BAX But Not BCL2 Is Necessary for Apoptosis in Neuroblastoma Cells Treated With Casiopeína® IIIia

Abstract

Background/aim: The emerging antineoplastics Casiopeínas^® induce uncoupling of the respiratory chain, production of reactive oxygen species (ROS), entry of Bax into mitochondria, and exit of Ca²⁺ and Bcl-2 from them, leading to apoptosis. This study aimed to elucidate whether BAX and BCL2 are necessary for apoptosis.

Materials and methods: We silenced BAX and BCL2 by CRISPR-Cas9, assessed ROS and calcium retention capacity (CRC) by spectrofluorometry, and caspase-3 with inmunoblotting in neuroblastoma (NB) cells and 3T3-L1 fibroblasts treated with cisplatin and Casiopeína IIIia (CasIIIia).

Results: We observed an increase in O^2•- production only in BCL2^KO NB cells treated with cisplatin (three-fold) and CasIIIia (five-fold), whereas the production of H₂O₂ in BCL2^KO NB cells treated with cisplatin and CasIIIia increased five-fold and three-fold, respectively. The baseline calcium-retention capacity (CRC) was 1.7 relative fluorescence units (RFU) in both cell types. In BAX^KO, cisplatin and CasIIIia increased CRC to ~2.3 RFU, and in BCL2^KO, they decreased CRC to ~1.1 RFU. We did not detect caspase-3 in BAX^KO NB cells.

Conclusion: Only BAX is essential for CasIIIia-induced apoptosis.

Keywords: BAX and BCL2 silencing; CRISPR/Cas9; Ca2+ retention capacity; Casiopeína IIIia; SK-N-SH cells; mPTP.