Ethanolic periwinkle leaf extract reduces telomerase expression in T47D cancer cells 79 *Department of Anatomy, Faculty of Medicine, YARSI University, Jakarta **Department of Biochemistry, Faculty of Medicine, YARSI University, Jakarta Correspondence: Prof. Dr. Endang Purwaningsih, MS, PA Department of Anatomy, Faculty of Medicine, University of YARSI Jln. Letjend Suprapto, Cempaka Putih, Jakarta10510 Phone: +6221-4206674/76 Email: endpurwaning@gmail.com/ endang.purwaningsih@yarsi.ac.id Univ Med 2015;34:79-86 DOI: 10.18051/UnivMed.2016.v35.79-86 pISSN: 1907-3062 / eISSN: 2407-2230 This open access article is distributed under a Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License ABSTRACT UNIVERSA MEDICINA May-August, 2015May-August, 2015May-August, 2015May-August, 2015May-August, 2015 Vol.34 - No.2 Vol.34 - No.2 Vol.34 - No.2 Vol.34 - No.2 Vol.34 - No.2 BACKGROUND Cancer cells have a relatively high telomerase activity and a lower p53 protein expression than normal cells, so that cancer cells have the ability to continue to proliferate and do not undergo apoptosis. One of the cancer treatments is chemotherapy using bioactive ingredients from synthesis or isolation of natural materials. One of the plants that have potential as anticancer agent is periwinkle (Catharanthus roseus L). The research objective was to evaluate the effect of ethanolic periwinkle leaf extract against p53 protein and telomerase expression in T47D cancer cells. METHODS An experimental study with controls was conducted involving T47D breast cancer cells. They were divided into 3 groups (control, ½ dose of IC50/ 26.849 µg/mL, and one dose of IC50/53.699 µg/mL) at a cell density of 1 x 104 cells/well. Expression of p53 and telomerase was measured by the immunohistochemistry method. Data were analyzed using one-way ANOVA followed by a multiple comparison test. RESULTS Periwinkle leaf extract significantly increased p53 protein expression (p<0.05) at both treatment doses, ½ IC50 and IC50, compared to the control group and it highly significantly reduced telomerase expression (p<0.01), in comparison with the control group at both treatment doses. CONCLUSION Periwinkle leaf extract has potential as an anti-breast cancer agent by increasing p53 protein expression and inhibiting telomerase expression. Keywords: Periwinkle, p53, telomerase, T47D cells Ethanolic periwinkle leaf extract reduces telomerase expression in T47D cancer cells Endang Purwaningsih*, Etty Widayanti*, and Yulia Suciati** DOI: http://dx.doi.org/10.18051/UnivMed.2015.v34.79-86 80 Purwaningsih, Widayanti, Suciati Periwinkle leaf extract reduces telomerase Ekstrak etanol daun tapak dara menurunkan ekspresi telomerase pada sel kanker T47D LATAR BELAKANG Kanker merupakan penyakit yang mendapat perhatian serius di dunia kedokteran. Hal ini disebabkan oleh jumlah penderitanya yang semakin meningkat dan belum ada cara efektif untuk mengobatinya. Sel kanker memiliki aktivitas telomerase yang relatif tinggi dan menunjukkan ekspresi protein p53 yang lebih rendah dibandingkan sel normal sehingga sel kanker memiliki kemampuan untuk terus berproliferasi dan tidak mengalami apoptosis. Salah satu pengobatan kanker adalah dengan kemoterapi menggunakan bahan-bahan bioaktif dari hasil sintesis atau isolasi bahan alam. Salah satu tanaman yang memiliki potensi sebagai antikanker adalah tapak dara (Catharanthus roseus L). Tujuan penelitian adalah untuk menilai efek ekstrak etanol daun tapak dara terhadap ekspresi protein p53 dan telomerase pada kultur sel kanker T47D. METODE Sebuah penelitian eksperimental dengan kontrol dilakukan deengan mengikut sertakan kultur sel kanker payudara T47D (cell line). Mereka secara acak dibagi menjadi 3 kelompok (kontrol, ½ dosis IC50/26.849 µg/mL, dan satu dosis IC50/53.699 µg/mL) dengan kepadatan sel 1 x 104 sel/sumuran. Ekspresi p53 dan telomerase diukur dengan metode immunohistochemistry. Data dianalisis menggunakan Anova satu jalan dilanjutkan dengan uji perbandingan berganda. HASIL Ekstrak daun tapak dara meningkatkan ekspresi protein p53 secara bermakna pada kedua dosis perlakuan (p<0,05) baik ½ IC 50 maupun 1 IC50 dibandingkan kelompok kontrol (p<0,05), dan menurunkan ekspresi telomerase secara sangat bermakna pada kedua dosis perlakuan (p<0,01). KESIMPULAN Ekstrak daun tapak dara memiliki potensi sebagai anti kanker payudara melalui peningkatan ekspresi protein p53 dan hambatan telomerase. Kata kunci : Tapak dara, p53, telomerase, sel T47D ABSTRAK INTRODUCTION Cancer is a health problem in various countries throughout the world and is a disease that attracts serious attention in the medical field. This is because the number of victims is increasing from year to year and no effective treatment method has been found. Medical cancer treatments that have been currently used are surgery, irradiation, and chemotherapy. The latter, which is currently of interest, comprises the use of synthetic or natural bioactive substances such as those from the rat tail rhizome (Typhonium flagelliforme) and periwinkle (Catharanthus roseus).