In Vitro and In Silico Investigation of BCI Anticancer Properties and Its Potential for Chemotherapy-Combined Treatments.

BACKGROUND: DUSP6 phosphatase serves as a negative regulator of MAPK kinases involved in numerous cellular processes. BCI has been identified as a potential allosteric inhibitor with anticancer activity. Our study was designed to test the anticancer properties of BCI in colon cancer cells, to characterize the effect of this compound on chemotherapeutics such as irinotecan and oxaliplatin activity, and to identify potential molecular targets for this inhibitor. METHODS: BCI cytotoxicity, proapoptotic activity, and cell cycle distribution were investigated in vitro on three colon cancer cell lines (DLD1, HT-29, and Caco-2). In silico investigation was prepared to assess BCI drug-likeness and identify potential molecular targets. RESULTS: The exposure of colorectal cancer cells with BCI resulted in antitumor effects associated with cell cycle arrest and induction of apoptosis. BCI exhibited strong cytotoxicity on DLD1, HT-29, and Caco-2 cells. BCI showed no significant interaction with irinotecan, but strongly attenuated the anticancer activity of oxaliplatin when administered together. Analysis of synergy potential further confirmed the antagonistic interaction between these two compounds. In silico investigation indicated CDK5 as a potential new target of BCI. CONCLUSIONS: Our studies point to the anticancer potential of BCI but note the need for a precise mechanism of action.

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides as an Important Scaffold for Anticancer Drug Discovery-In Vitro and In Silico Evaluation.

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides (MM-compounds) are a relatively new class of heterocyclic compounds that exhibit a wide variety of biological actions, including anticancer properties. Here, we used caspase enzyme activity assays, flow cytometry analysis of propidium iodide (PI)-stained cells, and a DNA laddering assay to investigate the mechanisms of cell death triggered by the MM-compounds (MM134, -6, -7, and -9). Due to inconsistent results in caspase activity assays, we have performed a bromodeoxyuridine (BrdU) incorporation assay, colony formation assay, and gene expression profiling. The compounds' cytotoxic and pro-oxidative properties were also assessed. Additionally, computational studies were performed to demonstrate the potential of the scaffold for future drug discovery endeavors. MM-compounds exhibited strong micromolar (0.06-0.35 µM) anti-proliferative and pro-oxidative activity in two cancer cell lines (BxPC-3 and PC-3). Activation of caspase 3/7 was observed following a 24-h treatment of BxPC-3 cells with IC50 concentrations of MM134, -6, and -9 compounds. However, no DNA fragmentation characteristics for apoptosis were observed in the flow cytometry and DNA laddering analysis. Gene expression data indicated up-regulation of BCL10, GADD45A, RIPK2, TNF, TNFRSF10B, and TNFRSF1A (TNF-R1) following treatment of cells with the MM134 compound. Moreover, in silico studies indicated AKT2 kinase as the primary target of compounds. MM-compounds exhibit strong cytotoxic activity with pro-oxidative, pro-apoptotic, and possibly pro-necroptotic properties that could be employed for further drug discovery approaches.

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 current study continues the evaluation of the anticancer potential of three de novo synthesized pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides-MM129, MM130, and MM131-against human cancer cells of HeLa, HCT 116, PC-3, and BxPC-3 lines. The pro-apoptotic activity of the investigated sulfonamides was shown by observations of changes in the mitochondrial transmembrane potential of the tested cells, externalization of phosphatidylserine on the cellular membrane surface, and cell morphology in microscopic imaging. The computational studies have shown that MM129 exhibited the lowest binding energy values when docked against CDK enzymes. In addition, the highest stability was shown for complexes formed between MM129 and CDK5/8 enzymes. All examined compounds induced cell cycle arrest in the G0/G1 phase in the BxPC-3 and PC-3 cells and simultaneously caused the accumulation of cells in the S phase in the HCT 116 cells. In addition, the increase in the subG1 fraction was observed in PC-3 and HeLa cells. The application of a fluorescent H2DCFDA probe revealed the high pro-oxidative properties of the tested triazine derivatives, especially MM131. In conclusion, the obtained results suggest that MM129, MM130, and MM131 exhibited strong pro-apoptotic properties towards investigated cells, mainly against the HeLa and HCT 116 cell lines, and high pro-oxidative potential as well. Moreover, it is suggested that the anticancer activity of the tested compounds may be associated with their ability to inhibit CDK enzymes activities.

