###### Abstract  
Cervical cancer is one of the most common types of cancer in women, with approximately 500,000 new cases and 250,000 deaths every year. Radiotherapy combined with chemotherapy represents the treatment of choice for advanced cervical carcinomas. The role of the chemotherapy is to increase the sensitivity of the cancer cells to irradiation. Cisplatin, the most commonly used drug for this purpose, has its limitations. Thus, we used a family of ferrocene derivatives (in addition, one new species was prepared using standard Schlenk techniques) and studied their effects on cervical cancer cells alone and in combination with irradiation. We applied colorimetric assay to determine the cytotoxicity of the compounds; flow cytometry to analyze the production of reactive oxygen species (ROS), cell cycle, and mitochondrial membrane potential (MMP); immunochemistry to study protein expression; and colony forming assay to evaluate changes in radiosensitivity. Treatment with ferrocenes exhibited significant cytotoxicity against cervical cancer cells, associated with increasing ROS production and MMP changes, suggesting the induction of apoptosis. The combined activity of ferrocenes and ionizing radiation highlighted ferrocenes as potential radiosensitizing drugs, while their higher single-agent toxicity in comparison with routinely used cisplatin could also be promising. Our results demonstrate antitumor activity of several tested ferrocenes both alone and in combination with radiotherapy.  
Keywords:ferrocenes; chemotherapy; cytotoxic effect; radiotherapy; radiosensitization; irradiation; cell death +
Footnote †: journal:  
radiotherapy [5]. Radiotherapy is usually used in more advanced cases, either to remove remaining microscopic disease after surgery (adjuvant therapy) or as a main treatment when surgery cannot be performed (curative therapy).  
Curative radiotherapy is often combined with chemotherapy to improve the treatment outcome [6]. The most commonly used chemotherapeutic drug in this case is cisplatin. Adding a weekly infusion of cisplatin to radiotherapy improves five-year overall survival by 6% and disease-free survival by 8% [6].  
Although platinum substances are widely used, they can cause serious side effects, including renal, neural, and gastrointestinal toxicity, that limit their usefulness [7]. Thus, not only the advantages but also the disadvantages of cisplatin stimulated further research into other types of compounds containing metal in their structure. Besides platinum complexes, species containing iron, titanium, ruthenium, gold, or palladium have been synthesized and tested [8; 9; 10; 11; 12; 13; 14]. Among the most intensively studied substances exhibiting radiosensitization effects are ruthenium compounds [15; 16; 17] or gold nanoparticles [16], as demonstrated also by in vivo screenings [18; 19; 20]. Nevertheless, despite a great deal of research into iron-containing antitumor compounds, there have been only a few investigations into the potential radiosensitizing effects of these compounds in cancer cells [21; 22].  
This article is focused on the series of ferrocene derivatives of the general formula [Fe(\(\eta^{5}\)-C\({}_{5}\)H\({}_{4}\)CH\({}_{2}\)(p-C\({}_{6}\)H\({}_{4}\))CH\({}_{2}\)(N-het))\({}_{2}\)] bearing either substituted or unsubstituted saturated five- and six-membered nitrogen-containing heterocycles (Figure 1). In vitro cytotoxicity analysis was performed on cell lines derived from cervical cancer. In the case of highly toxic ferrocenes, we focused especially on their mechanisms of action in terms of the disruption of cell metabolism and examination of cellular mechanisms leading to cell death. The combined effect of ferrocenes with ionizing radiation was also determined to test the possible use of these ferrocenes as potential radiosensitizing agents.  
Figure 1: Structures of the tested ferrocenes.