Watching the clock: endoplasmic reticulum-mediated control of circadian rhythms in cancer

Abstract

In the past 20 years both the circadian clock and endoplasmic reticulum (ER) stress signaling have emerged as major players in oncogenesis and cancer development. Although several lines of evidence have established functional links between these two molecular pathways, their interconnection and the subsequent functional implications in cancer development remain to be fully characterized. Herein, we provide an extensive review of the literature depicting the molecular connectivity linking ER stress signaling and the circadian clock and elaborate on the potential use of these functional interactions in cancer therapeutics.     

采用组织芯片技术研究宫颈癌中HPV与细胞周期调控因子p27表达的关系

目的研究宫颈癌组织中HPV感染与细胞周期调控因子p27表达的关系及意义。方法采用组织芯片技术制作60例宫颈癌组织芯片，同时用S—P免疫组织化学和原位分子杂交方法检测宫颈癌组织芯片中HPV、p27、p27mRNA的表达。结果60例宫颈癌组织中HPV、p27、p27mRNA的阳性率分别为48．3％、38．3％、51．7％。淋巴结转移阳性宫颈癌中p27和p27mRNA阳性率明显低于淋巴结转移阴性宫颈癌（P〈0．01）。淋巴结转移阳性宫颈癌中HPV阳性率明显高于淋巴结转移阴性宫颈癌（P〈0．05）。宫颈癌和CINⅢ级宫颈上皮内瘤变组织中HPV阳性率明显高于CINⅠ级和CINⅡ级（P〈0．05）。而p27和p27mRNA阳性率明显低于CINⅠ级和CINⅡ级宫颈上皮内瘤变组织（P〈0．05）。宫颈癌中HPV与p27及p27mR—NA表达呈明显负相关（r=-0．574，P〈0．05；r=-0．612，P〈0．01）。结论HPV感染和细胞周期调控因子p27低表达与宫颈癌密切相关。HPV感染与p27低表达有关。HPV和p27可作为评估宫颈癌预后的参考指标。应用组织芯片大规模高效检测临床组织样本是可行的，具有快速、方便、经济、准确的特点。     

Lobaplatin arrests cell cycle progression,induces apoptosis and alters the proteome in human cervical cancer cell Line CaSki

Cervical cancer is one of the most common gynecologic tumors. There is an upward trend in the incidence. The objective of this research was to explore the effect of lobaplatin on cervical cancer CaSki cells proliferation, cell cycle and apoptosis and analysis of the differential expressed proteins of CaSki cells after exposed to lobaplatin. Our findings have shown that lobaplatin inhibits cell proliferations in human cervical cancer CaSki cells in dose- and time-dependent manner. Flow cytometry assay confirmed that lobaplatin affected cervical cancer cell survival by blocking cell cycle progression in S phase and G0/G1 phase and inducing apoptosis in dose- and time-dependent manner. Lobaplatin treatment reduced polypyrimidine tract-binding protein 2, ribose-phosphate pyrophosphokinase, hypothetical protein, terminal uridylyltransferase 7, ubiquitin specific protease 16 and heterogeneous nuclear ribonucleoprotein A2/B1 expression and increase zinc finger protein 91, zinc finger protein, C-X-C motif chemokine 10 precursor, stromal cell protein and laminin subunit alpha-4 expression. Some of the differentially expressed proteins may be associated with antitumor effect of lobaplatin. Lobaplatin showed a good antitumour activity in in vitro models of human cervical cancer cells. These results indicate that lobaplatin could be an effective chemotherapeutic agent in human cervical cancer treatment by inducing apoptosis, cell cycle arrest and changing many kinds of protein molecule expression level.     

