Convolvulus galaticus, Crocus antalyensis, and Lilium candidum ExtractsShow Their Antitumor Activity Through Induction of p53-Mediated Apoptosis

on Human Breast Cancer Cell Line MCF-7 Cells

Onur Tokgun,1 Hakan Akca,1 Ramazan Mammadov,2 Candan Aykurt,3 and Gokhan Deniz3

1Department of Medical Biology, School of Medicine, and 2Department of Biology,Faculty of Arts & Sciences, Pamukkale University, Kınıklı, Denizli, Turkey.

3Department of Biology, Faculty of Arts & Sciences, Akdeniz University, Antalya, Turkey.

ABSTRACT Conventional and newly emerging treatment procedures such as chemotherapy, catalytic therapy, photody-

namic therapy, and radiotherapy have not succeeded in reversing the outcome of cancer diseases to any drastic extent, which

has led researchers to investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge

systems from various parts of the world are being re-investigated for their healing properties. It has been reported that several

members of the Convolvulaceae, Iridaceae, and Liliaceae families have antitumor activity against some tumor cell lines. Here

we first report that Convolvulus galaticus, Crocus antalyensis, and Lilium candidum species have cytotoxic activity on human

breast cancer cell line MCF-7 cells. Plant samples were collected and identified, and their cytotoxic effects on the MCF-7 cell

line were examined at different concentrations of methanol extracts. We found that all three plants have cytotoxic effects on

MCF-7 cells but that C. galaticus has the strongest cytotoxic effect even in the lowest extract concentration tested (0.32 lg/

mL). Our results indicate that these plant extracts have cytotoxic effects on human breast carcinoma cell line MCF-7 cells and

that this cytotoxic effect comes from p53-mediated stimulation of apoptosis.

KEY WORDS: � apoptosis � Convolvulus galaticus � Crocus antalyensis � Lilium candidum � MCF-7 cells � p53

� plant extracts

INTRODUCTION

Breast cancer is one of the most common form ofcancer and the leading cause of cancer mortality among

women, next to lung cancer, in the world. More than 10.5 newbreast cancer cases per 100,000 population occur worldwideannually, compared with nearly 5.8 per 100,000 population indeveloped countries.1 Breast cancer, like many other cancers,tends to spread throughout the body without any symptoms.By the time the tumor is detected, there is a high probabilitythat metastatic lesions might be present. This observation hasgenerated significant interest in the search for novel anti-cancer agents. Attention is currently focused on therapeuticregimens that are primarily based on apoptosis induction inthe cancer cells, in an attempt to significantly decrease ob-noxious side effects of traditional treatments. Regarding this,natural products/plants have been the main focus.2

Phytotherapy has been practiced since antiquity in Africa,Asia, Europe, and America. During the past 30 years, the use

of herbs and related products has increased from 34% in1990 to 42% in 1995. Throughout the centuries, severalplant extracts have been tested for antitumor potential.Plants have provided many effective anticancer agents incurrent use such as irinotecan, taxanes, topotecan, vinblas-tine, vincristine, etc.3–5 Plant-derived products are excellentsources for the discovery and development of new anti-cancer agents. Moreover, plant materials represent promis-ing sources of anticancer agents with lower side effectscompared with chemical drugs.

Lilium candidum L. belongs to the Liliaceae family,which grows throughout Mediterranean region and westernAsia. It probably originated from Syria and Persia. The es-sential oil, extracted from flowers (0.3%), is rich in vanillin(up to 2.5%), p-hydroxy-m-methoxytoluene (up to 50%), p-cresol, linalol, terpineol, phenylethyl alcohol, and its esters,with acetic, palmitic, benzoic, propionic, and cinnamic ac-ids.6 On the basis of usage in traditional medicine and oncontemporary experimental experiences, some authors referto the anti-inflammatory and healing effects of L. candidumL. Selected compounds isolated from the bulbs and flowersof L. candidum L. were investigated for potential anticar-cinogenic activity.6 L. candidum L. (Liliaceae), also called‘‘white Madonna lily,’’ is well known in folk medicine for

Manuscript received 20 February 2012. Revision accepted 8 June 2012.

