Antiproliferative effects of melatonin on the growth of rat pituitary prolactin-secreting tumor cells in vitro

Earlier studies showed that melatonin reduced the growth of 17-beta-estradiol (E(2))-induced rat pituitary prolactin-secreting tumor (prolactinoma) in vivo. The mechanisms of melatonin's inhibitory action on the prolactin-secreting tumor were further explored by investigating the in vitro effects of melatonin on the growth of pituitary prolactin-secreting tumor cells. Primary cultured prolactinoma cells from E(2)-induced rat pituitary prolactin-secreting tumor were treated with 10(-5), 10(-4) or 10(-3) m melatonin for 5 days. Apoptosis was evaluated using flow cytometry and the TdT-mediated dUTP nick-end labeling (TUNEL) method. In addition, cell viability was analyzed by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that incubation of prolactinoma cells with 10(-5), 10(-4) or 10(-3) m melatonin for 5 days inhibited cell growth and increased cell apoptosis. Furthermore, melatonin increased caspase-3 activity, Bax mRNA expression, and cytochrome c protein expression. Conversely, Bcl-2 mRNA expression and mitochondrial membrane potential were inhibited by melatonin treatment. Our results further suggest that melatonin inhibits tumor growth by inducing apoptosis of rat pituitary prolactin-secreting tumor directly via the damage of mitochondria.     

苦参素对小鼠肝细胞Bcl-2,Bax表达的影响

目的　探讨苦参素 (OM )阻断苯巴比妥钠 (PB)诱导的小鼠肝细胞凋亡的机理。方法　 84只小鼠随机分为 4组 :空白对照组 ,阴性对照组 ,阳性对照组和OM治疗组。用免疫组织化学法和末端标记技术 (TUNEL)检测小鼠肝组织Bcl 2 ,Bax表达情况和肝细胞凋亡情况。结果　TUNEL标记显示 :阳性对照组TUNEL标记阳性 ,其它 3组为阴性。Bcl 2蛋白在OM治疗组较阳性对照组表达显著 (P <0 0 5 )。在阳性对照组 ,Bax蛋白表达水平与其他 3组相比显著增强 (P <0 0 5 ) ,其他 3组间差异无显著性 (P >0 0 5 )。结论　OM可能通过促进Bcl 2表达来阻断苯巴比妥钠诱导的肝细胞凋亡 ,PB可能通过促进Bax表达来诱导肝细胞凋亡     

Melatonin inhibits growth of diethylstilbestrol-induced prolactin-secreting pituitary tumor in vitro: possible involvement of nuclear RZR/ROR receptors

Melatonin exerts a marked antiproliferative action in numerous experimentally-induced tumors in vivo as well as in both animal and human cell lines in vitro. However, the mechanisms of oncostatic action of melatonin is not clear, and the involvement of both membrane and nuclear receptors are suggested. Therefore, the aim of this study was to investigate effects of melatonin, and both agonist (CGP 52608), and antagonist (CGP 55644) of RZR/ROR nuclear receptors on the growth of diethylstilbestrol-induced rat prolactin-secreting pituitary tumor cells in vitro. Pituitary tumors were induced by subcutaneous implantation of a single silastic capsule containing 10 mg of diethylstilbestrol in 4-wk-old male Fischer 344 rats. Four months after the implantation of capsules the animals were killed by decapitation, pituitary tumors were aseptically removed, mechanically dispersed, and enzymatically digested with 0.2% collagenase and 0.2% hyaluronidase. The cells (6 x 105 cells/well) were incubated for 24 hr in the presence of melatonin, CGP 52608, CGP 55644 and CGP 55644 plus melatonin (at the concentrations of 107 and 10-9 m) at 37 degrees C in the humidified atmosphere of 95% air and 5% CO2. The group with the addition of solvent only served as control. The growth of cell was measured using the EZ4U system. Statistical analysis was performed using ANOVA followed by LSD test. Both melatonin and CGP 52608 significantly suppressed growth of tumor cells in vitro in both used concentrations. CGP 55644 stimulated growth of tumor cells and blocked the inhibitory effects of melatonin in vitro. Results of the present study as well as other experimental evidence strongly support the hypothesis that both membrane and nuclear receptors are involved in the oncostatic action of melatonin, and indicate that nuclear signalling plays an important role in this process.     

