Pretreatment Of Tumor Cells Research Articles

Soy isoflavones enhance radiotherapy in a metastatic prostate cancer model

We previously reported that genistein, the bioactive isoflavone of soybeans, acts as a radiosensitizer for prostate cancer. Pretreatment of tumor cells with genistein potentiated radiation-induced killing in vitro and in orthotopic models in vivo. However, pure genistein promoted increased lymph node metastasis, when administered alone in vivo. We investigated in vitro and in vivo the effects of soy isoflavones (genistein, daidzein and glycitein) as soy pills of similar composition are used in human interventions but not pure genistein. Soy isoflavones inhibited cell survival and potentiated radiation cell killing in PC-3 tumor cells, in vitro. Increased cell killing correlated with inhibition of antiapoptotic molecules Bcl-xL and survivin, upregulation of proapoptotic Bax molecule and PARP cleavage, suggesting activation of apoptotic pathways. In vivo, using the PC-3 orthotopic metastatic mouse model, soy isoflavones and prostate tumor irradiation led to enhanced control of primary tumor growth and metastasis, as observed with pure genistein and radiation. Interestingly, treatment with soy isoflavones did not increase metastasis to para-aortic lymph nodes in contrast to the consistent increase caused by pure genistein. Histologically prostate tumors, treated with soy isoflavones and radiation, showed tumor destruction and in situ tissue alterations, comparable with genistein and radiation effects. However, genistein, but not soy isoflavones, caused induction of HIF1-alpha in prostate tumors, suggesting that induction of hypoxia by pure genistein could contribute to increased metastasis. Our studies demonstrate the safety and potential role of soy isoflavones for enhancing the therapeutic effect of radiotherapy in prostate cancer.

Synthesis of Pyrophosphate-Containing Compounds that Stimulate VγVδ2 T Cells: Application to Cancer Immunotherapy

Human Vgamma2Vdelta2 T cells recognize nonpeptide antigens, such as isoprenoid pyrophosphomonoester intermediates, alkylamine compounds, and bisphosphonate drugs, as well as some tumor cells. Although attempts have been made to derive novel cancer immunotherapies based on the discovery of these unconventional antigens, effective therapies remain to be developed. Here, we synthesized a series of pyrophosphate-containing compounds and examined the chemical requirements for the recognition of pyrophosphomonoester antigens by gammadelta T cells. The structural analysis clearly demonstrated that a proximal methylene moiety plays a crucial role in the stimulatory activity of the antigens. For optimal gammadelta T cell proliferation, we find that the use of human serum albumin was preferred and that pyrophosphomonoesters were superior to nitrogen-containing bisphosphonate compounds. Using these techniques, we have successfully expanded gammadelta T cells from healthy donors as well as from cancer patients using one of the most active compounds, 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). The resulting expanded gammadelta T cells exhibited potent, cytotoxic activity against a wide variety of tumor cell lines. Even gammadelta T cells from a patient with advanced liver carcinoma efficiently responded to 2M3B1PP and exhibited strong cytotoxic activity against tumor cells. The pretreatment of tumor cells with nonpeptide antigens was essential for efficient cytotoxicity via TCR-gammadelta. The present study suggests a novel strategy for cancer immunotherapy using synthetic small pyrophosphate-containing compounds and nitrogen-containing bisphosphonates.

The Role of Soluble Fibrin and Fibrin Inhibitory Peptides in Cancer Metastasis.

