against Artemia salina and Inhibition Concentation (IC) 50

Original Article Anticancer properties of daily-consumed vegetables Amaranthus spinosus, Ipomoea aquatica, Apium graveolens, and Manihot utilisima to LNCaP prostate cancer cell lines Cristine D. Octaviani, Melinda Lusiana, Ade Zuhrotun 1, Ajeng Diantini, Anas Subarnas, Rizky Abdulah ABSTRACT Introduction: Prostate cancer is the second most common cancer in men worldwide. Efforts on fi nding potential sources from daily consumed foods that inhibit prostate cancer development are becoming important objective for scientists. Previous epidemiologic studies have consistently shown that consumption of fruit and vegetables was found to have signifi cant contribution in the prevention of cancer. Thus in this study, we investigated the anticancer potency of popular vegetables consumed in Indonesia, namely, Amaranthus spinosus, Ipomoea aquatica Forsk., Apium graveolens L., and Manihot utilisima to Lymph Node Carcinoma of the Prostate (LNCaP) human prostate cancer cell lines. Materials and Methods: The extracts were tested for Brine shrimp lethality test to guide their cytotoxic potencies, and methyl thiazolyl tetrazolium assay to LNCaP cells for their anticancer properties. Results: The results showed that I. aquatica Forsk has the most potential anticancer sources, with LC 50 against Artemia salina and Inhibition Concentation (IC) 50 against LNCaP being 266 g/ml and 400 g/ml, respectively. Conclusions: Our preliminary result suggest that I. aquatica Forsk. has the potential to be further investigated and developed as anticancer sources. Department of Pharmacology and Clinical Pharmacy, 1 Department of Biological Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia Key words: Brine shrimp lethality test, LNCaP, methyl thiazolyl tetrazolium assays, prostate cancer Access this article online Website: www.jnatpharm.org DOI: 10.4103/2229-5119.110366 Quick Response Code: Address for correspondence: Dr. Rizky Abdulah, Department of Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Universitas Padjadjaran, Jl. Raya Bandung Sumedang Km. 21, Jatinangor-45363, Indonesia. E-mail: abdulahrizky@ gmail.com INTRODUCTION As consequences of increases in welfare, public health, and nutrition as well as changes in population, there were also changes in patterns of degenerative diseases such as heart disease, diabetes, and cancer. According to Laborer s Health and Safety Fund of North America, one in every six men will get prostate cancer sometime in his life. Those who involve in heavy work and exposure to certain metals and chemicals are at high risk for prostate cancer. [1] Thus, although it has been money and time consuming, researches to find agents to use in the prevention or treatment of prostate cancer have become the main target of many scientists. [2] High intake of fruits and vegetables is consistently related to lower risk of cancer. [3] In most cancer, persons with low fruit and vegetable intake experience about twice the risk of cancer compared with those with high intake, even after control for potentially confounding factors. [4] Furthermore, the risk of prostate cancer increases with diet that is high in fat, low in fiber, and has few servings of vegetables. [1] Therefore, the National Cancer Institute encouraged to consume fruits and vegetables every day to reduce fat, and give high fiber diet. [5] In addition, scientific evidence have suggested that differences in diet and lifestyle may account in large part for the variability of prostate cancer incidences in different countries; thus, nutrition may play an important role in reducing the incidence and the risk of prostate cancer progression. [6] Herbal medicine has been universally accepted and has provided a great impact in the world of health and international trade. Therefore, it plays an important role to the human population worldwide. [2] The study of medicinal plants which have toxicity is the first step to find anticancer compounds. [7] One of the famous vegetables, broccoli, is known as anticancer as scientists have reported the benefits of its active compound sulforaphane in cancer prevention. [8,9] As part of efforts in finding potential sources that inhibit prostate cancer development from daily consumed Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 2013 67

