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1.  Targeting Multiple Conformations Leads to Small Molecule Inhibitors of the uPAR·uPA Protein-Protein Interaction that Block Cancer Cell Invasion 
ACS chemical biology  2011;6(11):1232-1243.
Interaction of the urokinase receptor (uPAR) with its binding partners including the urokinase-type plasminogen activator (uPA) at the cell surface triggers a series of proteolytic and signaling events that promote invasion and metastasis. Here, we report the discovery of a small molecule (IPR-456) and its derivatives that inhibit the tight uPAR·uPA protein-protein interaction. IPR-456 was discovered by virtual screening against multiple conformations of uPAR sampled from explicit-solvent molecular dynamics simulations. Biochemical characterization reveal that the compound binds to uPAR with sub-micromolar affinity (Kd = 310 nM) and inhibits the tight protein-protein interaction with an IC50 of 10 μM. Free energy calculations based on explicit-solvent molecular dynamics simulations suggested the importance of a carboxylate moiety on IPR-456, which was confirmed by the activity of several derivatives including IPR-803. Immunofluorescence imaging showed that IPR-456 inhibited uPA binding to uPAR of breast MDA-MB-231 tumor cells with an IC50 of 8 μM. The compounds blocked MDA-MB-231 cell invasion, but IPR-456 showed little effect on MDA-MB-231 migration, and no effect on adhesion, suggesting that uPAR mediates these processes through its other binding partners.
PMCID: PMC3220747  PMID: 21875078
Virtual screening; small molecule; protein-protein interaction; inhibitor; urokinase receptor; invasion; migration; metastasis; MDA-MB-231; cancer; breast cancer; urokinase-type plasminogen activator; uPAR; uPA; docking; scoring; flexible docking
2.  Small-Molecule Inhibition of the uPAR·uPA Interaction: Synthesis, Biochemical, Cellular, in vivo Pharmacokinetics and Efficacy Studies in Breast Cancer Metastasis 
Bioorganic & medicinal chemistry  2013;21(7):2145-2155.
The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 μM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5 hours and peak concentration of 5 μM. Similar levels of the inhibitor were detected in tumor tissue up to 10 hours. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.
PMCID: PMC3625246  PMID: 23411397
3.  Analogues of doxanthrine reveal differences between the dopamine D1 receptor binding properties of chromanoisoquinolines and hexahydrobenzo[a]phenanthridines 
Efforts to develop selective agonists for dopamine D1-like receptors led to the discovery of dihydrexidine and doxanthrine, two bioisosteric β-phenyldopamine-type full agonist ligands that display selectivity and potency at D1-like receptors. We report herein an improved methodology for the synthesis of substituted chromanoisoquinolines (doxanthrine derivatives) and the evaluation of several new compounds for their ability to bind to D1- and D2-like receptors. Identical pendant phenyl ring substitutions on the dihydrexidine and doxanthrine templates surprisingly led to different effects on D1-like receptor binding, suggesting important differences between the interactions of these ligands with the D1 receptor. We propose, based on the biological results and molecular modeling studies, that slight conformational differences between the tetralin and chroman-based compounds lead to a shift in the location of the pendant ring substituents within the receptor.
PMCID: PMC3264828  PMID: 22204903
dopamine D1receptor; agonist; dihydrexidine; doxanthrine
4.  Design, Synthesis, Biochemical Studies, Cellular Characterization, and Structure-Based Computational Studies of Small Molecules Targeting the Urokinase Receptor 
Bioorganic & medicinal chemistry  2012;20(15):4760-4773.
The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR•uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.
PMCID: PMC3437670  PMID: 22771232
5.  Heterolytic Cleavage of Dihydrogen by an Iron(II) PNP Pincer Complex via Metal–Ligand Cooperation 
Organometallics  2013;32(15):4114-4121.