(1-4) According to data from the World Health O rg a n i z a t i o n ( W H O ) , 8 0 % o f t h e w o r l d population still depends on plant-derived traditional medications for its health needs. A total of 25% of the commercially available modern drugs in the world are derived from active substances that have been isolated and developed from plants. At present the use of traditional medications is on the increase, despite the 81 Univ Med Vol. 34 No.2 substantial advances in modern medical therapeutics. The Indonesian community has recognized various medicinal plants, because Indonesia is known as one of the countries with the greatest biodiversity and as such has potential for the development of plant-based medications, particularly anticancer drugs.(5) One of the plants that contain potential anticancer compounds is the periwinkle plant (Catharanthus roseus (L) G Don), formerly known as Vinca rosea L. The leaves of this plant contain a number of alkaloids that are potential anticancer agents, i.e. vincristine, vinblastine, vindesine, vindoline, tabersonine, leurosine, catharanthine, and lochnerine.(4,6) Periwinkle leaf extract may be used as anticancer or antitumor agent, according to a number of previous in vivo studies on rats as well as in vitro studies using cultures of the MCF7 cancer cell line.(7,8) In addition, periwinkle leaf extract at a dose of 1.0 mL/kg has been shown to be able to significantly reduce serum HDL (high density lipoprotein) and LDL (low density lipoprotein) cholesterols and triglycerides in normal rats.(7) Furthermore it is known that there are more than 70 types of alkaloid in the roots, stems, leaves, and seeds of the periwinkle plant, including 28 bi-indole alkaloids. In addition to anticancer alkaloids, the periwinkle plant also contains blood glucose reducing alkaloids, i.e. leurosine, lochnerine, tetrahydroalstonine, vindoline and vindolinine.(6,9) An extract of its flowers also has hyperglycemic effects in diabetic rats and functions as antioxidant, antibacterial, anthelminthic, and antidiarrheal agent.(9-11) A number of cancer cells have a high telomerase activity and each type of cancer cell may have different degrees of telomerase expression according to the type or location of the cancer. This can be used as a basis for the treatment of cancer cells if there are substances that can inhibit the activity of the telomerase enzyme. In addition, cancer cells possess a high mitotic activity since they undergo uncontrolled cell division as a result of low p53 tumor suppressor protein expression. The p53 protein is responsible for the mechanism of apoptosis of cancer cells and has become the latest cancer treatment strategy.(12,13) A number of scientific data have demonstrated the benefits of periwinkle, such as inhibition of proliferation and triggering of apoptosis in cancer cells (11,14,15) as well as cytotoxicity against cancer cells.(8) The anti- apoptotic effect may be determined by counting the percentage of cells undergoing apoptosis by flow cytometer. The effect of the periwinkle plant on p53 or telomerase protein expression in cancer cells is still unknown. The objective of this study was to evaluate the effect of periwinkle (Catharanthus roseus L) leaf extract on p53 and telomerase expression in T47D breast cancer cells. METHODS Design of study This laboratory experimental study was carried out from June 2013 until November 2013 at the Department of Biochemistry, Faculty of Medicine, YARSI University. Study subjects The study subjects were cultures of the T47D cancer cell line, obtained from the Parasitology Laboratory, Faculty of Medicine, Gadjah Mada University. The cells were cultured on 24-well microplates using RPMI (Roswell Park Memorial Institute) medium, with