Cancers are the most common cause of death worldwide, and the number is still increasing in developing countries (Wild et al., n.d.). According to the International Agency for Research on Cancer report of 2020, the most common types of cancer in terms of incidence and mortality were lung cancer (2.1 million new cases; 1.8 million deaths in 2018), breast cancer (2.1 million new cases; 627,000 deaths in 2018), colorectal cancer (1.8 million new cases; 880,000 deaths in 2018). The goal of oncological treatment is to achieve a state in which the cancer can be controlled and is not life-threatening. It is extremely important to search for new active substances and therapeutic methods in order to extend the survival time of cancer patients, and above all to their complete recovery. (Geisslinger et al., 2019; Cooper, 2000; Kummar et al., 2006). Their treatment consists mainly of radiation therapy, surgical removal of the tumor and chemotherapy (Types of Cancer Treatment – NCI, n.d.). However, these methods are not very specific and selective, with a number of side effects for patients. Cytostatics act primarily on rapidly proliferating cells, and therefore also on myeloid cells, cells of the intestinal mucosa or hair cells (Nurgali et al., 2018; Demaria et al., 2017; Correa and Ahles, 2007). Also, neurological complications, cardiac complications, gastrointestinal disorders or secondary tumors are observed in patients. Many patients die from aftereffect caused by cytostatics, despite successful therapy. The main problem in the treatment of cancers is still too low differentiation of neoplastic cells in relation to healthy cells and the resulting lack of selectivity of the drugs used. Although scientists from around the world are conducting intensive research to develop substances that are highly selective for cancer cells, they have not been discovered so far. Therefore, it is extremely important to search for new, effective and safe substances that could be used in the development of anti-cancer strategies. Chemical synthesis is the basis for discovering new compounds with potential use in pharmacotherapy. Innovations in synthetic chemistry have contributed to the rapid increase in the number and variety of substances with new biological properties that can positively impact health and quality of patient life worldwide (Flick et al., 2022). The rapid increase in the number of compounds with potential pharmacological properties necessitates their efficient testing (Saeidnia et al., 2015).

Thiosemicarbazides are a group of organic compounds that, according to recent scientific reports, may act on several mechanisms involved in tumorigenesis. Studies have shown that they have effects on angiogenesis and on key enzymes for tumorigenesis: topoisomerases, ATP-ases, dihydroorthane dehydrogenase (Kozyra et al., 2022; Zhang et al., 2011; Kowalczyk et al., 2021). Thiosemicarbazide-derived compounds have also been shown to stimulate the process of apoptosis in tumor cells and exhibit anti-inflammatory and antioxidant effects (Bejarbaneh et al., 2020; Šarkanj et al., 2013; Geng et al., 2018; Tiperciuc et al., 2013; Malki et al., 2015). In addition, they have been shown to affect tumor cell migration and cell cycle, act at the molecular level by intercalating and damaging tumor cell DNA and altering the expression of genes key to the process of carcinogenesis (Kozyra et al., 2022; Pitucha et al., 2020; Xie et al., 2018). Despite the above findings, there are still no reports in the international literature of the discovery of a substance that exhibits effective and selective anticancer activity.

Since thiosemicarbazides have anti-inflammatory and antioxidant, anti-angiogenic, pro-apoptotic effects and affect many enzymes associated with carcinogenesis, they thus link the mechanisms that play a role in anticancer activity. In addition, there is reasonable expectation of their action at the molecular level by influencing interactions, damage, DNA synthesis and influence on the expression of genes related to carcinogenesis. Therefore, the aim of our study was to evaluate the cytotoxic properties and selectivity of chosen thiosemicarbazide derivatives (1a and 1b) on the following cell lines: human embryonic kidney (HEK293), fibroblasts (WS1), hepatocellular adenocarcinoma (HepG2), renal cell carcinoma (796-P) and non-small cell lung cancer (NCI-H1563) and glioblastoma multiforme (LN229). which will allow a preliminary assessment of the effect of thiosemicarbazide derivatives on different types of cancer cells. The influence of new derivatives on apoptosis and cell cycle has been determined. Also acute toxicity on Danio rerio embryos and larvae were evaluated. Danio rerio is an excellent vertebrate model for assessing the toxicity of new compounds due to external fertilization, transparent embryos, short embryo development cycle, as well as large clutch size (Ali et al., 2011; Di Paolo et al., 2015; Wernersson et al., 2015). In addition, it should be added that the zebrafish genome has been completely sequenced and is 83% identical to the human gene (with one gap) (Allende et al., 1996). Importantly, zebrafish embryos provide an alternative model system to adults as they are not believed to experience pain or any other discomfort (Strähle et al., 2012).