Alphavbeta3 Integrin Expression Research Articles

Uterine administration of C-X-C motif chemokine ligand 12 increases the pregnancy rates in mice with induced endometriosis

To study the effect of intrauterine injection of C-X-C motif chemokine ligand 12 (CXCL12), also known as a stem cell chemoattractant (stromal cell-derived factor 1), on fertility and endometrial receptivity in mice with endometriosis. Laboratory study. Academic Medical Center. Fifty-six mice underwent chemotherapy and bone marrow transplantation. Thirty-six of these mice underwent either surgery to induce endometriosis (n = 20) or sham surgery (n = 16). Injection of CXCL12 as a potential therapeutic agent to improve fertility in endometriosis. Pregnancy rate, bone marrow-derived cell (BMDC) recruitment and endometrial receptivity markers. The mice with or without endometriosis received a single uterine injection of either CXCL12 or placebo. Uterine injection of CXCL12 increased the pregnancy rates in a mouse model of endometriosis. Mice were euthanized after delivery, and implantation markers homeobox A11, alpha-v beta-3 integrin, and progesterone receptor were analyzed by immunohistochemistry, whereas green fluorescent protein positive BMDC recruitment was quantified by immunohistochemistry and immunofluorescence. The sham surgery groups without endometriosis had the highest cumulative pregnancy rate (100%) regardless of CXCL12 treatment. The endometriosis group treated with placebo had the lowest pregnancy rate. An increased pregnancy rate was noted in the endometriosis group after treatment with CXCL12. There was also an increase in BMDC recruitment and endometrial expression of progesterone receptor and alpha-v beta-3 integrin in the endometriosis group that received CXCL12 compared with that in the endometriosis group that received placebo. Uterine injection of CXCL12 increased the pregnancy rates in a mouse model of endometriosis. These results suggest that CXCL12 has a potential role as a therapeutic agent in women with infertility related to endometriosis and potentially other endometrial receptivity defects.

AlphaVBeta3 Integrin expression within uterine endometrium in unexplained infertility: a prospective cohort study

BackgroundImplantation defect is one of these contributing factors for unexplained infertility. In the mid-luteal phase, when implantation is expected to happen, Integrins expression is remarkably increased. So, Integrins could potentially serve as markers for the frame of the window of implantation. αVβ3 integrin could have a role as a potential receptor for embryonic attachment. The aim of the current study is to investigate whether the women with unexplained infertility have a pattern of expression of endometrial αvβ3 integrin that could differ from those who have normal fertility or not.MethodTwo groups of women have been included in this study. The first group was the Unexplained Infertility Group. This group included women diagnosed with unexplained primary infertility. The second group was the fertile Group, which included fertile parous women presented to the family planning clinic seeking contraception. 2D transvaginal ultrasound scan (TVS) was performed six days after detecting urinary LH surge. (TVS) was used to measure endometrial thickness, and subendometrial blood flow color Doppler Resistance Index (RI). On the same day of transvaginal ultrasound, endometrial samples were taken using the Endocell® office suction sampler for Immunohistochemistry (IHC) study using monoclonal mouse IgG antibodies to detect endometrial αvβ3 integrin.ResultsThirty-five fertile women with a diagnosis of unexplained infertility were included as a group I [Unexplained infertility Group] along with an equal number of fertile women as group II [Fertile Group]. The group of women with a diagnosis of unexplained infertility had a significantly lower αvβ3 integrin score when compared to the fertile group (median score 0, range:0–2 and median score 1, range: 1–3 and for infertile and fertile groups respectively, P < 0.0001). In addition, the unexplained infertility group had significantly higher subendometrial flow RI and Significantly thinner endometrial thickness.ConclusionThis study showed that Alpha v Beta 3 integrin is a significantly lower in endometrium in cases of unexplained infertility, which may suggest that underexpression of Alpha v Beta 3 integrin in human endometrium could be linked to defective uterine receptivity, and play a role as an unrecognized cause of infertility in this population of women. We need larger studies of adequate statistical power, ideally investigating more than one menstrual cycle in the same woman, to investigate the usefulness of using these molecular molecules in clinical practice.

