罗格列酮联用维甲酸在体内对直肠癌HCT-15细胞COX-2、MMP-7、TIMP-1表达的影响

目的：研究罗格列酮（ROS）联用全反式维甲酸（ATRA）对直肠癌裸鼠移植瘤HCT-15细胞COX-2、MMP-7、TIMP-1表达的影响,并初步探讨其抗肿瘤的机制。方法：建立直肠癌裸鼠移植瘤模型,荷瘤裸鼠随机分为未用药组、ROS组（ROS 25 mg.kg-1.2d-1）、ATRA组（ATRA 11mg.kg-1.2d-1）、ATRA联用ROS组[（ROS 25 mg＋ATRA 11 mg）.kg-1.2d-1]。灌胃40 d后,观察各组裸鼠移植瘤体积变化;利用免疫组化SP法观察移植瘤细胞中COX-2、MMP-7、TIMP-1的表达。结果：1）用药3组瘤体体积与未用药组比较均缩小,差别有统计学意义（P〈0.05）,ATRA联用ROS组的荷瘤体积缩小更明显（P〈0.05）;ROS组与ATRA组瘤体体积相当（P〉0.05）;2）用药3组移植瘤细胞内COX-2、MMP-7、TIMP-1的表达与未用药组比较均降低（P〈0.05）,且ROS组与ATRA组移植瘤细胞内COX-2、MMP-7、TIMP-1下降更明显（P〈0.01）,ROS组与ATRA组移植瘤细胞内三者的表达相当（P〉0.05）。结论：ROS与ATRA均有一定的抑瘤作用,ROS与ATRA联用可发挥协同抗肿瘤的作用,可能是通过抑制移植瘤细胞内COX-2、MMP-7、TIMP-1的表达而实现。     

罗格列酮联用维甲酸在体内对直肠癌HCT-15细胞COX-2、MMP-7、IMP-1表达的影响

目的:研究罗格列酮(ROS)联用全反式维甲酸(ATRA)对直肠癌裸鼠移植瘤HCT-15细胞COX-2、MMP-7、TIMP-1表达的影响,并初步探讨其抗肿瘤的机制.方法:建立直肠癌裸鼠移植瘤模型,荷瘤裸鼠随机分为未用药组、ROS组(ROS 25 mg·kg-1·2d-1),ATRA组(ATRA 11 mg·kg-1·2d-1)、ATRA联用ROS组f(ROS 25 mg+ATRA 11 mg),mg·kg-1·2d-1.灌胃40d后,观察各组裸鼠移植瘤体积变化;利用免疫组化SP法观察移植瘤细胞中COX-2、MMP-7、TIMP-1的表达.结果:1)用药3组瘤体体积与未用药组比较均缩小,差别有统计学意义(P< 0.05),ATRA联用ROS组的荷瘤体积缩小更明显(P<0.05);ROS组与ATRA组瘤体体积相当(P>0.05);2)用药3组移植瘤细胞内COX-2、MMP-7、TIMP-1的表达与未用药组比较均降低(P<0.05),且ROS组与ATRA组移植瘤细胞内COX-2、MMP-7、TIMP-1下降更明显(P<0.01),ROS组与ATRA组移植瘤细胞内三者的表达相当(P>0.05),结论:ROS与ATRA均有一定的抑瘤作用,ROS与ATRA联用可发挥协同抗肿瘤的作用,可能是通过抑制移植瘤细胞内COX-2、MMP-7、TIMP-1的表达而实现.     

全反式维甲酸对人结肠癌细胞亚株SW480/M5增殖和凋亡的影响

目的 观察全反式维甲酸(ATRA)对人结肠癌细胞亚株SW480/M5增殖和凋亡的影响.方法 噻唑蓝(MTT)比色法检测1、2、4、8μmol/L ATRA作用24、48、72 h对SW480/M5细胞的抑制率.流式细胞仪检测8 μmol/L ATRA作用24、48、72 h及2、4μmol/L ATRA作用48 h的肿瘤细胞周期和凋亡率.结果 ATRA抑制SW480/M5细胞增殖作用呈一定时效和量效依赖性;8μmol/LATRA作用72 h明显抑制肿瘤细胞增殖,抑制率接近30％.2、4μmol/L ATRA作用SW480/M5细胞48 h或8μmol/L作用24 h,肿瘤细胞G1期比例开始降低,S期比例增加(P＜0.05).8umol/L ATRA作用48 h后,S期比例进一步增高,G2/M期细胞比例不增加;作用72 h后,G1期细胞比例进一步下降,伴G2/M期细胞比例增加(P＜0.05).8μmol/L ATRA作用24 h无明显诱导SW480/M5细胞凋亡作用,作用48 h或72 h可诱导肿瘤细胞凋亡,但凋亡率差异无统计学意义(P＞0.05).结论 8μmoL/LATRA作用48 h或72 h通过阻滞SW480/M5细胞在S期或(和G2/M)期,并诱导肿瘤细胞凋亡,可明显抑制肿瘤细胞增殖.     

