Role of Integrin Receptors for Fibronectin,Collagen and Laminin in the Regulation of Ovarian Carcinoma Functions in Response to a Matrix Microenvironment

Integrins play an important role in cellular matrix interactions requisite for cancer cell adhesion, growth, migration and invasion. In this study, we have investigated the expression of integrin subunits α3, α6, αv and β1 in normal ovaries, benign ovarian tumors and ovarian carcinomas of different pathological grades. The expression of these integrins in ovarian cancer cell lines was also investigated, and their role in sustaining proliferation, adhesion, migration and invasion in cohort with the activation of signaling pathways in response to extracellular matrices (ECM) was evaluated. We demonstrate a differential expression pattern of α3, α6, αv and β1 integrin subunits in ovarian carcinomas compared to normal ovaries and benign ovarian tumors. Ovarian cancer cell lines (Hey, Ovcar3 and Peo.36) demonstrated significantly high expression of α3, α6, αv and β1 integrin subunits. A significant increase in proliferation and adhesion (P<0.05) in response to collagen 1 (Coll) and laminin (LM), ligands for integrin receptor α3β1 and α6β1 was observed in ovarian cancer cell lines. On the other hand, fibronectin (FN), a receptor for αvβ1 integrin, increased proliferation in all ovarian cancer cell lines studied but only enhanced adhesion in Hey cell line (P<0.05). Neutralizing antibodies against α3, α6, αv and β1 integrin subunits inhibited ECM-induced proliferation, but increased adhesion to ECM was inhibited by β1 integrin subunit antibody. No suppression of Coll, LM and FN-induced (Hey cells only) adhesion was observed in the presence of α3 or αv subunit antibodies but LM-induced adhesion was inhibited by blocking α6 subunit functions. LM, FN and Coll enhanced chemotactic migration in Hey cells, but direct invasion across ECM was observed only in the presence of LM and Coll. Blocking antibodies against α3, α6 and β1 integrin subunits inhibited both chemotactic migration and invasion of Hey cells in response to respective ECM. Adhesion of ovarian cancer cells to FN, Coll and LM activated Ras, Erk and Akt pathways. Neutralizing αv and β1 functions did not inhibit FN-induced activation of Ras and Erk pathways but inhibited the Akt pathway. On the other hand, antibodies against α6 and β1 subunits, but not α3 subunit, inhibited LM-induced activation of Ras but did not inhibit the downstream Akt pathway. Neutralizing β1 subunit function however, inhibited LM-induced Erk activation. Coll-induced activation of Ras, Erk and Akt pathways was inhibited by α3 and β1 integrin subunit antibodies. These results indicate that α3β1, αvβ1 and α6β1 integrin mediate proliferation, adhesion, migration and invasion of ovarian cancer cells in response to ECM and targeting these integrins to modulate integrin–ECM interactions in tumor cells may be a promising tool to reduce the dissemination of ovarian carcinoma in vivo.     

Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both 21 and 31 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequenc e intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhe-sion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients. © Rapid Science Ltd.     

Inhibitory effects of a specific phage-displayed peptide on high peritoneal metastasis of gastric cancer

Peritoneal dissemination in gastric cancer is the most frequent cause of the noncurative resection and recurrence after curative resection. We, therefore, evaluated the feasibility of a peptide, which was obtained by screening a random phage display library, in the treatment of peritoneal metastases of gastric cancer. In this study, a novel cell line, GC9811-P, with a high potential peritoneal metastasis of gastric cancer derived from its parental cell line, GC9811, was established. Using a phage display library, we isolated a specific peptide that selectively bound to GC9811-P cells rather than its parental GC9811cells. The isolated phage-displaying peptide, SMSIASPYIALE (named peptide PIII), was obtained after four rounds of selection, showing a tendency to preferentially bind to GC9811-P cells compared with a panel of other gastric cancer cell lines, and preferentially accumulate in peritoneal metastasis tumor tissue in comparison with control organs, peritoneum, liver, pancreas, spleen, lung, and kidney. Further study showed that synthetic peptide PIII could significantly inhibit adhesive and invasional ability of GC9811-P cells and could effectively block the corresponding phage binding to the GC9811-P cells, whereas, exposure of the cells to various concentrations of peptide PIII showed no obvious cell growth inhibition. Furthermore, a highly reproducible animal experimental model of gastric cancer with peritoneal dissemination was established in nude mice by injecting a suspension of the cell line into the gastric wall of nude mice. Animals intraperitoneally treated with peptide PIII in this model or another animal model of gastric cancer with peritoneal dissemination established using MKN45 cells showed suppressed tumor metastasis to peritoneum and significantly prolonged survival. In conclusion, the selected peptide PIII was a biologically active peptide and could effectively inhibit peritoneal dissemination of gastric cancer.     