Circulating soluble fibrin (sFn) is a marker for ongoing disseminated intravascular coagulation and may have prognostic significance, especially in metastatic cancers. Anti-coagulant therapies have been effective in reducing metastasis in several cancers, but with increased risk of bleeding. The authors have previously demonstrated that soluble fibrin (sFn), which is elevated in many cancer patients, enhances metastasis in an experimental model, and increases platelet/tumor cell adherence by cross-linking platelet aIIbb3 to tumor cell CD54 (a receptor for two of the leukocyte b2 integrins aLb2 and aMb2). sFn also binds to monocyte aMb2 (Mac1), and the peptide sequences of the fibrin(ogen) binding sites for aMb2 and CD54 have recently been identified. It was, therefore, hypothesized that sFn binding to these receptors would result in inhibition of monocyte/tumor cell adherence, and consequently cytotoxicity, which may be reversed by inclusion of blocking peptides. To test this, monocyte adherence to A375 melanoma cells was quantified at physiologically relevant shear rates (35–560 s−1) using a laminar flow perfusion chamber mounted on a Leica DMIRB inverted microscope equipped with a computer controlled digital imaging camera. Effector and target cells were untreated, or incubated with sFn (fibrinogen (Fg), 0.5 mg/ml; fibrin polymerization inhibitor Gly-Pro-Arg-Pro amide (GPRPa), 4 mM; and human thrombin (0.125 U/ml)) in the presence or absence of specific single or combined blocking peptides directed against the sFn binding sites on CD54 (P1) and aMb2 (P2), or the sFn g-chain binding sites for CD54 (P3) and aMb2 (P4). The effect of peptides on thrombin (0.125 U/ml) induced clotting of purified Fg (0.5 mg/ml) was assessed visually. The effect of sFn on monocyte cytotoxicity (effector: target 20:1) against green fluorescent protein (GFP) transfected A375 melanoma cells was measured by GFP release from lysed cells using a Perkin-Elmer Victor-3 96-well fluorescence plate reader. Pre-treatment of tumor cells with sFn significantly (P<0.01) increased monocyte adherence to 68.5 + 0.7% compared to the untreated control (32.9 +1.3%), whereas monocyte pre-treatment had no significant effect (P>0.05) on adherence. However, pre-incubation of both cells with sFn resulted in a significant (P<0.01) inhibition of adherence to 15.95 ± 1.0% (62% inhibition). sFn mediated inhibition of adherence was significantly reduced by pre-treatment of cells with a combination of P1 + P2 (to 22.3 ± 13.8% inhibition; P<0.01), and by pre-incubation of sFn with P3 + P4 (to 9.9 ± 3.6% inhibition; P<0.01). Single peptides blocked to an intermediate level, and controls performed appropriately. Furthermore, blocking peptides did not inhibit thrombin induced clotting of Fg. Pretreatment of both monocytes and A375 cells significantly inhibited specific cytotoxicity by 40% (P < 0.01 compared to untreated cytotoxicity − 28.6 ± 0.7%), and intermediate killing was observed when only one cell type was sFn treated. These results show that sFn incubation with both effector and target cells inhibits both cellular adherence and cytotoxicity by a mechanism involving monocyte aMb2 and tumor cell CD54. Adherence was restored by inclusion of sFn blocking peptides, which did not affect clotting. Clinically, these peptides may be effective therapeutically in reducing sFn mediated immunosuppression and may enhance the immune response to metastasizing cancer cells, without the risk of bleeding problems associated with other therapies.

Investigating the Combination of Trastuzumab and HER2/neu Peptide Vaccines for the Treatment of Breast Cancer

Trastuzumab, an anti-HER2/neu monoclonal antibody, is thought to promote HER2/neu receptor internalization and/or turnover. This study was designed to investigate the kinetics of trastuzumab treatment on tumor cells with varying levels of HER2/neu expression and to determine the effect of trastuzumab on HER2/neu-specific cytotoxic T lymphocyte-mediated lysis. Three cell lines with varying levels of HER2/neu expression were incubated with varying doses of trastuzumab at multiple time points. Trastuzumab binding and HER2/neu expression were determined. Peripheral blood mononuclear cells from three HLA-A2(+) healthy donors and four E75 peptide-vaccinated patients were stimulated with HER2/neu-derived peptides and tested in standard chromium release cytotoxicity assays with HER2/neu(+) tumor cells pretreated with trastuzumab. Treatment of tumor cells with 10 microg/mL of trastuzumab in an overnight incubation resulted in saturation of cell-surface HER2/neu receptors. At higher doses, trastuzumab staining and HER2/neu expression decreased in a time-dependent manner. Pretreatment of tumor cells with trastuzumab resulted in increases in specific cytotoxicity by peptide-stimulated cytotoxic T lymphocytes from HLA-A2(+) donors over untreated cells by an average of 5.6% and 15.3% (P = .0002) for doses of 10 and 50 microg/mL, respectively. In similar experiments involving peripheral blood mononuclear cells obtained from immunized patients, the average specific cytotoxicity for untreated cells was 34.2% +/- 1.3% vs. 40.6% +/- 2.5% (P = .035) and 40.7% +/- 1.6% (P = .0005) for those treated with 10 and 50 microg/mL, respectively. Our data suggest that pretreatment of breast cancer cells with trastuzumab induces turnover of the HER2/neu protein and enhanced killing by HER2/neu peptide-stimulated CTLs. This increased lysis occurs regardless of the degree of HER2/neu expression and seems more pronounced in vaccinated patients. These findings support further investigation into the use of combination immunotherapy with trastuzumab and HER2/neu peptide-based vaccines.