Address correspondence to: Hakan Akca, Department of Medical Biology, School ofMedicine, Pamukkale University, Kınıklı, Denizli, 20200, Turkey, E-mail: hakca@pau.edu.tr or hakanakca@yahoo.com

JOURNAL OF MEDICINAL FOODJ Med Food 15 (11) 2012, 1000–1005# Mary Ann Liebert, Inc., and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2012.0050

1000

the treatment of burns, ulcer, and inflammations and forhealing wounds. L. candidum L. extract contains variousbiologically active compounds.6,7

Crocus species are members of the family Iridaceae. Theplants in this family are herbs with rhizomes, corms, orbulbs. The family Iridaceae embraces about 60 genera and1500 species. The genus Crocus includes native speciesfrom Europe, North Africa, and temperate Asia and is es-pecially well represented in arid countries of southeasternEurope and Western and Central Asia. Crocus antalyensis,generally located in the Mediterranean region, is an endemicplant for Turkey.8,9 There is no report on medicinal uses ofC. antalyensis in the literature, but it is known that Crocusspecies are extensively used in traditional medicine forvarious purposes, as an aphrodisiac, antispasmodic, andexpectorant, for treatment of stomach ailments, for reducingstomachache, and for relieving tension.10 This study is thefirst to report antitumor activity of C. antalyensis.

There have been several chemical studies of both thefamily Convolvulaceae and the genus Convolvulus. Alka-loids have been reported from Convolvulus,11 and acylatedanthocyanis have been identified in Convolvulus.12 There isalso no report on medicinal uses of Convolvulus galaticus inthe literature, but it is known that Convolvulus species areextensively used in traditional medicine for various pur-poses as in ulcer treatment, diabetes, and tension.13

The aim of this study was to investigate the potentialantitumor effects of L. candidum, C. antalyensis, and C.galaticus on human breast cancer cell line MCF-7 cells.

MATERIALS AND METHODS

Plant materials

Plants were collected from around Turkey from differentlocations. C. galaticus was collected from around Kayseri,at an altitude of 1300 m above sea level. L. candidum wascollected from around Mugla, at an altitude of 30 m abovesea level. C. antalyensis was collected from around Antalya-Elmali, at an altitude of 750 m above sea level.

Plant extract preparation

After collection of plant samples, dried bulbs and leaveswere chopped with a blender, and methanol extracts wereprepared for the experiment. In this study, 10 g of the dried plantand 100 mL of solvent methanol (Merck, Darmstadt, Germany)were used for every sample.14 The mixture was extracted afterbeing heated in a vibrating water bath at 55�C. Having beenacquired as a result of extraction, the mixture was filteredthrough filter paper (Whatman No. 1), and the solvents wereevaporated in a rotary evaporator at 48–49�C. The water in eachextract was frozen in a freeze-drying machine and drawn out.

Cell lines and culture conditions

MCF-7 cells were cultured in RPMI 1640 medium (Sig-ma Aldrich, St. Louis, MO, USA) supplemented with 10%fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37�Cin a humidified incubator with 5% CO2.

Cell proliferation assay

The medium was aspirated when MCF-7 cells grown toabout 90% confluence. Cells were washed with phosphate-buffered saline, trypsinized, counted with a hemocytometer,and seeded into 96-well plates (3 · 104 cells/mL). After a 24-h incubation at 37�C in a 5% CO2 incubator, the mediumwas removed, and cells were treated with plant extractsadded to the medium in different concentrations (0.3, 0.6,1.25, 2.5, 5.0, 10.0, and 20.0 lg/mL) for 72 h. For the un-treated control group, cells were not treated with any ex-tracts. Plant extract concentrations were prepared in RPMI1640 medium that included 10% fetal calf serum and thensterilized by passage through a filter (pore size, 0.2 lm). Atthe end of incubation periods, medium was removed, andcytotoxicity in plant extract-treated and untreated controlgroups was measured by the luminometric method using aCytotoxGlo� kit (Promega, Madison, WI, USA). Values forthe concentration at which 50% inhibition occurred (IC50)were calculated for all three plant extracts.

