Cardiotoxin III suppresses hepatocyte growth factor–stimulated migration and invasion of MDA‐MB‐231 cells

The hepatocyte growth factor (HGF)/c‐Met signalling pathway is deregulated in most cancers and associated with a poor prognosis in breast cancer. Cardiotoxin III (CTX III), a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity. In this study, we use HGF as an invasive inducer to investigate the effect of CTX III on MDA‐MB‐231 cells. When cells were treated with non‐toxic doses of CTX III, CTX III inhibited the HGF‐promoted cell migration and invasion. CTX III significantly suppressed the HGF‐induced c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3k)/Akt and extracellular signal‐regulated kinase (ERK) 1/2. Additionally, CTX III similar to wortmannin (a PI3K inhibitor) and U0126 (an upstream kinase regulating ERK1/2 inhibitor) attenuated cell migration and invasion induced by HGF. This effect was paralleled by a significant reduction in phosphorylation of IκBα kinase and IκBα and nuclear translocation of nuclear factor κB (NF‐κB) as well as a reduction of matrix metalloproteinase‐9 (MMP‐9) activity. Furthermore, the c‐Met inhibitor PHA665752 inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of ERK1/2 and PI3K/Akt, suggesting that ERK1/2 and PI3K/Akt activation occurs downstream of c‐Met activation. Taken together, these findings suggest that CTX III inhibits the HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF/c‐Met‐dependent PI3K/Akt, ERK1/2 and NF‐κB signalling pathways, leading to the downregulation of MMP‐9 expression. Copyright © 2014 John Wiley & Sons, Ltd.     

Interleukin‐6 contributes to chemoresistance in MDA‐MB‐231 cells via targeting HIF‐1α

Chemoresistance is a critical challenge in the clinical treatment of triple‐negative breast cancer (TNBC). It has been well documented that inflammatory mediators from tumor microenvironment are involved in the pathogenesis of TNBC and might be related to chemoresistance of cancer cells. In this study, the contribution of interleukin‐6 (IL‐6), one of the principal oncogenic molecules, in chemoresistance of a TNBC cell line MDA‐MB‐231 was first investigated. The results showed that IL‐6 treatment could induce upregulation of HIF‐1α via the activation of STAT3 in MDA‐MB‐231 cells, which consequently contributed to its effect against chemotherapeutic drug‐induced cytotoxicity and cell apoptosis. However, knockdown of HIF‐1α attenuated such effect via affecting the expressions of apoptosis‐related molecules as Bax and Bcl‐2 and drug transporters as P‐gp and MRP1. This study indicated that targeting at IL‐6/HIF‐1α signaling pathway might be an effective strategy to overcome chemoresistance in TNBC therapy.     

Histone H1 inhibits the proliferation of MCF 7 and MDA MB 231 human breast cancer cells

Purified histone H1 exerts extracellular functions suggesting novel histone functions. The cytotoxic effects of histone H1 have lead to its choice as a pharmacological tool in breast cancer. Hence the present study was aimed at investigating the effect of exogenous histone H1 on the proliferation of estrogen receptor positive (MCF 7) and estrogen receptor negative (MDA MB 231) human breast cancer cells. Cells were incubated with various concentrations of histone H1 and antiproliferative activity was assessed by MTT assay. Proliferation of breast cancer cells was assessed from the activity of ornithine decarboxylase (ODC) using [(14)C] labeled ornithine. Histone H1-mediated cellular effects, such as anchorage dependent growth and apoptosis, were assessed by colony formation assay, fluorescence microscopy after acridine orange/propidium iodide staining and DNA fragmentation analysis. Histone H1 was significantly cytotoxic as it inhibited colony formation, ODC activity and induced apoptosis in both estrogen receptor positive and estrogen receptor negative cells. These results suggest that histone H1-induced antiproliferative effects on human breast cancer cells could possibly involve inhibition of ODC.     

Fucoxanthin inhibits tumour‐related lymphangiogenesis and growth of breast cancer

