Study on Anticancer Potential of Quinolinium Salts Cites Magic Red Caspase Assay

Congratulations to the ChEMTox lab at Universidad Metropolitana, directed by Dr. Beatriz Zayas, for publishing their evaluation of nitrobenzazolo [3,2-a]quinolinium salts as potential anticancer agents!

To assess apoptosis, the activation of caspases 3 & 7 in the A431 epidermoid carcinoma cells was monitored using the ImmunoChemistry Technologies Magic Red™ Caspase 3&7 Assay. Cells stained with this assay that contain activated caspases 3 & 7 will exhibit a red fluorescence that may be monitored via fluorescence detection methods. This substrate-based caspase assay does not require a wash step, permeabilization, or fixation.

The Magic Red Caspase 3&7 Assay utilizes a fluorogenic substrate MR-(DEVD)2, which is comprised of a disubstituted cresyl violet (aka Magic Red™, MR) fluorophore linked to two DEVD peptide molecules. Activated DEVDases caspase-3 and caspase-7, known as effector caspases in the apoptotic process, have an affinity for the peptide sequence DEVD and enact proteolytic cleavage of the peptide from the MR fluorophore. Once the peptide sequence DEVD is cleaved from one or both sides of the Magic Redb fluorophore, the molecule may emit fluorescence at 610 nm after excitation at 540 nm.

Novel Nitrobenzazolo[3,2-a]quinolinium Salts Induce Cell Death through a Mechanism Involving DNA Damage, Cell Cycle Changes, and Mitochondrial Permeabilization
Open Journal of Apoptosis, 2013, 2, 13-22.
doi:10.4236/ojapo.2013.22002 Published Online April 29, 2013
Open Access PDF (317KB)

Authors
Christian Vélez, Osvaldo Cox, Carlos A. Rosado-Berrios, Dennise Molina, Luz Arroyo, Sujey Carro, Anton Filikov, Vineet Kumar, Sanjay V. Malhotra, Marisol Cordero, Beatriz Zayas

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 mM. 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 Mitochondrial 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.

KEYWORDS
Apoptosis; A431; Quinolinium Salts; Mutagenesis; Caspase Activation