IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (2) 2011 Datura stram oium Extract Inhibits Mito sis in Cancer Cells A. H. M. Hussain, I . K. Abid Ali, A. A. Al-Naqeeb Departme nt of Medical Basic Sciences, College of Nursing, Unive rsity of Baghdad Received in: 21, July, 2010 Accepte d in: 14, December, 2010 Abstract The aim of this st udy was to evaluate the anti mitotic activity of ether extract p repared from Datura stramoium p lant on cancer cells. Recently oncology st udies are directed toward substances which act somehow to induce apop tosis or to p revent abnormal dividing of cell, that may p rovide therap eutic st rategies for the treatment of cancer. Thus, many research on various ty p es of p lants such as fruits, vegetables, ot her editable p lants and toxic p lants have been done in vitro and in vivo. But, few st udies on Datura p lant were occurred and there was no p revious study dealing with its effect on p roliferative activity of cancer cells. In current st udy , two ty p es of common malignant tumor were tested in vitro. These were mammary cancer and glioma. The cancer cells were exp osed to various concentration of ether extract of Datura stramoium 1000, 1250, 1500 g/ml. Then p ercentage of mitotic indexes were est imated. The results exhibited that there were decreases in p ercentages of mitotic index in both ty p es of cancer cells. However, the decrement was significant in the mammary cancer and insignificant in glioma cell line. Conclusi on: Conclusion obtained from this study indicated that ether extract st udy of Datura stramoium has ability to inhibit mitot ic activity of cancer cells. Key words: Datura stramoium, Ant iproliferative agent, Antimitot ic agents. Introduction Cell can be divided through entering cell cycle. The cell cycle consists of four distinct p hases: G1 p hase, S p hase, G2 p hase (cell actively known as interp hase, during which cell grows, accumulation nutrients needed for mitosis and dup lication its DNA) and M p hase in which the cell chromosomes are divided between two daughter cell and the cytop lasm divides forming distinct cell [1]. Any changes or aberration in this regulatory sy st em of cell division may lead to uncontrol cell division giving rise to malignancy defining a cancerous/neoplast ic growt h [2]. Thus sup p ression of abnormal cell division may p rovide therap eutic st rategies for the treatment of inap p rop riate cell p roliferation or decrease the rate of recurrence risk or increase rate of p atient survival [3]. Therefore the research is directed toward substances which act somehow to induce ap op tosis or t o p revent cell from division. These substances may be plant p oisoning or extracted from microorganism. Such as lunasin is a novel p eptide identified in soy bean has ability to sup p ress carcinogenesis of skin and other mammalian cells by chemical carcinogens [4]. Virgin olive p henol extract inhibits p roliferation of p romyelocytic leukemia cells (HL60) and induce apop tosis [5] whereas p henol and hexan extracts of M astic gum contain constituents which can induce P53 and P21 indep endent G1-p hase arrest followed by apop tosis of human HCT 116 colon cancer cells in vitro [6]. Studies of Nigella sativa and Crocus sativus extracts, showed that t hese plants have ability to inhibit t wo stages, initiation, IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (2) 2011 p romotion [dimethy benzena 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 p lant belongs to the family solanaceae and is a medicinal p lant with antinocicep tive, antioxidant, hy p olip idemic, anti-inflammatory , antirheomatoid and hy p oglycemic [9]. On the other hand, many st udies 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 (M DA- M B231), head, neck (FaDu) and lung (A549) cancer cell lines[10]. Whereas acetone and ether extract of D. stramoium seeds p roduced cytotoxicity on lary ngeal cancer cell (Hep 2), 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 p revented metastasis. M oreover, the prop hy lotic study showed that the extracts of D. stramoium reduce the percentage of mice bearing tumor in mice treated with these extracts p rior to imp lantation with mammary tumor [13]. The p urp ose of this st udy is to evaluate the effect of the ether extract of this p lant on cell division of cancer cell lines. Materials and Methods Preparation of se eds extract: The extract was p repared from Datura stramoium seeds, as described by Gasp ori-Comp ani et al.