β‐asarone and levodopa co‐administration protects against 6‐hydroxydopamine‐induced damage in parkinsonian rat mesencephalon by regulating autophagy: down‐expression Beclin‐1 and light chain 3B and up‐expression P62

In this study, we investigated Beclin‐1, light chain (LC)3B, and p62 expression in 6‐hydroxydopamine (6‐OHDA)‐induced parkinsonian rats after β‐asarone and levodopa (l ‐dopa) co‐administration. Unilateral 6‐OHDA injection into the medial forebrain bundle was used to create the models, except in sham‐operated rats. Rats were divided into eight groups: sham‐operated group; 6‐OHDA model group; madopar group (75 mg/kg, per os (p.o.)); l ‐dopa group (60 mg/kg, p.o.); β‐asarone group (15 mg/kg, p.o.); β‐asarone + l ‐dopa co‐administered group (15 mg/kg + 60 mg/kg, p.o.); 3‐methyladenine group (500 nmol, intraperitoneal injection); and rapamycin group (1 mg/kg, intraperitoneal injection). Then, Beclin‐1, LC3B, and p62 expression in the mesencephalon were detected. The mesencephalon was also observed by transmission electron microscope. The results showed that Beclin‐1 and LC3B expression decreased and that p62 expression increased significantly in the madopar, l ‐dopa, β‐asarone, and co‐administered groups when compared with the 6‐OHDA model. Beclin‐1 and LC3B expression in the β‐asarone and co‐administered groups were less than in the madopar or l ‐dopa groups, whereas p62 expression in the β‐asarone and co‐administered groups was higher than in the madopar or l ‐dopa groups. In addition, a significant decrease in autophagosome was exhibited in the β‐asarone and co‐administered groups when compared with the 6‐OHDA group. Our findings indicate that Beclin‐1 and LC3B expression decreased, whereas p62 expression increased after co‐administration treatment. In sum, all data suggest that the co‐administration of β‐asarone and l ‐dopa may contribute to the treatment of 6‐OHDA‐induced damage in rats by inhibiting autophagy activity.     

MLK3 silence induces cervical cancer cell apoptosis via the Notch‐1/autophagy network

Mixed‐lineage kinase 3 (MLK3), the mitogen‐activated protein kinase kinase kinase (MAP3K), has been recognized as a player in tumorigenesis and oncogenic signalling, yet its detailed functions and signalling in cervical cancer have not been fully elucidated. Here, we identify that cervical cancer cells display higher mRNA and protein levels of MLK3 than normal cervical epithelial squamous cells. In HeLa and SiHa cell, MLK3 knockdown using siRNA remarkably suppressed cell survival and promoted cell apoptosis, with increased expression of the apoptosis‐related protein Bax and reduced Bcl‐2. Moreover, MLK3 knockdown promoted cell autophagy, demonstrated by increased ratio of autophagy‐related proteins LC3II/LC3I and decreased p62 expression in MLK3 depletion cells. Furthermore, MLK3 knockdown remarkably abolished Notch‐1 expression in cervical cancer cells. By co‐treating Hela cells with MLK3 specific siRNA and pcDNA3.1‐Notch‐1 overexpression plasmid or autophagy inhibitor 3‐MA, we found that MLK3 played its role in cervical cancer cells via the Notch‐1/autophagy network. Our results demonstrate the importance of MLK3 in cervical cancer progression via modulating the Notch‐1/autophagy network, and suggest that MLK3 is a promising therapeutic target for cervical cancer.     

