IJMS Vol 43, No 1, January 2018 Original Article Antiproliferative Effects of Maritimum Extracts on Normal and Cancerous Cells

Ghaleb Tayoub1, PhD; Abstract Mohmad Al-Odat2, PhD; Amal Amer1, BSc; Background: are an important natural source of Abdulmunim Aljapawe1, BSc; compounds used in cancer therapy. Adnan Ekhtiar1,PhD contains potential anti-cancer agents such as alkaloids. In this study, we investigated the anti-proliferative effects of P. maritimum extracts on MDA-MB-231 human epithelial 1Department of Molecular Biology and Biotechnology, Atomic Energy adenocarcinoma cell line and on normal lymphocytes in vitro. Commission of , Damascus, Syria; Methods: Leaves, flowers, roots, and of P. maritimum 2Department of Radiation Protection and Safety, Atomic Energy Commission of were collected and their contents were extracted and diluted to Syria, Damascus, Syria different concentrations that were applied on MDA-MB-231 cells and normal human lymphocytes in vitro for different intervals. Correspondence: Ghaleb Tayoub, PhD; Cells viability, proliferation, cell cycle distribution, apoptosis, Atomic Energy Commission of Syria, and growth were evaluated by flow cytometry and microscopy. P. O. Box: 6091, Damascus, Syria Parametric unpaired t-test was used to compare effects of Fax: +963 11 6112289 Tel: +963 11 2132580 extracts on treated cell cultures with untreated control cell Email: [email protected] cultures. IC50 was also calculated. Received: 3 September 2016 Results: P. maritimum extract had profound effects on Revised: 15 October 2016 Accepted: 13 November 2016 MDA-MB-321 cells. It inhibited cell proliferation in a dose- and time-dependent manner. The IC50 values were 0.039, 0.035, and 0.026 mg/ml after 48, 72, and 96 hours of treatment with 0.1 mg/ml concentration of extract, respectively. Those values were 0.051 and 0.03 mg/ml after 72 and 96 hours for root extract, respectively, and 0.048 mg/ml after 96 hours for flower extract. There were no significant effects of P. maritimum bulb extracts on normal lymphocytes proliferation. Conclusion: P. maritimum extract has anti-proliferative effects on MDA-MB-231 cell line in vitro. The effects imply the involvement of mechanisms that inhibits cell growth and

arresting cells at S and G2/M phases. Cyclin B1, Bcl-2, and Ki67 expression was also affected. What’s Known Please cite this article as: Tayoub G, Al-Odat M, Amer A, Aljapawe A, Ekhtiar A. • P. maritimum has purgative, Antiproliferative Effects of Pancratium Maritimum Extracts on Normal and acaricidal, insecticidal, and antifungal Cancerous Cells. Iran J Med Sci. 2018;43(1):52-64. activities. • Previous studies on this plant have Keywords ● MDA-MB-231 ● Pancratium maritimum ● Gene led to the isolation of a variety of alkaloids belonging to different classes. Expression ● Cell Proliferation ● Lymphocytes • Clinical studies were conducted to investigate the use of alkaloids and their synthetic derivatives Introduction as a new anti-tumor drugs.

What’s New Cancer is one of the most common devastating disease affecting millions of people per year worldwide. Cancer has been estimated • We found that P. maritimum extract as the second leading cause of death in humans. According to the has a profound antiproliferative effect American Cancer Society,1 deaths arising from cancer constitute and arrested cancerous cycling cells at S 2-3% of the annual deaths recorded worldwide. Cancer is an and G2/M phases. It also affected Cyclin uncontrolled proliferation of a cell that might invade and destroy b1, Bcl-2, and Ki67 expression. other normal tissues and can eventually lead to patient death.

