Hepatocellular carcinoma (HCC) is one of the most common malignancies, with an increasing incidence. Despite the fact that systematic chemotherapy with a doxorubicin provides only marginal improvements in survival of the HCC patients, the doxorubicin is being used in transarterial therapies or combined with the target drug – sorafenib. The aim of the study was to evaluate the effect of natural flavonoids on the cytotoxicity of the doxorubicin against human hepatocellular carcinoma cell line HepG2. The effect of apigenin and its glycosides - cosmosiin, rhoifolin; baicalein and its glycosides – baicalin as well as hesperetin and its glycosides – hesperidin on glycolytic genes expression of HepG2 cell line, morphology and cells’ viability at the presence of doxorubicin have been tested. In an attempt to elucidate the mechanism of observed results, the fluorogenic probe for reactive oxygen species (ROS), the DNA oxidative damage, the lipid peroxidation and the double strand breaks were evaluated. To assess impact on the glycolysis pathway, the mRNA expression for a hexokinase 2 (HK2) and a lactate dehydrogenase A (LDHA) enzymes were measured. The results were analysed statistically with the one-way analysis of variance (ANOVA) and post hoc multiple comparisons. The apigenin and the hesperidin revealed the strongest effect on the toxicity of doxorubicin. Both flavonoids simultaneously changed the expression of the glycolytic pathway genes - HK2 and LDHA, which play a key role in the Warburg effect. Although separate treatment with doxorubicin, apigenin and hesperidin led to a significant oxidative DNA damage and double strand breaks, simultaneous administration of doxorubicin and apigenin or hesperidin abolished these damage with the simultaneous increase in the doxorubicin toxicity. The obtained results indicate the existence of a very effective cytotoxic mechanism in the HepG2 cells of the combined effect of doxorubicin and apigenin (or hesperidin), not related to the oxidative stress. To explain this synergy mechanism, further research is needed, The observed intensification of the cytotoxic effect of doxorubicin by this flavonoids may be a promising direction of the research on the therapy of hepatocellular carcinoma, especially in a chemoembolization.

In this study, three new organic ligands N'-(benzylidene)-6-chloropyrazine-2-carbohydrazonamide (L₁), 6-chloro-N'-(4-nitrobenzylidene)picolinohydrazonamide(L₂), and N'-(benzylidene)-4-chloropicolinohydrazonamide (L₃) and three copper coordination compounds (Cu(L₁)Cl₂, Cu(L₂)Cl₂ and Cu(L₃)Cl₂) based on them were synthesized. All obtained compounds were characterized using appropriate analytical techniques: elemental analysis (EA), thermogravimetric analysis (TG-DTG), Fourier transform infrared spectroscopy (FTIR) and flame-atomic absorption spectrometry (F-AAS). These methods of physicochemical analyses helped to assume that the complexation in three cases proceeds in a bidentate manner. The X-ray investigation confirmed the synthesis pathway and molecular structures for L₁ and L₃ ligands. The antimicrobial activity of the obtained compounds was then comprehensively investigated, where Cu(L₃)Cl₂ showed the strongest antibacterial properties against all tested bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli). LN229 human glioma cells and BJ human normal fibroblasts cells were treated with tested compounds and their cytotoxicity was evaluated with MTT test. The effect of complexing on antitumor activity has been investigated. The ligand L₁ and its complex showed similar activity against normal cells while complexation increases toxicity against cancer cells in concentrations of 50 and 100 μM. For the one pair of ligand/complex compounds the apoptosis detection, cell cycle analysis and gene expression analysis (qRT-PCR) were performed. Cu(L₁)Cl₂ showed the stronger toxic effect in comparison with L₁ due to the population of early apoptotic cells which revealed metabolic activity in MTT assay.

A series of thiosemicarbazone derivatives was prepared and their anti-tumor activity in vitro was tested. The X-ray investigation performed for compounds T2, T3 and T5 confirmed the synthesis pathway and assumed molecular structures of analyzed thiosemicarbazones. The conformational preferences of the thiosemicarbazone system were characterized using theoretical calculations by AM1 method. Selected compounds were converted into complexes of Cu (II) ions. The effect of complexing on anti-tumor activity has been investigated. The copper(II) complexes, with Schiff bases T1, T10, T12, T13, and T16 have been synthesized and characterized by chemical and elemental analysis, FTIR spectroscopy and TGA method. Thermal properties of coordination compounds were studied using TG-DTG techniques under dry air atmosphere. G361, A375, and SK-MEL-28 human melanoma cells and BJ human normal fibroblast cells were treated with tested compounds and their cytotoxicity was evaluated with MTT test. The compounds with the most promising anti-tumour activity were then selected and their cytotoxicity was verified with cell cycle analysis and apoptosis/necrosis detection. Additionally, DNA damages in the form of a basic sites presence and the expression of oxidative stress and DNA damage response genes were evaluated. The obtained results indicate that complexation of thiosemicarbazone derivatives with Cu (II) ions improves their antitumor activity against melanoma cells. The observed cytotoxic effect is associated with DNA damage and G2/M phase of cell cycle arrest as well as disorders of the antioxidant enzymes expression.

