Modified heparins inhibit integrin αIIbβ3 mediated adhesion of melanoma cells to platelets in vitro and in vivo

The adhesion of tumor cells with platelets is important in the process of tumor metastasis. A huge work has indicated that anti‐adhesion is an effective strategy for metastasis inhibition. In this article, we assess the role of platelet integrin α_IIbβ₃ in adhesion of melanoma cells to platelets and the effects of heparin and modified heparins on the adhesion in vitro and in vivo. We show that platelet integrin α_IIbβ₃ is involved in the interaction of human melanoma A375 cells with platelets, and the high affinity epitope resides on the α_IIb subunit rather than β₃ subunit. Heparin sulfate‐like proteoglycans on tumor cell surface are implicated in the adhesion of A375 cells to integrin α_IIbβ₃. We also show that RO‐heparin, CR‐heparin, N‐2,3‐DS‐heparin and 2,3‐O‐DS‐heparin can significantly inhibit A375 cells binding to the CHO cells expressing integrin α_IIbβ₃ under static and flow conditions, and remarkably inhibit the adhesion of A375 cells to the immobilized platelet layers under flow conditions. We find that A375 cells and B16F10 cells are arrested in the pulmonary vessels and adhered to platelets, and the initial interaction of tumor cells with platelets in lung vessel and long‐term establishment of metastatic foci can be inhibited by heparin as well as CR‐heparin and N‐2,3‐DS‐heparin. These data suggest that modified heparins can inhibit tumor cell‐platelet interaction mediated by platelet integrin α_IIbβ₃ and modified heparins may be a potential substitute for heparin in inhibiting tumor metastasis. © 2009 UICC     

Phenotypic properties of cultured tumor cells: Integrin αIIbβ3 expression,tumor-cell-induced platelet aggregation,and tumor-cell adhesion to endothelium as important parameters of experimental metastasis

The present study was undertaken to investigate the factors involved in determining the metastatic potential of cultured cells derived from solid tumors. We first investigated the effects of cell source and culture conditions on lung colony formation by i.v. injected B16a (B16 amelanotic melanoma) cells and inhibition of tumor colony formation by the thromboxane A2 syn-thase inhibitor, CGS14854. Prolonged culture resulted in a 10-fold decrease in the incidence of B16a lung colonies, whereas passage in vivo for 150 days did not affect lung colony formation by tumor cells isolated from enzymatic dispersates by centrifugal elutriation. Cultured BI6a cells maintained at low density (LD) and harvested at low passage (LP) formed significantly more lung colonies than B16a cells harvested at high densities (HD) or high passage (HP). Over-confluent tumor cells produced even lower number of lung colonies. Lung colony formation by elutriated BI6a cells (i.e., cells freshly isolated from tumor tissue) was consistently inhibited by CGS14854, whereas inhibition of lung colony formation by cultured B16a cells was dependent upon culture conditions. CGS 14854 was ineffective or less effective against HD/HP B16a cells. The differences in lung colony formation between LD, HD and elutriated B16a cells were not due to differential cell-cycle distribution. Mechanistic studies indicated that LD/LP tumor cells induced aggregation of homologous platelets, whereas HD/HP BI6a cells failed to induce significant platelet aggregation. Aggregation of homologous platelets correlated positively with lung-colonizing ability. Additionally, LD/LP cells demonstrated higher adhesion to endothelium than HD/HP BI6a cells. Finally, LD/LP BI6a cells expressed higher levels of α_IIbβ₃ integrins than HD/HP tumor cells, as determined by flow cytometry and immunofluorescence.     