Tumour lymphangiogenesis plays an important role in promoting the growth and lymphatic metastasis of tumours. The process is associated with cell proliferation, migration and tube‐like structure formation in lymphatic endothelial cells (LEC), but no antilymphangiogenic agent is currently used in clinical practice. Fucoxanthin is a material found in brown algae that holds promise in the context of drug development. Fucoxanthin is a carotenoid with variety of pharmacological functions, including antitumour and anti‐inflammatory effects. The ability of fucoxanthin to inhibit lymphangiogenesis remains unclear. The results of experiments performed as part of this study show that fucoxanthin, extracted from Undaria pinnatifida (Wakame), inhibits proliferation, migration and formation of tube‐like structures in human LEC (HLEC). In this study, fucoxanthin also suppressed the malignant phenotype in human breast cancer MDA‐MB‐231 cells and decreased tumour‐induced lymphangiogenesis when used in combination with a conditional medium culture system. Fucoxanthin significantly decreased levels of vascular endothelial growth factor (VEGF)‐C, VEGF receptor‐3, nuclear factor kappa B, phospho‐Akt and phospho‐PI3K in HLEC. Fucoxanthin also decreased micro‐lymphatic vascular density (micro‐LVD) in a MDA‐MB‐231 nude mouse model of breast cancer. These findings suggest that fucoxanthin inhibits tumour‐induced lymphangiogenesis in vitro and in vivo, highlighting its potential use as an antilymphangiogenic agent for antitumour metastatic comprehensive therapy in patients with breast cancer.     

UCH‐L1 promotes invasion of breast cancer cells through activating Akt signaling pathway

As a de‐ubiquitin enzyme, ubiquitin C‐terminal hydrolase (UCH)‐L1 has been shown to be overexpressed in several human cancers. However, the function of UCH‐L1 in invasion of breast cancers is still unclear. Here we report that the expression of UCH‐L1 is significantly higher in cancer cells with higher invasive ability. While ectopic UCH‐L1 expression failed to alter cell proliferation in MCF‐7 cells, it caused a significant upregulation of cellular invasion. Furthermore, siRNA mediated knockdown of UCH‐L1 led to suppression of invasion in UCH‐L1 overexpressing MCF‐7 cells. In order to identify molecular mechanisms underlying these observations, a novel in vitro proximity‐dependent biotin identification method was developed by fusing UCH‐L1 protein with a bacterial biotin ligase (Escherichia coli BirA R118G, BioID). Streptavidin magnetic beads pulldown assay revealed that UCH‐L1 can interact with Akt in MCF‐7 cells. Pulldown assay with His tagged recombinant UCH‐L1 protein and cell lysate from MCF‐7 cells further demonstrated that UCH‐L1 preferentially binds to Akt2 for Akt activation. Finally, we demonstrated that overexpression of UCH‐L1 led to activation of Akt as evidenced by upregulation of phosphorylated Akt. Thus, these findings demonstrated that UCH‐L1 promotes invasion of breast cancer cells and might serve as a potential therapeutic target for treatment of human patients with breast cancers.     

Arachidonic acid promotes FAK activation and migration in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n-6 polyunsaturated fatty acid that is present in an esterified form in cell membrane phospholipids, and it might be present in the extracellular microenvironment. In particular, AA promotes MAPK activation and mediates the adhesion of MDA-MB-435 breast cancer cells to type IV collagen. However, the signal transduction pathways mediated by AA have not been studied in detail. Our results demonstrate that stimulation of MDA-MB-231 breast cancer cells with AA promotes an increase in the phoshorylation of Src and FAK, as revealed by site-specific antibodies that recognized the phosphorylation state of Src at Tyr-418, and of FAK at tyrosine-397 and in vitro kinase assays. In addition, AA also induces an increase in the migration of MDA-MB-231 cells. In contrast, AA does not induce phosphorylation of FAK and an increase in cell migration of non-tumorigenic epithelial cells MCF10A. Inhibition of Gi/Go proteins, LOX and Src activity prevent FAK activation and cell migration. In conclusion, our results demonstrate, for the first time, that Gi/Go proteins, LOX and Src play an important role in FAK activation and cell migration induced by AA in MDA-MB-231 breast cancer cells.     

Profilin‐1 overexpression inhibits proliferation of MDA‐MB‐231 breast cancer cells partly through p27kip1 upregulation

Profilin‐1 (Pfn1), a ubiquitously expressed actin‐binding protein, has gained interest in epithelial‐derived cancer because of its downregulation in expression in various adenocarcinoma. Pfn1 overexpression impairs tumorigenic ability of human breast cancer xenografts thus suggesting that Pfn1 could be a tumor‐suppressor protein. The objective of the present study was to determine how Pfn1 overexpression affects cell‐cycle progression of breast cancer cells. We show that Pfn1 overexpression in MDA‐MB‐231 breast cancer cells causes cell‐cycle arrest in G1 phase and dramatically reduced proliferation in culture. Pfn1 overexpression results in increased protein stability of p27^kip1 (p27—a major cyclin‐dependent kinase inhibitor) and marked elevation in the overall cellular level of p27. Proliferation defect of Pfn1 overexpressers can be partly rescued by silencing p27 expression thus suggesting a critical role of p27 in Pfn1‐induced growth inhibition of MDA‐MB‐231 cells. Finally, Pfn1 overexpression was found to sensitize MDA‐MB‐231 cells to apoptosis in response to cytotoxic stimulus thus suggesting for the first time that survival of breast cancer cells can also be negatively influenced by Pfn1 upregulation. These findings may provide novel insights underlying Pfn1's tumor‐suppressive action. J. Cell. Physiol. 223:623–629, 2010. © 2010 Wiley‐Liss, Inc.     