Immunomodulatory impact of interferon-alpha in combination with chemoradiation of pancreatic adenocarcinoma (CapRI).

Data from a phase II trial combining chemoradiotherapy with interferon-alpha (IFN-alpha) (CapRI scheme) for adjuvant treatment of pancreatic carcinoma are very encouraging. Eight human ductal pancreatic carcinoma cell lines were treated with the CapRI scheme [5-fluorouracil (5-FU), Cisplatin, IFN-alpha and radiation]. Natural killer (NK) and T cells preincubated with IFN-alpha were tested in cytotoxicity assays against these cell lines and the mechanism of cell lysis was investigated. The induction of the immunoproteasome in tumour cells after IFN-alpha stimulation was analysed by immunoblot and RT-PCR. IFN-alpha activated NK cells and increased their cytotoxicity. This cytotoxicity was mediated as well by Fas-induced apoptosis as by perforin release. Pre-treatment of tumour cells with 5-FU and combinations showed a significant increase in the susceptibility of tumour cells against NK cells. Treatment of tumour cells with IFN-alpha induced a switch to the immunoproteasome and enhanced their vulnerability to T cells. This is the first description of this phenomenon in pancreatic carcinoma cells with implications for their immunogenicity. IFN-alpha activates NK cells against pancreatic carcinoma cells and 5-FU treatment makes tumour cells more susceptible. Furthermore, IFN-alpha induces the immunoproteasome with impact on the immunogenicity of pancreatic carcinoma cells. These mechanisms may be responsible for the improved clinical outcome of CapRI.

Vascular Endothelial Growth Factor Receptor-1 Activation Mediates Epithelial to Mesenchymal Transition in Human Pancreatic Carcinoma Cells

Our laboratory has shown that vascular endothelial growth factor receptor-1 (VEGFR-1) expression on human pancreatic cancer cell lines mediates cell migration and invasion. Because epithelial to mesenchymal transition (EMT) also plays a role in cell motility by altering the cell phenotype and morphology, we hypothesized that VEGFR-1 activation induces molecular alterations that mediate EMT. Our treatment of the human pancreatic cancer cell line L3.6pl with the VEGFR-1 ligands VEGF-A and VEGF-B led to morphologic changes characteristic of EMT, including loss of polarity, increased intercellular separation, and the presence of pseudopodia. Immunofluorescent staining with antibodies to E-cadherin and beta-catenin showed that VEGFR-1 activation led to translocation of E-cadherin and beta-catenin from their usual cell membrane-bound location to the cytoplasm and nucleus, respectively. Western blotting showed that VEGFR-1 activation led to decreased expression of the epithelial markers E-cadherin and plakoglobin, increased expression of the mesenchymal markers vimentin and N-cadherin, and increased nuclear expression of beta-catenin. Pretreatment of tumor cells with a VEGFR-1 blocking antibody inhibited the VEGFR-1-induced immunohistochemical and molecular changes in E-cadherin. VEGFR-1 activation led to an increase in expression of the EMT-associated transcription factors Snail, Twist, and Slug. The changes mediated by VEGFR-1 in this pancreatic carcinoma cell line are highly consistent with the changes characteristic of EMT. Given our previous finding of VEGFR-1-mediated tumor cell invasion and migration in pancreatic carcinoma cells, we hypothesize that VEGFR-1 plays a role in tumor progression in pancreatic cancer through the induction of EMT.