Aflatoxin B1 affects apoptosis and expression of Bax,Bcl‐2, and Caspase‐3 in thymus and bursa of fabricius in broiler chickens

Aflatoxin B₁ is known as a mycotoxin that develops various health problems of animals, the effects of AFB₁ on thymus and bursa of Fabricius in chickens are not clear. The objective of this study was to investigate the apoptosis of thymus and bursa of Fabricius in broilers fed with AFB₁. Two hundred Avian broilers were randomly divided into four groups of 50 each, namely control group and three AFB₁ groups fed with 0.15 mg, 0.3 mg, and 0.6 mg AFB₁/kg diet, respectively. In this study, flow cytometer and immunohistochemical approaches were used to determine the percentage of apoptotic cells and the expression of Bax, Bcl‐2, and Caspase‐3. The results showed that consumption of AFB₁ diets results in increased percentage of apoptotic cells and increased expression of Caspase‐3 in both thymus and bursa of Fabricius. The expression of Bax was increased and the expression of Bcl‐2 was decreased in the thymus, but no significant changes in Bax and Bcl‐2 expression were observed in the bursa of Fabricius when broilers fed with AFB₁. These findings suggest that adverse effects of AFB₁ on thymus and bursa of Fabricius in broilers were confirmed by increased apoptotic cells and abnormal expression of Caspase‐3. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1113–1120, 2016.     

PIK3R3 regulates ZO-1 expression through the NF-kB pathway in inflammatory bowel disease

Background and aimsInflammatory bowel disease (IBD) are the major risk factor for developing colitis associated cancer (CAC). Previously, we have reported that Phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) was overexpressed in colorectal cancer (CRC), but we don't know the role of PIK3R3 in IBD.MethodsWe investigated the differential expression of PIK3R3 and ZO-1 in IBD patients by using Immunohistochemical (IHC) and Gene Expression Omnibus (GEO) database analysis. Caco-2 cells were exposed to different conditions to assess protein level changes of PIK3R3 and ZO-1. Caco-2 cell monolayers were transfected with PIK3R3/siPIK3R3 to assess transepithelial electrical resistance. Tight junction protein integrity was assessed by immunoblot and immunofluorescence. For further, intestinal permeability and tight junction protein integrity were assessed in animal study to assess the treatment role of PIK3R3 specific inhibitor TAT-N 15 (N15).ResultsPIK3R3 was increased in IBD patients, and negatively controlled the expression of ZO-1. In vitro, PIK3R3 regulates ZO-1 by activating NF-kB pathway. Overexpression of PIK3R3 in Caco-2 cells decreased transepithelial electrical resistance (TEER), an opposite result was observed in siPIK3R3 cells. In animal study, inhibition of PIK3R3 by N15 contributed to amelioration of DSS-induced intestinal permeability. Mice treated with N15 exhibited less disruption of TJs in colon tissues.ConclusionsPIK3R3 was increased in clinical IBD patients with accompanying disruption of ZO-1 expression. Inhibition of PIK3R3 attenuated DSS-induced IBD symptoms in a mouse model. These findings indicated that PIK3R3 could be a therapeutic target for IBD.     

Di‐2‐ethylhexyl phthalate (DEHP) induces apoptosis and autophagy of mouse GC‐1 spg cells

As a widely used plasticizer in industry, di‐2‐ethylhexylphthalate (DEHP) can cause testicular toxicity, yet little is known about the potential mechanism. In this study, DEHP exposure dramatically inhibited cellviability and induced apoptosis of mouse GC‐1 spg cells. Furthermore, DEHP significantly increased the levels of autophagy proteins LC3‐II, Beclin1 and Atg5, as well as the ratio ofLC3‐II/LC3‐I. Transmission electron microscopy (TEM) further confirmed that DEHP induced autophagy of mouse GC‐1 spg cells. DEHP was also shown to induceoxidative stress; while inhibition of oxidative stress with NAC could increase cell viability and inhibit DEHP‐induced apoptosis and autophagy. These results suggested that DEHP induced apoptosis and autophagy of mouse GC‐1 spg cells via oxidative stress. 3‐MA, an inhibitor of autophagy, could rescue DEHP‐induced apoptosis. In summary, DEHP induced apoptosis and autophagy of mouse GC‐1 spg cells via oxidative stress, and autophagy might exert a cytotoxic effect on DEHP‐induced apoptosis.     