Characterization of platelet aggregation induced by human breast carcinoma and its inhibition by snake venom peptides,trigramin and rhodostomin

Summary MCF-7 cells, a metastatic human breast carcinoma line, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma (PRP). MCF-7 tumor cell-induced platelet aggregation (TCIPA) was almost blocked by apyrase (0.5 U/ml) and completely inhibited by hirudin (5 U/ml). This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 µM), but was limited by cell pretreatment with phospholipase A₂. MCF-7 cell suspension caused marked, dose-dependent decrease in plasma recalcification times using normal, Factor VIII-deficient, and Factor IX-deficient human plasma. This effect was potentiated in cell lysates but was inhibited in intact cells preincubated with sphingosine. MCF-7 cell suspension did not affect the recalcification time of Factor VII-deficient plasma. Taken together, these data suggest that MCF-7 TCIPA arises from MCF-7 tisssue factor activity expression. Trigramim and rhodostomin, RGD-containing snake venom peptides which antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented MCF-7 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against human tissue factor, platelet membrane glycoprotein IIb/IIIa and Ib prevented MCF-7 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC₅₀, 0.1 µM) and rhodostomin (IC₅₀, 0.03 µM), were about 5,000 and 18,000 times, respectively, more potent than GRGDS (IC₅₀, 0.54 mM).     

Inhibition of human malignant neuroblastoma cell DNA synthesis by lipoxygenase metabolites of arachidonic acid

In vivo studies have shown that inhibitors of cyclooxygenase metabolism of arachidonic acid may diminish growth and metastasis of certain tumors. Because cyclooxygenase inhibition may increase the production of lipoxygenase products of arachidonic acid metabolism, we have investigated the effect of two such products, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on tumor cell proliferation in vitro. When neuroblastoma cells (SK-N-SH) in culture were treated with 12-HETE for 18 hr, incorporation of [3H]thymidine was inhibited up to 64% at concentrations from 20 to 50 microM. Under the same conditions, 15-HETE resulted in inhibition of up to 46%, while arachidonic acid had no apparent effect. When evaluated in the presence of serum, 12-HETE at a concentration of 120 microM produced a 20.6 +/- 2.8% (S.E.) inhibition of the increase in total DNA content over 48 hr, while 15-HETE at this concentration produced a 16.5 +/- 5.3% inhibition. We conclude that 12-HETE, the product of platelet lipoxygenase, and 15-HETE, a product of neutrophil and lymphocyte lipoxygenases, can inhibit human neuroblastoma cell growth in vitro and may play a role in the effect of cyclooxygenase inhibitors on tumor growth in vivo.     

Bidirectional control of membrane expression and/or activation of the tumor cell IRGpIIb/IIIa receptor and tumor cell adhesion by lipoxygenase products of arachidonic acid and linoleic acid

Lewis lung carcinoma cells express a plasma membrane receptor (i.e., IRGpIIb/IIIa) which is immunologically and functionally related to the platelet aggregation receptor complex (i.e., GpIIb/IIIa). Both fluorescence microscopy and flow cytometric analysis reveal that surface expression and/or activation of this tumor cell receptor is enhanced by a phorbol ester [i.e., 12-O-tetradecanoylphorbol-13-acetate (TPA)] and a lipoxygenase metabolite of arachidonic acid; 12-hydroxyeicosatetraenoic acid (i.e., 12-HETE). TPA-enhanced expression appears to be mediated by a lipoxygenase metabolite, as this effect can be reversed by lipoxygenase inhibitors but not by cyclooxygenase inhibitors. In parallel with these results both TPA and 12(S)-HETE [but not 12(R)-HETE] enhance tumor cell adhesion to endothelial cells, subendothelial matrix and fibronectin, but not to type IV collagen. TPA-enhanced adhesion can be reduced by lipoxygenase inhibitors but not by cyclooxygenase inhibitors and in addition, stimulated adhesion can be blocked by pretreatment of tumor cells with specific polyclonal or monoclonal antibodies which react against IRGpIIb/IIIa. 12(S)-HETE-enhanced adhesion can also be inhibited by these same antibodies. In contrast, a lipoxygenase product of linoleic acid, 13(S)-hydroxyoctadecadienoic acid, inhibited TPA and 12(S)-HETE-enhanced tumor cell adhesion to endothelial cells, subendothelial matrix, and fibronectin. These results suggest that (a) IRGpIIb/IIIa is a multifunctional receptor which mediates tumor cell adhesion to a variety of biological substrata, (b) TPA enhances surface expression and/or activation of this receptor possibly via a lipoxygenase metabolite of arachidonic acid, and (c) these effects are opposed by a lipoxygenase metabolite of linoleic acid.     

