European Journal of Cancer (2014) 50, 1391– 1398

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Frondoside A enhances the antiproliferative effectsof gemcitabine in pancreatic cancer

0959-8049/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ejca.2014.01.002

Funding: These studies were funded by a grant from the Terry Fox Cancer Fund for Research in Cancer Therapy.⇑ Corresponding author: Address: Department of Physiology, College of Medicine and Health Sciences, United Arab Emirates Univers

Box 17666, Al Ain, United Arab Emirates. Tel.: +971 713 7551; fax: +971 7671966.E-mail address: tadrian@uaeu.ac.ae (T.E. Adrian).

J. Al Shemaili a,d, E. Mensah-Brown b, K. Parekh a, S.A. Thomas a, S. Attoub c,B. Hellman d, F. Nyberg d, A. Adem c, P. Collin e, T.E. Adrian a,⇑

a Department of Physiology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emiratesb Department of Anatomy, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emiratesc Department of Pharmacology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emiratesd Department of Pharmaceutical Biosciences, Uppsala University, Swedene Coastside Bio Resources, Stonington, Maine, USA

Available online 22 January 2014

KEYWORDS

Pancreatic cancerFrondoside AGemcitabine

Abstract Pancreatic cancer has a very poor prognosis. While gemcitabine is the mainstay oftherapy and improves quality of life, it has little impact on survival. More effective treatmentsare desperately needed for this disease. Frondoside A is a triterpenoid glycoside isolated fromthe Atlantic sea cucumber, Cucumaria frondosa. Frondoside A potently inhibits pancreaticcancer cell growth and induces apoptosis in vitro and in vivo. The aim of the present studywas to investigate whether frondoside A could enhance the anti-cancer effects of gemcitabine.Effects of frondoside A and gemcitabine alone and in combination on proliferation were inves-tigated in two human pancreatic cancer cell lines, AsPC-1 and S2013. To investigate possiblesynergistic effects, combinations of low concentrations of the two drugs were used for a 72 htreatment period in vitro. Growth inhibition was significantly greater with the drug combina-tions than their additive effects.Combinations of frondoside A and gemcitabine were tested in vivo using the athymic mousemodel. Xenografts of AsPC-1 and S2013 cells were allowed to form tumours prior to treat-ment with the drugs alone or in combination for 30 days. Tumours grew rapidly in pla-cebo-treated animals. Tumour growth was significantly reduced in all treatment groups. Atthe lowest dose tested, gemcitabine (4 mg/kg/dose), combined with frondoside A (100 lg/kg/day) was significantly more effective than with either drug alone.To conclude: The present data suggest that combinations of frondoside A and gemcitabinemay provide clinical benefit for patients with pancreatic cancer.� 2014 Elsevier Ltd. All rights reserved.

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1. Introduction

Pancreatic cancer is a disease with a dismal prognosisand little hope for cure because of delayed diagnosis andlack of effective therapies [1,2]. The mean survival timeafter diagnosis is about 5 months and even the subgroup(less than 20% of patients) in whom potentially curativesurgery is an option, those patients rarely survive morethan two or three years [2]. Gemcitabine is the mainstayof therapy for pancreatic cancer patients in neoadju-vant, adjuvant and palliative treatment protocols. How-ever, while this drug improves quality of life, survival isonly prolonged for about 1 month [3]. Thus, new thera-peutic strategies are urgently needed for pancreatic can-cer patients [3].

Frondoside A is a triterpenoid glycoside isolatedfrom the Atlantic Sea Cucumber, Cucumaria frondosa

[4,5]. Frondoside A has potent anti-proliferative, anti-invasive and anti-angiogenic effects on a variety of can-cers including adenocarcinomas of the pancreas, breast,lung, colon and prostate as well as in leukaemia [4–6].Frondoside A has been shown to block cancer cellgrowth and induce apoptosis both in vitro as well as inhuman cancer xenografts in athymic mice [4–6].

The aim of the present study was to investigatewhether the combination of frondoside A with gemcita-bine could be more efficacious than either drug alone inthe inhibition of growth of pancreatic cancer bothin vitro and in the xenograft model. The pancreatic can-cer cell lines chosen for these studies, AsPC-1 and S2013are both highly malignant and produce rapidly growingtumours in athymic mice.