The role S‐nitrosylation in manganese‐induced autophagy dysregulation in SH‐SY5Y cells

Overexposure to manganese (Mn) has been known to induce nitrosative stress. The dysregulation of autophagy has implicated in nitric oxide (NO) bioactivity alterations. However, the mechanism of Mn‐induced autophagic dysregulation is unclear. The protein of Bcl‐2 was considered as a key role that could participate to the autophagy signaling regulation. To further explore whether S‐nitrosylation of Bcl‐2 involved in Mn‐induced autophagy dysregulation, we treated human neuroblastoma (SH‐SY5Y) cells with Mn and pretreated cells with 1400 W, a selective iNOS inhibitor. After cells were treated with 400 μM Mn for 24 h, there were significant increases in production of NO, inducible NO synthase (iNOS) activity, the mRNA and protein expressions of iNOS. Interestingly, autophagy was activated after cells were treated with Mn for 0–12 h; while the degradation process of autophagy‐lysosome pathway was blocked after cells were treated with Mn for 24 h. Moreover, S‐nitrosylated JNK and Bcl‐2 also increased and phospho‐JNK and phospho‐Bcl‐2 reduced in Mn‐treated cells. Then, the affinity between Bcl‐2 and Beclin‐1 increased significantly in Mn‐treated cells. We used the 1400 W to neutralize Mn‐induced nitrosative stress. The results showed that S‐nitrosylated JNK and Bcl‐2 reduced while their phosphorylation were recovered to some extent. The findings revealed that NO‐mediated S‐nitrosylation of Bcl‐2 directly affected the interaction between Beclin‐1 and Bcl‐2 leading to autophagy inhibition.     

Beclin1、LC3和mTOR在肝癌中的表达及临床意义

目的 通过检测Beclin1、LC3和雷帕霉素靶蛋白(mTOR)在肝癌组织中的表达,并结合各相关临床病理指标,从而探讨Beclin1、LC3和mTOR的表达水平在肝癌发生发展中的作用及其相互之间的关系.方法 采用免疫组织化学技术,检测56例肝癌组织和40例正常肝脏组织中Beclin1、LC3和mTOR蛋白的表达情况,并分析二者与肝癌各相关临床病理特征之间的关系.结果 Beclin1、LC3和mTOR蛋白在肝癌组织中的阳性表达率分别为75％、70％和50％,均明显高于正常肝组织中的阳性表达率25％、30％、28％,差异有统计学意义(P<0.05).Beclin1与LC3在肝癌中表达呈正相关(r=0.643,P<0.01),与mTOR表达之间无明显相关性(r=-0.167,P>0.05).LC3与mTOR表达呈负相关(r=-0.386,P<0.01).Beclin1、LC3和mTOR的表达均与年龄、性别、肿瘤大小、HBSAg、AFP值等均无显著相关性(P>0.05),但与病理学分级和有无胆管癌栓有关(P<0.05).结论 Beclin1、LC3和mTOR的异常高表达可能与肝癌的发生关系密切.     

维生素K_3通过下调mTOR信号途径而诱导HeLa细胞自噬

目的观察VitK3(维生素K3,vitaminK3)对HeLa细胞损伤过程中的自噬相关蛋白Beclin1、LC3-Ⅱ表达的影响。方法以HeLa细胞为研究对象,采用MTT检测细胞存活率,用Western blot方法检测自噬相关蛋白Beclin1、LC3-Ⅱ表达的变化和蛋白激酶B(PKB,Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化-Akt/mTOR表达的变化。结果30μmol·L-1剂量以上的VitK3可明显抑制HeLa细胞的增殖(P<0.05)。VitK3作用12h后增加Beclin1和LC3-Ⅱ的蛋白表达水平(P<0.05),降低磷酸化的Akt/mTOR的蛋白表达水平(P<0.05)。结论VitK3能明显抑制HeLa细胞增殖并具有诱导HeLa细胞发生自噬的作用,其机制可能与下调mTOR信号有关。     

