Cancer Investigation, 27:171–177, 2009ISSN: 0735-7907 print / 1532-4192 onlineCopyright c© Informa Healthcare USA, Inc.DOI: 10.1080/07357900802210752

ORIGINAL ARTICLECellular and Molecular Biology

Inhibition of Cell Proliferation, Vascular EndothelialGrowth Factor and Tumor Growth by Albendazole

Mohammad Hossein Pourgholami,1 Zhao Yan Cai,1 Lisa Wang,1 Samina Badar,1 Matthew Links,2

and David Lawson Morris1

Cancer Research laboratories, University of New South Wales, Department of Surgery, St George Hospital (SESIAHS), Sydney, Australia1

Cancer Care Center, University of New South Wales, Department of Surgery, St George Hospital (SESIAHS), Sydney, Australia2

ABSTRACT

Vascular endothelial growth factor (VEGF) is the key molecule mediating tumor growth andmalignant ascites formation. We recently reported that, in an end stage OVCAR-3 xenograftmodel, albendazole (ABZ) suppresses ascites formation, but not tumor growth. Hence, in thepresent study, we assessed the effect of ABZ on in vitro OVCAR-3 cell proliferation plus in vivotumor growth, however, initiating ABZ treatment at mid stage (3 weeks post cell inoculation)rather than end stage disease. Here, ABZ treatment led to potent inhibition of cell proliferation,VEGF suppression, complete inhibition of ascites formation and most strikingly arrest of tumorgrowth.

INTRODUCTION

Malignant tumors are characterized by uncontrolled cellu-lar proliferation. Ongoing growth necessitates adequate bloodsupply to provide oxygen and nutrients, thus making it an an-giogenesis dependent event (1, 2). VEGF plays a central role inangiogenesis, tumor growth, metastasis and malignant ascitesformation (3–6). Through interaction with its tyrosine kinasereceptors, VEGF causes inhibition of apoptosis, cell prolifer-ation, sprouting, migration, tube formation, increased vascularpermeability leading to tumor growth, and malignant ascites for-mation (7). It is generally agreed that VEGFR-2 is the major me-diator of the mitogenic, angiogenic and permeability-enhancingeffects of VEGF (8). VEGF expression is correlated to highmitotic activity, FIGO stage (9), and tumor cell proliferation(10). Consequently, anti-VEGF agents such as VEGF anti-body,

Keywords: Albendazole, Angiogenesis, Microtubule, Ovariancancer, OVCAR-3, VEGFCorrespondence to:Professor David L. MorrisCancer Research laboratoriesUniversity of New South WalesDepartment of SurgerySydney, NSW 2217Australiaemail: david.morris@unsw.edu.au

VEGF-Trap, VEGFR-2 receptor antagonists, or inhibitors of theVEGFR-2 signaling pathways and in particular tyrosine kinaseinhibitors, have been shown to be effective inhibitors of angio-genesis and tumor growth (11–15).

ABZ is a widely used oral broad spectrum benzimidazolecarbamate (BZD) anthelmintic with a good safety record (16).We have previously reported antiproliferative properties for thedrug in hepatocellular and colorectal cancer animal models (17,18). However, our recent findings linking the drug to inhibitionof VEGF ascites formation has created optimism as evidencedby the recent literature (19, 20). Using female nude mice bear-ing highly advanced peritoneal carcinomatosis, we recently, re-ported that ABZ lowers malignant ascites formation (21), how-ever no effect in tumor growth was observed.

Overwhelming evidence suggests that effective inhibition ofVEGF leads to suppression of tumor growth (2, 22, 23). How-ever, the degree of inhibition is largely dependent on factors suchas tumor type, the anti-VEGF agent employed, and very impor-tantly, disease stage at initiation of drug therapy (22, 24–26). Atadvanced stages, secretion of a whole variety of other growthfactors and cytokines exacerbate tumor growth (27, 28) makingthe anti-VEGF agent less effective. These angiogenic moleculescan emanate from cancer cells, endothelial cells, stromal cells,blood and the extracellular matrix (29). To corroborate and ex-tend the evidence presented thus far, in the present study, wespecifically sought and employed a model that is used by otherresearchers (30–32). Additionally, we sought to investigate the

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correlation between the in vitro and the in vivo anti-tumor effects.Thus, before proceeding with the in vivo study, we first exam-ined the in vitro effect of ABZ on the proliferation of OVCAR-3cells freshly obtained from the ascites of carrier mice.