two replications for each group. The cultures were incubated for 72 hours or until confluence was reached, with a density of 1 x 104 cells/well. Upon confluence, the cultures were treated with the respective doses. Preparation of periwinkle leaf extract Periwinkle leaf extract was prepared as follows: fresh leaves were cleansed, cut into small strips, and air-dried. The dried strips were then blenderized into a powder. The powder was macerated in 80% ethanol in an Erlenmeyer flask, under continuous stirring. The ethanolic extract was then transferred to a rotary flask to 82 Purwaningsih, Widayanti, Suciati Periwinkle leaf extract reduces telomerase be concentrated in a rotary evaporator. The concentrated periwinkle leaf extract was dissolved in DMSO, diluted to give a number of concentrations, and filtered using a 0.2 µm filter before being placed on the 24-well plates. Intervention The cultures of the T47D cancer cell line were assigned to 3 groups, i.e. one control group that did not receive extract, one group receiving extract at a dose of one half the maximal inhibitory concentration (IC50) (53.699 µg/mL), and one group receiving a dose of ½ IC50 (26.849 µg/ mL). Before treatment with the extract, a test was performed to determine the IC50 of the periwinkle leaf extract against T47D cell cultures. The result of IC50 testing was used to determine the treatment dose to be used. The treated cell cultures on coverslips were incubated under 5% CO 2 at 37oC for 24 hours. Preparation of cell culture and cell growth media Preparation of Roswell Park Memorial Institute (RPMI) culture medium was by dissolving RPMI 1640 powder for one liter into approximately 800 ml of twice distilled water. After adding 2.0 grams of sodium bicarbonate and 2.0 grams of HEPES, twice distilled water was added to make up a volume of one liter. The solution was mixed with a magnetic stirrer for about 10 minutes until homogenous, then neutralized with 1 N HCl to give a pH of 7.2 – 7.4. Growth medium was prepared by mixing FBS 19 mL, penicilline streptomycin 2 mL, fungizone 0.5 mL, then diluted with RPMI 1640 culture medium to a volume of 100 mL. The solution was then aseptically filtered using a sterile 0.2 µm polyethylene sulfone filter and afterwards stored in the refrigerator in stoppered bottles. Immunohistochemical determination of p53 protein and telomerase expression T47D cell cultures at a density of 1x 104/ well that had attached to the coverslips were stained immunohistochemically according to standard procedures of the Parasitology Laboratory, Faculty of Medicine, Gadjah Mada University. Immunohistochemical determination of p53 protein and telomerase expression was as follows: the cell cultures on the coverslips, after being given the extract and incubated under 5% CO 2 at 37oC for 24 hours, were placed on poly-l- lysine slides and fixed in acetone or methanol for 10 minutes at -200C. The slides were then washed with PBS for 3 x 5 minutes, then 0.3% H 2 O 2 was dripped 20 minutes. Normal mouse serum (1:50) was dripped for 15 minutes and the liquid was discarded (without washing). Then p53 or telomerase antibody was dripped for 60 minutes. The slides were then washed with PBS for 3 x 5 minutes, incubated with biotinylated secondary antibody for 5-10 minutes, and again washed with PBS for 3 x 5 minutes. This was followed by incubating the slides with streptavidin-peroxidase for 5-10 minutes, washing with PBS for 3 x 5 minutes, incubating with the chromogen deaminobenzidine tetrahydrochloride (DAB) for 5-10 minutes (at a chromogen to substrate ratio of 1:20), and washing with distilled water. The slides were immersed in hematoxyllin for 3-5 minutes for counterstaining, washed with distilled water, dehydrated in 95% ethanol and immersed in xylene, for 10 minutes respectively. The slides were given a drop of Canada balsam as mounting medium and covered with a coverslip. The expression of p53 protein and telomerase was observed under the light microscope. Cells expressing p53 protein or telomerase showed brownish colored nuclei and cytoplasms, whereas cells without protein expression showed blue- violet nuclei and cytoplasms. Expression-positive cells were counted per 100 observed cells and counting was done in triplicate. The results were expressed as percentages. Data analysis The p53 protein expression and telomerase percentages were analyzed by one-way ANOVA, followed by least significant difference (LSD) multiple comparisons, using SPSS version 17. 