Ascites Increases Expression/Function of Multidrug Resistance Proteins in Ovarian Cancer Cells.

Chemotherapy resistance is the major reason for the failure of ovarian cancer treatment. One mechanism behind chemo-resistance involves the upregulation of multidrug resistance (MDR) genes (ABC transporters) that effectively transport (efflux) drugs out of the tumor cells. As a common symptom in stage III/IV ovarian cancer patients, ascites is associated with cancer progression. However, whether ascites drives multidrug resistance in ovarian cancer cells awaits elucidation. Here, we demonstrate that when cultured with ascites derived from ovarian cancer-bearing mice, a murine ovarian cancer cell line became less sensitive to paclitaxel, a first line chemotherapeutic agent for ovarian cancer patients. Moreover, incubation of murine ovarian cancer cells in vitro with ascites drives efflux function in these cells. Functional studies show ascites-driven efflux is suppressible by specific inhibitors of either of two ABC transporters [Multidrug Related Protein (MRP1); Breast Cancer Related Protein (BCRP)]. To demonstrate relevance of our findings to ovarian cancer patients, we studied relative efflux in human ovarian cancer cells obtained from either patient ascites or from primary tumor. Immortalized cell lines developed from human ascites show increased susceptibility to efflux inhibitors (MRP1, BCRP) compared to a cell line derived from a primary ovarian cancer, suggesting an association between ascites and efflux function in human ovarian cancer. Efflux in ascites-derived human ovarian cancer cells is associated with increased expression of ABC transporters compared to that in primary tumor-derived human ovarian cancer cells. Collectively, our findings identify a novel activity for ascites in promoting ovarian cancer multidrug resistance.

LMP-420, a small molecular inhibitor of TNF-α, prolongs islet allograft survival by induction of suppressor of cytokine signaling-1: synergistic effect with cyclosporin-A.

Inflammatory insults following islet transplantation (ITx) hinders engraftment and long-term function of the transplanted (Tx) islets. Using a murine model of ITx, we determined the role of LMP-420, a novel TNF-α inhibitor, both individually and in combination with the immunosuppressant cyclosporine A (CSA) in islet engraftment and survival. Diabetic C57BL/6 mice were Tx with 500 BALB/c islets under the kidney capsule. Four cohorts were used: LMP-420 only, CSA only, combination of LMP-420 and CSA (LMP+CSA), and control (n = 12 per cohort). Serial monitoring of blood glucose levels revealed that LMP+CSA (35 ± 5 days) prolonged stable blood insulin levels compared to control (6 ± 4 days). Immunohistology demonstrated that coadministration (LMP+CSA) results in a significant decrease in CD8(+) T-cell infiltration (LMP+CSA: 31 ± 18 vs. control: 224 ± 51 cells, p < 0.001). Serum cytokine analysis revealed that LMP-420 administration resulted in an increase in the anti-inflammatory cytokine IL-10 (2.5-fold), and a decrease in TNF-α (threefold) with no change in IL-2. However, coadministration resulted in a marked decrease in both IL-2 and TNF-α (threefold) along with increase in IL-10 (threefold). Coadministration also demonstrated increase of antiapoptotic SOCS-1 and Mn-SOD expression and significant reduction of donor-specific antibodies (p < 0.005). In conclusion, LMP-420 administration with CSA results in the upregulation of anti-inflammatory and antiapoptotic mechanisms which facilitate islet allograft engraftment and survival.

Anti-inflammatory and vasoprotective activity of a retroviral-derived peptide, homologous to human endogenous retroviruses: endothelial cell effects.

