Chk1/2 Inhibition by AZD7762 Enhances Andrographolide-Induced DNA Damage, Impairs DNA Repair, and Promotes Apoptosis in Head and Neck Squamous Cell Carcinoma CAL-33 Cells

Abstract

Background: Andrographolide (AG), a diterpenoid lactone and a major constituent of Andrographis paniculata, has shown promising antiproliferative effect on diverse cancer cell lines, including head and neck cancer. However, a significant anti-proliferative effect is observed only at high micromolar concentrations. Further, AG has been shown to have poor oral bioavailability. Therefore, it is essential to consider mechanism-based combinations that could synergistically enhance the therapeutic potential of this compound so that efficacy can be achieved in concentrations that are systemically achievable. Objective: The present study is aimed to evaluate the anticancer effects and associated molecular alterations of an AG and Chk1/2 inhibitor combination in head and neck squamous cell carcinoma (CAL-33) cells since the earlier studies have suggested ATM/ATR/Chk1/2-mediated activation of DNA damage checkpoints. Methods: Normal FHs74int cells and HNSCC cells (CAL-33 and FaDu) were used to assess cell proliferation and cell death. Cell cycle distribution and apoptosis were analyzed by flow cytometry and Acridine orange/EtBr, respectively, in CAL-33 cells. DNA damage in CAL-33 and FaDu cells was assessed by comet assay. Changes in the expression of proteins implicated in apoptosis, cell cycle progression, and DNA damage signaling were examined using Western blotting. Results: While AG did not show any significant changes in the growth or cell death of normal FHs74int cells, it strongly inhibited proliferation of HNSCC cells, concomitant with G2/M arrest and apoptosis. AG and AZD7762 showed an IC50 of 2.743 μM and 0.02600 μM, respectively, on cell proliferation in CAL-33 cells. Furthermore, AG caused an accumulation of intracellular ROS, which was inhibited by treatment with N-acetylcysteine. Chk1, Chk2 and γH2AX phosphorylation were induced in a dose-dependent manner by treatment with AG. The combination of AG with Chk1/2 inhibitor AZD7762 showed a synergistic effect in inhibiting cell proliferation with a CI of 0.4 through inhibition of DNA repair and premature mitotic entry, ultimately resulting in programmed cell death. Conclusions: These findings provide evidence that AG selectively targets DNA damage and repair in HNSCC CAL-33 cells via cell cycle arrest and apoptosis. Further, the anticancer potential of AG can be significantly enhanced by combination with the Chk1/2 inhibitor AZD7762, preventing repair of AG-induced DNA damage and amplifying checkpoint stress responses, resulting in enhanced apoptotic cell death. Together, these findings support the therapeutic potential of AG and highlight checkpoint kinase inhibition as a rational combination strategy for HNSCC treatment.