Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation

Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjugate Chem. 2008, 19, 290-8). In this study, sense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. The disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression by about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo.     

Design of cyclic RGD-conjugated Aib-containing amphipathic helical peptides for targeted delivery of small interfering RNA

To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to α_Vβ₃ integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the α_Vβ₃ integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0 μM PI/10 nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA.     

Multiphoton-absorption-induced-luminescence (MAIL) imaging of tumor-targeted gold nanoparticles

We demonstrate that multiphoton-absorption-induced luminescence (MAIL) is an effective means of monitoring the uptake of targeted nanoparticles into cells. Gold nanoparticles (AuNPs) with diameters of 4.5 and 16 nm were surface-functionalized with monocyclic RGDfK, an RGD peptide analogue that specifically targets the α(v)β? integrin, a membrane protein that is highly overexpressed in activated endothelial cells during tumor angiogenesis. To determine whether cyclic RGD can enhance the uptake of the functionalized AuNPs into activated endothelium, human umbilical vein endothelial cells (HUVECs) were used as a model system. MAIL imaging of HUVECs incubated with AuNPs demonstrates differential uptake of AuNPs functionalized with RGD analogues: RGDfK-modified nanoparticles are taken up by the HUVECs preferentially compared to AuNPs modified with linear RGD (GRGDSP) conjugates or with no surface conjugates. The luminescence counts observed for the AuNP-RGDfK conjugates are an order of magnitude greater than for AuNP-GRGDSP conjugates. Transmission electron microscopy shows that, once internalized, the AuNP-RGDfK conjugates remain primarily within endosomal and lysosomal vesicles in the cytoplasm of the cells. Significant aggregation of these particles was observed within the cells. MAIL imaging studies in the presence of specific uptake inhibitors indicate that AuNP-RGDfK conjugate uptake involves a specific binding event, with α(v)β? integrin-mediated endocytosis being an important uptake mechanism.     

Synthesis and evaluation of cyclic RGD-boron cluster conjugates to develop tumor-selective boron carriers for boron neutron capture therapy

Boron-containing agents play a key role in successful boron neutron capture therapy (BNCT). Icosahedral boron cluster-Arg-Gly-Asp (RGD) peptide conjugates were designed, synthesized, and evaluated for the biodistribution to develop tumor-selective boron carriers. Integrin αvβ3 is an attractive target for anti-tumor drug delivery because of its specific expression in proliferating endothelial and tumor cells of various origins. We, therefore, selected a c(RGDfK) moiety recognizing αvβ3 as an active tumor-targeting device to conjugate with icosahedral boron-10 clusters, disodium mercaptododecaborate (BSH) or o-carborane as a thermal neutron-sensitizing unit. Preparation of o-carborane derivatives involved microwave irradiation, and resulted in high yields in a short time. An in vitro cell adhesion assay on αvβ3-positive U87MG and SCCVII cells demonstrated the high binding affinity of conjugates to integrin αvβ3 (IC(50)=0.19-2.66 μM). Biodistribution experiments using SCCVII-bearing mice indicated that GPU-201 showed comparable tumor uptake and a significantly longer retention in tumors compared with BSH. These results suggest that GPU-201 is a promising candidate for use in BNCT.     

High expression of integrin αvβ3 enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors

The cell line OVCAR-4 was recently ranked as one of the most representative cell lines for high grade serous ovarian cancer (HGSOC). However, little work has been done to assess the susceptibility of OVCAR-4 cells and tumors to the more common types of molecular targeting. Proteome profiles suggest OVCAR-4 express high levels of integrin αvβ3 receptors. Using flow cytometry with fluorescent antibodies we determined that OVCAR-4 cells have high number of integrin αvβ3 receptors ([9.8?±?2.5]?×?10⁴/cell) compared to the well-characterized cell line U87-MG ([5.2?±?1.4]?×?10⁴/cell). However, OVCAR-4 cells also have roughly three times the surface area of U87-MG cells, so the average αvβ3 receptor density is actually lower (11?±?3 versus 18?±?6?receptors/µm²). A series of new fluorescent molecular probes was prepared with structures comprised of a deep-red squaraine fluorophore (～680?nm emission) covalently attached to zero, one, or two cyclic pentapeptide cRGD sequences for integrin targeting. Microscopy studies showed that uptake of the divalent probe into cultured OVCAR-4 cells was 2.2?±?0.4 higher than the monovalent probe, which in turn was 2.2?±?0.4 higher than the untargeted probe. This probe targeting trend was also seen in OVCAR-4 mouse tumor models. The results suggest that clinically relevant OVCAR-4 cells can be targeted using molecular probes based on αvβ3 integrin receptor antagonists such as the cRGD peptide. Furthermore, deep-red fluorescent cRGD-squaraine probes have potential as targeted stains of cancerous tissue associated with HGSOC in surgery and pathology settings.     

Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA

RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvβ3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvβ3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy.     

αvβ5/β6 integrin suppression leads to a stimulation of α2β1 dependent cell migration resistant to PI3K/Akt inhibition

Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins αvβ5/β6 and α2β1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our understanding of the consequences of αvβ5/β6 function on α2β1, we decreased the expression of αv integrins by either siRNA or lysosomal targeting strategies or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced α2β1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The αvβ5/β6-dependent limitation of α2β1 function could be overridden by TS2/16, an activating anti-β1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high α2β1-mediated cell migration observed in the absence of αv integrins or following activation of α2β1 integrins by the TS2/16. These results suggest that integrins αvβ5/β6 repress α2β1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of α2β1-mediated migration.     

Insulin receptor substrate 1 knockdown in human MCF7 ER+ breast cancer cells by nuclease-resistant IRS1 siRNA conjugated to a disulfide-bridged D-peptide analogue of insulin-like growth factor 1

IRS-1 overexpression has been associated with breast cancer development, hormone independence and antiestrogen resistance. IRS-1 is a major downstream signaling protein for insulin and IGF1 receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In estrogen-sensitive breast cancer cell lines, the widely used antiestrogen tamoxifen treatment reduces IRS-1 expression and function, thereby inhibiting IRS-1/PI-3K signaling. IRS-1 may serve as an alternative target to overexpressed IGF1R in breast cancer. While siRNA technology has become commonplace in many laboratories for in vitro gene knockdown studies, and in vivo stability issues are largely solved, its use in vivo is limited by an inability to efficiently and specifically deliver it to the intended site of action. We previously reported reduced survival of human MCF7 estrogen receptor positive breast cancer cells treated with a normal IRS1 siRNA delivered by a cationic lipid, plus an additive effect in combination with tamoxifen. We now report enhanced cellular uptake, relative to the unconjugated serum-stabilized IRS1 siRNA, of a serum-stabilized IRS1 siRNA conjugated with our previously characterized peptide mimetic of IGF1, D-(Cys-Ser-Lys-Cys), without the use of cationic lipids or electroporation, in MCF7 cells that overexpress IGF1R. Excess native IGF1 blocked uptake. An IRS1 siRNA cholesterol conjugate, targeted universally to cell membranes, was taken up by MCF7 cells as much as the peptide mimetic conjugate. IRS1 mRNA knockdown and IRS-1 protein knockdown were comparable for the IGF1 peptide and cholesterol conjugates. The unconjugated serum-stabilized IRS1 siRNA control showed negligible effects. Viability assays showed additive effects of siRNA treatment in combination with tamoxifen. In summary, we have taken the first step in converting an siRNA into a pharmacologically active agent for breast cancer.     

Src, PKCα, and PKCδ are required for αvβ3 integrin-mediated metastatic melanoma invasion

Background

Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor αvβ3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.

Results

Two melanoma cell lines C8161.9 and M14 both express high levels of αvβ3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an αvβ3-depenent manner. Elevated levels of PKCα and PKCδ, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed αvβ3-dependent invasion. Furthermore, over expression of Src or PKCα and PKCδ was sufficient to confer αvβ3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKCα expression, but not PKCδ, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKCα restored focal adhesion formation and partially restored stress fiber formation, while loss of PKCδ primarily restored stress fibers.

Conclusion

The misregulated expression of PKCα and PKCδ and elevated Src activity in metastatic melanoma cells is required for efficient αvβ3-mediated invasion. PKCα and Src enhance αvβ3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKCα influences focal adhesion formation, while PKCδ controls stress fibers.     

人黑色素瘤细胞裸鼠肺转移模型的建立

目的建立整合素αvβ3高表达的黑色素瘤细胞肺转移模型。方法通过将不同数目的M21细胞经尾静脉接种裸鼠,适时处死后计数肺表面癌结节。通过实时定量PCR和明胶酶谱的方法比较肺转移灶细胞与亲代M21细胞差异。结果M21细胞1×10^6、2×10^6、5×10^6尾静脉接种裸鼠,均能形成肺转移灶,癌结节数目均值分别为：84±8、70±6、88±12,三组之间没有明显差异,阴性对照组未形成转移灶。M21肺转移灶细胞与亲代细胞相比,增殖增快,MMP-2活性增高,整合素αv和β3mRNA表达水平明显增高。结论M21细胞1×10^6经尾静脉接种裸鼠50d内即可100%成瘤。M21肺转移灶细胞具有更快的增殖能力,整合素αvβ3和MMP-2表达水平明显增高。本实验建立了稳定的肺转移模型,为黑色素瘤和整合素αvβ3的研究提供重要的动物模型。     

Chemically programmed antibodies targeting multiple alpha(v) integrins and their effects on tumor-related functions in vitro