A novel αvβ3-blocking disintegrin containing the RGD motive, DisBa-01, inhibits bFGF-induced angiogenesis and melanoma metastasis

The integrin α_vβ₃ is involved in multiple aspects of malignant cancer, including tumor angiogenesis and metastasis, which makes the receptor a key target for the development of anti-cancer therapies. We report here on the production, the characterization and the in vivo anti-angiogenic and anti-metastatic properties of a novel α_vβ₃-binding disintegrin, DisBa-01, isolated from a cDNA library made with RNAs from the venom gland of Bothrops alternatus. The 11,637 Da-recombinant monomeric form of DisBa-01 displayed an RGD motif and interacted with purified α_vβ₃ integrin in surface plasmon resonance studies, in a dose-dependent and cation sensitive manner. A three-dimensional molecular model of DisBa-01 in complex with α_vβ₃ predicted a large surface of contacts with the β₃ subunit. DisBa-01 inhibited the adhesion of α_vβ₃-expressing human microvascular endothelial cell line-1 (HMEC-1) and murine melanoma cell line B16F10 to vitronectin (IC₅₀ = 555 nM and 225 nM, respectively), and transiently inhibited their proliferation without direct cell toxicity, but did not affect the binding nor the proliferation of a human breast cancer-derived cell line (MDA-MB-231) not expressing α_vβ₃. In vivo, DisBa-01 dose-dependently decreased bFGF-induced angiogenesis in a matrigel plug assay in athymic nude mice (IC₅₀ = 83 nM). When injected intravenously to C57BL/6 mice together with B16F10 melanoma cells, DisBa-01 time- and dose-dependently inhibited lung metastasis monitored by bioluminescent imaging. We conclude that DisBa-01 is a potent new inhibitor of α_vβ₃-dependent adherence mechanisms involved in neo-vascularization and tumor metastasis processes. Oscar H. P. Ramos and Alexandre Kauskot contributed equally to this work.     

Signaling by epidermal growth factor differentially affects integrin-mediated adhesion of tumor cells to extracellular matrix proteins

 The adhesion of different epidermal growth factor (EGF) receptor (EGFR) expressing cell lines to various extracellular matrix (ECM) proteins is influenced by EGF. To investigate a putative receptor cross-talk between EGFR and integrins we chose two cell lines for a more detailed analysis: the highly metastatic rat mammary carcinoma clone MTLn3 that showed increased adhesion to a panel of ECM proteins in the presence of 10 ng/ml EGF and the nonmetastatic human vulva carcinoma cell line A431 which showed a decreased adhesion under the same conditions. These EGF-mediated stimulatory or inhibitory effects on adhesion were observed within a few minutes. On human A431 cells the inhibitory effect was blocked by an EGFR-specific antibody that interferes with ligand binding. In cell adhesion assays performed in the presence of divalent cations MTLn3 and A431 cells exhibited the typical behavior described for integrin-dependent matrix adhesion: Mn²⁺ enhanced binding to collagen IV and fibronectin whereas Ca²⁺ inhibited adhesion to collagen IV but not to fibronectin. Adhesion-inhibition assays with anti-human integrin antibodies revealed that A431 cells adhere to collagen via α₁β₁ and α₂β₁, and that adhesion to fibronectin is mediated predominantly through α₅β₁. The interaction of MTLn3 cells with fibronectin was in part RGD dependent, indicating the involvement of either α₃β₁ or α₅β₁. Addition of EGF in these assays showed that affecting the integrin extracellular domains by addition of either bivalent cations, RGD peptides, or function-blocking integrin antibodies did not prevent the effects mediated by EGF. We conclude that signals downstream of EGFR can modulate integrin-mediated adhesion to ECM proteins in both an inhibitory and a stimulatory manner. Received: 8 May 1996 / Accepted: 1 July 1996     

Use of RNA interference to inhibit integrin (α6β4)-mediated invasion and migration of breast carcinoma cells