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Several chemo-preventive agents are used to Materials and Methods treat cancer, but many are toxic, which limits their full utilization in treatment regimes.2 Over Plant Material the past years, there has been an intense search Samples of P. maritimum (leaves, flowers, to find and develop novel anticancer drugs to roots, and bulbs) were collected in August and combat this disease. September 2011 during the flowering season More than 60% of currently used anticancer from its natural environment near Tartaus on agents are derived in one way or another from the Syrian Mediterranean Seacoast. Voucher natural sources. According to one estimate, specimens of this plant were deposited in the about 50% of breast cancer cases and 37% of herbarium of plant biotechnology department at prostate cancer patients are treated with plant the Atomic Energy Commission of Syria (AECS). derivative products.3 The medicinal value of Plant identity (voucher sample number 1116), plants lies in some chemical substances usually was approved by Al-Odat (plant taxonomist). secondary metabolites, that produce a definite physiological action in the human body. The most Preparation of the Plant Extract important of these bioactive compounds are Fresh plant tissues (100g leaves, 100g alkaloids, flavonoids, tannins, and phenolics.4 flowers, 50g roots, and 50g bulbs) were cut The Middle East region comprises about 700 into small pieces and extracted with 95% EtOH plant species that are known for their medicinal three times (48h each) at room temperature. The values.5 ethanol extracts were passed through Whatman Pancratium maritimum L. (Amaryllidaceae filter paper (No.1) in order to remove debris family) grows wild in sandy coastal habitats of and then concentrated with a rotary evaporator the Mediterranean.6 It has white flowers and very (BUCHI Heating Bath B-490). Concentrated large bulbs. This species was not registered in extract was lyophilized with a freeze-dryer. The the Syrian flora until recently where it has been dried matter was stored at −20 ºC until use. found near Tartaus-Syria by Al-Odat.7 Previous The yield of dried extracts from starting crude studies on this plant led to the isolation of a variety materials was 4.48g, 1.49g, 7.44g, and 7.43g of alkaloids.6,8-13 Those studies indicated that (w/w) for the leaves, roots, bulbs, and flowers, alkaloids and flavonoids contained in the bulbs respectively. P. maritimum have pharmaceutical properties. To study the anti-proliferative and the cytotoxic Pancratistatin, which is one of those alkaloids, has effects of the extract, lyophilized extracts were anticancer properties.14 P. maritimum extract has dissolved in dimethyl sulfoxide (DMSO) and purgative, acaricidal, insecticidal, anti-migratory, then different dilutions of the resultant solution antiviral, antimicrobial, immunostimulant, were applied on cell cultures. analgesic, antimalarial, antitumor, antifungal, and antioxidant activities.12,13,15,16 More recently, MDA-MB-321 Cell Line Cultures some studies have demonstrated a potent MDA-MB-321 human breast cancer cells, anticancer activity of Lycorine, Pancratistatin provided by Professor P. Bécuwe from the and Urngimionorine alkaloids.6,8 Cancer Research Unit (EA SIGRETO, Nancy, Pharmacological effects of Amaryllidaceae France), were aseptically cultured in 125 ml cell alkaloids fueled clinical studies to use them and culture flasks containing RPMI-1640 complete their synthetic derivatives in developing anti- medium (supplemented with 10% fetal bovine tumor17 and anti-Alzheimer’s therapeutics.18 serum (FBS)), 50 U/ml penicillin/streptomycin, Another study by Kaya19 has elucidated that and 2 mM L-glutamine. Cell cultures were kept P. maritimum extract has a cytotoxic activity on under optimum culturing conditions (37 ºC, 85% the brine shrimp (Artemia salina Leach). humidity, 5% CO2). Determined concentrations of In the present study, we investigated the the plant extracts were added to the cell cultures anti-tumor effects of P. maritimum extracts on when the density of cells reached 40-50%, as MDA-MB-231 cell line in vitro. This cell line is evaluated using Olympus converted microscope. an invasive adenocarcinoma cell line that is The tested concentrations of extracts were 0.1, ESR-, PR-, HER2-, and has non-functional p53. 0.02, 0.01, 0.001, or 0.0001 mg/ml. Treated cells This cell line was used for its availability in our were harvested for subsequent analyses after laboratories and because it represents a major 24, 48, 72, and 96-hour intervals in culture. In cancer type among women worldwide. This addition, we investigated the prolonged effects study also investigated the cytotoxic effects of the P. maritimum extract on MDA-MB-321 by of Pancratium maritimum extracts on normal treating three cell cultures with 0.1 mg/ml of bulb human lymphocytes. extract for 9 days.

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Human Lymphocytes Cell Cultures Bcl-2, Ki67, and cyclin-B1 expression in treated Normal human whole blood samples were and untreated cells by flow cytometry. For each cultured in a complete ready to use chromosome protein to be detected and for each sample, P medium with or without the presence of a 100 μl of cell suspension (~1x105 cells) were phytohemagglutinen as a mitogenic stimulant. transferred into a separate 5 ml tube. Cells were Culturing was conducted under sterile conditions washed twice with PBS containing 1% FCS and and cell cultures were kept under optimum growth 0.1% NaN3. Then, fixed and permeabilized with conditions. Plant extracts were then added to 250 µl of fixation/permeabilization buffer and lymphocytes cultures at the same concentrations incubated at 4ºC in the dark for 30 minutes. used for MDA-MB-231 cells. Treated cells were Cells were then washed twice with 1 ml of then harvested for subsequent analyses after permeabilization and washing buffer and 24, 48, 72, and 96-hour intervals in culture. suspended in 250 μl perm/wash buffer. A 20 μl of the fluorochrome-conjugated antibody was Preparing Cells for Analysis added and the sample was then incubated on Treated cells were harvested from flasks ice in the dark for 3 hours, washed with 1 ml by a brief treatment with trypsin and then PBS containing 1% FCS and 0.1% NaN3, and transferred into separate 50 ml conical then suspended in 500 µl of PBS and analyzed centrifuge tubes. After centrifugation for 5 min on the FACSCalibur. Appropriate isotypes and at 300×g, supernatant was discarded and cells autofluorescence controls were also included. were resuspended in 1 ml cell culture medium. A cell count was performed manually using a Cell Proliferation Assay hemacytometer and cell concentration was Bromodioxyuriden (BrdU) at 32.5 mM adjusted to ~5×106 cells/ml. Aliquots of those concentration was added into separate flasks cells were then subjected to subsequent assays. of treated and untreated cell cultures for 30 minutes before cells harvesting. Cells were Cell Viability Assay then washed with PBS containing 1% FCS