Integrins αvβ3 and αvβ6 are highly expressed on tumor cells and/or by the tumor vasculature of many human cancers, and represent promising targets for anticancer therapy. Novel chemically programmed antibodies (cpAbs) targeting these integrins were prepared using the catalytic aldolase Antibody (Ab) programming strategy. The effects of the cpAbs on cellular functions related to tumor progression were examined in vitro using tumor cell lines and their cognate integrin ligands, fibronectin and osteopontin. The inhibitory functions of the conjugates and their specificity were examined based on interference with cell-cell and cell-ligand interactions related to tumor progression. Cell binding analyses of the anti-integrin cpAbs revealed high affinity for tumor cells that overexpressed αvβ3 and αvβ6 integrins, and weak interactions with αvβ1 and αvβ8 integrins, in vitro. Functional analyses demonstrated that the cpAbs strongly inhibited cell-cell interactions through osteopontin binding, and they had little or no immediate effects on cell viability and proliferation. On the basis of these characteristics, the cpAbs are likely to have a broad range of activities in vivo, as they can target and antagonize one or multiple αv integrins expressed on tumors and tumor vasculatures. Presumably, these conjugates may inhibit the establishment of metastastatic tumors in distant organs through interfering with cell adhesion more effectively than antibodies or compounds targeting one integrin only. These anti-integrin cpAbs may also provide useful reagents to study combined effect of multiple αv integrins on cellular functions in vitro, on pathologies, including tumor angiogenesis, fibrosis, and epithelial cancers, in vivo.     

Characterization of conjugated and unconjugated bile acid transport via human organic solute transporter α/β

Bile acids are biosynthesized from cholesterol in hepatocytes and usually localize in the enterohepatic circulation system. This system is regulated by several transporters that are expressed in the liver and intestine. Organic solute transporter (OST) α/β, which is known as a bidirectional transporter for some organic anions, contributes to the transport of bile acids; however, the transport properties of individual bile acids are not well understood. In this study, we investigated the transport properties of five bile acids (cholic acid [CA], chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], ursodeoxycholic acid [UDCA], and lithocholic acid [LCA]) together with their glycine and taurine conjugates mediated by OSTα/β. Of the unconjugated bile acids, CA, CDCA, DCA, and LCA were taken up by OSTαβ/MDCKII cells more rapidly than mock cells, but no significant increase in the uptake of UDCA was observed. On the contrary, all glycine- and taurine-conjugated bile acids showed a significant increase in the uptake by OSTαβ/MDCKII cells. Saturable OSTα/β-mediated transports of CDCA, DCA, glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurochenodeoxycholic acid (TCDCA), and taurolithocholic acid (TLCA) were observed. The apparent Michaelis constants of CDCA, DCA, GCDCA, GDCA, GLCA, TCDCA, and TLCA for OSTα/β were 23.0 ± 4.0, 14.9 ± 1.9, 864.2 ± 80.7, 586.4 ± 43.2, 12.8 ± 0.5, 723.7 ± 4.8, and 23.9 ± 0.3 μM, respectively. However, the transport of other bile acids was not saturable. Our results indicate that OSTα/β has a low affinity but a high capacity for transporting bile acids.     

Alpha v integrin antagonists induce the disassembly of focal contacts in melanoma cells

In recent years, several antagonists of alpha(v)beta3 have been used to develop therapeutic approaches to the treatment of melanoma neoplasia. We studied the effects of anti-alpha(v)-integrin-blocking antibodies on attached M21 melanoma cells, the cellular distribution of alpha(v)-integrin and the molecular organization of focal structures. Anti-alpha(v)-integrin-blocking antibodies 17E6 and LM609, and an anti-alpha(v)beta3-integrin antagonist peptide cRGD 85189 induced detachment of M21 melanoma cells cultured for 24 hours on various substrates. cRGD was the most effective antagonist, reducing the number of adherent cells by 80%, while 17E6 reduced adhesion by only 30%. Light- and electron microscopy revealed attached cells with a flat shape and well-formed actin cytoskeleton. After treatment, cells became rounded and detached from the culture dish. alpha(v)-Integrins and focal-contact proteins were observed at adhesion sites in focal structures by immunocytochemistry. After treatment, however, cell rounding was accompanied by disorganization of the actin filaments and redistribution of alpha(v)-integrins and most of the focal proteins studied, except vinculin and tensin. Our results indicate that treatment of M21 melanoma cells with a(v)-integrin antagonists disrupts the actin cytoskeleton, redistributes a(v)-integrin and induces molecular disassembly of focal contacts.     

