Calcitonin inhibits invasion of breast cancer cells: involvement of urokinase-type plasminogen activator (uPA) and uPA receptor

There is a growing body of evidence indicating that calcitonin (CT) and calcitonin receptor (CTR) are involved in the regulation of cell growth, differentiation, and survival and in tissue development. However, the precise functional role of CT/CTR in breast cancer is still unknown. It is well established that the urokinase plasminogen activator (uPA) system plays an important role in breast cancer invasion and metastasis. The goal of this study was to investigate the effects of CT on regulation of the uPA system and invasive capacity of breast cancer cells. In the highly invasive MDA-MB-231 cell line, 10(-8) M CT decreased both uPA and uPAR mRNA and protein expression which was associated with inhibition of the extracellular signal-regulated kinase (ERK) 1/2 pathway. Furthermore, two weeks of CT administration to nude mice inhibited the expression of uPA mRNA in primary tumors by 25% (P<0.05), as compared to control, untreated animals. CT also inhibited the invasiveness of MDA-MB-231 cells by 37% (10(-8) M CT, P<0.05), as determined by a Matrigel invasion assay. To the best of our knowledge, this is the first report describing a direct effect of CT on breast cancer cell invasion. Our data might suggest a close link between CT signaling, the uPA-mediated pathway, and breast cancer invasion.     

uPA/uPAR downregulation inhibits radiation-induced migration, invasion and angiogenesis in IOMM-Lee meningioma cells and decreases tumor growth in vivo

Meningioma is a well-known tumor of the central nervous system, and is treated by surgical resection and/or radiation. Recently, ionizing radiation has been shown to enhance invasiveness of surviving tumor cells, and several proteolytic enzyme molecules, including urokinase plasminogen activator (uPA), seem to be upregulated after radiation. uPA and its receptor (uPAR) have been strongly implicated in tumor invasion, angiogenesis and progression. Hence, the tumor-associated uPA-uPAR system is considered a potential target for cancer therapy. In the present study, we show that radiation increases uPA levels in the IOMM-Lee meningioma cells, and subsequently, increases tumor invasion, migration and angiogenesis in vitro. Studies with signaling molecule inhibitors AG1478, U0126 and SB203580 (specific inhibitors of EGFR, MEK1/2 and p38 respectively) showed inhibition of uPA levels in both basal and irradiated-IOMM-Lee cells. The PI3K inhibitor (LY294002) and the AKT inhibitor (AKT inhibitor IV) also partially decreased uPA expression, whereas SP600125, a JNK inhibitor, did not affect uPA levels in either radiated or non-radiated cells. Further, a bicistronic plasmid construct with small interfering RNA (siRNA) against uPA and its receptor inhibited tumor invasion, migration and angiogenesis in radiation-treated IOMM-Lee cells. In addition, siRNA against uPA and its receptor inhibited subcutaneous tumor growth in athymic nude mice in combination with radiation in a synergistic manner. Thus, the specific targeting of proteases via RNA interference could augment the therapeutic effect of radiation and prevent the adverse effects resulting from tumor cells that receive sublethal doses of radiation within the tumor mass.     

Integrin signaling inhibits paclitaxel-induced apoptosis in breast cancer cells

Inherent or acquired drug resistance is one of the major problems in chemotherapy. The mechanisms by which cancer cells survive and escape the cytotoxic effects of chemotherapeutic agents are essentially unknown. In the present study, we demonstrate that in the MDA-MB-231 and MDA-MB-435 breast cancer cells, ligation of beta1 integrins by their extracellular matrix ligands inhibits significantly apoptosis induced by paclitaxel and vincristine, two microtubule-directed chemotherapeutic agents that are widely used in the therapy of breast cancer. We show that beta1 integrin signaling inhibits drug-induced apoptosis by inhibiting the release of cytochrome c from the mitochondria in response to drug treatment. Further, integrin-mediated protection from drug-induced apoptosis and inhibition of cytochrome c release are dependent on the activation of the PI 3-kinase/Akt pathway. Our results identify beta1 integrin signaling as an important survival pathway in drug-induced apoptosis in breast cancer cells and suggest that activation of this pathway may contribute to the generation of drug resistance.     