Anti-tumor efficacy of ultrasonic cavitation is potentiated by concurrent delivery of anti-angiogenic drug in colon cancer

This study investigated the efficacy of concurrent delivery of an anti-angiogenic drug and ultrasonic cavitation therapy in a mouse model of human colon cancer. A biotinylated form of the anti-angiogenic drug Endostar was conjugated to a streptavidin-coated microbubble (MB). Mice bearing subcutaneous tumors (HT29) were divided into 4 groups. Group 1 served as an untreated control. Group 2 served as a cavitation control and received naked microbubbles and sham ultrasonic cavitation (MB+sham cavitation). Group 3 received naked microbubbles and ultrasonic cavitation (MB+cavitation). Group 4 received Endostar loaded microbubbles and ultrasonic cavitation (Endostar-MB+cavitation). Ultrasonic cavitation was performed using a high-power custom built sonicator. Contrast-enhanced ultrasound imaging (CEUS) was used to measure tumor blood flow before and after ultrasonic cavitation. In vivo fluorescence imaging was performed to monitor changes in tumor volume. Immunohistochemistry was performed to assess CD31, VEGFR-2 and alpha-v beta-3 integrin expression within the tumor. Apoptosis of the tumor cells was determined by TUNEL assay, and ultrastructural changes within the tumor were examined by electron microcopy. Ultrasonic cavitation with Endostar-MB demonstrated a significantly greater inhibition of tumor blood flow on day 7 and tumor growth on day 16 compared with naked MB and control groups. The Endostar-MB treated mice showed significantly decreased expression VEGFR-2 and alpha-v beta-3 integrin, and increased apoptosis of tumor cells and degradation of the tumor ultrastructure. Our findings indicated that the anti-vascular and anti-tumor effects of ultrasonic cavitation could be potentiated by simultaneously delivering an anti-angiogenic drug in colon cancer.

Potential Significance of EMP3 in Patients with Upper Urinary Tract Urothelial Carcinoma: Crosstalk with ErbB2-PI3K-Akt Pathway

Upper urinary tract (pyelocalyceal cavities and ureter) urothelial carcinoma is a relatively rare neoplastic disease. Although diagnosis and treatment of this tumor variant have improved significantly, accurate risk stratification remains a challenge. To identify the putative oncogene involved in urothelial carcinoma progression we performed bioinformatics guided experimental investigation targeting chromosome 19q13. We investigated the effects of EMP3 on cancer cell growth, migration and adhesion in transfection and siRNA experiments in vitro. Crosstalk of integrins or ErbB2 with EMP3 was examined by reverse transcriptase-polymerase chain reaction and immunoblot. The potential involvement of epigenetic alterations of EMP3 in vitro and in vivo was analyzed by methylation specific polymerase chain reaction. To validate clinical relevance we measured EMP3 expression at the mRNA and protein levels in a cohort of 77 patients with upper urinary tract urothelial carcinoma and compared prognostic significance in relation to that of ErbB2 expression. We noted functional crosstalk between ErbB2 and EMP3 in vitro. EMP3 over expression promoted cancer cell proliferation and migration but suppressed cell adhesion in vitro. EMP3 activated the ErbB2-PI3K-AKT pathway to increase cell growth in vitro. In the clinical cohort Kaplan-Meier survival estimates showed that ErbB2 and EMP3 co-expression was the most important indicator of progression-free and metastasis-free survival in patients with upper urinary tract urothelial carcinoma (log rank test p = 0.018 and 0.04, respectively). EMP3 is an important prognostic indicator for selecting patients with upper urinary tract urothelial carcinoma for more intensive therapy. EMP3 is an innovative co-targeting candidate for designing ErbB2 based cancer therapy.