Cell viability of treated and untreated and 0.1% NaN3, suspended in a 250 µl of MDA-MB-231 cells was evaluated by BD cell fixation/permeabilization buffer for 30 minutes at viability kit as indicated in the company product 4ºC in the dark. Cells were then washed twice datasheet. Cell samples were analyzed directly with 1 ml of permeabilization and washing buffer. using BD FACSCalibur flow cytometer. Absolute They were incubated in 250μl cytoperm buffer cell numbers of live and dead cells were for 10 minutes and then washed. Cells were calculated as instructed in the product datasheet treated with 250 μl of DNase solution for one using Cellquest pro software. hour at 37 ºC in the dark and then washed with a permeabilization and washing buffer. A 20 μl Annexin-V Assay of the APC-anti-BrdU antibody was added to Annexin-V binding assay was used to evaluate each sample and incubated on ice in the dark apoptosis by flow cytometry according to the for 3 hours. Cells were washed with 1 ml of PBS manufacturer’s instructions and recommendations containing 1% FCS and 0.1% NaN3, and then indicated in the company product datasheet. resuspended in 500 µl of PBS containing 20 µl of 7-AAD. Cells were analyzed on the FACSCalibur. Proliferation and Cell Cycle Analysis Cycletest plus and APC-BrdU Flow kits from Statistical Analysis BD biosciences were used to analyze proliferation Statistical results were expressed as and cell cycle distribution. Cells of treated and mean±SD (standard deviation). The parametric control cell cultures were prepared and analyzed unpaired t-test was used to calculate the P value as indicated in the manufacturer’s products (MS-Excel 2007). The statistical significance datasheets. Apoptotic cells percentages were of difference (P<0.05) for the treated cultures determined from DNA content histograms, as the was determined relative to the control cultures. events in the channels below the sub G0/G1 peak. IC50 for treated cells was calculated using the Cell samples were analyzed on BD FACSCalibur following equation:20 flow cytometer with doublet discrimination IC50=(50%-LowInh%)/(HighInh%- module (DDM) on measuring pulse width versus LowInh%)×(HighCon-LowCon)+LowCon pulse area of FL2 in linear mode. Where:

LowInh%/HighInh%: % inhibition directly below/ Bcl-2, Cyclin-B1, and Ki67 Expression Analysis above 50% inhibition

Fluorochromes conjugated antibodies to LowCon/HighCon: Corresponding concentrations of Bcl-2 and cyclin-B1 were used to evaluate test compound

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Results The data are expressed as mean±SD for three independent experiments. P. maritimum extract has profound effects Data are expressed as the mean±SD for on MDA-MB-321 human breast cancer cells. three independent experiments. It inhibited cell growth and proliferation in a Figure 2 shows the results of prolonged dose- and time-dependent manner. It also treatment of MDA-MB-231 cells (9 days) with arrested cycling cells at S and G2/M phases and various concentrations of bulb extract. The affected cyclin B1, Bcl-2, and Ki67 expression. protracted treatment of MDA-MB-231 cells with P. maritimum bulb extracts lead to concentration- Effect of Extract on Cell Viability and Growth dependent inhibitory of the proliferation of Bulb or leaf extract of P. maritimum (at MDA-MB-231 cells at all concentrations. Viable a concentration of 0.1, 0.02, 0.01, 0.001, cells percentage reduced to 10%. The calculated or 0.001 mg/ml) inhibited the growth of concentration that reduced cell growth by 50% MDA-MB-231 cells. These inhibitory effects of (IC50) was 0.0146 mg/ml. both extracts were time- and concentration- dependent. The bulb extract was more Effect of Extract on the Cell Cycle effective (P=0.002) than leaf extract (P=0.004) To investigate the anti-proliferative action (figure 1A). The anti-proliferative effects of of P. maritimum bulb ethanol extracts further, the bulb extract were more evident at 0.1 and we tested its biological effects on the cell cycle 0.02 mg/ml concentrations compared with the in MDA-MB231 cancer cells. As shown in low concentrations (0.001 and 0.0001 mg/ml) figures 3 and 4, cells were significantly blocked (figure 1B). in S phase and G2/M phase after 48-96 hours of The concentration of 0.1 mg/ml was most treatment with various concentrations. It should effective (P=0.011) and caused growth inhibition be noted that the 0.1 mg/ml concentration by about 42%, 55%, 63%, and 68% after 24, significantly arrested cells at the S phase of cell 48, 72, and 96-hour intervals, respectively. cycle. The 0.1mg/ml concentration of the root extract caused growth inhibition by 30%, 47%, 64%, Effect of Extract on Bcl-2, Cyclin B1 and Ki67 and 73% after 24, 48, 72, and 96-hour intervals, Expression respectively. The leaf extract inhibited cell P. maritimum extracts affected the expression growth of MDA-MB-231 cells in the same of Bcl-2, cyclin B1 and Ki67 in MDA-MB-231 cells manner as bulb extract. This is also true for in a time- and concentration-dependent manner. flower extract. The low concentrations (0.001 The 0.1 mg/ml concentration decreased Bcl-2 and 0.0001 mg/ml) of the different plant parts expression significantly after 24 hours. However, extracts had no inhibitory effects; they actually Bcl-2 expression increased but remained below had some growth stimulating effects (P=0.28 and the normal levels by two-fold. While cyclin 0.20 for bulb extract at 0.001 and 0.0001 mg/ml, B1 expression decreased progressively with respectively). The IC50 after treatment with the time, Ki67 expression pattern was different. It highest concentration (0.1 mg/ml) were 0.039, decreased and then returned to previous levels 0.035, and 0.026 mg/ml after 48, 72, and 96 after 96-hour interval (figure 5). hours consecutively for bulb extract, 0.051 and 0.03 mg/ml after 72 and 96 hours consecutively Apoptosis Induction for root extract and 0.048 mg/ml after 96 hours P. maritimum bulb extracts showed minor for flower extract. effects on apoptosis induction, as evidenced by

