Ginkgo biloba exocarp extracts induces autophagy in Lewis lung cancer cells involving AMPK / mTOR / p70S6k signaling pathway

Abstract

ObjectivesGinkgo biloba L. is called a living fossil plant, and could be used for the treatment of cancer thousands of years ago in China. The extracts prepared from the Ginkgo biloba exocarp (Ginkgo biloba exocarp extracts, GBEE) has a significant anti-cancer effect. Autophagy plays an important role in the occurrence and development of cancer as programmed cell death (PCD) type II. Thus it would be interesting to study the effects and mechanisms of GBEE inducing autophagy in Lewis lung cancer (LLC) cells. MethodsMTT method was used to detect the inhibitory effect of GBEE on LLC cells. Monodansylcadaverine (MDC) staining method was applied to observe the formation of acidic vacuoles in cells. The ultrastructure of LLC cells was observed using transmission electron microscope (TEM) to confirm the formation of autophagosomes. Quantify reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA levels of Beclin1 and Atg5. Western Blot was used to detect the protein levels of Beclin1, Atg5, LC3I/II, p-AMPK, AMPK, p-mTOR, mTOR, p-p70S6k and p70S6K in LLC cells. ResultsGBEE (5–160μg/mL) inhibited the proliferation of LLC cells in vitro with the half maximal inhibitory concentration (IC50) value of 161.26μg/mL. The formation and activation of acidic vacuoleswere increased by the action of GBEE (10, 20 and 40μg/mL) on LLC cells. The autophagosomes were also increased. Meanwhile, it up-regulated both the mRNA and protein levels of Beclin1 and Atg5. The ratio of LC3-I/LC3-II protein was down-regulated. In addition, the protein level of p-AMPK was increased, and the p-mTOR and p-p70S6K was decreased. But the AMPK, mTOR and p70S6K proteins were not significantly changed. ConclusionsThe inhibitory effect of GBEE on LLC is associate with inducing autophagy in LLC cells, which may be closely relevant to the regulation of AMPK/mTOR/p70S6k signaling pathways.