Malignant and inflammatory tissues sometimes express endogenous retroviruses or their proteins. A highly-conserved sequence from retroviral transmembrane (TM) proteins, termed the "immunosuppressive domain (ID)", is associated with inhibition of immune and inflammatory functions. An octadecapeptide (MN10021) from the ID of retroviral TM protein p15E inhibits in vitro release of pro-inflammatory cytokines and increases synthesis of anti-inflammatory IL-10. We sought to determine if MN10021 has significant in vivo effects. MN10021, prepared by solid-phase synthesis, was dimerized through a naturally-occurring, carboxy-terminal cysteine. In vivo anti-inflammatory activity was determined using a murine model of sodium periodate (NaIO(4))-induced peritonitis. In vivo vasoprotective effects were determined using: (1) a carrageenan-induced model of disseminated intravascular coagulation (DIC) in mice; (2) a reverse passive Arthus model in guinea pigs; and (3) vasoregulatory effects in spontaneously hypertensive rats (SHR). In vitro studies included: (1) binding/uptake of MN10021 using human monocytes, cultured fibroblasts, and vascular endothelial cells (VEC); (2) gene expression by RT-PCR of MN10021-treated VEC; and (3) apoptosis of MN10021-treated VEC exposed to staurosporine or TNF-α. One-tenth nmol MN10021 inhibits 50 percent of the inflammatory response in the mouse peritonitis model. Furthermore, 73 nmol MN10021 completely protects mice in a lethal model of carrageenan-induced DIC and inhibits vascular leak in both the mouse DIC model and a guinea pig reverse passive Arthus reaction. MN10021 binds to and is taken up in a specific manner by both human monocytes and VEC but not by cultured human fibroblasts. Surprisingly, orally-administered MN10021 lowers blood pressure in SHR rats by 10-15% within 1 h suggesting a direct or indirect effect on the vascular endothelium. MN10021 and derived octapeptides induce iNOS (inducible nitric oxide synthase) mRNA in VEC and nitrate in VEC cell culture supernatants and protect VEC from induced apoptosis or necrosis. However, pretreatment of VEC with nitro-L-arginine methyl ester (L-NAME), while inhibiting the release of nitrate, does not block the anti-apoptotic effect of MN10021 and derived octapeptides suggesting that their potent vasoprotective and anti-inflammatory activity is not nitric oxide dependent.

Antigen delivery by alpha(2)-macroglobulin enhances the cytotoxic T lymphocyte response.

alpha(2)M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen-delivery vehicle, alpha(2)M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that alpha(2)M* encapsulation may also enhance antigen-specific CTL immunity. In this study, we demonstrate that alpha(2)M*-delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA(257-264) (SIINFEKL) demonstrated up to 25-fold greater IFN-gamma and IL-2 secretion when treated in vitro with alpha(2)M*-OVA compared with soluble OVA. The frequency of IFN-gamma-producing cells was increased approximately 15-fold, as measured by ELISPOT. Expansion of the OVA-specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA-specific cell-mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by alpha(2)M*-OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with alpha(2)M*-OVA. These alpha(2)M*-OVA-immunized mice demonstrated increased protection against a s.c.-implanted, OVA-expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that alpha(2)M*-mediated antigen delivery elicits specific CTL responses suggests the cross-presentation of antigen onto MHC class I. These results support alpha(2)M* as an effective antigen-delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.

SP-A preserves airway homeostasis during Mycoplasma pneumoniae infection in mice.

The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study, we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation, and cellular infiltration, were much greater in Mp infected SP-A(-/-) mice than wild-type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A(-/-) mice. Both Mp-induced biologic and physiologic changes were attenuated by pharmacologic inhibition of TNF-alpha. Our findings demonstrate that SP-A is vital to preserving lung homeostasis and host defense to this clinically relevant strain of Mp by curtailing inflammatory cell recruitment and limiting an overzealous TNF-alpha response.

Probing the stability of native and activated forms of alpha2-macroglobulin.

alpha2-Macroglobulin (alpha2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an approximately 40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in alpha2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in alpha2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of alpha2M complexes.

Alpha 2-macroglobulin binds CpG oligodeoxynucleotides and enhances their immunostimulatory properties by a receptor-dependent mechanism.

CpG oligodeoxynucleotides (ODN) stimulate the immune system and are under evaluation as treatments and vaccine adjuvants for infectious diseases, cancer, and immune system disorders. Although they have shown promising results in numerous clinical trials, the ultimate use of CpG ODN-based therapeutics may hinge on improved pharmacokinetics and reduced systemic side-effects. CpG ODN efficacy and potency might be enhanced greatly by packaging them into particles that protect them from degradation and specifically target them for uptake by immune-competent cells. The plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) binds numerous biologically active macromolecules, including cytokines, chemokines, and growth factors, and can modulate their activity. Molecules bound to alpha 2M are protected from interactions with neighboring macromolecules and are targeted for receptor-mediated uptake by immune-competent cells. Here, we report that activated alpha 2M (alpha 2M*) binds CpG ODN and enhances their immunostimulatory properties significantly. Murine macrophages treated with alpha 2M*-ODN complexes respond more rapidly and produce a greater cytokine response than induced by free CpG ODN. Using human PBMC, alpha 2M*-ODN complexes exhibit fourfold enhanced potency and 15-fold greater efficacy for stimulating production of inflammatory cytokines. alpha 2M* targets delivery of CpG ODN specifically to immune-competent cells, which endocytose the complexes sixfold more rapidly than free CpG ODN. CpG ODN bound to alpha 2M* are also protected from degradation by nucleases. This novel targeting technology may improve CpG ODN-based therapeutics by increasing efficacy at reduced doses, thus reducing side-effects and cost.