Involvement of p38 mitogen‐activated protein kinase pathway in honokiol‐induced apoptosis in a human hepatoma cell line (hepG2)

Background: Honokiol has been known to have antitumour activity. This study was conducted to evaluate the antiproliferative potential of honokiol against the hepG2 heptocellular cell line and its mechanism of action. Methods: hepG2 cells were treated with honokiol of 0–40 μg/ml concentration. The cytotoxic effect of honokiol was determined by a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The apoptosis was evaluated by flow cytometry. Western blots were used to analyse the expression of various proteins (procaspase‐9, procaspase‐3, cleaved caspase‐3, cytochrome c, Bcl‐2, Bax, Bad, Bcl‐X_L and p38). Results: Honokiol induced apoptosis with a decreased expression of procaspase‐3 and ‐9 and an increased expression of active caspase‐3. Exposure of hepG2 cells to honokiol resulted in the downregulation of Bcl‐X_L and Bcl‐2 expression and the release of mitochondrial cytochrome c to the cytosol. In addition, honokiol activated the p38 mitogen‐activated protein kinase (MAPK) pathway, and the inhibition of this pathway by SB203580 reduced honokiol‐induced apoptosis and activation of caspase‐3. Conclusion: Honokiol induces apoptosis of hepG2 human hepatocellular carcinoma cells through activation of the p38 MAPK pathway, and, in turn, activation of caspase‐3.     

糖尿病大鼠肾脏细胞凋亡与Bax和Bcl-2基因表达

目的　观察糖尿病大鼠肾脏细胞凋亡、Bax和 Bcl- 2表达及二者的相关性。　方法　单侧肾切除大鼠腹腔注射链脲佐菌素诱发糖尿病 ,采用原位末端标记法检测肾脏细胞凋亡 ;流式细胞术和免疫组化检测肾皮质 Bax和 Bcl- 2表达水平 ;原位杂交检测 Bax和 Bcl- 2 m RNA表达 ,并观察尿蛋白、BU N、尿肌酐等反映肾功能的有关指标。　结果　在制模后 2、4、8、12周时 ,糖尿病组大鼠较对照组肾小球、肾小管凋亡细胞数明显增多 ,Bax、Bcl- 2蛋白和 m RNA的表达显著增强 (P<0 .0 5 )。随着大鼠糖尿病病程延长 ,肾功能恶化 ,肾脏凋亡细胞数逐渐增多 ,Bax表达亦逐渐增强 ,Bax/Bcl- 2比增加 ,且肾脏凋亡细胞数与 Bax及 Bax/Bcl- 2比具有相关性 (P<0 .0 5 )。　结论　肾脏凋亡细胞的不断增加可能是糖尿病肾病发生、发展的原因之一 ,Bax和 Bcl- 2可能参与肾脏细胞凋亡的调控。     

Inhibition of growth of a prolactin-secreting pituitary tumor in rats by analogs of luteinizing hormone-releasing hormone and somatostatin.