Terminal transferase dUTP nick end-labelingapoptosis analysis

MCF-7 cells were trypsinized, counted with a hemocy-tometer, and then seeded into flasks (3 · 104/mL). For thedetection of the induction of apoptosis of each plant extract,MCF-7 cells were treated with IC50 values of each plant ex-tract (0.32 lg/mL for C. galaticus, 1.06 lg/mL for L. candi-dum, and 0.72 lg/mL for C. antalyensis) for 24 h at 37�C in ahumidified incubator with 5% CO2. At the end of the incu-bation period late apoptotic events were analyzed by terminaltransferase dUTP nick end-labeling (TUNEL) analysis usingthe In Situ Cell Death Detection Kit (Millipore, Billerica, MA,USA). Apoptotic cells were counted under the microscope.

Western blotting

At the end of the incubation period, MCF-7 cell lysatestreated with IC50 values of each plant extract were prepared inice-cold RIPA buffer (10 mM Tris-HCl [pH 7.5], 150 mMNaCl, 2 mM EDTA, 1% Nonidet P-40, 1% sodium deox-ycholate, and 0.1% sodium dodecyl sulfate). Cellular debriswas removed by centrifugation at 12,000 g for 5 min at 4�C.Proteins (100 lg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 2–15% gradient or7.5% polyacrylamide gels (Pierce, Rockford, IL, USA). Pro-teins were immunoblotted onto Hybond� polyvinylidenedifluoride membranes (Amersham-PharmaciaBiotech, Pis-cataway, NJ, USA) and labeled with related antibodies. Thep53 antibodies were obtained from Cell Signaling Technology(Danvers, MA, USA). Glyceraldehyde 3-phosphate dehydro-genase (GAPDH) and horseradish peroxidase–labeled anti-mouse secondary antibodies were obtained from Santa CruzBiotechnology (Santa Cruz Biotechnology, Santa Cruz, CA,USA). Primary and secondary antibody labeling blots weretreated with Super Signal West Pico chemiluminescent sub-strate (Pierce), exposed to Hyperfilm ECL� (Amersham-PharmaciaBiotech), and developed.

PLANT EXTRACTS INDUCE P53-MEDIATED APOPTOSIS 1001

Real-time reverse transcriptase–polymerase chainreaction analysis

Quantitative real-time reverse transcriptase (RT)–poly-merase chain reaction (PCR) analysis was applied to de-termine relative mRNA levels in plant extract-treated anduntreated control groups of MCF-7 cells. Cells were treatedwith IC50 values of each plant extract (0.32 lg/mL for C.galaticus, 1.06 lg/mL for L. candidum, and 0.72 lg/mL forC. antalyensis) for 24 h at 37�C in a humidified incubatorwith 5% CO2. At the end of the incubation time plant ex-tract-treated and untreated control group cells were washedwith ice-cold phosphate-buffered saline, and then total RNAfrom the MCF-7 cell line was prepared as described previ-ously.15 Expression of p53 and of its target genes for theproteins p21 and bax was assessed by quantitative real-timeRT-PCR analysis using a QuantiTect� Probe PCR kit(Qiagen, Hilden, Germany) and an ABI Prism� 7900HTsequence detection system (Applied Biosystems, FosterCity, CA, USA) according to the supplier’s protocols. Setsof TaqMan� probe and primers were purchased from Ap-plied Biosystems. A predeveloped TaqMan assay endoge-nous control of a GAPDH kit (Applied Biosystems) wasused for amplification of a GAPDH cDNA fragment. Therelative mRNA levels were calculated using the compara-tive Ct method. Levels of p21, bax, and p53 transcript wereevaluated. The transcript level of the GAPDH gene was usedas the endogenous reference.