重组人生长激素减少人结肠癌细胞株放疗诱导的细胞凋亡

目的：探讨重组人生长激素（rhGH）对结肠直肠癌细胞株放疗敏感性的影响,并研究其与细胞凋亡的关系。方法：应用流式细胞术及免疫荧光法检测9个人结肠直肠癌细胞株表面生长激素受体（GHR）的表达水平;应用克隆形成实验检测结肠直肠癌细胞经照射后的增殖能力,从而评估其放疗敏感性;应用流式细胞术（Annexin V-FITC染色）检测放疗诱导的细胞凋亡;应用Western blot方法检测rhGH干预后Akt磷酸化水平的变化。结果：从9个细胞株中选择HCT-8为GHR阳性表达细胞,LoVo细胞为阴性表达对照。rhGH显著提高了GHR阳性表达的HCT-8细胞经放疗后的克隆形成率[在高剂量8Gy照射下尤为明显,（52.1±2.9）%比（21.0±2.7）%,P〈0.001],同时减少了细胞凋亡（P〈0.05）;而对GHR阴性表达的LoVo细胞作用不明显（P〉0.05）。rhGH能诱导HCT-8细胞Akt快速磷酸化,呈PI-3K依赖（P〈0.001）。结论：rhGH使GHR阳性表达的结肠直肠癌细胞对放疗有抵抗作用,这种作用可能与其减少细胞凋亡有关。     

尼美舒利对结肠腺癌细胞MMP-2表达的影响

目的 探讨选择性环氧合酶-2（COX-2）抑制剂——尼美舒利对体外培养的结肠癌细胞的生长及对基质金属蛋白酶-2（MMP-2）表达的影响。方法实验分为尼美舒利组、二甲亚砜（DMSO）对照组及不接种细胞的空白对照组，采用MTT法检测不同浓度的尼美舒利对人结肠癌细胞株HT-29（COX-2高表达）及HCT-116（COX-2低表达）增殖的抑制效应；采用改良明胶酶谱分析法检测MMP-2的表达。结果尼美舒利对人结肠癌细胞株HT-29和HCT-116生长的抑制作用呈时间、剂量依赖性，且对HT-29细胞的抑制作用强于HCT-116细胞；尼美舒利抑制了HT-29细胞的MMP-2表达，而对HCT-116细胞MMP-2的表达的抑制作用不明显。结论尼美舒利可明显抑制COX-2高表达的结肠癌细胞株HT-29的增殖和生长，提示尼美舒利可能通过抑制COX-2活性而降低细胞MMP-2的表达。     

非甾体类抗炎药NS398诱导肾癌细胞凋亡的作用及其机制

目的观察COX-2选择性抑制剂NS398的对肾癌细胞增殖和凋亡作用的影响及其作用机制。方法采用标准的细胞培养方法对人肾癌786-0细胞进行培养，将NS398分别以25、50、100、150、200μmol／L的剂量加入细胞中作用24及48h后，应用MTT法检测NS398对肾癌细胞增殖的影响；将NS398以50、100及200μmol／L的浓度作用于肾癌786-0细胞24h后，用流式细胞仪测定细胞凋亡的情况；将NS398分别以25、50、100、150、200txmol／L的剂量加入细胞中作用24h后，应用RT．PCR和Western blotting分别检测肾癌786-0细胞中COX-2和bcl-2的mRNA及其蛋白表达的情况。结果NS398对肾癌786-0细胞具有较强的抑制作用，且这种抑制作用随浓度和时间的增加而增大，呈浓度依赖关系（P〈0．05）；NS398作用肾癌786-0细胞24h后，在细胞周期G0／G1期前出现明显的亚二倍体凋亡峰，随着浓度升高凋亡峰亦越来越增高（P〈0．05）；RT-PCR和Western Blot结果表明，不同浓度NS398作用下的肾癌786-0细胞中，COX-2和bcl-2无论在mRNA水平还是在蛋白水平均明显减弱，且呈剂量梯度下降。结论肾癌786-0细胞中存在着COX-2的过表达，选择性COX-2抑制剂NS398通过诱导凋亡来抑制肾癌786-0细胞的增殖；其机制可能是通过抑制COX-2的表达，导致bcl-2抗凋亡活性的降低来完成的。     