Sonoporation induces apoptosis and cell cycle arrest in human promyelocytic leukemia cells

Despite being a transient biophysical phenomenon, sonoporation is known to disturb the homeostasis of living cells. This work presents new evidence on how sonoporation may lead to antiproliferation effects including cell-cycle arrest and apoptosis through disrupting various cell signaling pathways. Our findings were obtained from sonoporation experiments conducted on HL-60 human promyelocytic leukemia cells (with 1% v/v microbubbles; 1 MHz ultrasound; 0.3 or 0.5MPa peak negative pressure; 10% duty cycle; 1 kHz pulse repetition frequency; 1 min exposure period). Membrane resealing in these sonoporated cells was first verified using scanning electron microscopy. Time-lapse flow cytometry analysis of cellular deoxyribonucleic acid (DNA) contents was then performed at four post-sonoporation time points (4 h, 8 h, 12 h and 24 h). Results indicate that an increasing trend in the apoptotic cell population can be observed for at least 12 h after sonoporation, whilst viable sonoporated cells are found to temporarily accumulate in the G₂/M (gap-2/mitosis) phase of the cell cycle. Further analysis using western blotting reveals that sonoporation-induced apoptosis involves cleavage of poly adenosine diphosphate ribose polymerase (PARP) proteins: a pro-apoptotic hallmark related to loss of DNA repair functionality. Also, mitochondrial signaling seems to have taken part in triggering this cellular event as the expression of two complementary regulators for mitochondrial release of pro-apoptotic molecules, Bcl-2 (B-cell lymphoma 2) and Bax (Bcl-2-associated X), are seen to be imbalanced in sonoporated cells. Furthermore, sonoporation is found to induce cell-cycle arrest through perturbing the expression of various cyclin and Cdk (cyclin-dependent kinase) checkpoint proteins that play an enabling role in cell-cycle progression. These bioeffects should be taken into account when using sonoporation for therapeutic purposes.     

异常光周期所致昼夜节律紊乱对大鼠腓肠肌时钟基因和葡萄糖摄取相关基因表达节律的影响

目的 探讨昼夜节律紊乱(CM)对大鼠腓肠肌时钟基因和葡萄糖摄取相关基因表达节律的影响。方法 雄性Wistar大鼠36只分为昼夜节律正常(CA)组和CM组,每组18只。造模第61天行ClockLab行为学检测。造模第85天行腹腔注射糖耐量试验和生理指标检测。第91~92天于8:00、12:00、16:00、20:00、24:00、4:00处死大鼠,收集腓肠肌组织,逆转录实时定量聚合酶链反应(RT-qPCR)检测Bmal1、Clock、Per2、Tbc1d1、Glut4、Pgc1α的mRNA。结果 CM组昼夜节律周期延长(t = -6.557, P < 0.001)、振幅下降(t = 2.326, P = 0.030),腹腔注射糖耐量试验血糖曲线下面积减少(t = -2.622, P = 0.016)。腓肠肌不同时间Bmal1 (F = 6.691, P < 0.001)、Clock(F = 4.188, P = 0.007)、Per2 (F = 10.893, P < 0.001)、Tbc1d1 (F = 3.411, P = 0.018)、Glut4 (F = 5.439, P = 0.002)、Pgc1α (F = 15.376, P < 0.001)表达有显著性差异,两组间腓肠肌Bmal1 (F = 5.020, P = 0.035)、Per2(F = 8.996, P = 0.006)、Tbc1d1 (F = 51.111, P < 0.001)、Pgc1α (F = 10.177, P = 0.004)表达有显著性差异,CM组腓肠肌24 h总Tbc1d1表达显著下降(t = 4.349, P < 0.001)。结论 异常光周期所致CM可导致大鼠糖耐量下降,这可能与大鼠腓肠肌Tbc1d1表达下降以及Bmal1、Per2、Pgc1α的转录节律改变有关。     