Abstract 424: Interference with alphaVbeta3 integrins via cyclic RGD peptides affects breast cancer cell behavior

Abstract Integrin alphavbeta3 is mainly expressed in stimulated endothelial cells, thus it has recently become the focus of intense research mainly to interfere with tumorangiogenesis. However, alphavbeta3 integrins are also expressed in a variety of tumor cells, such as melanoma, prostate cancer, breast cancer, cervix carcinoma or pancreatic cancer. While inhibition of integrin alphavbeta3 activity in endothelial cells has been studied extensively, any direct effects on integrin alphavbeta3 expressing tumor cells is hitherto unknown. In this study we examined alphavbeta3 integrin expression in breast cancer cell lines, where by MDA MB 231 revealed high integrin alphavbeta3 as well as VEGFR-2 and VEGF expression. While adhesion of MDA MB231 cells to an alphavbeta3 specific matrix such as fibrinogen was blocked by the addition of cyclic RGD peptides, sequentially addition of cyclic RGD peptides to already adherent MDA MB231 cells led to a diminished spreading in a time and dose dependent manner, starting at 20nM. Furthermore, tumor cell migration as well as transmigration through an EC monolayer upon VEGF stimulation was blocked whenever alphavbeta3 integrins were targeted by RGD mimetic peptide. Our data give first functional insights into the previously described effects of alphavbeta3 targeting small molecules, which led to a reduced breast cancer metastasis in vivo. Thus interfering with alphavbeta3 integrins on tumor cells might represent a promising therapeutic lead in certain malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 424. doi:10.1158/1538-7445.AM2011-424

Correlation of the Expression of Integrin αvβ3 in Endometrium and Peripheral Blood Lymphocytes in Infertile Patients

Integrin alphavbeta3 is an adhesion molecule thought to be relevant to blastocyst attachment. It is expressed by endometrial epithelial cells at the time of ''implantation window,'' and its expression may be altered in women with implantation failure. Integrin alphavbeta3 is also expressed by T lymphocytes. Because immune cells interact with endometrial tissues via integrins, we hypothesized that expression of integrin alphavbeta3 in endometrium and in peripheral blood lymphocytes may correlate. A total of 41 women with implantation failure underwent endometrial biopsy and blood collection at the time of implantation window. Integrin alphavbeta3 expression was evaluated in fresh frozen endometrial specimens using immunohistochemistry and on T lymphocytes using flow cytometry. Statistically significant negative correlation was found between integrin alphavbeta3 in endometrium and T lymphocytes. Integrin alphavbeta3 expression on T lymphocytes does not predict its endometrial expression.

Osteopontin and αvβ3 integrin as markers of endometrial receptivity: the effect of different hormone therapies

The osteopontin: alphavbeta3 integrin complex has been proposed as a means of distinguishing receptive from non-receptive endometrium in clinical practice, thus offering new directions for the development of contraceptive approaches targeted to the endometrium as well as a better understanding of occult causes of infertility in women. Histological dating and immunohistochemical study were performed in control and study cycles in seven groups of women including 10 subjects per group and who received clomiphene citrate, ovarian stimulation for IVF, oral contraception, dehydrogesterone for endometrial luteal phase defect, two different regimens of hormone replacement therapy, or no treatment. Ten healthy fertile women served as a general control group. Osteopontin and alphavbeta3 integrin expression in the human endometrium was closely related to endometrial maturation and this was irrespective of the endometrium being in-phase or out-of-phase and the hormonal treatment (or no treatment) received. In conclusion, immunohistochemical assessment of the endometrium indicates that the use of osteopontin and alphavbeta3 integrin or the osteopontin: alphavbeta3 integrin complex as targets for the development of contraceptive approaches or the understanding of the pathogenesis of female infertility offer little benefit compared with simple histological dating.

