RESEARCH ARTICLE

Disodium pentaborate decahydrate (DPD) induced apoptosisby decreasing hTERT enzyme activity and disrupting F-actinorganization of prostate cancer cells

Mehmet Korkmaz & Cigir Biray Avcı & Cumhur Gunduz &

Duygu Aygunes & Burcu Erbaykent-Tepedelen

Received: 30 July 2013 /Accepted: 16 September 2013# International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Animal and cell culture studies have showed thatboron and its derivatives may be promising anticancer agents inprostate cancer treatment. Thus, DU145 cells were treated withdisodium pentaborate decahydrate (DPD) for 24, 48, and 72 hin order to investigate the inhibitor effect and mechanisms ofDPD. Then, cell proliferation, telomerase enzyme activity, actinpolymerization, and apoptosis were detected by WST-1 assay,qRT-PCR, immunofluorescence labeling, and flow cytometry,respectively. We found that DPD inhibited the growth of hu-man prostate cancer cell line DU145 at the concentration of3.5 mM for 24 h. Our results demonstrated that 7 mM of DPDtreatment prevented the telomerase enzyme activity at the rateof 38 %. Furthermore, DPD has an apoptotic effect on DU145cells which were examined by labeling DNA breaks. With7 mM of DPD treatment, 8, 14, and 41 % of apoptotic cellswere detected for 24, 48, and 72 h, respectively. Additionally,immunofluorescence labeling showed that the normal organi-zation of actin filaments was disrupted in DPD-exposed cells,which is accompanied by the alteration of cell shape and byapoptosis in targeted cells. Taken together, the results indicatethat DPDmay exert its cytotoxicity at least partly by interferingwith the dynamic properties of actin polymerization and

decreasing the telomerase activity. Eventually, for the first time,the results of this study showed that DPD suppressed theactivity of telomerase in DU145 cells, and therefore, we sug-gested that DPD could be an important agent for its therapeuticpotential in the treatment of prostate cancer.

Keywords Prostate cancer . Boron . Pentaborate . hTERT .

Actin polymerization . Apoptosis

Introduction

Prostate tumors are one of the major types of cancer becauseof the high incidence and mortality rates [1, 2]. Due to highheterogeneity of the tumors and the high proliferation rate ofcells, this type of cancer has led to the investigation of manyanticancer molecules. However, drugs which are being used totreat tumors cannot be sufficient for reasons such as theheterogeneity of tumors, fundamentally different origin, andstages of the target cells. Therefore, new molecules that aremore efficient, more specific, and have better ranges of ther-apeutic effect are being explored.

Like other solid tumors, prostate cancer is thought to bedeveloped by a multistep process involving successive geneticevents that a normal cell becomes increasingly neoplastic [3,4]. After the formation of the prostatic intraepithelial neoplasia(PIN) lesions, prostate cancer has been developing a charac-teristic that leads to stromal invasion and following hormone-dependent or independent lymph node and bone metastases[5–7]. Different treatment options have been developed for thevarious stages of hormone-dependent and/or independent pros-tate cancer. Androgen ablation remains the most effectivetherapeutic tool for the treatment of prostate cancer, althoughandrogens today are largely inhibited by pharmaceutical agents

M. Korkmaz (*)Department of Medical Biology, Faculty of Medicine, Celal BayarUniversity, Manisa 45030, Turkeye-mail: mehmet.korkmaz@cbu.edu.tr

C. B. Avcı :C. Gunduz :D. AygunesDepartment of Medical Biology, Faculty of Medicine, EgeUniversity, Bornova Izmir 35100, Turkey

B. Erbaykent-Tepedelen (*)Department of Molecular Biology and Genetic, Faculty of Scienceand Letter, Avrasya University, Trabzon 61010, Turkeye-mail: burcu_erbaykent@yahoo.com

Tumor Biol.DOI 10.1007/s13277-013-1212-2

such as flutamide and bicalutamide [3, 8]. Initially, androgendeprivation therapy leads to a decrease in cell proliferation andtumor size, but later, hormone-independent metastatic prostatecancer reoccurs in 2–3 years which is highly aggressive [9–11].Hormone-independent prostate cancer, which leads to death,constitutes a problem to be solved in the clinic, and presently,there are no curative treatments available for resistant tumors,as the efficacy of standard cancer treatments decreases once atumor becomes independent [12, 13].