结肠直肠癌病人血浆金属蛋白酶及其组织抑制物的表达及意义

目的：探讨结肠直肠癌病人血浆中基质金属蛋白酶-9（MMP-9）及其组织抑制因子-1（TIMP-1）的表达与结肠直肠癌浸润、转移及预后的关系,以及手术、化疗对其表达的影响,以期在分子水平上更准确地判断结肠直肠癌的预后。方法：选取结肠直肠癌病人50例,于手术前、术后10d、6次化疗后2周,分别抽取病人4mL外周静脉血,采用酶联免疫分析法检测MMP-9、TIMP-1血浆浓度的变化。结果：低分化结肠直肠癌病人血浆MMP-9及TIMP-1水平高于高、中分化者（P〈0．01、P〈0．05）;TNMⅢ、Ⅳ期病人高于TNMⅠ、Ⅱ期者（P〈0．01）。手术后血浆MMP-9、TIMP-1水平显著下降,有统计学意义（P〈0．01、P〈0．05）,化疗后其浓度变化无显著性差异（P〉0．05）。结论：MMP-9和TIMP-1与肿瘤恶性程度有关;术前检测外周血MMP-9和TIMP-1浓度有可能成为结肠直肠癌辅助诊断及病情评估的较好血清学标志。MMP-9可能与肿瘤复发或转移存在一定关系,术后动态检测外周血MMP-9浓度可反映肿瘤负荷。从而对监测肿瘤复发提供一定帮助。     

1,25二羟维生素D3对胆管癌细胞系QBC939增殖和凋亡的影响

目的 探讨1,25二羟维生素D3[1,25(OH)2 D3]对胆管癌细胞系QBC939的体外增殖及凋亡的影响.方法 将不同浓度的1,25(OH)2 D3与胆管癌细胞系QBC939共同培养,采用MTT法测定细胞增殖能力、显微镜观察细胞形态学的改变、流式细胞仪检测细胞周期与凋亡、免疫细胞化学观察bcl-2的表达.结果 0.1～0.5μmol/L的1,25(OH)2 D3对胆管癌细胞QBC939有抑制作用,呈剂量依赖性.经1,25(OH)2 D3作用72h后细胞G1期比例升高,S期比例下降,其中0.5μmol/L组细胞G1期由(50.3±1.0)%上升至(65.5±3.2)%,S期由(39.4±0.5)%下降至(23.6±0.7)%;并且可诱导细胞产生凋亡,0.5μmol/L组作用后细胞凋亡率由0.5%上升至24.6%;bcl-2的表达下调.结论 1,25(OH)2 D3能抑制胆管癌细胞系QBC939增殖并诱导细胞凋亡,其引起凋亡的机制可能与下调bcl-2的表达相关.     

白藜芦醇对人胰腺癌PANC-1细胞增殖与侵袭的影响

目的 探讨白藜芦醇对人胰腺癌PANC-1细胞增殖与侵袭能力的影响.方法 实验分为空白对照组,0.1%DMSO组,白藜芦醇组(50、100、200 μmol/L).MTT法检测白藜芦醇对细胞增殖的影响;流式细胞仪检测细胞凋亡及周期变化;Transwell侵袭小室检测白藜芦醇对细胞侵袭的影响;荧光实时定量PCR和Western blot检测白藜芦醇对细胞Bax、Bcl-2、MMP-2、MMP-9表达的影响.数据以-x±s表示,多组比较采用方差分析.结果 (1)空白对照组抑制率为0;0.1%DMSO组细胞抑制率为3.25%±0.42%;白藜芦醇50 μmol/L组细胞抑制率为13.23%±1.68%;白藜芦醇100μmol/L组细胞抑制率为42.25%±3.20%;白藜芦醇200μmol/L组细胞抑制率为56.94%±5.31%.各组比较,差异有统计学意义(F=460.10,P<0.05).(2)空白对照组细胞凋亡率为0.05%±0.03%;0.1%DMSO组细胞为凋亡率为3.39%±1.77%;白藜芦醇50 μmol/L组细胞凋亡率为6.92%±1.85%;白藜芦醇100 μmol/L组细胞凋亡率为19.05%±2.01%;白藜芦醇200 μmol/L组细胞凋亡率为27.17%±6.43%.各组比较,差异有统计学意义(F=38.84,P<0.05).(3)0.1%DMSO组对细胞周期无显著影响.白藜芦醇引起PANC-1细胞G0/G1期和S期阻滞,G2/M期细胞减少.(4)空白对照组平均穿膜细胞数为61±13;0.1%DMSO组为54±13;白藜芦醇50 μmol/L组为48±15;白藜芦醇100 μmol/L组为23±6;白藜芦醇200 μmol/L组为18±7.各组比较,差异有统计学意义(F=69.08,P<0.05).(5)白藜芦醇可使PANC-1细胞Bax表达升高,Bcl-2表达下调.MMP-2、MMP-9的表达明显受到抑制,mRNA和蛋白水平变化一致.结论 白藜芦醇可明显抑制胰腺癌PANC-1细胞增殖,诱导细胞凋亡并抑制其侵袭能力.     

