Inhibition of stromal‐interacting molecule 1‐mediated store‐operated Ca2+ entry as a novel strategy for the treatment of acquired imatinib‐resistant gastrointestinal stromal tumors

Imatinib has revolutionized the treatment of gastrointestinal stromal tumors (GIST); however, primary and secondary resistance to imatinib is still a major cause of treatment failure. Multiple mechanisms are involved in this progression. In the present study, we reported a novel mechanism for the acquired resistance to imatinib, which was induced by enhanced Ca²⁺ influx via stromal‐interacting molecule 1 (STIM1)‐mediated store‐operated Ca²⁺ entry (SOCE). We found that the STIM1 expression level was related to the acquired resistance to imatinib in our studied cohort. The function of STIM1 in imatinib‐resistant GIST cells was also confirmed both in vivo and in vitro. The results showed that STIM1 overexpression contributed to SOCE and drug response in imatinib‐sensitive GIST cells. Blockage of SOCE by STIM1 knockdown suppressed the proliferation of imatinib‐resistant GIST cell lines and xenografts. In addition, STIM1‐mediated SOCE exerted an antiapoptotic effect via the MEK/ERK pathway. The results from this study provide a basis for further research into potential novel therapeutic strategies in acquired imatinib‐resistant GIST.     

Induction of HSP70 gene expression by modulation of Ca+2 ion and cellular p53 protein by curcumin in colorectal carcinoma cells

Curcumin (diferuloyl methane) is the major active yellow pigment of turmeric and curry. Studies in recent years have indicated that curcumin is a potent inhibitor of the initiation and promotion of chemical carcinogen-induced skin carcinogenesis in mice. When COLO205 colorectal carcinoma cells were treated with curcumin (60 μM), the appearance of apoptotic DNA ladders was delayed about 5 h, and G₁ arrest was detected. Further analysis of the endonuclease activities in these cells revealed that the activity of Ca⁺²-dependent endonuclease in COLO205 cells was profoundly inhibited and that the extent of inhibition depended on the degree of calcium depletion. The reduction of p53 gene expression was accompanied by the induction of HSP70 gene expression in the curcumin-treated cells. These findings suggest that curcumin may induce the expression of the HSP70 gene through the initial depletion of intracellular Ca⁺², followed by the suppression of p53 gene function in the target cells. © 1996 Wiley-Liss, Inc.     

Orai1对鼻咽癌细胞侵袭和上皮-间充质转化的影响及其机制

目的： 探讨Orai1对鼻咽癌细胞侵袭和上皮-间充质转化的影响及其作用机制。方法： 培养鼻咽上皮细胞NP69和4种鼻咽癌细胞系（CNE1、CNE2、HONE1和5-8F）,分别采用实时荧光定量PCR（qPCR）和Western blot法检测细胞中Orai1的mRNA和蛋白表达水平。采用靶向Orai1的短发夹RNA（shRNA）质粒,转染CNE2细胞,设转染对照质粒的CNE2细胞为阴性对照组,钙离子成像检测钙库调节的钙离子内流,Transwell小室检测细胞转移和侵袭,qPCR和Western blot法检测上皮-间充质转化（EMT）标志物E钙黏蛋白（E-cadherin）、N钙黏蛋白（N-cadherin）和波形蛋白（Vimentin）的mRNA和蛋白表达水平。结果： NP69、CNE1、CNE2、HONE1和5-8F细胞中均表达Orai1,且CNE2细胞中表达相对较高,故选择CNE2细胞进行后续试验。与阴性对照组相比,转染Orai1 shRNA后,CNE2细胞中Orai1的mRNA和蛋白水平均显著降低（P<0.05）;CNE2细胞的钙库调节钙离子内流能力、转移/侵袭能力和EMT相关标志蛋白E-cadherin、N-cadherin和Vimentin的mRNA和蛋白表达水平均显著下降（P<0.05）。结论： 抑制Orai1表达可抑制鼻咽癌细胞钙库调节钙离子内流、转移侵袭和EMT进程,提示Orai1可能成为治疗鼻咽癌转移的新靶标和新方向。     

