Inhibition of autophagy enhances apoptosis induced by bortezomib in AML cells

Bortezomib is a novel proteasome inhibitor, which has been successfully used to treat mantle cell lymphoma and multiple myeloma. However, the direct effects of bortezomib on acute promyelocytic leukaemia (APL) have not been fully investigated. In the present study, the WST-8 assay, western blotting, flow cytometry, monodansylcadaverine staining and transmission electron microscopy were performed. It was demonstrated that bortezomib treatment induced a time- and dose-dependent decrease in the viability of NB4 cells. Bortezomib treatment induced cell apoptosis in NB4 cells, as assessed by Annexin V/propidium iodide analysis, and the detection of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase, Bax and Bcl-2 expression. Furthermore, bortezomib treatment induced autophagy in NB4 cells, as indicated by autophagosome formation, p62 degradation, LC3-I to LC3-II conversion and formation of acidic autophagic vacuoles. Notably, autophagy induced by bortezomib was initiated prior to apoptosis. Inhibition of autophagy by knocking down Beclin-1 expression increased bortezomib-induced apoptosis in NB4 cells. Therefore, the present study revealed that the combination of bortezomib and autophagy inhibition may be a potential treatment strategy for APL.     

Apelin-13 inhibits apoptosis and excessive autophagy in cerebral ischemia/reperfusion injury

Apelin-13 is a novel endogenous ligand for an angiotensin-like orphan G-protein coupled receptor, and it may be neuroprotective against cerebral ischemia injury. However, the precise mechanisms of the effects of apelin-13 remain to be elucidated. To investigate the effects of apelin-13 on apoptosis and autophagy in models of cerebral ischemia/reperfusion injury, a rat model was established by middle cerebral artery occlusion. Apelin-13(50 μg/kg) was injected into the right ventricle as a treatment. In addition, an SH-SY5 Y cell model was established by oxygen-glucose deprivation/reperfusion, with cells first cultured in sugar-free medium with 95% N2 and 5% CO2 for 4 hours and then cultured in a normal environment with sugar-containing medium for 5 hours. This SH-SY5 Y cell model was treated with 10–7 M apelin-13 for 5 hours. Results showed that apelin-13 protected against cerebral ischemia/reperfusion injury. Apelin-13 treatment alleviated neuronal apoptosis by increasing the ratio of Bcl-2/Bax and significantly decreasing cleaved caspase-3 expression. In addition, apelin-13 significantly inhibited excessive autophagy by regulating the expression of LC3 B, p62, and Beclin1. Furthermore, the expression of Bcl-2 and the phosphatidylinositol-3-kinase(PI3 K)/Akt/mammalian target of rapamycin(mTOR) pathway was markedly increased. Both LY294002(20 μM) and rapamycin(500 nM), which are inhibitors of the PI3 K/Akt/mTOR pathway, significantly attenuated the inhibition of autophagy and apoptosis caused by apelin-13. In conclusion, the findings of the present study suggest that Bcl-2 upregulation and mTOR signaling pathway activation lead to the inhibition of apoptosis and excessive autophagy. These effects are involved in apelin-13-induced neuroprotection against cerebral ischemia/reperfusion injury, both in vivo and in vitro. The study was approved by the Animal Ethical and Welfare Committee of Jining Medical University, China(approval No. 2018-JS-001) in February 2018.     

Rapalogs induce non-apoptotic,autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Since mutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV-negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.     

Protective mechanism of Wnt4 gene on Parkinson’s disease (PD) transgenic Drosophila

Objective: The main pathological change of Parkinson’s disease (PD) is progressive degeneration and necrosis of dopaminergic neurons in the midbrain, forming a Lewy body in many of the remaining neurons. Studies have found that in transgenic Drosophila, mutations in the PTEN-inducible kinase 1 (PINK1) gene may cause indirect flight muscle defects in Drosophila, and mitochondrial structural dysfunction as well.

Methods: In this study, Wnt4 gene overexpression and knockdown were performed in PINK1 mutant PD transgenic Drosophila, and the protective effect of Wnt4 gene on PD transgenic Drosophila and its possible mechanism were explored. The Wnt4 gene was screened in the previous experiment; And by using the PD transgenic Drosophila model of the MHC-Gal4/UAS system, the PINK1 gene could be specifically activated in the Drosophila muscle tissue.