The bis-carbonyl Fe(II) complex trans-[Fe(PNP-iPr)(CO)2Cl]+ reacts with Zn as reducing agent under a dihydrogen atmosphere to give the Fe(II) hydride complex cis-[Fe(PNP-iPr)(CO)2H]+ in 97% isolated yield. A crucial step in this reaction seems to be the reduction of the acidic NH protons of the PNP-iPr ligand to afford H2 and the coordinatively unsaturated intermediate [Fe(PNPH-iPr)(CO)2]+ bearing a dearomatized pyridine moiety. This species is able to bind and heterolytically cleave H2 to give cis-[Fe(PNP-iPr)(CO)2H]+. The mechanism of this reaction has been studied by DFT calculations. The proposed mechanism was supported by deuterium labeling experiments using D2 and the N-deuterated isotopologue of trans-[Fe(PNP-iPr)(CO)2Cl]+. While in the first case deuterium was partially incorporated into both N and Fe sites, in the latter case no reaction took place. In addition, the N-methylated complex trans-[Fe(PNPMe-iPr)(CO)2Cl]+ was prepared, showing no reactions with Zn and H2 under the same reaction conditions. An alternative synthesis of cis-[Fe(PNP-iPr)(CO)2H]+ was developed utilizing the Fe(0) complex [Fe(PNP-iPr)(CO)2]. This compound is obtained in high yield by treatment of either trans-[Fe(PNP-iPr)(CO)2Cl]+ or [Fe(PNP-iPr)Cl2] with an excess of NaHg or a stoichiometric amount of KC8 in the presence of carbon monoxide. Protonation of [Fe(PNP-iPr)(CO)2] with HBF4 gave the hydride complex cis-[Fe(PNP-iPr)(CO)2H]+. X-ray structures of both cis-[Fe(PNP-iPr)(CO)2H]+ and [Fe(PNP-iPr)(CO)2] are presented.
PMCID: PMC3751463  PMID: 23990692
6.  Soluble urokinase receptor released from human carcinoma cells: a plasma parameter for xenograft tumour studies 
British Journal of Cancer  1999;81(2):203-211.
The urokinase plasminogen activator receptor (uPAR) plays a critical role in urokinase-mediated plasminogen activation and thereby in the process leading to invasion and metastasis. Soluble urokinase receptor (suPAR) is released from tumours, and in cancer patients the blood level of soluble receptor is increased. Using an enzyme-linked, immunosorbent assay (ELISA)-specific for the human urokinase receptor, release of soluble receptor was measured in cultures of human breast carcinoma cells, in tumour extracts and in plasma from mice with xenografted human tumours. Soluble human urokinase receptor (shuPAR) was released into culture supernatant during the growth of the human breast cancer cell line MDA-MB-231 BAG, and the level of shuPAR in conditioned medium determined by ELISA was a linear function of both viable cell number and time of incubation. Western blotting showed that the form of shuPAR measured by ELISA in conditioned medium consisted virtually exclusively of the three-domain full-length protein, while uPAR in cell lysates consisted of full-length uPAR as well as the domains (2+3) cleavage product. shuPAR was also released into the plasma of nude mice during growth of MDA-MB-231 BAG, MDA-MB-435 BAG and HCT 116 cells as subcutaneously xenografted tumours. Western blotting demonstrated that the shuPAR released from the xenografted human tumours into plasma consisted of the three-domain full-length protein, despite the finding of some cleaved uPAR in detergent extracts of tumour tissue. The levels of shuPAR determined by ELISA in the plasma of host mice during the growth of xenografted cell lines were highly correlated with tumour volume. © 1999 Cancer Research Campaign
PMCID: PMC2362879  PMID: 10496343
uPAR; soluble receptor; human cancer xenografts
7.  Down-Regulation of uPA and uPAR by 3,3′-Diindolylmethane Contributes to the Inhibition of Cell Growth and Migration of Breast Cancer Cells 
Journal of cellular biochemistry  2009;108(4):916-925.