不同转移潜能鼻咽癌细胞钙电流特征与细胞迁徙能力的相关性

背景与目的:肿瘤转移活性与瘤细胞以迁徙运动潜能为代表的生物力学特性有关,而其迁移运动的生物力学机制又受制于细胞内的钙离子活动和钙电流特征.本文探讨不同转移潜能鼻咽癌单克隆亚系细胞钙电流特征,及与细胞迁徙运动潜能的相关性.方法:人鼻咽癌高转移潜能细胞株5-8F及低转移潜能细胞株6-10B分别与10%含药血清(由黄芪、党参、白花蛇舌草等组成)共同培养,MTT法检测含药血清对细胞增殖的影响,Western blot检测nm23-H1蛋白表达水平.利用全细胞膜片钳制方法观察细胞钙电流特征(药物作用20 min),划痕实验观察细胞迁移能力(药物作用24 h),比较分析其相关性.结果:6-10B细胞的nm23-H1表达水平(2.9±0.4)较5-8F细胞(2.3±0.21)高(P＜0.05).5-8F细胞内钙释放激活的钙电流ICRAC为(-1.39±0.36)nA,与6-10B细胞lCRAC[(-0.66±0.40)nA]相比差异有统计学意义(P＜0.05).通过划痕区的5-8F细胞数(350±3)也显著高于6-10B细胞(246±1)(P＜0.05).应用含药血清干预后,5-8F和6-10B细胞增殖活性差异无统计学意义(P＞0.05),5-8F细胞的nm23-H1表达水平(3.9±0.1)明显高于6-10B细胞(1.0±0.1)(P＜0.05).干预后,5-8F细胞ICRAC降至(-1.27±0.35)nA,平均抑制幅度为(1.90±0.47)%;6-10B细胞则降至(-0.37±0.23)nA,平均抑制幅度为(0.46±0.12)%,两者差异有统计学意义(P＜0.05),并出现与钙电流变化特点平行的细胞迁移能力变化,通过划痕线的5-8F细胞数明显减少(94±6),而6-10B细胞则受影响甚小(229±6,P＜0.05).干预前后5-8F细胞迁移能力变化具有显著性(P＜0.05),6-10B细胞则无显著性差异(P＞0.05).结论:不同转移潜能鼻咽癌细胞钙电流特征及细胞迁移能力以及nm23-H1表达存在明显差异.应用含药血清干预后,高转移潜能细胞5-8F出现了与细胞钙电流ICRAC降低幅度相平行的细胞迁移能力抑制,药物血清可增加5-8F细胞的nm23-H1活性.     

Elevated Orai1 expression mediates tumor-promoting intracellular Ca2+ oscillations in human esophageal squamous cell carcinoma

Effective treatment as well as prognostic biomarker for malignant esophageal squamous cell carcinoma (ESCC) is urgently needed. The present study was aimed at identifying oncogenic genes involving dysregulated intracellular Ca²⁺ signaling, which is known to function importantly in cellular proliferation and migration. Tumors from patients with ESCC were found to display elevated expression of Orai1, a store-operated Ca²⁺ entry (SOCE) channel, and the high expression of Orai1 was associated with poor overall and recurrence-free survival. In contrast to the quiescent nature of non-tumorigenic epithelial cells, human ESCC cells exhibited strikingly hyperactive in intracellular Ca²⁺ oscillations, which were sensitive to treatments with Orai1 channel blockers and to orai1 silencing. Moreover, pharmacologic inhibition of Orai1 activity or reduction of Orai1 expression suppressed proliferation and migration of ESCC in vitro and slowed tumor formation and growth in in vivo xenografted mice. Combined, these findings provide the first evidence to imply Orai1 as a novel biomarker for ESCC prognostic stratification and also highlight Orai1-mediated Ca²⁺ signaling pathway as a potential target for treatment of this deadly disease.     