foods, in this research we investigate the anticancer properties of others daily consumed vegetables, namely, Amaranthus spinosus, Ipomoea aquatica Forsk., Apium graveolens L., and Manihot utilisima. Their cytotoxic activities were tested using Brine shrimp lethality test (BSLT) and compared with methyl thiazolyl tetrazolium (MTT) assay against prostate cancer cell lines LNCaP. This research aims to provide information about the anticancer potency of several daily consumed vegetables, to increase public awareness to their potential use in prostate cancer management. MATERIALS AND METHODS Samples Collection and Extraction The leaves of daily consumed vegetables A. spinosus, I. aquatica Forsk., A. graveolens L., and M. utilisima were obtained from local market and extracted using ethanol for 3 24 h by maceration. Liquid extracts were evaporated with a rotary evaporator under vacuum pressure at temperatures less than 50 C in order to obtain viscous extract, and calculated for their total yield. Brine Shrimp Lethality Test BSLT is the simple pharmacology guide for toxic screening of compounds using nauplii Artemia salina Leach as a convenient monitor for screening. The BSLT was performed following the method of mayer and coworkers. [7] Brieftly, the hatching eggs were prepared in artificial sea water (3.8% NaCl solution) for 48 h to mature shrimp called nauplii. The test samples (extract) were prepared by dissolving them in Dimethyl sulfoxide (DMSO) (not more than 50 L in 5 ml solution) added with artificial sea water to attain variable concentrations of each extract. A vial containing 50 L DMSO diluted to 5 ml was used as control. The nauplii of A. salina were applied to each of experimental vial and vial control. After 24 h of exposure, the dead naupliis were counted as percent of mortality. From these data, the percent of lethality of the brine shrimp nauplii and concentration variations were plotted into graphic and calculated for the LC 50. colorimetric MTT assay, as described previously. [10] Briefly, cells (2 10 4 in 50 l/well) were plated in 96-well plates. After the initial cell seeding, different concentrations of each extract were added and incubated for 24 h. Ten microliters of water-soluble tetrazolium (WST)-8 assay cell-counting solution (Dojindo Lab., Tokyo, Japan) was added to each well and incubated at 37 C for 3 h. After the addition of 100 l/well of 1 NHCl, the cell proliferation rate was then determined by measuring the absorbance at a wavelength of 450 nm, with a reference wavelength of 650 nm. The absorbance was read using a microtiter plate reader (Becton-Dickinson, NJ, USA). Results were derived from triplicate experiments. RESULTS Brine Shrimp Lethality Test Figure 1 shows that each of I. aquatica Forsk., A. graveolens L., A. spinosus, and M. utilisima extract reached 50% mortality at different concentrations. The LC 50 of each extract evaluated in this research, respectively, was 266 g/ml, 160 g/ml, 140 g/ml, and 10 g/ml. Cell Proliferation Inhibition LNCaP cells proliferation analysis in the presence of various concentrations of various extracts was performed using a colorimetric MTT assay. The results in Figure 2 show that all the extracts inhibited the LNCaP cell proliferation in a dose-dependent manner. Until the highest concentration tested, A. spinosus, A. graveolens L., and M. utilisima failed to reach 50% cell proliferation inhibition. Meanwhile, I. aquatica Forsk showed IC 50 concentration of 400 g/ml against LNCaP cells. Cell Culture and Treatment Human prostate cancer cell lines (LNCaP) were obtained from Dainippon Pharmaceutical (Tokyo, Japan). The cell lines were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma, MO, USA) supplemented with 10% fetal bovine serum and antibiotics (100 U/ ml penicillin and 100 g/ml streptomycin). For cell treatments, various concentrations of extracts were added to the cell culture medium for 24 h. Drug Sensitivity Assay Cell proliferation analysis was performed with cells in the presence of various concentrations of various extracts by a Figure 1: Brine shrimp lethality test results for daily consumed vegetables Amaranthus spinosus, Ipomoea aqua ca Forsk., Apium graveolens L., and Manihot u lisima. All extracts work in a dose-dependent manner, with Manihot u lisima extracts showing the strongest ac vity with LC 50 of 10 g/ml 68 Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 2013