塞来昔布协同顺铂P13K/Akt途径诱导骨肉瘤MG-63细胞凋亡的实验研究

目的 探讨环氧化酶-2(COX-2)选择性抑制剂塞来昔布对人骨肉瘤MG-63细胞增殖和凋亡的影响及作用机制.方法 塞来昔布(50 μmol/L或100 μmol/L)和(或)顺铂(10 μg/ml)作用骨肉瘤MG-63细胞48 h后,MTT法测定细胞增殖的抑制率,电子显微镜和流式细胞仪检测细胞凋亡,RT-PCR法检测基因水平COX-2表达变化,Western blot检测COX-2及凋亡相关蛋白表达变化.P13K抑制剂Wortmannin作用MG-63细胞48 h,检测蛋白表达变化.结果 塞来昔布导致MG-63细胞阻滞在G1期,通过激活半胱氨酸天冬氨酸蛋白酶(caspase)-9的内源性凋亡途径诱导细胞凋亡;顺铂单药作用后骨肉瘤MG-63细胞凋亡率为5.93%,而联合应用塞来昔布50μmol/L或100 μmol/L后,凋亡率分别为6.66%和37.15%,与顺铂联合具有明显的协同作用;COX-2蛋白表达未降低.塞来昔布联合顺铂明显降低P13K/Akt、survivin、Bcl-2的表达,检测到caspase-9、caspase-3的激活和PARP裂解片段.Wortmannin作用MG-63细胞48 h,检测到pAkt(Thr308)、Bcl-2、survivin表达下调.结论 塞来昔布可通过非COX-2途径诱导骨肉瘤MG-63细胞凋亡,与P13K/Akt、survivin、Bcl-2蛋白相关,并且PI3K/Akt途径在survivin、Bcl-2表达调控中发挥重要作用.这可能是塞来昔布药物干预的中心环节.     

表没食子儿茶素没食子酸酯对结肠癌细胞株HCT-8和HT29的抑制作用及影响机制

目的探讨绿茶提取物表没食子儿茶素没食子酸酯（EGCG）对结肠癌细胞株HCT－8细胞和HT29细胞增殖的抑制作用,研究其对HESl与JAGl基因表达的影响。方法体外培养HCT-8细胞和HT29细胞,采用不同浓度的EGCG（10、20、35mg／L）对其进行干预,MTT法检测EGCG对HCT-8细胞和HT29细胞增殖的抑制作用：用流式细胞仪检测EGCG对HCT-8细胞和HT29细胞细胞凋亡和细胞周期的影响。实时荧光定量PCR检测EGCG干预后的HCT-8细胞和HT29细胞的HESl与JAGl基因的表达情况。结果EGCG对HCT-8细胞和HT29细胞增殖及凋亡均有影响,3个EGCG浓度对HCT-8增殖抑制率分别为（28．894±5．076）％,（34．903±1．794）％和（39．028±0．105）％;对HT29的增殖抑制率分别为（14．682±4．244）％、（22．429±3．847）％和（29．840±5．076）％。EGCG能将HCT-8细胞阻滞在G／M期,阻碍其向M期转换,将HT29细胞阻滞在S期,阻碍其向G2期转换,抑制其细胞增殖。EGCG可下调两株细胞HESl的基因表达,但差异均无统计学意义（P〉0．05）：EGCG能上调两株细胞JAGl的基因表达,但只有HCT．8细胞的基因表达差异有统计学意义（O．201±0．018比0．440±0．077．P=0．029）。结论EGCG对体外培养的HCT-8细胞和HT29细胞的增殖有明显抑制作用．且能诱导细胞凋亡和影响细胞周期。其作用机制可能与上调JAGl的基因表达有关。     