A B Figure 1: (1A) Time-dependent anti-proliferative effects of 0.1 mg/ml of various extracts of P. maritimum (bulb, Leaf, flower and root) against MDA-MB-231 cells cell line after 24, 48, 72 and 96 hours of incubation. (1B) Effects of various concentrations (0.1, 0.02, 0.01, 0.001, and 0.0001 mg/ml) of P. maritimum bulb extracts on MDA-MB-231 cell line after 24, 48, 72, and 96 hours.

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Annexin-V binding assay using flow cytometry extracts did not significantly increase apoptosis (figure 6). in non-stimulated lymphocytes (P=0.07).

Effect of Extract on Normal Lymphocytes Discussion The effects of high concentrations (0.1 and 0.02 mg/ml) of P. maritimum bulb extracts were Carcinogenesis is known to involve many also tested on stimulated and non-stimulated signaling pathways such as modulation of normal human lymphocytes in vitro. Our transcription factors, apoptotic proteins, protein results did not indicate significant effects of kinases, cell cycle proteins (e.g. cyclins and P. maritimum bulb extracts on the distribution cyclin-dependent kinases), cell adhesion of stimulated lymphocytes on the various molecules, enzymes, and growth factor signaling phases of cell cycle (figure 7). P. maritimum pathways.21 bulb extracts induced apoptosis in stimulated Many plants, including bulbous species lymphocytes in a time- and dose-dependent contain many biologically active compounds manner. This effect was significant only after 72 that can be used as anticancer drugs.22,23 Many hours of treatment with 0.1 mg/ml concentration of these compounds are under investigation (P=0.02). Interestingly, P. maritimum bulb by various research groups, firms, and pharmaceutical companies. It is estimated that only 15% of this repertoire has been explored.24 These natural phytochemicals have potent and selective anti-cancer actions through mechanisms involving destabilizing microtubules formation in dividing cancer cells,25 inhibiting oncogenes expression, disrupting signal transduction pathways, preventing cell adhesion, and inhibiting enzymes. These actions also involve induction of cell differentiation, apoptosis, and tumor suppressor gene expression; in addition to induction of Figure 2: Anti-proliferative effect of prolonged treatment with detoxification molecules and enzymes such ethanol extract of P. maritimum bulb on MDA-MB-231 cell as phase II enzymes, glutathione, peroxidase, line. catalase, and superoxide dismutase. Natural

Figure 3: Results of cell cycle analysis of MDA-MB231 cells treated with various concentrations (0.1, 0.02, 0.01, 0.001, and 0.0001 mg/ml) of P. maritimum bulb ethanol extracts after 24, 48, 72, and 96 hours.

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A B

C D

E Figure 4: Flow cytometric dot plots showing the effects of P. maritimum extract (0.1 mg/ml) on the cell cycle/proliferation of MDA-MB-231 cells analyzed by BrdU incorporation assay: (A) control, (B) after 24h, (C) after 48h, (D) after 72h, and (E) after 96h of incubation. Cyan cluster represents apoptotic cells in the sub G0/G1 phase. Red cluster represents cells at the G0/G1 phase. Green cluster represents cells at the S phase. Pink cluster represents cells at the G2/M phase. phytochemicals are also known for their Alkaloids in particular may interact with many enhancement of immune response, preventing cellular molecules and interfere with the growth DNA adducts formation or DNA intercalation, and progression of the tumors.27 P. maritimum blocking angiogenesis, and regulating hormones contains up to 16 alkaloids, including lycorine, metabolism.26 maritidine, lycoramine, galanthamine

