Apricoxib upregulates 15-PGDH and PGT in tobacco-related epithelial malignancies.

BACKGROUND: Despite focused research in conventional therapies and considerable advances in the understanding of the molecular carcinogenesis of head and neck squamous cell carcinoma (HNSCC), the 5-year survival rate for patients with advanced disease remains ∼15-20%. The major causes of HNSCC-related deaths are cervical node and distant metastasis. E-cadherin has a key role in epithelial intercellular adhesion and its downregulation is a hallmark of epithelial-mesenchymal transition (EMT), which is associated with invasion, metastasis, and poor prognosis. Epithelial-mesenchymal transition is the major mechanism responsible for mediating invasiveness and metastasis of epithelial cancers. Recently, we reported the role of E-cadherin transcriptional repressors in the inflammation-induced promotion of EMT in HNSCC, which is mediated by COX-2. These findings suggest that therapies targeting the cyclooxygenase pathway may diminish the propensity for tumour metastasis in HNSCC by blocking the PGE2-mediated induction of E-cadherin transcriptional repressors. METHODS: Herein, we evaluate the efficacy of the COX-2 inhibitor, apricoxib, in HNSCC cell lines. Apricoxib is effective in preventing tumour cell growth in three-dimensional, and anchorage-independent growth assays, as well as decreasing the capacity for tumour cell migration. RESULTS: Herein, we evaluate the efficacy of the COX-2 inhibitor, apricoxib, in HNSCC cell lines. Apricoxib is effective in preventing tumour cell growth in three-dimensional, and anchorage-independent growth assays, as well as decreasing the capacity for tumour cell migration. Treatment of HNSCC cells with apricoxib also causes greater upregulation of E-cadherin and Muc1 expression and downregulation of vimentin, as compared with celecoxib treatment. This has significant implications for targeted chemoprevention and anti-cancer therapy because E-cadherin expression has been implicated as a marker of sensitivity to epidermal growth factor receptor tyrosine kinase inhibitor and other therapies. We show for the first time the molecular mechanisms underlying the efficacy of apricoxib in HNSCC cells. CONCLUSION: In addition to reversing EMT via inhibition of COX-2, apricoxib upregulates 15-prostaglandin dehydrogenase and the prostaglandin transporter, thereby reducing the levels of active PGE2 by both suppressing its synthesis and increasing its catabolism. These findings have significant implications for metastasis and tumour progression in HNSCC.

Suicide gene therapy with Herpes simplex virus thymidine kinase and ganciclovir is enhanced with connexins to improve gap junctions and bystander effects.

Connexins are proteins that form gap junctions between cells in various mammalian tissues. Because of their role in intercellular communication, connexins are important in the bystander cell death seen in Herpes simplex virus-thymidine kinase (HSV-TK) gene therapy for brain tumors. A selective review of connexin transduction/transfection studies with particular emphasis to central nervous system tumor cells is presented. In addition, specific references to studies with cell types that demonstrate low gap junction intercellular communication are presented. Data are included with the HT-29 colorectal tumor cell line to support the concept that enhancing gap junction protein expression in otherwise low gap junction communicating HT-29 cells increases bystander cell death and reduces tumor burden beyond what might be expected from HSV-TK and ganciclovir (GCV) treatment alone. Maximum in vitro bystander cell death was always produced when GCV treated co-cultures of TK-transduced and non-TK-transduced HT-29 cell lines were also transduced with connexin-43. When connexin was present in only one group of cells in the co-culture, there was more bystander cell death observed with connexin transduced into the non-TK-transduced cells, rather than the TK-transduced cells. The data presented reinforces conclusions made from earlier findings from cell line mixing experiments in which the non-TK-transduced cell population determined the level of bystander cell death (Burrows et al., 2002).

Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype.