Despite that the expected outcome of the antihormonetherapy is not adequate enough, some other anticancer mole-cules are combined to treatments for these tumors [14–16].Boron and its derivatives, which exist among these anticancermolecules, apparently have diverse effects through influenc-ing a cell signaling system or the formation and/or the activityof the molecules which are involved in many biochemicalprocesses [17]. The antiproliferative effect of boron deriva-tives by reducing cancer cell viability leads them to be partic-ularly suitable candidates for a promising new anticancertherapy.

Boron that was absorbed by the digestive and respiratorysystems almost at the rate of 100 % remained at fairly constantlevel in the blood and tissues via homeostatic mechanismarranged by the kidney while transformed into B(OH)3 (boricacid) and quite small amount of B(OH)4

− (borate) [17–19].Boric acid acts as a Lewis acid and has a strong tendency toform tetracovalent compounds with organic molecules thathave adjacent hydroxyl (cis-diols) groups, and therefore, thebiochemical essentiality of boron element is due to boric acid[17, 20, 21]. This characteristic of boric acid allows it to act asan effective inhibitor of enzymes such as peptidases, prote-ases, and proteasomes [13, 22, 23]. In recent years, epidemi-ological, animal, and laboratory studies have shown promis-ing results about the preventive and therapeutic effects ofboron derivatives for some types of cancer, especially of theprostate [20, 24]. In addition to the cellular functions of boron,these studies have also shown the anticarcinogenic feature ofboron derivatives [17, 20, 22, 23].

The early study of Barranco and Eckhert has reported thatboric acid completely inhibited the growth of DU145 prostatecancer cell line [25]. Then, Meacham et al. have shown thatboric acid markedly inhibited the growth of DU145 cells andpartially LNCaP cells at a concentration of 1 mM [26]. Inanother study, it was found that the concentration of boric acidfor inhibition of proliferation was neededmore than four timesin LNCaP cells, and therefore cellular mechanisms werefound to be important for antiproliferative effects [27].Recently, it has been reported that adhesion, migration, andinvasion capacity were decreased due to changes in F-actinorganization, and proliferative capacity was decreased withchanges in gene expression of MAPK proteins with boric acidtreatment in DU145 cells [20]. Furthermore, the changes inactin distribution and reduction in the activity of many

proteins such as Rho family of GTPases, which play a rolein cell migration and spread, have also been demonstrated inprostate cancer cells DU145 treated with boric acid and/orphenylboronic acid [13].

Previous studies from our lab have shown that disodiumpentaborate decahydrate has a greater potential as an inhibitorof cancer cell viability than its parent compound, boric acid.Therefore, the purpose of this study was to evaluate theantiproliferative effect of disodium pentaborate decahydrate(DPD) on prostate cancer and to find out whether the suppres-sion of proliferation was related to the blocking of telomeraseenzyme activity. The effects of DPD on telomerase enzymeactivity were shown by human telomerase reverse transcrip-tase (hTERT) Quantification Kit. In the hormone-independentprostate cancer cell line DU145, apoptotic effects of DPDwere investigated by flow cytometry. Our results demonstrat-ed that DPD had antiproliferative and apoptotic effects onprostate cancer cell line DU145. Cell morphology is deter-mined primarily by the arrangement of actin cytoskeleton, andthis provides an understanding on signal mechanism of amigrating cell. Structural alterations in shape and size likelycontribute to the inability of the cells to proliferate. Thus, wehypothesized that DPD could interfere with the dynamic stateof actin polymerization, and we found that DPD disrupt F-actin organization in a dose-dependent manner. For the firsttime, by this study, we showed that DPD suppressed theactivity of telomerase in prostate cancer cells. The results ofthis study showed the importance of DPD for its therapeuticpotential in the treatment of prostate cancer.

Materials and methods

Cell culture

Hormone-independent human prostate cancer cell line DU145was purchased from American Type Culture Collection(ATCC). DU145 cells (passage numbers 10–20) were propa-gated in DMEM HAM'S-F12 (Invitrogen, UK) mediumsupplemented with 10 % FBS (Invitrogen, UK), 1 %L-gluta-mine, and 1 % penicillin–streptomycin in a humidified incu-bator at 37 °C 5 % CO2. When the cells reach the confluency,they were removed from cell culture plates with trypsin–EDTA solution and subcultured for further experiments.

Cell treatment

A stock solution of disodium pentaborate decahydrate(Na2O·5B2O3·10H2O; National Boron Research Institute,BOREN) at a concentration of 0.5 M was prepared in dH2Oand stored at +4 °C until use. Required concentrations (0.25,0.5, 1, 2.5, 5, 7.5, and 10 mM for 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazoliom]-1.3-benzene disulfonate

Tumor Biol.