Triflavin, an Arg-Gly-Asp-containing Peptide, Inhibits Tumor Cell-induced Platelet Aggregation

In this study, we examined the effect of triflavin, an Arg-Gly-Asp (RGD)-containing snake venom peptide, on human cervical carcinoma (HeLa) cell- and B16-F10 mouse melanoma cell-induced platelet aggregation (TCIPA) in heparinized platelet-rich plasma. TCIPA appears to play an important role in the development of certain experimental tumor metastases. Two ADP-scavenging agents, apyrase (10 U/ml) and creatine phosphate (CP) (5 mM)/creatine phosphokinase (CPK) (5 U/ml) completely inhibited B16-F10 TCIPA, but hirudin (5 U/ml) had no effect. In contrast, apyrase and CP/CPK did not inhibit HeLa TCIPA while hirudin completely inhibited it. Furthermore, HeLa cells initially induced platelet aggregation and then blood coagulation at a later stage. In addition, HeLa cells shortened, in a concentration-dependent manner, the recalcification time of normal as well as factor VIII- and IX-deficient human plasma, but did not affect the recalciflcation time of factor VII-deficient plasma. This suggests that HeLa TCIPA occurs via activation of the extrinsic pathway, probably owing to tumor cell expression of tissue factor-like activity. HeLa cell-induced thrombin generation was confirmed by detection of amidolytic activity towards a chromogenic substrate, S-2238 (H-D-Phe-Pip-Arg-p-NA). Triflavin and GRGDS inhibited, in a dose-dependent manner, TCIPA caused by either cell line. On a molar basis, triflavin was 10,000–30,000 times more potent than GRGDS in this regard. Moreover, monoclonal antibodies raised against glycoprotein (GP) IIb/IIIa complex (i.e., 7E₃ and AP₂) and against GP Ib (i.e., AP₁) completely inhibited HeLa TCIPA. 7E₃ and AP₂ inhibited B16-F10 TCIPA by up to 80% whereas AP₁ showed only 30% inhibition of B16-F10 TCIPA. In conclusion, the inhibitory effect of triflavin on HeLa and B16-F10 TCIPA may be mediated principally by the binding of triflavin to the fibrinogen receptor associated with GP IIb/IIIa complex on the platelet surface. However, GP Ib is also involved in HeLa TCIPA as thrombin formation is the key factor in triggering platelet aggregation caused by HeLa cells.     

αv-Integrins in human melanoma: Gain of αvβ3 and loss OF αvβ5 are related to tumor progression in situ but not to metastatic capacity of cell lines in nude mice

We investigated the expression of α_v-integrins in different stages of human cutaneous melanocytic tumor progression. We observed that α_vβ₅ was the α_v-integrin expressed in all common nevocellular nevi, in 78% of dysplastic nevi, in 63% of early primary melanomas, in 43% of advanced primary melanomas, and in 33% of melanoma metastases. Hence, loss of α_vβ₅ expression was related to melanocytic tumor progression. In line with earlier reports, α_vβ₃ was exclusively detected in advanced primary melanomas and metastases (24% and 50% respectively). Staining with anti-α_v monoclonal antibodies (MAbs) in lesions where both α_vβ₃ and α_vβ₅ were absent showed that alternative α_v-integrins were expressed in advanced primary melanomas and metastases. By FACS analysis, we determined expression of α_vβ₅ and α_vβ₃ in 4 human melanoma cell lines with different metastatic capacities after s.c. inoculation into nude mice. One of the non-metastatic and both highly metastatic cell lines expressed α_vβ₅ at their surface. Surprisingly, α_vβ₃ was detected exclusively in the non-metastatic cell lines. Absence of α_vβ₃ in the highly metastatic cell lines was confirmed by lack of immunoprecipitation from ³⁵S-methionine-labeled cells and by absence of immunohistochemical staining on primary and metastatic xenograft lesions. Our findings indicate that α_vβ₅ expression is often lost in advanced stages of melanocytic tumor progression in situ, while α_vβ₃ is acquired, but that a decrease in α_vβ₅ and an increase in α_vβ₃ expression are not necessarily related to the metastatic behavior of human melanoma cells in nude mice. © 1995 Wiley-Liss, Inc.     

