NEW GOLD PINCER-TYPE COMPLEXES INDUCE CASPASE-DEPENDENT APOPTOSIS IN HUMAN CANCER CELLS IN VITRO NOVI KOMPLEKSI ZLATA PINCER-TIPA INDUKUJU KASPAZA-ZAVISNU APOPTOZU U HUMANIM ĆELIJAMA RAKA IN VITRO

Background / Aim. We investigated cytotoxicity of Au(III) complexes with pincer type ligands against cervical carcinoma cells (HeLa), breast cancer cells (MDA-MB-231 and 4T1) and colon carcinoma cells (HCT116 and CT26). We also examined the type and mechanism of cell death that these complexes induced in cancer cells. Methods. Cytotoxicity of Au(III) complexes was investigated by MTT assay. Apoptosis of treated cancer cells was measured by flow cytometry and applying Annexin V/7AAD staining. The expressions of active proapoptotic protein Bax, antiapoptotic protein Bcl-2 and the percentage of cells containing cleaved caspase-3 in treated cancer cells was determined by flow cytometry. Results. Complex 1 showed the most potent anti-tumor effect on HeLa cells, both compared to other two examined gold complexes and compared to cisplatin. The IC 50 values on HeLa cells after 72 hours were 1.3 ± 0.4 μM, 3.4 ± 1.3 μM, 5.7 ± 0.6 μM, 26.7 ± 6.5 μM for complexes 1 , 2 , 3 and cisplatin, respectively. Complex 1 also exhibited highest cytotoxicity against MDA-MB-231 and HCT116 cells compared to other tested compounds. The results of Annexin V/7AAD staining showed that all three complexes induced apoptosis in treated cells. Our gold(III) complexes induced apoptosis by caspase-dependent mechanism, but we did not observe that an activation of the internal pathway of apoptosis occurred in treated cancer cells. Conclusion. According to the results of our in vitro study, all three compounds and especially complex 1 are promising candidates for a new generation of anticancer drugs.


Introduction
In the United States, it is assumed that about 40% of the adults will be diagnosed cancer at certain age [1]. This is a worrying fact, but finding a cure for cancer is not easy.
There are major differences between tumors of various tissues and organs in cellular morphology, tumor aggressiveness and treatment of the tumors [1][2][3]. Such differences exist even among tumors of the same organ, such as breast tumors [3]. Even in the course of treatment, tumor cells are modified and may become resistant to therapy, which even further complicates the treatment [4]. Therefore, one universal cure for cancer will most likely never be found and testing of a substance that has chemotherapeutic potential against two or three types of tumors in vitro cannot provide sufficiently precise results.
Deregulation in apoptotic cell death machinery is a main characteristic of cancer [5]. Apoptosis is a process of cell degeneration that is not associated with inflammation and damage to surrounding healthy cells, and therefore a more favorable mechanism for reducing the number of tumor cells compared to necrosis [5,6]. Apoptosis happens spontaneously in malignant tumors, often noticeably delaying their growth, and it is increased in tumors responding to irradiation, chemotherapy, high temperature and hormone ablation [6]. Apoptosis alteration is responsible not only for tumor development and progression but also for tumor resistance to therapies [5]. On the other hand, much of the present attention in the process stems from the finding that it can be regulated by certain proto-oncogenes and the p53 tumor suppressor gene [5][6][7]. Two proteins located in 5 cytoplasm of the cells, B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and Bcl-2, are the activator and an inhibitor of apoptosis, respectively. It has been described, that Bax and Bcl-2, and their ratio, are predictive markers in different cancers [8]. Also, the significance of other molecules included in the apoptosis, such as caspases, had also been previously reported [9].
The use of cisplatin as a -standard‖ chemotherapeutic opened the door to the new metal-based drug research [10]. New complexes containing metals such as platinum, palladium, ruthenium and gold have recently been analyzed as potential antitumor agents [10][11][12]. Also, organometallic gold compounds occupied a valuable place in various anticancer researches due to their exceptional chemical characteristics with respect to gold coordination complexes [13]. Actually, many researchers have discovered that they could be utilized to create exceptionally capable metal-based drugs with potential relevance in the treatment of cancer [14].
However, these Au(III) complexes have not been investigated against other types of cancer cells and therefore further investigations were necessary. The aim of our research was to examine whether these Au(III) compounds with pincer type ligands might be utilized as potential antitumor agents. Therefore, we examined the antitumour potential of all three Au(III) complexes against cell lines of cervical carcinoma (HeLa), breast cancer (MDA-MB-231 and 4T1) and colon carcinoma (HCT116 and CT26). We also included mouse carcinoma cells in our research (4T1 and CT26) in order to prove future research potential of these complexes for in vivo studies. We investigated their cytotoxic effect against five types of cancer cells, the type and mechanism of cell death that these complexes induce in cancer cells.