A number of studies have confirmed anti-tumor activity of flavonoids and their ability to enhance the effectiveness of classical anticancer drugs. The mechanism of this phenomenon is difficult to explain because of the ambivalent nature of these compounds. Many therapeutic properties of these compounds are attributed to their antioxidant activity; however, it is known that they can act as oxidants. The aim of this study was to assess the influence of apigenin and hesperidin on MCF-7 breast cancer cells with doxorubicin. The cytotoxic effect was determined using an MTT test and cell cycle analysis. To evaluate the possible interaction mechanism, reduced glutathione levels, as well as the DNA oxidative damage and the double strand breaks, were evaluated. Additionally, mRNA expression of genes related to DNA repair was assessed. It was demonstrated that flavonoids intensified the cytotoxic effect of doxorubicin despite flavonoids reduced oxidative damage caused by the drug. At the same time, the number of double strand breaks significantly increased and expression of tested genes was downregulated. In conclusion, both apigenin and hesperidin enhance the cytotoxic effects of doxorubicin on breast cancer cells, and this phenomenon occurs regardless of oxidative stress but is accompanied by disorders of DNA damage response mechanisms.

In this paper, thiosemicarbazide derivatives were synthesized as potential anticancer agents. X-ray investigations for 1-(2,4-dichlorophenoxy)acetyl-4-(2-fluorophenyl) thiosemicarbazide, 1-(2,4-dichlorophenoxy)acetyl-4-(4-metylothiophenyl)thiosemicarbazide and 1-(2,4-di chlorophenoxy)acetyl-4-(4-iodophenyl)thiosemicarbazide were carried out in order to confirm the synthesis pathways, identify their tautomeric forms, analyze the conformational preferences of molecules, and identify intra- and intermolecular interactions in the crystalline state. TLC and RP-HPLC analyses were used to determine lipophilicity. The lipophilicity analysis revealed that the 4-substituted halogen derivatives of thiosemicarbazides showed greater lipophilicity compared with 2-substituted derivatives. The optimal range of lipophilicity for biologically active compounds logkw is between 4.14 and 4.78. However, as the analysis showed, it is not a decisive parameter. The cytotoxicity of the new compounds was evaluated against both the G-361 and BJ cell lines. Cytotoxicity analyses and cell-cycle and cell apoptosis assays were performed. The MTT test demonstrated that three compounds were cytotoxic to melanoma cells and not toxic to normal fibroblasts in the concentration range used. The cell cycle analysis showed that the compounds had no significant effect on the cell cycle inhibition. An extensive gene expression analysis additionally revealed that all compounds tested downregulated the expression of dihydroorotate dehydrogenase (DHODH). DHODH is a mitochondrial enzyme involved in the de novo synthesis of pyrimidines. Due to the rapid rate of cancer cell proliferation and the increased demand for nucleotide synthesis, it has become a potential therapeutic target.

Thiosemicarbazide is a useful structural moiety that has the biological potential. Optimization of this structure can result in groundbreaking discovery of a new class of therapeutic agents. In the light of this, 1-(2,4-dichlorophenoxy)acetyl-4-(1-naphthyl)thiosemicarbazide (1) and 1,4-bis[(2,4-dichlorophenoxy)acetylthiosemicarbazide]phenyl (2) were synthesized and characterized by spectroscopic method. Cytotoxicity of obtained compounds was evaluated on MKN74 gastric cancer cell line and human skin fibroblast BJ based on methylthiazolyldiphenyl-tetrazolium bromide (MTT) test. The apoptosis/necrosis and cell cycle analysis were conducted using image cytometry. Additionally, in DNA of treated cells, abasic sites (AP) and double strands breaks (DSB) presence were measured. Intercalating properties of active compounds were evaluated using the UV–spectroscopic method. Among newly synthesized derivatives, compound 2 showed toxic effects on gastric cancer cells with simultaneous lack of toxicity to normal fibroblasts. Cell cycle analysis revealed that both compounds influence cell division mainly at the stage of replication. Simultaneously with DNA synthesis disorders, DNA damages like AP-sites and DSBs were observed. Spectroscopic studies revealed possible DNA intercalating properties of tested compounds. Obtained results indicate that the newly synthesized thiosemicarbazide derivatives are a promising group of compounds with potential anticancer activity resulted from interactions with DNA and cell cycle interrupt.