Role of thrombin receptor in breast cancer invasiveness

Invasion, the ability of an epithelial cancer cell to detach from and move through a basement membrane, is a central process in tumour metastasis. Two components of invasion are proteolysis of extracellular matrix and cellular movement through it. A potential promoter of these two processes is thrombin, the serine proteinase derived from the ubiquitous plasma protein prothrombin. Thrombin promotes the invasion of MDA-MB231 breast tumour cells (a highly aggressive cell line) in an in vitro assay. Invasion by MDA-MB436 and MCF-7 cells, less aggressive cell lines, is not promoted by thrombin. Thrombin, added to the cells, is a stimulator of cellular movement; fibroblast-conditioned medium is the chemotaxin. Thrombin-promoted invasion is inhibited by hirudin. Stimulation of invasion is a receptor-mediated process that is mimicked by a thrombin receptor-activating peptide. Thrombin has no effect on chemotaxis in vitro. Thrombin receptor is detectable on the surface of MDA-MB231 cells, but not on the other two cell lines. Introduction of oestrogen receptors into MDA-MB231 cells by transfection with pHEO had no effect on thrombin receptor expression, in the presence or absence of oestradiol. This paper demonstrates that thrombin increases invasion by the aggressive breast cancer cell line MDA-MB231 by a thrombin receptor-dependent mechanism.     

Rabdosia rubescens inhibits breast cancer growth and angiogenesis

Investigators have shown that PC-SPES is a potent herbal mixture which has often been used by prostate cancer patients. In this study, we examined the inhibitory effects of certain individual components of PC-SPES on the in vitro proliferation of the human breast cancer cells MDA-MB231 and the human umbilical vein endothelial cells (HUVEC). Our data showed that individual components of PC-SPES had varying suppressive effects on cellular proliferation, and that Rabdosia rubescens appeared to be the most potent agent in these assays. Apoptosis was up-regulated by Rabdosia rubescens, as seen in the caspase-9 and TUNEL assays. These effects may be mediated via both the MAPK (mitogen-activated protein kinase) and the Akt kinase pathways. In mouse experiments, the extract from Rabdosia rubescens suppressed breast cancer xenograft size and decreased the tumor vessel density. We conclude that Rabdosia rubescens may potentially be used to treat or prevent breast cancer, and that the extract from this herbal source deserves further studies.     

Hsp27 overexpression inhibits doxorubicin-induced apoptosis in human breast cancer cells

Previously we demonstrated that heat shock protein 27 (hsp27) overexpression confers resistance to the chemotherapeutic agent doxorubicin in MDA-MB-231 breast cancer cells. Since induction of apoptosis is one underlying mechanism of chemotherapeutic drug action, we investigated the effect of hsp27 overexpression on doxorubicin-induced apoptosis, finding that hsp27 protects MDA-MB-231 cells from apoptosis. We also examined expression of the doxorubicin target, topoisomerase II (topo II), in control and hsp27-overexpressing stable transfectants, as topo II expression is important for both drug sensitivity and the initiation of apoptosis by doxorubicin. The relative levels of both topo IIalpha and beta were higher in the controls than the hsp27-overexpressing clones, suggesting that the apoptotic protective effect of hsp27 overexpression in MDA-MB-231 cells is associated with altered topo II expression.     

Caveolin-1 inhibits anchorage-independent growth, anoikis and invasiveness in MCF-7 human breast cancer cells

Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling and oncogenesis. Caveolin-1 is down-regulated in oncogene-transformed and tumor-derived cells. Antisense suppression of caveolin-1 or expression of a dominant negative form are sufficient for inducing cellular transformation. Expression of recombinant caveolin-1 inhibits anchorage-independent growth in cancer cells. The present study was designed to determine whether this is caused by inhibition of cancer cell survival or cell proliferation, and to test if another important property of cancer cells, i.e. matrix invasion, is modulated by expression of caveolin. Utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/Cav1), we demonstrate that caveolin-1 expression decreases MCF-7 cell proliferation rate and markedly reduces their capacity to form colonies in soft agar. The loss of anchorage-independent growth is not associated with stimulation of anoikis; in fact, MCF-7/Cav1 cells exhibit increased survival after detachment as compared with MCF-7 cells, indicating that in these cells caveolin-1 inhibits anoikis. Analysis of matrix metalloprotease release and matrix invasion revealed that expression of caveolin-1 inhibits also these important metastasis-related phenomena. Plating MCF-7 cells on a laminin matrix resulted in activation of ERK1/2, which was dramatically inhibited in MCF-7/Cav1 cells. We conclude that high expression level of caveolin-1 in human breast cancer cells exerts a negative modulatory effect on anchorage-independent growth by inhibiting cell proliferation even though matrix-independent cell survival is enhanced. Caveolin-1 expression inhibits also matrix invasion and blocks laminin-dependent activation of ERK1/2. The inhibitory effect of caveolin-1 on these transformation-dependent processes supports the hypothesis that caveolin-1 acts as a tumor suppressor protein which may impose major phenotypic changes when expressed in human cancer cells.     

High glucose and insulin enhance uPA expression,ROS formation and invasiveness in breast cancer-derived cells

Background

Accumulating evidence indicates that type 2 diabetes is associated with an increased risk to develop breast cancer. This risk has been attributed to hyperglycemia, hyperinsulinemia and chronic inflammation. As yet, however, the mechanisms underlying this association are poorly understood. Here, we studied the effect of high glucose and insulin on breast cancer-derived cell proliferation, migration, epithelial-mesenchymal transition (EMT) and invasiveness, as well as its relationship to reactive oxygen species (ROS) production and the plasminogen activation system.

Methods

MDA-MB-231 cell proliferation, migration and invasion were assessed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), scratch-wound and matrigel transwell assays, respectively. ROS production was determined using 2′ 7′-dichlorodihydrofluorescein diacetate. The expression of E-cadherin, vimentin, fibronectin, urokinase plasminogen activator (uPA), its receptor (uPAR) and its inhibitor (PAI-1) were assessed using qRT-PCR and/or Western blotting assays, respectively. uPA activity was determined using gel zymography.

Results

We found that high glucose stimulated MDA-MB-231 cell proliferation, migration and invasion, together with an increased expression of mesenchymal markers (i.e., vimentin and fibronectin). These effects were further enhanced by the simultaneous administration of insulin. In both cases, the invasion and growth responses were found to be associated with an increased expression of uPA, uPAR and PAI-1, as well as an increase in active uPA. An osmolality effect of high glucose was excluded by using mannitol at an equimolar concentration. We also found that all changes induced by high glucose and insulin were attenuated by the anti-oxidant N-acetylcysteine (NAC) and, thus, depended on ROS production.

Conclusions

From our data we conclude that hyperglycemia and hyperinsulinemia can promote breast cancer cell proliferation, migration and invasion. We found that these features were associated with increased expression of the mesenchymal markers vimentin and fibronectin, as well as increased uPA expression and activation through a mechanism mediated by ROS.

     

Gene silence-induced downregulation of survivin inhibits bladder cancer cells

Urinary bladder cancer accounts for approximately 3% of all cancers in humans. Treatment for urinary bladder is not satisfactory. The present study aims to elucidate the effect of gene silencing of survivin on the inhibition of bladder cancer cells. In this study, we constructed survivin shRNA-carrying lentiviral vectors. Bladder cancer cell lines, T24 cells and BJ cells, were transduced with the constructed shRNA of survivin. The frequency of apoptotic bladder cancer cells was assessed by flow cytometry. The results showed that transfection with survivin shRNA significantly inhibited cell proliferation of both T24 and BJ cells. Most T24 and BJ cells accumulated at the G2/M stage; a portion of them was at sub-G1 stage. An increase in the fraction of bladder cancer cells undergoing apoptosis was noted. Among eight apoptosis-associated proteins, the amounts of BAX and BAD were significantly increased in the survivin-deficient bladder cancer cells. The findings suggest that survivin may be a therapeutic target of bladder cancer to selectively inhibit cell proliferation of bladder cancer cells.     

RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth

The serine protease urokinase-type plasminogen activator (uPA) plays a significant role in tumor cell invasion and metastasis when bound to its specific receptor, uPAR (also known as CD87). In addition to the uPA-uPAR system, matrix metalloproteinases (MMPs) are involved in tumor cell invasion and metastasis. In this study, we achieved specific inhibition of uPAR and MMP-9 using RNAi technology. We introduced small interfering RNA to downregulate the expression of uPAR and MMP-9 (pUM) in breast cancer cell lines (MDA MB 231 and ZR 75 1). In vitro angiogenesis studies indicated a decrease in the angiogenic potential of the treated cells; in particular, a remarkable decrease was observed in the cells treated with bicistronic construct (pUM) in comparision to the controls. Additionally, bicistronic construct inhibited the formation of capillary-like structures in in vivo models of angiogenesis. Similarly, the invasive potential and migration decreased dramatically when treated with the bicistronic construct as shown by matrigel invasion and migration assays. These results suggest a synergistic effect from the simultaneous downregulation of uPAR and MMP-9. We also assessed the levels of phosphorylated forms of MAPK, ERK and AKT signaling pathway molecules and found reduction in the levels of these molecules in cells treated with the bicistronic construct as compared to the control cells. Furthermore, targeting both uPAR and MMP-9 totally regressed orthotopic breast tumors in nude mice. In conclusion, our results provide evidence that the simultaneous downregulation of uPAR and MMP-9 using RNAi technology may provide an effective tool for breast cancer therapy.     

他莫昔芬对乳腺癌MCF-7细胞侵袭能力的影响及其作用机制

目的 探讨他莫昔芬(TAM)对乳腺癌MCF-7细胞侵袭能力的影响及其作用机制.方法 采用对数生长期的乳腺癌MCF-7细胞,以无血清无酚红高糖DMEM培养基对其进行24 h的培养,然后将其分别接种并分为对照组(无血清无酚红高糖DMEM培养基)、TAM组(含有1μmol/L TAM的无血清无酚红高糖DMEM培养基),采用Western blot法、RT-PCR法检测两组MCF-7细胞基质金属蛋白酶-9(MMP-9)、MMP-2、血管内皮细胞生长因子(VEGF)蛋白及mRNA表达水平,采用Transwell法检测MCF-7细胞的侵袭能力.结果 TAM组MCF-7细胞中VEGF、MMP-9、MMP-2蛋白表达水平均明显高于对照组,VEGF、MMP-9、MMP-2 mRNA相对表达水平均明显高于对照组,差异均有统计学意义(P﹤0.01);TAM组MCF-7细胞的侵袭能力相对数明显高于对照组,差异有统计学意义(P﹤0.01).结论 TAM可能发挥类雌激素功能,促进乳腺癌MCF-7细胞侵袭能力,这与其提高MCF-7细胞中VEGF、MMP-9、MMP-2表达水平有关.     

Huaier aqueous extract inhibits proliferation of breast cancer cells by inducing apoptosis

Aqueous extract of Trametes robiniophila murr (Huaier) has been commonly used in China for cancer complementary therapy in recent years; however, the mechanisms of its anticancer effects are largely unknown. In the present study, we aim to investigate its inhibitory effect on both MCF‐7 and MDA‐MB‐231 cells, and explore the possible mechanisms of its anticancer effect. Cell viability and motility were measured by MTT and invasive assays, migration and scratch assays in vitro, respectively. The distribution of cell cycle, PI‐Annexin‐V staining and Rhodamine 123 assay were analyzed by flow cytometry, and western blot were used to test the apoptotic pathways. We found that Huaier extract could strongly inhibit cell viability of MCF‐7 and MDA‐MB‐231 cells in a time‐ and dose‐dependent manner; however, MDA‐MB‐231 cells showed more susceptibility to the treatment. Furthermore, cell invasiveness and migration were also suppressed with exposure to Huaier extract. We also indicated that Huaier could induce G0/G1 cell‐cycle arrest, p53 accumulation and activation selectively in MCF‐7 cells. Inspiringly, the PI‐Annexin‐V staining assay and western blot analysis confirmed cell apoptosis executed by caspase‐3. Decreased mitochondrial membrane potential by Rhodamine 123 assay and down‐regulation of Bcl‐2 and up‐regulation of BCL2‐associated X protein (BAX) indicated that Huaier induced apoptosis through the mitochondrial pathway. Caspase activation during Huaier‐induced apoptosis was confirmed by pan‐caspase inhibitor, Z‐VAD‐fmk. As expected, the inhibitor decreased Huaier‐induced apoptosis in both cell lines. Based on our findings, Huaier can induce cell apoptosis in both ER‐positive and ER‐negative breast cancer cell lines and is an effective complementary agent for breast cancer treatment. (Cancer Sci 2010; 101: 2375–2383)     

