迷迭香酸对乳腺癌MDA-MB-231细胞增殖、凋亡和迁移能力的影响

研究迷迭香酸对乳腺癌MDA-MB-231细胞增殖、凋亡和迁移能力的影响。采用磺酰罗丹明B(SRB)法测定迷迭香酸对乳腺癌MDA-MB-231细胞增殖的影响,Hoechst 33258荧光染色法观察细胞凋亡形态,Annexin VFITC/PI检测细胞凋亡率;同时,细胞划痕实验检测迷迭香酸对MDA-MB-231细胞体外迁移能力的影响,实时荧光PCR(qPCR)法检测Bax、Bcl-2、Caspase-3、MMP-2和MMP-9基因的表达水平。研究结果显示迷迭香酸能抑制乳腺癌MDA-MB-231细胞的增殖,且呈时间剂量依赖性;迷迭香酸处理后的MDA-MB-231细胞出现明显的凋亡形态,且细胞凋亡率明显增加,Bax和caspase-3 mRNA表达水平增加,而Bcl-2 mRNA表达水平降低。另外,迷迭香酸作用后可剂量依赖性地降低MDA-MB-231细胞的体外迁移能力;同时降低MMP-2和MMP-9 mRNA的表达。因此,迷迭香酸能有效的抑制MDA-MB-231细胞的增殖,诱导细胞凋亡,降低细胞迁移能力。     

辣椒碱对乳腺癌MDA-MB-231细胞迁移和侵袭的抑制作用及其机制

本研究旨在观察辣椒碱对人乳腺癌MDA-MB-231细胞迁移和侵袭的影响,并探讨其分子机制。乳腺癌MDA-MB-231细胞经不同浓度辣椒碱作用24 h后进行实验,采用CCK-8法检测细胞活性,采用细胞划痕实验测量细胞迁移率,采用Transwell侵袭实验检测细胞侵袭率,采用Western blot检测乳腺癌MDA-MB-231细胞中c-Src、p-c-Src(Tyr416)、黏着斑激酶(focal adhesion kinase,FAK)、p-FAK(Tyr576/577)、桩蛋白(Paxillin)、p-Paxillin(Tyr118)、基质金属蛋白酶2(matrix metalloproteinase 2,MMP2)和MMP9蛋白表达水平,采用RT-PCR法检测乳腺癌MDA-MB-231细胞中MMP2和MMP9的mRNA水平。结果显示,10~50μmol/L辣椒碱对MDA-MB-231细胞存活率无显著影响,100~500μmol/L辣椒碱显著降低MDA-MB-231细胞存活率(P0.05);与正常对照组比较,25和50μmol/L辣椒碱组MDA-MB-231细胞体外迁移率和侵袭率均显著降低(P0.05),c-Src、FAK和Paxillin蛋白的磷酸化水平均显著下降(P0.05),MMP2和MMP9的mRNA和蛋白表达水平均下降(P0.05);并且辣椒碱对MDA-MB-231细胞的上述作用具有剂量依赖性。以上结果提示,低浓度辣椒碱可剂量依赖性地抑制人乳腺癌MDA-MB-231细胞迁移和侵袭,其机制可能涉及辣椒碱对c-Src/FAK/Paxillin信号通路以及MMP2和MMP9表达的抑制。     

芹菜素对MDA-MB-231细胞增殖及侵袭转移的影响

目的:研究芹菜素对乳腺癌MDA-MB-231细胞增殖及侵袭转移能力的影响,探讨其作用 机制.方法:芹菜素处理MDA-MB-231细胞,MTT法及流式法检测细胞增殖、粘附和周期变化 ;Millicell小室检测新生血管形成、侵袭转移能力的改变;免疫细胞化学检测Bcl-2、Bax 、cyclinB1、VEGF、MMP-9、E-cd蛋白的表达差异.结果:芹菜素明显抑 制MDA-MB-231细胞增殖,诱导细胞凋,G2/M期细胞比例由4.79%增至36.12%(P＜0.05 );细胞的黏附、新生血管形成 能力均显著降低;侵袭转移穿膜细胞数明显低于对照组,分别由88.67、106.33降至45.67、 68(P＜0.05);Bcl-2、cyclinB1、VEGF、MMP-9蛋白表达明显降低;Bax,E-cd的表达则增 加.结论:芹菜素有效抑制MDA-MB-231细胞增殖,阻滞细胞于G2/M期, 诱导细胞凋亡;抑制 细胞粘附、新生血管形成、侵袭与转移的能力,其机制与抑制Bcl-2、cyclinB1、VEGF、MMP 9表达,促进Bax、E-cd表达有关.     