2. Material and methods

2.1. Cell lines and cell cultures

Frondoside A was produced from ethanol extracts ofthe epidermis of C. frondosa harvested from the Gulf ofMaine, with subsequent column chromatography as pre-viously described [7]. Two cell lines were used, AsPC-1and S2-013. AsPC-1 is a poorly differentiated cell linederived from a nude mouse xenograft initiated with cellsfrom the ascites of a patient with pancreatic cancer(American Type Culture Collection (Manassas, VA)[8]. S2013, a subclone of SUIT-2, is a well-differentiatedcell line derived from a liver metastasis of human pan-creatic cancer [9]. The S2-013 cells were cultured in Dul-becco’s Modified Eagles Medium (DMEM) and theAsPC-1 cells were grown in RPMI. Both media weresupplemented with 10% foetal bovine serum, penicillin(100 units/ml), streptomycin (100 lg/ml) (Gibco, GrandIsland, NY, United States of America (USA)) at 37 �Cin humidified air with 5% CO2 for 24 h. Cells were thenwashed twice with PBS, transferred into serum-freemedia and incubated for 24 h in 24-well plates, and

treated in fresh serum-free media with different concen-trations of frondoside A or gemcitabine, or combina-tions of both, and incubated for 72 h. Cells wereharvested by incubation in trypsin–ethylene diaminetetra-acetic acid (EDTA) solution for 15 min. Then cellswere centrifuged at 2000 g for 4 min and cell pellets weresuspended in fresh culture medium prior to measuringcell proliferation.

From pilot experiments it was clear that we couldonly see synergistic effects of the two drugs in vitro ifvery low concentrations of each drug were used. Con-centration response experiments were, therefore,designed to identify concentrations of frondoside Aand gemcitabine that would produce statistically signif-icant inhibition of proliferation of between 15% and25%.

2.2. Cell proliferation assays

Cell proliferation was measured by counting thenumber of viable cells (Guava ViaCount) on GuavaEasyCyte Plus cytometer (Millipore, Hayward, CA).

2.3. Animals and subcutaneous tumour cell implantation

AsPC-1 and S2013 cells were seeded into 75 cm2

flasks and cultured in a humidified atmosphere of 95%O2 and 5% CO2 at 37 �C, with media changes everyother day. The tumour cells were trypsinised, cell num-ber counted (Guava EasyCyte Plus Cytometer) andresuspended in PBS.

For cancer xenografts 6–8 week old athymic NMRInude mice (nu/nu, Charles River, Suizfeld, Germany)were bred in the animal facility. The mice were housedin micro-isolator cages in a filtered-air laminar flow cab-inet (EuroBioConcept, Paris, France), handled underaseptic conditions and fed with autoclaved laboratoryrodent food pellets. The animal protocol was approvedby the Institutional Animals Care and Use Committeeand all procedures were conducted to conform withInstitutional Guidelines that are in compliance with Col-lege of Medicine & Health Sciences, National and Inter-national Laws and Policies (EEC Council Directive 86/609, OJ L 358, 1, 12th December 1987; and NIH Guidefor Care and Use of Laboratory Animals, NIH Publica-tion No. 85–23, 1985).

Three million AsPC-1 or S2013 human pancreaticcancer cells were injected into both flanks of each ofthe athymic mice. Each individual experiment with a sin-gle cell line was carried out using mice of one sex. Oncetumours were established and had reached approxi-mately 100 mm3, 4–5 days after injection, the mice wererandomised into six mice/group: Group I (Control)received daily i.p. injections of vehicle (100 ll 0.2%DMSO); Group II (frondoside A) received daily i.p.injections of frondoside A 100 lg/kg/day (4 ll/g of

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25 lg/ml 0.2% DMSO); Group III (Gemcitabine)received five i.p. injections of gemcitabine on days 3, 6,9, and 12; Group IV (frondoside A + gemcitabine)received daily i.p. injections of frondoside A 100 lg/kg/day and five i.p. injections of gemcitabine. For thefirst experiment, each gemcitabine dose was 20 mg/kgand for the second experiment each gemcitabine dosewas 4 mg/kg. Animal weight and tumour size wererecorded every third day. The formula for calculatingtumour volume was: (length) � (width) � (length +width/2) � 0.526 = volume [10]. After 30 days of treat-ment the animals were euthanised and the tumours werecarefully dissected and tumour weights measured.