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.
PMCID: PMC3757474  PMID: 19693769
8.  Synthesis and cytotoxic evaluation of some new[1,3]dioxolo[4,5-g]chromen-8-one derivatives 
Homoisoflavonoids are naturally occurring compounds belong to flavonoid classes possessing various biological properties such as cytotoxicity. In this work, an efficient strategy for the synthesis of novel homoisoflavonoids, [1,3]dioxolo[4,5-g]chromen-8-ones, was developed and all compounds were evaluated for their cytotoxic activities on three breast cancer cell lines.
Our synthetic route started from benzo[d][1,3]dioxol-5-ol which was reacted with 3-bromopropanoic acid followed by the reaction of oxalyl chloride to afford 6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one. The aldol condensation of the later compound with aromatic aldehydes led to the formation of the title compounds. Five novel derivatives 4a-e were tested for their cytotoxic activity against three human breast cancer cell lines including MCF-7, T47D, and MDA-MB-231 using the MTT assay.
Among the synthesized compounds, 7-benzylidene-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4a) exhibited the highest activity against three cell lines. Also the analysis of acridine orange/ethidium bromide staining results revealed that 7-benzylidene-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4a) and 7-(2-methoxybenzylidene)-6,7-dihydro-8H-[1,3]dioxolo[4,5-g]chromen-8-one (4b) induced apoptosis in T47D cell line.
Finally, the effect of methoxy group on the cytotoxicity of compounds 4b-4d was investigated in and it was revealed that it did not improve the activity of [1,3]dioxolo[4,5-g]chromen-8-ones against MCF-7, T47D, and MDA-MB-231.
PMCID: PMC4019946  PMID: 24887061
Homoisoflavonoids; [1,3]dioxolo[4,5-g]chromen-8-one; Cancer; Cytotoxic activity
9.  3,3-Dimethyl-1,2,3,4,6,11-hexa­hydro­benzo[d]naphtho[2,3-b]furan-1,6,11-trione 
In the title compound, C18H14O4, the cyclo­hexene ring adopts a sofa conformation. In the crystalline state, the mol­ecules are linked into a chain by weak inter­molecular C—H⋯O hydrogen bonds.
PMCID: PMC2962159  PMID: 21203239
10.  Methyl 1-methyl-3-p-tolyl-1,2,3,3a,4,11c-hexa­hydro­benzo[f]chromeno[4,3-b]pyrrole-3a-carboxyl­ate 
In the title compound, C25H25NO3, the dihydro­pyran ring adopts a half-chair conformation, whereas the pyrrolidine ring is in a twist conformation. The tolyl group is oriented at an angle of 82.92 (7)° with respect to the napthalene ring system. In the crystal structure, mol­ecules are linked into centrosymmetric dimers by C—H⋯π inter­actions involving the benzene ring of the tolyl group.
PMCID: PMC2977094  PMID: 21583514
11.  Methyl 3-(4-bromo­phen­yl)-1-methyl-1,2,3,3a,4,9b-hexa­hydro­benzo[f]chromeno[4,3-b]pyrrole-3a-carboxyl­ate 
In the title compound, C24H22BrNO3, the dihydro­pyran ring adopts a half-chair conformation, whereas the pyrrolidine ring is in an envelope conformation. The bromo­phenyl group is oriented at an angle of 66.44 (4)° with respect to the naphthalene ring system. In the crystal structure, mol­ecules are linked into centrosymmetric dimers by C—H⋯π inter­actions and the dimers are connected via C—H⋯Br hydrogen bonds. The crystal structure is further stabilized by π–π inter­actions [centroid–centroid distance = 3.453 (1) Å].