Transforming Growth Factor-.BETA. Upregulates the Expression of Integrin and Related Proteins in MRC-5 Human Myofibroblasts

Myofibroblasts are defined as fibroblasts that express certain features of smooth muscle differentiation. Activation of myofibroblasts plays a central role in fibrosis. Transforming growth factor-beta (TGF-beta) is a potent activator of myofibroblasts; namely, TGF-beta causes changes in myofibroblast phenotypes including morphological alterations and the expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts. Because it has been well known that humoral factors, especially, TGF-beta, and extracellular matrix components cause myofibroblast activation, we examined the expression of integrin and related proteins during activation of MRC-5 human myofibroblasts with TGF-beta. Western blot analysis revealed that TGF-beta treatment for 3 days increased the expression of alpha-SMA, which was also immunocytochemically observed as actin stress fibers. In the early phase of TGF-beta treatment, fibronectin expression was greatly increased, followed by the increased expression of integrin alphav and alpha11 and integrin beta1 and beta3. Co-immunoprecipitation assays revealed that the integrin alphav subunit was co-precipitated with integrin beta1 and beta3, and that integrin beta1 was co-precipitated with alpha11, alphav, alpha2, and alpha5. The expression of focal adhesion kinase and integrin-linked kinase proteins was also upregulated by treatment with TGF-beta. In addition, the expression of type I collagen mRNA was increased by TGF-beta, but not type III collagen mRNA, as judged by real-time PCR analysis. These results suggest the possibility that TGF-beta induces fibronectin expression in MRC-5 cells, which subsequently induces the expression of integrin receptors, alphavbeta3, alphavbeta1, and alpha11beta1. This report also shows that expression of integrin alpha11 is upregulated during the TGF-beta-mediated activation of myofibroblasts.

Optical Imaging of Integrin αvβ3Expression with Near-Infrared Fluorescent RGD Dimer with Tetra(ethylene glycol) Linkers

Integrin alphavbeta3 plays great roles in tumor angiogenesis, invasion, and metastasis. We report here the noninvasive visualization of tumor integrin alphavbeta3 expression by using near-infrared fluorescence (NIRF) imaging of an IRDye800-labeled new cyclic RGD (arginine-glycine-aspartic acid) dimer with tetra(ethylene glycol) (PEG4) linkers (ie, E[PEG4-c(RGDfK)]2, PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) in a U87MG tumor model. Fluorescent dye-labeled E[PEG4-c(RGDfK)]2 were subjected to in vitro cell staining, in vivo NIRF imaging, ex vivo NIRF imaging, and histologic studies. The in vitro and in vivo characterization of dye-labeled E[PEG4-c(RGDfK)]2 were compared with dye-labeled RGD dimer without PEG4 linkers (namely, E[c(RGDfK)]2). Both Cy5.5-E[PEG4-c(RGDfK)]2 and Cy5.5-E[c(RGDfK)]2 exhibited integrin alphavbeta3 binding specificity in a cell-staining experiment. In vivo NIRF imaging showed higher tumor accumulation and tumor to background contrast of IRDye800-E[PEG4-c(RGDfK)]2 over IRDye800-E[c(RGDfK)]2. The tumor integrin alphavbeta3 specificity of IRDye800-E[PEG4-c(RGDfK)]2 was confirmed by successful inhibition of tumor uptake in the presence of an excess dose of c(RGDfK). Histologic examination revealed both tumor vasculature and tumor cell integrin alphavbeta3 binding of IRDye800-E[PEG4-c(RGDfK)]2 in vivo. In summary, NIRF imaging with IRDye800-E[PEG4-c(RGDfK)]2 offers an easy, fast, and low-cost way to detect and semiquantify tumor integrin alphavbeta3 expression in living subjects.

Expression of estrogen receptor, progesterone receptor and integrin alphaVbeta3 in endometrium of patients with luteinized unruptured follicle.