METHODS

Cell preparation

Cells were collected from the ascites of carrier mice inocu-lated with human ovarian carcinoma cell line OVCAR-3. Thesecells were originally obtained from the American Type Cul-ture Collection (ATCC), and initially prepared for in vivo s.c.xenografts which were then harvested and injected i.p. into car-rier mice. After development of ascites, ascitic tumor cells werecollected, washed with phosphate buffer, and used fresh for invitro cytotoxicity assays and in vivo tumor growth experiments(21).

To ascertain that the cells collected are epithelial ovarian can-cer cells, immunostaining for the detection of CA-125 was per-formed using the method described by McCormick et al. (33).For in vitro anti-proliferative assays, cells were maintained inRPMI 1640 medium with 2 mM l-glutamine, 2 g/L sodium bi-carbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyru-vate, 0.01 mg/ml bovine insulin, supplemented with 100 units/mlpenicillin and 100 units/ml streptomycin and 10% FBS in a hu-midified atmosphere at 37◦C.

Cytotoxicity assay

Sulforhodamine B (SRB) assay (34) was used to determinethe in vitro sensitivity of the OVCAR-3 tumor cells to ABZ.Briefly, cells were plated at a density of 500 cells/well in 96-wellplates and left in the incubator for 24 h. Following attachment,cells were treated with culture medium containing various con-centration of ABZ (0.01 to 1.0 micromoles/L). ABZ was initiallydissolved in ethanol and then diluted with the medium to providethe final desired ABZ concentration and a final ethanol contentof 1%. Control cells were treated with medium containing 1%ethanol. After 72 h of exposure to the drug, cells were fixed in10% (w/v) TCA for 0 min at 4◦C followed by tap water washing(× 5) and stained with 0.4% (w/v) SRB dissolved in 1% aceticacid. Unbound dye was removed by 5 washes with 1% aceticacid before air-drying.

Bound SRB was solubilized with 100 μl 10 mM Tris base(pH-10.5) and the absorbance read at 570 nm. Absorbance read-ings from the control wells were taken as 100% and absorbancefrom the ABZ treated wells are presented as % control (mean ±s.e.m.). Each drug concentration was tested in eight wells andeach experiment was repeated at least twice. To test the effect ofmultiple dosing and longer treatment periods, Trypan blue cellviability assay was used. Briefly, cells (20,000 per well) grownin 6 well plates were treated with various ABZ concentrations(0, 0.1 and 1.0 micromoles/L) for 6 to 9 days. The cell culturemedium was changed every other day. At the end of drug treat-ment period number of viable cells remaining were counted.

In vivo study

For all experiments, 6–8 weeks old female nude athymic BalbC nu/nu mice (Animal Resources Centre, Perth, Western Aus-tralia) were used. Animals were kept under specific pathogen-free conditions and fed autoclaved pellets and sterile water adlibitum. Health status of each animal was monitored daily. Thiswork had institutional (University of New South Wales) animalethics committee approval (ACEC 03/93).

Assessment of tumor growth

Mice were inoculated i.p. with 10 × 10 6 OVCAR-3 cellsisolated from the ascites of carrier mice and suspended in 1 mlof medium. Twenty one days later, a cohort (n = 6) was randomlyselected and euthanased as day 0 controls (C0). The remainingmice were randomly allocated to 2 groups (minimum of 6 pergroup) of vehicle (1 mL, 0.5% CMC given i.p.) or drug (ABZ150 mg/kg, 1 mL given i.p.) treated groups. Before initiatingdrug or vehicle treatment, 0.2 mL of blood was collected formeach animal (the leg vein), and then animals were subjected toperitoneal lavage (2 ml of sterile normal saline injected i.p. andaspirated after kneading). Cell free ascites fluid, ascites cells,plasma collected were stored at −80◦C for analysis.

Mice in the C0 group were euthanased and their tumors wereexcised and preserved immediately. Treatment was immediatelyinitiated in the remaining 2 groups and continued thrice weeklyfor 4 weeks. Abdominal circumference, body weight, and gen-eral health of each animal were checked each time before drugadministration. At the end of treatment period, blood was col-lected through cardiac puncture, animals were euthanased usinglethobarbital i.p. injection (VIRBAC, Sydney, Australia) and im-mediately subjected to a second peritoneal lavage as describedabove. Volume of ascites (volume of peritoneal wash aspiratedminus 2 ml), total number of viable tumor cells collected fromthe peritoneal lavage and the weight of tumors dissected fromthe peritoneal cavity were recorded. All collected samples wereappropriately stored at −80◦C for subsequent analysis.