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

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides constitute a novel group of heterocyclic compounds with broad biological activities including anticancer properties. The compounds investigated in this study (MM134, -6, -7, and 9) were found to have antiproliferative activity against BxPC-3 and PC-3 cancer cell lines in micromolar concentrations (IC50 0.11-0.33 µM). Here, we studied the genotoxic potential of the tested compounds with alkaline and neutral comet assays, accompanied by immunocytochemical detection of phosphorylated γH2AX. We found that pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides induce significant levels of DNA damage in BxPC-3 and PC-3 cells without causing genotoxic effects in normal human lung fibroblasts (WI-38) when used in their respective IC50 concentrations (except for MM134) and showed a dose-dependent increase in DNA damage following 24 h incubation of tested cancer cells with these agents. Furthermore, the influence of MM compounds on DNA damage response (DDR) factors was assessed using molecular docking and molecular dynamics simulation.

Preparation of Novel Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides and Their Experimental and Computational Biological Studies.

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides constitute a novel class of heterocyclic compounds with broad biological activity, including anticancer properties. Investigated in this study, MM-compounds (MM134, MM136, MM137, and MM139) exhibited cytotoxic and proapoptotic activity against cancer cell lines (BxPC-3, PC-3, and HCT-116) in nanomolar concentrations without causing cytotoxicity in normal cells (L929 and WI38). In silico predictions indicate that tested compounds exhibit favorable pharmacokinetic profiles and may exert anticancer activity through the inhibition of BTK kinase, the AKT-mTOR pathway and PD1-PD-L1 interaction. Our findings point out that these sulfonamide derivatives may constitute a source of new anticancer drugs after optimization.

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

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.

Amino- and polyaminophthalazin-1(2H)-ones: synthesis, coordination properties, and biological activity.

Amino- and polyaminophthalazinones were synthesized by the palladium-catalyzed amination (alkyl- and arylamines, polyamines) of 4-bromophthalazinones in good yields. The coordinating properties of selected aminophthalazinones towards Cu(II) ions were investigated and the participation of the nitrogen atoms in the complexation of the metal ion was shown. A biological screening of the potential cytotoxicity of selected synthesized compounds on HT-29 and PC-3 cell lines, as well as on the L-929 cell line, proved that some amino derivatives of phthalazinone show interesting anticancer activities. The detailed synthesis, spectroscopic data, and biological assays are reported.

Advances in DNA Repair-Emerging Players in the Arena of Eukaryotic DNA Repair.

Genomic DNA is constantly damaged by factors produced during natural metabolic processes as well as agents coming from the external environment. Considering such a wide array of damaging agents, eukaryotic cells have evolved a DNA damage response (DRR) that opposes the influence of deleterious factors. Despite the broad knowledge regarding DNA damage and repair, new areas of research are emerging. New players in the field of DDR are constantly being discovered. The aim of this study is to review current knowledge regarding the roles of sirtuins, heat shock proteins, long-noncoding RNAs and the circadian clock in DDR and distinguish new agents that may have a prominent role in DNA damage response and repair.

Mechanisms of Multidrug Resistance in Cancer Chemotherapy.

