DNA binding and apoptotic induction ability of harmalol in HepG2: Biophysical and biochemical approaches

• Harmalol shows maximum growth inhibition in HepG2 with GI50 of 14.2 μM.
• The alkaloid causes conformational changes and stabilization in CT-DNA.
• Induced DNA damage, alterations in cellular morphology and membrane potential.
• Alkaloid shows ROS dependent, p53 and caspase activated cytotoxicity in the cells.
• A significant increase in Sub Go/G1 population of HepG2 cell.

Harmalol administration caused remarkable reduction in proliferation of HepG2 cells with GI50 of 14.2 μM, without showing much cytotoxicity in embryonic liver cell line, WRL-68. Data from circular dichroism (CD) and differential scanning calorimetric (DSC) analysis of harmalol-CT DNA complex shows conformational changes with prominent CD perturbation and stabilization of CT DNA by 8 °C. Binding constant and stoichiometry was calculated using the above biophysical techniques. The Scatchard plot constructed from CD data showed cooperative binding, from which the cooperative binding affinity (K’ω) of 4.65 ± 0.7 × 105 M−1, and n value of 4.16 were deduced. The binding parameter obtained from DSC melting data was in good agreement with the above CD data. Furthermore, dose dependent apoptotic induction ability of harmalol was studied in HepG2 cells using different biochemical assays. Generation of ROS, DNA damage, changes in cellular external and ultramorphology, alteration of membrane, formation of comet tail, decreased mitochondrial membrane potential and a significant increase in Sub Go/G1 population made the cancer cell, HepG2, prone to apoptosis. Up regulation of p53 and caspase 3 further indicated the apoptotic role of harmalol.

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