Results: In PINK1 mutation transgenic fruit flies, the Wnt4 gene to study its implication on PD transgenic fruit flies’ wing normality and flight ability. We found that overexpression of Wnt4 gene significantly reduced abnormality rate of PD transgenic Drosophila and improved its flight ability, and then, increased ATP concentration, enhanced mitochondrial membrane potential and normalized mitochondrial morphology were found. All of these findings suggested Wnt4 gene may have a protective effect on PD transgenic fruit flies. Furthermore, in Wnt4 gene overexpression PD transgenic Drosophila, down-regulation autophagy and apoptosis-related proteins Ref(2)P, Pro-Caspase3, and up-regulation of Beclin1, Atg8a, Bcl2 protein were confirmed by Western Blotting.

Conclusion: The results imply that the restoring of mitochondrial function though Wnt4 gene overexpression in the PINK1 mutant transgenic Drosophila may be related to autophagy and/or apoptosis.     

Survivin-shRNA对视网膜母细胞瘤HXO-RB44细胞自噬及凋亡的影响

目的:研究Survivin-shRNA对视网膜母细胞瘤HXO-RB44细胞自噬及凋亡的影响。方法:培养视网膜母细胞瘤HXO-RB44细胞，构建Survivin-shRNA载体。按照处理不同分为Survivin-shRNA组、GFP组和CON组。分别采用流式细胞术检测Survivin-shRNA对HXO-RB44细胞凋亡和细胞周期的影响，应用MTT法检测Survivin-shRNA对HXO-RB44细胞活性的影响，Western blot法检测Survivin-shRNA对HXO-RB44 细胞自噬相关蛋白LC3、p62 与mTOR 表达的影响。结果:流式细胞术结果表明，与Control组和GFP组相比，Survivin-shRNA组细胞凋亡率增加，差异有统计学意义（P＜0.05）；随着作用时间的延长，S期出现阻滞，48 h阻滞最强，显著高于对照组（P＜0.05）。MTT结果发现Survivin-shRNA可抑制HXO-RB44细胞增殖活性，与Control组和GFP组相比，差异有统计学意义（P＜0.05）；Western blot结果发现，与对照组相比，LC3表达量显著增加（P＜0.01）；而mTOR表达量降低（P＜0.01）。结论:Survivin-shRNA可促进视网膜母细胞瘤HXO-RB44细胞的凋亡和自噬水平，进而特异性杀伤肿瘤细胞。     

基于“通肾络、益脾肾”治法研究足细胞凋亡及自噬

[目的]基于中医"通肾络、益脾肾"治法,探讨通络益肾方对体外高糖培养的小鼠肾小球足细胞自噬和凋亡蛋白SIRT1、BNIP3、P62、Bax表达的调控影响。[方法]成熟无特定病原体(SPF)级SD雄性大鼠40只,随机分为正常组、中药高、中、低剂量组各10只。按照人与大鼠体表面积折算方法计算出大鼠所需中药灌胃浓度:高剂量浓度4.76 g/mL、中剂量浓度2.38 g/mL、低剂量浓度1.19 g/m L,灌胃10 d取含药血清备用。足细胞分6组,正常组5.6 mmol/L葡萄糖+10%正常大鼠血清、高糖组30mmol/L葡萄糖+10%正常大鼠血清、通络益肾方含药血清高、中、低干预组30 mmol/L葡萄糖+10%高、中、低剂量大鼠血清、高渗组甘露醇24.5 mmol/L+10%正常大鼠血清。细胞培养48 h后收集,Hoechst33342荧光染色,观察各组足细胞凋亡状况及形态变化;流式细胞仪检测足细胞凋亡率;Western Blot检测细胞内自噬标志蛋白SIRT1、P62及促凋亡蛋白BNIP3、Bax表达水平。[结果]流式细胞仪检测结果显示通络益肾方中、低剂量组可降低高糖诱导的足细胞凋亡率(P0.01或P0.05),高剂量组不能改善凋亡情况(P0.05);Hoechst33342荧光染色观察结果也证实通络益肾方中、低剂量组可降低高糖诱导的足细胞凋亡率;蛋白印迹结果显示,与高糖组相比,通络益肾方中、低剂量组自噬标志蛋白SIRT1表达升高,自噬标志蛋白P62及促凋亡蛋白BNIP3、Bax蛋白表达下降(P0.05或P0.01),高剂量组SIRT1、BNIP3、P62、Bax蛋白表达未见明显改变(P0.05)。[结论]中剂量、低剂量通络益肾方能够启动细胞自噬减少高糖刺激下体外培养足细胞凋亡,降低细胞凋亡率及凋亡蛋白的表达。     

Novel cystamine-core dendrimer-formulation rescues ΔF508-CFTR and inhibits Pseudomonas aeruginosa infection by augmenting autophagy

Background: Cystic fibrosis (CF) is challenged with pathophysiological barriers for effective airway drug-delivery. Hence, we standardized the therapeutic efficacy of the novel dendrimer-based autophagy-inducing anti-oxidant drug, cysteamine.

