IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 Datura stramoium Extract Inhibits Mitosis in Cancer Cells A. H. M. Hussain, I. K. Abid Ali, A. A. Al-Naqeeb Department of Medical Basic Sciences, College of Nursing, University of Baghdad Received in: 21, July, 2010 Accepted in: 14, December, 2010 Abstract The aim of this study was to evaluate the anti mitotic activity of ether extract prepared from Datura stramoium plant on cancer cells. Recently oncology studies are directed toward substances which act somehow to induce apoptosis or to prevent abnormal dividing of cell, that may provide therapeutic strategies for the treatment of cancer. Thus, many research on various types of plants such as fruits, vegetables, other editable plants and toxic plants have been done in vitro and in vivo. But, few studies on Datura plant were occurred and there was no previous study dealing with its effect on proliferative activity of cancer cells. In current study, two types of common malignant tumor were tested in vitro. These were mammary cancer and glioma. The cancer cells were exposed to various concentration of ether extract of Datura stramoium 1000, 1250, 1500 g/ml. Then percentage of mitotic indexes were estimated. The results exhibited that there were decreases in percentages of mitotic index in both types of cancer cells. However, the decrement was significant in the mammary cancer and insignificant in glioma cell line. Conclusion: Conclusion obtained from this study indicated that ether extract study of Datura stramoium has ability to inhibit mitotic activity of cancer cells. Key words: Datura stramoium, Antiproliferative agent, Antimitotic agents. Introduction Cell can be divided through entering cell cycle. The cell cycle consists of four distinct phases: G1 phase, S phase, G2 phase (cell actively known as interphase, during which cell grows, accumulation nutrients needed for mitosis and duplication its DNA) and M phase in which the cell chromosomes are divided between two daughter cell and the cytoplasm divides forming distinct cell [1]. Any changes or aberration in this regulatory system of cell division may lead to uncontrol cell division giving rise to malignancy defining a cancerous/neoplastic growth [2]. Thus suppression of abnormal cell division may provide therapeutic strategies for the treatment of inappropriate cell proliferation or decrease the rate of recurrence risk or increase rate of patient survival [3]. Therefore the research is directed toward substances which act somehow to induce apoptosis or to prevent cell from division. These substances may be plant poisoning or extracted from microorganism. Such as lunasin is a novel peptide identified in soybean has ability to suppress carcinogenesis of skin and other mammalian cells by chemical carcinogens [4]. Virgin olive phenol extract inhibits proliferation of promyelocytic leukemia cells (HL60) and induce apoptosis [5] whereas phenol and hexan extracts of Mastic gum contain constituents which can induce P53 and P21 independent G1-phase arrest followed by apoptosis of human HCT116 colon cancer cells in vitro [6]. Studies of Nigella sativa and Crocus sativus extracts, showed that these plants have ability to inhibit two stages, initiation, IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 promotion [dimethybenzena anthracene/croton oil] skin carcinogenesis in mice [7]. While others repeated that six green tea catechins and caffein including antimutagenesis [8]. D. stramoium is a wild growing flowering plant belongs to the family solanaceae and is a medicinal plant with antinociceptive, antioxidant, hypolipidemic, anti-inflammatory, antirheomatoid and hypoglycemic [9]. On the other hand, many studies on D. stramoium showed that it has ability to act in vitro and in vivo against cancer cells. The aqueous D. stramoium leaf extracts showed cytotoxic effect on breast (MDA- MB231), head, neck (FaDu) and lung (A549) cancer cell lines[10]. Whereas acetone and ether extract of D. stramoium seeds produced cytotoxicity on laryngeal cancer cell (Hep2), mammary (AM N3) and glioma cell lines [11,12]. In vivo, D. stramoium seed extracts exhibited their ability to reduce the size and doubling rate of mammary cancer. In addition, ether extract prevented metastasis. Moreover, the prophylotic study showed that the extracts of D. stramoium reduce the percentage of mice bearing tumor in mice treated with these extracts p rior to implantation with mammary tumor [13]. The purpose of this study is to evaluate the effect of the ether extract of this plant on cell division of cancer cell lines. Materials and Methods Preparation of seeds extract: The extract was prepared from Datura stramoium seeds, as described by Gaspori-Compani et al.