NCX1在肝癌中的表达、调控Ca2+浓度及其对肝癌细胞增殖和迁移的影响

背景与目的：钠钙交换体亚型1(Na+-Ca2+ exchanger isoform 1,NCX1)可通过对细胞Ca2+平衡的调节参与癌症的发生,但是否参与肝癌的发生、发展及其作用机制的研究鲜见报道。该研究旨在探讨NCX1在肝癌中的表达变化,对人肝癌细胞HepG2增殖、迁移能力的影响及其可能的机制。方法：运用实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)和蛋白[质]印迹法(Western blot)检测NCX1 mRNA及蛋白在人正常肝细胞株LO2、肝癌细胞株HepG2、人正常肝组织和原发性肝细胞癌患者癌组织中的表达。采用共聚焦显微镜技术观察NCX1在细胞外无钠溶液刺激活化后,对正常肝细胞LO2及肝癌细胞HepG2中钙离子浓度的调控。采用MTT法、细胞划痕实验检测NCXl特异性的阻断剂KB-R7943对人肝癌细胞HepG2增殖、迁移的影响。结果：在肝癌细胞株HepG2和肝细胞癌组织中,NCX1 mRNA和蛋白质的表达均高于正常肝细胞株LO2和正常肝组织(P<0.05)。共聚焦显微镜实验发现,细胞外无钠溶液可以激活细胞内钙升高,正常肝细胞LO2及肝癌细胞HepG2细胞内钙离子浓度均增高,但肝癌细胞HepG2细胞内钙离子增高的幅值明显高于LO2细胞(P<0.05),NCXl特异性阻断剂KB-R7943可以显著阻断胞外无钠诱导的细胞内钙离子升高(P<0.05)。KB-R7943可以显著抑制肝癌细胞HepG2的增殖及迁移(P<0.05)。结论：原发性肝癌中NCX1的表达量上调,NCX1的活化可以调节细胞内钙变化,抑制NCX1的活性可以进一步抑制肝癌细胞的增殖和迁移。这提示NCX1可能在原发性肝癌的发生、发展中起了重要的作用。     

水杨酸钠诱导HL-60细胞凋亡及其与胞内游离Ca~(2+)浓度的关系

 目的　目的研究水杨酸钠对HL 6 0细胞的促凋亡活性及其与胞内游离Ca2 + 浓度的关系。方法Annexin V结合分析检测靶细胞磷脂酰丝氨酸外化 ;琼脂糖凝胶电泳检测断裂DNA ;激光共聚焦显微镜分析细胞内游离Ca2 + 浓度变化。结果　HL 6 0细胞经 5mmol/L(IC50 )水杨酸钠分别作用 10h、16h后 ,靶细胞的磷脂酰丝氨酸外化率显著高于对照组 (0 .2 4vs 0 .0 3) ,其基因组DNA在琼脂糖凝胶电泳中呈典型的梯状带型 ;此间 ,HL 6 0细胞内游离Ca2 + 浓度进行性升高。结论　水杨酸钠触发的HL 6 0细胞凋亡与胞内游离Ca2 + 浓度升高密切相关。     