GPER介导的丹参酮ⅡA抗三阴性乳腺癌细胞MDA-MB-231迁移作用机制研究

摘要 目的：基于体外细胞实验,探索丹参酮ⅡA对三阴性乳腺癌细胞MDA-MB-231迁移的抑制作用及其分子机制。方法：选取三阴性乳腺癌细胞MDA-MB-231,利用细胞增殖实验检测丹参酮ⅡA对MDA-MB-231细胞增殖的作用,并筛选适宜的药物浓度;应用划痕实验检测丹参酮ⅡA对MDA-MB-231细胞迁移率的影响;Western Blot法检测丹参酮ⅡA对G蛋白偶联雌激素受体（G protein-coupled estrogen receptor,GPER）及基质金属蛋白酶9（Matrix metalloprotein-9,MMP-9）表达的影响。结果：细胞增殖实验结果显示,丹参酮ⅡA可以抑制MDA-MB-231细胞增殖,且呈剂量依赖性（P＜0.05）;划痕实验结果显示,丹参酮ⅡA可以抑制MDA-MB-231细胞迁移,且呈剂量依赖性（P＜0.01）,加入GPER特异性抑制剂G15后迁移率有所上升（P＜0.01）。Western Blot结果显示,丹参酮ⅡA可以显著下调GPER和MMP-9蛋白的表达水平并呈剂量依赖性（P＜0.05）,加入GPER特异性抑制剂G15后,MMP-9表达有所上升（P＜0.01）。结论：丹参酮ⅡA可以抑制三阴性乳腺癌细胞MDA-MB-231迁移,其机制可能与抑制GPER介导的MMP-9表达相关。     

基于核因子相关因子2/血红素加氧酶-1信号通路探讨异荭草素对乳腺癌细胞恶性生物学行为的影响

摘要 目的：探讨异荭草素（ISO）对乳腺癌（BC）细胞恶性生物学行为及核因子相关因子2（Nrf2）/血红素加氧酶-1（HO-1）信号通路的影响。方法：体外培养人BC细胞系MDA-MB-231并分组：MDA-MB-231组、MDA-MB-231+ISO组（100 μmol/L ISO处理）、MDA-MB-231+ISO+OE-NC组（转染OE-NC后用100 μmol/L ISO处理）、MDA-MB-231+ISO+OE-Nrf2组（转染OE-Nrf2后用100 μmol/L ISO处理）。采用细胞计数试剂盒-8（CCK-8）检测MDA-MB-231细胞增殖;流式细胞术检测MDA-MB-231细胞周期和凋亡;Transwell实验检测MDA-MB-231细胞的侵袭和迁移能力;Western blot检测Nrf2/HO-1信号通路相关蛋白及凋亡蛋白表达。结果：与MDA-MB-231组相比,MDA-MB-231+ISO组、MDA-MB-231+ISO+OE-NC组细胞活力、S期和G2期细胞比例、迁移和侵袭能力、B淋巴细胞瘤-2（Bcl-2）、Nrf2、HO-1、基质金属蛋白酶-9（MMP-9）蛋白水平显著下降（P<0.05）,细胞凋亡率、G1/G0期细胞比例以及Bax、cleaved-Caspase-3蛋白水平显著上升（P<0.05）。与MDA-MB-231+ISO组相比,MDA-MB-231+ISO+OE-Nrf2组细胞活力、S期和G2期细胞比例、迁移和侵袭能力、Bcl-2、Nrf2、HO-1、MMP-9蛋白水平显著上升（P<0.05）,细胞凋亡率、G1/G0期细胞比例以及Bax、cleaved-Caspase-3蛋白水平显著降低（P<0.05）。结论：ISO可能通过抑制Nrf2/HO-1信号通路,抑制MDA-MB-231细胞恶性增殖、迁移和侵袭等行为。     

