## 3 Results and Discussion
### Promotion of Apoptosis by Ru(III) Complexes in LoVo-Treated Cells  
Apoptosis, known also as \"programmed cell death\", is a cellular \"suicide\" mechanism responsible for the control of the cell number in tissues and elimination of those predisposed to malignant transformation that present DNA mutations or have an aberrant cell cycle [60; 61; 62; 63]. During tumorigenesis, apoptosis is undermined through the systematic loss of regulatory control mechanisms, finally resulting in the generation of a malignant phenotype, and resistance to chemo- and radio-therapy [8; 64; 65]. The mechanisms of apoptosis might be of great importance for carcinogenesis, tumor evasion, and have practical implications for anticancer therapy, since many anticancer drugs act during the physiological pathways of apoptosis, in the end leading to tumor cell destruction [66; 67; 68; 69; 70]. Along with the inhibition of cellular proliferation, apoptosis is frequently evaluated in the biological response to treatment with various chemotherapeutic agents. After automatic data acquisition from the homogenous assays of real time of the compound-specific influence, and further of the end-point test of drug-mediated cytotoxicity, we calculated IC\({}_{50}\) values and determined the optimal doses to be used in further end-point experiments, such as evaluation of apoptosis induction. In order to study the potential role of Ru(III) complexes in the induction of apoptosis in colon cancer cells, the levels of apoptotic events were evaluated by flow cytometry techniques. LoVo colon cancer cells were cultured in complete medium for 24 h; then, the culture medium was changed, and cells were treated for an additional 24 h with 10 or 50 uM of Ru(III) complexes. Then, LoVo cells were detached with PBS/1 mM EDTA, sequentially washed with PBS and washing buffer, and centrifuged at 300\(\times\)\(g\)/5 min. Pellets were suspended in 400 uL binding buffer, and 100 uL was distributed in flow tubes and stained with 5 uL of Annexin-V/FITC and/or PI for 15 min/RT in the dark [7; 55]). As positive controls, cells treated with 10, 25, or 50 uM cisplatin were used, while the negative control (CTRL) was represented by untreated cells. The green and red fluorescences were measured by using a FACScan flow-cytometer (BD Biosciences) after acquiring 10,000 events. Then, the acquired data were analyzed using WinMDI2.9 and Kaluza softwares, and apoptotic events were expressed as cell percentages. \begin{table}
\begin{tabular}{c c c c} \hline
**Compound** & **IC50** & **IC75** & **IC90** \\ \hline Ru-cipro & 27.88 \(\pm\) 3.25 & 111.63 \(\pm\) 2.40 & 161.87 \(\pm\) 1.89 \\ Ru-nf & 4.70 \(\pm\) 1.36 & 108.91 \(\pm\) 0.88 & 171.44 \(\pm\) 2.23 \\ Ru-levo & 7.42 \(\pm\) 1.46 & 108.14 \(\pm\) 3.14 & 169.15 \(\pm\) 5.84 \\ Ru-oflo & 4.80 \(\pm\) 2.58 & 114.89 \(\pm\) 2.31 & 186.70 \(\pm\) 5.25 \\ Ru-enro & 39.63 \(\pm\) 1.97 & 165.63 \(\pm\) 7.70 & 241.23\(\pm\) 13.50 \\ Ru-enoxa & 62.44 \(\pm\) 0.06 & 174.14 \(\pm\) 2.21 & 252.16 \(\pm\) 3.50 \\ Ru-pip & 55.36 \(\pm\) 0.34 & 148.70 \(\pm\) 2.86 & 204.71 \(\pm\) 4.78 \\ Cis-Pt & 30.7 \(\pm\) 1.13 & 124.46 \(\pm\) 1.30 & 180.71 \(\pm\) 1.40 \\ \hline \end{tabular}  
* IC50, IC75, and IC90 values represent the concentrations of a tested compound required to inhibit 50%, 75%, or 90% of the cell growth, with respect to the control sample (in the absence of the tested compound), and they are presented as mean \(\pm\) SD of three independent experiments.  
\end{table} 
Table 2: Values (uM) of IC50, IC75, and IC90, for Ru(III) complexes and CisPt-mediated lysis. Figure 4 shows data from a significant experiment of induced apoptosis in LoVo cells by 24 h treatments with either Ru(III) complexes or CisPt compared to untreated cells. Total apoptosis took into account the labeled Annexin-V/FITC cells, and data are presented as histograms, the assay marker of positivity being set on non-treated cell champion. Figure 4: Induction of apoptosis in the LoVo cell line by Ru(III) complexes vs. CisPt.