Warfarin inhibition of metastasis: the role of anticoagulation.

Warfarin inhibits metastasis in animal models by mechanisms that remain unclear. A better understanding of this phenomenon may clarify processes underlying metastasis in human cancer. We have studied the effects of warfarin on metastasis in a rat model by intravenous injection of Mtln3 mammary carcinoma cells and subsequent counting of pulmonary seedlings. To determine whether warfarin acts principally on the tumour cells or the host, we pretreated either cells or animals with warfarin before intravenous injection of 10(4) Mtln3 cells. Pretreatment of tumour cells had no effect, whilst pretreatment of the host reduced median seedlings from 67 to 4.5 per animal (P less than 0.005). To determine whether warfarin was acting via its anticoagulant action, we reversed warfarin anticoagulation by intravenous injection of coagulation factors II, VII, IX and X. Restoration of coagulation for 12 h immediately after injection of cells completely reversed the warfarin effect (P less than 0.001), but if the injection of factors was delayed for 12 h it had no effect (P = 0.1753). We conclude that warfarin acts principally on the host, not the tumour cell, and that it acts via its effect on coagulation. The restriction of the effect to the first 12 h after tumour cell injection suggests a mechanism involving intravascular processes such as tumour cell survival or endothelial adhesion.

Method of laser activated nano-thermolysis for elimination of tumor cells

We describe novel ex vivo method for elimination of tumor cells from cell suspension, Laser Activated Nanothermolysis and propose this method for purging of bone marrow and blood transplants. K562 and human lympholeukemia cells were eliminated in experiments by laser-induced micro-bubbles that emerge inside individual target cells around selectively formed clusters of light-absorbing gold nanoparticles. Pretreatment of tumor cells with specific monoclonal antibodies and Ig-conjugated 30-nm gold particles allowed the formation of clusters of 10-20 on the surface of cell membrane. Electron microscopy found the nanoparticulate clusters inside the cells. Total (100%) elimination of K562 cells targeted with specific antibodies was achieved with single laser pulses with optical fluence of 5J/cm(2) at the wavelength of 532 nm without damage to the same cells targeted without specific antibodies. Total elimination of human lymphoblasts from suspension of normal stem cells was achieved by a single laser pulse with the optical fluence of 1.7J/cm(2), while the damage level of normal cells was 16%.

Cytotoxicity of combinations of IFN-beta and chemotherapeutic drugs.

Interferon-beta (IFN-beta) induces various antiproliferative activities. In solid tumor cells, IFN-beta inhibits cell cycle progression, which mainly occurs as S phase accumulation. The IFN-beta-induced cell cycle effect has been implicated in the antitumor effect of combinations of IFN-beta and chemotherapeutic drugs. In this report, we characterized the viability of various human tumor cells in vitro after combination treatment with IFN-beta protein and the chemotherapeutic drugs, cis-platinum (II) diamine dichloride (cisplatin), 5-fluorouracil (5-FU), paclitaxel (Taxol) and gemcitabine. IFN-beta could significantly potentiate the cytotoxicity of these chemotherapeutic drugs. The potentiating effect was observed after pretreatment of tumor cells with IFN-beta but did not require the constant presence of IFN-beta. The potentiating effect correlated with the sensitivity of the tumor cells to the IFN-beta-induced cytotoxicity. Furthermore, chemotherapeutic drugs also potentiated the cytotoxicity of IFN-beta. We conclude that the cell cycle effect per se did not determine the ability of IFN-beta to potentiate the cytotoxicity of chemotherapeutic drugs. We suggest that the combination of local IFN-beta gene therapy with chemotherapy could be an effective cancer treatment.