Ca2+在顺铂诱导人卵巢癌SKOV3细胞自噬反应中的作用

背景与目的：Ca2+在维持细胞生物活性方面扮演着很重要的角色,其在细胞内的储存、释放和摄取主要受内质网调节,细胞内Ca2+浓度的稳态是维持细胞生物能量代谢、蛋白质折叠和分泌的基础条件。本研究探讨Ca2+在顺铂诱导SKOV3细胞内质网应激-自噬反应中的作用机制。方法：取人卵巢癌SKOV3细胞系为研究对象,按以下步骤分组：①探讨顺铂诱导内质网应激与自噬反应,用6μg/mL的顺铂处理SKOV3细胞0、6、12和24 h;②了解顺铂和毒胡萝卜内酯(thapsigargin, TG)诱导内质网应激释放的Ca2+与自噬的关系,分别用TG和顺铂处理SKOV3细胞0、9和12 h;③探究Ca2+对自噬的作用机制,分成对照组、顺铂组、TG组、BAPTA-AM组、顺铂联合BAPTA-AM组和TG联合BAPTA-AM组。用蛋白[质]印迹法(Western blot)检测内质网应激相关蛋白GRP78和自噬标志性蛋白LC3蛋白的表达水平;用Fluo-4钙离子荧光探针检测细胞质中的Ca2+浓度变化;间接免疫荧光染色后,用共聚焦显微镜检测LC3蛋白的表达情况。结果：SKOV3细胞经6μg/mL顺铂作用6 h时GRP78灰度值(1.393±0.004)与其对照组(0.679±0.011)相比显著提高(t=113.2,P=0.000),在12 h时LC3灰度值(0.072±0.002)与其对照组(0.038±0.000)相比显著提高(t=25.5,P=0.000)。间接免疫荧光结果显示,顺铂(6μg/mL)组和TG(3μmol/L)组随作用时间的延长,细胞内LC3荧光斑点会逐渐增多,并伴随着细胞质Ca2+浓度上升。后经钙离子络合剂BAPTA-AM干预后,细胞内LC3荧光强度进一步增强。Westren blot结果显示,顺铂组LC3灰度值(0.039±0.000)小于顺铂联合BAPTA-AM组(0.071±0.001),TG组(0.035±0.001)小于TG联合BAPTA-AM组(0.065±0.001),差异有统计学意义(P=0.000)。结论：顺铂诱导SKOV3细胞内质网应激和自噬的发生,并伴随着细胞质内Ca2+浓度的上升。络合细胞质内Ca2+能增强顺铂诱导的自噬反应。     

Ca2＋荧光蛋白探针Cameleon和荧光染料探针fluo-3测定H2O2刺激的A549细胞中ca2＋浓度变化

目的：本研究旨在观察ca2＋荧光蛋白探针Cameleon和荧光染料探针fluo-3在H2O2刺激的A549细胞中的应用，实时测定细胞质ca2＋浓度（[ca2＋]i），并比较两种ca2＋探针在荧光曲线及[ca2＋]i测定值等方面的差异。方法：采用Ca“荧光蛋白探针CameleonYC3．6转染A549细胞，24h后用50mmol／LH2O2刺激细胞，激光扫描共聚焦显微镜实时测定选取细胞的[ca2＋]i变化。采用ca2＋荧光染料探针fluo-3负载细胞，1h后用50mmol／L H2O2刺激细胞，激光扫描共聚焦显微镜实时测定选取细胞的[ca2＋]i变化。采用DAPI染色试剂盒观察H2O2刺激后细胞凋亡情况。结果：采用Cameleon探针转染的细胞中，反映[ca2＋]i变化的R值曲线迅速升高后逐渐降至刺激前水平，通过公式计算发现[ca2＋]i变化范围是33．1—596．1nmol／L。采用fluo-3探针负载的细胞中，反映[ca2＋]i变化的荧光强度F值曲线逐渐升高至稳定，通过公式计算得到[ca2＋]i变化范围是131．8—695．6nmol／L。同时发现经H2O2刺激后，凋亡细胞百分比显著增加（P〈0．01）。结论：ca2＋荧光蛋白探针Cameleon YC3．6和荧光染料探针fluo-3均检测到H2O2诱导的细胞内[ca2＋]i变化．Cameleon YC3．6可能更灵敏地反映快速变化的钙信号。     

喉癌组织中HPV16／18E6、p53和EZH2的表达及其相关性分析

目的 检测HPV16／18E6、p53和EZH2在喉鳞状细胞癌（喉癌）组织中的表达,探讨它们在肿瘤发生发展过程中的相互作用。方法 采用免疫组化En Vision法检测78例喉癌、15例癌旁正常组织、20例声带息肉中HPV16／18E6、p53和EZH2蛋白的表达情况,分析它们的表达与喉癌临床病理特征之间的关系以及三者之间的相关性。结果 喉癌组织、癌旁正常组织、声带息肉中HPV16／18E6蛋白的阳性表达率分别为47.4％（37／78）、20.0％（3／15）和0（0／20）;p53蛋白阳性表达率为60.3％（47／78）、33.3％（5／15）和0（0／20）;EZH2蛋白阳性表达率为65.4％（51／78）、40.0％（6／15）和5.0％（1／20）,差异均具统计学意义（P＜0.05）。HPV16／18E6、EZH2的阳性表达率均随肿瘤T分期升高而增高（P＜0.05）;淋巴结转移组HPV16／18E6、p53阳性表达率高于无淋巴结转移组（P＜0.05）;p53阳性表达率随分化程度升高而增高（P＜0.05）。p53和EZH2阳性表达表达一致（kappa值=0.451,P＜0.05）。结论 HPV16／18E6、p53和EZH2蛋白表达与喉癌的发生关系密切。EZH2过表达与p53失活有关,二者在促进喉癌发展过程中发挥重要作用。     

