Melatonin promotes sorafenib‐induced apoptosis through synergistic activation of JNK/c‐jun pathway in human hepatocellular carcinoma

Melatonin has been shown to exert anticancer activity on hepatocellular carcinoma (HCC) through its antiproliferative and pro‐apoptotic effect in both experimental and clinical studies, and sorafenib is the only approved drug for the systemic treatment of HCC. Thus, this study was designed to investigate the combined effect of melatonin and sorafenib on proliferation, apoptosis, and its possible mechanism in human HCC. Here, we found that both melatonin and sorafenib resulted in a dose‐dependent growth inhibition of HuH‐7 cells after 48 hours treatment, and the combination of them enhanced the growth inhibition in a synergistic manner. Colony formation assay indicated that co‐treatment of HuH‐7 cells with melatonin and sorafenib significantly decreased the clonogenicity compared to the treatment with single agent. Furthermore, FACS and TUNEL assay confirmed that melatonin synergistically augmented the sorafenib‐induced apoptosis after 48 hours incubation, which was in accordance with the activation of caspase‐3 and the JNK/c‐jun pathway. Inhibition of JNK/c‐jun pathway with its inhibitor SP600125 reversed the phosphorylation of c‐jun and the activation of caspase‐3 induced by co‐treatment of HuH‐7 cells with melatonin and sorafenib in a dose‐dependent manner. Furthermore, SP600125 exhibited protective effect against apoptosis induced by the combination of melatonin and sorafenib. This study demonstrates that melatonin in combination with sorafenib synergistically inhibits proliferation and induces apoptosis in human HCC cells; therefore, supplementation of sorafenib with melatonin may serve as a potential therapeutic choice for advanced HCC.     

Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways

Aim:

To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action.

Methods:

Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting.

Results:

PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC₅₀ less than 20 μmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 μmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity.

Conclusion:

Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways.     

巨噬细胞移动抑制因子在妇科恶性肿瘤中的研究进展

巨噬细胞移动抑制因子(MIF)是近年研究非常广泛的细胞因子之一。生理情况下,MIF可以介导炎症及免疫反应,参与胚胎期的发育、组织创伤后的修复等。MIF还被证实在妇科恶性肿瘤,如宫颈癌、卵巢癌、子宫内膜癌等的发展过程中发挥重要作用,并与肿瘤的恶性程度密切相关。MIF可以激活多种信号通路,如c-Jun氨基末端激酶(JNK/cJun)、磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)等,通过促进细胞增殖、黏附、转移、血管新生、上皮间质转化和增强免疫抑制等方式加速肿瘤的发展。此外,许多研究都显示,抑制MIF的分泌及生物学活性可以有效减慢,甚至逆转肿瘤的发展,有效的MIF抑制剂有望成为治疗癌症的新希望。     

表里经配穴法对脑缺血再灌注损伤大鼠海马细胞凋亡及JNK信号通路的影响

【目的】观察表里经配穴法对脑缺血再灌注损伤大鼠海马细胞凋亡及c－jun氨基末端激酶（JNK）信号通路的影响,探讨其对脑缺血再灌注损伤保护作用的可能机制。【方法】将120只SD大鼠随机分为假手术组、模型组、表里经配穴电针组、抑制剂组;参照Longa法复制局灶脑缺血再灌注模型,表里经配穴电针组取表里经配穴的足三里、三阴交和尺泽、合谷行电针治疗,每日1次。采用神经行为学评定各组大鼠不同时段神经行为, TUNEL法检测海马细胞凋亡指数,免疫组织化学法检测p－JNK表达。【结果】表里经配穴电针组、抑制剂组神经功能缺损评分均显著低于模型组（P＜0．05）;模型组凋亡指数显著高于假手术组（P＜0．01）,表里经配穴电针组和抑制剂组凋亡指数均显著低于模型组（P＜0．05）;模型组各时段p－JNK表达较假手术组显著增加（P＜0．01）,表里经配穴电针组、抑制剂组p－JNK表达较模型组显著降低（P＜0．05）。【结论】表里经配穴法可不同程度地减轻缺血再灌注后大鼠的神经功能缺损,减少细胞凋亡,其机制可能与抑制JNK信号通路有关。     

