Natural antioxidants synergistically enhance the anticancer potential of AP9-cd, a novel lignan composition from Cedrus deodara in human leukemia HL-60 cells

Antioxidants have been used as adjuvant with anticancer therapy to synergize the potential of the anti-neoplastic therapeutics. Based on the fact, we have studied the effect of three natural antioxidants curcumin, silymarin and acteoside on AP9-cd (standardized lignan composition from Cedrus deodara) induced cytotoxicity in human leukemia HL-60 cells. The antioxidant potential of individual test compounds was first evaluated with ferric reducing antioxidant power (FRAP) test, which revealed that all four molecules behave as antioxidants. The apoptotic potential of AP9-cd was significantly enhanced in HL-60 cells in the presence of curcumin, silymarin and acteoside. It was confirmed by using various models like MTT assay, DNA fragmentation, nuclei condensation, sub-Go DNA population, Annexin-V-FITC binding, ROS depletion and immunoblotting in HL-60 cells. AP9-cd and individual antioxidants alone at low doses (10μg and 10μM, respectively) have meager or no cytotoxicity in HL-60 cells, whereas in mutual combinations, there were 2-3 times enhancement in Annexin-V-FITC and sub-Go DNA population. Moreover, prominent DNA ladders were observed at low doses of AP9-cd in combinations with various antioxidants. The Hoechst staining of the nucleus also revealed the same results for the HL-60 cells treated with AP9-cd and different antioxidants. The molecular diagnostics revealed that the combinations induced a strong antioxidant effect which was correlated with the downregulation of NF-κB expression in the nucleus. Out of the three antioxidants, curcumin was found to be more potent than acteoside and silymarin in terms of enhancing the apoptotic potential of AP9-cd. These results propose an important role of natural antioxidant as adjuvant to enhance the anticancer potential of AP9-cd and more likely other anti-neoplastic therapeutics.