The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the α6β4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the α6β4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced α6β4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of β4 expression in these cells augmented the formation of α6β1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the α6β4 integrin in invasion and migration that has been demonstrated previously by expression of the β4 subunit in β4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the α6β4 integrin may be a useful approach to prevent carcinoma cell progression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Angiogenesis and the growth potential of craniopharyngiomas

The present study was performed to evaluate the role of neovascularization on the behavior of craniopharyngiomas as well as the contribution of endothelial cell proliferation and migration in the remodeling and expansion of the vascular network associated with angiogenesis. Fourteen primary tumors were studied, all of the adamantinomatous type. CD34 immunostaining, an endothelial cell marker, localized vessels within the connective tissue stroma. MIB-1 immunopositivity was apparent in the nuclei of neoplastic cells, few endothelial cells, and stromal elements. MIB-1 counts were higher in epithelial than connective tissue cells. A positive correlation was found between the number of MIB-1 immunopositive cells and microvessel density (MVD). Immunohistochemistry demonstrated that integrin α_vβ3 expression was restricted to tumor vasculature; the tumor cells were immunonegative. Only 2.5% of vessels detected with CD34 were immunopositive for integrin α_vβ3. At present, no therapeutic implications can be drawn from our observations. More studies are needed to assess whether integrin α_vβ3 antagonists or drugs that arrest the cell cycle of endothelial cells can inhibit angiogenesis in craniopharyngiomas.     

Breast adenocarcinoma cell adhesion to the vascular subendothelium in whole blood and under flow conditions: effects of alphavbeta3 and alphaIIbbeta3 antagonists

Tumour cell adhesion to vascular extracellular matrix (ECM), an important step of metastatic progression, is promoted by platelets. The aim of our study was to investigate, in whole blood under venous and arterial shear conditions, the respective role of tumour cell α_vβ₃ and platelet α_IIbβ₃integrins in MDA-MB-231 breast adenocarcinoma cell adhesion to human umbilical vein endothelial cell ECM. For that purpose, blood containing MDA-MB-231 cells was incubated with non-peptide antagonists specific for platelet α_IIbβ₃ (lamifiban) or tumour cell α_vβ₃ (SB-273005). At 300 s⁻¹, each antagonist used alone did not modify tumour cell adhesion, whereas, at 1500 s⁻¹, tumour cell adhesion was decreased by 25% in presence of lamifiban indicating a role of platelet α_IIbβ₃ at higher shear rate. However, a combination of SB-273005 and lamifiban, or c7E3 Fab (a potent inhibitor of both α_IIbβ₃ and α_vβ₃) inhibited tumour cell adhesion by 40–45%, at either shear rate applied, indicating a cooperation between these two integrins in MDA-MB-231 cell adhesion to ECM, as well as the participation of other adhesive receptors on tumour cells and/or platelets. Thus, efficient anti-metastatic therapy should target multiple receptors on tumour cells and platelets. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of αvβ3 integrin reduces angiogenesis, bone turnover, and tumor cell proliferation in experimental prostate cancer bone metastases

The growth of metastatic prostate cancer cells in the bone involves an intimate interaction between the tumor cells and various elements of the bone microenvironment, resulting in increased rate of bone turnover and rapid tumor growth. The αvβ3 integrin has been shown to play an important role in tumor growth and angiogenesis, and is known to be critical to osteoclast formation and activity. This study was designed to examine the role of αvβ3 expressed by cells native to the bone in the growth and pathogenesis of prostate cancer bone metastases. Human prostate cancer cells which do not express αvβ3 or αIIbβ3 integrins were injected directly into human bone fragments previously implanted subcutaneously in SCID mice (SCID-human-bone model). At the same time treatment with anti-β3 antibody fragment (m7E3 F(ab′)₂) i.p. at 300 μg/dose 3× per week was initiated and continued for 2 weeks. In this system, m7E3 F(ab′)₂ only recognizes human bone-derived αvβ3. Antibody inhibition of αvβ3 integrin in vivo resulted in a specific reduction in the proportion of antigenically-human blood vessels within tumor-bearing bone implants (from 73.5% ± 3.93 in controls to 17.74% ± 5.64 in treated animals). Proliferation of the αvβ3-negative tumor cells was also reduced, although the overall vessel density was maintained by compensating mouse vasculature. Blockage of human bone-derived αvβ3 also significantly reduced the recruitment of osteoclasts in response to tumor cells, as well as degradation of calcified bone tissue. Together these observations confirm the importance of αvβ3 in bone metabolism and angiogenesis, and point to the role of these processes in controlling growth of metastatic prostate cancer cells in the bone. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Differential Effect of Endothelial Cell Factors on <Emphasis Type="Italic">In Vitro</Emphasis> Motility of Malignant and Non-malignant Cells