To investigate endometrium receptivity in patients with luteinized unruptured follicle (LUF) by measuring the expression of estrogen receptor (ER), progesterone receptor (PR) and integrin alphaVbeta3 in the endometrium. From May 2007 to Nov. 2007, 17 infertile women with LUF were selected as LUF group matched with 13 infertile cases with normal ovulation as control group. They all underwent frozen-thawed embryo transfer in Reproductive Medicine Center, Renmin Hospital of Wuhan University. Endometrial tissue in anterior and posterior wall of uterus of LUF group and control group were biopsied by a small curettage between 7 and 11 days after luteinizing hormone (LH) surge. The expression of ER, PR and integrin alphaVbeta3 in endometrium were detected by immunohistochemistry staining. The level of estrogen and progesterone were measured by chemiluminescence assay. Then, the relationship between alphaVbeta3 expression in endometrium and the level of estrogen/progesterone were analyzed in LUF patients. (1) There was no remarkable difference in the level of estrogen between LUF [(656 +/- 299) pmol/L] and control group [(727 +/- 275) pmol/L, P > 0.05]. However, the level of progesterone were (23 +/- 8) nmol/L in LUF group and (35 +/- 10) nmol/L in control group, which reached statistical difference (P < 0.01). (2) The expression of ER, PR in endometrium of LUF patients were 183.9 +/- 2.4 and 168 +/- 3, which were significantly higher than 109.4 +/- 6.3 and 106 +/- 4 in control group (P < 0.01). The expression of integrin alphaVbeta3 in endometrium of 115 +/- 11 in LUF group were significantly lower than 191 +/- 9 in control group (P < 0.01). (4) In LUF group, the expression of alphaVbeta3 in endometrium was correlated positively with the level of progesterone (r = 0.77, P < 0.01) and irrelevant with the level of estrogen (r = 0.01, P > 0.05). The higher expression of estrogen and progesterone and lower expression of integrin alphaVbeta3 might confer impaired receptivity of endometrium and interfere with embryo implantation.

Application of a novel method of double APAAP staining with subsequent quantitative image analysis to the examination of integrin expression in undifferentiated-type gastric carcinomas.

In undifferentiated-type gastric carcinoma (UGC), recognition of cancer cells is not easy, which has hampered its precise phenotypic analysis. To examine alterations of the integrin phenotype during the progression of UGC, we used double alkaline phosphatase anti-alkaline phosphatase staining and computer-aided image analyses for the expression of alpha1, alpha2, alpha3, alpha5, alpha6, alphaV, beta1, and beta4 integrin subunits and alphaVbeta3, alphaVbeta5, and alphaVbeta6 integrins in cytokeratin-positive cells in the mucosal, the submucosal, and the deeper parts of 10 early and 17 advanced UGCs, their non-neoplastic counterparts, and 9 lymph node (LN) metastases. We revealed declining expression of epithelial integrin subunits (alpha2, alpha3, alpha6, beta4) and increasing expression of mesenchymal integrin subunits (alpha1, alpha5) as the tumor invaded deeper, reflecting gradual epithelial-to-mesenchymal transition of the integrin phenotype during tumor invasion. Enhanced expression of the alphaV integrin subunit and alphaVbeta3 and alphaVbeta5 integrins correlated with tumor invasion, and that of alphaVbeta6 integrins with LN metastasis. Our results have demonstrated that the method we introduced is suitable for analysis of dynamic alterations of the integrin repertoire in UGC progression.

Stromal cell‐derived factor‐1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF‐κB‐dependent pathways

Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF-1 and CXCR4 (SDF-1 receptor). Treatment of osteosarcoma cells with SDF-1alpha increased the migration and cell surface expression of alphavbeta3 integrin. CXCR4-neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF-1alpha-induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF-1alpha-mediated migration and integrin expression. Stimulation of cells with SDF-1alpha increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited SDF-1alpha-mediated cell migration and integrin up-regulation. Stimulation of cells with SDF-1alpha induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the SDF-1alpha-mediated increasing kappaB-luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKalpha and IKKbeta mutants. Taken together, these results suggest that the SDF-1alpha acts through CXCR4 to activate MEK and ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of alphavbeta3 integrins and contributing the migration of human osteosarcoma cells.