BACKGROUND: Survivin is a mammalian protein that carries a motif typical of the inhibitor of apoptosis (IAP)proteins, first identified in baculoviruses. Although baculoviral IAP proteins regulate cell death, the yeast Survivin homolog Bir1 is involved in cell division. To determine the function of Survivin in mammals, we analyzed the pattern of localization of Survivin protein during the cell cycle, and deleted its gene by homologous recombination in mice. RESULTS: In human cells, Survivin appeared first on centromeres bound to a novel para-polar axis during prophase/metaphase, relocated to the spindle midzone during anaphase/telophase, and disappeared at the end of telophase. In the mouse, Survivin was required for mitosis during development. Null embryos showed disrupted microtubule formation, became polyploid, and failed to survive beyond 4.5days post coitum. This phenotype, and the cell-cycle localization of Survivin, resembled closely those of INCENP. Because the yeast homolog of INCENP, Sli15, regulates the Aurora kinase homolog Ipl1p, and the yeast Survivin homolog Bir1 binds to Ndc10p, a substrate of Ipl1p, yeast Survivin, INCENP and Aurora homologs function in concert during cell division. CONCLUSIONS: In vertebrates, Survivin and INCENP have related roles in mitosis, coordinating events such as microtubule organization, cleavage-furrow formation and cytokinesis. Like their yeast homologs Bir1 and Sli15, they may also act together with the Aurora kinase.

Purified herpes simplex thymidine kinase retroviral particles. II. Influence of clinical parameters and bystander killing mechanisms.

High-titer, purified herpes simplex virus thymidine kinase (HSV-TK) retroviral particles, followed with intraperitoneal ganciclovir (GCV) were tested in Fischer rats bearing 1-week established 9L gliosarcomas. 9L cells were infused intracranially through a cannula on day 0, given intracranial infusions of HSV-TK retroviral particles on days 7-12, and given 5 or 10 daily doses of intraperitoneal GCV starting at day 14. Tumor volumetric studies performed on rat brains at day 26 after tumor infusion revealed significant differences in experimental groups receiving HSV-TK retroviral particles plus 10-day GCV or the 100% transduced 9L-TK group receiving GCV versus control groups treated with either buffer, HSV-TK vector, or either 5- or 10-day regimens of GCV alone. The duration of GCV administration and the volume of tumor burden influenced efficacy. Adjuvant dexamethasone did not significantly affect efficacy. Experiments in which 9L rechallenge of animals treated with 9L-TK/GCV or 9L tumors treated with HSV-TK vector/GCV indicated that a host immune response was involved in rejecting the rechallenge tumor. Outcome was dependent upon the site and size of the rechallenge inoculum. In vitro, bystander effects were significant but were especially marked when cell-to-cell contact was maintained. Cumulatively, the data indicate that both the bystander effect and the host immune response are responsible for the efficacy observed in the suicide gene therapy paradigm using purified HSV-TK retroviral particles and GCV to treat smaller tumor burden in rats with 9L gliosarcoma.

Establishment of parameters for optimal transduction efficiency and antitumor effects with purified high-titer HSV-TK retroviral vector in established solid tumors.

Suicide gene therapy using the herpes simplex thymidine kinase gene and ganciclovir is an attractive strategy for solid tumors. Early animal studies involved intratumoral injection of retroviral producer cells or unprocessed supernatant to generate an antitumor effect. Xenotransplantation of producer cells proved effective in several models, but the crude supernatants from the same cells were of insufficient titer to produce antitumor effects. We have developed new non-murine producer lines that yield replication-defective retroviral vectors encoding thymidine kinase at high titer which are then further purified and processed, resulting in pharmaceutical grade retroviral vectors with titers of up to 10(8) cfu/ml. Purified, high-titer retroviral preparations were injected directly into solid tumors in two syngeneic mouse tumor models. Significant antitumor responses and some cures were observed following systemic ganciclovir therapy. Assays using monoclonal antibodies to measure thymidine kinase protein expression at the single cell level in vitro and in vivo were developed so that therapeutic transgene expression could be quantified. Intralesional delivery resulted in transduction of over 20% of tumor cells in a protocol designed to maximize transduction on the basis of separate analyses of route, dosage, and schedule of vector administration. A consensus strategy evolved in which the combined effects of increased titer and a longer duration of retroviral vector administration interact to maximize transduction efficiency. These results indicate that purified high-titer retroviral vectors have the potential to transfer effective quantities of therapeutic genes into solid tumors in human subjects and highlight some pharmacologic factors that could be valuable in the design of clinical gene therapy protocols.