We investigated the effects of [D-Trp6]LH-RH [agonistic analog of luteinizing hormone-releasing hormone (LH-RH)], N-Ac-[D-p-Cl-Phe1,2,D-Trp3,D-Phe6,D-Ala10]LH-RH (antagonistic analog), and [D-5-methoxy-Trp8]somatostatin (somatostatin analog) on the growth of the prolactin and corticotropin-secreting pituitary tumor 7315a in female Buffalo rats. Chronic administration of [D-Trp6]LH-RH in a dose of 25 micrograms/day, starting 18 days after inoculation with the tumor, inhibited the growth of the pituitary tumor. Tumor weight and volume also were reduced when this agonist was administered 3 days after inoculation. The antagonistic LH-RH analog, injected in a dose of 50-100 micrograms for 14-24 days, also significantly inhibited the growth of pituitary tumor. Chronic administration of the somatostatin analog in a dose of 25 micrograms twice a day likewise decreased tumor weights in comparison with controls. The inhibition of pituitary tumor growth by LH-RH agonist, LH-RH antagonist, and somatostatin analog was accompanied by a decrease in serum prolactin levels. It was concluded that LH-RH agonist, LH-RH antagonist, and somatostatin analog can inhibit the growth of estrogen-dependent prolactin/corticotropin-secreting pituitary tumor in rats.     

福辛普利对自发性高血压大鼠心室重塑时左室心肌细胞凋亡和Bcl_2及Bax基因表达的影响

目的 :研究福辛普利对自发性高血压大鼠 ( SHR)左室心肌细胞凋亡和 Bcl2 、Bax基因表达的影响。方法 :1使用左室重量指数作为大鼠左室重构的指标 ;2使用末端脱氧核苷酸转移酶标记技术和 DNA电泳技术 ,研究心肌细胞凋亡的发生情况 ;3使用核酸原位杂交技术研究左室心肌细胞 Bcl2 、Bax基因表达的变化。结果 :1福辛普利使 SHR左室重量指数显著降低 ,由 3 .3 1± 0 .17下降至 2 .5 6± 0 .2 5 ,P <0 .0 5 ;2福辛普利能显著减少左室心肌细胞凋亡的发生〔( 2 14± 2 8)下降至 ( 5 4± 11)个凋亡细胞 / 10 5细胞 ,P<0 .0 1〕;3福辛普利能显著增加细胞 Bcl2 基因的表达 ,同时减少 Bax的基因表达。福辛普利使切片的 Bcl2 与 Bax基因表达阳性面积百分数之比恢复正常 (均 P <0 .0 1)。结论 :血管紧张素转换酶抑制剂能有效地抑制心室重塑的发生以及此过程中的心肌细胞凋亡 ;福辛普利使心肌细胞 Bcl2 及 Bax基因表达阳性面积百分数之比恢复正常是其抗细胞凋亡作用的一个机制。     

Role of naofen in apoptosis of hepatocytes induced by lipopolysaccharide through mitochondrial signaling in rats

Aim: Lipopolysaccharide (LPS) causes apoptosis of hepatocytes, which is probably mediated by inflammatory substances released from Kupffer cells (KCs). Recently, we have reported that naofen, a newly found intracellular WD40-repeat protein, has a role in inducing the apoptosis in HEK293 cells. Hence, the present study was undertaken to investigate a role of naofen in the LPS-induced apoptosis of rat hepatocytes. Methods: Rats were treated with i.v. injections of LPS, and livers were extirpated to evaluate expression of naofen and apoptosis. In in vitro experiments, hepatocytes and KCs were separately isolated from rat livers. The incubation medium for KCs treated with LPS (KC-CM) was used for hepatocyte culture. Results: Intravenous injections of LPS enhanced the expression of naofen in the livers. Livers showed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive staining, and elevated caspase-3 activity. In isolated KCs or hepatocytes, LPS hardly affected naofen expression and caspase-3 activity, whereas incubation of hepatocytes with KC-CM enhanced both naofen expression and caspase-3 activation. Transfection of hepatocyte with naofen siRNA prevented such effects of KC-CM, and clearly eliminated KC-CM-induced reduction of Bcl-2 and Bcl-xL. In contrast, overexpression of naofen in hepatocytes downregulated Bcl-2 and Bcl-xL, released cytochrome c from mitochondria, and activated caspase-3. Conclusion: These results indicate that LPS may induce the hepatic apoptosis in association with enhanced naofen expression, and that naofen may mediate the activation of caspase-3 through downregulating the Bcl-2 and Bcl-xL expression, and releasing cytochrome c from mitochondria to cytoplasm.     