RNAi-mediated downregulation of urokinase plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth

In recent years, RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Several groups are actively exploring the use of small interfering RNA (siRNA) for therapeutic applications to treat cancer. Our previous studies have demonstrated the inhibition of various proteases, including serine proteases, cysteine proteases and matrix metalloproteases, via RNA interference (RNAi) in gliomas. Similar to gliomas, malignant meningiomas also exhibit elevated protease levels in comparison to normal brain and benign meningiomas. Here, we used siRNA to simultaneously target urokinase plasminogen activator (uPA) and its receptor, uPAR. A human CMV promoter-driven mammalian expression vector (pU2) was used to produce hairpin double-stranded RNA (hp RNA) to target uPA and uPAR. As determined by Western blotting and fibrin zymography, pU2 effectively inhibited uPAR protein levels and uPA enzymatic activity in meningioma cells (IOMM-Lee). In vitro studies (Matrigel invasion and spheroid migration) revealed reduced meningioma cell invasion and migration. Intratumoral injections of the plasmid vector expressing siRNA for uPA and uPAR resulted in regression of pre-established, subcutaneous tumors in mice. In addition, in vivo studies of mice injected with pU2-transfected meningioma cells revealed inhibition of intracranial tumor formation. These findings suggest that siRNA can be used as a potent and specific therapeutic tool for the treatment of malignant meningiomas in humans.     

EGF介导的NF-κB促进体外胰腺癌细胞的uPA表达与侵袭力

目的探讨表皮生长因子(EGF)促进胰腺癌细胞侵袭和转移的分子机制。方法用WST-1细胞增殖实验、细胞黏附实验和Transwell体外侵袭实验检测EGF对胰腺癌细胞系NOR-P1的增殖、黏附及侵袭能力的影响。采用Western blot和逆转录-聚合酶链反应(RT-PCR)检测uPA的表达。用凝胶电泳迁移实验检测核因子-κB(NF-κB)活性。结果EGF能够明显促进胰腺癌细胞的侵袭能力,但对胰腺癌细胞的黏附力及增殖并无明显影响。EGF明显上调胰腺癌细胞的NF-κB活性和尿激酶型纤溶酶原激活物(uPA)表达。NF-κB抑制物四氢化吡咯二硫代氨基甲酸酯(PDTC)能够明显抑制EGF所诱导的NF-κB活性,同时也抑制EGF所诱导的uPA表达及胰腺癌细胞的侵袭力。结论EGF通过活化NF-κB促进胰腺癌细胞的uPA表达和侵袭力,采用NF-κB抑制剂阻断NF-κB通路有利于胰腺癌的综合治疗。     

Roles of BIM induction and survivin downregulation in lapatinib-induced apoptosis in breast cancer cells with HER2 amplification