Determination of Maximum proliferationindex (MPI) and tumor marker levels

Immuno-histochemical staining of proliferating cells withKi67 antibodies has widely been used to assess tumor cellproliferation (35). To check the effect of ABZ on tumor, themethod described by McCormick et al. (33) was used withminor modifications. Briefly, paraffin embedded samples (5-μmthick) were placed on glass slides for Ki67 with staining primarymonoclonal mouse human antibodies (Dako, California, USA).

Samples were then deparaffinized in xylene, rehydratedin ascending series of ethanol and washed in Tris-bufferedphysiological saline. Ki67 positive cells were scored by usingZeiss AxioVision image analysis program (Carl ZeissVisionGMBH, Hallbergmoos, Germany). Ten randomly chosen areasfrom each sample were examined under a microscope at ×200 magnification. The cell was considered Ki67 positive ifthere was a clearly detectable brown color in the nucleus.

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The MPI was expressed as the percentage of stained tonon-stained cells in these tumor areas. In order to confirm adrug effect on the tumor growth, tumor marker levels (CA-125)in cell free ascites fluid (peritoneal wash) were determined bythe St. George Hospital Biochemistry laboratories (Sydney,Australia).

Determination of VEGF levels in plasma andascites fluid

VEGF levels in plasma and cell free ascites fluid were de-termined by means of an enzyme-linked immunosorbent assay(ELISA) according to manufacturer’s instructions, (Quanti kineR& D systems, Minneapolis, Minnesota, USA). VEGF concen-tration in the ascites fluid (peritoneal wash) was multiplied bythe volume of the aspirated peritoneal fluid collected to give thetotal VEGF value.

Protein assay

Protein content of the cell free ascites fluid (peritoneal wash)collected from mice prior to initiation of drug treatment and at theend of the drug treatment period (4 weeks later) were determinedusing a protein assay kit according to the manufacturer’s instruc-tions (Bio-Rad Laboratories, Australian subsidiary, Australia).

Data analysis and statistics

All data are reported as the mean ± S.E.M. In vitro cellproliferation data were analyzed using student’s t test followedby Tukeys. Animal data were analyzed using Mann-WhitneyU test. Effects were considered to be statistically significant atp < 0.05.

RESULTS

ABZ inhibits in vitro OVCAR-3 cellproliferation

In order to assess the effect of ABZ on tumor growth, wefirst examined the efficacy of the drug against the aggressiveOVCAR-3 cells isolated from the ascites of carrier mice bearingi.p. OVCAR-3 tumors and malignant ascites. Prior to this, im-munostaining of these cells were performed to confirm their ep-ithelial status and their high expression of CA-125 (Figure 1A).Incubation of these cells with various concentrations of ABZ inculture medium for 72 h resulted in dose-dependent inhibitionof cell proliferation (Figure 1B). Inhibition of cell proliferationwas highly significant (p = 0.001) in cells treated with 0.25micromoles/L ABZ. Cell viability of cells incubated for 3 dayswith the 1.0 micromoles/L ABZ was down to 2.07 ± 0.72% ofthe control (vehicle treated) cells (p < 0.001). Repeated treat-ment of cells with ABZ concentrations of 0.1 micromoles/L andhigher for, 3, 6 or 9 days revealed the dose and time dependencyof ABZ induced inhibition (Figure 1C).

Figure 1. OVCAR-3 cells isolated from the ascitic fluid of carriermice were used for in vitro tests. Initially these cells were immunos-tained and examined for CA-125 expression (magnification × 200).The brown staining confirms the epithelial nature of the tumor cells(A). Dose-dependent inhibition of proliferation of tumor cells in vitro.OVCAR-3 cells were treated with a single dose of ABZ (0.01–1.0micromoles/L) for 72 h and cell proliferation was measured usingMTT assay (B). Following 3, 6 or 9 days of treatment with ABZ (0.1micromoles/L or 1.0 micromoles/L), cell viability was assessed us-ing the Trypan Blue assay, (C). Columns, mean; bars, s.e.m.