[14]. Briefly, seeds were ground several times with diethy l ether in blender. The resulting p owder was extracted over night with 10 volume of cold 0.2M NaCl containing 0.005M - sodium p hosp hate buffer, p H 7.2. Aft er centrifugation, the sup ernatant was fractionated with (NH4)2SO4 at 0-40%, 40-60% and 60-100% saturation and the p recipitates obtaining were redissolved in p hosp hate buffered saline and dialysed against the same solution. Ce ll line s handling: Two ty p es of cancer cell lines were used in this st udy . These were mammary cancer (AM N3). It was p rovided kindly by Iraqi Center for Cancer and M edical Genetic Research and glioma cell lines p rovided kindly by Dr. A. Al-Shimary/Iraqi Center for Cancer and M edical 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 o C for 24 hours (using 2 falcons for each concentration and 2 for control). - By using growt h medium. Five ml at each concentration were p repared from extract. - The cell line was treated with extract after medium in each falcon was p oured 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 growt h 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 harvest ed by adding 2-3 ml of try p sin-versene and incubated at 37 o C for 2-3 minutes and put it in tube which containing media. - Centrifugation at 1500 rp m for 10 minutes was done. - The sup ernatant at each tube was decanted off by Past eur p ipett e and the cells were susp ended in 10 ml of 0.075M KCl. Pot assium chloride was added on the wall of tube with slowly mixing and tubes incubated in water bath at 37 o C for 30 minutes. - Aft er centrifugation at 1500 rp m for 10 minutes, sup ernatant were removed and cells washed many times with fixative solution (comp osed from 3 p arts methanol+1 p art glacial acetic acid). The fixative p repared at t he time of using and should be cooling before using. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (2) 2011 - Aft er solution became clear 2-3 drop s were drop p ed on wet chilled and grease free glass slide, and left slides for dry ing. - Slides were st ained using freshly mode working Giemsa st ain (p repared by adding 1 p art of Giemsa st ain to 4 p arts of Sorenson's buffer) which as app lied for 2 minutes then rapidly washed with Sorensen's buffer, thereafter left t o dry at room temp erature. - M icroscop ic examination under high magnification using 40X objective lens was p erformed 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]: M itot ic Index (%) = cellscountedTotal cellsdividingofnumber Re sults In mammary cancer cell line, the result exhibited that the higher p ercentage of mitotic index app eared in control 1.96+0.26% when there was no extract in the medium (zero concentration). Whereas the p ercentages became decrement in mitotic index when the cells exp osed to different concentration of Datura extract. The st atist ic analysis showed that there were insignificant decrement at 1000, 1500 g/ml 1.44+0.62 and 1.90+0.5%, resp ectively. While significantly decrement at level P<0.01 at concentration 1250g/ml 0.32+0.08% fig. (1). Fig. (2) illustrated the p ercentage of mitotic index of glioma cell line. There were insignificantly decrement in the mitotic index when the cells exp osed to extract were 1.29+0.25, 1.23+0.15 and 1.5+0.34% at 1000, 1250 and 1500 g/ml, resp ectively. 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 st udy was to invest igate whether the ether extract of D. stramonium acts as antip roliferative agent. The results reflected that the ether extract comp onents had ability to reduce mitosis of tested cancer cell lines (mammary cancer and glioma). However, the reduction was app eared significantly in mammary cancer and insignificantly in glioma. These observations indicate that t he ether extract was more effective on mammary cancer than glioma. The current investigation app eared that the ether extract has antitumor activity and exerted its activity by inhibit mitosis. Few st udies on this p lant had been done as antitumor [11], whereas t here were no previous studies on it as antip roliferative agent. The anticancer effect of Datura extract was sup p orted by p revious st udy in which app eared 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 st udies showed the same action of other ty p es of extract p repared from D. stramonium p lant against various ty p es of cancer cell lines [10,12]. Previous st udy illustrated that the comp onents of ether extract were flavenoids, tanrin, lectin and trace amounts of alkaloids[17]. Flavenoids are group of naturally occurring p olyp henole compounds p resent in fruits, vegetable and other edible p lants[18]. Research on p hy tochemicals showed that some flavenoids p rocess chemopreventive p rop erties and numerous studies were p erformed recently to assess the mechanisms whereby each flavenoids p revent cancer, including induction of cell cycle arrest, apop tosis and antip roliferation[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. S CI. VOL.24 (2) 2011 dependent kinase (CDK). That leads to decrease p hosp hory latron of retinoblast oma (Rb)[21]. Ot her ty p es 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]. Ot her flavoenoids inhibit p roliferation via inhibit epidermal growt h factor (EGF) induced and constitut ively active mitogen activated p rotein kinase (M APK) [22]. On the other hand, st udy on the p lant lectin showed lectin from p holiota adip osa had antip roliferative actively toward hepatoma Hep G2 cells and breast cancer M CF7 cells. But the inhibitory activities have not been determined [23]. In this st udy , we have demonst rated that ether extract of D. stramanium inhibited p roliferation of cancer cells. Furt her st udies are needed to determine the p recise p athway for antip roliferative action. Re ferences 1.Alberts, B.; Johnson, A.; Lwis, J.; Raff, M .; Roberts, K. and Watt er, K. (2008). M olecular 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). M olecular biology of the gene, (5 th ed.), San Francisco, Pearson/Benjamin: 510-12. 