Osteopontin stimulates invasion of NCI‐h295 cells but is not associated with survival in adrenocortical carcinoma

Gene array studies indicated that osteopontin (OPN) mRNA is highly expressed in adrenocortical carcinomas (ACCs). OPN enhances invasiveness, proliferation, and metastasis formation, and is associated with poor survival in some malignant diseases. Integrin alphavbeta3 has been shown to mediate OPN effects on invasion. In this study, we demonstrated OPN and integrin alphavbeta3 expression in normal adrenal glands and benign adenomas, with staining seen exclusively in adrenocortical cells as well as even stronger staining in ACC. Western blot analysis confirmed overexpression of OPN in ACC (p < 0.01). With Matrigel invasion assays, we have shown that OPN greatly stimulates the invasiveness of NCI-h295 cells (>six-fold increase, p < 0.001). Transfection with integrin alphavbeta3 further increased invasiveness after OPN stimulation (p < 0.001). This increase was reversed by the addition of an anti-integrin beta3 antibody, indicating a functional relationship of OPN and integrin alphavbeta3 in ACC. With tissue arrays, we confirmed high OPN expression in 147 ACC samples. However, no association with survival was seen in Kaplan-Meier analysis including 111 patients with primary tumours graded for OPN staining and follow-up data available. In conclusion, our in vitro data indicate that OPN and integrin alphavbeta3 may act as a functional complex facilitating the invasiveness of adrenocortical tumours. This relationship remains of relevance to our understanding of carcinogenesis, but further studies are needed to address the physiological and pathological function of OPN in adrenal tissue.

Leptin enhances cell migration in human chondrosarcoma cells through OBRl leptin receptor

Leptin, an adipocyte-derived cytokine that is closely associated with obesity, has recently been shown to be involved in carcinogenesis and cancer progression. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. In this study, we found that leptin increased the migration and the expression of alphavbeta3 integrin in human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the long form (OBRl) leptin receptor, which was higher than that in normal cartilage and human primary chondrocyte. Leptin-mediated migration and integrin upregulation were attenuated by OBRl receptor antisense oligonucleotide. Activations of insulin receptor substrate (IRS)-1, phosphatidylinositol 3-kinase (PI3K), Akt and nuclear factor-kappaB (NF-kappaB) pathways after leptin treatment were demonstrated, and leptin-induced expression of integrin and migration activity was inhibited by the specific inhibitor, small-interfering RNA and mutant of IRS-1, PI3K, Akt and NF-kappaB cascades. Taken together, our results indicated that leptin enhances the migration of chondrosarcoma cells by increasing alphavbeta3 integrin expression through the OBR1/IRS-1/PI3K/Akt/NF-kappaB signal transduction pathway.

Positive crosstalk between ERK and p38 in melanoma stimulates migration and in vivo proliferation

Melanoma is one of the most therapy-resistant cancers. Activating mutations in BRAF and NRAS are the source of extracellular signal regulated protein kinase (ERK) pathway activation. We show that melanoma cell lines, originating in different metastatic sites, with BRAF or NRAS mutations, in addition to active mitogen activated protein kinase (MAPK)-ERK, also have highly activated stress activated protein kinase (SAPK)-p38. This is in direct contrast to carcinoma cells in which the activity of the two kinases appears to be mutually exclusive; high level of p38 activity inhibits, through a negative feedback, ERK activity and prevents tumorigenesis. Melanomas are insensitive to ERK inhibition by p38 and utilize p38-signaling pathway for migration and growth in vivo. We found a positive functional loop linking the high ERK activity to surface expression of alphaVbeta3-integrin. This integrin, by interacting with vitronectin, induces p38 activity and increases IL-8 production, enhancing cell migration. Because the negative loop from p38 to ERK is lost, the two kinases can remain simultaneously activated. Inhibition of ERK and p38 activities is more effective in blocking in vivo growth than inhibition of each kinase individually. Future therapies might have to consider targeting of both pathways.