尼美舒利对人胃癌SGC7901细胞生长及细胞周期分布的影响

目的探讨尼美舒利对体外培养的胃癌细胞生长及细胞周期分布的影响。方法以人胃癌细胞株SGC7901为对象,用放射免疫分析法测定细胞培养上清PGE2水平,体外药物敏感实验（MTT）法检测尼美舒利对肿瘤细胞的增殖抑制效应,流式细胞术检测肿瘤细胞周期分布变化情况。结果尼美舒利可减少PGE2产生,尼美舒利对人胃癌细胞株生长的抑制作用呈时间、剂量依赖性效应,尼美舒利可改变胃癌细胞株细胞周期的分布,明显降低其增殖指数。结论尼美舒利可通过抑制环氧合酶-2（COX-2）活性抑制肿瘤细胞的分裂和增殖、阻止细胞周期进展,诱导其凋亡中起重要作用。     

顺铂作用于人肝癌细胞系HepG2后对肿瘤干细胞标志物的影响

目的研究顺铂作用于人肝癌细胞系HepG2后对肿瘤干细胞标志物的影响。方法培养HepG2人肝癌细胞,以对数生长期的细胞为研究对象,分成对照组和实验组,对照组不予特殊处理,实验组予以顺铂处理并测定细胞生长抑制率、移行愈合率,并测定相关肿瘤标记物P53、caspase-3、caspase-8、CD133、腺苷三磷酸结合盒转运体G2(ABCG2)、细胞间黏附分子-1(ICAM-1)等的表达变化。结果细胞生长抑制率:随着药物浓度增加而增强,与对照组比较,差异有统计学意义(P0.05)。细胞移行愈合率:实验组细胞移行愈合率明显低于对照组(P0.05),9μmol/L顺铂组移行愈合率明显低于6μmol/L顺铂组(P0.05)。相关肿瘤标记物P53、caspase-3、caspase-8随着药物浓度增加而各指标表达增强,与对照组相比差异有统计学意义(P0.05)。细胞免疫荧光方法显示随着顺铂浓度的增加,CD133、ABCG2、ICAM-1的表达增强。结论顺铂可能通过上调肿瘤标记物P53、caspase-3、caspase-8的表达,从而抑制人肝癌细胞HepG2的生长,并抑制HepG2细胞的迁移愈合,但会使其耐药性也增加。     

卡莫司汀对人脑肿瘤干细胞增殖及细胞周期的影响

目的:探讨卡莫司汀(BCNU)对人脑肿瘤干细胞在细胞增殖及细胞周期方面的影响。方法:取初发的室管膜瘤手术切除标本1例,取材后制备为单细胞,分别接种于无血清培养基获得肿瘤干细胞,接种于含血清培养基获得肿瘤细胞。不同浓度的BCNU作用于细胞72h后,MTT法检测细胞在体外对BCNU的敏感性,并用终浓度为0.01μg/mL的BCNU作用细胞不同的时间段,通过流式细胞仪检测BCNU对细胞周期的影响。结果:(1)MTT显示,BCNU能抑制肿瘤干细胞及肿瘤细胞的生长,其对肿瘤干细胞(IC50=0.157μg/mL)的抑制略低于对肿瘤细胞(IC50=0.072μg/mL)(P<0.05)。(2)细胞周期方面,与阴性对照比较,两类细胞均表现为随药物作用时间的延长,S期细胞比例增高,并伴有凋亡细胞的增多,但肿瘤干细胞的改变(24h)出现晚于肿瘤细胞(6h)。结论:BCNU对体外培养的脑肿瘤干细胞生长有明显的抑制作用,作用机制可能为通过干扰S期DNA的合成起作用;与脑肿瘤细胞相比,脑肿瘤干细胞在MTT及细胞周期的检测中,均表现出对BCNU有一定的耐药性。     

Initiation of apoptosis,cell cycle arrest and autophagy of esophageal cancer cells by dihydroartemisinin

Dihydroartemisinin (DHA) has recently been shown anti-tumor activity in various cancer cells. However, its effect on esophageal cancer remains unclear. In this study, for the first time, we demonstrated that DHA reduced viability of esophageal cancer cells in a dose-dependent manner. The mechanism was at least partially due to DHA induced apoptosis by upregulating the expression of Bax, downregulating Bcl-2, Bcl-xL and Procaspase-3, and increasing caspase-9 activation, induced cell cycle arrest by downregulating cyclin E, CDK2 and CDK4. Furthermore, we firstly found that DHA induced autophagy in cancer cells. We concluded DHA might be a novel agent against esophageal cancer.     

