Theaflavin and epigallocatechin‐3‐gallate synergistically induce apoptosis through inhibition of PI3K/Akt signaling upon depolymerizing microtubules in HeLa cells

Theaflavin (TF) and epigallocatechin‐3‐gallate (EGCG) both have been reported previously as microtubule depolymerizing agents that also have anticancer effects on various cancer cell lines and in animal models. Here, we have applied TF and EGCG in combination on HeLa cells to investigate if they can potentiate each other to improve their anticancer effect in lower doses and the underlying mechanism. We found that TF and EGCG acted synergistically, in lower doses, to inhibit the growth of HeLa cells. We found the combination of 50 µg/mL TF and 20 µg/mL EGCG to be the most effective combination with a combination index of 0.28. The same combination caused larger accumulation of cells in the G 2/M phase of the cell cycle, potent mitochondrial membrane potential loss, and synergistic augmentation of apoptosis. We have shown that synergistic activity might be due to stronger microtubule depolymerization by simultaneous binding of TF and EGCG at different sites on tubulin: TF binds at vinblastine binding site on tubulin, and EGCG binds near colchicines binding site on tubulin. A detailed mechanistic analysis revealed that stronger microtubule depolymerization caused effective downregulation of PI3K/Akt signaling and potently induced mitochondrial apoptotic signals, which ultimately resulted in the apoptotic death of HeLa cells in a synergistic manner. Theaflavin (TF) and epigallocatechin‐3‐gallate (EGCG) bind at different sites on tubulin simultaneously and cause potent depolymerization of microtubules in HeLa cells. This results in the induction of apoptosis by these two polyphenols in a synergistic manner through the inhibition of PI3K/Akt and the modulation of mitochondrial signaling.