83 Univ Med Vol. 34 No.2 RESULTS B e f o r e e v a l u a t i n g t h e e ff e c t o f t h e periwinkle leaf extract on the expression of p53 and telomerase, toxicity testing was performed on periwinkle leaf extract against cultures of T47D breast cancer cells. The toxicity tests yielded an IC50 value of 53.699 µg/mL. Assessment of periwinkle leaf extract effects on p53 protein and telomerase expression by immunohistochemistry was done by counting the percentages of expression-positive cells. The percentages of p53 and telomerase positive cells in the treatment groups receiving extract (at doses of ½ IC50 or 26.849 µg/mL and 1 IC50 or 53.699 µg/mL, respectively) are shown in Table 1. From the percentages of the expression- positive cells it is apparent that periwinkle leaf extract tended to increase p53 protein expression and to reduce telomerase expression. According to the results of Anova analysis on the expression of p53 protein and telomerase, there were substantially significant differences (p=0.000) between groups. Between-group LSD m u l t i p l e c o m p a r i s o n t e s t r e s u l t s o n t h e expression of p53 and telomerase are presented in Table 2. The LSD multiple comparison test results on p53 protein expression showed significant differences (p<0.05) between the control group and the group receiving a dose of one-half IC50, and also between the group receiving a dose of one-half IC50 and the group receiving a dose of one IC50. There were also highly significant differences (p<0.01) between the control group and the group receiving a dose of one IC50. The LSD multiple comparison test results on telomerase expression showed highly significant differences between the control group and the one-half IC50 group, between the control group and the one IC50 group, and between the one- half IC50 group and the one IC50 group. DISCUSSION From the results of this study, it is apparent that ethanolic periwinkle leaf extract is able to reduce telomerase expression and increase p53 protein expression in T47D breast cancer cells. T h e r e f o r e t h e c o m p o u n d s c o n t a i n e d i n periwinkle leaf extract are able to inhibit telomerase expression in breast cancer cells, or in other words, the extract may act as a telomerase inhibitor. Previous investigators have reported that chloroform and methanolic extracts of the periwinkle (Catharanthus roseus L) possess cytotoxic effects against HCT-116 colon cancer cells.(16) In addition crude aqueous periwinkle extract also has cytotoxic effects against leukemic T cells and induces the proliferation of normal blood cells.(17) There have been no reports on the effect of periwinkle extract on telomerase expression in colon cancer cells and leukemic T cells. It has also been reported that Table 1. Expression of p53 protein and telomerase in T47D cell cultures by treatment group IC50 : one half the maximal inhibitory concentration Table 2. Results of LSD test on expression of p53 and telomerase, by treatment group IC50: one half the maximal inhibitory concentration 84 Purwaningsih, Widayanti, Suciati Periwinkle leaf extract reduces telomerase curcumin, a component of turmeric (Curcuma longa L) rhizomes, has in vitro anti-proliferative activity against Bel7402, HL60 and SGC7901 cancer cell lines and demonstrates in vivo antitumor effects in laboratory rats. In addition, curcumin at a dose of 1 ì M is able to inhibit telomerase activity in cancer cells in vitro and to induce apoptosis.(18) It is known that various cancer cells have relatively higher telomerase activities than normal cells and that there is a correlation between telomerase activity and the development of cancer cells. Telomerase is the principal key to cellular immortality and tumorogenesis. Telomerase is activated in 80–90% of human cancer cells, but not in somatic cells (normal cells). Furthermore it has been reported that telomerase may be a biomarker for early detection of cancers and cancer monitoring, and has sufficient potential for a basis of development of cancer treatment.(19) The p53 protein is a tumor suppressor protein that is responsible for the mechanism of apoptosis in cancer cells. The mechanism comprises the repair of cells that have been induced to become cancerous. Expression of specific p53 protein occurs in abnormally replicating cells and requires programmed cell death called apoptosis. Higher p53 protein expression is associated with a higher tendency to apoptosis. The p53 protein is expressed upon the occurrence of DNA damage.