[14]. Briefly, seeds were ground several times with diethyl ether in blender. The resulting powder was extracted over night with 10 volume of cold 0.2M NaCl containing 0.005M- sodium phosphate buffer, pH 7.2. After centrifugation, the supernatant was fractionated with (NH4)2SO4 at 0-40%, 40-60% and 60-100% saturation and the precipitates obtaining were redissolved in phosphate buffered saline and dialysed against the same solution. Cell lines handling: Two types of cancer cell lines were used in this study. These were mammary cancer (AM N3). It was provided kindly by Iraqi Center for Cancer and Medical Genetic Research and glioma cell lines provided kindly by Dr. A. Al-Shimary/Iraqi Center for Cancer and Medical Genetic Research. Three concentrations of the ether extract were used 0, 1000, 1250, 1500 g/ml for treating the cells. The handling of each cell line for each concentration was occurred according to[15] with slight modification. -The cell line was cultured in falcon by using RPM I media containing 10% of fetal calfe serum at 37 oC for 24 hours (using 2 falcons for each concentration and 2 for control). - By using growth medium. Five ml at each concentration were prepared from extract. - The cell line was treated with extract after medium in each falcon was poured off then incubated at 37 o C for 72 hrs i.e. when confluent monolayer was formed. - The medium containing extract was replaced (in each falcon) by growth medium containing 20% fetal calfe serum, then after 6 hours from changing medium, 0.2 ml colchicin was added to 5 ml of medium. - At the end time, cells were harvested by adding 2-3 ml of trypsin-versene and incubated at 37 o C for 2-3 minutes and put it in tube which containing media. - Centrifugation at 1500 rpm for 10 minutes was done. - The supernatant at each tube was decanted off by Pasteur pipette and the cells were suspended in 10 ml of 0.075M KCl. Potassium chloride was added on the wall of tube with slowly mixing and tubes incubated in water bath at 37 oC for 30 minutes. - After centrifugation at 1500 rpm for 10 minutes, supernatant were removed and cells washed many times with fixative solution (composed from 3 parts methanol+1 part glacial acetic acid). The fixative prepared at the time of using and should be cooling before using. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 - After solution became clear 2-3 drops were dropped on wet chilled and grease free glass slide, and left slides for drying. - Slides were stained using freshly mode working Giemsa stain (prepared by adding 1 part of Giemsa stain to 4 parts of Sorenson's buffer) which as applied for 2 minutes then rapidly washed with Sorensen's buffer, thereafter left to dry at room temperature. - Microscopic examination under high magnification using 40X objective lens was performed to determine mitotic index by counting the number of dividing cell was counted with the total number of cells (more than 1000 cells were counted). The mitotic index was calculated as follows[16]: Mitotic Index (%) = cellscountedTotal cellsdividingofnumber Results In mammary cancer cell line, the result exhibited that the higher percentage of mitotic index appeared in control 1.96+0.26% when there was no extract in the medium (zero concentration). Whereas the percentages became decrement in mitotic index when the cells exposed to different concentration of Datura extract. The statistic analysis showed that there were insignificant decrement at 1000, 1500 g/ml 1.44+0.62 and 1.90+0.5%, respectively. While significantly decrement at level P<0.01 at concentration 1250g/ml 0.32+0.08% fig. (1). Fig. (2) illustrated the percentage of mitotic index of glioma cell line. There were insignificantly decrement in the mitotic index when the cells exposed to extract were 1.29+0.25, 1.23+0.15 and 1.5+0.34% at 1000, 1250 and 1500 g/ml, respectively. As shown in figure (3), there was significant differences at level P<0.05 in mitotic index between mammary cancer cell line and glioma cell line 0.32+0.08, 1.23+0.15% at concentration 1250 g/ml. Discussion A major objective of this study was to investigate whether the ether extract of D. stramonium acts as antiproliferative agent. The results reflected that the ether extract components had ability to reduce mitosis of tested cancer cell lines (mammary cancer and glioma). However, the reduction was appeared significantly in mammary cancer and insignificantly in glioma. These observations