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

Institute of Oncology 14, 11000 Belgrade, Serbia

and Radiology of Serbia 2 IFBV, 10 rue Kommes L-6988, Hostert, Luxembourg
3Galenika fitofarmacija a.d. , Batajnica road b.b., 11080

Belgrade, 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.
 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.
Results

Figure 2. Survival of
Figure 1. Survival of HeLa, BG, Fem-x, LS, MDA-MB-361 and unstimulated and PHA-
K562 cells grown for 72 hours in the presence of increasing stimulated PBMC grown for 72
concentrations of Artemisia annua tea, determined by MTT test hours in the presence of
increasing concentrations of
Artemisia annua tea,
determined by MTT test

Table 1. Concentrations of Artemisia annua tea which induced 50 % decrease in target cell survival
Artemisia HeLa BG Fem-x LS MDBA- K 562 PBMC PBMC+
annua IC MB-361 PHA
50
mg/ml 3.06+/- 3.20+/- 3.76+/- 10.45+/- 8.86+/- 1.33+/- 10.38+/- 9.27+/-
0.62 0.65 1.20 0.26 0.42 0.38 0.49 0.54
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

Selectivity coefficient in the antitumor

action

IC50 PBMC / IC50 HeLa 3.39

HeLa IC50 PBMC+PHA / IC50
HeLa 3.03

IC50 PBMC / IC50 K562 7.8

IC50 PBMC+PHA / IC50
K562 6.97

IC50 PBMC / IC50 BG 3.24

LS174 IC50 PBMC+PHA / IC50 BG 2.9

IC50 PBMC / IC50 Fem-x 2.76

IC50 PBMC+PHA / IC50
Fem-x 2.46
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.
 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.
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