Novel Nitrobenzazolo[3,2-a]quinolinium Salts Induce Cell Death through a Mechanism Involving DNA Damage, Cell Cycle Changes, and Mitochondrial Permeabilization

Abstract

This study reports the capacity of three nitro substituted benzazolo[3,2-a]quinolinium salts NBQs: NBQ 95 (NSC-763304), NBQ 38 (NSC 763305), and NBQ 97 (NSC-763306) as potential antitumor agents. NBQ’s are unnatural alkaloids possessing a positive charge that could facilitate interaction with cell organelles. The anticancer activities of these compounds were evaluated through the National Cancer Institute (NCI) 60 cell line screening which represents diverse histologies. The screening was performed at 10 µM on all cell lines. Results from the NCI screening indicated cytotoxicity activity on six cell lines. In order to explore a possible mechanism of action, a detailed biological activity study of NBQ 95 and NBQ 38 was performed on A431 human epidermoid carcinoma cells to determine an apoptotic pathway involving, cell cycle changes, DNA fragmentation, mutations, mitochondrial membrane permeabilization and caspases activation. DNA fragmentation, cell cycle effects, mutagenesis, mitochondrial permeabilization and activation of caspases were determined by fluorimetry and differential imaging. Our data showed that A431 growth was inhibited with an average IC50 of 30 µM. In terms of the mechanism, these compounds interacted with DNA causing fragmentation and cell cycle arrest at sub G0/G1 stage. Mutagenesis was higher for NBQ 38 and moderate for NBQ 95 Mitochon-drial permeabilization was observed with NBQ 38 and slightly for NBQ 95. Both compounds caused activation of Caspases 3 and 7 suggesting an apoptotic cell death pathway through an intrinsic mechanism. This study reports evidence of the toxicity of these novel compounds with overlapping structural and mechanistic similarities to ellipticine, a known anti-tumor compound.

Xylazine as a Drug of Abuse and Its Effects on the Generation of Reactive Species and DNA Damage on Human Umbilical Vein Endothelial Cells

“…Determination of DNA fragmentation is used as an indicator of apoptosis in drug toxicity assessment. This assay measures cells containing less than 1DNA equivalent (Sub-G1) after degradation of DNA triggered by endonucleases [ 35 ]. Cells were harvested after drug treatment and implementing the previously described conditions, fixed with 70% ethanol, incubated 24 hours at 4°C, stained with 500 μ l of 1 μ g/ml DAPI and incubated for 5 minutes, according to manufacturer's specifications, and analyzed by image analysis with NucleoCounter NC-3000 instrument.…”

“…DNA fragmentation content in EA.hy926 cells after 24 h of exposure to vehicle (negative control group, NC), camptothecin (positive control group, PC, 50 μ M), xylazine (XYL, 60 μ M), cocaine (COC, 160 μ M), 6-monoacetylmorphine (6-MAM, 160 μ M), XYL/COC (50 μ M), XYL/6-MAM (50 μ M), and XYL/COC/6-MAM (40 μ M) was evaluated in each phase independently and compared to negative control group. DNA fragmentation content assay was performed to measure cells containing less than 1DNA equivalent (Sub-G1) after degradation of DNA triggered by endonucleases or drug interaction [ 35 ]. Cells were harvested after drug treatment, fixed with 70% ethanol, incubated 24 hours at 4°C, stained with 1 μ g/ml DAPI (5 min incubation), and image-analyzed with NucleoCounter NC-3000 instrument.…”