## 4 Discussion  
In recent articles, syntheses of some ferrocenes have been published [23; 24]. Several of these compounds showed a clear cytotoxic effect that was equal or even greater when compared with cisplatin, which has become a mainstay of cancer therapy. We hypothesize that ferrocenes, similarly to cisplatin, may, in combination with ionizing radiation, show a synergistic cytotoxic effect. This radiosensitization effect could be particularly beneficial in the treatment of chemoresistant malignancies, e.g., cervical tumors that are generally perceived as resistant to cisplatin [45; 46]. In previous studies, the mechanisms of transport of substituted ferrocenes were investigated using differential pulse voltammetry and inductively coupled plasma mass spectrometry. We identified membrane transferrin-receptor-mediated endocytosis of transferrin-bound ferrocene as the major mechanism of cellular uptake. Importantly, the rate of ferrocene accumulation in cancer cells is proportional to its cytotoxic effects [24; 47]. To assess the exact mode of action of the studied ferrocenes, several methods were used. First, while no changes in cell cycle distribution were observed in SiHa cells, HeLa cells showed increased accumulation of cells in the S phase in response to treatment with all tested ferrocenes. \begin{table}
\begin{tabular}{c c c c|c c c} \hline \hline  & \multicolumn{2}{c|}{**HeLa Cells**} & \multicolumn{4}{c}{**SiHa Cells**} \\ \hline
**Conc.** & **1b** & **2a** & **3** & **Conc.** & **1b** & **2a** & **3** \\
0.5 \(\upmu\)M & 0.9 \(\pm\) 0.08 & 1.0 \(\pm\) 0.1 & 0.9 \(\pm\) 0.08 & 1 \(\upmu\)M & 1.4 \(\pm\) 0.19 & 1.3 \(\pm\) 0.07 & 0.9 \(\pm\) 0.08 \\
1 \(\upmu\)M & 1.6 \(\pm\) 0.13 & 0.9 \(\pm\) 0.2 & 1.2 \(\pm\) 0.08 & 2 \(\upmu\)M & 1.8 \(\pm\) 0.5 & 1.4 \(\pm\) 0.17 & 1.2 \(\pm\) 0.08 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Dose-modifying factors for the selected ferrocenes in HeLa and SiHa cells. Figure 8: Graphical interpretation of the surviving fraction (SF) for the combination of selected ferrocenes with different doses of irradiation in (**a**) HeLa cells exposed to 1 \(\upmu\)M ferrocenes and (**b**) SiHa cells treated with 2 \(\upmu\)M ferrocenes. However, the increase in proportion of cells in the S phase predominantly after treatment with **1b** and **2a** was significantly lower when compared to that in cisplatin-treated cells. This could be of potential interest, since the doses of cisplatin used probably slow down DNA synthesis due to repairing its damage, which is associated with the accumulation of cells in the S phase [48], and cells in S phase are generally considered to be more radioresistant [49]. Thus, even though cisplatin is commonly used with radiotherapy, the effect of ionizing radiation in this combination is probably partly attenuated because cisplatin actually increases the proportion of cells in the S phase [50; 51]. Therefore, treatment with ferrocenes resulting in the induction of cell cycle arrest in the S phase to a much lesser extent compared to cisplatin would be more effective, bringing greater benefit to these patients. Second, a potent increase in reactive oxygen species was observed upon treatment with the selected ferrocenes. This can be explained by the basic chemical structure of the ferrocene core that is known as a catalyst in a Fenton reaction generating both hydroxyl radicals and higher oxidation states of the iron. Thus, in the presence of transition metals, H\({}_{2}\)O\({}_{2}\), a product of mitochondrial oxidative respiration, is reduced inside the cells; this generates oxygen radicals responsible for damage to all macromolecules, including DNA, proteins, and membrane phospholipids that are damaged by the peroxidation of unsaturated fatty acids in exposed cells [52; 53; 54]. In recent years, several studies have confirmed potent induction of ROS by a range of organometallic complexes, including ferrocenes, in cancer cells [55; 56; 57; 58; 59; 60; 61; 62]. These studies imply their possible utilization in cancer research and treatment.