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ABZ inhibits growth of OVCAR-3 tumors

This study was designed in a way where tumor growth inABZ treated mice could be compared to both the pretreatmentvalues (group C0, where a group of mice where euthanased justprior to initiation of drug treatment) and also to the group treatedwith the vehicle (Cv) for the same length of time as the drug.Mean tumor weights in the control untreated C0 group, controlvehicle treated group and ABZ group were, 0.44 ± 0.06 g, 1.96± 0.2 g and 0.65 ± 0.10 g, respectively (Figure 2). Mean tumorweight in the vehicle treated group increased dramatically overthe 4 week treatment period (p < 0.001). In contrast, over thesame period, tumor growth in ABZ treated mice was highlysuppressed (P = 0.068 compared to C0 control group and P =0.0002 compared to vehicle treated group).

ABZ reduces Ki67 staining of cells

Immunostaining of cells with Ki67 antibodies has widelybeen used for assessing tumor cell proliferation. A reduction ofKi67-defined cell proliferation has been found in patients withgood response to chemotherapy for ovarian cancer (36). To gaininsight into the action of ABZ on highly proliferating tumorcells, tumors collected from the experiment were subjected toKi67 antibody immunostaining. Percentage of proliferating cellsin ABZ treated tumors were 4. ± 5.2 % compared to 26.1 ± 7.1% in vehicle treated tumors (p < 0.05).

Total suppression of ascites production

Both tumor growth and even more so, accumulation of ma-lignant ascites fluid are VEGF dependent processes which are

Figure 2. Tumor weights in nude mice inoculated i.p. with 10 millionOVCAR-3 cells and either euthanased at 3 weeks (C0) or treatedwith the vehicle (1 ml, i.p.) or ABZ (150 mg/kg; 1 mL, i.p.) thriceweekly for 4 weeks. Values represent mean ± s.e.m. and the me-dian for each group.

inversely associated with survival (37). Here, in contrast to thevehicle treated mice, the ABZ treated animals had no macro-scopically visible sign of malignant ascites. Whereas, an aver-age ascites volume (peritoneal wash – 2 mL) of 1.95 ± 0.12 ml(mean ± s.e.m.) was collected from each of the vehicle treatedmice, 5 out of 6 mice receiving ABZ treatment were almostascites free (Figure 3A).

Inhibition of ascitic cell number, tumormarker and protein levels

At euthanasia (end of treatment period), the number of tumorcells collected from the peritoneal lavage were 144.8 millioncells and 0.12 million per mice in vehicle and ABZ treated mice,respectively (Figure 3B). Depicted in Figure 3C are the CA-125levels in the peritoneal wash of these mice prior to initiationof vehicle or ABZ therapy and at the end of the treatment pe-riod. At euthanasia CA-125 levels in the ABZ treated mice were1680 ± 116 units/ml compared to 450 ± 5284 units/ml in the

Figure 3. Ascites volume (A), number of floating tumor cells (B), tu-mor marker level (C), protein concentration (D), in vehicle (VEH orABZ treated mice, as described in Fig. 2). Numbers 1 and 2 denotepre (at 3 weeks) and post (at 7 weeks) treatment values in the samegroup of animals, respectively. Mice treated with ABZ scarcely pro-duced any ascites during the treatment period. Columns, mean;bars, s.e.m. (Continued)

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Figure 3. Continued

vehicle treated mice (p < 0.001). Excessive protein loss into theperitoneal cavity is a feature of malignant ascites brought aboutby the microvasculature hyper-permeability. Protein concentra-tions found in the cell free peritoneal wash of vehicle and ABZtreated mice are presented in Figure 3D. As expected, asciticprotein levels in the ABZ mice were extremely low.

Suppression of plasma and ascitic fluidVEGF levels

VEGF plays a central role in both tumor growth and ascitesproductions. Using a standard ELISA kit, VEGF levels weremeasured in the plasma and the peritoneal wash collected fromanimals just before initiation and at the end of drug treatment(Figures 4A and 4B, respectively). VEGF levels were highlysuppressed in both the plasma and the peritoneal ascitic fluid(p < 0.001). In vehicle treated mice, compared to pre-treatmentvalues, plasma VEGF levels had significantly surged during the4 week treatment period. In contrast, in ABZ treated mice, therewas sharp decline in plasma VEGF levels. Even more striking,while the peritoneal wash VEGF levels in the vehicle treatedmice had dramatically increased from 21840 ± 2195 pg/mL to81560 ± 19510 pg/mL, VEGF concentration in the peritonealwash of ABZ treated mice had declined from 34120 ± 11320pg/mL to levels below the assay detection limit.