铁皮石斛对2型糖尿病大鼠胰岛组织JNK、AKT蛋白磷酸化表达的影响

目的:研究铁皮石斛有效部位对2型糖尿病大鼠胰岛组织JNK蛋白、AKT蛋白磷酸化表达的影响。方法:采用正常大鼠、高脂高糖-链脲佐菌素致糖尿病(STZ-T2DM)模型,研究铁皮石斛降糖机制,Western blot法检测JNK、ph JNK、AKT、ph AKT在大鼠胰岛组织中的表达。结果:与正常对照组相比较,模型组大鼠胰岛组织JNK蛋白磷酸化程度显著增加(P<0.01),Aktser473磷酸化减弱(P<0.01)。与模型组相比较,铁皮石斛TP、TF、TE显著降低大鼠胰岛组织JNK(Thr183/Tyr185)磷酸化,增加Aktser473磷酸化水平。结论:铁皮石斛有效部位具有抑制T2DM大鼠胰岛组织JNK Thr183/Tyr185磷酸化水平,增加AKT ser473磷酸化水平的作用。     

c-jun末端激酶在亚砷酸钠致人胚胎肝细胞损伤中的表达

目的探讨亚砷酸钠染毒对人胚胎肝(L-02)细胞中c-jun末端激酶(JNK)的变化。方法将处于对数生长期的L-02细胞分别暴露于终浓度为0(对照)、50、100、150μmol/L的亚砷酸钠溶液中培养24 h,采用四甲基偶氮唑蓝(MTT)法检测细胞生长情况,采用流式细胞术检测染毒24 h时的细胞周期及细胞凋亡率;采用蛋白杂交(Western-blot)法检测JNK及p-JNK的蛋白表达水平。结果与对照组相比,各浓度亚砷酸钠染毒组L-02细胞的存活率及G0-G1期构成比均较低,JNK、p-JNK的蛋白表达水平和S期构成比及凋亡率均较高;而G2-M期构成比在50μmol/L亚砷酸钠染毒组较低,在100、150μmol/L亚砷酸钠染毒组均较高,差异均有统计学意义(P0.05)。且随着亚砷酸钠染毒浓度的升高,L-02细胞凋亡率及JNK和p-JNK蛋白的表达水平均呈上升趋势,细胞存活率呈下降趋势。结论亚砷酸钠诱导L-02细胞凋亡可能与JNK及p-JNK表达的增加有关。     

肉碱棕榈酰转移酶1对60Co γ射线照射大鼠小肠上皮细胞IEC-6增殖的影响及相关机制研究

目的探索60Co γ射线诱导大鼠小肠上皮细胞(IEC-6)中CPT1A和CPT1B蛋白表达变化, 并进一步研究肉碱棕榈酰转移酶1(CPT1)变化对受照细胞增殖的影响及相关分子机制。方法 IEC-6细胞经棕榈酸、血清饥饿以及血清饥饿联合棕榈酸处理后, 给予0、5、10或15 Gy60Co γ射线照射, 照射后24 h收集细胞并提取蛋白, 利用Western blot方法检测CPT1A和CPT1B蛋白的表达水平变化;ETO是CPT1的小分子抑制剂, 利用克隆形成率实验和CCK-8实验分析ETO抑制CPT1对60Co γ射线照射IEC-6细胞存活及增殖的影响;利用Western blot方法检测5 Gy60Co γ射线照射ETO处理IEC-6细胞48 h后细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基末端激酶(JNK)蛋白表达及磷酸化水平变化。结果棕榈酸处理组中, CPT1A蛋白在15 Gy γ射线照射后表达量显著增加(t=-2.82, P<0.05)。血清饥饿处理组中, CPT1A蛋白在5、10和15 Gy γ射线照射后表达量明显升高(t=-3.28、-8.72、-8.67...