Apoptosis‐inducing effect of garcinol is mediated by NF‐κB signaling in breast cancer cells

Garcinol, obtained from Garcinia indica in tropical regions, is used for its numerous biological effects. Its anti‐cancer activity has been suggested but the mechanism of action has not been studied in‐detail, especially there is no report on its action against breast cancer cells. Here we tested our hypothesis that garcinol may act as an anti‐proliferative and apoptosis‐inducing agent against breast cancer cell lines. Using multiple techniques such as MTT, Histone‐DNA ELISA, Annexin V‐PI staining, Western blot for activated caspases and cleaved PARP, homogenous caspase‐3/7 fluorometric assay and EMSA, we investigated the mechanism of apoptosis‐inducing effect of garcinol in ER‐positive MCF‐7 and ER‐negative MDA‐MB‐231 cells. We found that garcinol exhibits dose‐dependent cancer cell‐specific growth inhibition in both the cell lines with a concomitant induction of apoptosis, and has no effect on non‐tumorigenic MCF‐10A cells. Our results suggested induction of caspase‐mediated apoptosis in highly metastatic MDA‐MB‐231 cells by garcinol. Down‐regulation of NF‐κB signaling pathway was observed to be the mechanism of apoptosis‐induction. Garcinol inhibited constitutive NF‐κB activity, which was consistent with down‐regulation of NF‐κB‐regulated genes. This is the first report on anti‐proliferative and apoptosis‐inducing action of garcinol against human breast cancer cells and the results suggest that this natural compound merits investigation as a potential chemo‐preventive/‐therapeutic agent, especially against breast cancer. J. Cell. Biochem. 109: 1134–1141, 2010. © 2010 Wiley‐Liss, Inc.     

Polarised Clathrin‐Mediated Endocytosis of EGFR During Chemotactic Invasion

Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.      

Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration

Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11-amino acid sequence between amino acids 717 and 727 of Tiam1 ((717)GEGTDAVKRS(727)L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1).Using an Escherichia coli-derived calmodulin-binding peptide (CBP)-tagged recombinant Tiam1 (amino acids 393-728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393-738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1-ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)-tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1-ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin-Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.     

Down‐regulation of uPA and uPAR by 3,3′‐diindolylmethane contributes to the inhibition of cell growth and migration of breast cancer cells

3,3′‐Diindolylmethane (DIM) is a known anti‐tumor agent against breast and other cancers; however, its exact mechanism of action remains unclear. The urokinase plasminogen activator (uPA) and its receptor (uPAR) system are involved in the degradation of basement membrane and extracellular matrix, leading to tumor cell invasion and metastasis. Since uPA‐uPAR system is highly activated in aggressive breast cancer, we hypothesized that the biological activity of B‐DIM could be mediated via inactivation of uPA‐uPAR system. We found that B‐DIM treatment as well as silencing of uPA‐uPAR led to the inhibition of cell growth and motility of MDA‐MB‐231 cells, which was in part due to inhibition of VEGF and MMP‐9. Moreover, silencing of uPA‐uPAR led to decreased sensitivity of these cells to B‐DIM indicating an important role of uPA‐uPAR in B‐DIM‐mediated inhibition of cell growth and migration. We also found similar effects of B‐DIM on MCF‐7, cells expressing low levels of uPA‐uPAR, which was due to direct down‐regulation of MMP‐9 and VEGF, independent of uPA‐uPAR system. Interestingly, over‐expression of uPA‐uPAR in MCF‐7 cells attenuated the inhibitory effects of B‐DIM. Our results, therefore, suggest that B‐DIM down‐regulates uPA‐uPAR in aggressive breast cancers but in the absence of uPA‐uPAR, B‐DIM can directly inhibit VEGF and MMP‐9 leading to the inhibition of cell growth and migration of breast cancer cells. J. Cell. Biochem. 108: 916–925, 2009. © 2009 Wiley‐Liss, Inc.     

Src42A modulates tumor invasion and cell death via Ben/dUev1a-mediated JNK activation in Drosophila

Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, we have identified Src42A, the ortholog of mammalian Src, as a key modulator of both Ras^V12/lgl^−/− triggered tumor invasion and loss of cell polarity gene-induced cell migration. Our genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, we showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with Ras^V12 to promote tumor overgrowth and invasion. In addition, we found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. Our data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a.