胃癌组织Beclin1表达与幽门螺杆菌感染的相关性

目的:探讨胃癌组织自噬相关蛋白Beclin1的表达和临床意义,及与幽门螺杆菌(HP)感染的相关性.方法:应用免疫组化法检测120例胃癌和配对正常胃组织中自噬相关蛋白Beclin1的表达;分析Beclin1的表达和临床病理学参数的关系;分析Beclin1的表达和无疾病进展生存(disease free survival,DFS)的相关性;应用碳14呼气试验检测胃癌患者幽门螺杆菌的感染,并分析Beclin1的表达和HP感染的相关性.结果:Beclin1在胃癌和正常胃组织的高表达率分别为59.2%(71/120)、36.7%(44/120),胃癌组织Beclin1的高表达率显著高于正常胃组织,差异有统计学意义(P=0.004).Beclin1的高表达与胃癌患者pTNM分期(P<0.001)和区域淋巴结转移(P<0.001)有显著负相关性.与Beclin1高表达的患者比较,Beclin1低表达患者的中位DFS显著缩短(21 vs 25个月,P=0.032).HP阳性胃癌组织的Beclin1高表达率显著高于HP阴性者(71.0%vs 43.1%,P=0.002);Spearmen相关性检验显示HP感染和Beclin1表达有显著相关性(γ=0.280,P=0.002);logistic回归分析显示HP感染对Beclin1表达有显著影响(P=0.009).结论:胃癌组织Beclin1表达水平增高;Beclin1高表达提示较好的预后;HP感染与Beclin1表达有显著相关性.     

自噬相关蛋白 Beclin1、LC3在甲状腺癌合并颈部淋巴结转移中的表达及临床意义

目的:探究分析与细胞自噬相关的蛋白Beclin1及LC3在甲状腺癌合并颈部淋巴结转移中的表达及其在临床治疗中的意义。方法:采用免疫组织化学方法检测,选取48例我院2012年2月至2014年2月收集的甲状腺癌组织、30例癌旁正常组织用以检测Beclin1及LC3蛋白表达,另取38例甲状腺癌转移淋巴结以及30例的正常淋巴结临床标本,用以检测Beclin1及LC3蛋白表达。结果:在取材的所有样本中均有表达Beclin1,但在甲状腺癌组织中含量低于癌旁正常的甲状腺组织(P<0.01),转移淋巴结中Beclin1水平明显低于正常淋巴结组(P<0.01);免疫组化结果显示在所有样本中也检测到LC3蛋白表达,在甲状腺癌组织中的含量最低,并且显著低于癌旁甲状腺组织(P<0.01),在发生转移的淋巴结组织中,LC3含量低于正常淋巴结组织(P<0.01)。结论:自噬功能相关蛋白Beclin1与LC3的表达与甲状腺癌是否发生转移密切相关,是肿瘤向囊外扩展的直接标记。该蛋白具有作为甲状腺癌淋巴结转移的术前评估指标的潜在可能,具有一定的指导意义。     

沉默Beclin1基因对β-榄香烯抑制人胃癌SGC-7901细胞增殖与诱导自噬的影响

目的探讨Beclin1基因对β-榄香烯抗人胃癌SGC-7901细胞增殖及自噬诱导的影响。方法用前期实验构建成功的GV112-Beclin1-shRNA1(short hairpin RNA,短发夹RNA)慢病毒载体感染人胃癌SGC-7901细胞。采用四甲基偶氮唑盐微量酶反应比色法(MTT)检测β-榄香烯对SGC-7901细胞增殖的抑制作用,以及沉默Beclin1基因后对β-榄香烯抗增殖能力的影响。免疫蛋白印迹(Western blot)检测自噬相关蛋白LC3-II、P62的表达,评价β-榄香烯作用SGC-7901后的自噬水平和Beclin1基因沉默对β-榄香烯诱导自噬的影响。结果 MTT法显示,β-榄香烯能够抑制胃癌SGC-7901细胞增殖,抑制作用随浓度的增加而递增(浓度为20、50、100μg/mL比0μg/mL时,抑制率为21.5%、31.7%、37.4%比0,P<0.05);沉默Beclin1基因后β-榄香烯抑制胃癌SGC-7901细胞抑制率增加(42.3%比36.1%,P<0.05)。Western blot法显示,β-榄香烯能诱导SGC-7901细胞发生保护性自噬,自噬相关蛋白LC3-Ⅱ表达上调,P62蛋白表达下降,沉默Beclin1基因可抑制β-榄香烯对SGC-7901细胞保护性自噬的增强,LC3-Ⅱ蛋白表达水平下降,P62蛋白表达增加。结论β-榄香烯可诱导胃癌SGC-7901细胞发生保护性自噬,下调Beclin1基因有助于降低保护性自噬的发生,从而增强β-榄香烯的抗癌效果。     