Synthesis of [2‐13C, 4‐13C]‐(2R,3S)‐catechin and [2‐13C, 4‐13C]‐(2R,3R)‐epicatechin

The first synthesis of doubly labeled, [2‐¹³C, 4‐¹³C]‐(2R,3S)‐catechin 15 and [2‐¹³C, 4‐¹³C]‐(2R,3R)‐epicatechin 18 starting from labeled 2‐hydroxy‐4, 6‐bis(benzyloxy)acetophenone 3 and labeled 3, 4‐bis(benzyloxy)‐benzaldehyde 7 are described. Copyright © 2010 John Wiley & Sons, Ltd.     

Multisite phosphorylation of Bcl‐2 via protein kinase Cδ facilitates apoptosis of hypertrophic cardiomyocytes

Activated protein kinase Cδ (PKCδ) associated with cardiac hypertrophy moves from the cytoplasm to the mitochondria and subsequently triggers the apoptotic signalling pathway. The underlying mechanisms remain unknown. The aim of the present study was to investigate whether mitochondrial translocation of PKCδ phosphorylates multiple sites of Bcl‐2, resulting in an imbalance between Bcl‐2 and Bak or Bax, thus enhancing the susceptibility of hypertrophic cardiomyocytes to angiotensin II (AngII)‐induced apoptosis. Chronic pressure overload was induced by transverse aortic constriction (TAC) in rats. The apoptotic rate increased in hypertrophied cardiomyocytes. In AngII‐treated hearts (10 nmol/L, 60 min), there was an increase in the number of TERMINAL deoxyribonucleotidyl transferase‐mediated dUTP–digoxigenin nick end‐labelling (TUNEL)‐positive cells; PKCδ inhibition with 500 nmol/L δV1‐1 for 60 min prevented the AngII‐induced increase in apoptosis. In the hypertrophied myocardium, PKCδ expression increased, whereas that of Bcl‐2 decreased compared with the synchronous control. Treatment of hearts with 10 nmol/L AngII for 60 min activated PKCδ and induced translocation of PKCδ to the mitochondria, where activated PKCδ facilitated the phosphorylation of Bcl‐2 at serine‐87 and serine‐70 sites. The multisite phosphorylated Bcl‐2 was released from the mitochondria, and exhibited reduced affinity for Bak and Bax. The imbalance between Bcl‐2 and Bak/Bax induced the release of mitochondrial cytochrome c and then activated the caspase 3 apoptotic pathway during AngII stimulation (10 nmol/L, 60 min) of hypertrophied cardiomyocytes. Inhibition of PKCδ reduced these effects of AngII. The results suggest that PKCδ can counteract the anti‐apoptotic effect of Bcl‐2 and may promote cardiomyocyte apoptosis through multisite phosphorylation of Bcl‐2 in hypertrophied cardiomyocytes.     

