Katarzyna Błaszczak-Świątkiewicz

Biological approach of anticancer activity of new benzimidazole derivatives

BACKGROUND A series of new benzimidazole derivatives, earlier synthesized, was tested in vitro as new bioreductive prodrugs with the potential anticancer activity. Their effect on the DNA destruction and growth inhibition into selected tumor cell lines at normoxia and hypoxia conditions was determined. METHODS The human lung adenocarcinoma A549 cell line was used to determine the anticancer activity of the analyzed compounds by using WST-1 assay. The apoptosis test (caspase 3/7 assay) was used to define the cytotoxic way of tumor cells death. Additionally test In situ DNA Damage Assay Kit was applied to recognize the DNA destruction. RESULTS Four of the examined compounds (1, 3, 7, 9) show a very good antiproliferative effect and three of them are specific for hypoxia conditions (2, 4, 8). CONCLUSION Compound 8 is the most cytotoxic against human lung adenocarcinoma A549 cells at hypoxic conditions. Hypoxia/normoxia cytotoxic coefficient of compound 8 (4.75) is close to hypoxia/normoxia cytotoxic coefficient of tirapazamine (5.59) - reference substance in our experiments and this parameter locates it between mitomycin C and 2-nitroimidazole (misonidazole). The screening test of the caspase-dependent apoptosis proved that the exposure of compounds 1-2 and 7-8 against A549 cells for a 48 h promote apoptotic cell death. Additionally, the test of the DNA damage established that compounds 1, 2, 7, 8 are specific agents for the hypoxia-selective cytotoxicity of nitrobenzimidazoles [6,26].

Some characteristics of activity of potential chemotherapeutics – benzimidazole derivatives

In this work, the biological activity of some benzimidazoles and benzimidazole-4,7-diones was compared. These two groups of compounds were evaluated as potential chemotherapeutics and their characteristic relationship structure to biological activity was discussed. The authors compared their effect into the cytotoxic, apoptosis and DNA destruction approach. Their cytotoxic effect on the human lung adenocarcinoma A549 cells line was determined by WST-1 test. Next the cytotoxic way of tumor cells death was determined by caspase 3/7 test. The last point referred to the DNA destruction of A549 cells and test in situ DNA Assay Kit was applied. Two of the examined compounds (B2 and D2) show a very good correlation of the cytotoxic effect normoxia to hypoxia and they have been found as the potential agents of the DNA damage. The most cytotoxic feature possesses N-oxide benzimidazole derivatives (D and B groups). The screening test of the DNA damage established that N-oxide benzimidazole derivatives (D and B groups) can be more potent as the hypoxia-selective agents for tumor cells than benzimidazole derivatives (A and C groups). Additionally, the test of the caspase-dependent apoptosis proved that the exposure of benzimidazole-4,7-diones against A549 cells, especially in hypoxia, promotes apoptotic cell death.

Novel tetrahydroacridine derivatives inhibit human lung adenocarcinoma cell growth by inducing G1 phase cell cycle arrest and apoptosis

Lung cancer is not only the most commonly diagnosed cancers worldwide but it is still the leading cause of cancer-related death. Acridine derivatives are a class of anticancer agents with the ability to intercalate DNA and inhibit topoisomerases. The aim of this study was to evaluate the effect of sixteen new tetrahydroacridine derivatives on the viability and growth of human lung adenocarcinoma cells. We compared anticancer activity of a series of eight compounds with 4-fluorobenzoic acid and eight compounds with 6-hydrazinonicotnic acid differed from each other in length of the aliphatic chain containing from 2 to 9 carbon atoms. Interestingly, tetrahydroacridine with 4-fluorobenzoic acid (compounds 9-16) showed higher anticancer activity than derivatives with 6-hydrazinonicotnic acid (compounds 1-8) and their efficacy was correlated with increasing number of carbon atoms in the aliphatic chain. The results showed that inhibition of cancer cell growth by the most effective compounds 15 and 16 was associated with induction of G1 phase cell cycle arrest followed by caspase-3 dependent apoptosis. Our findings suggest that tetrahydroacridine with 4-fluorobenzoic acid containing 8 and 9 carbon atoms may be potential candidate for treatment of lung cancer.

Biological evaluation of the toxicity and the cell cycle interruption by some benzimidazole derivatives

In this work, the in vitro tests of biological activity of benzimidazoles were conducted. This group of benzimidazole derivatives was evaluated as potential bioreductive agents and their characteristic pro-apoptosis activity and cell cycle interruption on the human lung adenocarcinoma A549 cells were discussed. Their toxicity on the healthy human erythrocytes and their influence on the healthy human erythrocytes acetylcholinesterase enzyme (AChE) were established. Their apoptosis activity on A549 cells line was determined by Annexin V-APC test, and it was visualized by Hoechst test. In the next stage, their influence on the cell cycle interruption was determined by using the ribonuclease reagent. The AChE inhibition test was defined by the Ellman method, and the red blood cell lysis was defined by erythrotoxicity test. The results proved the pro-apoptosis properties of all tested compounds in normoxia and hypoxia. The DNA content assay showed that the benzimidazoles possess the ability to interrupt S phase of tumor cell cycle. The best activity in this action was presented by compound 1, especially in hypoxia, and it proves that the N-oxide analogs are predispositioned to the hypoxic target. In this study, the benzimidazoles were found as potentially biocompatible and their inhibition of acetylcholinesterase was lower than tirapazamine and much lower than tacrine which constitutes their desired effect of potential biological activity.