MTT assay
MTT assay is the colorimetric test used to examine viability of cells. The basic principle of this assay is based on the ability of viable or living cells to reduce yellow tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the purple formazan [16]. Namely, living cells contain NAD(P)H-dependent oxidoreductase enzymes that have this ability, so the purple color of the cellular suspension after the 9 Therefore, we added 1 ml of cell suspension containing 10 6 cells/ml of complete medium in each well of 24-well plates. After 24 hours of incubation in an atmosphere containing 5% CO2 and at 37ºC, the medium was removed and 1 ml of the medium containing the half maximal inhibitory concentration (IC50) of tested substances was added.
Each substance was tested in triplicate and in three independent experiments. The control population consisted of cells supplemented with complete medium with the same concentration of DMSO as in treated cells. The cells were then incubated for 24 hours in order to detect substance-induced cellular changes [17]. After the incubation in an atmosphere containing 5% CO2 and at 37ºC, the cells were trypsinised, washed three times in ice-cold PBS (Phosphate Buffer Saline) and 1x10 5  and triplicates ± standard deviation (SD). We analyzed statistical significance between the percentages of cells found in early and late apoptosis against the percentage of necrotic cells for each test substance and for each type of tumor cells [18,19].

Analysis of key proteins involved in the apoptosis process
The next step in our research was to examine the mechanism by which the process of apoptosis in the treated cells was activated. We wanted to determine if there was a change in the amount or activity of the key proteins involved in the apoptosis process. Therefore, we examined the cellular levels of the proapoptotic protein active-Bax, the antiapoptotic protein Bcl-2, and the percentage of the cells in which caspase-3 is active [18,19]. We compared the populations of cells treated with the half maximal inhibitory

Statistical analysis
The distributions of the obtained data were evaluated for normality using the Shapiro-Wilk test. The values of MTT and Annexin assays were presented as mean ± standard deviation (SD). The values of apoptotic proteins were presented as medians due to large standard deviations and distribution of data that was not normal. All experiments were performed in triplicates and in three separate repetitions. Commercial SPSS version 20.0 for Windows was used for statistical analysis. Statistical evaluation was performed by Student's T-test for paired observations, or one-way ANOVA depending on data 11 distribution. P values less than 0.05 were considered to indicate a statistically significant difference.

Results
The cytotoxicity of complex 1 and its ligand was partially tested on cell lines of colon carcinoma (LS-174), lung carcinoma (A549) and melanoma (A375) cell lines. The results of this study have shown that complex 1 exhibited stronger anti-tumor effect on all three types of tumor cells tested compared to cisplatin [15]. Since some compounds may show delayed toxicity, it is concluded that the analysis of cytotoxicity should be performed after an interval of at least 48 hours [20]. Therefore, we chose to analyze cytotoxicity after 72 hours similarly to previous experiments [21][22][23].   . 1(HCT116), 2(CT26) vs. 2(HCT116), 3(CT26) vs.
We showed that our gold(III) complexes, and especially complex 1, displayed strong antitumor effects in vitro against all three types of tested human cancers cells, i.e.
cervical cancer cells, breast cancer cells and colon cancer cells. Therefore, the next step of our research was to investigate the mechanism, or the type of cell death resulting in a decrease the viability of treated cells.
The results of Annexin V/7AAD staining showed that the apoptosis was induced by our gold complexes and cisplatin in all five types of cancer cells tested (Fig. 2, p<0.05). In all cases, less than 4% of the total population of cells had been necrotic, while the rest of the non-viable cell population was in different stages of apoptosis. In general, a higher percentage of cells had entered early apoptosis phase, and a slightly lower percentage of cells had already entered the late stage of apoptosis (Fig. 2). Consequently, the next step of our research was to investigate whether our gold complexes influenced the cytoplasmic concentration of antiapoptotic protein Bcl-2, the activation of the proapoptotic protein Bax and the activation of the caspase cascade in HeLa cancer cells. We decided to investigate these events in HeLa cells considering that all three complexes exhibited the strongest cytotoxicity against this type of cancer cells, and these results were presented in Fig. 3. The results of our research have shown that all three gold complexes and cisplatin insignificantly reduced the amount of antiapoptotic protein Bcl-2 (Fig. 3A, p>0.05).
Although this decrease was not statistically significant, it appeared as a noticeable trend. In addition, we have shown that there has been no statistically significant change in the activation of proapoptotic protein Bax in the groups of cancer cells cultivated in the presence of Au(III) complexes (Fig. 3B, p>0.05). However, in cisplatin-treated cells, statistically significant increase of active-Bax was detected in comparison to untreated cells  (Fig. 3D, p<0.05).