全反式维甲酸诱导膀胱癌Ｔ２４细胞株凋亡的研究

目的 :探讨全反式维甲酸 (ATRA)对人膀胱癌 T2 4细胞株的凋亡诱导作用。方法 :应用荧光显微镜、流式细胞仪及 DNA琼脂糖凝胶电泳等技术研究 T2 4细胞凋亡。结果 :用 3× 10 - 6 m ol/ L 的 ATRA处理 T2 4细胞6天 ,荧光显微镜下计数 T2 4细胞凋亡率为 15 .2 0 % ,对照组为 0 .0 1% (P <0 .0 5 ) ;流式细胞仪测定 T2 4细胞凋亡率为 15 .31% ,对照组为 1.49% ;T2 4细胞 DNA琼脂糖凝胶电泳呈现凋亡特征性梯形条带。结论 :ATRA对人膀胱癌 T2 4细胞株有凋亡诱导作用 ,为 ATRA应用于膀胱癌临床治疗提供了实验依据     

Cyclic stretch enhances apoptosis in human lumbar ligamentum flavum cells via the induction of reactive oxygen species generation

Objective: The lumbar ligamentum ?avum (LF) is an important part of the spine to maintain the stability of the spine. In this study we aimed to examine whether mechanical force by cyclic stretch could induce apoptosis in human LF cells and investigate the underlying mechanism.

Methods: LF cells were isolated from six young patients undergoing spinal surgery and then cultured in vitro. LF cells were subjected to cyclic stretch and the poptosis was detected by flow cytometry. The level of intracellular reactive oxygen species (ROS) and caspase-9 activity were measured.

Results: Cyclic stretch at a frequency of 0.5?Hz with 20% elongation induced the apoptosis of human LF cells in vitro, and this was correlated with increased ROS generation and activation of caspase-9.

Conclusion: Our study suggests that cyclic stretch-induced apoptosis in human LF cells may be mediated by ROS generation and the activation of caspase-9.     

Retinoic acid slows progression and promotes apoptosis of spontaneous prostate cancer

BACKGROUND: All-trans retinoic acid (ATRA) promotes terminal differentiation in epithelial cells and anti-angiogenesis and thus, may have beneficial effects in an intervention therapy for prostate cancer. METHODS: We used the autochthonous spontaneous transgenic adenocarcinoma of the mouse prostate (TRAMP) model system to test the ability of ATRA to prevent initiation and progression of prostate cancer in a pre-clinical setting. RESULTS: Initial studies demonstrated that exposure of TRAMP-derived C2N prostate tumor cells to ATRA in vitro decreased total viable cell numbers with a concomitant decrease in the fraction of cells in S phase. When TRAMP mice were treated in vivo with ATRA for either 6 or 8 weeks at low, medium, or high dose, mice on average presented with lower grade and more differentiated tumors. However, ATRA therapy conferred no significant protection on incidence of tumors or frequency of metastasis at any dose. Nevertheless, we were able to observe a significant decrease in the expression of synaptophysin, a marker of neuroendocrine differentiation, in tumors of mice receiving the highest dose of ATRA. As well, expression of the cell cycle inhibitor p21 was found to be elevated only in well-differentiated tumors of mice, treated with ATRA while expression of p27, was found to be elevated only in the poorly differentiated tumors. CONCLUSIONS: Collectively, our in vitro and in vivo data demonstrates that ATRA was able to slow prostate tumor cell proliferation, induce apoptosis, and block the emergence of the neuroendocrine phenotype. Furthermore, our study suggests the differential regulation of p21 and p27 as a molecular mechanism whereby ATRA intervention therapy can inhibit the natural history of spontaneous prostate cancer.     