(12,13) The p53 protein is a tumor suppressor protein, with a molecular weight of 53 kilodalton, that is activated upon the occurrence of DNA damage or certain types of stress in the cells. This protein can promote apoptosis by increased expression of the Bax gene, which codes for the Bax protein that plays a role in apoptosis. Detection of damaged DNA is regulated by the p53 tumor suppressor. When DNA damage occurs, p53 prevent the cells from entering the next phase of cell division and gives time to the DNA to repair itself, or if the damage is sufficiently severe, p53 will initiate programmed cell death (apoptosis). Loss of a number of molecular checkpoints are to be found in the development of several tumors or cancers.(20) Administration of the leaf extract of periwinkle (Catharantus roseus) can increase p53 protein expression, such that this extract may be used to increase apoptosis of breast cancer cells. The periwinkle plant produces secondary metabolites that are active as alkaloid anticancer agents. (9,14) On the other hand, periwinkle extract has been shown to reduce telomerase expression, as has been shown in T47D cell cultures in the treatment group at ½ I C 5 0 a n d 1 I C 5 0 . Te l o m e r a s e i s a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomers. Telomerase is a structure that is responsible for the maintenance of telomers, being a TTAGGGn nucleotide replication. In most human somatic cells, such a s f i b r o b l a s t s , t e l o m e r a s e e x p r e s s i o n i s repressed, the telomers become progressively shorter and at each somatic cell division the cells undergo programmed cell death or apoptosis. In contrast, the majority of human tumor or cancer cells express telomerase, such that the telomer length is stable and the cells do not die by apoptosis. This observation shows that telomer maintenance is essential for tumor or cancer cell proliferation.(21) Telomerase activity determines cell proliferation, both of cancer and normal cells, in vitro as well as in vivo conditions.(22,23) Telomerase activity in cancer cells increase after certain stages, i.e. when there is a loss of a number of substances or telomer subunits in the cells. Finally the telomerase is activated to stabilize the telomers, the cancer cells become immortal and proliferate continuously. (24) Telomerase plays a role in cell replication, since it is able to maintain telomer length in the cell progeny. Therefore cancer cells without sufficient telomerase will cease to grow when this enzyme cannot any longer continuously maintain telomer length as a result of abnormal control of proliferation. Lack of telomerase may inhibit 85 Univ Med Vol. 34 No.2 cancer growth by continuous cell division such that the telomers become shorter and the cells die before the occurrence of further damage. However, if the cancer cells are able to synthesize telomerase then these cells will proliferate continuously and become immortal.(23) Periwinkle leaf extract contains more than 100 monoterpenoids that exert cytotoxic effects and are used in cancer chemotherapy. Alkaloids such as vincristin and vinblastin in periwinkle leaf extract may function as potent antineoplastic agents (25) by inhibiting DNA synthesis and blocking mitosis in metaphase or anaphase.(26) A study using periwinkle leaf extract on T47 D cancer cells found that the extract had anticancer effects through induction of apoptosis, but that it did not show antioxidant activity in T47D cells.(11) One limitation of this study is that it did not investigate other proteins that play a role in apoptosis, such as Bax and BcL-2, after administration of periwinkle leaf extract to T47D breast cancer cells. As a result of this study, a natural raw material with chemopreventive actions against breast cancer cells will be available from the periwinkle plant (Catharanthus roseus). CONFLICT OF INTEREST There are no conflict of interest. CONCLUSION Compounds in periwinkle leaf extract are potential agents against breast cancer by increasing p53 protein expression leading to apoptosis and by telomerase inhibition. ACKNOWLEDGEMENT The authors wish to express their profound gratitude to the Directorate General of the Ministry of Higher Education (Direktorat Jendral Dikti) for support of this study through the HPEQ research fund, allowing us to conduct this study. REFERENCES 1. Hamid IS, Nazar DS, Ratnani. Hambatan ekspresi vascular endothelial growth factor oleh ekstrak daun sambung nyawa pada endotel membran korioalantois. J Veteriner 2013;14:85- 90. 