indicate that the ether extract was more effective on mammary cancer than glioma. The current investigation appeared that the ether extract has antitumor activity and exerted its activity by inhibit mitosis. Few studies on this plant had been done as antitumor [11], whereas there were no previous studies on it as antiproliferative agent. The anticancer effect of Datura extract was supported by previous study in which appeared that high concentration of the ether extract exhibited cytotoxic effect on different cancer cell lines, but not on normal cell line [12]. And few other studies showed the same action of other types of extract prepared from D. stramonium plant against various types of cancer cell lines [10,12]. Previous study illustrated that the components of ether extract were flavenoids, tanrin, lectin and trace amounts of alkaloids[17]. Flavenoids are group of naturally occurring polyphenole compounds present in fruits, vegetable and other edible plants[18]. Research on phytochemicals showed that some flavenoids process chemopreventive properties and numerous studies were performed recently to assess the mechanisms whereby each flavenoids prevent cancer, including induction of cell cycle arrest, apoptosis and antiproliferation[16,19]. Some flavenoids induce G1 and G2 arrest, by binding to and inhibiting the activity of cycline IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 dependent kinase (CDK). That leads to decrease phosphorylatron of retinoblastoma (Rb)[21]. Other types of flavoenoids can decrease E2F-1 level which may contributed to cell cycle arrest at G1, whereas their activity to decrease CdC2 make cell cycle arrest at G2[21]. Other flavoenoids inhibit proliferation via inhibit epidermal growth factor (EGF) induced and constitutively active mitogen activated protein kinase (MAPK) [22]. On the other hand, study on the plant lectin showed lectin from pholiota adiposa had antiproliferative actively toward hepatoma HepG2 cells and breast cancer MCF7 cells. But the inhibitory activities have not been determined [23]. In this study, we have demonstrated that ether extract of D. stramanium inhibited proliferation of cancer cells. Further studies are needed to determine the precise pathway for antiproliferative action. References 1.Alberts, B.; Johnson, A.; Lwis, J.; Raff, M.; Roberts, K. and Watter, K. (2008). Molecular biology of the cell, (5 th ed.), Garland Science, New York: 315-16. 2.Watson, J. D.; Baker, D. A.; Bell, S. P.; Gann, A.; Levine, M.; and Losick, R. (2004). Molecular biology of the gene, (5 th ed.), San Francisco, Pearson/Benjamin: 510-12. 3.Lary, S. ; Blanchette, M. and Michael-Levesque, J. (2006). Delphinidin, a dietary, anthocyanidin, inhibits vascular endothelial growth factor receptors phospharylation, Carcinogenesis, 27(5): 989-996. 4. Jeong, H .; Lam, Yi. and Delumen, B. (2002). Barley lunasin suppresses ras-induced colony formation and inhibits core histone acety lation in mammalian cell, J. Agric. Food Chem., 50(21): 5903-5908. 5. Fabiani, R.; DeBartolomeo, A. ; Rosignoli, P.; Servili, M.; Selvaggini, R.; Francesco, G.; Disarerio, and Morozzi, G., (2006). Virgin olive oil phenol inhibit proleferation of human promyelocytic leukemia cells (HL60) by inducing apoptosis and differentiation, J. Nut, 136,614-619. 6.Dimas, K.; Hatziantoniou, S.; Wyche, J. and Pantazis, P., (2009). A mastic gum extract induces suppression of growth of human colorectal tumor xenegrafts in immunodeficient mice, INT.J.Exp. & Cli. Pathophysiol. Drug Res., 23(1): 63-68. 7. Salomi, M. J.; Nair, S. C.; and Panikkar, K. R. (1991). Inhibitory effect of Nigella sativa and Saffron (Crocus satirus) on chemical carcino genesis in mice, Nutr Cancer, 16(1): 67- 72. 8. Valeic, S.; Timmermann, B. N.; Alberts, D. S.; Wacher, G. A.; Krutzch, M.; Wymer, J. and Guillen, J.M. (1996). Inhibitory effect of six green tea catechine & caffeine on the growth of four selected human tumor cell lines, Anticancer Drugs, 7(4): 461-8. 9. Gharaibeh, M. N.; Elayan, H. H. and Slahab, A. S., (1988). Hypoglycemic effected Teucrium Polium, J., Ethano Pharmacol, 24: 93-99. 10. Ahmad, I.; Abdalla, M.; Mustafa, N.; Qnais, E. and Abdalla, F. (2009). Datura aqueous leaf extract enhances cytotoxicity via metabolic oxidative stress on different human cancer cells, Jordan Journal of Biological Sciences, 2(1): 9-14. 11. Sasaki, T.; Yamazaki, K.; Yamori, T. and Endo, T. (2002). Inhibition of proliferation and induction of differentiation of glioma cells with Datura stramonium agglutinin, British Journal of Cancer, 87: 918-923. 