Rosin Derivative IDOAMP Inhibits Prostate Cancer Growth via Activating RIPK1/RIPK3/MLKL Signaling Pathway.

Rosin derivatives such as dehydroabietic acid and dehydroabietic amine belonging to diterpenoids have similar structure with androgen that inhibited the occurrence and development of prostate cancer. In this study, the effects and possible mechanism of the rosin derivative IDOAMP on prostate cancer were investigated. Our results showed that IDOAMP effectively inhibited cell viabilities of LNCaP, PC3, and DU145 prostate cells. After the treatment with IDOAMP, the levels of cleaved-PARP, LC3BII/I, and HMGB1 were increased, whereas the expression of GPX4 was decreased. Interestingly, cell viability was reversed by the supplements of necrostatin-1 and necrosulfonamide. Meanwhile, the IDOAMP downregulated the expression of human Aurora kinase A that was overexpressed in prostate cancer. In addition, co-IP results showed that IDOAMP inhibited the binding of Aurora kinase A to the receptor-interacting serine/threonine kinase 1 (RIPK1) and RIPK3. However, the binding of RIPK1 to FADD, RIPK3, or MLKL was significantly promoted. Further studies showed that the phosphorylation levels of RIPK1, RIPK, and MLKL were increased in a concentration-dependent manner. In in vivo model, IDOAMP reduced the tumor volumes and weights. In conclusion, IDOAMP directly inhibited Aurora kinase A and promoted the RIPK1/RIPK3/MLKL necrosome activation to inhibit the prostate cancer.

Moderate activation of autophagy regulates the intracellular calcium ion concentration and mitochondrial membrane potential in beta-amyloid-treated PC12 cells.

Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease. Aggregated beta-amyloid (Aß) disturbs Ca(2+) homeostasis and causes mitochondrial dysfunction and finally underlies AD. Recent evidence suggests that autophagy initiation by Beclin-1 protein might be involved in the pathogenesis of AD. However, the effects of Beclin-1 dependent autophagy on intracellular calcium ion concentration ([Ca(2+)]i) and mitochondrial membrane potential (MMP) is unclear. The effects of Beclin-1 dependent autophagy that were activated by a gradient concentration of autophagy activator rapamycin or inhibited by autophagy inhibitor 3-methyladenine (3-MA) on cell viability and cell morphology were examined. Pretreatment with rapamycin significantly up-regulated the expression of Beclin-1 in response to Aß1-42 application, but after pretreatment with 3-MA it was significantly down-regulated. Moderate activation of Beclin-1 dependent autophagy had an up regulation effect on cell viability and could maintain the original morphology of cells. Furthermore, rapamycin or 3-MA on [Ca(2+)]i and MMP in Aß1-42 treatment of PC12 cells were evaluated. We also report that PC12 cells treated with Aß1-42 showed an increase in [Ca(2+)]i but a decrease in MMP when compared to the normal control. However the application of rapamycin prior to this prevented the increase in [Ca(2+)]i and the decrease in MMP in response to Aß1-42. When 3-MA was applied this exacerbated the effect of Aß1-42 on the [Ca(2+)]i and the MMP. This shows that moderate activation of Beclin-1 dependent autophagy by rapamycin can modulate Ca(2+) homeostasis and maintain MMP in response to Aß1-42 induced cytotoxicity and so may have a preventive function in AD.

Beta-asarone attenuates amyloid beta-induced autophagy via Akt/mTOR pathway in PC12 cells.