Tumor necrosis is associated with increased alphavbeta3integrin expression and poor prognosis in nodular cutaneous melanomas

BackgroundTumor necrosis and apoptotic activity are considered important in cancer progression, but these features have not been much studied in melanomas. Our hypothesis was that rapid growth in cutaneous melanomas of the vertical growth phase might lead to tissue hypoxia, alterations in apoptotic activity and tumor necrosis. We proposed that these tumor characteristics might be associated with changes in expression of cell adhesion proteins leading to increased invasive capacity and reduced patient survival.MethodsA well characterized series of nodular melanoma (originally 202 cases) and other benign and malignant melanocytic tumors (109 cases) were examined for the presence of necrosis, apoptotic activity (TUNEL assay), immunohistochemical expression of hypoxia markers (HIF-1 α, CAIX, TNF-α, Apaf-1) and cell adhesion proteins (αvβ3 integrin, CD44/HCAM and osteopontin). We hypothesized that tumor hypoxia and necrosis might be associated with increased invasiveness in melanoma through alterations of tumor cell adhesion proteins.ResultsNecrosis was present in 29% of nodular melanomas and was associated with increased tumor thickness, tumor ulceration, vascular invasion, higher tumor proliferation and apoptotic index, increased expression of αvβ3 integrin and poor patient outcome by multivariate analysis. Tumor cell apoptosis did also correlate with reduced patient survival. Expression of TNF-α and Apaf-1 was significantly associated with tumor thickness, and osteopontin expression correlated with increased tumor cell proliferation (Ki-67).ConclusionTumor necrosis and apoptotic activity are important features of melanoma progression and prognosis, at least partly through alterations in cell adhesion molecules such as increased αvβ3 integrin expression, revealing potentially important targets for new therapeutic approaches to be further explored.

Microglial expression of αvβ3 and αvβ5 integrins is regulated by cytokines and the extracellular matrix: β5 Integrin null microglia show no defects in adhesion or MMP‐9 expression on vitronectin

As the primary immune effector cells in the CNS, microglia play a central role in regulating inflammation. The extracellular matrix (ECM) protein vitronectin is a strong inducer of microglial activation, switching microglia from a resting into an activated potentially destructive phenotype. As the activating effect of vitronectin is mediated by alphav integrins, the aim of the current study was to evaluate the requirement of the alphavbeta5 integrin in mediating microglial adhesion and activation to vitronectin, by studying these events in beta5 integrin-null murine microglia. Surprisingly, beta5 integrin null microglia were not defective in adhesion to vitronectin. Further analysis showed that microglia express the alphavbeta3 integrin, in addition to alphavbeta5. Flow cytometry revealed that microglial alphav integrin expression is regulated by cytokines and ECM proteins. alphavbeta3 integrin expression was downregulated by IFN-gamma, TNF, LPS, and TGF-beta1. alphavbeta5 expression was also reduced by IFN-gamma, TNF, and LPS, but strongly increased by the antiactivating factors TGF-beta1 and laminin. Gel zymography revealed that beta5 integrin null microglia showed no deficiency in their expression of matrix metalloproteinase (MMP)-9 in response to vitronectin. Taken together, these data show that microglia express two different alphav integrins, alphavbeta3 and alphavbeta5, and that expression of these integrins is independently regulated by cytokines and ECM proteins. Furthermore, it reveals that the alphavbeta5 integrin is not essential for mediating microglial adhesion and MMP-9 expression in response to vitronectin.