PMCID: PMC2977109  PMID: 21583696
12.  Methyl 1-methyl-3-phenyl-1,2,3,3a,4,9b-hexa­hydro­benzo[f]chromeno[4,3-b]pyrrole-3a-carboxyl­ate 
In the title compound, C24H23NO3, the dihedral angle between the naphthalene ring system and the phenyl ring is 76.82 (6)°. The pyrrolidine ring adopts an envelope conformation. In the crystal, weak inter­molecular C—H⋯O and C—H⋯π inter­actions are observed.
PMCID: PMC2977716  PMID: 21583852
13.  Methyl 3-(4-chloro­phen­yl)-1-methyl-1,2,3,3a,4,11c-hexa­hydro­benzo[f]chromeno[4,3-b]pyrrole-3a-carboxyl­ate 
In the title compound, C24H22ClNO3, the dihedral angle between the naphthalene ring system and the chloro­phenyl ring is 67.44 (4)°. The pyrrolidine and dihydro­pyran rings exhibit envelope and half chair conformations, respectively. In the crystal structure, weak C—H⋯π inter­actions are observed.
PMCID: PMC2983657  PMID: 21580370
14.  Methyl 3-(4-methoxy­phen­yl)-1-methyl-1,2,3,3a,4,11b-hexa­hydro­benzo[f]chromeno[4,3-b]pyrrole-3a-carboxyl­ate 
In the title compound, C25H25NO4, the pyrrolidine ring exhibits an envelope conformation and the tetra­hydro­pyran ring exhibits a half-chair conformation. The crystal structure is stabilized by inter­molecular C–H⋯π inter­actions.
PMCID: PMC2983994  PMID: 21580710
15.  4-(4-Bromo­phen­yl)-2,3,3a,4,5,11c-hexa­hydro­benzo[f]furo[3,2-c]quinoline 
In the title compound, C21H18BrNO, both heterocyclic rings, viz. the hydro­pyridine ring and the adjacent hydro­furan ring, adopt envelope conformations. These two heterocycles make a dihedral angle of 37.3 (1)°. The dihedral angle between the hydro­pyridine and benzene rings is 69.6 (1)°. In the crystal, adjacent mol­ecules are linked by pairs of inter­molecular C—H⋯O hydrogen bonds, forming centrosymmetric dimers.
PMCID: PMC3200627  PMID: 22065710
16.  rac-Ethyl 6-hy­droxy-6-methyl-3-oxo-4-phenyl-1,3,4,5,6,7-hexa­hydro­benzo[c][1,2]oxazole-5-carboxyl­ate 
In the title compound, C17H19NO5, the cyclo­hexene ring is in a half-chair conformation and the isoxazole ring in an envelope conformation with the N atom as the flap. The C atoms in the 4- and 6-positions are of the same absolute configuration, whereas the C atom in the 5-position is of the opposite configuration, i.e. (4S*,5R*,6S*). The methyl fragment of the eth­oxy­carbonyl group at position 5 is disordered over two sets of sites in a 0.60:0.40 ratio. The crystal packing displays inter­molecular N—H⋯O and O—H⋯O hydrogen bonds.
PMCID: PMC3247417  PMID: 22220035
17.  2-Amino-4-(4-chloro­phen­yl)-5,6,7,8,9,10-hexa­hydro­benzo[8]annulene-1,3-dicarbonitrile 
In the title compound, C20H18ClN3, the cyclo­octene ring exhibits conformational disorder of two methyl­ene groups with a site-occupation factor of 0.859 (6) for the major occupied site. In the crystal, mol­ecules are connected into inversion dimers via pairs of weak N—H⋯N hydrogen bonds, forming an R 2 2(12) graph-set motif. These dimers are further connected via weak N—H⋯Cl inter­actions into chains running along [011]. There are also C—H⋯N interactions present in the crystal.