Statistical analysis

All experiments were performed in replicates of three andrepeated independently to confirm the results. Significanceof the differences in the means was determined using Stu-dent’s t test and considering P < .05 to be statistically sig-nificant.

RESULTS

For the detection of specific cytotoxic activity of eachplant extracts, luminometric analysis was used as describedin Materials and Methods. MCF-7 cells were incubated withseven different concentrations (0.3, 0.6, 1.25, 2.5, 5.0, 10.0,and 20.0 lg/mL) of total methanol extracts of C. galaticus,C. antalyensis, and L. candidum for 72 h. Figure 1 clearlyindicates that total methanol extracts of C. galaticus, C.antalyensis, and L. candidum have cytotoxic effects onMCF-7 cellular proliferation. C. antalyensis and C. galati-cus have a stronger cytotoxic effect than L. candidum (Fig.1). We also calculated IC50 values for each plant extractfrom Figure 1 and represented in Table 1. According toTable 1, C. galaticus extract has the lowest IC50 values(0.32 lg/mL) compared with C. antalyensis (0.72 lg/mL)and L. candidum (1.06 lg/mL); the IC50 values are in goodagreement with our cytotoxicity results.

Because extensive cell death was observed in proliferat-ing MCF-7 cells after treatments with the plant extracts, wehave investigated if the plant extract treatment-induced celldeath occurred through apoptosis. MCF-7 cells were incu-

bated with IC50 values of total methanol extracts ofC. galaticus, C. antalyensis, and L. candidum for 24 h. Atthe end of the incubation time, cells were harvested andassayed for induction of apoptosis with use of the TUNELmethod. The TUNEL assay was developed as a method toidentify individual cells that were undergoing apoptosis bylabeling the ends of degraded DNA with the polymeraseterminal deoxynucleotidyl transferase. Figure 2 shows thatC. galaticus, C. antalyensis, and L. candidum extracts havethe ability to induce apoptosis on MCF-7 cell lines. Amongthe three plant extracts C. galaticus has the biggest effect oninduction of apoptosis on MCF-7 cells, which is positivelyrelated to the cytotoxic effects of the plants.

The p53 tumor suppressor gene regulates cell cycle pro-gression and cell survival in response to cellular stress. DNAdamage or oncogenic stress induces p53 protein levels, al-lowing elimination of incipient tumor cells by apoptosis. Todetermine if the plant extracts can induce apoptosis throughp53, we detected p53 protein expression by western blotting

FIG. 1. Convolvulus galaticus, Crocus antalyensis, and Liliumcandidum extracts have cytotoxic effects on MCF-7 cells. Humanbreast carcinoma cell line MCF-7 cells were seeded at a density of3 · 104/mL in 96-well plates. After 24 h, the cells were washed withphosphate-buffered saline, fresh growth medium was added, and thencells were treated with C. galaticus, C. antalyensis, and L. candidumextracts in different concentration for 72 h. At the end of the incu-bation, cell viability was determined in plant extract-treated anduntreated control groups by the luminometric method. Data aremean – SD values (n = 3). *P < .001 by Student’s t test for cytotoxiceffects of plant extract compared with the untreated control group.

Table 1. 50% Inhibition Concentrations

of the Three Plant Extracts Tested

Plant species IC50 (lg/mL)

Convolvulus galaticus 0.32Lilium candidum 1.06Crocus antalyensis 0.72

IC50, concentration at which 50% inhibition occurs.