125Ⅰ粒子组织间植入对大肠癌的抑制作用及其机制

目的探讨125Ⅰ粒子抑制大肠癌生长的作用及其机制.方法采用雄性BALB/C裸小鼠建立人类大肠癌HCT-8细胞株的裸小鼠皮下移植瘤模型,将荷瘤鼠分5组(每组10只),对照组、空剂量组(0 Bq)、低剂量组(7.4×106 Bq)、中剂量组(14.8×106 Bq)、高剂量组(29.6×106 Bq),原位末端标记法检测肿瘤细胞凋亡的情况,免疫组织化学SP法染色检测血管内皮生长因子(VEGF),行微血管密度记数(MVD),同时检测p53蛋白表达情况.结果125Ⅰ粒子组织间植入治疗大肠癌,高、中、低剂量组抑瘤率分别为46.2%、34.0%、21.5%.在高剂量组中,p53蛋白表达增加,凋亡细胞增多,MVD及VEGF减少.结论(1)确证了125Ⅰ粒子对大肠癌治疗的有效性.(2)125Ⅰ粒子的推荐剂量14.8×106～29.6×106Bq.(3)125Ⅰ粒子组织间植入抑制肿瘤新生血管生成,同时p53蛋白表达量增加,细胞凋亡增多.     

Synergistic effects in apoptosis induction by taurolidine and TRAIL in HCT-15 colon carcinoma cells.

Induction of apoptosis in tumor cells by TRAIL (tumor necrosis factor [TNF]-related apoptosis-inducing ligand) is a promising therapeutic principle in oncology, although toxicity and resistance against TRAIL are limiting factors. Taurolidine (TRD), an antineoplastic agent with low toxicity, is a potential candidate for combined therapy with TRAIL. The aim of this study was to evaluate the apoptotic effects of a combined treatment with TRD and TRAIL in a human HCT-15 colon carcinoma cell line. HCT-15 cells were incubated with increasing concentrations of recombinant human TRAIL (50 ng/mL to 500 ng/mL) or TRD (50 micromol/L to 1000 micromol/L). In a second experiment, cells were furthermore exposed to a combination of both substances (TRAIL 50 ng/mL and TRD 100 micromol/L). At various time points (3 h to 36 h), cell viability, apoptosis, and necrosis were quantified by FACS analysis (propidium iodide/annexin V-FITC) and confirmed by TUNEL assay. Incubation with TRD resulted in cell death induction with maximum effects observed at 100 micromol/L and 1000 micromol/L after 36 h. TRAIL application led to dose-dependent cell death induction as early as 6 h. Combined treatment of TRD (100 micromol/L) and TRAIL (50 ng/mL) caused a sustained induction of apoptosis that was superior to single-agent application, exceeding a merely additive effect. Combinatory treatment of human colon carcinoma cells with TRD and TRAIL results in a synergistic effect on apoptosis induction with a significant increase of the apoptotic index. Combination of TRAIL with the nontoxic TRD might represent a novel therapeutic strategy in oncological therapy.     

Thapsigargin modulates osteoclastogenesis through the regulation of RANKL-induced signaling pathways and reactive oxygen species production.

The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase-3 activity, cytosolic Ca2+, and RANKL-induced activation of NF-kappaB and AP-1 activities. INTRODUCTION: Apoptosis and differentiation are among the consequences of changes in intracellular Ca2+ levels. In this study, we investigated the effects of the endoplasmic reticular Ca2+-ATPase inhibitor, thapsigargin (TG), on osteoclast apoptosis and differentiation. MATERIALS AND METHODS: Both RAW264.7 cells and primary spleen cells were used to examine the effect of TG on RANKL-induced osteoclastogenesis. To determine the action of TG on signaling pathways, we used reporter gene assays for NF-kappaB and activator protein-1 (AP-1) activity, Western blotting for phospho-extracellular signal-related kinase (ERK), and fluorescent probes to measure changes in levels of intracellular calcium and reactive oxygen species (ROS). To assess rates of apoptosis, we measured changes in annexin staining, caspase-3 activity, and chromatin and F-actin microfilament structure. RESULTS: At concentrations that caused a rapid rise in intracellular Ca2+, TG increased caspase-3 activity and promoted apoptosis in osteoclast-like cells (OLCs). Low concentrations of TG, which were insufficient to measurably alter intracellular Ca2+, unexpectedly suppressed caspase-3 activity and enhanced RANKL-induced osteoclastogenesis. At these lower concentrations, TG potentiated ROS production and RANKL-induced NF-kappaB activity, but suppressed RANKL-induced AP-1 activity and had little effect on ERK phosphorylation. CONCLUSION: Our novel findings of a biphasic effect of TG are incompletely explained by our current understanding of TG action, but raise the possibility that low intensity or local changes in subcellular Ca2+ levels may regulate intracellular differentiation signaling. The extent of cross-talk between Ca2+ and RANKL-mediated intracellular signaling pathways might be important in determining whether cells undergo apoptosis or differentiate into OLCs.