LNCaP prostate cancer cell lines. Figure 2 shows LNCaP cells proliferation properties of all extracts. I. aquatica Forsk. showed IC 50 concentration of 400 g/ml against LNCaP cells. However, M. utilisima, A. spinosus, and A. graveolens L. failed to reach 50% cell proliferation inhibition at the highest concentration tested. Figure 2: An cancer ac vi es of Amaranthus spinosus, Ipomoea aqua ca F., Apium graveolens L., and Manihot u lisima against LNCaP human prostate cancer cell lines. Ipomoea aqua ca F. showed the most potent ac vi es with IC 50 of 400 g/ml DISCUSSIONS BSLT was choosen in this study because it is widely known as a simple biological test to guide pharmacologic activities. In this study, it was showed that M. utilisima reached 100% mortality of nauplii A. salina at the concentration of 200 g/ml, whereas A. graveolens L. and I. aquatica Forsk. reached 100% mortality at concentration of 400 g/ml and 1,000 g/ml, respectively. However, A. spinosus failed to reach LC 100 until the highest concentration tested. This bioassay also used as a common guideline because of its certain correlation with the cancer cell lines, whereas LC 50 value less than 1,000 g/ml is considered to have significant pharmacologic activity and could be used as in vitro cancer preliminary study. [7] Based on its LC 50 results, it was suggested that all extracts had the potential to be further investigated for their anticancer properties. All the extracts showed a dose-dependent manner of effects with their LC 50 less than 1,000 g/ml. The LC 50 of M. utilisima, A. graveolens L, A. spinosus, and I. aquatica Forsk. were 10 g/ml, 140 g/ml, 160 g/ml, and 266 g/ ml, respectively. Other studies have reported an LC 50 of 920 g/ml for A. spinosus extract, [11] 385 g/ml for I. aquatic, [12] and 900 g/ml for A. graveolens extract. [11] Apart from the different of environmental condition during the analysis, these dissimilarities with our study might because of the differences in geography, climate, and environment where the plants have grown that will influence its secondary metabolites. Based on our BSLT results, we performed the anticancer analysis of the extracts in vitro by using MTT assays to There are two types of cancer cells in the human prostate gland: androgen dependant and androgen independant cells. [13] Androgens are hormones that activate the production of testosterone. LNCaP prostate cancer cell is androgen-influenced cancer cell. [14] In the body, the formation of excessive androgen hormone can induce excessive cell proliferation, resulting in cancer. [15] Therefore, it is tempting to speculate that the mechanism of I. aquatica Forsk. Against prostate cancer LNCaP cell proliferation might involve the androgen pathway of the protate cancer cells. In our BSLT results, it was M. utilisima that showed the most toxic extract with LC 50 10 g/ml; however, in an in vitro study against LNCaP cancer cell lines, it did not reach their IC 50 until the highest concentration tested. On the contrary, it was I. aquatica Forsk., the least toxic extract on BSLT with LC 50 of 266 g/ml, that showed the best anticancer activity against LNCaP cancer cell lines with IC 50 400 g/ml. In this research, we found little correlation of BSLT study with its in vitro study. This may be influenced by the secondary metabolites in I. aquatica Forsk. extract that may have antagonistic activities in BSLT. Prostate cancer is a kind of solid-typed cancer that may have less sensitivity compared