桥本甲状腺炎中细胞凋亡调节蛋白Bcl-2和Bax的免疫组织化学研究

目的：了解细胞凋亡调节蛋白Bcl-2和Bax在桥本甲状腺炎（HT）中的人布变化及其意义。方法：以非毒性甲状腺肿（NTG）为对照（17例）,采用免疫组织化学方法,检测17例桥本甲状腺炎患者甲状腺标本中Bcl-2和Bax的表达及分布,结果：免疫染色半定量分析及图像分析结果显示,HT中Bcl-2和Bax的免疫染色强度均显著高于对照组（P＜0．01）, 其中Bax免疫染色强阳性甲状腺滤泡细胞多分布于浸润淋巴滤泡附近,Bcl-2免疫染色强阳性细胞则多分布于远离浸润淋巴泡的区域,但在Bcl-2免疫反应阴性的淋巴滤泡周围亦有少量分布。结论：HT中细胞凋亡调节蛋白Bcl-2和Bax在甲状腺滤泡细胞中呈有特征性分布的高表达,其表达部位及比较的改变可能对甲状腺滤泡萎缩,破坏具有调控作用。     

Histone deacetylase 9 downregulation decreases tumor growth and promotes apoptosis in non‐small cell lung cancer after melatonin treatment

Histone deacetylase 9 functions as an oncogene in a variety of cancers, but its role on non‐small cell lung cancer (NSCLC) has not been reported. Melatonin was proven to possess anticancer actions, whereas its effect on NSCLC and underlying mechanisms remains poorly understood. In this study, 337 patients with complete clinicopathologic characteristics who underwent NSCLC surgery were recruited for the study. We found that NSCLC patients with high HDAC9 expression were correlated with worse overall survival and poor prognosis. HDAC9 knockdown significantly reduced NSCLC cell growth and induced apoptosis both in vivo and in vitro. Melatonin application also markedly inhibited cell proliferation, metastasis, and invasion and promoted apoptosis in NSCLC cells. Moreover, RNA‐seq, real‐time quantitative polymerase chain reaction, and western blot analyses showed that melatonin treatment decreased the HDAC9 level in NSCLC cells. A mechanistic study revealed that HDAC9 knockdown further enhanced the anticancer activities of melatonin treatment, whereas HDAC9 overexpression partially reversed the melatonin's anticancer effects. Additionally, the in vivo study found melatonin exerted anti‐proliferative and pro‐apoptotic effects on xenograft tumors which were also strengthened by HDAC9 knockdown. These results indicated that HDAC9 downregulation mediated the anti‐NSCLC actions of melatonin, and targeting HDAC9 may be the novel therapeutic strategy for NSCLC.     

Darbepoetin alfa exerts a cardioprotective effect in autoimmune cardiomyopathy via reduction of ER stress and activation of the PI3K/Akt and STAT3 pathways