(WST)-1 assay) were freshly prepared by diluting the stocksolution in culture medium immediately before use.Twenty-four hours after seeding, the cells were treatedcontinuously with appropriate concentrations of DPD for24, 48, and 72 h in culture conditions (37 °C 5 % CO2).Control cells were incubated under the same conditions with-out DPD.

WST-1 assay

Determination of IC50 dose of DPD in DU145 cells wereperformed by using WST-1 assay (Roche Diagnostics) asindicated in the manufacturer's instruction. Cell culture wascarried out in 96-well plates, and cells were incubated for 24 hwithout DPD. After addition of DPD, cells were incubated for24, 48, and 72 h, and cell proliferation was assessed by using aWST-1 kit. The colorimetric assay is based on the cleavage ofthe tetrazolium salt, WST-1, by mitochondrial dehydroge-nases in viable cells. WST-1 solution (20 μL/well) was addedto cells in 96-well plates followed by incubation for 2 h at37 °C. The plate was read spectrophotometrically at 440 nmusing a microplate reader (Bio-Rad, Coda, Richmond, CA).The viability of the cells was calculated as the percentage ofWST-1 reduction. The absorbance of control cells (subjectedto the same procedure) was assumed as 100 %. The 50 %lethal doses were calculated by the GraphPad Prism 5.0 pro-gram (GraphPad Software).

Determination of hTERT activity

Fifty microliters of total RNA was isolated from the cellculture of DU145 cells which were untreated and treated with3.5 and 7mM for 24, 48 and 72h by using High Pure RNAIsolation Kit (Roche, Germany). hTERT mRNA quantifica-tion was performed with commercially available LightCyclerTeloTAGGGG hTERT Quantification Kit (Roche AppliedScience) by using the qRT-PCR. All subsequent quantificationsteps were carried out according to the manufacturer'sinstructions.

Immunofluorescence labeling and microscopy

For the detection of F-actin distribution, DU145 cells grownon cover slips were treated with increasing concentrations ofDPD for 48 h. At the end of the incubation time, cells wererinsed in PBS and fixed in 4 % paraformaldehyde for 1 h atRT. After rinsing with PBS, cells were permeabilized with0.2 % triton X-100 containing PBS and were blocked beforeprimary antibody incubation for 5 min with 1 % BSA in PBS.Rinsed cells were labeled with F-actin by using phalloidin-conjugated to Alexa Fluor 488 (5 U/ml; Invitrogen, MolecularProbes) for 1 h. Cells were washed four times with PBS,mounted with 0.5 μg/ml 4,6-diamidino-2-phenyindole

(DAPI)) containing 30% glycerol in PBS, and analyzed usinga Leica DMIL fluorescent microscope (Leica, Germany)immediately.

Evaluation of apoptosis by flow cytometry

To evaluate apoptosis in DPD-treated cells, a single-stepstaining assay for labeling DNA breaks with FITC-dUTPwas done using an APO-DIRECT Kit (BD Pharmingen).For this experiment, DU145 cells were seeded in six-wellplates. After 24 h, the media were removed and replaced withDMEM-F12 containing DPD at a concentration of 3.5 and7 mM. Control and DPD-treated cells were incubated for 24,48, and 72 h and, at the end of each time point, cells wereharvested using a trypsin–EDTA solution. After centrifuga-tion, the cell pellet was resuspended in 500 μL PBS.Paraformaldehyde (1 %) in PBS was then added to the cellsuspension and incubated for 15min on ice. After two times ofwashing with PBS, the cell pellet again was resuspended in70 % ethanol and stored at −20 °C. Cell samples were thenpelleted, washed, and resuspended in 50 μL of staining solu-tion containing a reaction buffer, terminal deoxynucleotidyltransferase enzyme, FITC-labeled deoxyuridine triphosphatenucleotides, and deionized water. After 60 min of incubationat 37 °C, cells were again washed, pelleted, and resuspendedin 250 μL of propidium iodine/RNase solution and incubatedfor 30 min in RT. The protocol was provided by the manufac-turer and followed accordingly including the use of positiveand negative controls. Apoptosis was evaluated using BDAccuri C6 Flow Cytometry (Becton–Dickinson, USA) utiliz-ing a 488-nm argon laser light source for excitation and a 530-nm band-pass filter for FITC fluorescence (FL-1A) and afluorescence detector equipped with a 585/42 band-pass filterfor FL2A. A total of 20,000 events were acquired for analysisusing Cell Quest Software.