BMP9结合ALK2受体激活BMPs/SMAD抑制乳腺癌MDA-MB-231增殖侵袭和迁移

为探讨骨形态发生蛋白9是通过结合受体ALK1还是ALK2来抑制人乳腺癌癌细胞MDA-MB-231的增殖、侵袭和迁移,本研究采用RT-PCR检测ALK1、ALK2在MDA-MB-231中内源性表达,同时用RT-PCR检测显性负性突变ALK2腺病毒和BMP9腺病毒共感染MDA-MB-231后,DNALK2表达。采用MTT、细胞划痕实验、Transwell侵袭实验检测DNALK2对BMP9作用下MDA-MB-231增殖、侵袭、迁移的影响。RT-PCR检测DNALK2和BMP9腺病毒共感染MDA-MB-231后CTGF m RNA表达,Western blot检测BMPs/SMAD信号通路中SMAD1/5/8总的蛋白和磷酸化蛋白以及CTGF表达。结果显示,MDA-MB-231只存在ALK2表达,DNALK2和BMP9腺病毒共感染MDA-MB-231后,DNALK2 m RNA表达明显升高;MDA-MB-231/GFP/DNALK2组吸光度值(0.392±0.044)较MDA-MB-231/BMP9/DNALK2组(0.433±0.045)吸光度值无明显改变(p0.05);MDA-MB-231/GFP/DNALK2组划痕愈合率(67.3±8.6)%与MDA-MB-231/BMP9/DNALK2组划痕愈合率(59.9±6.4)%无明显改变(p0.05),MDA-MB-231/GFP/DNALK2组穿膜细胞数为(21.7±3.4)个与MDA-MB-231/BMP9/DNALK2组穿膜细胞数为(17.4±5.4)个无明显改变(p0.05)。Western blot显示:DNALK2能阻断BMP9上调磷酸化SMAD1/5/8和下调人结缔组织生长因子CTGF表达。由此得出结论,BMP9可通过结合ALK2受体激活BMPs/SMAD信号通路来抑制MDA-MB-231增殖、侵袭和迁移。     

SB-431542抑制乳腺癌细胞BT-549的增殖和侵袭及其机理研究

肿瘤转移是导致肿瘤患者死亡的最主要原因,TGF-β超家族成员Nodal分子被证实参与肿瘤细胞的增殖和转移,因而基于Nodal信号为靶标开展抗肿瘤研究成为可能。该研究应用Western blot检测乳腺癌细胞株BT-549、T-47D、MCF-7、SK-BR-3和MDA—MB-231中的Nodal和基质金属蛋白酶-2（matrix metalloproteinase-2,MMP-2）的表达水平,发现它们在BT-549细胞中表达量最高。然后采用不同浓度_Nodal信号抑制剂SB．431542（1-50μmol／L）处理BT-549细胞48h,利用MTT法揭示20～50gmol／L的SB-431542抑制该细胞增殖。进一步利用细胞划痕和Transwell实验证明,10μmol／L的SB-431542可抑制乳腺癌细胞的迁移和侵袭。最后,通过明胶酶谱和Westernblot显示,10～30gmol／L的sB．431542可剂量依赖性地抑制MMP-2的表达和活性。上述结果说明,SB-431542通过阻断Nodal信号通路可效抑制乳腺癌细胞BT-549的增殖、迁移和侵袭,其作用机制可能与降低MMP-2的表达和活性有关。     

