Objective: To study the effects of Cucurbitacin E, extracted from Ecballium elaterium L. A.Rich. on ovarian cancer cell lines in vitro.  Materials and Methods: Human ovarian cancer cells (OV_95_CC3) and human lymphocytes were treated with Cucurbitacin E (CuE), a tetracyclic triterpenoid. Morphological changes were examined under the microscope, using the Papanicolau staining procedure, after one and twenty-four hours incubation. Results: Marked effects were observed in treated ovarian cancer cells. With previously studied cell lines, reversible budding of cells and thread formation were observed. In the present study, cells treated with CuE demonstrated more dramatic changes, which were irreversible and more pronounced after twenty-four hours. These changes were not observed in untreated ovarian cells and normal lymphocytes treated with the same compound. Conclusion: From these results obtained, it can be observed that Cucurbitacin E is toxic to ovarian cancer cells but not to normal peripheral lymphocytes.peer-reviewe

Attard, Everaldo

Brincat, Mark P.

Cuschieri, Alfred

OAR@UM

JOURNAL  OF  NATURAL  REMEDIES1. IntroductionIn our preliminary investigation [1], ovarian andstomach cancer cell viability was observedwith various concentrations of Cucurbitacin E(CuE). CuE was isolated from Ecballiumelaterium  L. (squirting cucumber). A series ofexperiments were initiated to determine thepotential cytotoxic effects of CuE in vitro  onthe two mentioned cancer cell lines. It washighly effective against ovarian cancer cellswhile busulphan was more effective againststomach cancer cells. Therefore the resultsobtained prompted further analysis.Several Cucurbitacins have been previouslytested. In their studies Gitter and co-workers[2] reported that the Cucurbitacins (i.e. D, I, Eand CuE methylether) produced blistering andMorphological effects induced by Cucurbitacin  Eon ovarian cancer cells in vitroE. Attard1*, A. Cuschieri2, M.P. Brincat31. Institute of Agriculture, University of Malta, Msida, MSD 06, Malta2. Department of Anatomy, Faculty of Medicine and Surgery, University of Malta, Medical School, Msida, MSD06, Malta3. Department of Obstetrics and Gynaecology, Faculty of Medicine and Surgery, University of Malta, MedicalSchool, Msida, MSD 06, MaltaAbstractObjective: To study the effects of Cucurbitacin E, extracted from Ecballium elaterium  L. A.Rich. on ovariancancer cell lines in vitro.  Materials and methods: Human ovarian cancer cells (OV_95_CC3) and humanlymphocytes were treated with Cucurbitacin E (CuE), a tetracyclic triterpenoid. Morphological changes wereexamined under the microscope, using the Papanicolau staining procedure, after one and twenty-fourhours incubation. Results: Marked effects were observed in treated ovarian cancer cells. Reversible buddingof cells and thread formation were observed in previous studies. However, in the present study, cells treatedwith CuE demonstrated more dramatic changes, which were irreversible and more pronounced after twenty-four hours. These changes were not observed in untreated ovarian cells and normal lymphocytes treatedwith the same compound. Conclusion: The results obtained indicate that Cucurbitacin E is toxic to ovariancancer cells but not to normal peripheral lymphocytes.Key words: Ecballium elaterium L., Cucurbitacin E, ovarian cancer, lymphocytes, morphology.thread formation of Ehrlich ascites and sarcomatumour cells. However, no other changes suchas swelling or vacuolisation were observed.Further morphological studies were carried outon CuD and CuI [3], using Ehrlich ascites andSarcoma Black tumour cells. The results werein accordance with the previous study [2].Shohat and co-workers [4] worked on theeffects of CuI on human leukaemic andnormal lymphocytes. It was observed thatblister formation occurred in response to CuI.These three studies claimed that CuI was themost active compound among theCucurbitacins. No other morphologicalinvestigations have been carried out involvingCucurbitacins, since the investigation ofShohat and co-workers [4].The cytotoxicity of CuE is related to itsstructure. There are two sites of interest inthe molecule; the 5α-cucurbitanehydrocarbon skeleton and the aliphatic sidechain at C-17.  