Lobetyol activate MAPK pathways associated with G1/S cell cycle arrest and apoptosis in MKN45 cells in vitro and in vivo

AimThe agent lobetyol, which is isolated from Lobelia chinensis, was previously shown to be cytotoxicity againts several cancer cell lines in published report. Today, we perform a study in vitro and in vivo to analyze its anti-carcinoma effect in MKN45 cells and to explore the molecular mechanism.Main methodsThe growth inhibition of lobetyol on MKN45 cells was analyzed with MTT and flow cytometry. Hoechst 33342 staining and TUNEL cover glass staining were used to provide the visual evidence of apoptosis. Western blotting assay was performed to study the activation or blocking of related signaling pathways.Key findingsLobetyol induce apoptosis and cell cycle arrest in a time- and dose-dependent manner in MKN45 cells in our study. This process is mediated by the MAPK signaling pathways.This study confirmed the cytotoxicity of lobetyol in MKN45 cells and provided an insight into the molecular mechanism, which demonstrates the potential of lobetyol as an anti-tumor agent.     

Cell proliferation and cell cycle control: a mini review

Tumourigenesis is the result of cell cycle disorganisation, leading to an uncontrolled cellular proliferation. Specific cellular processes-mechanisms that control cell cycle progression and checkpoint traversation through the intermitotic phases are deregulated. Normally, these events are highly conserved due to the existence of conservatory mechanisms and molecules such as cell cycle genes and their products: cyclins, cyclin dependent kinases (Cdks), Cdk inhibitors (CKI) and extra cellular factors (i.e. growth factors). Revolutionary techniques using laser cytometry and commercial software are available to quantify and evaluate cell cycle processes and cellular growth. S-phase fraction measurements, including ploidy values, using histograms and estimation of indices such as the mitotic index and tumour-doubling time indices, provide adequate information to the clinician to evaluate tumour aggressiveness, prognosis and the strategies for radiotherapy and chemotherapy in experimental researches.     

siRNA靶向沉默hTERT基因对人胰腺癌Capan-2细胞增殖、凋亡和周期的影响

目的研究hTERT—siRNA对人胰腺癌Capan-2细胞增殖、凋亡、细胞周期的影响。方法利用RNAi技术靶向沉默Capan-2的hTERT基因表达，应用细胞直接计数法和流式细胞术（FCM）检测Capan-2细胞的增殖、凋亡和细胞周期的变化。结果在hTERT-siRNA的使用浓度为50nm、Lipo转染浓度为3μl／2ml转染体积和hTERT沉默效率为100％的条件下，转染hTERT—siRNA24h后，细胞生长已明显减慢，抑制细胞增殖率为26．39％（P〈0．05），第2、3、5、7天抑制细胞增殖率分别为46．77％、70．61％、84．71％和85．99％（P〈0．001）。随着转染细胞按常规培养时间的延长，早期凋亡细胞明显增多（P〈0．001），以24h后更明显；活性细胞明显减少（P〈0．001），而损伤细胞明显增多（P〈0．001），以6h后明显；死亡细胞明显增多（P〈0．001），以24h后更明显。siRNA组与阴性对照组和Lipo对照组均有明显差异（P〈0．001）。G0-G1期细胞明显增多（P〈0．01），S期细胞明显减少（P〈0．01），以24h后明显；G2～M期细胞明显减少（P〈0．01），以48h后明显；晚期凋亡细胞增多（P〈0．05），以48h后明显。结论hTERT—siRNA可抑制人胰腺癌Capan-2细胞生长，使较多的细胞停留在G0～G1期，而进入G2～M期和S期的细胞减少，可促进细胞凋亡。     