Motility of cancer cells plays a critical role in tumor metastasis, and as such is a target for intervention. The motility of malignant Calu-1 human lung epithelial carcinoma cells is upregulated when placed on a human umbilical vein endothelial cell monolayer, while that of non-malignant L132 human lung epithelial cells is not. To dissect the factor(s) causing such differential behaviors, the motile responses of both cell lines to endothelial cell factors—secreted to the media, on the endothelial cell surface, and secreted to the extracellular matrix—and to individual extracellular matrix proteins were compared. Cell motility was quantified by tracking the cell movement on a surface with time-lapse video microscopy, which was analyzed with the persistent random walk model of motility. None of the factors tested had a remarkable effect on L132 cell motility, but the Calu-1 cell motility was significantly upregulated by endothelial cell extracellular matrix and by laminin, fibronectin, collagen I and collagen VI individually. Flow cytometry analysis revealed significantly higher expression levels of integrin subunits β1, α2, α3, and α6, which are known receptors for these extracellular matrix proteins, on the Calu-1 than L132 cells, implicating a role of these integrins in the observed motile behaviors of these cell lines.     

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A^*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8×A2.1/K^b mouse-derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81–88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81–88), we found that the heterodimeric CD8αβ coreceptor, but not normally expressed homodimeric CD8αα, is required for tetramer binding and functional redirection of TCR-transduced human T cells. CD8⁺T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 antibody and required more peptide than wild-type (WT) MDM2 TCR⁺ T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) αβ domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expresion, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease. These two authors contributed equally to this work.     

IQGAP1, a negative regulator of cell-cell adhesion, is upregulated by gene amplification at 15q26 in gastric cancer cell lines HSC39 and 40A

Our previous comparative genomic hybridization (CGH) study revealed a novel amplified region at 15q26 in two cell lines established from diffuse types of gastric cancer (GC). In this amplified region, FES and IGF1R, known targets on 15q26, were located telomeric to the amplicon in the two cell lines, HSC39 and 40A, suggesting that another tumor-associated gene exists in this region. While screening expressed sequence tags (ESTs) for novel genes in this region, we identified the IQGAP1 amplification. IQGAP1 has been reported to encode a ras GAP-related protein, and its interaction with cadherin and/or β-catenin induces a dissociation of β-catenin from the cadherin-catenin complex, one of the mechanisms for cell-cell adhesion. Northern and Western blot analyses revealed that amplification of this gene was accompanied by corresponding increases in mRNA and protein expression. Moreover, immunocytochemical staining showed that overexpressed IQGAP1 accumulated at the membrane, suggesting its colocalization with β-catenin. Taken together, these findings suggest that IQGAP1 may be one of the target genes in the 15q26 amplicon correlated with a malignant phenotype of gastric cancer cells, such as diffuse and invasive characteristics, through the disruption of E-cadherin-mediated cell-cell adhesion. Received: September 20, 2000 / Accepted: October 17, 2000     

Modulation of acute visceral nociception and bladder inflammation by plant triterpene, α, β-amyrin in a mouse model of cystitis: role of tachykinin NK<Subscript>1</Subscript>-receptors,and K<Superscript>+</Superscript><Subscript>ATP</Subscript> channels

Objective and design: We previously described the visceral antinociceptive property of α, β-amyrin in a mouse model of cystitis induced by cyclophosphamide (CPM). This study examined the contribution of vanilloid-1 (TRPV1), peripheral NK1 receptors to CPM-evoked nociceptive behaviors and bladder edema, and its possible modulation by α, β-amyrin. Methods: The effect of α, β-amyrin (10, 30, and 100 mg/kg, p. o.) and N-acetylcysteine (NAC) on CPM (400 mg/kg, i. p.)-induced cystitis was studied in mice. Sensory deafferentation was done by a high dose capsaicin. The parameters analysed were: CPM-evoked noxious behaviors, bladder edema, vascular permeability, and NK₁ immunoreactivity. To assess the role of K⁺ _ATP channels in α, β-amyrin effect, animals were pretreated with glibenclamide. Results: α, β-amyrin (30 and 100 mg/kg) and NAC significantly (p < 0.01) suppressed the visceral pain-related behaviors and NK₁ immunoreactivity, but bladder edema was reduced weakly. Glibenclamide reversed the effects of α, β-amyrin. Sensory deafferentation by capsaicin significantly reduced the nociceptive responses and the NK₁ immunoreactivity to noxious stimulation by CPM. Conclusions: α, β-amyrin attenuates CPM-induced visceral pain and bladder edema by mechanisms that involve, at least in part, a block either of Substance P release or its receptor function, and partly by opening K⁺ _ATP channels. Received 13 February 2007; returned for revision 13 April 2007; accepted by G. Geisslinger 14 May 2007     

