Objective: Many experiments have revealed the anti-tumor activity of spiro-quinazolinone derivatives
on different cell types. Exposing KG1-a cells to N-(4- tert- butyl- 4'- oxo- 1'H- spiro [cyclohexane- 1, 2'- quinazoline]-
3'(4'H)- yl)- 4- methyl benzenesulfonamide (4t-CHQ), as an active sub-component of spiroquinazolinone
benzenesulfonamides, the experiment investigated the possible mechanisms that manifest the role
of 4t-CHQ in leukemic KG1-a progenitor cells. Mechanistically, the inhibitory effects of 4t-CHQ on KG1-a
cells emerge from its modulating function on the expression of Bax/Bcl2 and survinin proteins.
Methods: Cell viability was assessed using MTT assay. The IC50 value of cells was calculated to be 131.3μM,
after 72h-incubation with 4t-CHQ, ranging from 10 to 150μM. Apoptotic changes were studied using Acridine
Orange/Ethidium Bromide (AO/EB) staining. DNA fragmentation was analyzed by agarose gel electrophoresis
method. To evaluate the percentage of apoptotic cells and cell growth dynamic apoptotic features, we performed
Annexin V/PI double staining assay and cell cycle analysis by flow cytometry.
Results: According to the results, apoptosis induction was initiated by 4t-CHQ in the KG1-a cells (at IC50
value). Cell dynamic analysis revealed that the cell cycle at the G1 phase was arrested after treatment with 4t-
CHQ. Western blotting analysis showed enhancement in the expression ratio of Bax/Bcl-2, while the expression
of survinin protein decreased in a time-dependent manner in the KG1-a cells. According to the docking simulation
data, the effectiveness of 4t-CHQ on KG1-a cells commenced by its reactions with the functional domain of
BH3 and Bcl2 and BIR domains of survivin protein.
Conclusion: These results demonstrate a remarkable role of 4t- CHQ in arresting leukemia KG1-a stem cells
both by induction of apoptosis as well as by down-regulating survivin and Bcl2 proteins.