Role of Bcl‐2 in tumour cell survival and implications for pharmacotherapy

Objectives Bcl‐2 is a protein that inhibits apoptosis, leading to cell survival. The Bcl‐2 family has six different anti‐apoptotic proteins, three pro‐apoptotic proteins that are similar in structure, and other integrating proteins that function as promotors or inhibitors in the progression of apoptosis. In this discussion paper, we provide an overview of apoptosis, the role of Bcl‐2 in normal cellular and molecular processes, and the role of Bcl‐2 in tumour cell survival. It focuses primarily on anti‐apoptotic Bcl‐2, its activation in cancer, the manner in which it regulates the intrinsic and extrinsic mechanisms of apoptosis, and its broad molecular interactions with other critical proteins in the cell. Certain cancer treatments are reviewed and related directions for the future are presented. Key findings Apoptosis is common to all organisms – for eukaryotes it is a normal process of development and regeneration. The rate at which apoptosis occurs is critical to the survival of the organism, as too much can lead to the onset of degenerative diseases such as dementia, and too little may lead to cancer. FKBP‐38 is a binding protein that has been discovered to be upregulated in highly aggressive cancers and binds to Bcl‐2 rather than the pro‐apoptotics to induce a state of hyper‐mitosis. A short binding protein (Nur‐77) provides new insights into Bcl‐2 ‘masking’. Nurr‐77 binds to Bcl‐2 and exposes the BH3 domain, transforming it from a cancer promoter to an unorthodox cancer inhibitor. This presents in itself an interesting and exciting opportunity – increasing the rate of apoptosis in neoplastic cells that are usually protected by Bcl‐2 activity at the mitochondria. Summary Development of drugs in the form of BH3‐only and BH123 mimetic drugs provide a interesting avenue for cancer therapy for the future. Drugs that can either promote, or mimic anti‐IAP activity such as Smac/Diablo would certainly be productive, thereby inducing apoptosis. Medicinal usage which can effectively suppress FKBP38 in Bcl‐2‐dependent cancers would provide further arsenal to combat apoptotic irregularities, particularly a treatment that is more dominant than kinetin riboside. WAVE‐1 inhibitors may effectively suppress the phosphorylation of Bcl‐2, thereby potentially reducing hyper‐mitosis and increasing apoptosis. Recent findings shed molecular light on PDT, namely ER stress, and potential for anti‐cancer therapy via either apoptosis or autophagy. A drug that can effectively upregulate Nurr‐77, thereby masking the anti‐apoptotic properties of Bcl‐2, would indeed be life‐saving for cancer patients.     

Palmitic acid induces autophagy in hepatocytes via JNK2 activation

Aim： Free fatty acid-induced lipotoxicity plays a crucial role in the progression of nonalcoholic fatty liver disease （NAFLD）. In the present study we investigated the effects of a high-fat diet and free fatty acids on the autophagic process in hepatocytes in vivo and in vitro and the underlying mechanisms.
Methods： LC3-II expression, a hallmark of autophagic flux, was detected in liver specimens from patients with non-alcoholic steatohepatitis （NASH） as well as in the livers of C57BL/6 mice fed a high-fat diet （HFD） up to 16 weeks. LC3-II expression was also analyzed in human SMMC-7721 and HepG2 hepatoma cells exposed to palmitic acid （PA）, a saturated fatty acid. PA-induced apoptosis was detected by Annexin V staining and specific cleavage of PARP in the presence and absence of different agents.

Results： LC3-II expression was markedly increased in human NASH and in liver tissues of HFD-fed mice. Treatment of SMMC-7721 cells with PA increased LC3-II expression in time- and dose-dependent manners, whereas the unsaturated fatty acid oleic acid had no effect. Inhibition of autophagy with 3MA sensitized SMMC-7721 cells to PA-induced apoptosis, whereas activation of autophagy by rapamycin attenuated PA-induced PARP cleavage. The autophagy-associated proteins Beclin1 and Atg5 were essential for PA-induced autophagy in SMMC-7721 cells. Moreover, pretreatment with SP600125, an inhibitor of JNK, effectively abrogated PA-mediated autophagy and apoptosis. Specific knockdown of JNK2, but not JNK1, in SMMC-7721 cells significantly suppressed PA-induced autophagy and enhanced its pro-apoptotic activity; whereas specific knockdown of JNK1 had the converse effect. Similar results were obtained when HepG2 cells were tested.

Conclusion： JNK1 promotes PA-induced lipoapoptosis, whereas JNK2 activates pro-survival autophagy and inhibits PA lipotoxicity. Our results suggest that modulation of autophagy may have therapeutic benefits in the treatment of lipid-related metabolic diseases.     

