

The cyclic voltammogram of 1.0 mmol L−1 of BWCN wasdetected in a pH 4 (0.4 mol L−1 CH3COONa + CH3COOH)buffer solution (Fig. S7†). The compound (vs. Ag/AgCl) at ascan rate of 20 mV s−1 appeared with mean peak potentials[E1/2 = (Epa + Epc)/2] of +831(I–I′) and −437(I–I′) mV. The Redoxpeaks I–I′ and II–II′ corresponded to two consecutive one-elec-tron processes of the W atoms.49

Stability studies

To investigate the stability of the compound, BWCN wasdetected in a pH 6.8 aqueous solution by situ UV spectroscopy.As shown in Fig. S8,† the UV spectrum of BWCN in anaqueous solution displayed two absorption bands centered atca. 210 nm and 250 nm, which respectively was attributed tothe Od→M and Ob, Oc→M charge transfer transitions. Withthe prolonged time of the placed solution, the UV spectroscopyFig. 1 The position of the organic imi molecule in compound BWCN.

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of BWCN from day 1 to day 14 had no obvious change. Thisresult indicated that BWCN could stably exist for the researchon drugs at ambient temperature. It is well known that POMsare commonly sensitive to the pH value of the studied media.The influences of the pH value on the stability of BWCN in anaqueous solution have also been elaborately probed by meansof UV spectroscopy (Fig. S9†) Scrutinizing the variable pro-cesses adjusted using HCl or NaOH solution allows us to ascer-tain that BWCN is stable in the pH range of 5.53–8.05.

The growth inhibition effect of BWCN on human carcinomacells

The effects of BWCN on the growth of three selected cancercells were examined using an MTT assay. BWCN could inhibitthe growth of human carcinoma cells, including SGC-7901,HT-29 and HepG-2 cells (Fig. 2). Due to the fact that coloncancer increases annually, the wide spread crowd, the therapyresistance and the scarcity of efficient drugs, HT-29 cells weretested in this study. As shown in Fig. 2a, the comparative

results showed that the inhibitory rates of BWCN in HT-29cells was greater than that in the HepG-2 cells, and less thanthat in the SGC-7901 cells treated with the same concen-trations (40, 80, 160, 240 and 320 μmol L−1) for 24 h. Theinhibitory rates of the HT-29 cells were increased by 16.20,46.94, 78.23, 89.11 and 91.84% in 40, 80, 160, 240 and320 μmol L−1 of BWCN, respectively, when compared with thecontrol group. Moreover, the inhibitory rate of BWCN in theHT-29 cells was significantly higher than those of tungstates,bismuthates, cobalt and the organo-ligand imi. It seems thatthe high inhibitory effects of BWCN are less caused by initialmetallic or organic materials (Fig. 2b). In addition, the inhibi-tory effect of BWCN in HT-29 cells was also used to compareothers such as 5-fluorouracil (5-Fu) and oxaliplatin (OXA). Thedata clearly indicates that BWCN at the same dose level signifi-cantly inhibits the cell viability in comparison with 5-Fu andOXA (Fig. 2c).

The effect of BWCN on morphological changes in HT-29cells. Apoptosis is a mode of programmed cell death and is animportant biological consequence of exposure to extrinsicagents.50–54 It is a complex process regulated by a variety offactors.55,56 The cell morphologic observation of BWCN wasperformed using inverted light microscope and fluorescencemicroscopy. After observations with an inverted light micro-scope treatment with 160 μmol L−1 of BWCN for 24 h, HT-29cells began to show shrinkage, rounding, and fragmentationin comparison with the control cells (Fig. 3a). In addition,morphologic changes in the nuclei were observed after stain-ing with Hoechst 33342 by fluorescence microscopy (Fig. 3b).Typical morphologic changes of apoptosis including chroma-tin condensation, nuclear fragmentation, and apoptotic bodyformation appeared in HT-29 cells treated with 160 μmol L−1

of BWCN for 24 h.