Ectopic αIIbβ3 Integrin Signaling Involves 12-Lipoxygenaseand PKC-mediated Serine Phosphorylation Events in Melanoma Cells

Megakaryocytic genes such as alphaIIbbeta3 can be expressed by malignant cells as part of the disturbances in their gene regulation. However, the function of the gene product greatly depends on the interaction of the ectopic protein with the new environment. The outside-in signaling of the ectopically expressed alphaIIbbeta3 integrin was studied in B16a murine melanoma cells using a monoclonal antibody, specifically directed to the activated conformation of alphaIIbbeta3, PAC-1 and the physiological ligand, fibrinogen. Ligation of alphaIIbbeta3 induced down-regulation of FAK but serine phosphorylation of three protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that the ligation of the receptor in B16a cells induces approximately 50% increase in phosphoserine positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling pathway, since ligation of alphaIIbbeta3 induces 12-HETE production within 5 min and pretreatment of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated rearrangement of actin filaments upon alphaIIbbeta3 ligation paralleled by downregulation of p125FAK and phoshotyrosine+ adhesion plaques and translocation of PKCalpha to stress fibers and cortical actin. PKC appears to be the major effector serine kinase of the alphaIIbbeta3-coupled signaling pathway, since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents the ligation-induced serine phosphorylation. Previous studies have indicated a role for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor invasion, therefore the involvement of this cascade in the signaling of the ectopic alphaIIbbeta3 integrin may partially explain its role in tumor progression.

Inhibitory effects of evodiamine on in vitro invasion and experimental lung metastasis of murine colon cancer cells.

We have previously reported that evodiamine had a marked inhibitory activity on tumor cell migration in vitro. To extend our study, the effects of evodiamine on invasion, growth, and metastatic development of colon 26-L5 cells were examined here. Evodiamine inhibited the invasion of tumor cells into Matrigel in a concentration-dependent manner, and achieved 70% inhibition at 10 microg/ml. Treatment of tumor cells with evodiamine for 24 h showed little effect on tumor growth at concentrations of less than 10 microg/ml, whereas an over 48-h treatment resulted in a concentration- and time-dependent inhibition. Pretreatment of tumor cells with 10 microg/ml evodiamine before inoculation into mice caused 70% reduction in their lung metastasis formation. When evodiamine at 10 mg/kg was administered into mice from the 6th day after tumor inoculation, the number of tumor nodules in lungs was decreased by 48% as compared to control. The inhibition rate was equivalent to that produced by cisplatin, a potent anti-cancer drug. Evodiamine did not affect the body weight of mice in the experimental period, whereas cisplatin caused serious weight loss. These results suggest that evodiamine may be regarded as a promising agent in tumor metastasis therapy.

Targeting human prostatic carcinoma through basic fibroblast growth factor receptors in an animal model: Characterizing and circumventing mechanisms of tumor resistance

BACKGROUND Basic fibroblast growth factor receptors on DU145 human prostatic carcinoma xenografts serve as targets for the delivery of a growth factor-toxin chimera, basic fibroblast growth factor-saporin (bFGF-SAP), which produces significant antitumor activity in a nude mouse model. However, DU145 tumors often become resistant to prolonged treatment. METHODS Nude mice bearing DU145 xenografts were intravenously administered bFGF-SAP (0.05 μg/kg weekly for 4 weeks), and a panel of eight tumors was isolated from the treated animals and established in monolayer culture. RESULTS In cell-survival assays, sensitivity of the treated tumor-derived cell lines to bFGF-SAP (IC50 = 12–100 nM) varied widely from cells derived from a vehicle-treated control tumor (IC50 = 10 nM). A significant inverse correlation was observed between increased IC50 values in vitro and increased tumor growth delay in vivo. Pretreatment of tumor cells with suramin or neutralizing antibodies to bFGF or keratinocyte growth factor (KGF) circumvented resistance in one of the tumor lines, confirming autocrine-mediated resistance. In another tumor subline, a 3-fold decrease in bFGF high-affinity receptor sites, which concurred with a 4-fold decrease in ability to internalize the bFGF ligand, was consistent with a decrease in total cellular expression of the FGF2 receptor (Bek). Resistance was circumvented by alternatively targeting FGF1 receptor (Flg) on these cells with a saporin immunotoxin. CONCLUSIONS These studies identify alterations in the ligand-targeted receptor as a frequent contributor to resistance arising in DU145 tumors to in vivo treatment with a bFGF receptor-directed-toxin chimera, and provide the basis for designing methods to circumvent resistance for the purpose of enhancing efficacy of receptor-directed therapies in the treatment of prostate cancer. Prostate 40:178–191, 1999. © 1999 Wiley-Liss, Inc.

