蛇床子素诱导内质网应激抑制SMMC7721人肝癌细胞增殖的机制研究＊

目的 研究蛇床子素抑制肝癌细胞增殖的机制。方法 采用20 - 100 μM蛇床子素干预SMMC7721人肝癌细胞24 h、48 h和72 h，运用MTT方法检测细胞增殖；采用20 - 80 μM蛇床子素干预SMMC7721肝癌细胞24 h，运用流式细胞术检测细胞凋亡及Western blot检测Bax和Bcl-2蛋白表达；采用 60 μM蛇床子素干预SMMC7721肝癌细胞48 h，运用流式细胞术检测细胞周期，以实时荧光定量PCR技术检测GRP78、DDIT3、GADD34、ATF4、EIF2A、GDF15、CDKN1B、FOXO3、RICTOR、SGK1基因表达，Western blot方法检测CyclinD1、CyclinE1、p27^Kip1和CDK4细胞周期蛋白及GRP78、GRP94、PERK、XBP-1s、CHOP、p-eIF2α(Ser51)内质网应激蛋白表达。各实验均以0.1% DMSO为对照组，并以0.1 μM毒胡萝卜素为内质网应激诱导剂组。结果 与对照组比较，100 μM蛇床子素干预24 h显著抑制SMMC7721肝癌细胞增殖，其抑制率为36.76%（P < 0.05）；60 - 100 μM蛇床子素干预48 h后显著抑制SMMC7721肝癌细胞增殖，抑制率为30.84%-52.49%（P < 0.05）；40 - 100 μM蛇床子素干预72 h后显著抑制SMMC7721肝癌细胞增殖，抑制率为32.7%-73.15%（P < 0.05）。大于60 μM蛇床子素可诱导SMMC7721肝癌细胞晚期凋亡且显著抑制促凋亡蛋白Bax表达（P < 0.05）。60 μM蛇床子素干预SMMC7721肝癌细胞48 h后，G1期细胞数增加、G2期和S期减少，且CyclinD1、CyclinE1和p27^Kip1蛋白表达显著被抑制（P < 0.05）。20 - 80 μM蛇床子素处理SMMC7721肝癌细胞24 h后，均显著促进GRP78、DDIT3（CHOP）、ATF4、FOXO3、RICTOR和SGK1基因表达（P < 0.05）； 20 μM蛇床子素显著促进GADD34和EIF2A基因表达（P < 0.05）；80 μM蛇床子素显著促进GDF15基因表达（P < 0.05），且CHOP、XBP-1s蛋白表达显著增强。结论 蛇床子素通过诱导内质网应激反应以抑制SMMC7721肝癌细胞增殖，从而发挥抗肝癌活性。     

Autophagy and immunological aberrations in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disease, in which immune defects can occur at multiple points of the cascading auto‐aggressive immune reactions, resulting in a striking heterogeneity of clinical presentations. The clinical manifestations of such autoimmune response can be severe: common manifestations symptoms include rash and renal inflammation progressing to kidney failure. Autophagy, the cellular “self‐digestion” process, is a key factor in the interplay between innate and adaptive immunity. Dysregulation of autophagy has been implicated in numerous autoimmune diseases. Several lines of evidence from genomic studies, cell culture systems, animal models, and human patients are emerging to support the role of autophagy in progression and pathogenesis of SLE. In this review, we summarize recent key findings on the aberrations of autophagy in SLE, with a special focus on how deregulated autophagy promotes autoimmunity and renal damage. We will also discuss how the observed findings may be translated into therapeutic settings.     

Melatonin activates cell death programs for the suppression of uterine leiomyoma cell proliferation

The circadian nature of melatonin has a protective effect on the progression of female reproductive cancers, including breast and ovarian cancers. However, the effect of melatonin on the growth of uterine leiomyoma is still unclear. In this study, we found that the growth of uterine leiomyoma ELT3 cells was reduced by treatment with melatonin. Treatment with melatonin increased the distribution of sub-G1 phase and increased DNA condensation in ELT3 cells. Melatonin-induced apoptosis and autophagy cell death progression were observed in ELT3 cells. Melatonin exerts a highly selective effect on primary normal human uterine smooth muscle (UtSMC) cells. The UtSMC cell cycle was arrested by melatonin treatment through up-regulation of p21, p27, and PTEN protein expression, but melatonin did not further promote apoptosis program activation. Melatonin reduced cell proliferation in ELT3 cells underlying the activation of melatonin MT1 and MT2 receptors, which in turn down-regulated the Akt-ERK1/2-NFκB signaling pathway. Melatonin reduced ELT3 tumor growth in both xenograft and orthotopic uterine tumor mice models. The extracellular matrix of the tumor was also reduced by melatonin treatment. Taken together, these results suggest that melatonin potentially plays a role in suppression of uterine leiomyoma growth.     