沉默RAGE对人卵巢癌细胞株增殖、凋亡能力及周期分布的影响

目的 研究沉默晚期糖基化终产物受体(receptor for advanced glycosylation end products,RAGE)对人卵巢癌细胞株增殖、凋亡能力及周期分布的影响.方法 人上皮性卵巢癌SKOV-3细胞经过慢病毒载体构建,分为空白组、过表达组和沉默组.进行细胞增殖、细胞凋亡、细胞周期实验,We...     

过表达SOCS5对非小细胞肺癌增殖、迁移和侵袭的作用及机制研究

摘要：目的?分析细胞因子信号传导抑制因子5(SOCS5)对非小细胞肺癌(NSCLC)细胞增殖、迁移和侵袭能力的影响，并探讨其可能的作用机制。方法?应用癌症基因组图谱(TCGA)数据库分析SOCS5在肺癌组织中的表达情况； western blot检测SOCS5在NSCLC细胞系PC-9、A549和SPC-A-1及正常支气管上皮细胞系16HBE中的表达水平；选择SOCS5表达水平最低的NSCLC细胞系进行SOCS5过表达质粒转染，实验设对照(NC)组和SOCS5过表达质粒转染(oe-SOCS5)组。采用MTS试验检测各组细胞增殖能力；划痕试验检测各组细胞迁移能力；Boyden试验检测各组细胞侵袭能力；western blot检测各组细胞中PI3K/Akt/mTOR信号通路活性。结果?TCGA数据库分析结果显示，SOCS5?mRNA在肺腺癌和肺鳞癌组织中的表达水平显著低于正常肺组织(P<0.05)。western blot结果显示，SOCS5蛋白在PC-9、A549和SPC-A-1细胞中的表达水平均低于其在16HBE细胞中的表达水平(P<0.05)；选择表达水平最低的PC-9细胞进行SOCS5过表达质粒转染，结果发现与NC组相比，oe-SOCS5组PC-9细胞增殖、迁移和侵袭能力均降低(P<0.05)；western blot结果显示，与NC组相比，oe-SOCS5组细胞pPI3K、pAkt和pmTOR蛋白的表达水平均降低(P<0.05)。结论?SOSC5在NSCLC中呈异常低表达，过表达SOCS5可抑制NSCLC细胞的增殖、迁移和侵袭能力，抑制PI3K/Akt/mTOR信号通路的激活可能是作用机制之一。     

虫草素调节非小细胞肺癌细胞株细胞周期、凋亡及相关抑癌基因的实验研究

目的 研究虫草素对非小细胞肺癌细胞株H358细胞周期、凋亡及相关抑癌基因的调节作用。方法 培养非小细胞肺癌细胞株H358并分组,空白对照组用不含药物的DMEM处理,虫草素组用含有50μmol/L、100μmol/L、200μmol/L虫草素的DMEM处理,处理24 h后测定细胞周期分布、细胞凋亡率、抑癌基因表达水平。结果 不同剂量虫草素处理后,H358细胞的S期比率、凋亡率及PTEN、p53、p16及Caspase-3、Caspase-8、Caspase-9的mRNA表达水平明显高于空白对照组,G2/M期比率明显低于对照组(P <0. 05),且200μmol/L虫草素处理后,H358细胞的S期比率、凋亡率及PTEN、p53、p16及Caspase-3、Caspase-8、Caspase-9的mRNA表达水平明显高于其他浓度虫草素处理的细胞,G2/M期比率明显低于其他浓度虫草素处理的细胞(P <0. 05)。结论 虫草素处理非小细胞肺癌细胞株能够使细胞周期停滞、细胞凋亡加剧,增加抑癌基因表达是虫草素发挥以上调节作用的分子途径,并呈剂量依赖性。     

