| Objective:To investigate the effect of protein kinase B inhibitor perifosine and ceramide C6 on the proliferation, apoptosis,and autophagy of glioma cells, and try to disclose intrinsic synergistic molecular mechanism of combined treatment, and to provide the theoretical basis for molecular targeted therapy for glioma.Methods:1. Detecting the effect of perifosine on different concentrations and different action times in cultured glioma cells by MTT assay and clonogenicity assay. Detecting the apoptosis under perifosine-treated by Annexin V/PI and Histone DNA EILISA method. The expression of AKT1,p-AKT,p-S6,p-AMPKα,p-ACC and ATG-5,LC-3B,Becllin-1were analyzed by Western Blot. Measure cellular ceramide levels of perifosine-treated in cultured glioma cells on different action times by high performance liquid chromatography.2. Detecting the effect of ceramide(C6) on different concentrations and different action times in cultured glioma cells by MTT assay. Detecting the effect under perifosine, ceramide(C6) and perifosine+C6 treated by MTT assay and Trypan blue staining in glioma cells. After combined treated, the expression of ERK,p-AKT,p-S6,p-S6 K,p ERK and ATG-5,LC-3B,Beclin-1 were analyzed by Western Blot.3. Detecting the effect of autophagy inhibitor such as 3-Methyaldenine and Cq and caspase inhibitor z-VAD-fmk in cultured glioma cells by MTT assay. Detecting the effect under perifosine, 3-MA and perifosine +3-MA treated in cultured glioma cells by MTT assay.Detecting the apoptosis of cells under combined treated by Annexin V/PI and Histone DNA EILISA method.4. Transfect ATG5-si RNA to HEK 293 cells to establish an autophagy defects cells. Detecting perifosine induced cytotoxicity and apoptosis in autophagy defects cells.RESULTS:1. MTT assay and clonogenicity assay showed perifosine significantly reduced the cellar viability of giloma cells in a concentration-and time-dependent maner. The expression of p-AKT was sharply suppressed by perifosine treatment while the expression of ATG-5、LC-3B、Beclin-1 were up-regulated by Western Blot. Histone DNA ELISA and Annexin V/PI showed perifosine-induced apoptosis but it is modest. Perifosine can increase cellular ceramide levels limitedly.2. MTT assay and Trypan blue staining indicated that cytotoxicity of perifosine+C6 is much more than single agent,and apoptosis enhanced significantly by Histone DNA ELISA and Annexin V/PI method.The level of p-AKT is no change while the expressiong of ATG-5、LC-3B、Beclin-1 was significantly reduced in response to combined treatment.3. MTT assay showed autopathy inhibitor 3MA and Cq reduced the cellar viability of giloma cells, but it can be blocked by z-VAD-fmk. Autopathy inhibitor 3-MA enhanced perifosine-induced cytotoxicity in glioma cells. Compared with single agent,apoptosis enhanced in perifosine+C6 group by Histone DNA ELISA and Annexin V/PI method. The synergistic cytotoxicity can be blocked by z-VAD-fmk.4. Establishing autophagy defects cells successfully, perifosine significantly reduce autophagy defects cells viability. Annexin V/PI showed that apoptosis enhanced under perifosine treatment. It makes no difference between perifosine and perifosine+C6 on the suppression of autophagy-deficient(ATG-5 RNAi) HEK293 cellsConclusions:1. Perifosine is expected to be one of the molecular targeted candidate for glioma treatment and has potential clinical application value.2. Ceramide(C6) can enhance perifosine-induced cytotoxicity,suggesting the synergistic effect.3. The mechanism of synergistic effect is that Perifosine can induce significantly autophaty, which inhibit apoptosis. Ceramide(C6) restores perifosine-induced apoptosis and induce autophaty inhibition facilitates perifosine-induced cytotoxicity in glioma cells without affecting AKT-m TOR signaling... |
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