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A B

C D

E F Figure 5: Representative flow cytometric profiles showing the effects of P. maritimum bulb extract (0.1 mg/ml) on the expression of Bcl-2, cyclin B1, and Ki67 in MDA-MB-231 cells before (A, C, E) and after (B, D, F) 96 hours of incubation. Black histogram represents the cells expressing the molecule tested. Other overlapping histograms represent the isotype control. narciclasine, lycoricidine, and pancratistatin. In this study, we investigated the Some of these alkaloids and their congeners antiproliferative effects of P. maritimum extract have demonstrated potent cytotoxicity against on the MDA-MB-231 cell line in vitro. This cell cancer cell lines in vitro and potent antitumor line is an invasive adenocarcinoma cell line that activity in vivo.6,11,12 Pancrimatine B and is ESR-, PR-, HER2-, and has non-functional N-methyl-8, 9-methylenedioxyphenanthridine p53. Our results showed that P. maritimum extracted from P. maritimum have antiproliferative extracts inhibited the growth and the proliferation and antimigratory activity against the highly of MDA-MB-231 breast cancer cell line. This metastatic human prostate cancer cell line inhibition was dose- and time-dependent. As 11 PC-3 cells without cytotoxicity. evident from IC50 values, the inhibition effect

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A B

C D

E Figure 6: Flow cytometric dot plots showing the effects of P. maritimum extract (0.1 mg/ml) on cell viability of MDA231 cells analyzed by Annexin-V binding assay: (a) control, (b) after 24h, (c) after 48h, (d) after 72h, and (e) 96h of incubation. Green cluster represents live cells, red cluster is the dead cells, blue dots represent necrotic cells, and pink dots are the apoptotic cells. of bulb extract on MDA-MB-231 cell growth extract can be explained by differences in the was superior to that of the root, flower, and content of active substances among different leaf extracts sequentially. However, lower parts of the plant. concentrations of different parts extracts did not The low concentrations of P. maritimum inhibit cell growth. The increased effect of bulb extracts, apparently, did not interfere with

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Figure 7: The effects of two concentrations of bulb extract on the proliferation of normal stimulated (PHA) and non-stimulated (no PHA) normal human lymphocytes for different time intervals. Results are expressed as the mean±SD. cell viability. This is consistent with the results extracts suppress the growth and proliferation obtained by Ryu28 and Jeong29 on Orostachys of glioblastoma multiforme cancer cells by japonicas extracts. inhibiting protein synthesis pathways.35 Our P. maritimum extracts arrested cycling results showed that P. maritimum extracts down-

MDA-MB-321 cells at S and G2/M phases. This regulated expression of cyclin B1 (proliferation selective blocking of cancerous cells in specific regulating molecule) and Ki67 (nuclear antigen cell cycle phase is a favorable characteristic of expressed by cancerous proliferating cells). anti-cancer drugs.30 The mechanism involved in The cyclin B1 and Cdc2 kinase regulate the this arrest might be through interfering with DNA entry and progression of the mitotic phase in synthesis pathways31 or blocking the formation eukaryotic cells. Cyclin B1 and Cdc2 kinase of mitosis spindle.32 Many studies have shown activity is known to be involved in the G2/M that alkaloids induce depolymerization of phase transition of the cell cycle. This G2/M microtubules in cancer cells.33,34 Recent studies transition requires the accumulation of cyclin also suggest that, at nanomolar concentrations, B1 and Cdc2.36,37 Here, we demonstrated that narciclasine and alkaloid found in P. maritimum treatment of MDA MB-321 cells cancer cells