Dilated human cardiomyopathy is associated with suppression of the prosurvival phosphatidylinositol-3-kinase (PI3K)/Akt and STAT3 pathways. The present study was carried out to determine if restoration of the PI3K/Akt and STAT3 activity by darbepoetin alfa improved cardiac function or reduced cardiomyocyte apoptosis in rabbit autoimmune cardiomyopathy induced by a peptide corresponding to the second extracellular loop of the ß₁-adrenergic receptor (ß₁-EC_II). We found that ß₁-EC_II immunization produced progressive LV dilation, systolic dysfunction and myocyte apoptosis as measured by TUNEL, single-stranded DNA antibody, and active caspase-3. These changes were associated with activation of p38 mitogen-activated protein kinase (MAPK), endoplasmic reticulum stress markers (GRP78 and CHOP), and increased cleavage of procaspase-12, as well as decreased phosphorylation of Akt and STAT3, and decreased Bcl2/Bax ratio. As expected, darbepoetin alfa treatment increased phosphorylation of Akt and STAT3. It also increased the myocardial expression of erythropoietin receptor which was reduced in the failing myocardium, and improved cardiac function in the ß₁-EC_II-immunized animals. The latter was associated with reductions of myocyte apoptosis and cleaved caspase-3, as well as reversal of increased phosphorylation of p38-MAPK, increased ER stress, and decline in Bcl2/Bax ratio. The anti-apoptotic effects of darbepoetin alfa via Akt and STAT activation were also demonstrated in cultured cardiomyocytes treated with the anti-ß₁-EC_II antibody. These effects of darbepoetin alfa in vitro were prevented by LY294002 and STAT3 peptide inhibitor. Thus, we conclude that darbepoetin alfa improves cardiac function and prevents progression of dilated cardiomyopathy probably by activating the PI3K/Akt and STAT3 pathways and reducing ER stress.     

急性心肌梗死心肌细胞中Bax和Bcl-2基因的mRNA表达量及其与凋亡的关系

目的 :探讨急性心肌梗死心肌细胞中Bax和Bcl 2基因的mRNA表达量及其与凋亡的关系。方法 :新西兰兔 4 8只 ,随机分为不结扎冠状动脉的正常对照 (oh)及结扎冠状动脉后 1h ,2h ,3h ,4h ,6h ,8h及 12h共8组。TUNEL法检测梗死区心肌细胞凋亡 ,逆转录PCR法检测Bax和Bcl 2基因在心肌细胞中的mRNA表达量 ,探讨它们之间的相互关系。结果 :兔梗死区心肌细胞凋亡千分率在冠状动脉结扎后 1h开始增多 ,4h达高峰 ,其后逐渐下降 ,到 12h降至正常。Bax基因mRNA的表达量在冠状动脉结扎后 2h开始升高 ,4h达高峰 ,持续 6h后逐渐下降 ,至 12h接近正常。Bcl 2基因mRNA表达量在冠状动脉结扎后 2h开始明显下降 ,4h达最低点 ,持续 2h后开始回升 ,到 12h时已恢复到比正常高的水平。两种基因mRNA表达量的对数值之间呈负相关 ,心肌细胞凋亡数与Bac/Bcl 2基因mRNA比值呈正相关。结论 :兔梗死区心肌细胞存在明显的凋亡现象 ,Bax基因使其上调、Bcl 2基因使其下调 ,Bax/Bcl 2基因mRNA表达比值调控细胞凋亡     

Inhibitory effect of norcantharidin on the growth of human gallbladder carcinoma GBC-SD cells in vitro

Introduction P rimary gallbladder carcinoma is a lethal and aggressive malignant neoplasm with a dormant course, difficult diagnosis, early metastasis or recurrence, and poor prognosis.[1-3] The only potentially curative therapy for gallbladder carcinoma is surgical resection. Unfortunately, most patients with this type of cancer present with advanced andunresectable disease-only 10%-30% of patients in culture medium overnight, they were treated with can be considered for surgery on presentati…     

神经生长因子对体外培养的垂体催乳素瘤细胞的抑制作用

目的 研究神经生长因子（ＮＧＦ）和溴陷亭（ＢＣ）对体外培养的垂体乳素（ＰＲＬ）分泌腺瘤细胞的激素分泌量和增殖的影响。方法 每例体外培养的垂体ＰＲＬ分泌腺瘤细胞分成４组;对照组、ＮＧＦ组、ＢＣ组和ＮＧＦ＋ＢＣ组,对不同的药物干预,观察培养液不激素分泌量和＾３Ｈ－胸腺啶脱氧核苷（ＴｄＲ）摄取率的改变。结果 在８例培养成活的垂体ＰＲＬ分泌腺瘤细胞中,与对照组相比,ＮＧＦ对ＰＲＬ分泌量的抑制作用不明显,但     