Effects of prostacyclin on tumor cell-induced platelet aggregation

Prostacyclin has been evaluated for its ability to inhibit tumor cell-induced platelet aggregation (TCIPA) induced by several rodent tumor lines: B16a (melanoma); 3LL (carcinoma); 15091A (adenocarcinoma); and W256 (carcinosarcoma). Aggregation of human platelets by all four lines was inhibited by prostaglandin I2 (PGI2) in a dose-dependent manner, with complete inhibition observed at 10 ng/ml. However, higher PGI2 concentrations were required to inhibit aggregation of homologous rat platelets induced by W256 cells. Prostacyclin was compared to other icosanoids known to inhibit platelet aggregation and was found to be 100-fold more potent than either prostaglandin E1 or prostaglandin D2 and 1000-fold more potent than its stable nonenzymatic metabolite (6-ketoprostaglandin F1 alpha). Prostaglandin E2 in contrast to prostaglandin E1 and prostaglandin D2, did not inhibit TCIPA; however, both prostaglandin E2 and its enzymatic metabolite (13,14-dihydro-15-ketoprostaglandin E2) prevented PGI2 inhibition of TCIPA. The addition of prostaglandin I2 (100 ng/ml) after initiation of TCIPA (50% of maximum response) resulted in immediate arrest of TCIPA followed by reversal of platelet aggregation. Prostacyclin partially reversed platelet aggregation when added at 100% of maximum response. Platelets enhanced the adhesion of [125I]uridine-labeled W256 cells to plastic culture dishes under both aggregatory and nonaggregatory conditions. Prostacyclin in vitro inhibited platelet-facilitated tumor cell adhesion. These in vitro results demonstrated that PGI2 is a potent inhibitor of TCIPA and of tumor cell adhesion; we suggest that these are possible mechanisms to explain the antimetastatic effects of PGI2 in vivo [Honn, K. V., Cicone, B., and Skoff, A. Science (Wash. D.C.), 212: 1270-1272, 1981].     

Matrix metalloproteinase 2 in tumor cell-induced platelet aggregation: regulation by nitric oxide

A correlation exists between the ability of tumor cells to aggregate platelets and their tendency to metastasize. Tumor cell-induced platelet aggregation (TCIPA) facilitates the embolization of the vasculature with tumor cells and the formation of metastatic foci. It is well documented that matrix metalloproteinases (MMPs) play an integral part in tumor spread and the metastatic cascade. Therefore, we have examined the role of MMPs during TCIPA and its regulation by nitric oxide (NO) in vitro. Human HT-1080 fibrosarcoma and A549 lung epithelial cancer cells induced TCIPA in a concentration-dependent manner that was monitored by aggregometry. This aggregation resulted in the release of MMIP-2 from platelets and cancer cells, as measured by zymography. HT-1080 cells released significantly more MMP-2 than A549 cells and were more efficacious in inducing TCIPA. Inhibition of MMP-2 with phenanthroline (1-1000 microM), a synthetic inhibitor of MMPs, and by neutralizing anti-MMIP-2 antibody (10 microg/ml) reduced TCIPA induced by HT-1080 cells. TCIPA was abolished by simultaneous inhibition of platelet function with acetylsalicylic acid (100 microM; thromboxane pathway inhibitor), apyrase (250 microg/ml; ADP pathway inhibitor), and phenanthroline. NO donors such as S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione (both at 0.01-100 microM) inhibited TCIPA and MMP-2 release from platelets and tumor cells. The inhibitory actions of S-nitroso-n-acetylpenicillamine and S-nitrosoglutathione were reversed by 1H-[1,2,4]oxadiazole[4,3]quinoxalin-1-one (0.01-30 microM), a selective inhibitor of the soluble guanylyl cyclase. We conclude that (a) human fibrosarcoma cells aggregate platelets via mechanism(s) that are mediated, in part, by MMP-2; (b) NO inhibits TCIPA, in part, by attenuating the release of MMP-2; and (c) these effects of NO are cGMP-dependent.     