Discussion
Seeking new solutions to increase selectivity and specificity of chemotherapy in cancer cells has attracted much attention in science, recently. It was a widespread opinion that the cytotoxic effects of metal complexes are the result of direct damage to nuclear DNA [24]. However, gold(III) complexes exert their cytotoxic activities through mechanisms that are considerably diverse from those of platinum drugs [24] [25][26][27] who also synthesized gold(III) complexes that are significantly more effective against cancer cells in comparison to cisplatin. However, cisplatin showed statistically higher cytotoxicity against CT26 cells compared to complex 1.
The results of our study showed that gold(III) complexes exhibit strong anticancer activity in vitro, which is in agreement with the results of some previous studies [25][26][27]. Au(III) complexes was examined against HeLa cells under the same conditions in comparison to our study [22]. Their IC50 values were similar or even significantly lover in comparison to the results that we obtained, and the proposed mechanism that decreases viability of treated cells was apoptosis [22].
The results of recent studies pointed toward the induction of apoptosis as a major cytotoxic mechanism of gold(III) complexes against cancer cells [22,28]. Apoptosis is mediated by two main pathways, an extrinsic pathway that involve cell surface receptors, and an intrinsic pathway via mitochondria and the endoplasmic reticulum. The results of our research were completely in agreement with the results of other studies, where it had also been shown that complexes of gold displayed cytotoxicity against tested cancer cells by induction of apoptosis [26]. This is very important because substances acting cytotoxicly by induction of apoptosis do not induce changes in surrounding healthy tissue; there is no process of inflammation or other adverse effects [6].
Therefore, the next step in our research was to examine the mechanism of apoptosis in the group of treated cancer cells. It has already been shown that substances acting through the mitochondrial, internal pathway of apoptosis changed the activity or concentration of proapoptotic and antiapoptotic proteins [18,19]. In addition, it has already been shown that gold complexes could induce apoptosis by activating the mitochondrial pathway of apoptosis [26,29]. Activation of caspase-8 is typical for the external (receptor) pathway of apoptosis, while increased active Bax and/or decreased Bcl-2 is rather connected with the internal (mitochondrial) pathway. In both cases, downstream activation of caspase-3 occurs. Afterward, caspase-3 initiates apoptotic DNA fragmentation by proteolytically inactivating the DFF45/ICAD protein complex [30].
The results of our research indicated that the most probably there had been no induction of apoptosis due to the effect of the complex 1-3 through the internal pathway of apoptosis. Further research are necessary in order to confirm the following assumption, but we may suggest that the activation of the external pathway of apoptosis is the most probable cause of Au(III) complexes'-induced apoptosis [6,31]. On the other hand, we undoubtlessly concluded that all three gold(III) complexes induced apoptosis by activating the caspase cascade, as there had been statistically significant increase in the amount of active caspase-3 in cells treated with IC50 values of gold(III) complexes 1-3 compared to untreated cells (p<0.05).
Although it has been shown that gold(III) complexes have a different mechanism of action in comparison to cisplatin, the precise mechanism of cytotoxic activity of gold(III) complexes has not yet been fully clarified.

20
It has been shown that gold(III) complexes could induce apoptosis by activating the internal pathway of apoptosis and activating the caspase cascade. The results of our research have shown that our gold(III) complexes induced apoptosis by the caspasedependent mechanism, but we did not observe that an activation of the internal pathway of apoptosis occurred in treated cancer cells. The most probable cytotoxic mechanism of investigated gold(III) complexes was activation of the external pathway of apoptosis, but this assumption must be proven. However, all three investigated gold(III) complexes, especially complex 1, showed strong cytotoxicity against human cancer cells in vitro and induced apoptosis by caspase-dependent mechanism. It is therefore necessary to further investigate the mechanism of cytotoxicity of these complexes. According to the results of our in vitro study, and if some further in vivo investigations show promising results, complex 1 may be a good candidate for a new generation of anticancer drugs.