2. Chadidjah, Nasihun T, Widayati E, et al. Typhonium flagelliforme decreases protein expression in murine breast cancer. Univ Med 2014;33:161-70. 3. Purwaningsih E, Widayanti E, Suciati Y. Cytotoxicity assay of Typhonium flagelliforme Lodd against breast and cervical cancer cells. Univ Med 2014;33:75-82. 4. Sain M, Sharma V. Catharanthus roseus (an anti- cancerous drug yielding plant): a review of potential theurapeutic properties. Int J Pure App Biosci 2013;1:139-42. 5. Farida Y, Rahayu L, Faizatun. Aktivitas antioksidan serbuk n-heksana dan metanol hasil pengeringan semprot ekstrak keladi tikus (Typhonium flagelliforme L) Decue. Kongres Ilmiah XVIII Ikatan Apoteker Indonesia 2010. Makasar, 10–12 Desember 2010. 6. Man S, Gao W, Wei C, et al. Anticancer drugs from traditional toxic Chinese medicines: review. Phytother Res 2012;26:1449-65. 7. Antia BS, Otokan JE. Effect of leaf juice of Catharanthus roseus Linn on cholesterol, triglyceride, and lipoprotein levels in normal rats. Indian J Pharmacol 2005;37:401-2. 8. Ruskin S, Aruna SR. In vitro and in vivo antitumor activity of Catharanthus roseus. IRJPAS 2014;4:1-4. 9. Tiong SH, Yeng Looi C, Hazni H, et al. Antidiabetic and antioxidant properties of alkaloids from Catharanthus roseus (L) G. Don. Molecules 2013;18:9770-84. 10. Natarajan A, Syed Ahmed KSZ, Sundaram S, et al. Effect of aqueous flower extract of Catharanthus roseus on alloxan induced diabetes in male Albino rats. IJPSDR 2012;4:150-3. 11. Widowati W, Mozef TJ, Risdian C, et al. Apoptosis and antioxidant activities of Catharanthus roseus (L) G. Don extract on breast cancer cell line. Ind J Cancr Chem 2010;1:99- 107. 12. Bai L, Zhu WG. P53: structure, function and therapeutic applications. J Cancer Mol 2006;2: 141-53. 13. Amaral JD, Xavier JM, Steer CJ, et al. The role of p53 in apoptosis. Discovery Med 2010;9:145- 52. 86 Purwaningsih, Widayanti, Suciati Periwinkle leaf extract reduces telomerase 14. Widowati W, Mozef T, Risdian C, et al. Anticancer and free radical scavenging potency of Catharanthus roseus, Dendropthoe petandra, Piper betle and Curcuma mangga extracts in breast cancer cell lines. Oxid Antioxid Med Sci 2013;2:137-42. 15. Looi CY, Arya A, Cheah FK, et al. Induction of apoptosis in human breast cancer cells via caspase pathway by vernodalin isolated from Centratherum anthelminticum (L.) seeds. PLoS ONE 2013;8:e56643. doi:10.1371/journal.pone. 005664. 16. Siddiqui MJ, Ismail Z, Aisha AFA, et al. Cytotoxic activity of Catharanthus roseus (Apocynaceae) crude extracts and pure compounds against human colorectal carcinoma cell line. Int J Pharmacol 2010;6:43-7. 17. Ahmad NH, Rahim RA, Mat I. Chatarhanthus roseus aqueous extract is cytotoxic to Jurkat leukaemic T cells but induce the proliferation of normal peripheral blood mononuclear cells. Trop Life Sci 2010;21:105-15. 18. Chui SX, Qu KJ, Xie YY, et al. Curcumin inhibits telomerase activity in human cancer cell lines. Int J Mol Med 2006;18:227-31. 19. Chen CH, Cheng RJ. Prevalence of telomerase activity in human cancer. J Formos Med Assoc 2011;110:275-89. 20. Rivlin N, Brosh R, Oren M, et al. Mutations in the p53 tumor suppressor gene: important milestones at the various steps of tumorogenesis. Genes Cancer 2011;2:466-74. 21. Quellette MW, Wright WE, Shay JW. Targeting telomerase-expressing cancer cells. J Cell Mol Med 2011;15:1433-42. 22. Shay JW, Wright WE. Senescence and immortalization: role of telomere and telomerase. Carcinogenesis 2005;26:867-74. 23. Artandi SE, DePinho RA. Telomeres and telomerase in cancer. Carcinogenesis 2010;3:9- 18. 24. Blackburn E. Telomerase and cancer. Mol Cancer Res 2005;3:9. DOI: 110.1158/1541-7786 -MCR-05-0147. 25. Gajalaksmi S, Vijayalaksmi S, Rajeswari D. Pharmacological activities of Catharanthus roseus: a perspective. Int J Pharm Bio Sci 2013;4: 431-9. 26. Aqubi AE, Al-Naimi RS, Al Taee EH. Comparative pathological and cytogenetical study of ethanolic extract Vinca rosea L and vinblastine in treating mammary gland adenocarcinoma implanted mice. Kufa J Vet Med Sci 2011;2:146-63.