Inhibition of TPA and 12(S)-HETE-stimulated tumor cell adhesion by prostacyclin and its stable analogs: Rationale for their antimetastatic effects

We have investigated the regulatory role of PGI₂ and its stable analogs, i.e., iloprost and cicaprost, on 12(S)-HETE- and TPA-enhanced tumor cell integrin expression and adhesion. Walker 256 carcinosarcoma cells express αllb β3 integrin receptors, which mediate their adhesion to endothelium, subendothe-lial matrix and fibronectin. Adhesion is enhanced by treatment with exogenous 12(S)-HETE but not 12(R)-HETE or other lipoxygenase-derived hydroxy fatty acids, as well as by TPA. Both 12(S)-HETE and TPA enhanced αllb β3 expression on W256 cells. PGI₂ iloprost and cicaprost inhibited both 12(S)-HETE- and TPA-enhanced adhesion to endothelium and suben-dothelial matrix as well as αllb β3 expression on W256 cells. The mechanism responsible for the effect of PGI₂ was explored. Prostacyclin treatment of W256 cells resulted in an enhanced production of cAMP in a time- and dose-dependent manner. Pre-treatment of tumor cells with increasing concentrations of adenosine resulted in a dose-dependent decrease in the PGI₂ effect on TPA or 12(S)-HETE-enhanced adhesion, suggesting that the PGI₂ effect is mediated through PKA. Dibutyryl cAMP also blocked the 12(S)-HETE- or TPA-enhanced adhesion, and adenosine pre-treatment did not result in an inhibition of the dibutyryl cAMP effect. Collectively, our results suggest that the cyclooxygenase metabolite PGI₂ can antagonize the lipoxygenase metabolite 12(S)-HETE- and TPA-enhanced αllb β3 expression and tumor cell adhesion via activation of adenylate cyclase and elevation of intracellular levels of cAMP. © 1995 Wiley-Liss, Inc.     

Identification of a novel platelet antagonist that binds to CLEC-2 and suppresses podoplanin-induced platelet aggregation and cancer metastasis

Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCμ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective.     

Expression of α3β1 integrin receptor and its ligands in human lung tumors

Increasing experimental evidence demonstrates that malignant transformation is associated with changes in the repertoire of expression of the integrin family of molecules, which mediate cell-matrix and cell-cell interactions. We have analyzed immuno-histochemically and immunochemically the expression of VLA-3 integrin and its known ligands, namely, laminin (LM), fibronectin (FN), collagen type IV (Coll IV), nicein (NIC), and entactin/nidogen (ENT), in lung tumors of various histological types. α₃β₁ was detectable in normal bronchial epithelium and along basement membranes of alveolar walls. In non-small cell lung carcinomas (NSCLC) the integrin was expressed in 82% of the cases, independently of histological type and degree of differentiation of the tumors. On the other hand, only 13% of the small cell lung carcinomas (SCLC) displayed a weak and heterogeneous distribution of the α₃β₁ complex. Our findings were confirmed immunochemically using long-term tumor cell lines. While the expression of both α₃β₁ and ligands LM, FN, Coll IV, and Ent correlated in NSCLC with the presence of basement membranes, FN was the only ligand detectable in the stroma of SCLCs. A selective loss of nicein in basement membranes was demonstrated in NSCLC indicating an impairment of expression of this glycoprotein following malignant transformation. © 1995 Wiley-Liss, Inc.     