Targeting human prostatic carcinoma through basic fibroblast growth factor receptors in an animal model: characterizing and circumventing mechanisms of tumor resistance.

Basic fibroblast growth factor receptors on DU145 human prostatic carcinoma xenografts serve as targets for the delivery of a growth factor-toxin chimera, basic fibroblast growth factor-saporin (bFGF-SAP), which produces significant antitumor activity in a nude mouse model. However, DU145 tumors often become resistant to prolonged treatment. Nude mice bearing DU145 xenografts were intravenously administered bFGF-SAP (0.05 microg/kg weekly for 4 weeks), and a panel of eight tumors was isolated from the treated animals and established in monolayer culture. In cell-survival assays, sensitivity of the treated tumor-derived cell lines to bFGF-SAP (IC50 = 12-100 nM) varied widely from cells derived from a vehicle-treated control tumor (IC50 = 10 nM). A significant inverse correlation was observed between increased IC50 values in vitro and increased tumor growth delay in vivo. Pretreatment of tumor cells with suramin or neutralizing antibodies to bFGF or keratinocyte growth factor (KGF) circumvented resistance in one of the tumor lines, confirming autocrine-mediated resistance. In another tumor subline, a 3-fold decrease in bFGF high-affinity receptor sites, which concurred with a 4-fold decrease in ability to internalize the bFGF ligand, was consistent with a decrease in total cellular expression of the FGF2 receptor (Bek). Resistance was circumvented by alternatively targeting FGF1 receptor (Flg) on these cells with a saporin immunotoxin. These studies identify alterations in the ligand-targeted receptor as a frequent contributor to resistance arising in DU145 tumors to in vivo treatment with a bFGF receptor-directed-toxin chimera, and provide the basis for designing methods to circumvent resistance for the purpose of enhancing efficacy of receptor-directed therapies in the treatment of prostate cancer.

Selectins induced by interleukin-1 β on the human liver endothelial cells act as ligands for sialyl Lewis X-expressing human colon cancer cell metastasis

We have previously reported that colon cancer cells metastasized to the liver expressed an increased amount of sialyl Lewis X (SLeX) antigen compared to their corresponding primary lesions. It is now well known that SLeX antigen and sialyl Lewis A (SLeA) antigen are ligands for the selectins expressed on the endothelial cells. Therefore, it is assumed that SLeX-rich colon cancer cells could be easily adhered to the endothelial cells that express selectins. In this report we have tried to induce selectin expression on the human liver sinusoidal endothelial cells and have examined the adhesion of SLeX-high or -low expressing colon cancer cells to the interleukin-1 β (IL-1 β)-treated liver specimens using Stamper–Woodruff assay. These human colon cancer cells are termed KM12HX or KM12LX cells, respectively. A significantly increased number of KM12HX cells adhered to the IL-1 β-treated liver specimens compared to KM12LX cells. The adhesion of KM12HX cells was inhibited by the pretreatment of tumor cells with anti-SLeX antibody or by the pretreatment of liver specimens with anti-selectin antibodies. Selectin expression on the liver sinusoidal endothelial cells and endothelial cells of blood vessels after IL-1 β treatment was confirmed by immunohistochemically using anti-selectin monoclonal antibodies (MAbs). These findings strongly suggest that SLeX-expressing cancer cells could adhere to the sinusoidal endothelial cells via an SLeX–selectin interaction system and this could be a first step for colon cancer cells that metastasize to the liver. The mechanism by which these selectins can be induced in vivo is the next problem to be considered.

Low density lipoproteins and Lovastatin modulate the organ-specific transendothelial migration of primary and metastatic human colon adenocarcinoma cell lines in vitro.