2.4. Histological analysis

Slides of wax embedded sections were deparaffinisedand hydrated to buffer. Sections were routinely stainedwith haematoxylin and eosin (H and E). For detectionof apoptosis, sections were transferred into 0.1 M citratebuffer pH 6.0, placed in a microwave oven at 700 Wattsand boiled for 1 min. After incubation with 3% hydro-gen peroxide in 0.1 M PBS to block endogenous perox-idase reaction and 1% BSA in PBS +0.05% tween 20 toblock non-specific binding, sections were treated withanti-rabbit active caspase 3 (Cat No: 3015–100,BioVision, Malpitas, CA, USA) diluted 1:200 withDako reale antibody diluents, (code S2022, DakoCopenhagen, Denmark at room temperature for30 min. Subsequent staining was done using the DAKOReale Envision Detection System, Peroxidase/DAB+,Rabbit/Mouse (Code K5007, Dako Copenhagen,Denmark) according to manufacturer’s instruction.Sections were then counterstained with haematoxylinfor 30 s and examined by light microscopy.

2.5. Statistical analysis

All data were analysed using analysis of variance(ANOVA) with Bonferroni’s or Dunnett’s correctionsfor multiple comparisons as appropriate. The growthcurves for tumour growth in vivo were analysed as areaunder the curve for statistical purposes. Differences wereconsidered statistically significant when P 6 0.05.Graphs were created using the GraphPad PrismSoftware.

3. Results

Both frondoside A and gemcitabine markedly inhib-ited growth of human AsPC-1 and S2013 cells. In preli-minary experiments it became clear that the only way todetect synergistic effects of the two agents was to usevery low concentrations of each drug. From concentra-tion response assays, it was revealed that concentrationsthat produced a statistically significant (�15–25%)

reduction in proliferation over 72 h were: gemcitabine50–100 nM for AsPC-1 and 2 nM for S2013, for frondo-side A it was 20–50 nM for AsPC-1 and 100–200 nM forS2013 (Fig. 1). These concentrations were used for sub-sequent experiments to investigate the synergistic effectsof the two agents.

The effects of combinations of frondoside A and gem-citabine were tested in parallel with the drugs used aloneversus control. The inhibition of proliferation of thecombination was then compared with the calculatedadditive effects of the drugs used alone. In AsPC-1 cells,a combination of 50 nM gemcitabine with 20 nMfrondoside A produced greater inhibition of prolifera-tion than the calculated additive effects of the drugsgiven alone, but narrowly failed to reach statistical sig-nificance (P < 0.066, Fig. 2). However, the other concen-tration combinations tested, 50 nM gemcitabine/50 nMfrondoside A; 100 nM gemcitabine/20 nM frondosideA; and 100 nM gemcitabine/50 nM frondoside A, allproduced significantly greater inhibition of proliferationthan the calculated additive effects (P < 0.02, P < 0.002,and P < 0.0001, respectively, Fig. 2). Similarly, in S2013cells combinations of 2 nM gemcitabine with either 100or 200 nM frondoside A also produced significantlygreater inhibition of proliferation than the calculatedadditive effects (P < 0.001 and P < 0.0002, respectively,Fig. 2).