木犀草素负向调节核糖体蛋白S12表达抑制MDA-MB-231细胞增殖与侵袭CSCD

木犀草素(luteolin,Lut)对多种肿瘤细的生长具有抑制作用,但对乳腺癌细胞的生物学行为的影响尚不明确,本研究旨在探讨Lut对乳腺癌细胞增殖与侵袭的影响及其作用机制。首先体外培养乳腺癌细胞(MDA-MB-231和MCF7),免疫印迹实验检测细胞静息状态下核糖体蛋白S12(ribosomal protein S12,RPS12)表达水平。并采用MTT法、免疫印迹法分别测定不同浓度Lut处理后细胞的增殖水平,胞内RPS12及c-Myc的表达,以及PI3K/Akt、mTOR和S6K的磷酸化。同时采用c-Myc及PI3K/Akt、mTOR抑制剂处理组细胞,分别检测细胞增殖水平以及细胞内RPS12以及c-Myc表达。随后构建RPS12启动子报告基因,研究Lut对其转录的影响。最后在细胞内过表达c-Myc,或采用siRNA沉默RPS12表达,检测细胞侵袭和迁移的变化。结果显示,RPS12在乳腺癌细胞系MDA-MB-231和MCF7细胞系中均呈高水平表达。采用不同浓度Lut处理细胞后,其增殖均明显降低,PI3K/Akt、mTOR及S6K磷酸化水平与对照组相比有所减弱,c-Myc和RPS12表达也显著受到抑制,PI3K/Ak和mTOR抑制剂也具有类似结果。此外,抑制c-Myc后可显著降低乳腺癌细胞内RPS12表达以及转录活性,但细胞过表达c-Myc后RPS12水平显著增高,而沉默RPS12则可以显著抑制细胞的侵袭和迁移。以上结果表明Lut能够抑制乳腺癌细胞系MDA-MB-231的增殖与侵袭,其机制可能与抑制PI3K/Akt/mTOR信号通路进而下调c-Myc的表达,最终抑制RPS12表达有关。     

BMP9对人乳腺癌细胞MDA-MB-231骨转移的影响及可能机制

探讨BMP9对人乳腺癌MDA-MB-231细胞骨转移能力的影响及其可能的机制。扩增高滴度的BMP9表达腺病毒,感染MDA-MB-231细胞,制备表达BMP9的重组MDA-MB-231/ BMP9细胞,以此作为实验组;同时以含GFP的空载腺病毒感染该细胞为MDA-MB-231/GFP,联合MDA-MB-231共同作为对照组;RT-PCR及Western blot检测重组MDA-MB-231/ BMP9细胞中BMP9以及磷酸化Smad1(PSmad1)的表达;定量PCR及Western blot检测三组细胞中CTGFmRNA和蛋白水平的表达情况,最后结合X片运用免疫组织化学染色的方法检测三组标本中CTGF的表达。结果发现重组MDA-MB-231/ BMP9细胞中存在BMP9的表达;与对照组细胞相比,MDA-MB-231/ BMP9细胞中存在PSmad1的活化增强及CTGF的表达下调;X片发现实验组裸鼠胫骨溶骨性缺损减少;瘤体组织免疫组化发现实验组CTGF表达下调。所以BMP9可以在体内抑制乳腺癌MDA-MB-231细胞的骨转移并且这种抑制作用有可能是通过下调CTGF来实现的。     

瑞戈非尼抑制三阴性乳腺癌MDA-MB-231细胞增殖的研究

目的:利用裸鼠皮下成瘤动物模型及裸鼠肝脏原位多发与弥散模型,探索新型分子靶向药物瑞戈非尼对三阴性乳腺癌MDA-MB-231增殖的抑制作用,确定其分子机制。方法:培养获得人高侵袭性三阴性乳腺癌细胞MDA-MB-231,经BALB/c裸鼠皮下注射形成皮下肿瘤,或经肝门静脉注射形成肝脏多发、弥散的肿瘤模型。给予瑞戈非尼灌胃给药治疗,3周后解剖,测量肿瘤体积并称重;或进行PET/CT检测,对核素强度、散发肿瘤占肝脏面积进行定量。通过定量PCR(q PCR)实验验证上皮-间质转化标志物与转移、侵袭相关标志物的表达水平。结果:在裸鼠皮下成瘤模型以及裸鼠肝脏原位多发与弥散肿瘤模型中,瑞戈非尼隔日灌胃给药持续3周能够显著抑制MDA-MB-231的增殖;皮下肿瘤体积、重量,以及裸鼠肝脏PET/CT检测显示瑞戈非尼持续给药组的相对核素强度均明显低于对照组;q PCR实验反映出瑞戈非尼能够抑制MDA-MB-231的上皮-间质转化与转移、侵袭作用。结论:揭示了瑞戈非尼对三阴性乳腺癌细胞系MDA-MB-231的增殖抑制作用,初步探索了其分子机制,为瑞戈非尼应用于三阴性乳腺癌的治疗提供参考。     