12. Mirza, A.; Al-Jeboori, K. and Yaseen, N. (2010). Investigation of the phytochemicals presence in the local Datura stramonium seeds by using two extraction method, Iraq, J. of Veter. Med., 34(1) .Under Press. 13. Mirza, Hussain, A.; H., (2009). Investigation of the effect of the thorn apple (Datura stramonium)extracts\ on mammary adenocarcinoma by In vitro and In vivo study .Adisseration submitted to the council of college of Veterinary Medicine, Baghdad University . IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 14. Gasperi-Campani, A.; Barbieri, L.; Morell, P.; and Stripe, F., (1980). Seed extracts inhibiting protein synthesis in vitro, Biochemi, J., 186, 439-441. 15. Masters, J. R. (2000). Animal cell culture, a Practical Approach, 3 rd ed., Oxford University Press: 205-6. 16. Ikedo, K. ; Pant, B. ; Mishiro, A. et al., (2000). A convenient method for evaluation of anti-tumor agents affecting the cell cycle, J. Bio. Sci. Bio eng., 90(5): 574-576. 17. Vayalil, P. K. ; Mittal, A. and Katiyar, C. (2004). Proanthecyanidius from grape seed inhibit expression of matrix metalloproteinase in human prostate carcinoma cells which is associated with the inhibition of activation of MAPK. Carcinogenesis, 25(6): 987-995. 18. Mirza, A.; Al-Jeboori, K. and Yaseen, N. (2010). Cytotoxic affect of Datura stramonium extraction cancer cell lines, Iraq , J. of Veter. Med., 34(1) .Under Press. 19. Harborne, J. B. (1986). The Flavonids advances in research science, (1 st ed.), Chapman & Hall/CRC. 20. Birt, D. F.; Herdrichs, and Wang, W. (2001). Dietary agents in cancer prevention flavenoids and ISO Flavenoid, Pharmacol. Ther., 90: 157-177. 21. Lu, X.; Durg, J.; Cho, H.J. (2005). Fisetin inhibits the activities of cyclin dependent- kinases leading to cell cycle arrest in HT-29 Human Colon Cancer Cells, Amer. Society , N.J. 22. Tyaqi, A.; Agarwal, R. and Agarwal, C. (2003). Grape seed extract inhibit EGF-induced and constitutively active mitogenic signaling but activate JNK in human prostate carcinoma DU145 cells possible role in approliferation and apoptosis: Oncogene: 29, 1302-1316. 23. Zhang, G. O.; Sun, D.; Wang, HX and NgTB, (2009). A noval lectin with antiproliferation activity from the medicinal mashroom pholiota adiposa. acta Bio. Chem. Pd., 56 (3): 415- 21. Fig. (1) Mitotic index of mammary cancer cell line after exposure to different concentrations of the ether extract of Datura stramonium. * Means There is a significant difference at level P IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.24 (2) 2011 Fig. (2) Mitotic index of glioma cell line after exposure to different concentrations of the ether extract of Datura stramonium. Fig. (3) Comparing between mammary cancer cell line and glioma cell line in mitotic index after exposure to the ether extract of Datura stramonium. 2011) 2( 24مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة المجلد تثبیط انقسام الخالیا السرطانیة بمستخلص الداتورة سترامونیوم أسمهان عدنان النقیب،عبد علي ابتسام خلف ، آالء حسن مرزة حسین جامعة بغداد،كلیة التمریض،قسم العلوم الطبیة األساسیة 2010،تموز،21 :استلم البحث في 2010، كانون االول، 14 :قبل البحث في الخالصة ـــرطانیة ــــ ــــا الســــ ــــ ــــــي الخالیــ ــــ ـــري فـ ــــ ــــداتورة االثیـــ ــــ ـــتخلص الـــ ــــ ـة مســـ ــــ ــــ ــیم فعالیـ ــــ ــــ ــى تقیـ ــــ ــــ ة إل ــــ ــــ ـــــذه الدراســـ ــــ ــــدف هـ ــــ . تهــ مواد تعمل بطریقة ما فـي الحـث علـى إحـداث عملیـة المـوت المبـرمج أو لمنـع الخالیـا استعمالالحدیثة باتجاه تتجه الدراسات ـة أنــواع اذمــن االنقســام غیــر الطبیعــي وبهــذا تــوفر ســتراتیجیة لعــالج الســرطان، ه بعــض الدراســات والبحــوث إلــى دراسـ تتجــ والنباتـات األخـرى الصـالحة لألكـل منهـا والسـامة فـي الزجـاج وفـي الجسـم إّال ان ،والخضـر ،الفواكهمثل ،مختلفة من النباتات أثیر نبـات الــداتورة فـي الخالیــا السـرطانیة والتوجــد دراسـات حــول تأثیرهـا اثر فــي الدراسـات قلیلــة حـول تــ عملیـة االنقســام وتكــ . الخالیا السرطانیة بیثـة المزروعـة التـي تتمثـل بخـط مزرعـة خالیـا الغـدة اللبنیـة وكـذلك تتضمن هذه الدراسة نوعین من خطوط الخالیا الورمیة الخ ، 1250، 1000خـط مزرعــة سـرطان الــدماغ والتــي تـم تعریضــها إلـى تراكیــز مختلفــة مـن مســتخلص الـداتورة االثیــري وهــي . مللتر ومن ثم تم تقییم النسبة المئویة النقسام الخالیا/مایكروغرام 1500 كـان االنخفـاض معنویـًا فــي اذخفاضـًا فـي مؤشـرات االنقسـام فـي كـال النـوعین للخالیـا السـرطانیة ن هنـاك انأظهـرت الدراسـة ا . سرطان الغدة اللبنیة وفي مزرعة سرطان الدماغ ـام ـا الدراســة ان المســـتخلص االثیــري لنبــات الــداتورة یمتلــك القــدرة علــى تثبــیط انقســ ـتنتاجات التــي توصــلت الیهـ مــن أهــم االسـ . ةالخالیا السرطانی .داتورة سترامونیوم، مضادات االنقسام الخلوي، مضادات التكاثر الخلوي: الكلمات المفتاحیة