Synthesis of (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐ 3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate‐1‐14C

γ‐Cyhalothrin ( 1a ), (S)‐cyano(3‐phenoxyphenyl)methyl (1R,3R)‐3‐[(1Z)‐2‐chloro‐3,3,3‐trifluoro‐1‐propenyl]‐2,2‐dimethylcyclopropanecarboxylate, is a single‐isomer, synthetic pyrethroid insecticide marketed by Pytech Chemicals GmbH, a joint venture between Dow AgroSciences and Cheminova A/S. As a part of the registration process there was a need to incorporate a carbon‐14 label into the cyclopropyl ring of this molecule. A high yielding radiochemical synthesis of γ‐cyhalothrin was developed from readily available carbon‐14 labeled N‐t‐Boc protected glycine. This seven step synthesis, followed by a preparative normal phase HPLC separation of diastereomers, provided 21.8 mCi of γ‐cyhalothrin‐1‐¹⁴C ( 1b ) with >98% radiochemical purity. Copyright © 2007 John Wiley & Sons, Ltd.     

Tetrandrine induces apoptosis Via caspase‐8, ‐9, and ‐3 and poly (ADP ribose) polymerase dependent pathways and autophagy through beclin‐1/ LC3‐I,II signaling pathways in human oral cancer HSC‐3 cells

Tetrandrine is a bisbenzylisoquinoline alkaloid that was found in the Radix Stephania tetrandra S Moore. It had been reported to induce cytotoxic effects on many human cancer cells. In this study, we investigated the cytotoxic effects of tetrandrine on human oral cancer HSC‐3 cells in vitro. Treatments of HSC‐3 cells with tetrandrine significantly decreased the percentage of viable cells through the induction of autophagy and apoptosis and these effects are in concentration‐dependent manner. To define the mechanism underlying the cytotoxic effects of tetrandrine, we investigated the critical molecular events known to regulate the apoptotic and autophagic machinery. Tetrandrine induced chromatin condensation, internucleosomal DNA fragmentation, activation of caspases‐3, ‐8, and ‐9, and cleavage of poly (ADP ribose) polymerase (PARP) that were associated with apoptosis, and it also enhanced the expression of LC3‐I and ‐II that were associated with the induction of autophagy in human squamous carcinoma cell line (HSC‐3) cells. Tetrandrine induced autophagy in HSC‐3 cells was significantly attenuated by bafilomycin A1 (inhibitor of autophagy) pre‐treatment that confirmed tetrandrine induced cell death may be associated with the autophagy. In conclusion, we suggest that tetrandrine induced cell death may be through the induction of apoptosis as well as autophagy in human oral cancer HSC‐3 cells via PARP, caspases/Becline I/LC3‐I/II signaling pathways. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 395–406, 2016.     

Microwave‐assisted synthesis of (RS) methyl‐2‐([2′‐14C]4,6‐dimethoxypyrimidin‐2′‐yloxy)‐2‐phenyl [1‐14C]ethanoate

We report here a facile synthesis of (RS) methyl‐2‐([2′‐¹⁴C]4,6‐dimethoxypyrimidin‐2′‐yloxy)‐2‐phenyl [1‐¹⁴C]ethanoate under microwave irradiation. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis of 14C‐labelled myosmine, [2′‐14C] ‐3‐(1‐pyrrolin‐2‐yl)pyridine

¹⁴C‐Labelled myosmine ([2′‐¹⁴C]‐3‐(1‐pyrrolin‐2‐yl)pyridine) was synthesized for autoradiography studies starting from [carboxyl‐¹⁴C]‐nicotinic acid by initial esterification of the latter in the presence of 1,1,1‐triethoxyethane. Without any purification the ethyl nicotinate formed was directly reacted with N‐vinyl‐2‐pyrrolidinone in the presence of sodium hydride, yielding ¹⁴C‐labelled myosmine. The product was purified by silica gel column chromatography. The radiochemical yield was 15% and the specific activity 55.2 mCi/mmol. Copyright © 2003 John Wiley & Sons, Ltd.