6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells

Gingerol (Zingiber officinale Roscoe, Zingiberaceae) is one of the most frequently and heavily consumed dietary condiments throughout the world. The oleoresin from rhizomes of ginger contains [6]-gingerol (1-[4′-hydroxy-3′-methoxyphenyl]-5-hydroxy-3-decanone) and its homologs which are pungent ingredients that have been found to possess many interesting pharmacological and physiological activities, such as anti-inflammatory, antihepatotoxic and cardiotonic effects. However, the effects of [6]-gingerol on metastatic processes in breast cancer cells are not currently well known. Therefore, in this study, we examined the effects of [6]-gingerol on adhesion, invasion, motility, activity and the amount of MMP-2 or -9 in the MDA-MB-231 human breast cancer cell line. We cultured MDA-MB-231 cells in the presence of various concentrations of [6]-gingerol (0, 2.5, 5 and 10 μM). [6]-Gingerol had no effect on cell adhesion up to 5 μM, but resulted in a 16% reduction at 10 μM. Treatment of MDA-MB-231 cells with increasing concentrations of [6]-gingerol led to a concentration-dependent decrease in cell migration and motility. The activities of MMP-2 or MMP-9 in MDA-MB-231 cells were decreased by treatment with [6]-gingerol and occurred in a dose-dependent manner. The amount of MMP-2 protein was decreased in a dose-dependent manner, although there was no change in the MMP-9 protein levels following treatment with [6]-gingerol. MMP-2 and MMP-9 mRNA expression were decreased by [6]-gingerol treatment. In conclusion, we have shown that [6]-gingerol inhibits cell adhesion, invasion, motility and activities of MMP-2 and MMP-9 in MDA-MB-231 human breast cancer cell lines.     

Matrine suppresses breast cancer cell proliferation and invasion via VEGF-Akt-NF-<Emphasis Type="Italic">κ</Emphasis>B signaling

Matrine has shown therapeutic and/or adjuvant therapeutic effects on the treatment of some patients with breast cancer. However, its mechanisms of action are largely unknown. To disclose the mechanisms, we investigated in vitro and ex vivo effects of matrine on the cancer cells. Our results confirmed that matrine significantly suppressed the proliferation of highly-metastatic human breast cancer MDA-MB-231 cell line. Matrine displayed synergistic effects with existing anticancer agents celecoxib (the inhibitor of cyclooxygenase-2), trichostatin A (the histone deacetylase inhibitor) and rosiglitazone against the proliferation and VEGF excretions in MDA-MB-231 cells. Matrine induced the apoptosis and cell cycle arrest by reducing the ratios of Bcl-2/Bax protein and mRNA levels in the cancer cells. Matrine significantly reduced the invasion, MMP-9/MMP-2 activation, Akt phosphorylation, nuclear factor κB p-65 expression and DNA binding activity, and mRNA levels of MMP-9, MMP-2, EGF and VEGFR1 in MDA-MB-231 cells. Collectively, our results suggest that matrine inhibits the cancer cell proliferation and invasion via EGF/VEGF-VEGFR1-Akt-NF-κB signaling pathway.     

Bone morphogenetic protein-4 induced by NDRG2 expression inhibits MMP-9 activity in breast cancer cells

In the current study, we examined the function of N-myc downstream-regulated gene 2 (NDRG2) expression in breast cancer cells, especially focusing on the role of bone morphogenetic protein-4 (BMP-4) induced by NDRG2. NDRG2 expression in MDA-MB-231 cells inhibited the mRNA expression of several matrix metalloproteinases (MMPs) and the gelatinolytic activity of MMP-9. Interestingly, a specific induction of active BMP-4 was exclusively observed in MDA-MB-231-NDRG2 cells but not in MDA-MB-231-mock cells. Neutralization of BMP-4 in MDA-MB-231-NDRG2 cells resulted in the rescue of MMP-9 mRNA expression and migration capacity. In addition, treatment with recombinant BMP-4 dramatically suppressed MMP-9 mRNA expression, gelatinolytic MMP-9 activity, migration, and invasion capacity both in MDA-MB-231 and PMA-treated MCF-7 cells. Collectively, our data show that BMP-4 induced by NDRG2 expression inhibits the metastatic potential of breast cancer cells, especially via suppression of MMP-9 activity.     