miR‐149* induces apoptosis by inhibiting Akt1 and E2F1 in human cancer cells

microRNAs (miRNAs) play vital roles in several biological processes, including apoptosis, by negatively regulating the expression of target genes. The molecular mechanisms of the key survival signal, Akt family, have been widely explored. However, it remains to be ascertained whether Akt1, the predominant isoform in most tissue, is a direct target of miRNA. In this study, we identified Akt1 and E2F1 to be two direct targets of miR‐149* and b‐Myb to be an indirect target by reporter assays and Western blot analyses. Ectopic expression of miR‐149*‐induced apoptosis in Be2C, a neuroblastoma cell line, and in HeLa cells. Silencing of Akt1 or E2F1 expression also led to similar apoptotic changes in these two cell lines, suggesting that the pro‐apoptotic effects of miR‐149* were exerted by repressing Akt1 and E2F1 expressions. Importantly, analysis of primary neuroblastoma samples revealed a significant inverse correlation of miR‐149* with E2F1 expressions (P = 0.026). Interestingly, using the reporter assays, excess miR‐149 introduced by transfection to simulated its preponderance in the in vivo condition, could not overcome the repressive function of miR‐149* on the target genes. This implies that the pro‐apoptotic function of miR‐149* may not be dampened by its predominant cognate, miR‐149, in vivo. Our findings not only provided the first evidence that Akt1 is a direct target of miRNA but also demonstrated that miR‐149* is a pro‐apoptotic miRNA by repressing the expression of Akt1 and E2F1. © 2010 Wiley‐Liss, Inc.     

Ca2+-induced changes in energy metabolism and viability of melanoma cells.

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. We show here that a rise in intracellular-free calcium ion (Ca2+), induced by Ca2+-ionophore A23187, exerted a deleterious effect on glycolysis and viability of B16 melanoma cells. Ca2+-ionophore caused a dose-dependent detachment of phosphofructokinase (EC 2.7.1.11), one of the key enzymes of glycolysis, from cytoskeleton. It also induced a decrease in the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis. All these changes occurred at lower concentrations of the drug than those required to induce a reduction in viability of melanoma cells. We also found that low concentrations of Ca2+-ionophore induced an increase in adenosine 5'-triphosphate (ATP), which most probably resulted from the increase in mitochondrial-bound hexokinase, which reflects a defence mechanism. This mechanism can no longer operate at high concentrations of the Ca2+-ionophore, which causes a decrease in mitochondrial and cytosolic hexokinase, leading to a drastic fall in ATP and melanoma cell death. The present results suggest that drugs which are capable of inducing accumulation of intracellular-free Ca2+ in melanoma cells would cause a reduction in energy-producing systems, leading to melanoma cell death.     

Bcl-2和Beclin-1在肝癌组织中的表达及其相关性

目的:探讨Bcl-2和Beclin-1在肝癌(hepatocellular carcinoma,HCC)患者术后复发中的作用,并分析肝癌组织中Bcl-2和Beclin-1的蛋白表达是否具有相关性。方法:收集肝癌组织114例,采用免疫组织化学法检测各组中 Bcl-2蛋白和Beclin-1蛋白的表达情况。与患者临床资料进行统计学分析。结果:利用χ2检验发现Bcl-2在HCC中的表达仅与患者的肿瘤个数程度相关,Beclin-1在HCC中的表达仅与患者的肿瘤分化程度相关,差异具有统计学意义。HCC中Bcl-2和Beclin-1蛋白的表达呈负相关（r =-0.61,P＜0.000 1）。Bcl-2表达阳性影响患者的预后(Log-rank=8.892,P=0.002 9),Beclin-1表达阴性影响患者的预后(Log-rank=5.350,P=0.020 7)。多因素分析显示肿瘤的肝内复发与肝内肿瘤个数、Bcl-2表达有关。结论:Bcl-2和Beclin-1在肝癌中具有相关性,共同影响患者的预后。     