Fig. 2 The inhibitory rates of BWCN in HT-29, SGC-7901 and HepG-2cells. The cells were exposed to different concentrations of BWCN for24 h, and the cell viability was determined by an MTT assay. The data arepresented as mean ± S.D. (n = 5). (a) The inhibitory rates of BWCN inHT-29, SGC-7901 and HepG-2 cancer cells. (b) The inhibitory rates ofBWCN and others in HT-29 cells. (c) The inhibitory rates of BWCN, 5-Fuand OXA in HT-29 cells. * p < 0.05, compared to the control group ofthe organic imi molecule in the BWCN compound.

Fig. 3 The morphologic changes of BWCN in HT-29 cells. (a) Theimages were captured by an inverted light microscope (Leica DM IRB).The HT-29 cells began to show shrinkage, rounding and fragmentationin contrast with the untreated cells (original magnification 200×). (b) TheHT-29 cells were treated with 160 μmol L−1 of BWCN for 24 h. The cellswere stained with Hoechst 33342 and visualized under a fluorescencemicroscope (original magnification 200×).

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The effect of BWCN on the ultramicrostructural changes inHT-29 cells

The ultramicrostructure of BWCN in HT-29 cells was alsoinvestigated in this study. As shown in Fig. 4, the SEM electro-micrographs (Fig. 4a and b) showed distinct morphologicalchanges corresponding to a typical cellular surface mor-phology of apoptosis, which includes cell membrane blebbing,microvilli disappearance or reduction, and separated apoptoticbodies. Untreated HT-29 cells have shown a restoration of thetypical morphological features of colon carcinoma cells whichinclude numerous microvilli on their surface with membraneconnections. Cell shrinkage, increased cellular granularity, theformation of apoptotic bodies, dilated nuclear membranes,vacuoles in the cytoplasm and DNA fragmentation wereobserved in the apoptotic cells by TEM (Fig. 4c and d). Simi-larly, the characteristic morphology of apoptosis was alsoobserved in those cells treated with 160 μmol L−1 of BWCN.Untreated cells showed normal morphology with randomly dis-tributed organelles and nuclei with finely granular and uni-formly dispersed chromatin and a single large electron-densenucleolus.

The effect of BWCN on HT-29 cell apoptosis

Single cell gel electrophoresis (SCGE) was assessed basing onthe impairment levels of DNA in the cell nuclear by stainingthe cells. DNA damage in the nuclei was observed by a cometassay (Fig. 5). The HT-29 cells were treated with 160 μmol L−1

of BWCN for 24 h and the DNA damage was evaluated follow-ing ethidium bromide staining using fluorescence microscopy.Compared with the control cells, the BWCN-treated cells

induced DNA effusion in the HT-29 cells show a large comettail length for typical characteristics of apoptotic cell death.

Caspase-3 expression

Cleaved-caspase-3 is the final executant for cell death in apop-tosis induction by various triggers. Therefore, cleaved-caspase-3 expression was measured in HT-29 colon carcinoma cellsafter BWCN treatment for 24 h by Western blotting. As show inFig. 6, cells susceptible to BWCN-induced apoptosis showedactivation of caspase-3 indicated by the increased expressionin the level of procaspase-3 and procaspase-3 cleavage. Inresponse to apoptotic stimuli, procaspase-3 becomes cleavedinto a 20 kDa fragment, and the subsequent autocatalytic reac-tion leads to the formation of the 17 kDa fragment.

Colon cancer is the third most common cancer in theworld, especially in developed countries, and at least 40% ofpatients with colorectal cancer develop metastases.57,58

Chemotherapeutic agents, which can lead to the activation ofcell death programs, should prove effective for the moleculartherapy of cancer.59–63 Our study also showed the prospects ofPOMs in molecular therapy of colon carcinoma, and revealedthat POMs of trilacunary Keggin-type tungstobismuthates withimi suppressed cell proliferation and the possible correlationswith their ability to induce apoptosis.

Apoptosis, known as a programmed cell death, is an impor-tant biological consequence of exposure to extrinsicagents.50,51,53 The induction of apoptosis is an importanttarget for cancer therapy.64,65 In this study, caspase-3expression was determined in human colon carcinoma HT-29cells treated with POMs. The results demonstrate that afterexposure to BWCN, cleaved-caspase-3 expression was found tobe up-regulated. Our data suggested that BWCN induces apop-tosis in HT-29 cells eventually through the activation ofcaspase-3.