Purified herpes simplex thymidine kinase Retrovector particles. I. In vitro characterization, in situ transduction efficiency, and histopathological analyses of gene therapy-treated brain tumors.

Replication-defective, highly purified retroviral vectors (Retrovector), at titers of 10(8) colony forming units/mL, were prepared that conferred either beta-galactosidase or herpes simplex thymidine kinase (HSV-TK) activity. 9L gliosarcoma cells, transduced efficiently in vitro, were highly sensitive to ganciclovir (GCV). The mean frequency of in situ transduction, measured by flow cytometry of single-cell tumor suspensions isolated from rat brains, was 3.2 +/- 0.6%; similar assessments were made by staining of beta-galactosidase or by immunohistochemistry with anti-HSV-TK. In vitro HSV-TK-transduced and G418-selected 9L-TK gliosarcoma tumors treated with GCV were eradicated in approximately 53% of the animals (10/19) at day 26, however, 89% (17/19) histologically showed < 1% tumor volume. Histologic evaluation at day 26 of animals with established 9L tumors treated with intralesional injection of HSV-TK vector followed by GCV treatment showed that 29% (4/14) had no tumor; 50% (7/14) had < 1% tumor volume. Regression of tumors proceeded over the time since the complete rate was increased at day 60. Neither HSV-TK vector particles nor GCV alone altered the histological profile of 9L tumors, but substantial numbers of CD4+ and CD8+ lymphocytes infiltrated the tumors of animals treated with both. In cured animals, the former tumor bed contained cell debris, immune cells, and fibroblasts and was without damage to adjacent brain. The efficacy of suicide gene therapy for rat gliosarcoma using highly purified virion vectors approaches that of packaging cell lines.

Up-regulation of endoglin on vascular endothelial cells in human solid tumors: implications for diagnosis and therapy.

We have characterized a murine IgM monoclonal antibody, TEC-11, that recognizes endoglin and may be suitable for targeting cytotoxic agents to human tumor vasculature. TEC-11 strongly stains endothelial cells in a broad range of solid human tumors while staining endothelial cells in the majority of normal, healthy adult tissues relatively weakly. Human umbilical vein endothelial cells (HUVECs) in sections of the umbilical vein react weakly with TEC-11, whereas proliferating HUVECs in tissue culture react strongly and uniformly. HUVEC cultures grown to confluence and then rested contain two subpopulations having high and low levels of endoglin expression. Flow cytometry revealed that a significant proportion of cells with high endoglin expression are cycling, having markedly increased levels of cellular protein, RNA, and DNA by comparison to low endoglin-expressing cells, which appear to be noncycling. Taken together, the increased binding of TEC-11 to tumor vasculature and to dividing as opposed to noncycling HUVECs in vitro suggests that endoglin is an endothelial cell proliferation-associated marker. An immunotoxin [TEC-11.deglycosylated ricin A chain (dgA)] composed of TEC-11 and dgA was 3000-fold more potent at inhibiting protein synthesis in proliferating HUVEC cultures than in confluent cultures. The confluent cells were no more sensitive to TEC-11.dgA than they were to an isotype-matched immunotoxin of irrelevant specificity. These findings suggest that TEC-11.dgA might have therapeutic value in the treatment of solid tumors in humans by selectively killing dividing endothelial cells which are prevalent in such tumors.

Antibody-directed targeting of the vasculature of solid tumors.

An attractive strategy for the therapy of carcinomas and other solid tumors would be to target cytotoxic agents or host immune effectors to the endothelial cells of the tumor vasculature rather than to the tumor cells themselves. The key advantage of this approach is that the endothelial cells are freely accessible through the blood whereas the tumor cells are, for the most part, inaccessible. Also, endothelial cells are similar in different tumors, making it feasible to develop a single reagent for treating numerous types of cancer. In this chapter, we review progress in this "vascular targeting" approach, from the validation of the concept in a mouse model to the characterization of the TEC-11 antibody against endoglin, an endothelial cell proliferation marker that is upregulated on endothelial cells in miscellaneous human solid tumors. In addition, we review other tumor endothelial cell markers that are candidates for vascular targeting in man.

