Compound Berberine Induced Autophagyvia P13K/AKT/mTOR Signaling Pathway in CNE2 NPC Cells

Objective: To investigate the autophagy-inducing effect of Compound Berberine (CBBR) on CNE2 nasopharyngeal carcinoma (NPC) cells and its possible intervening targets in P13K/AKT/mTOR signaling pathway via an experimental study.

Methods: CNE2 cells at exponential growth phase were taken as the target cells in this study. IC50 concentration for CBBR was determined by MTT assay at firstly. Then, 3 different concentrations of CBBR, i.e. 0.25 mg.mL-1, 0.50 mg.mL-1 and 1.00 mg.mL-1 around the concentration of IC50, were taken for following intervention experiments respectively. Fluorescein labeling method was utilized to assay the inducing effect of CBBR on the autophagy of CNE2 cells, followed by Western blot procedure to explore the changes of key messenger molecules in the autophagy-related signaling pathway of P13K/AKT/mTOR, both combined with 3-MA block test in a comparative way and carried out through detecting the expressive levels of Beclin 1, LC3-? and LC3-? as well as the ratio of LC3-?:LC3-?.

Results: IC50 of CBBR was determined at the level of0.5mg.mL-1. The inducing effect of CBBR on autophagy of CNE2 cells was shown occurred in various modes, not only a simple concentration-dependent tendency, with its effect minimal at the concentration of 0.25mg.mL-1 and maximal at the concentration of 0.50mg.mL-1, while only a little remarkable higher at the concentration of 1.00mg.mL-1 than that of 0.5mg.mL-1. Although its inducing effect was weakened a little following the pretreatment by 3-MA, the effect combined with CBBR was still significantly higher than that of simply blocked by 3-MA. Moreover, changes in the expressive levels of Beclin1, LC3-? and LC3-? as well as LC3-?:LC3-? all showed a tendency corresponding to the changed autophagic features of CNE2 cells (P<0.05 or P<0.01), given more supporting evidences for the inducing effect of CBBR on autophagy of CNE2 cells.

Conclusions: CBBR can bring about inhibiting effect on the proliferating activity of CNE2 cells through inducing increased autophagic activity via intervening targets in P13K/AKT/mTOR signaling pathway, and this effect could not be completely blocked by the antagonist 3-MA. It might be possible that activated autophagic progress by some factors, such as miR-125b via activation of CXCL 12/CXCR4 axis, could function epigenetically as a mediator to cause epithelial-mesenchymal transition (EMT), promote tumor cell invasion, and induce chemical drugs resistance by contraries and CBBR could be utilized to inhibit the proliferating activity of NPC cells via intervening such a linking target as well.