盐酸埃克替尼诱导肺癌HCC827细胞周期阻滞及其机制研究

目的 研究国家一类新药盐酸埃克替尼（Icotinib）对人非小细胞肺癌细胞HCC827的增殖抑制及细胞周期的影响,并探讨其作用机制.方法 实验分为空白组、对照组和Icotinib处理组,采用四甲基偶氮唑盐法（MTT）检测Icotinib对人肺癌HCC827细胞增殖的影响;流式细胞仪检测细胞周期变化;Western blot检测相关蛋白表达,应用SPSS 13.0进行统计学分析.结果 Icotinib以时间-剂量依赖的方式抑制HCC827细胞增殖,48 h的IC50为0.60 μmol/L,72 h的IC50为0.06 μmol/L;Icotinib诱导HCC827细胞发生明显的G1期阻滞并具有剂量依赖性.进一步对周期相关蛋白检测发现,Icotinib处理组较对照组相比,显著上调p21蛋白表达,抑制cyclin D1及cyclin A表达,但对cyclin E作用不明显.检测还发现Icotinib处理组明显下调ERK的磷酸化水平.结论 Icotinib能够明显抑制HCC827细胞增殖,引起细胞G1期阻滞,其机制可能与上调p21及抑制cyclin D1、cyclin A蛋白表达水平相关.并且MAPK/ERK信号通路在其介导的生物学效应中起重要作用.     

基质金属蛋白酶9在鉴别肾细胞癌和肾盂移行细胞癌中的意义

目的：探讨基质金属蛋白酶-9在鉴别肾细胞癌和肾盂移行细胞癌中的应用。方法：采用酶联免疫法对肾细胞癌患者,肾盂移行细胞癌患者和正常人血清及尿中基质金属蛋白酶-9的浓度进行测定。结果：肾细胞癌和肾盂移行细胞癌组患者尿基质金属蛋白酶-9均高于正常对照组（P〈0.05）;肾盂移行细胞癌组尿基质金属蛋白酶-9的浓度亦明显高于肾细胞癌组（P〈0.05）;而各组血清中基质金属蛋白酶-9值差异无统计学意义（P〉0.05）。结论：检测尿中基质金属蛋白酶-9的浓度对于鉴别肾细胞癌和肾盂移行细胞癌有重要意义。     

Inhibition of G1 cell cycle arrest in human gingival fibroblasts exposed to phenytoin

Gingival overgrowth is caused in response to the antiepileptic drug phenytoin (PHT). PHT‐induced gingival overgrowth is characterized by the proliferation of fibroblasts and increased collagen formation in gingiva. Fibroblast proliferation is regulated through the cell cycle. Thus, in the present study, we examined the effects of PHT on the cell cycle, the expression of cell cycle control proteins and the proliferation in human gingival fibroblasts (hGFs). Cells were stimulated in serum‐free DMEM with or without 0.25 μm PHT. Subsequently, the cell cycle phase distribution and the protein expression after 24 h and the cell proliferation after 24, 48 and 72 h were evaluated. PHT significantly inhibited synchronization at the G₀/G₁ phase of the cell cycle in hGFs through serum starvation. Stimulation with PHT for 48 and 72 h significantly induced a proliferative response in hGFs. PHT decreased the expression of the Cdk‐inhibitory proteins p21 and p27 and increased the levels of the S phase‐promoting proteins phospho‐Thr160‐Cdk2 and phospho‐Ser807/811‐Rb in serum‐free DMEM. The inhibition of G₁ cell cycle arrest in hGFs may result from an increase in phosphorylated Cdk2 and Rb proteins and decreased levels of p21 and p27 proteins by PHT. The gingival overgrowth may be caused by the failure of the G1 cell cycle arrest in GFs exposed to PHT.