The mechanism for the neuroprotective effect of melatonin against methamphetamine‐induced autophagy

Abstract: Methamphetamine (METH) is a common drug of abuse that induces toxicity in the central nervous system and is connected to neurological disorders such as Parkinson’s disease. METH neurotoxicity is induced by reactive oxygen species (ROS) production and apoptosis. Moreover, autophagy is an alternative to cell death and a means for eliminating dysfunctional organelles. In other cases, autophagy can end up in cell death. Nonetheless, it is not clear whether autophagy is also correlated with apoptotic signaling in drug‐induced neurotoxicity. Therefore, we hypothesized that METH‐generated toxicity associated with initiating the apoptotic signaling cascade can also increase the autophagic phenotype in neuronal cells. Using the SK–N–SH dopaminergic cell line as our model system, we found that METH‐induced autophagy by inhibiting dissociation of Bcl‐2/Beclin 1 complex and its upstream pathway that thereby led to cell death. We uncovered a novel function for the anti‐apoptotic protein Bcl‐2, as it played a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Furthermore, Bcl‐2 was activated by c‐Jun N‐terminal kinase 1 (JNK 1), which is upstream of Bcl‐2 phosphorylation, to induce Bcl‐2/Beclin 1 dissociation. Furthermore, we demonstrated a novel role for melatonin in protecting cells from autophagic cell death triggered by the Bcl‐2/Beclin 1 pathway by inhibiting the activation of the JNK 1, Bcl‐2 upstream pathway. This study provides information regarding the link between apoptosis and autophagy signaling, which could lead to the development of therapeutic strategies that exploit the neurotoxicity of drugs of abuse.     

自噬基因Beclin 1在宫颈鳞癌中的蛋白表达及其临床意义

目的探讨自噬基因Beclin 1在宫颈鳞癌中的蛋白表达以及与肿瘤发生发展的关系。方法采用免疫组化SP法检测81例宫颈鳞癌、20例宫颈上皮内瘤样病变（CIN,Ⅱ～Ⅲ级）和20例正常宫颈组织中自噬基因Beclin 1的蛋白表达。结果宫颈鳞癌组织中Beclin 1阴性、弱阳性、强阳性表达率依次是43．2％（35／81）、34．6％（28／81）、22．2％（18／81）,与正常宫颈和宫颈上皮内瘤样病变组相比,Beclin 1在宫颈鳞癌中的表达显著下调（P=0．011）。宫颈鳞癌中Beclin 1的表达在不同FIGO分期、年龄、宫颈肌层浸润深度、宫颈肿瘤大小、大体类型间的差异无统计学意义。但在有无盆腔淋巴结转移、不同肿瘤分化程度间差异有统计学意义（P〈0．05）。结论自噬基因Beckn 1在宫颈鳞癌中蛋白表达下调,并与宫颈鳞癌盆腔淋巴结转移和肿瘤分化程度有关。     

Autophagy in tumour suppression and promotion

Autophagy, a well-described cellular mechanism for lysosomal degradation of cytoplasmic content, has emerged as a tumour suppression pathway. Recent evidence indicates that the tumour suppressor function of autophagy is mediated by scavenging of damaged oxidative organelles, thereby preventing accumulation of toxic oxygen radicals that would cause genome instability. Paradoxically, however, in some cases autophagy can also promote the survival of cancer cells once tumours have developed. This is attributed to the ability of autophagy to promote cell survival under conditions of poor nutrient supply, as often faced by solid tumours and metastasising cancer cells. In addition, autophagy is frequently upregulated in tumours as a response to therapy and may protect tumours against therapy-induced apoptosis. In this review we discuss the mechanisms that link autophagy to tumour suppression and promotion and provide examples of the dual functions of autophagy in cancer.     

The significance of autophagy in cancer

Autophagy, a vacuolar process of cytoplasmic degradation, is implicated in a form of programmed cell death distinct from apoptosis. The regulation of autophagy is complex and signalling pathways such as the target of rapamycin (Tor) kinase pathway, play important roles in tumourigenesis. Beclin 1, an autophagic protein, has been found to be a tumour suppressor. Conversely, cancer cells may exploit autophagy as a means to adapt to hypoxic and nutrient-limiting environments. The relative importance of autophagic cell death and apoptosis in carcinogenesis remains to be established, and the Bcl-2 family of proteins may be instrumental in coordinating the two pathways of programmed cell death.     