PMCID: PMC3435713  PMID: 22969584
18.  Crystal structure of 3-(3-oxo-2,3,4,4a,5,6-hexa­hydro­benzo[h]cinnolin-2-yl)propionic acid 
The asymmetric unit of the title compound, C15H16N2O3, contains two independent mol­ecules, which present a different conformation of the carb­oxy­lic acid side chain [C—C—C—OH torsion angles = 65.3 (7) and −170.1 (5)°]. In both mol­ecules, the di­hydro­pyridazinone ring adopts a geometry inter­mediate between a twisted-boat and a half-chair conformation, while the central six-membered ring is almost in a half-boat conformation. In the crystal, mol­ecules are linked by O—H⋯Ok (k = ketone) hydrogen bonds, generating [01-1] chains. Aromatic π–π stacking contacts between the benzene and the di­hydro­pyridazinone rings [centroid–centroid distance [3.879 (9) Å] are also observed.
PMCID: PMC4257230  PMID: 25484687
crystal structure; pyridazinone moiety; stat3 inhibitor
19.  Main Group Multiple C–H/N–H Bond Activation of a Diamine and Isolation of A Molecular Dilithium Zincate Hydride: Experimental and DFT Evidence for Alkali Metal–Zinc Synergistic Effects 
Journal of the American Chemical Society  2011;133(34):13706-13717.
The surprising transformation of the saturated diamine (iPr)NHCH2CH2NH(iPr) to the unsaturated diazaethene [(iPr)NCH=CHN(iPr)]2- via the synergic mixture nBuM, (tBu)2Zn and TMEDA (where M = Li, Na; TMEDA = N,N,N′,N′-tetramethylethylenediamine) has been investigated by multinuclear NMR spectroscopic studies and DFT calculations. Several pertinent intermediary and related compounds (TMEDA)Li[(iPr)NCH2CH2NH(iPr)]Zn(tBu)2 (3), (TMEDA)Li[(iPr)NCH2CH2CH2N(iPr)]Zn(tBu) (5), {(THF)Li[(iPr)NCH2CH2N(iPr)]Zn(tBu)}2 (6), and {(TMEDA)Na[(iPr)NCH2CH2N(iPr)]Zn(tBu)}2 (11), characterized by single-crystal X-ray diffraction, are discussed in relation to their role in the formation of (TMEDA)M[(iPr)NCH=CHN(iPr)]Zn(tBu) (M = Li, 1; Na, 10). In addition, the dilithio zincate molecular hydride [(TMEDA)Li]2[(iPr)NCH2CH2N(iPr)]Zn(tBu)H 7 has been synthesized from the reaction of (TMEDA)Li[(iPr)NCH2CH2NH(iPr)]Zn(tBu)23 with nBuLi(TMEDA) and also characterized by both X-ray crystallographic and NMR spectroscopic studies. The retention of the Li–H bond of 7 in solution was confirmed by 7Li–1H HSQC experiments. Also, the 7Li NMR spectrum of 7 in C6D6 solution allowed for the rare observation of a scalar 1JLi–H coupling constant of 13.3 Hz. Possible mechanisms for the transformation from diamine to diazaethene, a process involving the formal breakage of four bonds, have been determined computationally using density functional theory. The dominant mechanism, starting from (TMEDA)Li[(iPr)NCH2CH2N(iPr)]Zn(tBu) (4), involves the formation of a hydride intermediate and leads directly to the observed diazaethene product. In addition the existence of 7 in equilibrium with 4 through the dynamic association and dissociation of a (TMEDA)LiH ligand, also provides a secondary mechanism for the formation of the diazaethene. The two reaction pathways (i.e., starting from 4 or 7) are quite distinct and provide excellent examples in which the two distinct metals in the system are able to interact synergically to catalyze this otherwise challenging transformation.
PMCID: PMC3662402  PMID: 21777000
20.  Probing Binding and Cellular Activity of Pyrrolidinone and Piperidinone Small Molecules Targeting the Urokinase Receptor 
ChemMedChem  2013;8(12):1963-1977.