Tumor cell arrest and tumor migration are two of the critical steps in the metastatic cascade. We hypothesized that these steps may be facilitated by the low density lipoprotein (LDL)-induced activation of microvessel endothelial cells (MVEC). The purpose of our study was to investigate the biological effects of an LDL-enriched milieu and the effects of the anticholesterol drug Lovastatin on metastatic behavior. The SW480 and SW620 are primary and metastatic human colonic adenocarcinoma cell lines derived from the same patient. We investigated the effect of LDL on adhesion and migration of the two tumor cell lines across human brain, lung, liver and dermal endothelial monolayers. Adhesion and migration assays were done before and after pretreatment of the MVEC or tumor cells with LDL (100 microg/ml) for 24 h. Although metastatic SW620 cells were more adherent to MVEC compared with primary SW480 cells, LDL pretreatment of SW480 and SW620 cells did not affect tumor cell adhesion to MVEC. In contrast, tumor cell migration was significantly increased across endothelial monolayers when MVEC were pretreated with LDL. Transendothelial cell migration was not significantly affected by pretreatment of the tumor cells with LDL. Lovastatin is an inhibitor of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. It has been shown to have anti-tumor activity in vitro. We investigated the effect of Lovastatin on tumor cell kinetics and tumor cell migration across MVEC. Growth curves and migration assays were done before and after pretreatment of the tumor cells with Lovastatin (30 microg/ml). Migration assays were also done after treatment of unstimulated or LDL-stimulated MVEC (100 microg/ml) for 24 h with Lovastatin. Lovastatin inhibited the in vitro growth of the metastatic SW620 cell line to a greater extent than the invasive SW480E cell line. On the other hand, pretreatment of tumor cells with Lovastatin (30 microg/ml) did not suppress transendothelial tumor cell migration of tumor cells. Finally, Lovastatin given to mice effectively suppressed the number of MCA-26 tumor colonies in the liver of Balb/c mice compared with untreated mice.

Inhibition of tumor invasion and metastasis by calcium spirulan (Ca-SP), a novel sulfated polysaccharide derived from a blue-green alga, Spirulina platensis.

We have investigated the effect of calcium spirulan (Ca-SP) isolated from a blue-green alga, Spirulina platensis, which is a sulfated polysaccharide chelating calcium and mainly composed of rhamnose, on invasion of B16-BL6 melanoma, Colon 26 M3.1 carcinoma and HT-1080 fibrosarcoma cells through reconstituted basement membrane (Matrigel). Ca-SP significantly inhibited the invasion of these tumor cells through Matrigel/fibronectin-coated filters. Ca-SP also inhibited the haptotactic migration of tumor cells to laminin, but it had no effect on that to fibronectin. Ca-SP prevented the adhesion of B16-BL6 cells to Matrigel and laminin substrates but did not affect the adhesion to fibronectin. The pretreatment of tumor cells with Ca-SP inhibited the adhesion to laminin, while the pretreatment of laminin substrates did not. Ca-SP had no effect on the production and activation of type IV collagenase in gelatin zymography. In contrast, Ca-SP significantly inhibited degradation of heparan sulfate by purified heparanase. The experimental lung metastasis was significantly reduced by co-injection of B16-BL6 cells with Ca-SP. Seven intermittent i.v. injections of 100 microg of Ca-SP caused a marked decrease of lung tumor colonization of B16-BL6 cells in a spontaneous lung metastasis model. These results suggest that Ca-SP, a novel sulfated polysaccharide, could reduce the lung metastasis of B16-BL6 melanoma cells, by inhibiting the tumor invasion of basement membrane probably through the prevention of the adhesion and migration of tumor cells to laminin substrate and of the heparanase activity.

T-cell activation induced by anti-CD3 x anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand.

T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific antibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy.