1002 TOKGUN ET AL.

in the plant extracts of treated samples. Western blot ana-lyses showed that levels of tumor suppressor p53 proteinwere significantly increased after the 24-h incubation withC. galaticus, C. antalyensis, and L. candidum extracts inMCF-7 cells (Fig. 3). The results indicate that these plantextracts induce apoptosis via induction of cellular accumu-lation of p53 in the MCF-7 cell line. Because tumor sup-pressor p53 is a transcriptional activator, we want toexamine the effects of p53 expression on its target genes,p21 and bax. Therefore, we analyzed p53, p21, and baxmRNA expressions in plant extract-treated and untreatedMCF-7 cells with quantitative real-time PCR. Figure 4clearly shows that levels of p53 and its target genes p21 andbax mRNA increased after all three plant extract treatments.

FIG. 3. C. galaticus, C. antalyensis, and L. candidum extracts in-duce accumulation of p53. MCF-7 cells were cultured in RPMI 1640medium with 10% fetal calf serum and incubated with C. galaticus,C. antalyensis, and L. candidum extracts with IC50 values for 24 h.Cell lysates were fractioned on 10% sodium dodecyl sulfate–poly-acrylamide gel electrophoresis, and western blots were probed withantibody directed against human p53, striped off, and reprobed withanti-human glyceraldehyde 3-phosphate dehydrogenase (GAPDH)antibody for even loading. Results are representative of three inde-pendent experiments. Densitometric analysis was also performed.Band intensities of p53 were normalized against that of GAPDH todetermine fold increase in the amount of p53.

FIG. 2. C. galaticus, C. antalyensis, and L.candidum extracts induce apoptosis in MCF-7cells. Human breast carcinoma cell lines MCF-7 cells (3 · 104/mL) were incubated with IC50

values of C. galaticus, C. antalyensis, and L.candidum extracts for 24 h. After the end of theincubation time cells were washed with phos-phate-buffered saline and then assayed by ter-minal transferase dUTP nick end-labelinganalysis using an In Situ Cell Death DetectionKit (Millipore) system to indicate cellular ap-optosis: (i) untreated control, (ii) cells treatedwith C. galaticus extract (0.32 lg/mL), (iii)cells treated with C. antalyensis extract(0.72 lg/mL), and (iv) cells treated with L.candidum extracts (1.06 lg/mL). Arrows indi-cate dark-stained nuclei, which indicate DNAfragmentation and nuclear condensation.*P < .001 by Student’s t test for cytotoxic ef-fects of plant extract compared with the un-treated control group.

FIG. 4. C. galaticus, C. antalyensis, and L. candidum extracts in-duce p53, p21, and bax mRNA expressions. Data are mean – SDvalues of three analyses by quantitative reverse transcriptase–poly-merase chain reaction.

PLANT EXTRACTS INDUCE P53-MEDIATED APOPTOSIS 1003

DISCUSSION

Cancer remains one of the leading causes of death aroundthe world. Breast cancer is the second highest incidence in theworld after lung cancer. Chemotherapy is the most commonand effective method for the treatment of breast cancer.Various cancer therapies have currently been examined, in-cluding the use of natural products from plants. The need todevelop more effective and less toxic anticancer drugs hasprompted researchers to explore new sources of pharmaco-logically active compounds. Natural products have long beenused to prevent and treat diseases, including cancers, andmight be good candidates to develop anticancer drugs.16

Plants have substantial potential for the discovery of activeanticancer compounds as most chemotherapeutic drugs, liketaxol and vincristine, were already isolated from plants.Therefore, attempts to search for plant-derived active com-pounds for new anticancer treatments might be promising.

