普洱茶水提物对巨噬细胞分泌肿瘤坏死因子-α的影响

目的探讨普洱茶水提物对巨噬细胞分泌肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)的影响。方法以佛波酯处理THP-1细胞48 h,分化得到巨噬细胞,以其为炎症细胞模型,用不同浓度的普洱茶水提物(125、250μg/ml)与终浓度为100 EU/ml的脂多糖的混合物作用于巨噬细胞,ELISA法检测细胞培养液中TNF-α的含量。结果普洱茶水提物能有效抑制巨噬细胞炎症因子TNF-α的分泌,且浓度为250μg/ml的普洱茶水提物对TNF-α分泌的抑制作用强于浓度为125μg/ml的普洱茶水提物,呈剂量依赖性。结论普洱茶水提物对巨噬细胞炎症因子TNF-α的分泌具有抑制作用。这种效果与已知的绿茶抗炎的主要成分EGCG无关。     

普洱茶提取物对宫颈癌HeLa细胞的诱导凋亡作用

目的探讨普洱茶提取物对人宫颈癌HeLa细胞系的诱导凋亡作用及其分子机制。方法应用不同浓度的普洱茶提取物(15、30、60、120、250、500μg/ml)作用HeLa细胞后,采用MTT法检测其对细胞增殖的影响;DAPI染色法分析细胞凋亡形态学变化;Annexin V-FITC/PI双染流式细胞术检测细胞早期凋亡;Western blot分析pro-caspase-3和pro-caspase-9的变化。结果普洱茶提取物对HeLa细胞的增殖具有明显的抑制作用,且呈浓度依赖性;经普洱茶提取物处理的细胞荧光显微镜下可见典型的凋亡形态学变化,细胞早期凋亡率显著高于未处理的细胞(P<0.05);经普洱茶提取物处理的细胞pro-caspase-3和pro-caspase-9均被活化。结论普洱茶提取物具有可以诱导HeLa细胞凋亡的天然活性成分,且凋亡途径可能依赖于caspase-3、caspase-9相关的线粒体凋亡途径。     

Potential Antitumor Effects of 6-Gingerol in p53-Dependent Mitochondrial Apoptosis and Inhibition of Tumor Sphere Formation in Breast Cancer Cells

Hormone-specific anticancer drugs for breast cancer treatment can cause serious side effects. Thus, treatment with natural compounds has been considered a better approach as this minimizes side effects and has multiple targets. 6-Gingerol is an active polyphenol in ginger with various modalities, including anticancer activity, although its mechanism of action remains unknown. Increases in the level of reactive oxygen species (ROS) can lead to DNA damage and the induction of DNA damage response (DDR) mechanism, leading to cell cycle arrest apoptosis and tumorsphere suppression. Epidermal growth factor receptor (EGFR) promotes tumor growth by stimulating signaling of downstream targets that in turn activates tumor protein 53 (p53) to promote apoptosis. Here we assessed the effect of 6-gingerol treatment on MDA-MB-231 and MCF-7 breast cancer cell lines. 6-Gingerol induced cellular and mitochondrial ROS that elevated DDR through ataxia-telangiectasia mutated and p53 activation. 6-Gingerol also induced G0/G1 cell cycle arrest and mitochondrial apoptosis by mediating the BAX/BCL-2 ratio and release of cytochrome c. It also exhibited a suppression ability of tumorsphere formation in breast cancer cells. EGFR/Src/STAT3 signaling was also determined to be responsible for p53 activation and that 6-gingerol induced p53-dependent intrinsic apoptosis in breast cancer cells. Therefore, 6-gingerol may be used as a candidate drug against hormone-dependent breast cancer cells.     

USP22 Promotes NSCLC Tumorigenesis via MDMX Up-Regulation and Subsequent p53 Inhibition

Increasing evidence suggests that ubiquitin-specific protease 22 (USP22) has great clinicopathologic significance in oncology. In this study, we investigated the role of USP22 in human NSCLC tumorigenesis along with the underlying mechanisms of action. First, we determined the expression of USP22 in human NSCLC, as well as normal tissues and cell lines. We then studied the effects of USP22 silencing by shRNA on NSCLC cell growth in vitro and tumorigenesis in vivo, along with the effect on the p53 pathway. We found that USP22 is overexpressed in human NSCLC tissues and cell lines. USP22 silencing by shRNA inhibits proliferation, induces apoptosis and arrests cells at the G0/G1 phases in NSCLC cells and curbs human NSCLC tumor growth in a mouse xenograft model. Additionally, USP22 silencing downregulates MDMX protein expression and activates the p53 pathway. Our co-immunoprecipitation analysis shows that USP22 interacts with MDMX in NSCLC cells. Furthermore, MDMX silencing leads to growth arrest and apoptosis in NSCLC cells, and over-expression of MDMX reverses the USP22 silencing-induced effects. Taken together, our results suggest that USP22 promotes NSCLC tumorigenesis in vitro and in vivo through MDMX upregulation and subsequent p53 inhibition. USP22 may represent a novel target for NSCLC treatment.     

Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse

Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2) mouse (nax—naked-ataxia mutant mouse) correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc) degeneration. Loss of Pcs in the nax cerebellum is compartmentalized and harmonized to the classic pattern of gene expression of the cerebellum in the wild type mouse. Usually, degeneration starts in the anterior and posterior zones and continues to the central and nodular zones of cerebellum. Studies have suggested that the p75 neurotrophin receptor (NTR) plays a role in Pc degeneration; thus, in this study, we investigated the p75NTR pattern and protein expression in the cerebellum of the nax mutant mouse. Despite massive Pc degeneration that was observed in the nax mouse cerebellum, p75NTR pattern expression was similar to the HSP25 pattern in nax mice and comparable with wild type sibling cerebellum. In addition, immunoblot analysis of p75NTR protein expression did not show any significant difference between nax and wild type sibling (p > 0.5). In comparison with wild type counterparts, p75NTR pattern expression is aligned with the fundamental cytoarchitecture organization of the cerebellum and is unchanged in the nax mouse cerebellum despite the severe neurodevelopmental disorder accompanied with Pc degeneration.     

The Anti-Tumor Activity of the NEDD8 Inhibitor Pevonedistat in Neuroblastoma

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53^MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53^WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.     

Regulation of p53 and Cancer Signaling by Heat Shock Protein 40/J-Domain Protein Family Members

Heat shock proteins (HSPs) are molecular chaperones that assist diverse cellular activities including protein folding, intracellular transportation, assembly or disassembly of protein complexes, and stabilization or degradation of misfolded or aggregated proteins. HSP40, also known as J-domain proteins (JDPs), is the largest family with over fifty members and contains highly conserved J domains responsible for binding to HSP70 and stimulation of the ATPase activity as a co-chaperone. Tumor suppressor p53 (p53), the most frequently mutated gene in human cancers, is one of the proteins that functionally interact with HSP40/JDPs. The majority of p53 mutations are missense mutations, resulting in acquirement of unexpected oncogenic activities, referred to as gain of function (GOF), in addition to loss of the tumor suppressive function. Moreover, stability and levels of wild-type p53 (wtp53) and mutant p53 (mutp53) are crucial for their tumor suppressive and oncogenic activities, respectively. However, the regulatory mechanisms of wtp53 and mutp53 are not fully understood. Accumulating reports demonstrate regulation of wtp53 and mutp53 levels and/or activities by HSP40/JDPs. Here, we summarize updated knowledge related to the link of HSP40/JDPs with p53 and cancer signaling to improve our understanding of the regulation of tumor suppressive wtp53 and oncogenic mutp53 GOF activities.     

3种茶提取物对MCF-7细胞增殖、凋亡的影响及其与蛋白激酶B的相关性

目的 探讨红茶、绿茶及普洱茶提取物对人乳腺癌细胞MCF-7增殖、凋亡的影响及其与蛋白激酶B(Protein ki-nase B,PKB)的相关性。方法用不同浓度的3种茶提取物处理MCF-7细胞不同时间,MTT法检测其对MCF-7细胞增殖的影响;Annexin V-FITC/PI双染流式细胞术检测其对细胞凋亡的影响;实时定量PCR检测细胞PKB基因mRNA的转录水平。结果 3种茶提取物对MCF-7细胞的增殖均具有明显的抑制作用,且呈时间、剂量依赖性;3种茶提取物处理的细胞早期凋亡率均高于未处理细胞,PKB基因mRNA的表达量明显降低。结论 3种茶提取物均含有可以抑制PKB转录活性的天然成分,并可诱导MCF-7细胞凋亡;其中绿茶诱导MCF-7细胞凋亡效果最明显。     

人类小细胞肺癌细胞转染野生型p53基因发生的凋亡

为了探索p5 3抑癌基因在临床应用中的潜在价值 ,以带有突变型p5 3基因的人类小细胞肺癌细胞株 (LTEP P)为研究对象 ,开发了一种新型的基因转染方法。将构建有野生型p5 3基因cDNA的质粒与阳离子脂质体和转铁蛋白结合成三元复合物 ,再用于LIEP P细胞的基因转染。通过显微观察、吖啶橙荧光染色、琼脂糖凝胶电泳和切口末端标记试验 ,证实细胞发生了凋亡。表明在体外实验中 ,该三元复合物可有效地转染肿瘤细胞 ,而野生型p5 3基因能够诱导肿瘤细胞发生凋亡。     

YueF Overexpression Inhibits Cell Proliferation Partly through p21 Upregulation in Renal Cell Carcinoma