沉默beclin1基因对缺氧缺糖/复氧复糖HT22细胞凋亡的影响

目的 探讨beclin1基因对缺氧缺糖/复氧复糖小鼠海马神经元细胞系HT22细胞凋亡的影响。方法 取对数生长期的HT22细胞,随机分为4组：正常组（normal）、缺氧缺糖/复氧复糖（OGD/R）模型组（model）、beclin1基因沉默组（beclin1^-/-）、转染对照组（control）。除normal组外,其余各组细胞均在缺氧缺糖6 h后进行复氧复糖。利用RNAi技术,针对小鼠cDNA序列设计beclin1干扰序列,用脂质体Lipo2000包裹后转染至HT22细胞。于转染48 h后用荧光显微镜观察转染效率,Western blotting检测细胞beclin1 表达情况。各组细胞均于复氧复糖24 h后采用CCK-8法检测细胞活力,乳酸脱氢酶（LDH）法检测细胞损伤情况,免疫荧光染色检测Bax、Bcl-2表达及比值变化,Western blotting检测LC3、P62及Caspase-3表达。SPSS 19.0 统计学软件进行数据分析。结果 与normal组相比,model组细胞活力及P62蛋白表达显著降低（P<0.01）,乳酸脱氢酶（LDH）漏出率、LC3Ⅱ/LC3Ⅰ、Caspase-3表达及Bax/Bcl-2均显著升高（P<0.01）。与model组相比,beclin1^-/-组细胞活力及LC3Ⅱ/LC3Ⅰ 表达显著降低（P<0.01）,LDH漏出率、Bax/Bcl-2及P62、Caspase-3表达显著升高（P<0.01）;control组与model组比较差异无显著性。结论 沉默beclin1抑制细胞自噬可使缺氧缺糖/复氧复糖处理的HT22细胞损伤加重,细胞凋亡进一步增加。     

Aerosol delivery of beclin1 enhanced the anti-tumor effect of radiation in the lungs of K-rasLA1 mice

Radiotherapy alone has several limitations for treating lung cancer. Inhalation, a non-invasive approach for direct delivery of therapeutic agents to the lung, may help to enhance the therapeutic efficacy of radiation. Up-regulating beclin1, known as a tumor suppressor gene that plays a major role in autophagy, may sensitize tumors and lead to tumor regression in lungs of K-ras^LA1 lung cancer model mice. To minimize the side-effects of radiotherapy, fractionated exposures (five times, 24-h interval) with low dose (2 Gy) of radiation to the restricted area (thorax, 2 cm) were conducted. After sensitizing the lungs with radiation, beclin1, complexed with a nano-sized biodegradable poly(ester amine), was prepared and delivered into the murine lung via aerosol three times/week for four weeks. In a histopathological analysis, animals treated with beclin1 and radiation showed highly significant tumor regression and low progression to adenocarcinoma. An increase in the number of autophagic vacuoles and secondary lysosomes was detected. Dissociation of beclin1-bcl2 stimulated autophagy activation and showed a synergistic anti-tumor effect by inhibiting the Akt-mTOR pathway, cell proliferation and angiogenesis. The combination of radiation with non-invasive aerosol delivery of beclin1 may provide a prospect for developing novel therapy regimens applicable in clinics.     

Hepatitis B virus x protein induces autophagy via activating death‐associated protein kinase

Hepatitis B virus x protein (HBX), a product of hepatitis B virus (HBV), is a multifunctional protein that regulates viral replication and various cellular functions. Recently, HBX has been shown to induce autophagy; however, the responsible mechanism is not fully known. In this study, we established stable HBX‐expressing epithelial Chang cells as the platform to study how HBX induced autophagy. The results showed that the overexpression of HBX resulted in starvation‐induced autophagy. HBX‐induced autophagy was related to its ability to dephosphorylate/activate death‐associated protein kinase (DAPK). The block of DAPK by its siRNA significantly counteracted HBX‐mediated autophagy, confirming the positive role of DAPK in this process. HBX also induced Beclin 1, which functions at the downstream of the DAPK‐mediated autophagy pathway. Although HBX could activate JNK, a kinase known to participate in autophagy in certain conditions, the change in JNK failed to influence HBX‐induced autophagy. In conclusion, HBX induces autophagy via activating DAPK in a pathway related to Beclin 1, but not JNK. This new finding should help us to understand the role of autophagy in HBX‐mediated pathogenesis and thus may provide targets for intervening HBX‐related disorders.