| Objective: Acori Graminei Rhizoma is a kind of aromatic herb that opens orifices and revives consciousness and widely used in brain diseases. Volatile oil is one of the main active ingredient of Acori Graminei Rhizoma. Though it is frequently prescribed in formulations for brain tumors, the anti-glioma effect has not been examined. In this study, we focused on the volatile oil of Acori Graminei Rhizoma(VOA) and its anti-glioma effect and the potential mechanisms have been preliminarily investigated.Methods: First, VOA was obtained by the technique of supercritical fluid extraction(SFE),and its main components were determined by HPLC/MS. In the characterization of the role of anti malignant brain glioma: Different cell lines including human glioblastoma cells such as A172, U87,U251,U118, Mouse embryonic fibroblasts NIH/3T3 and human brain glial HEB were first determined of their IC50 by using SRB assay. And the effect of VOA on the long-term proliferation of glioma cells A172 and U251 was next detected by clone formation assay. In the mechanism terms: Annexin V/PI Flow cytometry detection combined with hoechst 33342 staining detected the apoptosis effect of VOA and LC3-GFP transfection fluorescence combined with acridine orange(AO) staining experiments also examined the autophagy effect of VOA after 48 hours treatment in both cells. Western Blot detected the apoptosis-retated cleaved caspase 3, cleaved caspase 8, cleaved caspase 9 proteins and autophagy-related LC3II/I, p62,atg 5, beclin 1 proteins. At the same time caspase apoptosis pathway and AMPK/m TOR autophagy pathway were also determined. To further determined whether p53 mediated the apoptosis and autophagy pathway in A172 cells. Inhibition of p53 expression by sip53 and p53 inhibitor PFT- α were used to observe the effects of A172 cell on autophagy and apoptosis related proteins. Finally, autophagy inhibitor 3-MA and CQ were used to inhibit autophagy, then the effect of inhibiting autophagy on cell viability was detected by SRB assay, and protein cleaved caspase 3 was detected by Western Blot and immunofluorescence methods.Results: The malignant brain tumor cells of A172, U87, U251, U118 were treated with VOA for 48 hours and the IC50 values of them were 121, 162, 223, 190 μg/ml. For 72 hours the IC50 values of them were 97, 109, 137, 207 μg/ml. For NIH/3T3 and HEB cells, no matter for 48 or 72 hours the IC50 values of them were all > 250 μg/ml. Clonogenic assay also showed that the inhibition concentration of VOA in A172 cells significantly lower than that in U251 cells. After A172 and U251 cells were treated with different concentrations of VOA for 48 hours, the inhibitory effect in both cells determined by flow cytometry showed a significantly increase of apoptosis cells compared with control group. And Hoechst 33342 staining exhibited much more cells with enhanced blue fluorescence with condensed and fragmented nuclei than those treated with vehicle alone. We further transfected GFP-LC3 into both cells and a diffuse localization of LC3 fluorescence was observed in both untreated A172 and U251 cells, whereas a punctate pattern of LC3 fluorescence was detected in both VOA-treated cells. Further acridine orange staining showed that there was obvious formation of acidic vesicular organelles(AVOs) in VOA treated both cells. And for autophagy-related LC3II/I, atg5, beclin 1 proteins. Western Blot results showed an increase expression of them while a decrease in p62 protein. Western Blot detected apoptosis-related Bax, Bcl-2, pro-caspase 3, cleaved caspase 3, cleaved caspase 8, cleaved caspase 9 proteins. And there was an increase of Bax/Bcl-2 ratio and a decrease of pro-caspase 3. For cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, they were all increased compared with control groups while this phenomenon did not happen in U251 cells. Meanwhile, we also detected autophagy-related LC3II/I, atg 5, beclin 1, p-AMPK, AMPK, p-p70S6 K, p70S6 K, p-m TOR, m TOR proteins. In the two cell lines, with the increase of the concentration of VOA, the ratio of p-AMPK/AMPK showed an increasing trend. But for p-p70S6K/p70S6 K, p-m TOR/m TOR ratio test showed a weakening trend in A172 cells. For U251 cells, the ratio of p-p70S6K/p70S6 K, p-m TOR/m TOR did not significantly reduced, while the ratio of p-ULK1/ULK1 was significantly increased. Western Blot method was used to detect the expression of p53 in A172 and U251 cells. It was found that with the increase of the concentration and time, the p53 on A172 cells showed a decreasing trend, while in U251 cells, p53 had no significant change. After using sip53 and PFT-α in A172 cells, autophagy related protein LC3II/I, AMPK were increased, while m TOR and p62 showed a decreasing trend, and the expression of apoptosis related protein cleaved caspase 3 was increased. Autophagy inhibitor 3-MA and CQ were used to inhibit autophagy in both cells, SRB assay results show that after the inhibition of autophagy in A172 and U251 cells, it was more toxic on cell proliferation. At the same time, Western blot combined with immunofluorescence detection showed after inhibition autophagy by 3-MA or CQ, there was an increase of cleaved caspase 3 expression in A172 cells and there was no obvious change of cleaved caspase 3 in U251 cells.Conclusion: In this study, VOA exhibited obvious growth inhibitory effect on human malignant glioma cells, which seems to be closely related to p53 status. Apoptotic cell death and protective autophagy were induced by VOA in both A172 and U251 cells. Moreover, the underlying mechanisms are cell type specific and dependent on p53 status. For cell apoptosis mechanism, VOA induced a caspase dependent apoptotic pathway in A172 cells and a caspase-independent pathway in U251 cells. For cell autophagy mechanism, VOA induced AMPK/m TOR pathway of autophagy in A172 cells and AMPK/ULK1 pathway of autophagy in U251 cells. At last, we found that inhibit of autophagy can promote apoptosis effect in both A172 and U251 cells. |
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