Results

DPD shows an antiproliferative effect on DU145 cells

The sensitivity of DU145 cells to the cytotoxic effect of DPDwas investigated by WST-1 assay. Our results indicated thatcell proliferation and viability decreased significantly as DPDconcentrations increased when compared to untreated controlsshown in Fig. 1. IC50 values (half-maximal concentration ofgrowth inhibition) of DPD in DU145 cell line were calculatedas 3.5 mM for 24 h and 2.1 mM for 48 and 72 h by theGraphPad Prism 5.0 program. Consequently, a dose- andtime-dependent antiproliferative effect was observed in thehormone-independent DU145 cell line exposed to DPD.

Tumor Biol.

DPD leads to a decrease in the hTERT activity

Telomerase activity in DU145 cell line shows decreasingpatterns with the treatment of DPD compared to untreatedcontrol cells (Fig. 2). While there was no significant change intelomerase activity caused by IC50 dose of 3.5 mM, 7 mM ofDPD treatment has resulted in a decrease at the rate of 38 and25 % for 48 and 72 h, respectively (p ≅0.048). However,telomerase activity shows insignificant amounts of increasesfor 24-h treatments (p ≅0.254).

DPD disrupts F-actin organization in a dose-dependentmanner

Previous studies have demonstrated that boric acid and deriv-atives such as phenylboronic acid havemultiple mechanisms ofaction on prostate and breast cancer cells that involve cellularand morphological changes or decreased cell migration andviability [13, 20, 23]. Therefore, we hypothesized that DPD caninterfere with the dynamic state of actin polymerization, and

immunofluorescence labeling were performed by staining fila-mentous (F)-actin (phalloidin) to determine intracellular cyto-skeletal actin distribution. As shown in Fig. 3, randomly dis-tributed F-actin condensation emerged in the cells treated withincreasing concentrations of DPD (Fig. 3b–f), while controlcells (Fig. 3a) possessed a developed actin cytoskeleton withclearly visible, smooth structure. We observed distinct changesin cell morphology and organization of actin filaments withDPD treatment. Many cells reduced their volume (Fig. 3b, c)and intercellular contacts which have few stress fibers(Fig. 3d(I)) and show diffuse actin staining (Fig. 3e(III)) anddisrupted actin organizaton (Fig. 3c(II)). DPD treatment in-duced a flattened and angular morphology in DU145 cells thatcould indicate an effect on cell migration and metastasis(Fig. 3e, f). The differences in cell size and actin distributionare direct effects of the DPD exposure because cells wereimaged at the same magnification and exposure settings. As aresult, it was suggested that DPD may be limiting the tumormetastasis by preventing cancer cell migration via disruption ofactin cytoskeleton.

DPD-induced apoptosis in a dose- and time-dependentmanner

To determine whether the inhibition of cell growth decreasesin hTERT activity or disruption of F-actin organization wasclosely related to apoptosis, we analyzed apoptotic cells by asingle-step staining method for labeling DNA breaks usingflow cytometry. DU145 cells were exposed to 3.5 and 7 mMDPD for 24, 48, and 72 h. According to a 7-mM DPDconcentration, the percentage of cells staining positive in-creased to 8, 14, and 41 % for 24, 48, and 72 h, respectively,whereas at 3.5 mM DPD, 6, 7, and 32 % apoptotic cells weredetected for 24, 48, and 72 h, respectively (Fig. 3a, b).Eventually, evaluation of flow cytometry results showed thatthe percentage of apoptotic cells increased gradually togetherwith DPD treatment in time- and dose-dependent manner.

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Fig. 1 Effects of DPD onDU145cell viability assessed by theWST-1 assay for 24, 48, and 72 h.Increasing concentrations of DPDlead to a significant decrease incell growth compared withcontrols. Cell viability expressedas percentage of control values.The WST-1 assay was performedusing triplicate samples in at leasttwo independent experiments

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Fig. 2 Effects of DPD on hTERT mRNA levels. Expression levels ofhTERT were determined by qPCR (LC-480, Roche) in DU145 cellstreated with 3.5 and 7 mM DPD for 24, 48, and 72 h, and variations inthe telomerase activity for each group are plotted as relative ratios byusing GraphPad program. qPCR analysis were performed at least threetimes for each concentration

Tumor Biol.