Oxygen-mediated regulation of gelatinase and tissue inhibitor of metalloproteinases-1 expression by invasive cells.

The relative expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) is an important determinant in trophoblast invasion of the uterus and tumor invasion and metastasis. Our previous studies have shown that low oxygen levels increase the in vitro invasiveness of trophoblast and tumor cells. The present study examined whether changes in oxygen levels affect TIMP and MMP expression by cultured trophoblast and breast cancer cells. Reverse zymographic analysis demonstrated reduced TIMP-1 protein secretion by HTR-8/SVneo trophoblast cells as well as MDA-MB-231 and MCF-7 breast carcinoma cells cultured in 1% vs 20% oxygen for 24 h. While gelatin zymography revealed no changes in the levels of MMP-9 secreted by HTR-8/SVneo trophoblasts cultured under various oxygen concentrations for 24 h, human MDA-MB-231 breast carcinoma cells displayed increased MMP-9 secretion and human MCF-7 breast cancer cells exhibited reduced secretion of this enzyme when cultured under similar conditions. In contrast, MMP-2 levels remained unchanged in all cultures incubated under similar conditions. Western blot analysis of MMP-9 protein in cell extracts confirmed the results of zymography. To assess the contribution of enhanced MMP activity to hypoxia-induced invasion, the effect of an MMP inhibitor (llomastat) on the ability of MDA-MB-231 cells to penetrate reconstituted extracellular matrix (Matrigel) was examined. Results showed that MMP inhibition significantly decreased the hypoxic upregulation of invasion by these cells. These findings indicate that the increased cellular invasiveness observed under reduced oxygen conditions may be due in part to a shift in the balance between MMPs and their inhibitors favoring increased MMP activity.     

Effects of laver extracts on adhesion,invasion, and migration in SK-Hep1 human hepatoma cancer cells

The laver (Porphyra tenera), red seaweed, has been reported to have anticancer activity, but little is known about its molecular mechanisms of action. The objective of this study was to determine the effects of laver extract on cancer cell proliferation, invasion, and metastasis in SK-Hep1 cells using migration and invasion assays. We also investigated the relationship of MMP-2/-9 and TIMP-1/-2 expression at both the protein and gene level in SK-Hep1 human hepatoma carcinoma cells after laver extract treatment. Laver extract inhibited cancer cell growth in a dose-dependent manner. In an invasion assay conducted in Transwell chambers, laver extract showed 19.6 and 27.2% inhibition of cancer cell at 200 and 400 μg/mL, respectively, compared to the control. The mRNA levels of both MMP-2 and MMP-9 were down-regulated by laver extract treatment in a dose-dependent manner. Laver extract, at 400 μg/mL, was inhibited by MMP-2 and MMP-9 expressions by 70.1 and 77.0%, respectively. An inverse relationship in the mRNA contents of MMP-2/-9 and TIMP-1/-2 expressions in SK-Hep1 cells was found by laver extract treatment. Our results demonstrate antimetastatic properties of laver extract in inhibiting the adhesion, invasion, and migration of SK-Hep1 human hepatoma cancer cells.     