The effect of frondoside A and gemcitabine ongrowth, measured as both tumour volume and tumourweight was investigated on AsPC-1 and S2013 pancre-atic cancer xenografts in athymic mice. The first experi-ment with both cell lines used gemcitabine at aconcentration of 20 mg/kg. This concentration wasone fifth of that used by the Eli Lilly company and othergroups in the original xenograft work with this drug[11–14]. In these first experiments, 100 lg/kg/dayfrondoside A and 20 mg/kg/dose gemcitabine bothcaused significant inhibition of growth of the xenografts,measured as area under the curve or as tumour weight(Fig. 3). However because gemcitabine at this dosecaused near total inhibition of tumour growth it wasnot possible to see any synergistic effect when it wascombined with frondoside A (Fig. 3). The experimentwas then repeated in both cell lines using 4 mg/kg/dosegemcitabine with the same concentration of frondosideA (100 lg/kg/day). Once again, all treatments causedsignificant inhibition of tumour growth in this secondexperiment (Fig. 4). However, at 4 mg/dose, only 4%of that used in early experiments, gemcitabine was lesseffective than at 20 mg/kg/dose. In these studies thecombinations of frondoside A and gemcitabine were sig-nificantly more effective than either drug alone (Fig. 4).Importantly, when measured as area under the curve thecombination was more effective than gemcitabine alone(AsPC-1 cells: P < 0.05; S2103 cells: P < 0.005, Fig. 4).Similarly, when measured as tumour weight the

Fig. 1. Effect of low doses of gemcitabine (Gem) and frondoside A (Fron), alone and in combination, on viable cell number of A; AsPC-1 and B:S2013 human pancreatic cancer cells. Significant differences from control are indicated by: *<0.05; ***<0.0005.

Fig. 2. Effect of combinations of gemcitabine and frondoside A on reduction of viable cell number compared with the calculated additive effects ofthe two agents added alone in A, B, C and D: AsPC-1 cells and E and F: S2013 cells. Significant differences between observed effects of thecombination and that calculated from the addition of the single effects are indicated by: *<0.05; **<0.005; ***<0.0005.

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Fig. 3. Effects of placebo (vehicle), gemcitabine (20 mg/kg/dose on the days indicated by the arrows), frondoside A (100 lg/day) and thecombination on growth of xenografts of A, C, E and G: AsPC-1 and B, D, F and H: S2013 human pancreatic cancer cells in athymic mice. Panels Aand B show calculated tumour volumes recorded every third day. Panels C and D show calculated areas under curves from data in panels A and B.Panels E and F show tumour weights recorded when the animals were euthanised on day 30. Panels G and H show body weights of the animalsrecorded every third day. Significant differences between groups are indicated by: *<0.05; **<0.005; ***<0.0005.

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combination was more effective than gemcitabine alone(both cell lines: P < 0.05, Fig. 4).

There was no significant difference in body weightsbetween the control and any of the treatment groupsthroughout the entire treatment period in eitherexperiment.

Histological analysis included both H and E sectionsand staining with active caspase 3 antibodies. The H andE sections of both cell lines revealed central necrosis inmany tumours, particularly in the control untreatedgroup. Otherwise the H and E staining was unremark-able for both cell lines.

Fig. 4. Effects of placebo (vehicle), gemcitabine (4 mg/dose on the days indicated by the arrows), frondoside A (100 lg/day) and the combinationon growth of xenografts of A, C, E and G: AsPC-1 and B, D, F and H: S2013 human pancreatic cancer cells in athymic mice. Panels A and B showcalculated tumour volumes recorded every third day. Panels C and D show calculated areas under curves from data in panels A and B. Panels E andF show tumour weights recorded when the animals were euthanised on day 30. Panels G and H show body weights of the animals recorded everythird day. Significant differences between groups are indicated by: *<0.05; **<0.005; ***<0.0005.

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In AsPC-1 xenografts, the caspase 3 staining revealeda marked increase in the proportion of apoptotic cellsapoptosis in tumour cells in the treated groups. Thiswas more prominent in the gemcitabine-treated thanthe frondoside A-treated tumours and was particularly

marked in the combination treatment group. In addi-tion, there was extensive apoptosis in the infiltratingcells of the perivascular cuff which was particularlyevident in the frondoside A and drug combination-trea-ted tumours. Representative pictures of the caspase 3

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staining are available online as supplementalinformation.

In the sections from the S2013 xenografts, stainingfor active caspase 3 revealed necrosis in all three treatedgroups which was very extensive in the combinationtreatment group. There were also scattered apoptoticcells in the tissues from each of the treated groups.The extensive necrosis is likely to reflect extensive apop-tosis following treatment of the xenografts of this cellline. Representative pictures of the caspase 3 stainingare available online as supplemental information.