The 23 - 24 double bond andthe 25-acetyl [5, 6] moiety on the aliphaticside chain is are related to the alkylatingactivity on the DNA [7]. The 5α-cucurbitanehydrocarbon skeleton is related to thereceptor binding at the cell membrane [8].As a consequence, it may be postulated thatthe Cucurbitacins are effective againsthormone-responsive tumours, which includeovarian, breast, prostate and others.More recent investigations were carried out onCucurbitacins, one of which was the NCIprotocol for CuE [9]. CuE was also tested onprostate cancer PC3 in vitro [10], and otherCucurbitacins were tested on the two-stagecarcinogenesis of skin tumour [11, 12]. Despiteall these investigations, no morphologicalstudies were conducted. In the present study,morphological investigations were pursued todetermine the effects of CuE on a hormone-responsive cell line (OV_95_CC3).2. Materials and methods2.1 ChemicalsCuE was isolated from Ecballium elateriumfruit by solvent extraction and tested for itspurity by various analytical methods against aknown standard [1].2.2 Cell CulturesHuman ovarian (OV_95_CC3) cell line andhuman lymphocytes were obtained from theDepartment of Anatomy, University of Malta.The ovarian cells were cultured and subculturedto propagate the cell line. The lymphocyteswere isolated from the blood of a healthy humanmale volunteer, using Histopaque-1077 (Sigma,U.S.A).2.3 Application of the cytotoxic agentCuE serial dilutions ranging from 0.18 to 180µM were used as treatments for the two celltypes cultured. Aliquots of cell suspension (2ml) were pipetted in the wells of a six-wellplate, containing a sterile Thermanox (Nunc,Denmark) coverslip at the base. The cells wereallowed to adhere to the coverslips byincubation at 37°C and 5% CO2 for 2 days.Meanwhile, the separated lymphocyte fractionwas resuspended in RPMI with PHA and 2 mlaliquots were transferred to universal containers(Nunc, Denmark). The cells were incubated for2 days to determine any lymphocyte activation.Both ovarian cells and lymphocytes wereexposed to 2 ml of the four different CuEconcentrations, leaving only one sample of eachcell line untreated, as a control of cell viability.One set of cells was incubated for 1 h while theother set for 24 h at 37°C and 5% CO2. This wasperformed in triplicate containers.2.4 Morphological observationsAfter the specified time interval, lymphocyteswere precipitated onto the slides using theCytospin 2 (Shandon, UK) and fixed in alcohol.All fixed specimen were then stained by the1b) showed normal cell morphology, with noabnormal alterations in the cytoplasm, nucleusor cell membrane. The cells were viable at 1and 24 h. CuE did not induce lymphocytedamage (Fig. 1c).In fact, the morphology remained unalteredand the cells were viable even after 24 h. Verylow CuE concentrations (i.e. 0.18 µM),produced significant alterations in the ovariancancer cells as opposed to lymphocytes anduntreated ovarian cancer cells. Abnormalmorphology characterised by budding wasobserved after 1 h (Fig. 1d) and irreversibledamage, which involved the formation ofapoptotic bodies was noted after 24 h (Fig. 1e).The cells, which remained intact but showedaltered morphology, took up trypan blue,confirming their death. The percentageabnormal morphological changes are tabulatedbelow (Table 1).The untreated lymphocytes had an irregularmorphology, which is typical of activated T-lymphocytes. The treated lymphocytes were notaffected by the drug as cell viability was stillhigh after 1 and 24 h (Attard, 1996 unpublishedobservations). The finding that the lymphocyteswere unaffected by the drug is in accordancewith the study of Shohat and co-workers [4].polychromic Papanicolau staining technique[13]. After staining, the slides were mountedin D.P.X. and examined under high powermicroscope (oil-immersion) using a Zeisscompound microscope.Differential counts were performed by takingseveral photomicrographs under the low power.At least four replicates were prepared. Cellswith abnormal morphology were counted as agroup and the values obtained were insertedin the following equation to obtain percentagecounts:2.5 Statistical analysisThe values are expressed in mean±SEM (n=10,lymphocytes; n=4, ovarian cancer cells). Thedata was analysed by using the Kruskal-Wallisone way analysis of variance (ANOVA) todetermine non parametric statisticalsignificance (P≤0.05).3. Results and discussionThe untreated lymphocytes (Fig. 1a) remainedunaltered and viable after 1 and 24 h timeintervals. Untreated ovarian cancer cells (Fig.Table 1.Percentage abnormal morphological changes, on lymphocytes and ovarian cancer cellsobserved after 1 h and 24 h, at the four concentrations of CuE used.            Percentage Abnormal Morphological Changes       [Mean ± SEM]Concentration Peripheral Lymphocytes Ovarian cancer cells(µM) 1h 24 h 1h 24 h0 0.000±0.000 0.000±0.000 0.000±0.000 0.000±0.0000.18 0.000±0.000 0.000±0.000 4.355±0.802 †27.913±2.1351.8 0.000±0.000 0.000±0.000 †18.102±2.769 †19.957±0.88318 0.000±0.000 0.000±0.000 †24.900±1.063 ‡31.710±1.083*180 0.000±0.000 0.000±0.000 †23.730±1.666 ‡35.378±0.889*Each point is the mean ± SEM (ANOVA: *p<0.05, v=9 against control for each cell type and at eachseparate time interval) :  † denotes budding and threading while ‡ denotes formation of apoptotic bodies.  