60 Iran J Med Sci January 2018; Vol 43 No 1 Antiproliferative effects of Pancratium maritimum with P. maritimum extracts resulted in the apoptosis in several different types of cancer decrease of cyclin B1 levels as evidenced by including colon, prostate and breast carcinomas, the flow cytometric expression profile of cyclin leukemia, melanoma and glioblastoma, with B1. In particular, down-regulation of cyclin B1 minimal toxicity to non-cancerous counterpart expression is the supporting evidence of the cells.47,48 Pancratistatin selectively targets inhibiting effects of P. maritimum extracts on cancer cell mitochondria and induces apoptosis MDA-MB-321 cells proliferation. independent of p53, a frequently mutated tumor P. maritimum extracts also affected the suppressor in human carcinomas.49 expression pattern of Bcl2 (anti-apoptosis Our results showed that P. maritimum extract regulating molecule). Many studies on the has no capacity to enhance the proliferation extracts of other plant species,28,38 also of stimulated human lymphocytes. However, reported changes in the expression patterns stimulated lymphocytes were more susceptible of Bcl2 and other molecules involved in the to apoptosis; the reason for the extract being apoptosis pathways in treated cells. Alkaloids more effective on stimulated lymphocytes. have been shown to induce cell growth arrest, phosphorylation of Bcl-2, increased Bax Conclusion protein levels, and finally cell death.39,40 Bcl-2 is known to play an important role in the intrinsic The results show that P. maritimum extract apoptosis pathway and protects the microtubule at 0.1 mg/ml concentration has a profound integrity.18 Pancratistatin, a major alkaloid in and significant antiproliferative effect on P. maritimum extracts, may also contribute to MDA-MB-231 cancerous cell line in vitro. This cell death by indirect activation of pro-apoptotic the first report regarding the antitumor effects Bcl-2 proteins and/or interfere with antioxidant of P. maritimum extracts on MDA-MB-231 response mechanisms.41 Pancratistatin also cell line. The antiproliferative effect implies induces an autophagic response in p53-mutant the involvement of mechanisms that leads to prostate cancer cells that might be due in part cell proliferation inhibition, arresting cells at S to possible down-regulation of Bcl-2 proteins by and G2/M phases, and cyclin B1, Bcl-2, Ki67 pancratistatin.42 Lycorine, another alkaloid found expression down-regulation. In addition to in P. maritimum extracts, has been implicated in testing the effects of those fractions in vivo, we down-regulation of anti-apoptotic Bcl-2 family intend to conduct future studies on fractionated member Mcl-1.43 Recent studies have indicated compounds of bulb extract and test their effects that lycorine also activates caspase-3, -8, -9 and on different neoplastic cell lines to identify the modulates the expression of anti-apoptotic Bcl-2 most effective fraction. The mechanism of family member, Mcl-1, leading to apoptosis in observed effects also requires further detailed leukemia cells.43,44 It is therefore possible that investigation. P. maritimum extract may be modulating the anti- apoptotic effect of Bcl-2 proteins to contribute to Acknowledgement cell death. However, in this study, P. maritimum extracts showed minor effects on apoptosis The authors would like to thank the Director induction. This indicates that the inhibition General of the AECS and the head of Molecular of MDA-MB-231 cells growth is not through Biology and Biotechnology Department for their triggering apoptosis induction pathways or that support. the cell line is resistant to apoptosis induction. This is in agreement with the findings of Ryu.28 Conflict of Interest: None declared. In general, our results are in accord with the findings of studies carried out on different plant References species extracts and on different cancerous cell lines.45,46 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, The effects of P. maritimum extract on Murray T, et al. Cancer Statistics. CA normal lymphocytes proliferation was minor Cancer J Clin. 2008;58:71-96. doi:10.3322/ and nonspecific. This is consistent with Griffen CA.2007.0010. PMID: 18287387. findings that pancratistatin, a major alkaloid in 2. Richardson MA. Biopharmacologic and P. maritimum extract, is selective in inducing herbal therapies for cancer: research update apoptosis specifically in Jurkat cells (a human from NCCAM. J Nutr. 2001;131:3037s-40s. lymphoma cells) and in clinical leukemia PubMed PMID: 11694644. samples with minimal effect on non-cancerous 3. Kathiresan K, Boopathy NS, Kavitha S. peripheral blood mononuclear cells.42 Studies Coastal vegetation—an underexplored have indicated that pancratistatin induces source of anticancer drugs. Nat Prod Rad.