Apoptotic cell death is controlled by pro-apoptotic caspases,proteases that are synthesized as inactive precursors and acti-vated by proteolytic processing.66 The apoptotic cascade ampli-fication reactions of caspases are initiated via the followingtwo major pathways: a mitochondrial pathway (the release ofcytochrome C from the mitochondria)67 and the death recep-tor pathway (the activation of death receptors in response to

Fig. 5 The effect of BWCN on DNA damage in HT-29 cells by SCGE. (a)Untreated HT-29 cells (original magnification 400×). (b). HT-29 cellstreated with 160 μmol L−1 of BWCN for 24 h show a large comet taillength.

Fig. 4 The ultrastructural changes of BWCN in HT-29 cells. (a) The SEMshows untreated HT-29 cells (original magnification 10 000×). (b) SEMshows HT-29 cells treated with 160 μmol L−1 of BWCN for 24 h. Thevolume of treated cells decreased, and microvillus on the surface elimi-nated. Invaginations and globular projections emerged on the surface ofapoptotic cells. Bulks of spherule formation split from apoptotic cells(original magnification 7000×). (c) TEM shows untreated HT-29 cells(original magnification 10 000×). (d) TEM shows HT-29 cells treated with160 μmol L−1 of BWCN for 24 h. The treated cells became small andround. The nuclear volume decreased. The chromatin became con-densed and diffused in the nuclei. Finally, the cell membrane dis-appeared and the entocyte spilled (original magnification 5000×).

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ligand binding).68 Upon the triggering of either pathway, cas-pases, the final executioners of apoptosis, are activated,causing the degradation of cellular proteins.69 The majority ofchemotherapeutic agents trigger the mitochondrial pathway.70

Among the proteins in the caspase family, caspase-3 is thefinal executioner enzyme associated with cell death duringstimuli-induced apoptosis.71 Once activated, caspase-3 is freeto initiate the various processes involved in apoptosis.72–74 Inthe present study, BWCN at 0.1 and 160 μmol L−1 for 24 h pro-moted the expression and activation of caspase-3, and caspase-3 produced cleaved caspase-3 (17 KD and 20 KD) fragments inHT-29 cells. These results indicate that BWCN increased thecaspase-3 activity in the HT-29 cells. Some similar activationsof caspase-3 have been reported in colon carcinoma cells inthe presence of other chemotherapeutic agents.75 However, thecaspase-3 protein has not previously been involved in POM-induced apoptosis studies in tumor cells by in vivo andin vitro. These results suggest that POM-induced apoptosis byactivation of caspase-3 would contribute to the novel approachof chemotherapy for colon carcinoma. Further study needs tobe conducted which correlates the mitochondrial pathway forapoptosis and the development of novel drug-delivery systemsusing POMs.

Conclusions

In summary, a novel cobalt-based polyoxometalate, (Himi)2-[Bi2W20O66(OH)4Co2(H2O)6Na4(H2O)14]·17H2O (BWCN), was

synthesized and structurally characterized, based on cobalt-based polyoxometalate of trilacunary Keggin-type tungstobis-muthates modified with imi. BWCN could inhibit the cell via-bility of human cancer cells. BWCN also induced apoptosis incolon carcinoma HT-29 cells by morphological changes andthe activation of caspase-3. Thus, new potential candidatetherapeutics from POM chemicals is an important field fordevelopment into novel colon cancer therapies. However, thepossible mechanism of BWCN on apoptotic induction inHT-29 cells needs to be further studied.

Acknowledgements

This work was supported the National Natural Science Fund ofChina (NSF, no. 20971032 and 21271056), the SpecializedResearch Fund for the Doctoral Program of Higher Education(20122329110001), the NSF of Heilongjiang Province (no.C201037, B201216), the Postdoctoral Science Foundation ofHeilongjiang (LBH-Z10292). Bai-Bin Zhou and Jia-Ren Liu areco-corresponding authors. The authors declare no competingfinancial interest.

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Fig. 6 The expression of caspase-3 and β-actin in the HT-29 cells treated with different concentrations of BWCN for 24 h. The cell lysates wereseparated on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, and probed with anti-β-actin, or anti-cleaved-caspase-3 antibodies.The protein content was normalized by β-actin. * p < 0.05, compared to the control group.

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