Institute of Oncology and Radiology of Serbia

1. In vitro study of antitumor effect of Artemisia annua tea Zorica Juranić1, Pierre Lutgen2, Ivana Matić1, Milan Juranić3 1Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia 2IFBV, 10 rue Kommes L-6988, Hostert, Luxembourg 3Galenika fitofarmacija a.d. , Batajnica road b.b., 11080 Belgrade, Serbia Institute of Oncology and Radiology of Serbia Introduction One of the main goals in modern cancer research is to discover chemotherapeutic agents that selectively inhibit proliferation and survival of malignant cells, with minimal cytotoxicity against nontransformed healthy cells, especially immunocompetent peripheral blood mononuclear cells. Bioactive phytochemicals from Chinese medicinal plant species Artemisia annua, commonly known as sweet wormwood, have been reported to exhibit important pharmacological and medicinal properties, including antimalarial, antibacterial, antiviral and anticancer effect 1-4. The bioactive constituents of Artemisia annua are artemisinin, arteannuin, artenuic acid and flavonoids. Numerous studies demonstrate that artemisinin, a sesquiterpene lactone and its bioactive derivatives dihydroartemisinin, artesunate, artemether and arteether exert potent anticancer action 2-4. Furthermore, cancer supressive action of flavonoids has been well documented4. It is considered that those bioactive compounds could suppress initiation of malignant transformation, promotion and progression of cancer. The anticarcinogenic potential of phytochemical artemisinin, its derivatives and some flavonoids as well could be attributed to their influence on molecular targets of intracellular signal transduction pathways that regulate cell cycle, differentiation, apoptosis, cell migration and angiogenesis 2-4. The aim of this in vitro cytotoxic study was to elucidate whether the tea prepared from Chinese medicinal plant Artemisia annua has antitumor potential. Cytotoxicity of Artemisia annua tea was evaluated against selected malignant cell lines: human cervix adenocarcinoma HeLa, human malignant melanoma Fem-x and BG, human myelogenous leukemia K562, human breast adenocarcinoma MDA-MB-361 and human colon carcinoma LS174. In order to determine the selectivity in the antitumor action, the cytotoxic effect of tea was investigated against normal human immunocompetent peripheral blood mononuclear cells (PBMC), unstimulated and stimulated to proliferate by mitogen phytohemagglutinin.

2. Materials and methods Preparation of tea Artemisia annua grown in Luxembourg had an average artemisinin content of 0.15%. Tea for each experiment was prepared by adding 100 ml of boiling distilled water to 5 g of dry herb leaves. The mixture was covered, stayed for 10 min and the leaves were removed by filtration. After cooling at room temperature, the tea was filtered through Millipore filter, 0.22 µm, before use. Treatment of malignant cell lines HeLa and Fem-x (2,000 cells per well), K562 (5,000 cells per well), BG and LS (7,000 cells per well) and MDA-MB-361 cells (10,000 cells per well) were seeded into 96-well microtiter plates and 20 hours later five different concentrations of Artemisia annua tea were added to the cells. Nutrient medium was added to the cells in control wells. All experiments were done in triplicate. Treatment of PBMC PBMC (150,000 cells per well) were seeded into nutrient medium only or enriched with phytohemagglutinin in 96-well microtiter plates. After 2 hours Artemisia annua tea was added to the wells to five different final concentrations, except to the control wells where nutrient medium only was added to the cells. Determination of target cell survival After 72 h of continuous action of investigated tea, the survival of target cells was determined by MTT test.

3. Results Figure 2. Survival of unstimulated and PHA-stimulated PBMC grown for 72 hours in the presence of increasing concentrations of Artemisia annua tea, determined by MTT test Figure 1. Survival of HeLa, BG, Fem-x, LS, MDA-MB-361 and K562 cells grown for 72 hours in the presence of increasing concentrations of Artemisia annua tea, determined by MTT test Table 1. Concentrations of Artemisiaannua tea which induced 50 % decrease in target cell survival

4. Figure 3 . Photomicrographs of K562, HeLa and LS174 cells obtained 72 hours after continuous Artemisia annua tea activity control Artemisia annua tea c=16 mg/ml Table 2. Selectivity in the antitumor action of Artemisia annua tea K562 HeLa LS174

5. Discussion Tea prepared from Artemisia annua dry leaves exhibited selective dose-dependent cytotoxic effect against malignant cell lines and both unstimulated and stimulated PBMC. The strongest cytotoxic action was observed against K562 cells. Moreover, the tea exerted pronounced cytotoxic effect on melanoma BG and Fem-x cells and on HeLa cells, which showed similar sensitivity to the action of the investigated tea. Cytotoxic activity was found to be weaker against MDA-MB-361 and LS174 cells. On the other hand, cytotoxicity of tea was very weak significantly weaker against human healthy immunocompetent PBMC than against K562, HeLa, Fem-x and BG malignant cells. In adittion, cytotoxicity was slightly weaker on unstimulated PBMC in comparison to stimulated PBMC. Observed differences in intensities of cytotoxic action of Artemisia annua tea against selected malignant cell lines suggest that specific tea constituents might affect different target molecules of oncogenic signaling pathways of specific cell type. Pronounced cytotoxic effect to unstimulated and stimulated PBMC might be attributed to the direct inhibitory action of phytochemicals towards specific sensitive subpopulations of immunocompetent cells i.e. regulatory cells which suppress the antitumor immune response. Identifying these PBMC subpopulations could be important in order to elucidate whether the subpopulations of healthy PBMC included in the direct antitumor immune response remain unsensitive to activity of these agents.

6. Conclusions Results from present research clearly demonstrate stronger and highly selective antitumor effect of Artemisia annua tea to leukemia K562 cells in comparison to healthy PBMC. To melanoma BG and Fem-x cells and to cervix adenocarcinoma HeLa cells tea was also selective in its antitumor action but to a less extent. Cytotoxic effect was weaker against breast adenocarcinoma MDA-MB-361 and colon carcinoma LS174 cells. Observed cytotoxicity was not as pronounced to healthy PBMC as to some malignant cell lines. Further research is needed in order to investigate anticancer potential of Artemisia annua tea. • References • De Ridder S, Van der Kooy F, Verpoorte R.: Artemisia annua as a self-reliant treatment for malaria in developing countries. J Ethnopharmacol 2008; 120: 302-314. • Efferth T.: Mechanistic perspectives for 1,2,4 – trioxanes in anti-cancer therapy. Drug Resist Updates 2005; 8: 85-97. • Firestone GL, Sundar SN.: Anticancer activities of artemisinine and its bioactive derivatives. Expert Rev Mol Med 2009; 11: e32. • Ferreira JFS, Luthria DL, Sasaki T, Heyerick A.: Flavonoids from Artemisia annua L. as Antioxidants and Their Potential Synergism with Artemisinin against Malaria and Cancer. Molecules 2010; 15: 3135-3170.