The present study was designed to investigate the po-tential therapeutic abilities of C. galaticus, L. candidum, andC. antalyensis extracts in human breast cancer cell lineMCF-7 cells. Before this study, there is no report on theseplant species on anticancer abilities in the literature. This isthe first to report that C. galaticus, L. candidum, and C.antalyensis have strong cytotoxic effects on human breastcancer cell line MCF-7 cells in a dose-dependent manner.Among the extracts of the three plants, C. galaticus extracthas the strongest cytotoxic effects on MCF-7 cells in a dose-dependent manner. We also detected strong cytotoxic ef-fects of the extracts of C. antalyensis, which is an endemicplant in Antalya, Turkey, on MCF-7 cells. It was reportedthat saffron, which also belongs to the Crocus genus, caninduce apoptosis in MCF-7 cells17 and in HeLa andHepG218 cell lines. Our results are in good agreement withpreviously published results. Anticancer effects of C. an-talyensis are also observed in other species of the Crocusgenus as reported for saffron. Also, some reports werepublished on the anticancer effects of Lilium species onhuman cancer cell lines,7,19 but until our study, there hasbeen no report on the antitumor activity of L. candidum onany human cancer cell lines in the literature.

Apoptosis is an active physiological process resulting incellular self-destruction that involves specific morphologi-cal and biochemical changes in the nucleus and cyto-plasm.20–22 Agents that suppress the proliferation ofmalignant cells by inducing apoptosis may represent auseful mechanistic approach to both cancer chemopreven-tion and chemotherapy.23 As many anticancer agents havebeen developed, unfavorable side effects and resistancebecome serious problems.24 Thus, there is growing interestin the use of plant materials for the treatment of variouscancers and the development of safer and more effectivetherapeutic agents.25 p53 is an extremely efficient inhibitorof cell growth, inducing cell cycle arrest and/or apoptoticcell death, depending on cell type and environment.26

Therefore, regulation of p53 activity is critical to allownormal cell division. The tumor-suppressive function of p53must be dampened sufficiently to allow normal growth and

development. There are many mechanisms through whichp53 is regulated.27 The major mechanisms include regula-tion of p53 protein levels, control of the localization of p53protein, and modulation of the activity of p53, in particularits ability to function as a sequence-specific transcriptionfactor.28,29 We detected with western blot analysis that C.galaticus, L. candidum, and C. antalyensis total extracts leadto increased p53 protein accumulation (Fig. 3). Because wewant to examine whether p53 accumulation comes fromincreased p53 transcriptional activity or not, the effects ofthe three plant extracts on p53 mRNA expression were alsoexamined by quantitative real-time PCR. Our results indi-cate that total extracts of C. galaticus, L. candidum, and C.antalyensis induced p53, p21, and bax mRNA expressions inhuman breast carcinoma cell line MCF-7 cells. p53 is atranscriptional activator, and it is well known that p53 caninduce expression of apoptosis-mediator genes.30 Hastaket al.30 reported that p53 can also induce the expressions ofp21 and bax mRNAs. Our results are in good agreementwith previously published results.

Here we report that C. galaticus, L. candidum, and C.antalyensis total extracts have strong cytotoxic effects oncellular proliferation, and this cytotoxicity resulted throughthe p53-mediated apoptosis. However, it has been reportedthat p53 gene mutation rate is 20–30% in breast cancer,31–33

which means that p53 is in its wild-type form in 70–80% ofbreast carcinoma patients.33 p53-mediated apoptosis is avery important pathway to kill the breast carcinoma cells.Because oncologists have very limited weapons for thebattle against breast cancer, it is very important to find newcompounds that potentially can be used as a new drug oncancer treatment. Our results did not show that C. galaticus,C. antalyensis, and L. candidum total extracts can be used asa treatment against breast carcinoma, but clearly indicatethat these three plant extracts potentially include an che-mical compound or compounds against breast cancer cells.Therefore, these compounds still need to be isolated andidentified in future studies.

CONCLUSIONS

Our data indicate that C. galaticus, C. antalyensis, and L.candidum have strong anticancer effects on human breastcancer cell line MCF-7 cells and that these effects comefrom induction of apoptosis through accumulation of p53.Our data indicate that these three plant species potentiallymay include active compounds that may be improved as atherapeutic agent for human breast cancer, after isolationand identification studies in the future.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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