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2006;5:115-9. plant extracts from Amaryllidaceae. Phytother 4. Edeoga H, Okwu D, Mbaebie B. Res. 2003;17:1220-3. doi: 10.1002/ptr.1346. Phytochemical constituents of some PubMed PMID: 14669261. Nigerian medicinal plants. Afr J Biotechnol. 17. Rinner U, Hudlicky T. Synthesis of 2005;4:685-8. Amaryllidaceae constituents-an update. 5. Azaizeh H, Saad B, Khalil K, Said O. The state Synlett. 2005;2005:365-87. of the art of traditional arab herbal medicine 18. Soltan MM, Hamed AR, Hetta MH, in the eastern region of the mediterranean: Hussein AA. Egyptian Pancratium a review. Evid Based Complement Alternat maritimum L. flowers as a source of anti- Med. 2006;3:229-35. doi: 10.1093/ecam/ Alzheimer’s agents. Bulletin of Faculty of nel034. PubMed PMID: 16786053; PubMed Pharmacy, Cairo University. 2015;53:19-22. Central PMCID: PMCPMC1475945. 19. Kaya Gİ, Sarıkaya B, Çiçek D, Ünver 6. Berkov S, Evstatieva L, Popov S. Alkaloids Somer N. In vitro Cytotoxic Activity of in Bulgarian Pancratium maritimum L. Sternbergia sicula, S. lutea and Pancratium Z Naturforsch C. 2004;59:65-9. PubMed maritimum Extracts. Hacettepe Univ J PMID: 15018055. Faculty Pharm. 2010;30:41-8. 7. Al-Oudat M, Qadir M. The halophytic flora 20. Bhaumik A, Das S, Acharjee S, Das P, Mani G, of Syria. International Center for Agricultural Swarnalatha J. The bioactive molecule Research in the Dry Areas, Aleppo, Syria. resveratrol (RVTL) obtained from the black 2011. grapes (Vitis vinefera) act as potential 8. Bastida Armengol J, Berkov S, Torras hepatocytes regenerators and cytotoxic Claveria L, Pigni NB, Andradre JPd, agent. Der Pharma Chemica. 2015;7:112-27. Martínez V, et al. Chemical and biological 21. Aggarwal BB, Shishodia S. Molecular targets aspects of Amaryllidaceae alkaloids. Recent of dietary agents for prevention and therapy of Advances in Pharmaceutical Sciences, cancer. Biochem Pharmacol. 2006;71:1397- 2011;2011:65-100. 421. doi: 10.1016/j.bcp.2006.02.009. 9. Bastida J, Lavilla R, Viladomat F. Chemical PubMed PMID: 16563357. and biological aspects of Narcissus alkaloids. 22. Fennell CW, van Staden J. Crinum species Alkaloids Chem Biol. 2006;63:87-179. in traditional and modern medicine. PubMed PMID: 17133715. J Ethnopharmacol. 2001;78:15-26. doi: 10. Jin Z. Amaryllidaceae and Sceletium 10.1016/s0378-8741(01)00305-1. PubMed alkaloids. Nat Prod Rep. 2009;26:363-81. PMID: 11585683. doi: 10.1039/b718044f. PubMed PMID: 23. Evidente A, Kireev AS, Jenkins AR, 19240946. Romero AE, Steelant WF, Van 11. Ibrahim SR, Mohamed GA, Shaala LA, Slambrouck S, et al. Biological evaluation Youssef DT, El Sayed KA. New alkaloids of structurally diverse amaryllidaceae from Pancratium maritimum. Planta Med. alkaloids and their synthetic derivatives: 2013;79:1480-4. doi: 10.1055/s-0033- discovery of novel leads for anticancer 1350741. PubMed PMID: 23970422. drug design. Planta Med. 2009;75:501-7. 12. Youssef D. Bioactive principles of the doi: 10.1055/s-0029-1185340. PubMed flowers of Pancratium maritimum. Bulletin of PMID: 19235683; PubMed Central PMCID: Pharmaceutical Sciences-Assiut University. PMCPMC3125136. 2003;26:171-8. 24. Louw CA, Regnier TJ, Korsten L. Medicinal 13. Taie HAA, Esawy MAE-T, Mohamed MAE-M. bulbous plants of South and their Antioxidant and antimicrobial activities of traditional relevance in the control of different parts of Pancratium maritimum. infectious diseases. J Ethnopharmacol. Planta Medica. 2015;81:PM_166. doi: 2002;82:147-54. PubMed PMID: 12241989. 10.1055/s-0035-1565543. 25. Prasain JK, Barnes S. Metabolism 14. Nikopoulos D, Alexopoulos AA. In vitro and bioavailability of flavonoids in propagation of an endangered medicinal chemoprevention: current analytical plant: Pancratium maritimum L. J Food Agric strategies and future prospectus. Mol Pharm. Environ. 2008;6:393-98. 2007;4:846-64. doi: 10.1021/mp700116u. 15. Georgiev V, Ivanov I, Berkov S, Pavlov A. PubMed PMID: 18052086. Alkaloids biosynthesis by Pancratium 26. Liu RH. Potential synergy of phytochemicals maritimum L. shoots in liquid culture. Acta in cancer prevention: mechanism of action. Physiol Plant. 2011;33:927-33. J Nutr. 2004;134:3479s-85s. PubMed PMID: 16. Sener B, Orhan I, Satayavivad J. Antimalarial 15570057. activity screening of some alkaloids and the 27. Chen C, Kong AN. Dietary chemopreventive