Targeted albumin-based nanoparticles for delivery of amphipathic drugs

We report the preparation and physical and biological characterization of human serum albumin-based micelles of approximately 30 nm diameter for the delivery of amphipathic drugs, represented by doxorubicin. The micelles were surface conjugated with cyclic RGD peptides to guide selective delivery to cells expressing the α(v)β(3) integrin. Multiple poly(ethylene glycol)s (PEGs) with molecular weight of 3400 Da were used to form a hydrophilic outer layer, with the inner core formed by albumin conjugated with doxorubicin via disulfide bonds. Additional doxorubicin was physically adsorbed into this core to attain a high drug loading capacity, where each albumin was associated with about 50 doxorubicin molecules. The formed micelles were stable in serum but continuously released doxorubicin when incubated with free thiols at concentrations mimicking the intracellular environment. When incubated with human melanoma cells (M21+) that express the α(v)β(3) integrin, higher uptake and longer retention of doxorubicin was observed with the RGD-targeted micelles than in the case of untargeted control micelles or free doxorubicin. Consequently, the RGD-targeted micelles manifested cytotoxicity at lower doses of drug than control micelles or free drug.     

Cellular delivery and biological activity of antisense oligonucleotides conjugated to a targeted protein carrier

Targeted delivery can potentially improve the pharmacological effects of antisense and siRNA oligonucleotides. Here, we describe a novel bioconjugation approach to the delivery of splice-shifting antisense oligonucleotides (SSOs). The SSOs are linked to albumin via reversible S-S bonds. The albumin is also conjugated with poly(ethylene glycol) (PEG) chains that terminate in an RGD ligand that selectively binds the alphavbeta3 integrin. As a test system, we utilized human melanoma cells that express the alphavbeta3 integrin and that also contain a luciferase reporter gene that can be induced by delivery of SSOs to the cell nucleus. The RGD-PEG-SSO-albumin conjugates were endocytosed by the cells in an RGD-dependent manner; using confocal fluorescence microscopy, evidence was obtained that the SSOs accumulate in the nucleus. The conjugates were able to robustly induce luciferase expression at concentrations in the 25-200 nM range. At these levels, little short-term or long-term toxicity was observed. Thus, the RGD-PEG-albumin conjugates may provide an effective tool for targeted delivery of oligonucleotides to certain cells and tissues.     

Cyclic RGD functionalized gold nanoparticles for tumor targeting

Integrin α(v)β(3) is an adhesion molecule involved in physiological and pathological angiogenesis as well as tumor invasion and metastasis. Therefore, it is considered an important target for molecular imaging and delivery of therapeutics for cancer, and there is a strong interest in developing novel agents interacting with this protein. Nevertheless, the interaction of individual ligands is often still weak for efficient tumor targeting, and many research groups have synthesized multivalent displays in order to overcome this problem. Gold nanoparticles can be considered a smart platform for polyvalent presentation on account of their globular shape, tunable size, facile surface chemistry, and biocompatibility. Moreover, their unique physical properties render gold nanoparticles ideal candidates for tumor diagnosis and therapy. Here, we report the synthesis and characterization of gold nanoparticles functionalized with cRGD integrin ligand and their employment for targeting human cancer cells expressing α(v)β(3) integrin.     

An efficient chemical approach to bispecific antibodies and antibodies of high valency

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (d-Lys⁶)-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (d-Lys⁶)LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (d-Lys⁶)-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors α(v)β(3) and α(v)β(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M-21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.     

Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells

The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 μM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin αv and α5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that αvβ1 and αvβ3 integrins were involved in adhesion of cells to Vn, and αvβ3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, α5β1 and αvβ1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.     

Osteopontin increases migration and MMP‐9 up‐regulation via αvβ3 integrin,FAK, ERK,and NF‐κB‐dependent pathway in human chondrosarcoma cells

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, invasion and proliferation via interaction with its receptor, that is, αvβ3 integrin. However, the effect of OPN on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that OPN increased the migration and expression of matrix metalloproteinase (MMP)‐9 in human chondrosarcoma cells (JJ012 cells). RGD peptide, αvβ3 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the OPN‐induced increase of the migration and MMP‐9 up‐regulation of chondrosarcoma cells. OPN stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal‐regulated kinase (ERK). In addition, treatment of JJ012 cells with NF‐κB inhibitor (PDTC) or IκB protease inhibitor (TPCK) inhibited OPN‐induced cell migration and MMP‐9 up‐regulation. Stimulation of JJ012 cells with OPN also induced IκB kinase α/β (IKK α/β) phosphorylation, IκBα phosphorylation, p65 Ser⁵³⁶ phosphorylation, and κB‐luciferase activity. The OPN‐mediated increases in MMP‐9 and κB‐luciferase activities were inhibited by RGD peptide, PD98059 or FAK and ERK2 mutant. Taken together, our results indicated that OPN enhances the migration of chondrosarcoma cells by increasing MMP‐9 expression through the αvβ3 integrin, FAK, MEK, ERK and NF‐κB signal transduction pathway. J. Cell. Physiol. 221: 98–108, 2009. © 2009 Wiley‐Liss, Inc