In vitro effects of eicosanoid synthesis inhibitors in the presence of linoleic acid on MDA-MB-231 human breast cancer cells

We investigated the effects of cyclooxygenase and lipoxygenase inhibitors in the presence of linoleic acid (LA), as well as the direct effects of prostaglandin E (PGE) and leukotriene B (LTB) on a human breast cancer cell line (MDA-MB-231)in vitro. Piroxicam, esculetin, and nordihydroguaiaretic acid (NDGA) suppressed cell growth and thymidine incorporation. However, a low concentration (1 µg/ml) of indomethacin (INDO) stimulated cell growth and thymidine incorporation, while a high concentration of INDO (30 µg/ml) inhibited both. Esculetin and NDGA reduced the secretion of LTB, whereas piroxicam reduced the secretion of PGE. INDO reduced the secretion of PGE, but a low concentration of INDO increased the secretion of LTB. Consequently, cell growth was correlated with the PGE and/or LTB concentrations when the cells were treated with these cyclooxygenase or lipoxygenase inhibitors. On the other hand, exogenous PGE₂ partially reversed the inhibition of thymidine incorporation caused by INDO, whereas LTB₄ exerted a similar effect in the case of esculetin or NDGA. The reversibility of the piroxicam effect with PGE₂ is not convincing. Therefore, it is suggested that the growth of MDA-MB-231 cellsin vitro is affected by both the lipoxygenase and cyclooxygenase products, probably the other eicosanoids rather than PGE₂ and LTB₄.     

The capacity of human malignant B-lymphocytes to disseminate in scid mice is correlated with functional expression of the fibronectin receptor α5β1 (CD49E/CD29)

The α₅β integrin (CD49e/CD29), a heterodimeric membrane protein, is the “classical” fibronectin receptor on many cell types. During B-cell ontogeny, expression of the α₅-subunit is developmentally regulated. The αβ₁ is decisive for migration on fibronectin substrate and very likely cooperates with other adhesion molecules in transvascular trafficking. To test whether α₅β influences local growth vs. disseminative spread of neoplastic B-cells in vivo, human B-cell lines mimicking different maturational stages were transferred s.c. into severe combined immunodeficiency (SCID) mice and examined for α₅β expression and for adherence on fibronectin substrate in vitro and ex vivo. All cell lines were locally tumorigenic. Dissemination was observed in all animals carrying Nalm-6 tumors, in one animal with a BL 60 and in 2 mice carrying a Raji tumor. By contrast, Daudi, BJAB and U266 tumors did not disseminate. As evidenced by immunohistochemistry and flow cytometry, all lines and their tumors were to various extents β₁-positive but showed considerable differences in α₅ expression. The functional surface expression of α₅β₁correlated with fibronectin adherence of the lines. Daudi expressed α₅β₁ in a non-functional configuration which was rendered functional only upon applying high concentrations of Mg⁺⁺ and Mn⁺⁺. B-cell lines functionally expressing α₅β₁ at high or moderate levels disseminated in SCID mice while α₅-negative lines and Daudi did not. These results support the conclusion drawn from an earlier in situ analysis of human B-cell lymphomas/ leukemias that the α₅β₁ integrin contributes to the disseminative phenotype of malignant B cells.     

Transformation and tumor progression are frequently associated with expression of the α3/β1 heterodimer in solid tumors

The ability of tumor cells to interact with the extracellular matrix (ECM) is functionally mediated by a variety of receptor molecules among which the integrins are a well-characterized family of mediators. In this study we have investigated immuno-histochemically the in vivo expression of the α/β₁ promiscuous receptor for ECM constituents in a variety of human solid malignancies. Although the receptor appears to undergo changes in distribution patterns, its expression is maintained in a high percentage of primary (76%) and metastatic (82%) tumors. Furthermore, the comparative immunohistochemical evaluation of α/β₁ and its ligands, in a selected number of tumors of different histotypes, demonstrated that the expression of this integrin correlates with the presence of at least one ligand, either around nests of neoplastic cells or at the epithelial-stromal interface. The highly conserved expression of α/β₁ shown in this study suggests that this receptor may play a role in tumor growth at the primary as well as at the metastatic site.     