Biological Evaluation of the Activity of Some Benzimidazole-4,7-dione Derivatives

The study presented here is a follow up of the authors’ interest in the approach to selective and cytotoxic bioreductive anticancer prodrugs. The current work is devoted to explore both the biological activity of some previously obtained compounds and the search for an explanation of their target(s) in hypoxic pathways. In this work the biological activity of some benzimidazole-4,7-diones was evaluated. These compounds were examined as potential bioreductive agents specific for the hypoxic environment found in tumor cells. The main aim was concerned with establishing their cytotoxic properties by using proliferation, apoptosis and DNA destruction tests on selected tumor cells. Their cytotoxic effects on two tumor cell lines (human lung adenocarcinoma A549 cells line and human malignant melanoma WM115) was compared by means of a WST-1 test. Next, the mode of cytotoxicity behind the selected tumor cells’ death was determined by the caspase 3/7 test. The last point referred to the DNA destruction of A549 and WM115 cells and the in situ DNA Assay Kit test was applied. The cytotoxic tests confirmed their activity against the tumor cells and target hypoxia (compounds 2b, 2a, 2d). The screening test of the caspase-dependent apoptosis proved that the exposure of the tested tumor cells in hypoxia to these benzimidazole-4,7-diones promoted the apoptotic cell death. Additionally, the DNA damage test established that benzimidazole-4,7-diones can be potential hypoxia-selective agents for tumor cells, especially compound 2b. All results classify the tested benzimidazole-4,7-diones as promising, lead molecules and provide a rationale for further molecular studies to explain their usefulness as potential inhibitors of the hypoxia-inducible factor 1 (HIF1).

APPLICATION OF HPLC METHOD FOR INVESTIGATION OF STABILITY OF NEW BENZIMIDAZOLE DERIVATIVES

Obtained benzimidazole derivatives, our newly synthesized heterocyclic compounds, belong to a new group of chemical bondings with potential anticancer features. A biochemical study determining anticancer activity of benzimidazole derivatives was conducted in the environment of 0.2% DMSO (dimethyl sulfoxide). The HPLC method was used to determine stability of these compounds in a biochemical study environment. Optimization of the chromatographic system and validation of the established analytical method were performed. Stability analysis of the derivatives was examined in the reversed phases RP-8 system, where pure acetonitrile was used as an eluent for the analysis of compound 1–4. The HPLC analysis revealed that the compounds studied are homogeneous in the environment of 0.2% DMSO up to 96 hr.

Virus-directed enzyme prodrug therapy and the assessment of the cytotoxic impact of some benzimidazole derivatives

Virus-directed enzyme prodrug therapy is one of the major strategy of increasing cytotoxicity of bioreductive agents. This research intended to examine new selected benzimidazole derivatives as a substrate for nitroreductase, the enzyme involved in nitroreduction which is responsible to the production of cytotoxic metabolites. In this way, the selectivity and strength of cytotoxicity can be raised. The effect of benzimidazoles on virus transfected cells and non-virus transfected cells A549 cell line was established by Annexin V + propidium iodide test, western blot, and polymerase chain reaction analysis of specific pro- and anti-apoptotic proteins in the corresponding gene expression and additionally nitroreductase gene expression. Our results proved the pro-apoptotic properties of all tested compounds in normoxia and hypoxia, especially according to virused A549 cells where the time of exposition was reduced from 48 to 4 h. In this shorten period of time, the strongest activity was shown by N-oxide compounds with nitro-groups. The apoptosis was confirmed by generation of BAX gene and protein and reduction of BCL2 gene and protein.

Determination of the Stabilities of New Quinazoline Derivatives by HPLC

Abstract The quinazoline derivatives reported in this work belong to a new group of compounds with a type of chemical bonding that has potential anticancer features. A biochemical study determining anticancer activity of quinazoline derivatives and N3‐quinazoline oxide will be conducted in the medium of 0.2% DMSO. The HPLC method was used to determine stabilities of these compounds in a biochemical study environment. Optimisation of the chromatographic system and validation of the established analytical method was performed. Analysis of the stabilities of the derivatives examined was performed in the reversed phase RP‐18 system, in which pure acetonitrile was used as an eluent for the analysis of compounds 1 and 3, and a mixture of acetonitrile/methanol in the ratio of 95/5% for compounds 2 and 4. The HPLC analysis revealed that the compounds studied are homogeneous in the 0.2% DMSO medium, up to 96 hours.