Aim: To investigate the effect of 7-(4-methoxyphenyl)-5, 8a-diphenyl-1, 2, 3, 7, 8, 8a-hexahydroimidazo[1, 2-a]pyridine (TIP-6) on cell proliferation in human hepatoma cell line HepG2 and human normal hepatocyte cell line L02.
Methods: Typan blue assay was used to check the effect of TIP-6 on cell proliferation. The changes of cell morphology were observed by the phase contrast microscope. Flow cytometry (FCM) was used to check cell cycle. Autophagy and autophagic cell death were detected after acridine orange (AO) staining under fluorescent microscopy. Apoptosis was analyzed by Annexin V/7-AAD, DAPI staining and DNA ladder. NF-kappaB expression was detected with cellular immunochemistry.
Results: Cell proliferation inhibiting effect was appeared when treated with TIP-6 from 60 mumol/L to 200 mumol/L, which was correlated with treated concentrations and time. The proliferation rates were just 12.10% and 18.75% (vs control) under 200 mumol/L 72 h in HepG2 and L02 respectively. Vacuolization were found more and more frequently with the increasing of TIP-6 concentrations and treated time prolonged. FCM results indicated that cells were blocked in G2/M phase, and more sensitive were found in HepG2 than L02. AO staining results indicated that the phenomenon of autophagy and autophagic cell death were occurred and appeared more potent with more TIP-6 and longer time treated. No apoptosis markers were found with Annexin V/7-AAD and DAPI staining, and no DNA ladders were found either, these indicated that TIP-6 didn't induce apoptosis in these cells. NF-kappaB was found increased after treated with TIP-6, and the autophagic vacuole became more and more with the increasing of NF-kappaB protein, but the proliferation rates decreased at the same time.
Conclusion: TIP-6 inhibited cell proliferation and induced autophagy and autophagic cell death in HepG2 and L02 cells. NF-kappaB activation may be involved in these effects.