Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as Novel Potential Anticancer Agents: Cytotoxic and Genotoxic Activities In Vitro

Abstract

In this paper, we present for the first time the evaluation of cytotoxicity and genotoxicity of de novo synthesized pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides MM129, MM130, and MM131 in human tumor cell lines: HeLa, HCT 116, PC-3, and BxPC-3. Cytotoxic and genotoxic properties of the tested compounds were estimated using the MTT assay, comet assay (alkaline and neutral version), and γ-H2AX immuno-staining. Examined sulfonamides exhibited strong anticancer properties towards tested cells in a very low concentration range (IC50 = 0.17–1.15 μM) after 72 h exposure time. The results of the alkaline and neutral version of the comet assay following 24 h incubation of the cells with tested compounds demonstrated the capability of heterocycles to induce significant DNA damage in exposed cells. HCT 116 cells were the most sensitive to the genotoxic activity of novel tricyclic pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides in the neutral version of the comet assay. Immunocytochemical detection of γ-H2AX showed an increase in DNA DSBs level in the HCT 116 cell line, after 24 h incubation with all tested compounds, confirming the results obtained in the neutral comet assay. Among all investigated compounds, MM131 showed the strongest cytotoxic and genotoxic activity toward all tested cell types. In conclusion, our results suggest that MM129, MM130, and MM131 exhibit high cytotoxic and genotoxic potential in vitro, especially towards the colorectal cancer cell line HCT 116. However, further investigations and analyses are required for their future implementation in the field of medicine.

Genotoxicity of Novel Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides in Normal and Cancer Cells In Vitro

“…Furthermore, incubation of colon cancer cells with MM131 —another pyrazolo[4,3- e ]tetrazolo[1,5- b ][1,2,4]triazine sulfonamide—induced apoptosis of DLD-1 and HT-29 cells with observed down-regulation of mTOR kinase, soluble intercellular adhesion molecule-1 (sICAM-1), and cathepsin, together with up-regulation of beclin-1 B [ 33 ]. We have also shown, with the use of an alkaline/neutral version of the comet assay and γ-H2AX staining, that MM129 , MM130 , and MM131 sulfonamides exhibited genotoxic activity in four investigated cancer cell lines (HeLa, HCT-116, PC-3, and BxPC-3) [ 34 ]. The occurrence of DNA damage following exposure to genotoxic agents would normally trigger the DDR responsible for the activation of DNA repair mechanisms or elimination of the severely damaged cell.…”

“…The molecular dynamics simulation suggested that the tested compounds bind strongly and durably with molecular targets and may exert an anti-cancer effect through their inhibition [ 19 ]. Furthermore, structurally similar compounds MM129 , MM130 , and MM131 exhibited cytotoxic activity with IC 50 concentrations of 0.17–1.15 μM in four cancer cell lines (HeLa, HCT 116, PC-3, and BxPC-3) and genotoxic activity, as indicated by alkaline/neutral comet assays and γH2AX staining [ 34 ].…”

“…The majority of the neutral comet assay steps were performed analogously to the alkaline version except for the electrophoresis step. In contrast to the alkaline comet assay, electrophoresis was performed in a buffer comprising 100 mM TRIS and 300 mM sodium acetate with the pH of the solution adjusted to 9.0 by glacial acetic acid, as described previously by Bukowski et al [ 34 ]. Electrophoresis was run at 12 V, 50 mA for 60 min.…”

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as Novel Potential Anticancer Agents: Apoptosis, Oxidative Stress, and Cell Cycle Analysis

“…The high pro-oxidative activity of tested triazine derivatives is consistent with the data obtained from our previous studies, where examined compounds exhibited high genotoxicity against the same cancer cell lines. Similarly to the genotoxicity studies, MM131 showed the most potent pro-oxidative potential among the tested compounds [63]. Furthermore, research has shown that the accumulation of ROS-related damages in lipids, proteins, and DNA may be responsible for the suppression of tumor cell proliferation and growth via cell cycle arrest and apoptosis.…”

“…Importantly, the investigated compounds were noticeably less cytotoxic toward normal cells-human foreskin fibroblast (Hs27) and human peripheral blood mononuclear cells (PBMCs). The IC 50 values obtained in the MTT assay were shown in the Supplementary Material (Table S1) [63]. Therefore, we decided to investigate the pro-oxidative and pro-apoptotic properties of three novel derivatives of pyrazolo-triazine sulfonamides MM129, MM130, MM131 against four adherent human cancer cell lines-HeLa, HCT 116, PC-3, and BxPC-3.…”