Ca2+信号对肝癌细胞基质金属蛋白酶分泌与活化的影响

目的　探讨Ca2 信号调节对肝癌细胞基质金属蛋白酶 (MMPs)分泌与活化的影响。方法　在体外人SMMC 772 1肝癌细胞培养体系中 ,引入Ca2 及其激动剂和抑制剂。应用SDS 聚丙烯酰胺 (SDS PAGE)蛋白电泳和明胶酶谱电泳分析方法 ,观察肝癌细胞培养上清中MMPs的分泌与活化水平的变化。结果　人SMMC 772 1肝癌细胞MMP 2和MMP 9的分泌和活化随着培养体系中Ca2 浓度升高而升高 ,在 0 .8mmol/LCa2 浓度时 ,达稳定水平 ,其中以MMP 2的分泌和活化最明显 ,较 0mmol/LCa2 组升高 (10 9.71± 2 7.93) % (P <0 .0 0 1)。在无血清培养条件下 ,肝癌细胞分泌蛋白经电泳鉴定结果提示主要为MMPs。细胞内Ca2 库释放诱导剂毒胡萝卜内酯 (Tg ,4 μmol/L)可明显诱导MMP 2和MMP 9的分泌和活化 ,MMPs的分泌和活化总量较对照组升高 (5 8.6 3± 31.0 4 ) % (P <0 .0 5 ) ;而抑制剂S 亚硝基 乙酰青霉胺 (SNAP ,2 0 0 μmol/L)则抑制了Tg的诱导作用。 结论　肝癌细胞内Ca2 信号的调控在MMPs的分泌活化过程中发挥重要的作用     

A strategy for selective anti-cancer drug concentration increase in rat glioma tissue with Ca2+-channel blocker co-administration: calcium kinetics in intra-glioma arteriolar smooth muscle cells

Summary A rat glioma model was employed to estimate the Ca²⁺ kinetics in the tumor arteriolar smooth muscle cells. Electron microcytochemistry revealed that the density of intracellular Ca²⁺ deposits in the intra-tumor arteriolar smooth muscle cells was significantly greater, with slightly higher membrane Ca²⁺-adenosine triphosphatase (ATPase) activity, compared to the contralateral cerebral arterioles. Furthermore, the administration of tyrphostin, a tyrosine kinase inhibitor, specifically increased only the intra-tumor blood flow. These findings suggest that the condition of the intra-tumor arteriole alters the susceptibility to contraction by the accelerated Ca²⁺ influx into the cytoplasm mediated through the tyrosine kinase pathway. After the administration of diltiazem, which also has a blocking effect on the Ca²⁺-channel mediated through this pathway, the local intra-tumor blood flow showed an increase of 39% with a marked decrease of intracellular Ca²⁺ concentration of the arteriolar smooth muscle cells in the tumor, while the blood flow in the basal ganglia increased by only 8%. The intra-tumor concentration of Nimustine-HCI (ACNU) with co-administration of diltiazem was significantly increased compared to that without the co-administration. Co-administration of diltiazem may be a valuable strategy in chemotherapy for glioma in affording the selective increase of intra-tumor concentration of the anti-cancer drug.     

