IJMS Vol 43, No 1, January 2018 Original Article Antiproliferative Effects of Pancratium Maritimum Extracts on Normal and Cancerous Cells Ghaleb Tayoub1, PhD; Abstract Mohmad Al-Odat2, PhD; Amal Amer1, BSc; Background: Plants are an important natural source of Abdulmunim Aljapawe1, BSc; compounds used in cancer therapy. Pancratium maritimum 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 Syria, Damascus, Syria; Methods: Leaves, flowers, roots, and bulbs 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 plant 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 bulb 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 amaryllidaceae 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. 52 Iran J Med Sci January 2018; Vol 43 No 1 Antiproliferative effects of Pancratium maritimum 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. Iran J Med Sci January 2018; Vol 43 No 1 53 Tayoub G, Al-Odat M, Amer A, Aljapawe A, Ekhtiar A 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.