Catabolic pathways regulated by mTORC1 are pivotal for survival and growth of cancer cells expressing mutant Ras

Oncogenic Ras stimulates macropinocytosis, a clathrin-independent endocytosis that increases the uptake of extracellular fluid. However, the functional significance of and regulatory mechanisms driving macropinocytosis in cancer cells remain largely unknown. Here, we show that extracellular macromolecules, such as albumin, internalized by Ras-expressing cells can support growth and survival under the nutrient-deprived conditions like those found in tumors. Moreover, we demonstrate that autophagy, a lysosome-mediated catabolic pathway, is required for the uptake and degradation of macropinocytic vesicles. Intracellular metabolites derived from macropinocytosis and autophagy directly influence the activity and localization of mTOR, which is ultimately responsible for the restoration of cell growth. Surprisingly, suppression of mTORC1, which typically triggers anabolic processes, facilitates macropinocytosis and thus supports cell growth and survival under the nutrient-deprived conditions. In a mouse xenograft model of pancreatic ductal adenocarcinoma, concomitant inhibition of macropinocytosis/autophagy and mTOR activity resulted in antitumor effects. These data suggest that novel anti-cancer strategies interrupting these metabolic processes and related signaling molecules may represent promising therapeutic avenues.     

Antagonistic effects of chloroquine on autophagy occurrence potentiate the anticancer effects of everolimus on renal cancer cells

Renal cell carcinoma is an aggressive disease often asymptomatic and weakly chemo-radiosensitive. Currently, new biologic drugs are used among which everolimus, an mTOR inhibitor, that has been approved for second-line therapy. Since mTOR is involved in the control of autophagy, its antitumor capacity is often limited. In this view, chloroquine, a 4-alkylamino substituted quinoline family member, is an autophagy inhibitor that blocks the fusion of autophagosomes and lysosomes. In the present study, we evaluated the effects of everolimus alone or in combination with chloroquine on renal cancer cell viability and verified possible synergism. Our results demonstrate that renal cancer cells are differently sensitive to everolimus and chloroquine and the pharmacological combination everolimus/chloroquine was strongly synergistic inducing cell viability inhibition. In details, the pharmacological synergism occurs when chloroquine is administered before everolimus. In addition, we found a flow autophagic block and shift of death mechanisms to apoptosis. This event was associated with decrease of Beclin-1/Bcl^-2 complex and parallel reduction of anti-apoptotic protein Bcl^-2 in combined treatment. At last, we found that the enhancement of apoptosis induced by drug combination occurs through the intrinsic mitochondrial apoptotic pathway activation, while the extrinsic pathway is involved only partly following its activation by chloroquine. These results provide the basis for new therapeutic strategies for the treatment of renal cell carcinoma after appropriate clinical trial.     

Autoantibodies against the autophagic protein microtubule-associated light-chain 3 (LC3): Immunocharacterization of an atypical ANA pattern

Abstract

Autoantibodies to nuclear and cytoplasmic antigens are commonly detected by indirect immunofluorescence (IIF) on HEp-2 cells, and three major staining patterns (nuclear, cytoplasmic, and mitotic) are distinguished. Here, we report an atypical cytoplasmic pattern, not described so far, observed in the serum of a patient with a controversial diagnosis of systemic lupus erythematosus (SLE). Moreover, for the first time, we have revealed the presence of autoantibodies against the microtubule-associated light-chain 3 (LC3) protein, which plays a key role in the autophagic process. The target antigen has been identified in IIF by means of a competition test using purified anti-LC3 antibodies on HEp-2 cells, and confirmed by Western blot analysis using cellular or recombinant LC3 as antigen, immunoreacted with the patient’s serum. The identification of this atypical pattern and the related autoantibody-antigen system sheds new light on autophagy, which is increasingly considered to be involved in the etiopathogenesis of autoimmune disorders, and could contribute to select more personalized therapies.     