Figure 4. Depicted in section A are the VEGF levels in plasma(pg/mL) before (VEH-1, ABZ-1) and after (VEH-2, ABZ-2) initiationof treatment (vehicle or ABZ). Total VEGF values in the peritonealwash before initiation of therapy and at the end of therapy (B).Columns, mean; bars, s.e.m.

DISCUSSION

This study was primarily aimed at examining the effectof ABZ on proliferation, growth, and VEGF production by achemo-resistant and highly VEGF dependant human ovariancancer cell line. ABZ was first assessed in vitro for its poten-tial anti-proliferative effects on OVCAR-3 cells, freshly isolatedfrom the ascites of carrier mice. In vitro, treatment of these cellswith various concentrations of ABZ resulted in dose-dependentinhibition of cell proliferation. Following this, the effect of ABZon in vivo tumor growth was tested. Here, treatment was initi-ated 3 weeks post cell inoculation as described by Hu et al. (24,32).

Additionally, and in order to have better assessment of theeffect of the drug on tumor growth, two sets of controls wereset up, one, which was euthanased just prior to initiation of drugtherapy (C0) and the group which were euthanased at the endof vehicle treatment (Cv). This design allowed assessment ofthe extent of VEGF and tumor growth happening during the

Inhibition of OVCAR-3 Tumor Growth by Albendazole 175

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course of treatment and hence a more precise evaluation of thedrug efficacy. Results obtained demonstrate that ABZ treatmentled to profound suppression of tumor growth. Consistent withthis, tumor proliferation index and the tumor marker levels wereboth profoundly lower in the ABZ treated mice. Furthermore,in mice receiving ABZ, plasma and ascites fluid (or peritoneallavage fluid) and the VEGF levels were all highly suppressed.

It has been shown the effect of anti-VEGF agents on tumorgrowth is variable and dependent on the extent of tumor VEGFdependency, the pharmacological manipulation, stromal VEGFcontribution and stage of the disease at the initiation of drugtherapy (27). At advanced disease stages, tumors begin toproduce a wider array of angiogenic molecules both VEGFrelated such as VEGF-C, placental growth factor (PIGF) andalso a range VEGF-independent molecules thus leading toextensive tumor growth and the eventual development ofresistance to anti-VEGF therapy (28, 38).

Our results demonstrate that, how over time both ascites andplasma VEGF levels increase in these mice. On the other hand,while tumor weights in ABZ treated mice were much lowerthan the weights in vehicle treated group (Cv), they were notsignificantly different from the pretreatment group (C0 controlgroup). This indicates that, while ABZ treatment had suppressedfurther tumor growth but it had not induced regression of theexisting tumor mass. This seems consistent with the suggestionthat, the greatest role of antiangiogenic agents may be in theprevention of new blood vessel formation rather than resorptionof existing tumor vessels (39).

In addition to inhibition of tumor growth, other highly VEGFdependent factors such as, ascites formation, ascitic tumor cellnumber, ascitic protein concentration, were all dramatically sup-pressed in ABZ treated mice. Malignant ascites formation is ahighly VEGF dependent process, and a consequence of neo-vascularization and increased vascular permeability originat-ing not only from tumor vessels but also from the vascula-ture of the peritoneal lining (5, 40). Here, at the end of thetreatment period, mice treated with ABZ were largely free ofascites. While this can be partly attributed to suppression ofthe tumor growth, the degree of inhibition seen here is rathersuggestive of impediment of protein and fluid loss through thevessels lining the peritoneal cavity, a consequence of the low-ered VEGF levels in both the peritoneal cavity and the systemiccirculation.

In summary, we have shown for the first time, that the po-tent inhibitory effect of ABZ on OVCAR-3 tumor growth,correlate well with both its in vitro antiproliferative effectsand its in vivo anti-VEGF property. These results indicate adual anti-tumor role for ABZ, affecting both tumor cell pro-liferation as well as potently inhibiting VEGF formation. Onthis basis, ABZ may be of value in a regimen for treat-ment of women with ovarian cancer and malignant ascites.This notion is supported by the recent developments showingstriking increased interest in the synthesis and evaluation ofnew benzimidazole analogues as anti-VEGF anti-cancer agents(19, 20).

ACKNOWLEDGMENT

This work was supported in part by a grant-in-aid from the“Lady Fairfax Foundation.”

ABBREVIATIONS

ABZ albendazole;MPI maximum proliferation indexSRB Sulforhodamine B assay;VEGF vascular endothelial growth factor;

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