Bcl?2与Bax基因在结直肠癌合并血吸虫病患者体内表达及临床病理意义

目的　探讨Bcl?2和Bax基因在结直肠癌（CRC）合并血吸虫病患者体内的表达水平及其临床病理意义。方法　以2016年6月—2020年6月在大理大学第一附属医院接受手术治疗的CRC患者作为研究对象,采用随机数字表法从单纯CRC患者和CRC合并血吸虫病患者中各随机抽取30例作为CRC组和CRC?S组。收集两组患者癌组织和CRC组患者癌旁组织,采用实时荧光定量PCR法和免疫组织化学法检测样本中Bcl?2和Bax mRNA和蛋白表达水平,应用HE染色法观察并比较两组患者癌组织细胞凋亡率。结果　共纳入研究对象60例,其中CRC组和CRC?S组均30例,两组患者性别构成（[χ2] = 0.271,P＞0.05）、平均年龄（t = -0.596,P＞0.05）、结直肠肿瘤生长方式（[χ2] = 0.275,P＞0.05）、肿瘤部位（[χ2] = 4.008,P＞0.05）、肿瘤浸润深度（[χ2] = 0.608,P＞0.05）、肿瘤分化程度（[χ2] = 0.364,P＞0.05）及肿瘤是否脉管转移（[χ2] = 1.111,P＞0.05）差异均无统计学意义,但肿瘤组织学类型、肿瘤是否淋巴结转移和TMN分期差异均有统计学意义（[χ2] = 5.963、8.297、5.711,P均＜0.05）。与CRC组患者癌旁组织相比,CRC组和CRC?S组患者癌组织中Bcl?2、Bax mRNA和蛋白表达水平均升高（P均＜0.05）;与CRC组相比,CRC?S组患者癌组织中Bcl?2 mRNA和蛋白表达水平均升高（P均＜0.05）,Bax mRNA和蛋白水平均降低（P均＜0.05）。CRC组和CRC?S组患者肿瘤组织细胞凋亡率分别为42.00%和25.35%,差异有统计学意义（[χ2] = 41.500,P = 0.000）。结论　血吸虫病可能通过影响细胞凋亡信号通路中Bcl?2和Bax基因表达而参与CRC发生与进展。     

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.     

盐酸美金刚对血管性痴呆大鼠CREB和细胞色素c表达的影响

目的 探讨盐酸美金刚对血管性痴呆(vascular dementia,VaD)大鼠学习记忆功能的影响和可能机制.方法 采用双侧颈总动脉永久结扎法建市大鼠VaD模型.36只SD大鼠分为假手术组、VaD组和美金刚组(灌胃给予盐酸美金刚,1次/d,连续8周),每组12只.用Y型迷官试验观察其行为学改变,用免疫组化技术检测大鼠海马CA1区和皮质神经元环磷酸腺苷反应元件结合蛋白(cAMP responsive element binding protein,CREB)和细胞色素c(cytochrome c,CytC)表达变化.TUNEL染色检测神经元凋亡情况.结果 美金刚组学习和记忆成绩分别为(50.17±5.56)次和(7.08±0.90)次,显著优于VaD组的(66.36±5.41)次和(4.64±1.03)次(P均＜0.01);海马CA1区和皮质CREB平均吸光度分别为147.51±7.11和155.37±4.52,显著高于VaD组的135.11±8.52和142.39±5.24(P均＜0.01);海马CA1区和皮质CytC平均吸光度分别为116.53±4.91和113.51±6.05,显著低于VaD组的136.65±7.43和138.09±4.88(P均＜0.01);美金刚组细胞凋亡程度也较VaD组明显改善.结论 盐酸美金刚可能通过上调CREB表达、抑制CytC释放和神经元凋亡来改善VaD大鼠的学习记忆功能.