Helichrysetin and TNF-α synergistically promote apoptosis by inhibiting overactivation of the NF-κB and EGFR signaling pathways in HeLa and T98G cells

Abstract

Tumor necrosis factor-α (TNF-α) has different effects on apoptosis depending on activation or inactivation of the nuclear factor-κB (NF-κB) and epidermal growth factor receptor (EGFR) signaling pathways. Helichrysetin, a natural chalcone, inhibits NF-κB nuclear translocation in mouse pancreatic β cells. The present study aimed to iden- tify the effect of helichrysetin on activation of the NF-κB and EGFR signaling pathways induced by TNF-α, and the synergistic effect of helichrysetin and TNF-α on apoptosis of HeLa and T98G cells. Cell proliferation was measured by Cell Counting Kit-8 assay, while apoptosis was measured by Hoechst 33258 and Annexin V/PI staining. NF-κB activity was detected by luciferase assay, protein expression was measured by western blotting and mRNA expression was detected by quantitative PCR assay. The results revealed that in HeLa and T98G cells helichrysetin blocked the increased phosphorylation of NF-κB p65 induced by TNF-α. Although helichrysetin alone decreased cell viability, helichrysetin and TNF-α synergistically decreased cell viability. Helichrysetin, not TNF-α, promoted apoptosis, while the combination of helichrysetin and TNF-α synergistically increased apoptosis. In addition, helichrysetin and TNF-α synergistically enhanced the activation of caspase-3 and poly-(ADP-ribose)-polymerase compared with helichrysetin alone. Helichrysetin inhibited the phosphorylation of transforming growth factor-β activated kinase (TAK1), IκB kinase-α/β (IKK-α/β), NF-κB p65 and EGFR induced by TNF-α. Consistent with the inhibition of NF-κB activation, the increased TNF-α-induced mRNA expression levels of TNF-α, IL-1β, CCL2, CCL5 and CXCL10 were significantly downregulated by helichrysetin. Therefore, helichrysetin and TNF-α synergistically promoted apoptosis by inhibiting TAK1/IKK/NF-κB and TAK1/EGFR signaling pathways in HeLa and T98G cells, indicating a potential thera- peutic strategy for cancer.