Discussion

Recent studies provide evidence that boron may haveanticarcinogenic and anti-inflammatory properties [17,20–23]. Boron that is absorbed by the digestive and respira-tory systems almost at a rate of 100 % present in aqueoussolution as either boric acid (B(OH)3) or borate (B(OH)4

-)[17–19]. It was evidenced that more than 90 % of boroncompounds which were absorbed by the organism are rapidlyexcreted via urine within a few days [18, 28]. Therefore thepharmacological properties of boron and/or boric acid such aslow toxicity and not to accumulate in the body as well as anatural constitute of the human blood leads to be an attractiveanticancer agent [13, 23].

DPD, which is a new boron derivative, is thought to bea more effective anticancer agent than boric acid dependingon a higher rate of boron content. Thus, initially theantiproliferative effect of DPD on prostate cancer cell line

DU145 was investigated, and cytotoxicity was assessed bythe colorimetric assay in which viable cells cleaved theWST-1tetrazolium salt. The results, which were obtained from thecalculation of the cell viability as the percentage of WST-1reduction, showed the extent of growth inhibition in a time-and dose-dependent manner via DPD treatment. AlthoughIC50 values were calculated as 3.5 mM for 24 h and 2.1 mMfor 48 and 72 h, survival of DU145 cells was suppressed bymore than 70 % in 7 mM of DPD treatment. In this study, wewanted to have information about the toxicity limits of thisnew boron compound, DPD, in cell culture studies aswell as clarify the mechanism of antitumoral activity.Eventually, determination of the dose to be used inin vivo studies was carried out by taking into consider-ation the data obtained from both this manuscript andprevious epidemiological and animal studies. In thismanner, the dose level which can be adapted in vivostudies will be determined.

Control 0.5mM DPD 1mM DPD

3.5mM DPD 5mM DPD 7mM DPD

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DAPIDAPI DAPI

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Fig. 3 DPD-induced changes in F-actin distribution following 48 h ofexposure in DU145 cells. DU145 cells plated on cover slips were treatedwith increasing concentrations of DPD. Cells were stained with 488-phalloidin for F-actin (green) and DAPI for the nucleus (blue). Cellswere imaged at a ×60 objective at identical exposure settings withimmunofluorescence microscope. Images of DPD-treated (0.5–7 mM)

cells show F-actin retraction. a Untreated control cells. b–f Images fromcells treated with 0.5, 1, 3.5, 5, and 7 mM DPD, respectively. Immuno-fluorescence experiments were performed twice, and at least 20 imageswere taken for each treatment. (I) Loss of stress fibers, (II) disrupted actinorganization, and (III) enlarged cell with a diffuse actin staining

Tumor Biol.

Telomerase is a ribonucleoprotein enzyme which has anactivity of leading uncontrolled cell proliferation and immor-tality by constantly adding telomeric repeats to the 3′ strand ofchromosomes. Many studies have indicated positive correla-tion between telomerase activity and hTERT which is a cata-lytic subunit of telomerase [29, 30]. We have found that 7 mMof DPD treatment inhibited the expression of telomerase bydecreasing hTERT level. hTERT ratio of the incubatedDU145 cell line with DPD decreased at the rate of 38 and25 % for 48 and 72 h, respectively (Fig. 2). However, telo-merase activity showed insignificant amounts of increases inthe first 24 h; hTERT ratio gradually and significantly de-creased in the following days. An increase in telomeraseactivity can be observed due to an alternative mechanismdeveloped by the cells to bypass the growth inhibition ofDPD. Additionally, protein phosphatase 2A is a negativeregulator of telomerase activity, and PP2A activation resultsin the downregulation of telomerase activity [30]. Therefore,the other reason can be suppression of PP2A activity. On the

other hand, DPD shows the most effective antiproliferativeeffect particularly in 48 h, the period where hTERT ratiopresents the most prominent decrease (Figs. 1 and 2).

Suppression of telomerase activity was not the sole mecha-nism by which DPD affected cell proliferation. F-actin is animportant cytoskeletal factor in cell migration and invasion, andits disruption can induce apoptosis [20, 31]. Cell morphology isdetermined primarily by the arrangement of the actin polymer-ization and can offer insight into the effect on metastasis-relatedaspects of cancer cells [13, 20]. Additionally, Barranco et al.demonstrated that prolonged exposure to pharmacologicallyrelevant levels of boric acid induced the morphological andbiochemical changes in DU145 cells, which were characteristicof apoptosis [20]. We observed distinct changes in cell mor-phology and organization of actin filaments with DPD treat-ment (Fig. 3) and is accompanied by increased amounts ofapoptosis (Fig. 4). In our study, the time- and dose-dependentincreases of apoptotic cells were clearly visible in APO-DIRECT analysis (Fig. 4a, b). These data indicate that DPD