Integrin α3β1 can promote adhesion and spreading of metastatic breast carcinoma cells on the lymph node stroma

We have reported that metastatic human melanoma cells utilize the α_vβ₃ integrin to adhere to lymph node vitronectin (VN). In the present study, the adhesion of human and rat breast carcinoma cells to lymph node tissue was analyzed. We have previously shown a correlation between the metastatic potential of breast carcinoma cells and an RGD-mediated adhesion to cryostat sections of peripheral lymph nodes; this adhesion could be blocked by an antibody to the integrin β₁ subunit. Here, we show that the metastatic breast carcinoma cell were significantly more adherent to fibronectin (FN) expressed by lymph node-derived stromal cells than non-metastatic cells. Metastatic cells also spread more rapidly than non-metastatic cells on FN-coated substrates. Using a combination of immunofluorescence microscopy, immunoprecipitation and blocking assays with integrin-specific antibodies, we found (i) that expression of the α₃β₁ integrin on metastatic mammary carcinoma cells was specifically increased in comparison to non-metastatic cells and (ii) that the α₃β₁ receptor was involved in the increased adhesion of metastatic cells to lymph node FN and in cell spreading on FN-coated substrates. Our data also suggest that the α₅β₁ integrin, which is also expressed on the metastatic cells, did not contribute to this increase in adhesion. Our data implicate the α₃β₁ integrin in adhesion to lymph node stromal cell FN and suggest that metastatic cells of different tissue origins (e.g., melanoma and breast carcinoma) may utilize distinct integrin-ligand combinations to colonize the same target organ. © 1996 Wiley-Liss, Inc.     

Integrin expression in cutaneous malignant melanoma: Association of the α3/β1 heterodimer with tumor progression

The cell-surface heterodimers of the integrin family of molecules, which mediate cell-cell and cell-substratum interactions, are likely to be functionally relevant in local and metastatic tumor growth. In the present study we have analyzed whether the α₃/β₁ receptor for collagen, laminin and fibronectin undergoes changes in expression during tumor progression in cutaneous malignant melanoma (CMM). The results of this study have demonstrated that, while low levels of VLA₃ expression are detectable in benign lesions, in primary melanomas the heterodimer undergoes progressive increase in expression which correlates with the degree of dermal invasiveness. Metastatic lesions were found VLA₃ positive in 82% of cases. Furthermore, the heterodimer is homogeneously expressed in multiple autologous metastases. The presence of VLA₃ correlates with detection of at least one of the ligands in 45% of the cases studied. These findings provide additional evidence that tumor progression in CMM is associated with changes in integrin phenotypes which include the α₃/β₁ heterodimer.     

Differential localization of transforming growth factor-β isoforms in human gastric mucosa and overexpression in gastric carcinoma

Transforming growth factor β (TGF-β) isoforms comprise a family of multifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. We examined TGF-β expression in normal human gastric mucosa and carcinoma. The distribution and expression of TGF-β isoforms in 4 normal mucosa samples from organ donors, in 12 normal mucosa samples adjacent to gastric cancer and in 12 gastric carcinomas were examined using immunohistochemistry and Northern blot analysis. Because TGF-βs regulate collagen expression, collagen type 1 α₁ mRNA amounts were also examined. Immunohistochemical analysis of normal human gastric tissue samples indicated that TGF-β₁ localized principally in parietal cells but also in some surface mucus cells, TGF-β₂ was present exclusively in chief cells and TGF-β₃ was present in parietal, chief and mucus cells. In the gastric cancers, strong colocalization of TGF-β₁, -β₂ and -β₃ was evident in the cancer cells. Northern blot analysis indicated that, compared to normal gastric tissue, gastric cancers showed a 4.8- and 6-fold increase in mRNA amounts encoding TGF-β₁, and TGF-β₃, respectively. In contrast, TGF-β₂ mRNA amounts were comparable in both groups. Northern blot analysis showed a 10-fold increase in human collagen type 1 α₁ mRNA amounts compared to normal gastric tissue. These findings imply a role for TGF-βs in normal human gastric mucosa function, and raise the possibility that the aberrant colocalization and overexpression of all 3 TGF-β isoforms in human gastric cancer cells in vivo may bcontribute to the pathobiology of gastric carcinoma. Int. J. Cancer 71:131–137, 1997. © 1997 Wiley-Liss, Inc.