Desmoplastic small round cell tumor of the central nervous system: report of two cases and review of the literature

Desmoplastic small round cell tumor (DSRCT) is a malignant tumor often involving the abdominal and/or pelvic peritoneum. Only one fully documented example has arisen in the central nervous system (CNS). Herein, we describe two additional examples, fulfilling the morphologic, immunohistochemical, and molecular criteria (EWS/WT1 translocation) of DSRCT. Both arose in the cerebellopontine angle (CPA) and underwent spinal dissemination. Patient 1, a 37-year-old male, underwent a subtotal resection, and 2 years later died of recurrent disease with spinal dissemination. Patient 2, a 39-year-old man, presented with cerebellar and CPA lesions as well as spinal leptomeningeal deposits. After 27 months of adjuvant therapy, he is alive with progressive disease. In conclusion, CNS DSRCT follows a similar aggressive course as do peritoneal examples. Although rare, DSRCT warrants consideration in the differential diagnosis of “malignant small blue cell tumors” of the CNS. For consideration of publication in Virchows Archiv.     

Kakkalide and Its Metabolite Irisolidone Ameliorate Carrageenan-Induced Inflammation in Mice by Inhibiting NF-κB Pathway

The anti-inflammatory activities of kakkalide, a major constituent of the flower of Pueraria thunbergiana, and irisolidone, a metabolite of kakkalide produced by intestinal microflora, against carrageenan-induced inflammation in air pouches on the backs of mice and in lipopolysaccharide (LPS)-stimulated peritoneal macrophages were investigated. Kakkalide and irisolidone down-regulated the gene expression of cytokines [tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β)] and cyclooxygenase-2 (COX-2) and the production of pro-inflammatory cytokines, TNF-α and IL-1β, and inflammatory mediators, NO and prostaglandin E₂ (PGE₂), in LPS-stimulated peritoneal macrophages. These agents also inhibited the phosphorylation of IκB-α and the nuclear translocation of nuclear factor-kappa B (NF-κB). Orally administered kakkalide and irisolidone significantly reduced carrageenan-induced inflammatory markers, leukocyte number, and protein amount in the exudates of the air pouch. These constituents also inhibited PGE₂ production and COX-2 inducible nitric oxide synthase, IL-1β, and TNF-α expression. These agents also inhibited NF-κB activation. The anti-inflammatory effects of irisolidone were more potent than those of kakkalide. Based on these findings, kakkalide and irisolidone may inhibit inflammatory reactions via NF-κB pathway, and irisolidone, a metabolite of kakkalide, may more potently inhibit these inflammatory reactions.     

Effects of HIF-1α on human gastric cancer cell apoptosis at different CO2 pressures

The effects and potential molecular mechanisms underlying carbon dioxide (CO₂) pneumoperitoneum on gastric cancer cell apoptosis are not fully understood. In this study, we assessed the effects of CO₂ pneumoperitoneum on the apoptosis of MKN-45 gastric cancer cells. Additionally, we investigated the role of HIF-1α in CO₂ pneumoperitoneum-induced apoptosis of gastric cancer cells. MKN-45 cells were cultured in CO₂ or air pneumoperitoneum at 0, 12 and 15 mmHg pressures for 4 h. We observed a change in cells morphology and increasing apoptotic ratios in MKN-45 cells when they were put into a 15 mmHg CO₂ pneumoperitoneum environment. However, there was no significant difference between the 0, 12 mmHg CO₂ pneumoperitoneum and the control groups. Exposure to 15 mmHg CO₂ pneumoperitoneum significantly enhanced the expression levels of HIF-1α and Bax, while it attenuated Bcl-2 expression levels. When we inhibited HIF-1α by small interfering RNA (siRNA), we found that the apoptotic ratio of MKN-45 cells decreased in 15 mmHg CO₂ pneumoperitoneum. This treatment markedly elevated Bcl-2 levels and decreased Bax expression. These data suggest that CO₂ pneumoperitoneum may accelerate the apoptosis of MKN-45 cells at higher pressures. HIF-1α is a crucial factor that affects gastric cancer cell apoptosis by downregulating the Bcl-2/Bax ratio.     