Investigation of the Relationship between the Intracellular Ca^2＋ Levels and Caspases-3 and 8 Expression in Rat Mammary Gland Carcinoma Undergoing Apoptosis

OBJECTIVE To investigate the relationship between the level of cas-pase-3 and 8 expression and intracellular Ca2 levels in BCML-TA299 breast cancer cells in the process of apoptosis. METHODS Mice were divided into three IFNа-treated groups and one control group as follows: an intratumoral injection, subcutaneous injection, preventive injection, and a control without injection. The cellular DNA content, changes in the cell cycle and the relationship between the cell Ca2 concen-trations and the expression of caspase-3 and 8 were examined. RESULTS After injection of IFN-α-2b by different routes, the morphologic transformation of the breast cancer cells in each group was observed. There was a typical apoptotic response in the intratumoral-injection group. The expression of caspase-3 and 8 was diverse among the experimental groups, and correlated with the cellular Ca2 concentration. Caspase-3 and 8 expres-sion and the cellular Ca2 level were higher following intratumoral injection compared to the other treatments (P<0.01). Among the experimental groups, the cell cycle displayed definitive changes. CONCLUSION a) Both caspase-3 and 8 and the intracellular Ca2 are elevated in the process of cell apoptosis in BCML-TA299 breast cancer tis-sues. These changes may play important roles in the occurrence and devel-opment of breast cancer; b) Variation in the route of IFN-α-2b administration can produce different responses in the expression of caspase-3 and 8 and the concentration of Ca2 in apoptotic tumor cells.     

The MEK/ERK pathway mediates COX-2-selective NSAID-induced apoptosis and induced COX-2 protein expression in colorectal carcinoma cells

Nonsteroidal antiinflammatory drugs (NSAIDs) can prevent colorectal tumorigenesis in humans and in rodents. In vitro and in vivo studies indicate that one of their principal antineoplastic avenues is the induction of apoptosis. We have shown previously that NS-398, which selectively inhibits cyclooxygenase-2 (COX-2) over cyclooxygenase-1, induces apoptosis of colorectal tumour cells and elevates COX-2 protein expression. Here, we have determined that the extracellular signal-regulated kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway mediates these effects of NS-398. Treatment of HT29 colorectal carcinoma cells with 75 microM NS-398 caused activation of ERK-1/-2 but not of the p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. This was apparent at 24 hr and maintained at 72 hr. U0126, a specific inhibitor of the ERK-activating kinases MEK-1/-2, prevented the activation of ERK induced by NS-398 and blocked the increase in COX-2 protein expression seen when HT29 cells were treated with NS-398 alone. The activation of ERK by NS-398 preceded and accompanied a decrease in attached cell yield and an increase in apoptosis. U0126 dose-dependently protected HT29 cells from these antiproliferative effects of NS-398, indicating an antiproliferative role for sustained ERK-1/-2 activation in response to this NSAID. These results point to a key role for the MEK/ERK signalling pathway in mediating the effects of a COX-2-selective NSAID on colorectal carcinoma cells.     

Influence of the Calmodulin Antagonist EBB on Cyclin B1 and Cdc2-p34 in Human Drug-resistant Breast Cancer MCF-7/ADR Cells

OBJECTIVE To investigate the influence of O-(4-ethoxyl-butyl)- berbamine(EBB)on the expression of cyclin B1 and cdc2-p34 in the human drug-resistant breast cancer MCF-7/ADR cell line. METHODS The MTT assay was used to assess the cytotoxicity of EBB.Different levels of EBB were added to different cell lines at series of time points solely or combined with doxorubicin(DOX) to detect the effect on the expression of cyclinB1 and cdc2-p34 by Western blots.cdc2-p34 tyrosine phosphorylation was detected by immunoprecipitation.In addition,apoptosis and cytoplastic Ca 2 concentrations were systematically examined by laser scanning confocal microscopy(LSCM). RESULTS EBB showed little inhibitory activity on human umbilical vein endothelial cells(ECV304),whereas EBB inhibited cell growth(IC50 range,4.55~15.74μmol/L)in a variety of sensitive and drug-resistance cell lines.EBB also down-regulated the expression of cyclin B1 and cdc2-p34 in a concentration and time dependent manner,which was an important reason for the G2/M phase arrest.EBB was shown to induce apoptosis of MCF-7/ADR cells while increasing the level of cytoplastic Ca 2 . CONCLUSION The low cytotoxicity of EBB suggests it may be useful as a rational reversal agent.The effect of EBB on cell cycle arrest and related proteins,apoptosis,and cytoplastic Ca 2 concentration may be involved in reversing multidrug resistance.