A high-throughput Gaussia luciferase reporter assay for screening potential gasdermin E activators against pancreatic cancer

It is discovered that activated caspase-3 tends to induce apoptosis in gasdermin E (GSDME)-deficient cells, but pyroptosis in GSDME-sufficient cells. The high GSDME expression and apoptosis resistance of pancreatic ductal adenocarcinoma (PDAC) cells shed light on another attractive strategy for PDAC treatment by promoting pyroptosis. Here we report a hGLuc-hGSDME-PCA system for high-throughput screening of potential GSDME activators against PDAC. This screening system neatly quantifies the oligomerization of GSDME-N to characterize whether pyroptosis occurs under the stimulation of chemotherapy drugs. Based on this system, ponatinib and perifosine are screened out from the FDA-approved anti-cancer drug library containing 106 compounds. Concretely, they exhibit the most potent luminescent activity and cause drastic pyroptosis in PDAC cells. Further, we demonstrate that perifosine suppresses pancreatic cancer by promoting pyroptosis via caspase-3/GSDME pathway both in vitro and in vivo. Collectively, this study reveals the great significance of hGLuc-hGSDME-PCA in identifying compounds triggering GSDME-dependent pyroptosis and developing promising therapeutic agents for PDAC.

Akt Inhibitor Perifosine Prevents Epileptogenesis in a Rat Model of Temporal Lobe Epilepsy / 神经科学通报·英文版

Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by various factors. We previously reported that pretreatment with perifosine, an inhibitor of Akt (also called protein kinase B), abolishes the rapamycin-induced paradoxical increase of S6 phosphorylation in a rat model induced by kainic acid (KA). Since Akt is an upstream target in the mTOR signaling pathway, we set out to determine whether perifosine has a preventive effect on epileptogenesis. Here, we explored the effect of perifosine on the model of temporal epilepsy induced by KA in rats and found that pretreatment with perifosine had no effect on the severity or duration of the KA-induced status epilepticus. However, perifosine almost completely inhibited the activation of p-Akt and p-S6 both acutely and chronically following the KA-induced status epilepticus. Perifosine pretreatment suppressed the KA-induced neuronal death and mossy fiber sprouting. The frequency of spontaneous seizures was markedly decreased in rats pretreated with perifosine. Accordingly, rats pretreated with perifosine showed mild impairment in cognitive functions. Collectively, this study provides novel evidence in a KA seizure model that perifosine may be a potential drug for use in anti-epileptogenic therapy.

Perifosine regulates human brain glioma U251 cell proliferation, apopto-sis and autophagy through suppression of PI3 K/Akt pathway / 中国病理生理杂志

AIM:To investigate the effect of perifosine, an inhibitor of protein kinase B ( PKB/Akt) , on the cell cycle, apoptosis and autophagy in human brain glioma U251 cells, and to determine the relationship between perifos-ine-induced autophagy and apoptosis of glioma.METHODS:The cell growth inhibition was determined by MTT assay. The cell cycle distribution of U251 cells was examined by flow cytometry.The cell apoptosis was analyzed by Annexin V-FITC apoptosis detection kit.The protein expression of P21, P27, cyclin B1, caspase-9 and PARP was examined by Wes-tern blot analysis.The distribution and expression of LC3-Ⅱ, an autophagy marker, was observed to determine the effect of perifosine-induced autophagy.RESULTS:Perifosine inhibited the cell viability in a dose-dependent manner.In perifos-ine-treated U251 cells, the cell cycle was arrested in G2 phase and the expression of cyclin B1 was inhibited.Perifosine in-duced apoptosis of U251 cells through activation of caspase-9 cleavage, PARP cleavage and survivin inhibition.In addi-tion, suppression of autophagy by chloroquin, an inhibitor of autophagy, increased the number of apoptotic cells.CON-CLUSION:Perifosine inhibits cell proliferation and triggers apoptosis and autophagy in human U251 cells.Blocking auto-phagy magnifies perifosine-induced glioma cell apoptosis.

Akt phosphorylation and mechanism of TRAIL-resistance in A549 cells / 中国免疫学杂志

Objective:To investigate mechanism of TRAIL-resistance in A549 cells ( a cell line of non-small cell lung carcino-ma cells) due to Akt phosphorylation .Methods:A549 cells were treated with Akt inhibitor Perifosine and rsTRAIL protein individual-ly and in combination.The expressions of Akt phosphorylation(p-Akt),c-FLIPLL and caspase-8 were detected by Western blot.The apoptotic rate of the A549 cells treated was detected by flow cytometry and the cell proliferation was evaluated by MTT assay .Results:A549 cells showed the increased level of Akt phosphorylation mediated by rsTRAIL protein .Treatment with the Akt inhibitor Perifosine induced a suppression of Akt activation in A 549 cells and a concomitant decrease in the expression of c-FLIPLL .As a result, Perifosine significantly enhanced TRAIL-induced apoptosis rate of (76.5 ±3.02)%and cytotoxic rate of (83.2 ±2.54)%by promoting the activ-ity of caspase-8.Conclusion:Akt activity promotes A549cells survival against TRAIL-induced apoptosis and that the cytotoxic effect of rsTRAIL protein can be enhanced by modulating the Akt phosphorylation in human non -small cell lung carcinoma cells .