Incorporation of low molecular weight molecules into alpha(2)-macroglobulin by nucleophilic exchange.

alpha(2)-Macroglobulin (alpha(2)M) is a proteinase inhibitor that functions by a trapping mechanism which has been exploited such that the receptor-recognized, activated form (alpha(2)M( *)) can be employed to target antigens to antigen-presenting cells. Another potential use of alpha(2)M( *) is as a drug delivery system. In this study we demonstrate that guanosine triphosphate, labeled with Texas red (GTP-TR) formed complexes with alpha(2)M( *) following activation by proteolytic or non-proteolytic reactions. Optimal incorporation occurred with 20 microM GTP-TR, pH 8.0 for 5h at 50 degrees C. NaCl concentration (100 or 200 mM) had little effect on incorporation at this pH or temperature, but was significant at sub-optimum temperature and pH values. Maximum incorporation was 1.2 mol GTP-TR/mol alpha(2)M( *). PAGE showed that 70-90% of the GTP-TR is bound in a SDS/2-mercaptoethanol resistant manner. Guanosine, adenosine, and imidazole competed with GTP-TR to form complexes with alpha(2)M( *).

LMP-420, a small-molecule inhibitor of TNF-alpha, reduces replication of HIV-1 and Mycobacterium tuberculosis in human cells.

BACKGROUND: Co-infections of human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (M. Tb) are steadily increasing and represent a major health crisis in many developing countries. Both pathogens individually stimulate tumor necrosis factor-alpha (TNF) release from infected cells and TNF, in turn, enhances the replication of each. A recent report on a Phase I clinical trial suggested that etanercept (soluble TNF receptor) might be beneficial in treating HIV/M. Tb co-infected patients. We sought to determine if a small molecule inhibitor of TNF synthesis and activity could block replication of either organism and thus be a potential adjunct to existing drugs targeting these agents. RESULTS: LMP-420, a novel anti-inflammatory agent that inhibits TNF, was tested for HIV-1 inhibition both alone and in combination with AZT (3' -azido-3-deoxythymidine). LMP-420 alone was tested against M. Tb. HIV-1 infected human peripheral blood mononuclear cells (PBMC) or M. Tb-infected human alveolar macrophages (AM) were treated with a single dose of LMP-420 and viral or bacterial replication determined after 7 or 5 days respectively. Viral replication was determined from supernatant p24 levels measured by ELISA. M. Tb replication was determined by bacterial culture of macrophage lysates. LMP-420 alone inhibited HIV replication over 7 days with an IC50 of approximately 300 nM. Combination of LMP-420 with AZT doubled the level of HIV inhibition observed with AZT alone. LMP-420 alone inhibited the replication of virulent M. Tb by >80%, more than that observed with anti-TNF antibody alone. CONCLUSION: Inhibition of TNF with inexpensive, small-molecule, orally-active drugs may represent a useful strategy for enhancing the activity of currently-available antiviral and anti-M. Tb agents, particularly in those areas where co-infections with these pathogens act to synergistically enhance each other.

The properties of rabbit alpha1-macroglobulin upon activation are distinct from those of rabbit and human alpha2-macroglobulins.