Research design and methods: Human primary-CF epithelial-cells, CFBE41o-cells were used to standardize the efficacy of the dendrimer-cystamine in correcting impaired-autophagy, rescuing ΔF508-CFTR and Pseudomonas-aeruginosa (Pa) infection.

Results: We first designed a novel cystamine-core dendrimer formulation (G4-CYS) that significantly increases membrane-ΔF508CFTR expression in CFBE41o-cells (p < 0.05) by forming its reduced-form cysteamine, in vivo. Additionally, G4-CYS treatment corrects ΔF508-CFTR-mediated impaired-autophagy as observed by a significant decrease (p < 0.05) in Ub-LC3-positive aggresome-bodies. Next, we verified that in non-permeabilized CFBE41o-cells, G4-CYS significantly (p < 0.05) induces ΔF508-CFTR’s forward-trafficking to the plasma membrane. Furthermore, cysteamine’s known antibacterial and anti-biofilm properties against Pa were enhanced as our findings demonstrate that both G4-CYS and its control DAB-core dendrimer, G4-DAB, exhibited significant (p < 0.05) bactericidal-activity against Pa. We also found that both G4-CYS and G4-DAB exhibit marked mucolytic-activity against porcine-mucus (p < 0.05). Finally, we demonstrate that G4-CYS not only corrects the autophagy-impairment by rescuing ΔF508-CFTR in CFBE41o-cells but also corrects the intrinsic phagocytosis defect (p < 0.05).

Conclusions: Overall, our data demonstrates the efficacy of novel cystamine-dendrimer formulation in rescuing ΔF508-CFTR to the plasma membrane and inhibiting Pa bacterial-infection by augmenting autophagy.     

Adaptive response of resistant cancer cells to chemotherapy

Despite advances in cancer therapeutics and the integration of personalized medicine, the development of chemoresistance in many patients remains a significant contributing factor to cancer mortality. Upon treatment with chemotherapeutics, the disruption of homeostasis in cancer cells triggers the adaptive response which has emerged as a key resistance mechanism. In this review, we summarize the mechanistic studies investigating the three major components of the adaptive response, autophagy, endoplasmic reticulum (ER) stress signaling, and senescence, in response to cancer chemotherapy. We will discuss the development of potential cancer therapeutic strategies in the context of these adaptive resistance mechanisms, with the goal of stimulating research that may facilitate the development of effective cancer therapy.     

Cytoprotective autophagy induction by withaferin A in prostate cancer cells involves GABARAPL1

Withaferin A (WA) is a naturally occurring steroidal lactone with proven cancer chemopreventive activity in preclinical models of different cancers including prostate adenocarcinoma. Previously we compared the RNA-seq data from control and WA-treated 22Rv1 human prostate cancer cells to identify mechanistic targets of this phytochemical. The Gene Ontology pathway analysis of the RNA-seq data revealed significant upregulation of genes associated with autophagy upon WA treatment in 22Rv1 cells. In this study, we extended these findings to investigate the mechanism underlying WA-induced autophagy. Initially, we confirmed autophagy induction by WA treatment by transmission electron microscopy using three prostate cancer cell lines (LNCaP, 22Rv1, and PC-3). Fourteen common genes altered by 8- and 16-hour exposure to WA were identified from human autophagy PCR array and these results were consistent with the RNA-seq data. Two key autophagy markers (LC3BII and SQSTM1) were robustly increased in WA-exposed LNCaP, 22Rv1, and PC-3 cells as determined by immunoblotting, and this effect was elevated in the presence of autophagy inhibitor bafilomycin A1 (BafA1). BafA1 treatment augmented WA's cytotoxicity and subsequently its proapoptotic potential. WA treatment induced GABARAPL1 (ATG8L) protein expression in all three cell lines and its knockdown by RNA interference attenuated WA-mediated apoptosis. WA-induced autophagy was not affected in the presence of an antioxidant (EUK134). Taken together, the present study reveals that WA-mediated autophagy is cytoprotective and mediated by GABARAPL1.