Adhesion of HT-29 colon carcinoma cells to endothelial cells requires sequential events involving E-selectin and integrin β4

HT-29 colon carcinoma cells attach to TNFα-activated human umbilical vein endothelial cells (HUVECs) by their specific binding to E-selectin. This interaction activates, in the cancer cells, the MAPK SAPK2/p38, which leads to their transendothelial migration (Laferrière et al., J Biol Chem 2001; 276: 33762). In this study, we investigated the role of E-selectin in activating integrins to modulate adhesion and regulate integrin-mediated events. Blocking the integrins from HT-29 cells (α2, α3, α6, αvβ5, β1 and β4) with specific antibodies revealed a role for β4 integrin in their adhesion to TNFα-treated HUVEC. The β4 integrin-dependent adhesion was maximal after 30 min, whereas the-E-selectin-dependent adhesion was maximal after 15 min. Integrin β4 became quickly phosphorylated upon addition of HT-29 cells to endothelial cells and the effect was independent of the expression of E-selectin. Moreover, a recombinant E-selectin/Fc chimera did not induce the phosphorylation of β4. The phosphorylation of β4 is not required for adhesion since adhesion was not affected in HT-29 cells that express a truncated form of β4 that is deleted from its cytoplasmic phosphorylatable domain. However, the expression of the non-phosphorylatable deletant of β4 was associated with decreased transendothelial cell migration underscoring the key role for the cytoplasmic domain of β4 in cell migration. We suggest: 1) that the adhesion of HT-29 cells to activated endothelial cells follows at least two essential sequential steps involving the binding of E-selectin to its receptor on carcinoma cells and then the binding of β4 to its own receptor on endothelial cells; 2) that the phosphorylation of integrin β4 contributes to enhance the motile potential of cancer cells and increase their trans-endothelial migration. Overall, our results indicate that the interaction of metastatic cancer cells with endothelial cells implies a specific sequence of signaling events that ultimately leads to an increase in their efficient transendothelial migration. Abbreviations: ERK – extracellular signal-regulated kinase; GFP – green fluorescent protein; ICAM – intercellular adhesion molecules: JNK – c-Jun NH₂-terminal kinase; MAPK – mitogen-activated protein kinase; MAPKAP K2, MAP kinase-activated protein kinase 2; PI3K – phosphatidyl inositol 3-kinase; SAPK – stress-activated protein kinase; TNFα– tumor necrosis factor-α. This revised version was published online in July 2006 with corrections to the Cover Date.     

Shear Stress Induced Release of Von Willebrand Factor and Thrombospondin-1 in Uvec Extracellular Matrix Enhances Breast Tumour Cell Adhesion

Endothelial cells in vivo are exposed to blood shear forces and flow perturbations induce their activation. Such modifications of hemodynamic can be observed in patients with cancer. We have submitted endothelial cells (HUVEC) to shear stress (13 dynes/cm²) and isolated their extracellular matrix (ECM) prior perfusion with breast adenocarcinoma cells (MDA-MB-231) in whole blood at a shear rate of 1500 s⁻¹. Exposure of HUVEC to 13 dynes/cm² (τ₁₃) for 2 h enhanced the secretion of von Willebrand factor (vWF) and thrombospondin-1 (TSP-1) in the ECM. Moreover, MDA-MB-231 cell adhesion was enhanced to such treated-ECM. This over-adhesion was inhibited by pre-incubating the ECM with anti-vWF or anti-TSP-1 antibodies, or by blocking tumour cell α_vβ₃ integrin. Although blood platelets were involved in tumour cell adhesion to ECM, blockade of platelet GPIb or α_IIbβ₃ receptors did not specifically inhibit the enhanced tumour cell adhesion observed on τ₁₃. ECM. These findings indicate that shear stress can modulate the expression of adhesive proteins in ECM, which favours direct tumour cell adhesion via α_vβ₃ and other receptors.