Effects of 4,5,4'-trihydroxy-3,3'-dimethoxybibenzyl from Dendrobium ellipsophyllum on induction of apoptosis and inhibition of cancer stem-like cells in human lung cancer H460 cells

Abstract

Failure of current chemotherapeutic drugs leads to the recurrence of tumors and increased mortality in lung cancer patients. This study investigated 4,5,4'-trihydroxy-3,3'-dimethoxybibenzyl (TDB), a bibenzyl extracted from Dendrobium ellipsophyllum Tang and Wang as a novel anticancer agent for lung cancer. Selective anticancer activity of TDB against human lung cancer cells was demonstrated with a high half maximal inhibitory concentration (IC50) approximately > 300 µM in dermal papilla cells, while IC50 in human lung cancer H460 cells was approximately 100 ± 5.18 µM. Flow cytometry confirmed that TDB (50 µM) treatment for 24 h, caused significant early and late apoptosis but not necrosis in H460 cells. The up-regulation of p53, a tumor-suppressor protein, was observed in human lung cancer cells treated with 10-50 µM of TDB. The mechanism for apoptosis was attributed to p53 activation, which was related to up-regulation of pro-apoptotic Bax, reduction of anti-apoptosis Bcl-2 and Mcl-1 proteins and suppression on protein kinase B (Akt) survival pathway. Another possible mechanism involved in TDB-induced apoptosis in H460 cells was the modulation of reactive oxygen species (ROS), as intracellular ROS level was decreased by TDB (50 µM) treatment for 3-6 h as detected by 2',7'-dichlorofluorescein diacetate (DCFH2-DA) fluorescent probe. Moreover, Hoechst33342 staining showed that apoptosis was significantly decreased in H460 cells incubated with 100 µM of hydrogen peroxide (H2O2) for 30 min prior exposure to TDB (50 µM) compared with the cells only treated with TDB. Additionally, TDB inhibited cancer stem cell (CSC)-like characteristics in H460 cells as evidenced in spheroid assay and flow cytometry analysis, whereby CSCs, CD133-positive cells markedly decreased after TDB treatment. TDB caused a decrease in protein levels of the stemness-related transcription factors; Nanog and Sox2 while there was no alteration of Oct4. There was the reduction of related regulator proteins, β-catenin. In conclusion, TDB extracted from D. ellipsophyllum demonstrated stemness suppression and selective apoptosis induction via intracellular ROS reduction in human lung cancer cells. The results obtained from this study strengthen the potential development of TDB as an anticancer compound with a favorable safety profile.