Mechanism Of Autophagic Cell Death In B16Cells Induced By TNF-α

ObjectivePrevious studies found that tumor necrosis factor(TNF-α) could induce mouse B16melanoma cells apoptosis, whether it could induce autophagy of B16cells is not clear. This study aim to elucidate this question to analyze whether TNF-α can induce B16cells autophagy, the cell viability and the quantity of the autophagosome were observed under microscope, to analyse the key factors of autophagy in B16cells induced by TNF-α. The expression level of autophagy related genes and proteins were detected by western blotting. The signaling pathway of autophagy of B16cells induced by TNF-α was analyzed by detecting the marker genes and proteins of classic autophagic signal path.Methods1. The mouse melanoma B16cells were cultured in vitro, they are divided into three groups according to different treating methods:the control group, TNF-α treated group (12hours and24hours after treated with20ng/ml TNF-α) and3-methyladenine(3-MA)+TNF-α treated group(pretreat2hours at10mmol/L3-MA and then12hours and24hours after treated with20ng/ml TNF-α).2. The cell viability was obversed under phase contrast microscopy before and after TNF-α treatment.3. The quantity of the autophagosome in B16cells were observed under transmission electron microscope (TEM) before and after TNF-α treatment.4. The total RNA of B16cells were extracted. And the mRNA expression level of autophagy related gene Atg5, Atg7, Atg12and the key gene of endoplasmic reticulum stress Chop were analysed by real-time polymerase chain reaction (PCR).5. Collecting and lysing cells, extracting its total protein. The autophagy mark protein LC3-Ⅱ, autophagic cell death mark protein Beclinl were detected by western blot; the phosphorylation level of key protein p70S6K and Akt of mTOR and P13K pathway were analysed by western blotting.Results1. Compare with the control group, the growth of B16cells was inhibitted. We can see that the ratios of died cells were about10%at12hours and30%at24hours after TNF-a treatment.2. There are many autophagosome of different stages in B16cells after TNF-α treatment.3. Compared with the control, the expression levels of autophagy mark protein LC3-Ⅱ and autophagic cell death mark Beclin1are significantly increased. In3-MA+TNF-α treated group, the expression level of LC3-Ⅱ and Beclinl was lower than TNF-α treated group.4. The mRNA expression level of autophagy related gene Atg5, Atg7, Atg12increased in TNF-α treated group. The mRNA expression level of the key gene Chop of endoplasmic reticulum stress significantly up regulated in time dependent manner.5. Compared with the control, phosphorylation level of p70S6K was down regulated in TNF-α treated group, phosphorylation level of Akt significantly also down regulated. In3-MA+TNF-α treated group, phosphorylation level of p70S6K was higher than TNF-α treated group at12hours and lower than TNF-α treated group at24hours; phosphorylation level of Akt is the same as TNF-α treated group.Conclusion1. TNF-α can induce the autophagy of B16cells.2. Wene the amount of autophagsome of B16cells induced by TNF-α reach to a certain level, the degradation of them increase gradually, leading to the decline of of the amount autophagosome.3. TNF-α can induce the autophagic cell death of B16cells.4. TNF-α could induce the autophagy and autophagic cell death of B16cells through inhibit the mTOR and Akt pathway.5. TNF-α can induce the endoplasmic reticulum stress of B16cells. But whether endoplasmic reticulum stress could trigger autophagy induced by TNF-α need to be elucidated.