Vascular targeting--a new approach to the therapy of solid tumors.

An attractive approach to the therapy of solid tumors is to attack the endothelial cells of the tumor vascular bed rather than the tumor cells themselves, which circumvents the problem of poor penetration of tumor masses by monoclonal antibodies and other macromolecules. In this review, we will discuss the drawbacks of targeting solid tumors and the advantages of the 'vascular targeting' approach, describe the validation of the concept in a mouse model and summarize the properties of tumor endothelial cell markers, which are candidates for vascular targeting in humans.

Potent antitumor effects of an antitumor endothelial cell immunotoxin in a murine vascular targeting model.

Immunotoxins and other antibody-based therapeutic reagents have proved effective agonist lymphomas and leukemias, but results with carcinomas and other solid tumors have thus far been less impressive. A major reason for this difference is that macromolecules penetrate poorly and unevenly into solid tumors. A solution to this problem would be to attack the endothelial cells of the tumor vascular bed rather than the tumor cells themselves. We have developed a murine model of this vascular targeting approach where transfection of the tumor cells with a cytokine gene causes them to induce the expression of an experimental marker (MHC Class II) on tumor endothelium. In this report we show that an anti-Class II-deglycosylated Ricin A-chain immunotoxin kills IFN-gamma-activated endothelial cells in culture and, when injected into tumor-bearing Balb/c nude mice, causes complete thrombosis of the tumor vasculature, widespread infarction, and dramatic regressions of large solid tumors. These findings suggest that immunoconjugates prepared with recently described antibodies against human tumor endothelium could provide a broad-based therapy for variety of solid cancers in humans.

Cytotoxicity of a novel anti-ICAM-1 immunotoxin on human myeloma cell lines.

We have generated a murine monoclonal antibody (UV3) which recognizes an epitope on ICAM-1 expressed on myeloma cells. By flow cytometric analysis, the epitope on ICAM-1 recognized by this antibody is strongly expressed on human myeloma cells, pre-B leukemia cells and Burkitt's lymphoma cell lines. Most human T cell lines are weakly positive. The antibody does not react with red blood cells, polymorphonuclear leukocytes (PMNs) or resting B lymphocytes from normal donors, and reacts very weakly with resting T cells. Immunohistochemical assays indicate that the antibody does not react with normal liver, kidney, heart, brain, thymus or lung. An immunotoxin (IT) was prepared by coupling UV3 to deglycosylated ricin A-chain (dgA). In protein synthesis inhibition assays it was highly cytotoxic to the human myeloma cell lines HS-SULTAN (IC50 = 1 x 10(-11)M) and ARH-77 (IC50 = 9 x 10(-11)M), but not to cell lines of T cell lineage or most cell lines of the B lineage. Our results suggest that the UV3-dgA may have therapeutic potential for the treatment of human multiple myeloma.

Eradication of large solid tumors in mice with an immunotoxin directed against tumor vasculature.

Antibody-based therapy of solid tumors has met with limited success, chiefly because solid tumors are relatively impermeable to macromolecules. This problem could be circumvented by attacking the readily accessible endothelial cells of the tumor vascular bed. We have developed a model to test this "vascular targeting" approach in which cytokine gene transfection of the tumor cells causes them to induce an experimental marker selectively on tumor vascular endothelium. An anti-tumor endothelial cell immunotoxin caused complete occlusion of the tumor vasculature and dramatic regressions of large solid tumors. By contrast, a conventional anti-tumor cell immunotoxin of equivalent in vitro potency produced only minor, transient antitumor effects but, when combined, the two immunotoxins induced permanent complete remissions in over half of the animals. These experiments indicate that immunotoxins directed against recently described markers on vascular endothelial cells in human tumors could provide a general treatment for solid tumors in humans.

