br Materials and methods br Results br Discussion More exper

Materials and methods
Results
Discussion More experiments were performed in order to explain the mechanism that is involved in ras inhibitor death both PC3 and Hep G2 cells using compound 5. Results showed that this compound inhibits cell viability inducing apoptosis in a concentration-dependent manner. Further experiments exhibited that compound 5 was also able to promote autophagy. Impaired autophagy was implicated in the pathogenesis of many diseases, including cancer, diabetes, Crohn's etc. [48,49]. Generally, autophagy inhibits the apoptosis, and apoptosis-associated caspase activation inhibits, in turn, the autophagic process. Some authors reported that sometimes, autophagy might induce apoptosis or necrosis producing autophagic cell death [50,51]. Consequently, in this cells compound 5 inducing the autophagic process increases the apoptosis. More interestingly, compound 5 treatment also results in mitochondrial dysfunction and ROS accumulation and it is more active than Cl-IB-MECA inducing ROS level greater that Cl-IB-MECA. Probably this ROS accumulation is essential for autophagy and apoptosis induced by compound 5. The intracellular ROS levels is strictly connected with functional status of autophagy in complex ways influencing tumorigenesis in specific manners that modulate autophagy for cancer prevention and treatment [52]. Ability of this compound to involve both autophagic and apoptotic mechanisms via ROS production could be a possible explanation of its antitumor action mechanism. Moreover, this compound was tested in the migration of the cancer cells. Usually the cancer metastasis represents the principal cause of cancer-related deaths appearing an advanced stage of malignancy. Metastasis includes migration and invasion of cancer cells using the scratch test. All the processes require the involvement of a wide range of cellular mechanisms. Cell migration is a highly integrated process that includes development of cytoplasmic protrusions, attachment, and traction. Results showed that compound 5, such as Cl-IB-MECA are able to inhibit the migration of the cancer cells at low concentration. Various studies have shown the function of A3AR and downstream signalling pathways in various tumour cell lines. However, very limited number of novel compounds expressed the anticancer properties against multiple cancer cell types. Taken together, our results for the first time indicated that stimulation of A3AR induces cell growth arrest and apoptosis in PC3, Hep G2, and Caco-2. Therefore, the novel A3AR ligand 5 can be considered as a potential compound for further biological analysis in vivo and towards the development of new anticancer drugs.
Author contributions
Conflict of interest
Acknowledgment MK and OYH acknowledge Tampere University of Technology (85482 and 85486) for Instrumental facility grant support. We also thank Prof. Matti Karp for providing the access to Fluorescence Plate reader instrumental facility at TUT.
Introduction Cancer is a complex set of diseases characterized by different molecular alterations. Cancer accumulates subsequent mutations in genes involved in the regulation of cell proliferation, apoptosis and metabolism [1], [2]. One mechanism employed by the carcinogenic process is angiogenesis. Pro-angiogenic and chemotactic factors such as VEGF and IL-8 correlate with tumor aggressiveness [3], [4]. The aggressiveness of cancer depends on its ability to redirect immune cell activity to its own advantage [5]. Eventually, tumor cells acquire the ability to migrate from the tumor ambient to distal areas, a process called metastasis which is associated with an unfavorable outcome. [6], [7]. To orchestrate these processes, different types of cancers may use different mediators and receptors [8], [9]. Adenosine is a nucleoside that can act as a signaling molecule binding to adenosine receptors. In humans there are four adenosine receptors (AR): A1, A2a, A2b, and A3. Other species express these receptors playing an important physiological role, these species include mice [10], [11], mouse [12], [13], [14], rat [14], [15] and rabbit [16], [17], [18]. Both A1 and A3 receptors are linked to Gi protein [19], [20], activation triggers a decrease in cAMP levels [21], [22]. Moreover, A2a and A2b receptor are coupled to Gs and can induce an increase in cAMP levels [23], [24]. In addition A2b can link to the Gq protein triggering the activation of phospholipase C and mobilization of calcium in different cellular models [25]. A2b receptor is the adenosine receptor with the lowest affinity for adenosine, for this reason it has been underestimated in terms of its physiological importance compared to other adenosine receptors. However it is relevant under certain conditions, it has a protective role against inflammation, ischemia and hypoxia [26], [27], [28], [29], [30]. It may also play a protective role in the progression of type 2 diabetes [31], and is involved in bone development [32]. A2b receptor activation has also been reported in diseases, including participation in carcinogenic processes. In this sense, an increase in the expression of A2b receptor has been found in biopsies of patients who develop cancer [33], [34], [35]. This article will be reviewing the contribution of A2b receptor in cancer progression by means of mechanisms linking immunomodulation, angiogenesis and metastasis. Finally the potential use of A2b receptor as a therapeutic target will be analyzed.