Alzheimer's disease (AD) is an age related and progressive neurodegenerative disease. Autophagy is a self-degradative process and plays a critical role in removing long-lived proteins and damaged organelles. Recent evidence suggests that autophagy might be involved in the pathogenesis of AD. ß-asarone have various neuroprotective effects. However, the effect of ß-asarone on autophagy in amyloid ß-peptide (Aß) induced cell injury is unclear, and little is known about the signaling pathway of ß-asarone in autophagy regulation. The aim of the present study was to determine whether ß-asarone protects cells from Aß1-42 induced cytotoxicity via regulation of Beclin-1 dependent autophagy and its regulating signaling pathway. We examined effects of ß-asarone on cell morphology, cell viability, neuron specific enolase (NSE) levels, autophagosomes and regulating Beclin-1, p-Akt and p-mTOR expressions in Aß1-42 treated PC12 cells. We found that ß-asarone could maintain the original morphology of cells and increase cell viability and decrease NSE levels significantly. Meanwhile, ß-asarone decreased Beclin-1 expression significantly. In addition, ß-asarone can increase levels of p-Akt and p-mTOR. These results showed that ß-asarone protected cells from Aß1-42 induced cytotoxicity and attenuated autophagy via activation of Akt-mTOR signaling pathway, which could be involved in neuroprotection of ß-asarone against Aß toxicity. Our findings suggest that ß-asarone might be a potential preventive drug for AD.

Dynamic of neurochemical alterations in striatum, hippocampus and cortex after the 6-OHDA mesostriatal lesion.

Immediate neurochemical alterations produced by 6-OHDA could explain the general toxic pattern in the central nervous system. However, no evidences describe the effects of 6-OHDA on early changes of neurotransmitters in rats' striatum, cortex and hippocampus. In our study, unilateral 6-OHDA injection into medial forebrain bundle (MFB) was used in rats, then five neurotransmitters were analyzed at 3, 6, 12, 24, 48 and 72 h, respectively. Results showed that 6-OHDA injection caused a sharp decline of striatal dopamine (DA) levels in the first 12h followed by a further reduction between 12 and 48 h. However, striatal levels of homovanillic acid (HVA) were stable in the first 12h and showed a marked reduction between 12 and 24h. Striatal levels of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) decreased linearly for 72 h, whereas levels of norepinephrine (NE) showed a slight reduction in the first 48 h, and returned back to normal afterwards. Striatal HVA/DA ratio increased significantly in the first 12h, but 5-HIAA/5-HT ratio showed a sharp increase between 12 and 72 h. Besides, neurochemical alterations were also found in hippocampus and cortex, and the correlations of neurotransmitters were analyzed. Our study indicated that NE system had little influence in the early phase of 6-OHDA injection, moreover, early neurochemical alterations were involved with striatum, hippocampus and cortex.

Dynamic expressions of Beclin 1 and tyrosine hydroxylase in different areas of 6-hydroxydopamine-induced Parkinsonian rats.

Beclin 1, a regulator of the autophagy pathway, plays an important role in Parkinson's disease (PD). However, the crucial mechanism of Beclin 1 in PD remains unclear. Therefore, we investigated dynamic expressions of Beclin 1 and tyrosine hydroxylase (TH) in different brain areas of 6-OHDA-induced rats. Beclin 1 and TH expressions were analyzed by flow cytometry and immunohistochemistry, respectively. The results showed that Beclin 1 expressions were low in the sham group, but rose significantly after 6-OHDA injection. In the striatum and cortex, Beclin 1 increased at 3 h, peaking at 12 h, while in the hippocampus, it increased at 3 h and peaked at 24 h, then it declined slowly and remained steady at 72 h. Beclin 1 expression in the striatum and cortex areas was higher than that of the hippocampus area at 12 h. In addition, the time-course of TH expression in the striatum was similar to that in the mesencephalon. TH expression declined dramatically between 0 and 12 h. Pearson analysis showed significant negative correlations between TH and Beclin 1 expression in the areas we analyzed. While TH expression declined gradually between 12 and 72 h, significant positive correlations between TH and Beclin 1 were detected during that interval. This indicated that activation of Beclin 1-dependent autophagy may inhibit the loss of TH-positive neurons.

Establish a flow cytometric method for quantitative detection of Beclin-1 expression.