Lapatinib, a dual tyrosine kinase inhibitor of the epidermal growth factor receptor and human epidermal growth factor receptor 2 (HER2), is clinically active in patients with breast cancer positive for HER2 amplification. The mechanism of this anti-tumor action has remained unclear, however. We have now investigated the effects of lapatinib in HER2 amplification-positive breast cancer cells with or without an activating PIK3CA mutation. Lapatinib induced apoptosis in association with upregulation of the pro-apoptotic protein Bcl-2 interacting mediator of cell death (BIM) through inhibition of the MEK-ERK signaling pathway in breast cancer cells with HER2 amplification. RNA interference (RNAi)-mediated depletion of BIM inhibited lapatinib-induced apoptosis, implicating BIM induction in this process. The pro-apoptotic effect of lapatinib was less pronounced in cells with a PIK3CA mutation than in those without one. Lapatinib failed to inhibit AKT phosphorylation in PIK3CA mutant cells, likely because of hyperactivation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway by the mutation. Depletion of PIK3CA (a catalytic subunit of PI3K) revealed that survivin expression is regulated by the PI3K pathway in these cells, suggesting that insufficient inhibition of PI3K-survivin signaling is responsible for the limited pro-apoptotic effect of lapatinib in HER2 amplification-positive cells with a PIK3CA mutation. Consistent with this notion, depletion of survivin by RNAi or treatment with a PI3K inhibitor markedly increased the level of apoptosis in PIK3CA mutant cells treated with lapatinib. Our results thus suggest that inhibition of both PI3K-survivin and MEK-ERK-BIM pathways is required for effective induction of apoptosis in breast cancer cells with HER2 amplification.     

MDA-7 results in downregulation of AKT concomitant with apoptosis and cell cycle arrest in breast cancer cells

The melanoma differentiation-associated gene-7 (mda-7) is a known mediator of apoptosis in cancer cells but not in normal cells. We hypothesized that MDA-7 interferes with the prosurvival signaling pathways that are commonly altered in cancer cells to induce growth arrest and apoptosis. We also identified the cell signaling pathways that are antagonized by MDA-7 leading to apoptosis. Using an adenoviral expression system, mda-7 was introduced into the breast cancer cell lines SKBr3, MCF-7 and MDA-MB-468, each with a different estrogen receptor (ER) and HER-2 receptor status. Downstream targets of MDA-7 were assessed by reverse phase protein array analysis, western blot analysis and immunofluorescence confocal microscopy. Our results show that MDA-7-induced apoptosis was mediated by caspases in all cell lines tested. However, MDA-7 modulates additional pathways in SKBr3 (HER-2 positive) and MCF-7 (ER positive) cells including downregulation of AKT-GSK3β and upregulation of cyclin-dependent kinase inhibitors in the nucleus. This leads to cell cycle arrest in addition to apoptosis. In conclusion, MDA-7 abrogates tumor-promoting pathways including the activation of caspase-dependent signaling pathways ultimately leading to apoptosis. In addition, depending on the phenotype of the breast cancer cell, MDA-7 modulates cell cycle regulating pathways to mediate cell cycle arrest.     

GnRH analogs reduce invasiveness of human breast cancer cells

Objective Bone, besides lung and liver, is one of the most preferential metastatic target sites for breast cancers. Although the precise molecular mechanisms underlying this preference need to be elucidated, it appears that bone microenvironments possess unique biological features that enable circulating cancer cells to home, survive and proliferate, and destroy bone. The majority of human breast cancers and in addition most breast cancer cell lines express GnRH receptors. Their proliferation is time- and dose-dependently reduced by GnRH-I and GnRH-II agonists by counteracting of the mitogenic signal transduction.Methods We have established a coculture system of different breast cancer cell lines stable transfected with red fluorescence (DS-Red) and human primary osteoblasts (hOB) or MG63 human osteosarcoma cells to analyze tumor cell invasion to bone.Results We could show that breast cancer cell invasion was increased when cocultured with hOB or MG63. Treatment with GnRH-I and GnRH-II analogs reduced the ability to invade a reconstituted basement membrane (Matrigel) and to migrate in response to the cellular stimulus. Searching for the molecular mechanisms we found that GnRH treatment reduces expression of the osteoblast derived chemokine SDF-1 by hOB or MG63 cells cocultured with breast cancer cells.Conclusion These data represent the first report that the activation of tumor GnRH receptors reduces the metastatic potential of breast cancer cells. The crosstalk between metastatic breast cancer cells and bone is critical to the development and progression of bone metastases. Disruption of this interaction will allow us to design mechanism-based effective and specific therapeutic interventions for bone metastases.