YueF is a novel putative tumor suppressor gene that can inhibit proliferation and induce apoptosis in hepatoma cells, but its role in renal cell carcinoma (RCC) remains unclear. Here, we examined the expression of the YueF gene in RCC tissues and the effect of YueF on cell proliferation in RCC 786-0 cells. The results showed that YueF was expressed at high levels in normal kidney tissues and cell lines but was reduced or absent in RCC tissues and 786-0 cells. Lentivirus-mediated YueF overexpression in RCC 786-0 cells caused cell-cycle arrest in the G1 phase and dramatically reduced proliferation in culture. YueF overexpression resulted in increased protein levels of p53 and p21(WAF1/Cip1), whereas the protein levels of cyclin D1 and pRb were decreased. The proliferation defects caused by YueF overexpression could be partially rescued by the expression of p21 siRNA. These findings suggest a critical role for p21 in the YueF-induced growth inhibition of 786-0 cells and provide novel insights into the mechanism underlying the tumor-suppressive action of YueF.     

Defective Autophagosome Formation in p53-Null Colorectal Cancer Reinforces Crocin-Induced Apoptosis

Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53−/− cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G₁ (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53−/− after 24 h. Crocin induced inefficient autophagy in HCT116 p53−/− cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53−/− after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT116 wild-type cells. Baf-exposed HCT116 p53−/− cells did not, however, show any enhancement of apoptosis induction despite an increase in the DNA damage-sensor accumulation, γH2AX indicating that crocin induced an autophagy-independent classical programmed cell death.     

Overexpression of MicroRNA-30b Improves Adenovirus-Mediated p53 Cancer Gene Therapy for Laryngeal Carcinoma

MicroRNAs play important roles in laryngeal carcinoma and other cancers. However, the expression of microRNAs in paracancerous tissue has been studied less. Here, using laser capture microdissection (LCM), we detected the expression of microRNAs in paracancerous tissues. Among all down-regulated microRNAs in the center area of tumor tissues, only miR-30b expression was significantly reduced in paracancerous tissues compared to surgical margins. Therefore, to further investigate the effect of miR-30b on laryngeal carcinoma, we stably overexpressed miR-30b in laryngeal carcinoma cell line HEp-2 cells. It was found that although there was no significant difference in cell viability between miR-30b overexpressed cells and control HEp-2 cells, p53 expression was obviously enhanced in miR-30b overexpressed cells. Whether miR-30b could improve the anti-tumor effect of adenovirus-p53 (Ad-p53) in laryngeal carcinoma and other cancer cell lines was also evaluated. It was found that in miR-30b overexpressed HEp-2 cells, p53-mediated tumor cell apoptosis was obviously increased both in vitro and in vivo. MDM2-p53 interaction might be involved in miR-30b-mediated anti-tumor effect. Together, results suggested that miR-30b could modulate p53 pathway and enhance p53 gene therapy-induced apoptosis in laryngeal carcinoma, which could provide a novel microRNA target in tumor therapy.     

骨髓间充质干细胞对辐射诱发胸腺损伤修复中cyclin-D1和p53基因mRNA转录水平的影响

目的探讨骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)对辐射诱发小鼠胸腺损伤修复过程中cyclin-D1和p53基因mRNA转录水平的影响,为阐明BMSCs在肿瘤发生发展过程中的作用提供理论依据。方法全骨髓贴壁法体外分离、培养C57BL/6小鼠BMSCs。将C57BL/6小鼠随机分为3组:正常对照组、辐射组和辐射+BMSCs组,辐射组和辐射+BMSCs组行1.75 Gy X线全身照射,每周1次,连续4周,复制电离辐射诱发的胸腺损伤模型;辐射+BMSCs组末次照射后,经尾静脉注入BMSCs,0.2 ml/只,细胞浓度为2.0×106个/ml。3个月后处死各组小鼠,取胸腺组织,采用RT-PCR法检测各组小鼠胸腺组织cyclin-D1和p53基因mRNA的转录水平,流式细胞术检测细胞周期。结果辐射组小鼠胸腺组织cyclin-D1基因mRNA的转录水平高于正常对照组,但差异无统计学意义(P>0.05),而p53基因mRNA的转录水平明显高于正常对照组,且差异有统计学意义(P<0.05)。辐射+BMSCs组小鼠胸腺组织cyclin-D1和p53基因mRNA的转录水平低于辐射组,但差异无统计学意义(P>0.05)。辐射组小鼠胸腺G1期细胞百分比(29.81%)明显低于正常对照组(55.39%),辐射+BMSCs组小鼠胸腺G1期细胞增多(72.08%)。结论 BMSCs可通过抑制cyclin-D1的过度表达和p53基因的突变,使损伤小鼠胸腺细胞停留在G1期进行修复,进而促进组织的修复,抑制肿瘤的增殖。