3.Lary , S. ; Blanchette, M . and M ichael-Levesque, J. (2006). Delphinidin, a dietary, anthocy anidin, inhibits vascular endothelial growt h factor receptors p hosp harylation, Carcinogenesis, 27(5): 989-996. 4. Jeong, H .; Lam, Yi. and Delumen, B. (2002). Barley lunasin sup p resses 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 M orozzi, G., (2006). Virgin olive oil p henol inhibit p roleferation of human p romyelocytic leukemia cells (HL60) by inducing apop tosis and differentiation, J. Nut , 136,614-619. 6.Dimas, K.; Hatziantoniou, S.; Wyche, J. and Pantazis, P., (2009). A mast ic gum extract induces sup p ression of growt h of human colorectal tumor xenegrafts in immunodeficient mice, INT .J.Exp . & Cli. Pathop hy siol. 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, Nut r Cancer, 16(1): 67- 72. 8. Valeic, S.; Timmermann, B. N.; Alberts, D. S.; Wacher, G. A.; Krut zch, M .; Wymer, J. and Guillen, J.M . (1996). Inhibitory effect of six green tea catechine & caffeine on the growt h of four selected human tumor cell lines, Ant icancer Drugs, 7(4): 461-8. 9. Gharaibeh, M . N.; Elayan, H. H. and Slahab, A. S., (1988). Hy p oglycemic effected Teucrium Polium, J., Ethano Pharmacol, 24: 93-99. 10. Ahmad, I.; Abdalla, M .; M ust afa, N.; Qnais, E. and Abdalla, F. (2009). Datura aqueous leaf extract enhances cytotoxicity via metabolic oxidative st ress 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 p roliferation and induction of differentiation of glioma cells with Datura stramonium agglutinin, British Journal of Cancer, 87: 918-923. 12. M irza, A.; Al-Jeboori, K. and Yaseen, N. (2010). Investigation of the p hy tochemicals p resence in the local Datura stramonium seeds by using two extraction method, Iraq, J. of Veter. M ed., 34(1) .Under Press. 13. M irza, Hussain, A.; H., (2009). Investigation of the effect of the thorn app le (Datura stramonium)extracts\ on mammary adenocarcinoma by In vitro and In vivo st udy .Adisseration submitt ed to the council of college of Veterinary M edicine, Baghdad University . IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 (2) 2011 14. Gasp eri-Camp ani, A.; Barbieri, L.; M orell, P.; and Stripe, F., (1980). Seed extracts inhibiting p rotein sy nthesis in vitro, Biochemi, J., 186, 439-441. 15. M asters, J. R. (2000). Animal cell culture, a Practical Ap p roach, 3 rd ed., Oxford University Press: 205-6. 16. Ikedo, K. ; Pant, B. ; M ishiro, 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. ; M itt al, A. and Katiyar, C. (2004). Proanthecy anidius from grape seed inhibit exp ression of matrix metalloproteinase in human p rost ate carcinoma cells which is associated with t he inhibition of activation of M APK. Carcinogenesis, 25(6): 987-995. 18. M irza, A.; Al-Jeboori, K. and Yaseen, N. (2010). Cy totoxic affect of Datura stramonium extraction cancer cell lines, Iraq , J. of Veter. M ed., 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 p revention 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. Ty aqi, A.; Agarwal, R. and Agarwal, C. (2003). Grap e seed extract inhibit EGF-induced and const itut ively active mitogenic signaling but activate JNK in human p rost ate carcinoma DU145 cells p ossible role in app roliferation and apop tosis: Oncogene: 29, 1302-1316. 23. Zhang, G. O.; Sun, D.; Wang, HX and NgTB, (2009). A noval lectin with antip roliferation activity from the medicinal mashroom pholiota adiposa. acta Bio. Chem. Pd., 56 (3): 415- 21. Fig. (1) Mitoti c index of mammary cancer cel l line after exposure to different concentrations of the ethe r extract of Datura stramonium. * Means There i s a signi ficant difference at l evel P IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.24 ( 2) 2011 Fig. (2) Mitotic index of glioma cel l line after exposure to different concentrations of the ethe r extract of Datura stramonium. Fig. (3) Comparing between mammary cancer cel l line and gli oma cel l line in mi totic index after exposure to the ethe r 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 ـــــــتخلص الـــ ـــــة مســـ ــــ ــــــیم فعالیـ ــــ ــــــى تقیـ ــــة إلــــ 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 تتجــه بعــض الدراســات والبحــوث إلــى دراســة أنــواع اذمــن االنقســام غیــر الطبیعــي وبهــذا تــوفر ســتراتیجیة لعــالج الســرطان، والنباتـات األخـرى الصـالحة لألكـل منهـا والسـامة فـي الزجـاج وفـي الجسـم إّال ان ،والخضـر ،الفواكهمثل ،مختلفة من النباتات عملیـة االنقســام وتكــاثر فــي الدراسـات قلیلــة حـول تــأثیر نبـات الــداتورة فـي الخالیــا السـرطانیة والتوجــد دراسـات حــول تأثیرهـا . 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 مــن أهــم االســتنتاجات التــي توصــلت الیهــا الدراســة ان المســـتخلص االثیــري لنبــات الــداتورة یمتلــك القــدرة علــى تثبــیط انقســـام . ةالخالیا السرطانی .داتورة سترامونیوم، مضادات االنقسام الخلوي، مضادات التكاثر الخلوي: الكلمات المفتاحیة