with other liquid-typed cancer cells; thus, it needs further studies using other type of cancer cell lines to support its anticancer potential. Although much remains to be done, our work generates important findings to support the hypothesis that daily consumed vegetables may contribute to the prevention of certain types of cancer. There are two limitations that need to be acknowledged and addressed regarding this study. The first limitation concerns the active compounds of the extracts, whereas the second is its anticancer mechanism. Both are currently being investigated in our laboratory. CONCLUSIONS Among the daily consumed vegetables investigated in this preliminary study, I. aquatica Forsk. was the most potential extract compared with others for its anticancer potency against LNCaP human prostate cancer cell lines. Its LC 50 against A. salina and IC 50 against LNCaP were 266 g/ml and 400 g/ml, respectively. Our result suggests that I. aquatica Forsk. has the potential to be further investigated and developed as anticancer sources. Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 2013 69

ACKNOWLEDGMENTS We thank Meiliana Thamrin and Rano K. Sinuraya for their technical assistance. REFERENCES 1. Laborer s Health and Safety Fund of North America. Avoid prostate cancer, eat your vegetables! Available from: http://www.lhsfna.org/ index.cfm?objectid=9449dcd2-d56f-e6fa-922c4ae21546be84. [Last cited 2012 Jan 2]. 2. Madhuri S, Pandey G. Some anticancer from medicinal plants of foreign origin. Curr Sci 2009;96:779-83. 3. Steinmetz KA, Potter JD. Vegetables, fruit, and cancer prevention: A review. J Am Diet Assoc 1996;96:1027-39. 4. Block G, Patterson B, Subar A. Fruit, vegetables, and cancer prevention: A review of the epidemiological evidence. Nutr Cancer 1992;18:1-29. 5. Heimendinger J, Chapelsky D. The National 5 A Day for Better Health Program. Adv Exp Med Biol 1996;401:199-206. 6. Ledesma N. Nutrition & Prostate Cancer. Available from: http://www.psarising.com/download/nutrition_pca_ucsf.pdf. [Last cited 2012 Jan 1]. 7. Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE, McLaughlin JL. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med 1982;45:31-4. 8. Zhang Y, Talalay P, Cho CG, Posner GH. A major inducer of anticarcinogenic protective enzymes from broccoli: Isolation and elucidation of structure. Proc Natl Acad Sci U S A 1992;89:2399-403. 9. Shankar S, Ganapathy S, Srivastava RK. Sulforaphane enhances the therapeutic potential of TRAIL in prostate cancer orthotopic model through regulation of apoptosis, metastasis, and angiogenesis. Clin Cancer Res 2008;14:6855-66. 10. Abdulah R, Kobayashi K, Yamazaki C, Koyama H. Molecular targets of selenium in prostate cancer prevention (Review). Int J Oncol 2011;39:301-9. 11. Krishnaraju AV, Rao TV, Sundararaju D, Mulabagal V, Hsin-Sheng T, Gottumukakala VS. Assessment of bioactivity of Indian medicinal plants using Brine Shrimp (Artemia salina) Lethality Assay. Int J Appl Sci Eng 2005;2:125-34. 12. Balasuriya BM, Dharmaratne HR. Cytotoxicity and antioxidant activity studies of green leafy vegetables consumed in Sri Lanka. J Nat Sci Found Sri Lanka 2007;35:255-8. 13. Wang H, Sun D, Ji P, Mohler J, Zhu L. An AR-Skp2 pathway for proliferation of androgen-dependent prostate-cancer cells. J Cell Sci 2008;121:2578-87. 14. Xiao L, Gonzalez-Guerrico A, Kazanietz MG. PKC-mediated secretion of death factors in LNCaP prostate cancer cells is regulated by androgens. Mol Carcinog 2009;48:187-95. 15. Swerdloff RS, Ng JC. Gynecomastia: Etiology, Diagnosis, and Treatment, 2011. Available from: http://www.endotext.org/male/male14/ maleframe14.htm. [Last cited 2012 Jan 1]. Cite this article as: Octaviani CD, Lusiana M, Zuhrotun A, Diantini A, Subarnas A, Abdulah R. Anticancer properties of daily-consumed vegetables Amaranthus spinosus, Ipomoea aquatica, Apium graveolens, and Manihot utilisima to LNCaP prostate cancer cell lines. J Nat Pharm 2013;4:67-70. Source of Support: Nil. Conflict of Interest: None declared. 70 Journal of Natural Pharmaceuticals, Volume 4, Issue 1, January-June, 2013