The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here we evaluate the binding and biological activity of a new class of pyrrolidinone (3) and piperidinone (4) compounds, along with derivatives of previously-identified pyrazole (1) and propylamine (2) compounds. Competition assays revealed that the compounds displaced a fluorescently-labeled peptide (AE147-FAM) with inhibition constant Ki ranging from 6 to 63 μM. Structure-based computational pharmacophore analysis followed by extensive explicit-solvent molecular dynamics simulations and free energy calculations suggested pyrazole-based 1a and piperidinone-based 4 adopt different binding modes, despite their similar two-dimensional structures. In cells, compounds 1b and 1f showed significant inhibition of breast MDA-MB-231 and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but 4b exhibited no cytotoxicity even at concentrations of 100 μM. 1f impaired MDA-MB-231 invasion, adhesion, and migration in a concentration-dependent manner, while 4b inhibited only invasion. 1f inhibited gelatinase (MMP-9) activity in a concentration-dependent manner, while 4b showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1α and NF-κB signaling, while pyrrolidinone and piperidinone (3 and 4b) had no effect. Annexin V staining suggested that the effect of pyrazole-based 1f on proliferation was due to cell killing through an apoptotic mechanism.
PMCID: PMC4058332  PMID: 24115356
21.  Identification of the functional role of peroxiredoxin 6 in the progression of breast cancer 
The molecular mechanisms involved in breast cancer metastasis still remain unclear to date. In our previous study, differential expression of peroxiredoxin 6 was found between the highly metastatic MDA-MB-435HM cells and their parental counterparts, MDA-MB-435 cells. In this study, we investigated the effects of peroxiredoxin 6 on the proliferation and metastatic potential of human breast cancer cells and their potential mechanism.
Expression of peroxiredoxin 6 in the highly metastatic MDA-MB-231HM cells was investigated by RT-PCR, real-time PCR and western blot. A recombinant expression plasmid of the human peroxiredoxin 6 gene was constructed and transfected into MDA-MB-231 and MDA-MB-435 cells. The effects of peroxiredoxin 6 on the proliferation and invasion of MDA-MB-231 and MDA-MB-435 cells were investigated by the Cell Counting Kit-8 method, colony-formation assay, adhesion assay, flow cytometry and invasion assay in vitro. miRNA was used to downregulate the expression of peroxiredoxin 6. Genes related to the invasion and metastasis of cancer were determined by RT-PCR, real-time PCR and western blot. The tumorigenicity and spontaneously metastatic capability regulated by peroxiredoxin 6 were determined using an orthotopic xenograft tumor model in athymic mice.
Overexpression of peroxiredoxin 6 in MDA-MB-231HM cells compared with their parental counterparts was confirmed. Upregulation of peroxiredoxin 6 enhanced the in vitro proliferation and invasion of breast cancer cells. The enhancement was associated with decreasing levels of tissue inhibitor of matrix metalloproteinase (TIMP)-2 and increasing levels of the urokinase-type plasminogen activator receptor (uPAR), Ets-1 (E26 transformation-specific-1), matrix metalloproteinase (MMP)-9 and RhoC (ras homolog gene family, member C) expression. The results were further demonstrated by RNA interference experiments in vitro. In an in vivo study, we also demonstrated that peroxiredoxin 6-transfected breast cancer cells grew much faster and had more pulmonary metastases than control cells. By contrast, peroxiredoxin 6 knockdown breast cancer cells grew more slowly and had fewer pulmonary metastases. Effects similar to those of peroxiredoxin 6 on the uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression observed in in vitro studies were found in the in vivo study.
Overexpression of peroxiredoxin 6 leads to a more invasive phenotype and metastatic potential in human breast cancer, at least in part, through regulation of the levels of uPAR, Ets-1, MMP-9, RhoC and TIMP-2 expression.
PMCID: PMC2246172  PMID: 17980029
22.  siRNA-mediated Simultaneous downregulation of uPA and its receptor inhibits angiogenesis and invasiveness triggering apoptosis in breast cancer cells. 
International journal of oncology  2006;28(4):831-839.