We have characterized native and activated forms of rabbit alpha1M and compared them to rabbit and human alpha2M. Similar to human alpha2M, rabbit alpha1M is a tetramer associated via disulfide bonds and non-covalent interactions that exhibits autolysis into two fragments when heated. Like human alpha2M, rabbit alpha1M is cleaved by trypsin at one site; however, rabbit alpha1M shares characteristics with rabbit alpha2M that are different from the properties of human alpha2M. Amine or trypsin treatment of rabbit alpha-macroglobulins does not result in a significant conformational change or cleavage of four thiolester bonds. Full thiolester cleavage is only observed for rabbit alpha1M after exposure to both trypsin and a small amine. Additionally, amine-treated rabbit alpha-macroglobulins retain trypsin inhibitory potential and do not fully shield bound proteinases. Methylamine and trypsin treatment of rabbit alpha1M results in two dissimilar conformations that display differing exposure of the receptor-recognition site. While ammonia- and methylamine-modified rabbit alpha1M bind to macrophages with similar affinity to that of human alpha2M, trypsin-treated rabbit alpha1M exhibits dramatically lower affinity. This suggests that rabbit alpha1M may not play the same proteinase-inhibiting physiological role as human alpha2M.

Inhibition of endothelial activation: a new way to treat cerebral malaria?

BACKGROUND: Malaria is still a major public health problem, partly because the pathogenesis of its major complication, cerebral malaria (CM), remains incompletely understood. However tumor necrosis factor (TNF) is thought to play a key role in the development of this neurological syndrome, as well as lymphotoxin alpha (LT). METHODS AND FINDINGS: Using an in vitro model of CM based on human brain-derived endothelial cells (HBEC-5i), we demonstrate the anti-inflammatory effect of LMP-420, a 2-NH2-6-Cl-9-[(5-dihydroxyboryl)-pentyl] purine that is a transcriptional inhibitor of TNF. When added before or concomitantly to TNF, LMP-420 inhibits endothelial cell (EC) activation, i.e., the up-regulation of both ICAM-1 and VCAM-1 on HBEC-5i surfaces. Subsequently, LMP-420 abolishes the cytoadherence of ICAM-1-specific Plasmodium falciparum-parasitized red blood cells on these EC. Identical but weaker effects are observed when LMP-420 is added with LT. LMP-420 also causes a dramatic reduction of HBEC-5i vesiculation induced by TNF or LT stimulation, as assessed by microparticle release. CONCLUSION: These data provide evidence for a strong in vitro anti-inflammatory effect of LMP-420 and suggest that targeting host cell pathogenic mechanisms might provide a new therapeutic approach to improving the outcome of CM patients.

A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

Increased immunogenicity of HIV envelope subunit complexed with alpha2-macroglobulin when combined with monophosphoryl lipid A and GM-CSF.

Critical to the success of HIV-1 subunit vaccines is the development of strategies to augment vaccine immunogenicity. Successful adjuvants must not only improve immunogenicity above current adjuvant levels, but must also decrease the dose of immunogen required for optimal immunogenicity. We have evaluated activated alpha2-macroglobulin (alpha2M*) and a squalene-based stable emulsion containing monophosphoryl lipid A (MPL-SE) with granulocyte-macrophage colony stimulating factor (GM-CSF) as adjuvants to enhance the immunogencity of candidate HIV immunogens. Balb/c mice were subcutaneously immunized on days 0, 14 and 28 with 100-0.1 microg of HIV-1 envelope gp120 C4-V3 immunogens from either HIV IIIB (C4-V3(IIIB)) or SHIV 89.6P (C4-V3(89.6P)). Immunogens were tested covalently coupled to alpha2M*, formulated with MPL-SE/GM-CSF, or as a combination of both. Using CFA/IFA, only 50 and 100 microg, but not lower doses of C4-V3(IIIB) peptides, induced antibody responses. In contrast, peak antibody responses were detected in mice immunized with 10 microg of C4-V3 peptide coupled to alpha2M* (alpha2M*-peptide). Similar to CFA/IFA, MPL-SE/GM-CSF induced optimal antibody responses at 50 and 100 microg of C4-V3 immunogen. However, the combination of MPL-SE/GM-CSF with alpha2M*-C4-V3 peptide decreased the dose of C4-V3 required for optimal response to 5 microg for C4-V3(IIIB), and to 0.1 microg for C4-V3(89.6P). Taken together, HIV envelope gp120 C4-V3 peptides covalently complexed with alpha2M* and formulated with MPL-SE/GM-CSF resulted in a subunit HIV immunogen capable of inducing anti-HIV envelope antibody responses at doses up to 100-fold less than those needed with CFA/IFA or MPL-SE/GM-CSF alone.