4. Discussion

The cytidine analogue gemcitabine (20,20-dif-luorodeoxycytidine) is the standard palliative treatmentin unresectable pancreatic cancers and is the usual base-line therapeutic in adjuvant and neoadjuvant clinical tri-als attempting to improve on the desperate situation ofpancreatic cancer patients [2,3]. However the search ison for drugs to improve upon the very minimal clinicalresults of gemcitabine [3]. In the present study, frondo-side A exhibited synergistic effects with gemcitabine,which were greater than the calculated additive effectsof the two drugs used alone. However, because of theefficacy of gemcitabine in vitro and in the mouse xeno-graft model, this only became evident when low dosesof gemcitabine were used. In the animal studies, thecombined therapy of gemcitabine and frondoside Apotently inhibited the growth of two very rapidly grow-ing and aggressive pancreatic adenocarcinomas, eventhough to see this effect we needed to lower the gemcit-abine dose to only 4% of what had been used in the stan-dard pre-clinical studies with this drug [11–14].

It was remarkable that with the higher dose of gem-citabine and with the combination of the low dose ofgemcitabine with frondoside A, we actually saw tumourregression in the early treatment period. Tumour regres-sion is difficult to achieve in pancreatic cancer because ofthe marked desmoplastic reaction. Even gemcitabinedoes not induce tumour regression in most patients withthis disease. Body weight did not significantly differbetween the experimental groups at any time duringthe experiment. Moreover, frondoside A shows noapparent toxicity. Previous studies have not shown anyeffects on complete blood count, liver enzymes or bodyweight [6]. Histochemical analysis of active caspase 3in AsPC-1 xenografts revealed that both drugs inducedapoptosis in the tumour cells, with the degree of apopto-sis most marked in the group receiving the combinedtreatment. Apoptosis was also seen in the S2013 xeno-grafts, however in this cell line the extent of positivestaining was more marked with areas that appearednecrotic.

According to the present study, frondoside A may beparticularly valuable for patients undergoing potentially

curative surgery, but may also be useful for patients des-perately looking for better palliative treatment strategiesthan gemcitabine alone. High compliance of cancerpatients can be assumed because of the lack of sideeffects.

It should be noted, however, that the subcutaneousxenograft model is not a very good mimic of the humandisease. Indeed, no animal model fully recapitulates theclinical situation. Further studies using other modelsshould be completed before these findings could betranslated to the clinic. Several previous studies haveshown that frondoside A induces cell cycle arrest andapoptosis through the mitochondrial pathway [4–6].At present the molecular mechanism by which frondo-side A inhibits tumour growth and triggers apoptosisis not clear and we are currently investigating the roleof several proteins whose expression is markedly alteredsoon after exposure of tumour cells to the agent. We doknow that frondoside A causes a marked up-regulationof the cyclin-dependent kinase inhibitor, p21 and thatthis has to be through a mechanism other than involve-ment of p53, since the latter protein is non-functional inall of the pancreatic cancer cells we have used for ourfrondoside A investigations thus far [4].

In conclusion, frondoside A improves the efficacy ofgemcitabine in two pancreatic cancer cell lines in vitro

and when xenografted into athymic mice. Thus com-bined therapy of gemcitabine with frondoside A couldbe a promising approach in treating pancreatic adeno-carcinoma. This therapeutic strategy should be consid-ered for neoadjuvant, adjuvant and palliative clinicaltrials in pancreatic cancer patients since there is a signif-icant beneficial effect of adding frondoside A to gemcit-abine treatment.

Conflict of interest statement

PC and TEA are named inventors on a patent(US2005288239; WO2005072528) on the use of frondo-side A for cancer therapy.

Acknowledgements

These studies were funded by a grant from the TerryFox Cancer Fund for Research in Cancer Therapy. Sup-port for the development of frondoside A came from theNational Cancer Institute, Bethesda, MD and from theMaine Technology Institute, Gardiner, Maine S2-013cells were provided by Dr. M.A. Hollingsworth, EppleyCancer Institute, Omaha, NE.

Appendix A. Supplementary data

Supplementary data associated with this article canbe found, in the online version, at http://dx.doi.org/10.1016/j.ejca.2014.01.002.

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