Cisplatin modulates tumor cell susceptibility to tumor-infiltrating lymphocytes in vivo

We investigated the in vivo augmentation of susceptibility of tumor cells to tumor-infiltrating lymphocytes (TILs) with cisplatin (CDDP). TILs showed cytotoxicity against autologous and established tumor cells. Pretreatment of tumor cells with CDDP 2 mu g/ml for 12 h enhanced the susceptibility of tumor cells to TILs in vitro. TILs and autologous tumor cells were obtained from malignant ascites of patients, before and after the intraperitoneal administration of CDDP. TILs had higher cytotoxicity against autologous tumor cells of CDDP treated as compared to untreated control tumor cells, providing direct evidence of in vivo immunomodulatory effect of CDDP in cancer patients.

Decreased expression of ICAM-1 and its induction by tumor necrosis factor on breast-cancer cells in vitro.

In order to study adhesion-molecule expression and its consequences for cellular recognition, the presence of adhesion molecules ICAM-1, VCAM-1, VLA-4, LFA-1, alpha, LFA-1 beta, LFA-3, beta1-integrin and beta3-integrin was studied on specimens from breast tissue by immunohistochemistry and on cells from breast cell lines propagated in vitro. Breast-cancer tissue and the breast-cancer cell lines MCF-7, SK-BR-3 and ZR-75-1 showed expression of ICAM-1 and VLA-4 significantly lower than that of benign breast cells or normal breast epithelium. Of various cytokines tested, including recombinant human (rh) interleukin-6 (IL-6), rh tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), granulocyte/macrophage-colony-stimulating-factor (GM-CSF), interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma), only TNF was able to re-induce expression of ICAM-1 on cells from MCF-7, SK-BR-3 and ZR-75-1. Further, the ability of either unstimulated or lymphokine-stimulated killer (LAK) cells to recognize and lyse native or TNF-stimulated breast-cancer cells was studied. Whereas neither unstimulated lymphocytes or LAK cells were able to lyse untreated breast-cancer cells deficient for ICAM-1 expression, pre-treatment of tumor cells with TNF led to increased tumor-cell lysis. Anti-ICAM-1 antibodies, and pre-treatment of tumor cells with anti-TNF-receptor antibodies, abrogated these findings, corroborating their specificity. We thus conclude that the defective expression of ICAM-1 in our model might constitute a mechanism by which breast-cancer cells escape immunologic recognition and lysis by appropriate effector cells.

Regulation of 12(S)-HETE production in tumor cells.

12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] is a major 12-lipoxygenase (12-LOX) metabolite of arachidonic acid (AA) (Chen et al., 1992a). Previously, we have shown that exogenous 12(S)-HETE increases tumor cell adhesion to endothelium (Grossi et al., 1989; Honn et al., 1992), induces large vessel and microvessel endothelial cell retraction (Honn et al., 1989; Tang et al., 1992) and stimulates tumor cell spreading on matrix (Timar et al., 1992). Tumor cells express a variety of adhesion molecules. Among them, integrin αIIbβ3 is an important receptor for tumor cell-host cell and tumor cell-matrix interaction (Chen et al., 1992b; Chang et al., 1992a). Pretreatment of tumor cells with exogenous 12(S)-HETE enhances their adhesion to and spreading on fibronectin, which is mediated exclusively by αIIbβ3 receptors and in a cytoskeleton-dependent manner (Chopra et al., 1991; Timar et al., 1992). Besides the effects on integrins, 12(S)-HETE also appears to regulate other factors which are important to tumor cell metastasis. Pretreatment of tumor cells with 12(S)-HETE stimulates their motility (Timar et al., 1993) and release of cathepsin B (Honn et al., 1994) a proteinase associated with tumor cell invasiveness. Interestingly, we observed that treatment of W256 cells with 12(S)-HETE induced an increase in membrane-associated PKC activity (Liu et al., 1991). This activation of PKC by 12(S)-HETE also was observed in B16a cells (Liu et al., unpublished). Since only the PKCα isozyme was found in B16a cells by western blotting, it suggests that 12(S)-HETE can up-regulate PKCα activity in these cells.KeywordsBiosynthetic EnzymeTumor Cell AdhesionW256 CellLipoxygenase MetaboliteA431 Epidermoid Carcinoma CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.