Discovery of lesser known flavones as inhibitors of NF-κB signaling in MDA-MB-231 breast cancer cells—A SAR study

Seventeen flavonoids with different substitutions were evaluated for inhibition of nuclear factor-κB (NF-κB) signaling in the invasive breast cancer cell line MDA-MB-231. They were screened using an engineered MDA-MB-231 cell line reporting NF-κB activation. The modulation of expression of two NF-κB regulated genes involved in tumorigenesis, matrix metalloproteinase-9 (MMP-9), and cyclooxygenase-2 (COX-2) were also analyzed in these cells. Among the compounds tested, all except gossypetin and quercetagetin inhibited the activation of NF-κB, and the expression of MMP-9 and COX-2 to different degree. Methylated flavone, chrysoeriol (luteolin-3′-methylether), was found to be the most potent inhibitor of MMP-9 and COX-2 expressions. The effect of chrysoeriol on cell proliferation, cell cycle, apoptosis and metastasis was analyzed by established methods. Chrysoeriol caused cell cycle arrest at G2/M and inhibited migration and invasion of MDA-MB-231 cells. The structure–activity relations amongst the flavonoids as NF-κB signaling inhibitors was studied. The study indicates differences between the actions of various flavonoids on NF-κB activation and on the biological activities of breast cancer cells. Flavones in general, were more active than the corresponding flavonols.     

Unravelling the antimetastatic potential of pentoxifylline, a methylxanthine derivative in human MDA-MB-231 breast cancer cells

Pentoxifylline (PTX), a methylxanthine derivative is a non-steroidal immunomodulating agent with unique hemorheologic properties. It is used in the treatment of intermittent claudication as it increases the amount of oxygen reaching tissues by increasing the flexibility of red blood cells. Recently, it has also shown to exhibit anti-metastatic and anti-angiogenic activities in B16F10 melanoma cells both in vitro as well as in vivo. As per the reports, the choice of drug in the treatment of breast cancer is paclitaxel, but the major limitation is its toxicity. However, the effects of PTX on metastatic processes in breast cancer are not currently known. Therefore, in this study, we have examined the effect of PTX in MDA-MB-231 human breast cancer cells. The MTT assay showed dose- and time-dependent decreases in cellular proliferation. The non-toxic concentration of PTX selected were 1, 2.5 and 5 mM for 24 h. PTX induced a G0-G1 cell-cycle arrest leading to apoptosis. Further, it affected adhesion to both the matrigel and collagen type-IV in a time- and dose-dependent manner. The PTX impeded the migration of MDA-MB-231 cells and also decreased the activities of both MMP-2 and MMP-9. Thus, PTX at non-toxic doses affected cellular proliferation, adhesion, migration and invasion. These results demonstrate its anti-metastatic effect on MDA-MB-231 cells, and further studies need to be carried out to understand the mechanism of action.     

Toll-like receptor 9 agonists promote cellular invasion by increasing matrix metalloproteinase activity

Toll-like receptor 9 (TLR9) recognizes microbial DNA. We show here that TLR9 protein is expressed in human breast cancer cells and clinical breast cancer samples. Stimulation of TLR9-expressing breast cancer cells with the TLR9 agonistic CpG oligonucleotides (1-10 mumol/L) dramatically increased their in vitro invasion in both Matrigel assays and three-dimensional collagen cultures. Similar effects on invasion were seen in TLR9-expressing astrocytoma and glioblastoma cells and in the immortalized human breast epithelial cell line MCF-10A. This effect was not, however, dependent on the CpG content of the TLR9 ligands because the non-CpG oligonucleotides induced invasion of TLR9-expressing cells. CpG or non-CpG oligonucleotide-induced invasion in MDA-MB-231 cells was blunted by chloroquine and they did not induce invasion of TLR9(-) breast cancer cells. Treatment of MDA-MB-231 cells with CpG or non-CpG oligonucleotides induced the formation of approximately 50-kDa gelatinolytic band in zymograms. This band and the increased invasion were abolished by a matrix metalloproteinase (MMP) inhibitor GM6001 but not by a serine proteinase inhibitor aprotinin. Furthermore, CpG oligonucleotide treatment decreased tissue inhibitor of metalloproteinase-3 expression and increased levels of active MMP-13 in TLR9-expressing but not TLR9(-) breast cancer cells without affecting MMP-8. Neutralizing anti-MMP-13 antibodies inhibited the CpG oligonucleotide-induced invasion. These findings suggest that infections may promote cancer progression through a novel TLR9-mediated mechanism. They also propose a new molecular target for cancer therapy, because TLR9 has not been associated with cancer invasiveness previously.