安氟醚减轻心脏手术中小鼠的疼痛作用研究

目的研究安氟醚对心肌缺血小鼠心肌细胞保护作用的潜在机制。方法(1)用随机数字表法将成模后小鼠分为2组:对照组和安氟醚组,每组20只。小鼠经腹腔注射异丙肾上腺素3 mg·kg^-1制备心肌缺血模型。在心脏搭桥术前,对照组小鼠用异戊巴比妥(150 mg·kg^-1)麻醉,搭桥完成后腹腔注射等体积生理盐水;安氟醚组用安氟醚(1.0 mg·kg^-1)麻醉,术后4 h腹腔注射(0.2 mg·kg^-1)安氟醚处理;每天1次,连续3 d。用视觉模拟评分(VAS)评估小鼠术后疼痛程度。(2)处死2组小鼠后获取心肌细胞,将细胞分为2组:安氟醚组和对照组;另外,将心肌细胞分为4组:对照组(等体积PBS)、安氟醚组(安氟醚1 mg·mL^-1)、安氟醚+磷酸肌醇3激酶(PIK3)抑制剂组(安氟醚+PI3K抑制剂,均1 mg·mL^-1)和PI3KI抑制剂组(PI3K抑制剂1 mg·mL^-1)。通过细胞计数试剂盒法检测细胞活力(光密度值),MTT法测定细胞增殖(光密度值),缺口末端标记法和流式细胞仪分别检测2组和4组的细胞凋亡情况,蛋白质印迹法检测免疫球蛋白结合蛋白(BIP)、CCAAT增强子结合蛋白(CCAAT enhancer binding protein,CHOP)和PI3KI/蛋白激酶B(Akt)通路相关蛋白表达水平。结果(1)安氟醚组和对照组的VAS分别为(6.53±1.37)和(13.21±2.35)分,组间比较差异有统计学意义(P<0.05)。(2)安氟醚组和对照组的BIP蛋白相对表达水平分别为0.23±0.03和1.04±0.12;这2组的CHOP蛋白表达分别为0.47±0.01和0.98±0.09;这2组的caspase-3蛋白表达分别为0.34±0.01和1.00±0.12;这2组的caspase-8蛋白表达分别为0.27±0.01和1.03±0.08;这2组的p-PI3K蛋白表达分别为1.53±0.16和0.98±0.08;这2组的p-Akt蛋白表达分别为1.69±0.19和0.99±0.11;这2组的心肌细胞存活率分别为(0.87±0.12)%和(0.62±0.03)%;这2组的细胞凋亡率分别为(0.33±0.01)%和(1.08±0.09)%,安氟醚组与对照组比较,上述指标差异均有统计学意义(均P<0.05)。安氟醚+PI3K抑制剂组和对照组的细胞存活率分别为(0.91±0.05)%和(0.98±0.06)%;这2组心肌细胞凋亡率分别为(1.12±0.12)%和(1.02±0.09)%;安氟醚+PI3K抑制剂组与对照组比较,上述指标的差异均无统计学意义(均P>0.05)。结论安氟醚有助于减轻心脏手术中小鼠的疼痛并通过PI3K/Akt信号通路抑制心肌细胞的凋亡。     

内质网应激在三种常见精神疾病中的研究进展

精神疾病常常表现为慢性病程,具有反复发作、慢性迁延及致残的特点,是严重影响人类健康的疾病之一.近年来精神疾病的患病率逐年升高,给社会带来了严重的疾病负担.但是,目前大部分精神疾病的发病机制尚未完全阐明.不断增加的研究证据表明,内质网应激(ERS)与精神疾病的发生发展有着密切联系,这些疾病以精神分裂症、双相障碍及抑郁障碍这三种常见精神疾病为代表.同时,研究也发现一些相关药物可能通过靶向ERS而发挥作用,提示ERS可能是精神疾病的潜在作用靶点.因此,未来深入探讨ERS在精神疾病中的具体作用机制,可为精神疾病的治疗提供新的思路.     

Zearalenone Induces Apoptosis and Cytoprotective Autophagy in Chicken Granulosa Cells by PI3K-AKT-mTOR and MAPK Signaling Pathways

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and testicular toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in granulosa cells. This study investigated the effects of ZEA on chicken granulosa cells. The results show that ZEA at different doses significantly inhibited the growth of chicken granulosa cells by inducing apoptosis. ZEA treatment up-regulated Bax and downregulated Bcl-2 expression, promoted cytochrome c release into the cytosol, and triggered mitochondria-mediated apoptosis. Consequently, caspase-9 and downstream effector caspase-3 were activated, resulting in chicken granulosa cells apoptosis. ZEA treatment also upregulated LC3-II and Beclin-1 expression, suggesting that ZEA induced a high level of autophagy. Pretreatment with chloroquine (an autophagy inhibitor) and rapamycin (an autophagy inducer) increased and decreased the rate of apoptosis, respectively, in contrast with other ZEA-treated groups. Autophagy delayed apoptosis in the ZEA-treated cells. Therefore, autophagy may prevent cells from undergoing apoptosis by reducing ZEA-induced cytotoxicity. In addition, our results further show that the autophagy was stimulated by ZEA through PI3K-AKT-mTOR and MAPK signaling pathways in chicken granulosa cells.