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Fig. 4 APO-DIRECT analysis of DPD-treated DU145 cells by flowcytometry. A total of 20,000 events were acquired for analysis using CellQuest Software. FL-1A scale shows FITC-conjugated dUTP incorpora-tion in apoptotic DNA, and FL2A scale shows propidium iodide (PI)staining of DNA molecule. a DU145 cells staining positive for apoptosisare included in the R2 regions of the histograms. The small background is

population of control (untreated) cells staining positive. A dose-depen-dent population shift was observed from control to DPD-treated cells for24, 48, and 72 h. b Graphs show the percentage of DU145 cells stainingpositive for apoptosis obtained from APO-DIRECT analysis, and thepopulation of apoptotic cells was increased with dose and timedependence

Tumor Biol.

could be selectively influence actin polymerization, and thesemorphological changes could be due to the reduction of theforce required for cell adhesion and migration. Therefore, theobserved disruption of F-actin organization in DPD-treatedcells suggests a reduced capacity of cell invasion and metasta-sis, and the structural alterations are likely contributing to theinability of the cells to proliferate. Combining the results of thisstudy, it can be suggested that one of the mechanisms of theantitumor activity of DPD could be through the induction ofapoptosis via inhibiting the expression of telomerase ordisrupting F-actin organization of prostate cancer cells.Therefore, we suggest that DPD may be an important agentfor its therapeutic potential in treatment of prostate cancer byinducing apoptosis. Although the complete mechanism forapoptosis is not clear, for the first time, we have shown by thisstudy that DPD suppresses the activity of telomerase in DU145cells. Cytotoxic effect on tumor cells or decreasing the activityof proteins such as focal adhesion kinase (FAK), RhoA, Rac1,Cdc42, and MAPK are some of the suggested mechanisms toexplain the apoptotic effect of DPD [13, 17]. However,further studies are needed to show which signal mechanismsare responsible for apoptosis and what are the direct target(s)of DPD.

Additionally, boric acid acts as a Lewis acid and formsester complexes with hydroxyl groups of organic compoundswhen the hydroxyl groups are in cis orientation and adjacent.In this way, boron interacted with the molecules on the cellsurface and may have affected the integrity of the cell mem-brane and signaling pathways. This property also results inboron as boric acid-forming complexes with several biologi-cally important sugars, including ribose. Cell culture studiesshow that boric acid binds to and is a reversible inhibitor ofcyclic ADP ribose [17]. Actin is one of the most abundantproteins in eukaryotes, where it is found throughout the cyto-plasm. Themost commonly found forms of actin, compared toall the possible combinations, are ATP-G-Actin and ADP-F-actin [32, 33]. Additionally, plant findings also have led to thehypothesis that boron could act as a cellular signal that inter-acts with transcription factors and thus affects the expressionof some genes [17]. Consequently, we thought that there is aneed for higher doses or longer time exposures for transcrip-tional repression mediated by transcription factors in nucleusand a decrease in the level of hTERT mRNA compared toalteration of ADP-F-actin in the cytoplasm. Hence, DPDeffect on actin polymerization is observed with reduced con-centration (3.5 mM). To date, studies have indicated thatmembrane organizations such as focal adhesion formationand actin filament organization were altered by telomeraseknockdown [34, 35]. Also, they have found that antisense-hTERTmay render the cancer cells more vulnerable to stimuliand induce apoptosis [35]. Therefore, we do not consider thatthe events are independent. However, it should be noted thatthe direct link between the hTERT and F-actin organization

must be more complex, so further studies are required toclarify this complex situation.

Furthermore, the feature of boron that forms ester com-plexes act as an effective inhibitor for enzymes such as prote-ase, peptidase, and nitric oxide synthase [13, 22, 23]. Thus, itwas thought that the migration and invasion capacity weredecreased because of the destruction of extracellular matrixwhich was prevented due to the inhibition of serine proteasesby DPD. Eventually, the varying levels and activities of ex-tracellular matrix proteins, such as Tenascin-C, and serinproteases, such as Hepsin, should be assessed in further stud-ies in the presence of DPD.

Acknowledgments We would like to thank the National Boron Re-search Institute, BOREN, for providing DPD. This research was support-ed with grants (2009-92 and 2010-91) from BAP projects by the CelalBayar University.

Conflicts of interests No competing interests are declared by any of theauthors.

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