Flow cytometry is an advanced technology for efficient, rapid, specific and multi-parameter analysis of single cells in various basic research fields including cytobiology, immunology, genetic, hematology and other basic research. Beclin-1 protein is an important indicator in monitoring autophagic activity. However, quantitative flow cytometry had been rarely reported till now to be applied in the detection of Beclin-1 expression. The present study was aimed to establish a flow cytometric method for quantitative detection of Beclin-1 expression by employing the autophagy inhibitor 3-methyladenine as the control. A multi-parameter optimal method for Beclin-1 protein staining is as follows. 2 % bovine serum albumin in phosphate buffered saline was used for sample block. Concentration of primary antibody was 0.004 µg/µL. Samples were incubated at room temperature (25 °C) for 30 min. The prepared samples had better to be detected immediately or to be stored at 4 °C and detected within 6 h, otherwise the samples should be fixed in 1 % paraformaldehyde storing at 4 °C and detected within 3 d. Furthermore, we employed the immunohistochemistry to validate the method in vivo, the results confirmed flow cytometric method. The established flow cytometric analysis for Beclin-1 protein has the advantage of simpleness, speediness, sensitivity and reproducibility.

Increased expression of Beclin-1-dependent autophagy protects against beta-amyloid-induced cell injury in PC12 cells [corrected].

Alzheimer's disease (AD) is an age-related and progressive neurodegenerative disease. Beta-amyloid (Aß) plays an important role in the pathogenesis of AD. Autophagy is a self-degradative process and its related protein Beclin-1 is involved in the initiation of autophagy. However, the role of Beclin-1 in the pathogenesis of AD is rarely reported. In this study, we examined cell viability and medium levels of neuron-specific enolase (NSE) in PC12 cells incubated with gradient concentrations of Aß(1-42) (0.625, 1.25, 2.5, 5, 10 µM) for 6, 12, 24, 48, and 72 h, drew the index changes curves, and investigated the correlation between them. The result showed that cell viability was negatively correlated with NSE levels. Based on this study, Beclin-1 expression was quantitatively detected in Aß1-42-treated PC12 cells and the dynamic changes curve of Beclin-1 was drawn from 3 to 72 h. Beclin-1 expression was positively correlated with cell viability. Furthermore, both autophagy inhibitor 3-methyladenine (3-MA) and autophagy activator rapamycin were used to investigate the effect of autophagy on Aß(1-42)-induced cell injury. Aß(1-42)-induced Beclin-1 expression was further upregulated by rapamycin but was downregulated by 3-MA. Moreover, cell viability was increased by rapamycin but was decreased by 3-MA, and NSE was decreased by rapamycin but was increased by 3-MA, suggesting that activation of Beclin-1-dependent autophagy before the damage occurred can prevent neuronal cell death, while inhibition of Beclin-1-dependent autophagy can hastened cell death. These findings indicate that increasing Beclin-1-dependent autophagy may have a preventive effect before the AD occurred.

Beta-asarone attenuates ischemia-reperfusion-induced autophagy in rat brains via modulating JNK, p-JNK, Bcl-2 and Beclin 1.

Beta-asarone has significant pharmacological effects on the central nervous system. It can attenuate neuronal apoptosis, but its effects on the brain ischemia-reperfusion-induced autophagy have not been reported yet. Our study was a two-stage procedure: evaluation of ß-asarone effects on the autophagy at first, and then analysis of the possible mechanism. The middle cerebral artery occlusion (MCAO) model was adopted to make the brain injure and Beclin 1 was used to evaluate the autophagy. We hypothesized that the mechanism might be related to c-Jun N-terminal kinases (JNK), phospho-JNK (p-JNK), Bcl-2 and Beclin 1. To test this hypothesis, we evaluated JNK, p-JNK, Bcl-2 and Beclin 1 levels with flow cytometry. Additionally, we divided the brain into three regions: ischemic region, ischemic penumbra, and normal region, and analyzed them respectively. We found, compared to both groups II (model control) and III (low dose), Beclin 1 levels in groups IV (medium dose) and V (high dose) were significantly decreased. Beclin 1, JNK and p-JNK levels in groups VII (ß-asarone) and VIII (JNK inhibitor) were significantly decreased, but Bcl-2 levels were significantly increased. Additionally, Beclin 1, JNK, p-JNK and Bcl-2 levels among the three regions had no significant differences. We conclude that ß-asarone can attenuate the autophagy in a dose-dependent manner. The mechanism is likely that ß-asarone can decrease JNK and p-JNK levels at first, and then increase Bcl-2 level, finally interfere with the functions of Beclin 1 during the execution of autophagy. Additionally, ß-asarone can attenuate autophagy in a widespread manner.