Expression of Kallikrein 7 Diminishes Pancreatic Cancer Cell Adhesion to Vitronectin and Enhances Urokinase-Type Plasminogen Activator Receptor Shedding

To examine the effects of kallikrein 7 (KLK7) expression in pancreatic cancer cells on cell adhesion to extracellular matrix proteins. Kallikrein 7 was overexpressed in the pancreatic cancer cell line BxPC-3, and in vitro cell adhesion assays were performed to determine alterations in cell adhesion to collagen I, fibronectin, and vitronectin compared with vector-transfected cells. Changes in cell surface expression of alphavbeta3 integrin were examined by flow cytometry, and the release of soluble urokinase-type plasminogen activator receptor (uPAR) was monitored by Western blot analysis. The expression of KLK7 in BxPC-3 cells led to reduced adhesion to vitronectin but not collagen I or fibronectin. Loss of cell adhesion to vitronectin was not caused by changes in the level of alphavbeta3 integrin cell surface expression; however, a significant increase in the amount of uPAR shed was observed in KLK7-expressing cells compared with vector-transfected control cells. The aberrant expression of KLK7 in pancreatic cancer cells leads to a reduction in cell adhesion to vitronectin that may result from the cleavage of uPAR from the cell surface. Kallikrein 7 expression, therefore, may play an important role in the dissemination of tumor cells through the production of soluble uPAR in pancreatic cancer and perhaps other human malignancies.

The influence of 1,25-dihydroxyvitamin D3 and 1,24-dihydroxyvitamin D3 on alphavbeta3 integrin expression in cancer cell lines.

Integrins are cell-surface receptors engaged in important cancer invasion processes, such as adhesion, migration, proliferation and differentiation. The aim of this study was to evaluate the effect of 1,25-dihydroxyvitamin D3 (calcitriol) and its metabolite 1,24-dihydroxyvitamin D3 (PRI-2191) on alphavbeta3 integrin expression in various cancer cell lines. The expression levels of the beta3 and alphav integrins were reduced only in the WEHI-3 and LLC cell lines by the two compounds. Calcitriol or PRI-2191 treatment caused differentiation of WEHI-3 mouse leukemia cells, but apoptosis of LLC cells. WEHI-3 and LLC cells exposed to calcitriol or PRI-2191 lost their migratory and adhesive potentials. The inhibition of migratory potential was higher in the LLC cells than in the WEHI-3 cells and appeared to correlate with the increased down-regulation of alphavbeta3 integrin by calcitriol or PRI-2191. The observed in vivo effects (antitumor and antimetastatic) in mice bearing subcutaneously transplanted LLC cancer are possibly associated with inhibited migratory potential as a consequence of the lowered integrin expression caused by calcitriol or PRI-2191.

Stromal cell‐derived factor‐1 increase αvβ3 integrin expression and invasion in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. The stromal cell-derived factor 1 (SDF-1), constitutively secreted by human lung epithelium cells, has been shown to function in a key role for recruitment of neutrophils. Here, we found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of CXCR4 (SDF-1 receptor), which was higher than normal cartilage and human chondrocyte. SDF-1alpha and lung epithelium cells conditioned medium (LECM) induced the invasiveness of chondrosarcoma cells. SDF-1 siRNA inhibited LECM-induced invasion of chondrosarcoma cells and SDF-1alpha also directly induced the cell surface expression of alphavbeta3 but not alpha2beta1 and alpha5beta1 integrin. Activations of ERK and NF-kappaB pathways after SDF-1 treatment was demonstrated, and SDF-1alpha-induced expression of alphavbeta3 integrin and invasion activity was inhibited by the specific inhibitor and mutant of ERK and NF-kappaB cascades. Taken together, our results indicate that lung derived-SDF-1alpha enhances the invasiveness of chondrosarcoma cells by increasing alphavbeta3 integrin expression through the CXCR4/ERK/NF-kappaB signal transduction pathway.