A murine model for antibody-directed targeting of vascular endothelial cells in solid tumors.

An attractive approach to the therapy of solid tumors would be to target cytotoxic agents or coagulants to the vasculature of the tumor rather than to the tumor cells themselves. This strategy has 3 advantages: (a) it should be applicable to many types of solid tumors because all require a blood supply for survival and growth; (b) the target endothelial cells are directly accessible through the blood and are normal cells, making the outgrowth of resistant mutants unlikely; and (c) there is an in-built amplification mechanism because thousands of tumor cells are reliant on each capillary for nutrients and oxygen. Despite its theoretical attractions, the approach of tumor vascular targeting has not been testable because antibodies that recognize tumor vascular endothelial cell antigens with adequate specificity are currently not available. In this study, we developed a model system in which to investigate the antibody-directed targeting of vascular endothelial cells in solid tumors in mice. A neuroblastoma transfected with the mouse interferon-gamma gene, C1300(Mu gamma), was grown in antibiotic-treated BALB/c nude mice. The interferon-gamma secreted by the tumor induces the expression of major histocompatibility complex Class II antigens on the tumor vascular endothelium. Class II antigens are absent from the vasculature of normal tissues, although they are present on B-lymphocytes, cells of monocyte/macrophage lineage, and some epithelial cells. Anti-Class II antibody administered i.v. strongly stains the tumor vasculature, whereas an antitumor antibody directed against a major histocompatibility complex Class I antigen of the tumor allograft produces classical perivascular tumor cell staining. This model should enable the theoretical superiority of tumor vascular targeting over conventional tumor cell targeting to be tested.

Influence of tumor-derived interleukin 1 on melanoma-endothelial cell interactions in vitro.

Human melanoma cell lines that express high constitutive levels of the metastasis-associated marker intercellular adhesion molecule 1 (ICAM-1) were found to secrete interleukin 1 (IL-1) in vitro. Experiments with neutralizing antibodies showed that this cytokine was responsible for their expression of ICAM-1 but not that of two other progression/metastasis markers, Muc-18 and Gp IIb/IIIa. The IL-1 present in melanoma-conditioned medium induced the expression of vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 on human umbilical vein endothelial cells (ECs) in culture and increased the rate at which melanoma cells and ECs adhered to each other. IL-1-producing melanoma lines adhered significantly more rapidly to ECs than did non-IL-1-producing lines, and this enhancement was reduced by prior incubation of the melanoma cells with neutralizing anti-IL-1 antibodies. Similarly, endothelial cells treated with conditioned medium from IL-1-producing melanoma lines adhered significantly more rapidly to melanoma cells than did ECs treated with medium from non-IL-1-producing melanoma lines, and this enhancement was abolished by addition of anti-IL-1 antibodies to EC cultures in conditioned medium. Blocking antibodies to endothelial vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule 1, and ICAM-1 failed to inhibit melanoma-EC adhesion, but an antibody to tumor cell GpIIb/IIIa did block adhesion by up to 44%. The ability to secrete IL-1 could increase the metastatic potential of melanoma cells by stimulating tumor cell-EC adhesion.

The rat placenta expresses paternal class I major histocompatibility antigens.

The expression of paternally inherited class I MHC antigens on the placental trophoblast of the rat has been investigated using a mouse anti-rat monoclonal antibody (MN4-91-6) in an indirect immunoperoxidase labelling assay on cryostat sections. Strong specific staining was obtained on the spongy zone trophoblast of the mature placenta from DA male (RT1a) X PVG female (RT1c) matings. In marked contrast, no staining was observed on the labyrinthine trophoblast nor on the trophoblastic giant cells at any stage of gestation from 8 to 19 days post-coitum. None of the trophoblastic cell populations at any stage of gestation were reactive with an anti-class II monoclonal antibody. Class I positive endovascular cytotrophoblast cells were present in the maternal arterial sinusoids of the decidua. These findings imply that maternal immunoregulatory mechanisms must be essential for the survival of the placenta and fetus.