Cancer is one of the main causes of death worldwide. Despite the significant development of methods of cancer healing during the past decades, chemotherapy still remains the main method for cancer treatment. Depending on the mechanism of action, commonly used chemotherapeutic agents can be divided into several classes (antimetabolites, alkylating agents, mitotic spindle inhibitors, topoisomerase inhibitors, and others). Multidrug resistance (MDR) is responsible for over 90% of deaths in cancer patients receiving traditional chemotherapeutics or novel targeted drugs. The mechanisms of MDR include elevated metabolism of xenobiotics, enhanced efflux of drugs, growth factors, increased DNA repair capacity, and genetic factors (gene mutations, amplifications, and epigenetic alterations). Rapidly increasing numbers of biomedical studies are focused on designing chemotherapeutics that are able to evade or reverse MDR. The aim of this review is not only to demonstrate the latest data on the mechanisms of cellular resistance to anticancer agents currently used in clinical treatment but also to present the mechanisms of action of novel potential antitumor drugs which have been designed to overcome these resistance mechanisms. Better understanding of the mechanisms of MDR and targets of novel chemotherapy agents should provide guidance for future research concerning new effective strategies in cancer treatment.

DNA damage and methylation induced by organophosphate flame retardants: Tris(2-chloroethyl) phosphate and tris(1-chloro-2-propyl) phosphate in human peripheral blood mononuclear cells.

Phosphorus flame retardants are a group of chemicals that are used to slow or prevent the spread of fire. These compounds have been detected in different environments including human organism. In the present study, we have investigated DNA-damaging potential and effect on DNA methylation of tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP) in human peripheral blood mononuclear cells (PBMCs). In order to determine DNA damage and repair, the alkaline and neutral versions of the comet assay were used. The level of DNA methylation was determined with specific antibodies against methylated DNA. PBMCs were exposed to TCEP and TCPP at the concentrations in the range of 1-1000 µM for 24 h. We have observed that TCEP and TCPP induced DNA damage-DNA breaks and alkali-labile sites. All DNA damages were effectively repaired during 120-min repair incubation. The results have also shown that TCEP and TCPP decreased the level of DNA methylation in PBMCs. In the case of TCEP, this effect was observed at a very low concentration of 1 µM.

[Polymorphism of genes encoding proteins of DNA repair vs. occupational and environmental exposure to lead, arsenic and pesticides]. / Polimorfizm genów kodujacych bialka naprawy DNA a zawodowe i srodowiskowe narazenie na olów, arsen i pestycydy.

Genetic polymorphism is associated with the occurrence of at least 2 different alleles in the locus with a frequency higher than 1% in the population. Among polymorphisms we can find single nucleotide polymorphism (SNP) and polymorphism of variable number of tandem repeats. The presence of certain polymorphisms in genes encoding DNA repair enzymes is associated with the speed and efficiency of DNA repair and can protect or expose humans to the effects provoked by xenobiotics. Chemicals, such as lead, arsenic pesticides are considered to exhibit strong toxicity. There are many different polymorphisms in genes encoding DNA repair enzymes, which determine the speed and efficiency of DNA damage repair induced by these xenobiotics. In the case of lead, the influence of various polymorphisms, such as APE1 (apurinic/apyrimidinic endonuclease 1) (rs1130409), hOGG1 (human 8-oxoguanine glycosylase) (rs1052133), XRCC1 (X-ray repair cross-complementing protein group 1) (rs25487), XRCC1 (rs1799782) and XRCC3 (X-ray repair cross-complementing protein group 3) (rs861539) were described. For arsenic polymorphisms, such as ERCC2 (excision repair cross-complementing) (rs13181), XRCC3 (rs861539), APE1 (rs1130409) and hOGG1 (rs1052133) were examined. As to pesticides, separate and combined effects of polymorphisms in genes encoding DNA repair enzymes, such as XRCC1 (rs1799782), hOGG1 (rs1052133), XRCC4 (X-ray repair cross-complementing protein group 4) (rs28360135) and the gene encoding the detoxification enzyme PON1 paraoxonase (rs662) were reported. Med Pr 2018;69(2):225-235.