62 Iran J Med Sci January 2018; Vol 43 No 1 Antiproliferative effects of Pancratium maritimum

compounds and ARE/EpRE signaling. Identification of tubulin as the molecular target Free Radic Biol Med. 2004;36:1505-16. of proapoptotic pyrrolo-1,5-benzoxazepines. doi: 10.1016/j.freeradbiomed.2004.03.015. Mol Pharmacol. 2006;70:60-70. doi: 10.1124/ PubMed PMID: 15182853. mol.105.021204. PubMed PMID: 16571652. 28. Ryu DS, Lee HS, Lee GS, Lee DS. Effects 37. Dash BC, El-Deiry WS. Phosphorylation of of the ethylacetate extract of Orostachys p21 in G2/M promotes cyclin B-Cdc2 kinase japonicus on induction of apoptosis through activity. Mol Cell Biol. 2005;25:3364-87. the p53-mediated signaling pathway in doi: 10.1128/mcb.25.8.3364-3387.2005. human gastric cancer cells. Biol Pharm Bull. PubMed PMID: 15798220; PubMed Central 2012;35:660-5. PubMed PMID: 22687398. PMCID: PMCPMC1069593. 29. Jeong JH, Ryu DS, Suk DH, Lee DS. Anti- 38. Sun Y, Xu HJ, Zhao YX, Wang LZ, Sun LR, inflammatory effects of ethanol extract Wang Z, et al. Crocin Exhibits Antitumor from Orostachys japonicus on modulation Effects on Human Leukemia HL-60 Cells In of signal pathways in LPS-stimulated RAW Vitro and In Vivo. Evid Based Complement 264.7 cells. BMB Rep. 2011;44:399-404. Alternat Med. 2013;2013:690164. doi: doi: 10.5483/BMBRep.2011.44.6.399. 10.1155/2013/690164. PubMed PMID: PubMed PMID: 21699753. 23573146; PubMed Central PMCID: 30. Singh NP, Lai HC. Artemisinin induces PMCPMC3615578. apoptosis in human cancer cells. Anticancer 39. Vitale I, Antoccia A, Cenciarelli C, Crateri P, Res. 2004;24:2277-80. PubMed PMID: Meschini S, Arancia G, et al. Combretastatin 15330172. CA-4 and combretastatin derivative induce 31. Rezaei PF, Fouladdel S, Ghaffari SM, mitotic catastrophe dependent on spindle Amin G, Azizi E. Induction of G1 cell cycle checkpoint and caspase-3 activation in arrest and cyclin D1 down-regulation in non-small cell lung cancer cells. Apoptosis. response to pericarp extract of Baneh in 2007;12:155-66. doi: 10.1007/s10495-006- human breast cancer T47D cells. Daru. 0491-0. PubMed PMID: 17143747. 2012;20:101. doi: 10.1186/2008-2231-20- 40. Liou JP, Wu CY, Hsieh HP, Chang CY, 101. PubMed PMID: 23351343; PubMed Chen CM, Kuo CC, et al. 4- and Central PMCID: PMCPMC3556048. 5-aroylindoles as novel classes of 32. Yuan SY, Lin CC, Hsu SL, Cheng YW, Wu JH, potent antitubulin agents. J Med Chem. Cheng CL, et al. Leaf Extracts of Calocedrus 2007;50:4548-52. doi: 10.1021/jm070557q. formosana (Florin) Induce G2/M Cell Cycle PubMed PMID: 17685504. Arrest and Apoptosis in Human Bladder 41. Dong LF, Freeman R, Liu J, Zobalova R, Cancer Cells. Evid Based Complement Marin-Hernandez A, Stantic M, et al. Alternat Med. 2011;2011:380923. doi: Suppression of tumor growth in vivo by 10.1155/2011/380923. PubMed PMID: the mitocan alpha-tocopheryl succinate 21760824; PubMed Central PMCID: requires respiratory complex II. Clin Cancer PMCPMC3132470. Res. 2009;15:1593-600. doi: 10.1158/1078- 33. Checchi PM, Nettles JH, Zhou J, Snyder JP, 0432.ccr-08-2439. PubMed PMID: Joshi HC. Microtubule-interacting drugs 19223492. for cancer treatment. Trends Pharmacol 42. Griffin C. Evaluation of the Selective Anti- Sci. 2003;24:361-5. doi: 10.1016/s0165- Cancer Activity of Natural and Synthetic 6147(03)00161-5. PubMed PMID: Alkaloids [dissertation]. [Ontario. Canada]: 12871669. University of Windsor. 2011. Chapter 3: 34. Honore S, Pasquier E, Braguer D. Electronic Theses and Dissertations; Understanding microtubule dynamics for p. 60-70. improved cancer therapy. Cell Mol Life Sci. 43. Liu XS, Jiang J, Jiao XY, Wu YE, Lin JH, 2005;62:3039-56. doi: 10.1007/s00018-005- Cai YM. Lycorine induces apoptosis and 5330-x. PubMed PMID: 16314924. down-regulation of Mcl-1 in human leukemia 35. Lefranc F, Sauvage S, Van Goietsenoven G, cells. Cancer Lett. 2009;274:16-24. doi: Megalizzi V, Lamoral-Theys D, Debeir O, 10.1016/j.canlet.2008.08.029. PubMed et al. Narciclasine, a plant growth PMID: 18829157. modulator, activates Rho and stress fibers 44. Liu J, Hu WX, He LF, Ye M, Li Y. Effects in glioblastoma cells. Mol Cancer Ther. of lycorine on HL-60 cells via arresting 2009;8:1739-50. doi: 10.1158/1535-7163. cell cycle and inducing apoptosis. FEBS mct-08-0932. PubMed PMID: 19531573. Lett. 2004;578:245-50. doi: 10.1016/j. 36. Mulligan JM, Greene LM, Cloonan S, Mc febslet.2004.10.095. PubMed PMID: Gee MM, Onnis V, Campiani G, et al. 15589827.

Iran J Med Sci January 2018; Vol 43 No 1 63 Tayoub G, Al-Odat M, Amer A, Aljapawe A, Ekhtiar A

45. Reagan-Shaw S, Eggert D, Mukhtar H, mitochondria and reduces growth of human Ahmad N. Antiproliferative effects of colon tumor xenografts. Mol Cancer Ther. apple peel extract against cancer cells. 2011;10:57-68. doi: 10.1158/1535-7163. Nutr Cancer. 2010;62:517-24. doi: mct-10-0735. PubMed PMID: 21220492. 10.1080/01635580903441253. PubMed 48. Chatterjee SJ, McNulty J, Pandey S. PMID: 20432173. Sensitization of human melanoma cells 46. Ghate NB, Hazra B, Sarkar R, Mandal N. In by tamoxifen to apoptosis induction by vitro anticancer activity of Spondias pinnata pancratistatin, a nongenotoxic natural bark on human lung and breast carcinoma. compound. Melanoma Res. 2011;21:1-11. Cytotechnology. 2014;66:209-18. doi: doi: 10.1097/CMR.0b013e328337abff. 10.1007/s10616-013-9553-7. PubMed PubMed PMID: 20300039. PMID: 23686547; PubMed Central PMCID: 49. Fantin VR, Leder P. Mitochondriotoxic PMCPMC3918266. compounds for cancer therapy. Oncogene. 47. Griffin C, Karnik A, McNulty J, Pandey S. 2006;25:4787-97. doi: 10.1038/ Pancratistatin selectively targets cancer cell sj.onc.1209599. PubMed PMID: 16892091.

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