Abnormal cellmorphology (%) =Number ofabnormal cells Total numberof cellsX 100In the latter case, lymphocytes were treatedwith CuI (0.4 and 1.2 µM). These observationsare in accordance with the postulations madeby Dougherty and co-workers [14]. These statedthat a steroid-like compound with no hydroxylgroup at C17, no ketone group at C20 and aketone group at C11 only, is practically non-toxic to lymphocytes. This configuration fitswith that of CuE.The untreated ovarian cancer cells had a regularmorphology, i.e. round cells with an ovalnucleus. However, with drug treatment, therewere unusual morphological changes, whichappeared after 1 h, for allconcentrations except the 0.18 µMconcentration. The cells manifestedbudding, which consisted ofcytoplasmic protrusions. These budsdetach from the cell to formisolated apoptotic bodies.At the same time, the nucleusshrunk with occasional cellsshowing disintegration of thenucleoli. Cell disintegrationoccurred only with the highconcentrations (18 µM and 180µM) after 24 h (P<0.05). This is thelate phase of apoptosis. Thesechanges were irreversible, withblebbing showing early signs ofcell death. No blistering wasobserved as described by theresearch teams of Gitter [2],Gallily [3] and Shohat [4].Moreover, the morphologicalchanges described in previousstudies were reversible after 24 h.This indicates, that in these studies,the effects of the Cucurbitacins wasonly temporary while in the presentstudy with ovarian cancer cells, theeffects were more pronounced after24 h, suggesting, the induction ofcell death through apoptosis, by Cucurbitacinson this cell line.On the other hand, the budding effect, onlymphocytes, was described by Doughtery andco-workers [14] when treated withcorticosteroids. King and co-workers [15]described the presence of cytoplasmic blebsin Ehrlich ascites cells, when treated with amercurial metabolic inhibitor (salyrgin). Thesecytoplasmic blebs presumably correspond tocell budding.The freeing of the nuclei, in the presentinvestigation is in accordance with the findingsFig. 1. The effects of CuE on ovariancancer cells and lymphocytes:a. untreated lymphocytes after24 h.b. untreated ovarian cancer cellsafter 24 h.c. CuE (180mm) - treatedlymphocytes after 24 h.d. CuE (0.18mm)-treated ovariancancer cells after 1 h.e. CuE (0.18mm)-treated ovariancancer cells after 24 h (scale:10 mm).1. Attard E, Scicluna-Spiteri A, Grixti M, Cuschieri A.(1996) Xjenza.  1(1): 29-34.2. Gitter S, Gallily R, Shohat B, Lavie D. (1961) CancerRes.  21(4): 516-521.3. Gallily R, Shohat B, Kalish J, Gitter S, Lavie D. (1962)Cancer Res.  22(9): 1038-1045.4. Shohat B, Gitter S, Lavie D. (1962) J. Nat. CancerRes. 38: 1-9.5. Kupchan SM, Smith RM, Aynehchi Y, MaruyamaM. (1970) J. Org. Chem.  359(9): 2891-2894.6. Kupchan SM, Tsou G. (1973) J. Org. Chem.  38(5):1055-1056.7. Sartorelli AC, Johns DG. (1974) Antineoplastic andImmunosuppressive Agents I,  Springer-Verlag:New York; 37.8. Witkowski, A. and Koopa, J. (1951) Biochim.Biophys. Acta.  674: 246-255.9. Fuller RW, Cardellina JH, Cragg GM, Boyd MR.(1994) J. Nat. Prod.  57(10):1442 - 1445.10. Duncan KL, Senderowicz AM, Malspeis L, GreverMR, Sausville EA. (1994) Proc. Am. Assoc.Cancer Res.  35(2438): 409.11. Konoshima T, Takashi M, Kozuka M, Nagao T,Okabe H, Irino N, Nakasumi T, Tokuda H,Nishino H. (1995) Biol. Pharm. Bull.  18(2):284 - 287.12. Konoshima T, Takashi M, Tatsumoto T, KozukaM, Kasai R, Tanaka O, Nie R.-L, Tokuda H,Nishino H, Iwashima A. (1994) Biol. Pharm.Bull.  17(5):668 - 671.13. Boon ME, Drijver JS. (1986) Routine CytologicalStaining Techniques,  Macmillan EducationLTD, New York; 44-45.14. Dougherty TF, Berliner DL, Berliner ML. (1961)Symposium: Adrenal Steroids Corticosteroid-Tissue Interactions. Metabolism. 10:966-989.15. King DW, Paulson SR, Puckett NL,. Krebs AT.(1959) Am. J. Path.  35:1067-1079.16. Palmer CG, Hodes ME, Warren AK. (1961) Exp.Cell Res.  24:429-439.Referencesof Palmer and co-workers [16], who describedthis effect when sodium lauryl sulphate wasapplied to Ehrlich ascites cells atconcentrations of 0.6 and 0.7 mM.This study shows that Cucurbitacins may havea potential effect on cells, that are hormone-responsive, and hence prompts further studiesin this direction.

Morphological effects induced by Cucurbitacin E on ovarian cancer cells in vitro

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Morphological effects induced by Cucurbitacin E on ovarian cancer cells in vitro

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