A wide variety of tumor cells exhibit overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR). In breast cancer, expression of uPA and uPAR is essential for tumor cell invasion and metastasis. It is also known that uPA binds to uPAR and activates the RAS extra-cellular signal regulated kinase (ERK) signaling pathway. In our study, we have introduced small interfering RNA (siRNA) to downregulate the expression of uPA and uPAR in two breast cancer cell lines (MDA MB 231 and ZR 75 1). uPA and uPAR were downregulated individually using single constructs, and in combination using a bicistronic construct driven by a CMV promoter in a pcDNA-3 mammalian expression vector. Reverse transcription PCR (RT-PCR) and Western blot analyses indicated downregulation at both the mRNA and protein levels. In vitro angiogenesis studies using conditioned medium in HMEC-1 cells indicated a decrease in the angiogenic potential of conditioned media from treated cells when compared to the controls. This decrease in angiogenic potential was remarkably higher with the bicistronic construct. Similarly, the invasive potential of these cells decreased dramatically when treated with the bicistronic construct, thereby suggesting a synergistic effect from the downregulation of both uPA and uPAR. Furthermore, when uPA and uPAR were downregulated simultaneously, the apoptotic cascade was triggered as indicated by the upregulation of both initiator and effector caspases as well as other pro-apoptotic molecules. A mitochondrial permeability assay and FACS analysis revealed an increase in apoptotic cells in the uPA/uPAR treatment as compared to the other treatments. This overexpression of pro-apoptotic caspases in relation to the RNAi-induced downregulation of uPA and uPAR clearly suggests the involvement of the uPA-uPAR system in cell survival and proliferation in addition to their role in tumor progression.
PMCID: PMC1398074  PMID: 16525631
breast cancer cells; invasion; angiogenesis; uPA; uPAR
23.  CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity 
Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.
CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.
Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.
HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.
PMCID: PMC3446347  PMID: 22621373
24.  Maspin Regulates Hypoxia-Mediated Stimulation of uPA/uPAR Complex in Invasive Breast Cancer Cells 
Cancer biology & therapy  2005;4(4):400-406.
Maspin, a unique serine proteinase inhibitor (serpin), plays a key role in mammary gland development and is silenced during breast cancer progression. Maspin has been shown to inhibit tumor cell motility and invasion in cell culture, as well as growth and metastasis in animal models. In this study, we investigated the effect of maspin on the regulation of hypoxia-induced expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), with respect to invasive potential in metastatic breast cells MDA-MB-231. We hypothesized that maspin can neutralize or mitigate hypoxia- induced expression of uPA/uPAR in metastatic breast cancer cells, resulting in suppression of their invasive potential. To test our hypothesis, we employed the highly invasive MDA-MB-231 breast cancer cells that are devoid of maspin, and transfected them with the maspin gene, and then determined the effect of hypoxia on uPA/uPAR expression. Normal mammary epithelial cells 1436N1 were used as a control. Our findings demonstrate that maspin downregulated the basal and hypoxia-induced uPA/uPAR expression and reduced the stimulatory effect of hypoxia on the in vitro invasive ability of MDA-MB-231-cells. In addition, maspin also inhibited the enzymatic activity of secreted and cell associated uPA in MDA-MB-231 cells. These results indicate that maspin inhibits hypoxia-induced invasion of metastatic breast cancer cells by blocking the uPA system, thus illuminating an important molecular pathway for therapeutic consideration.
PMCID: PMC3175738  PMID: 15846059
uPA/uPAR; hypoxia; invasion; maspin; breast cancer
25.  RNAi-mediated Downregulation of Urokinase Plasminogen Activator Receptor (uPAR) and Matrix Metalloprotease-9 (MMP-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 (siRNA) 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.
PMCID: PMC2396459  PMID: 17657740
RNAi; uPAR; MMP-9; Invasion; Angiogenesis; Tumor growth

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