Evaluation the Effect of Hydroalchoholic Extract of Artemisia siberi and Artemisia vulgaris L on the Induction of Apoptosis in B16 Cell Lines

Cancer is the second leading cause of death worldwide after cardiovascular disease in spite of a fact that a decrease in cancer mortality rate has been reported (1). Although, cancer is the third cause of death in IRAN after cardiovascular diseases and road accidents and it`s mortality rate are more than developed conteries. Cancer chemotherapy of cytotoxic drugs and radiotherapy are accompanied by highly deleterious and often life-threatening side effects. In recent years, the use of medicinal plants in the treatment of diseases such as cancer has developed significantly (2). Among them, Artemisia is a large genus of plant, around 200 and 400 species which is grow in temperate climates of dry or semiarid habitats. The most well known species include A. vulgaris, A. tridentata ,A. annua and A. absinthium. Most species have aromas and bitter tastes from terpenoids and sesquiterpene lactones and bioactive phytochemicals that have been reported to exhibit sveral human health effects including  antimalarial, antioxidant, neuroprotective, anti-inflammatory, and antimicrobial activities (3). Recently, artemisinin derivative (artemisinin-combination therapies) are accepted as a gold standard treatment worldwide for malaria caused by Plasmodium falciparum. In the case of cancer treatment, the plant species and its bioactive compounds target multiple pathways. Also, due to the promising features of herbal plant its anti-cancer activity has been investigated for use in modern medicine. In this case, artemisinins have shown potent anticancer activity in multiple cancers in vitro and in vivo via different mode of action including autophagy, cell cycle arrest, and apoptosis.A well-known type of programed cell death which described in 1842 is apoptosis. The cells initiates intracellular apoptotic signaling in response to many factors including stress, heat, radiation, viral infection as well as   hypoxia, Several proteins andmediators are involved, but two main methods of regulation have been identified: the intrinsic pathway is also known as the mitochondrial pathway and extrinsic pathway is identified by TNF and Fas pathways.Apoptosis is one of the of programmed cell death that occurs in multicellular organisms such as mamals and can be activated by either intrinsic or extrinsic factors. There are at least two kind of signaling pathways that lead to apoptosis, the intrinsic pathway of apoptosis and the extrinsic pathway of apoptosis.  The extrinsic pathway of apoptosis is triggered by the extracellular environment and death signals. The intrinsic pathway of apoptosis pathway is linked to mitochondria and DNA damage. In both the intrinsic and extrinsic pathway of apoptosis, signaling results in the activation of a family of Cys (Cysteine) proteases, named caspases. In the mitochondrial-dependent phatway Cyto C release lead to formation of a catalytic multiprotein protein so called apoptosome that can activates Caspase-9. Activated Caspase-9 then is able to cleave Caspase-3and apoptosis cascade. In the other hand, during apoptosis which is initiated by mitochondria, cytochrome c is released through the activation of Bax and Bak proteins. Upon cytochrome c is released it binds with Apoptotic protease activating factor – 1 also called Apaf-1 The Apaf-1 then bind to pro-caspase-9 to create a protein complex known as an apoptosome. Active from of initiator caspase of 9 is cleaved by apoptosome and start to cleaves and activates effector pro-caspase into the effector caspase-3 (4).
In several studies have shown that different types of Artemisia Sp. might have anticancer effect but the mode of action of its anticancer activity is not well known. In this regard, the aim of this study was to evaluate the effect of hydroalchoholic extract of Artemisia siberi Besser. and Artemisa vulgaris L. on the induction of apoptosis in B16 cell lines.

For this purpose, the hydroalchoholic extracts of these two plants were prepared by percolation method. Then the B16 melanoma cell lines were grown on Dulbecco's Modified Eagle Medium (DMEM) cell culture and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( MTT) method was utilized to obtain the appropriate concentrations of Artemisia siberi Besser. and Artemisa vulgaris L. for determination of The half maximal inhibitory concentration (IC50) of each extract. After calculation of IC50, the amount of released cytochrome C and activity of caspase 3 were measured by using standard kits. In these experiments, duxorubicine was used as positive control. For possible identification of active ingredient of extracts, Quersetin was used as flavonoid marker.

The half maximal inhibitory concentration (IC50 value) of the Artemisia siberi Besser. and  Artemisa vulgaris L.  was determined by constructing dose-response curves. According to the IC50 values, their potencies can be ranked as follows:Artemisia siberi Besser> Quercetin>Artemisa vulgaris L. According to the MTT test, IC50 values were 480 µg/ml, 500 µg/ml and 5000µg/ml for Artemisia siberi Besser, Quercetin and Artemisia. Vulgaris L, respectively.The amount of cytochrome C release for Artemisia siberi Besser. and Artemisa vulgaris L.  was significantly increased 1.53 and 1.93 time more than control group respectively.  There was a significant difference between the increases of activities of caspase3 by Artemisia siberi Besser. and Artemisa vulgaris L compared to the negative and positive controls (1.63 and 4.7times more than negative control respectively). There was also significant difference between the increases activities of caspase3 by Artemisa vulgaris L compared to positive control.

According to the outcomes, it is concluded that the hydroalchoholic extracts of A. vulgaris and A. siberi can induce apoptosis via mitochondrial-dependent pathway as a considerable underlying mechanism.The IC50 value of Artemisia siberi Besser is comparible with Quercetin. Also, the caspase 3 activity induced by Artemisa vulgaris L is notable. However, inorder to show that this phytochemical have anticancer activity, fractionation and separation of active ingredients as well as more in vitro and in vivo studies seems to be necessary.