ApoG2 Shows Anticancer And Radiosensitizing Effects In Nasopharyngeal Carcinoma Cells By Inducing Autophagy

Objective:Autophagy is one of the responses of cancer cells to various therapies, including ionizing radiation. we have known that radiation induces autophagy, but not apoptosis, in various malignant tumor cell lines. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2 anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity. apogossypolone (ApoG2) is a novel derivate of gossypol. a novel small-molecule inhibitor of antiapoptotic Bcl-2 family proteins. we hypothesized that ApoG2 has both an Bcl-2 inhibitors and radiosensitizing effect by activating autophagy.Methods:MTT assay was conducted to determine the inhibition rate of ApoG2 in CNE1, CNE2, SUNE1 and NP69 cells. IC50 value was 30.5μM and 17.3μM in CNE1 and SUNE1 cells respectively. The subjects were divided into 4 groups: control group, ApoG2 group, radiation group, and ApoG2+radiation group. The subjects were disposed with proper concentration, and then treated with X-ray after 4h. The radiosensitizing effect of ApoG2 in CNE2 cells were detected by cell colony assay. Colony assay was also used to evaluate the effect of Knock down Atg5 CNE2 cells on the survival curve of ApoG2+radiation group in both cell lines. Inhibition of the binding of Bcl-2 and Beclin 1 by ApoG2 in vitro in CNE2 cells detected by Immunoprecipitation assays. GFP-LC3 was transinfected into cells to detect autophagy measuring by confocal microscopy. The expression of related proteins LC3 and P62 were detected using Western blot analysis. We further found that ApoG2 effectively suppressed tumor growth of CNE2 xenografts in nude mice and enhanced the antitumor effect of radiation on NPC cells in vivo.Results:1. ApoG2 enhance radiosensitizing effects in CNE1, CNE2 and SUNE1 cellsCell proliferation inhibition was evaluated by MTT cell viability assay. After treated with ApoG2 for 72 h at concentration ranging from 0 to 100μM, the IC50 value was calculated as 2.84,5.64,2.18μM, it is no use for NP69 cells. All of the DEF (dose enhancement factor) value was larger than 1 in different dose duration group of ApoG2+radiation by colony formation assay. The DEF of radiation+2,1 or 0.5μM ApoG2 continuous is 1.92,1.52 and 1.13. These results indicated that ApoG2 can enhance radiosensitization in CNE2 cells in a time-dependent manner.2. Inhibition of the binding of Bcl-2 and Beclin 1 by ApoG2 in vitro in CNE 2 cellsBeclin 1 (BH3-only family), the first identified mammalian autophagy protein, can function as a tumor suppressor in mammals. The Beclin 1 protein could play a role in recruiting proteins from the cytosol for autophagic degradation or in supplying the autophagic pathway with membrane components. Immuno-precipitation assays were performed to demonstrate whether ApoG2 treatment affected the binding of Beclin 1 and Bcl-2. CNE 2 cells lysates served as a positive control.3. ApoG2 induce autophagy in CNE 2 cells after radiationTo measure autophagy, CNE2 cells were transfected with an expression construct for LC3 fused to green fluorescent protein (GFP-LC3). In control cells GFP-LC3 was evenly distributed in the entire cytoplasm. After treatment of radiation+20μM ApoG2, ring-shaped structures were detectable in the cytosol, indicating the association of GFP-LC3 with autophagosomal membranes which showed an induction of autophagy. After ApoG2+radiation treatment, LC3 was detected by immunoblotting analysis. The results indicated that the expression of LC3-II was gradually upregulated. This confirmed the results of GFP-LC3 transfection and indicated that ApoG2+ radiation could further induce autophagy cell death in CNE2 cells. CNE2 cells were treated with ApoG2,2Gy and ApoG2+2Gy in 48h.4. knock down Atg5 can enhance survival rate of CNE 2 treated with radiation+ApoG2To confirm that ApoG2 induced autophagic cell death in NPC cell, CNE2 cells were treated with shRNA Atg5. Colony formation assay indicated that shRNA Atg5 significantly reduced cell death induced by radiation and ApoG2. The result shows that the rate of cell survival at VEC group and shAtg5 group is 67%,82%.5. ApoG2's effects on tumor growth of CNE 2 xenografts in nude miceWe further found that ApoG2 effectively suppressed tumor growth of CNE2 xenografts in nude mice and enhanced the antitumor effect of radiation on NPC cells in vivo. The rate of inhibition in ApoG2,2Gy and ApoG2+2Gy groups is 46.89%,19.34% and 61.64%. Immunohistologic staining demonstrate LC3 was gradually upregulated.Conclusions:1. ApoG2 induce autophagy in NPC cells.2. ApoG2 enhance radiosensitizing effects in NPC cells.3. Inhibition of the binding of Bcl-2 and Beclin 1 and activite its downstream signalling is a potential radiosensitizing mechanism of ApoG2 in NPC cells.4. ApoG2 enhanced the antitumor effect of radiation of CNE2 xenografts in nude mice.