James Warren

Can Homeopathic Treatment Slow Prostate Cancer Growth

Background: Homeopathy is a complementary medicine widely used around the world. Despite extensive use of homeopathy for cancer and other serious conditions with reported success, clinical and laboratory research has been equivocal, and no rigorous research has been done on cancer. In 1999, the US National Cancer Institute evaluated the effects of homeopathic treatment of cancer from a clinic in India and has released a request for protocols to conduct further research into this treatment. Therefore, the authors conducted a series of carefully controlled laboratory studies evaluating the effects of commonly used homeopathic remedies in cell and animal models of prostate cancer. Study Design: One hundred male Copenhagen rats were randomly assigned to either treatment or control groups after inoculation with prostate tumor cells. Methods: Prostate tumor cells DU-145, LNCaP, and MAT-LyLu were exposed to 5 homeopathic remedies. Male Copenhagen rats were injected with MAT-LyLu cells and exposed to the same homeopathic remedies for 5 weeks. In vitro outcomes included tumor cell viability and apoptosis gene expression. In vivo outcomes included tumor incidence, volume, weight, total mortality, proliferating cell nuclear antigen (PCNA) expression, apoptotic cell death (terminal deoxynucleotidyl transferase mediated d-uridine triphosphate nick end labeling), and gene expression (rAPO-multiprobe). Results: There were no effects on cell viability or gene expression in 3 prostate cell lines with any remedies at any exposure time. There was a 23% reduction in tumor incidence (P < .0001), and for animals with tumors, there was a 38% reduction in tumor volume in homeopathy-treated animals versus controls (P < .02). At time of killing, experimental animals with tumors had a 13% lower average tumor weight (P < .05). Tumors in these treated animals showed a 19% increase in apoptotic cell death (P < .05) and reduced PCNA-positive cells. Conclusions: The findings indicate that selected homeopathic remedies for the present study have no direct cellular anticancer effects but appear to significantly slow the progression of cancer and reduce cancer incidence and mortality in Copenhagen rats injected with MAT-LyLu prostate cancer cells.

N-glycosylation is required for binding of murine pregnancy-specific glycoproteins 17 and 19 to the receptor CD9.

Murine pregnancy‐specific glycoproteins (PSGs) are encoded by 17 different genes. Different family members have different expression levels at different stages of embryonic development. It is currently unknown whether all members of this family of placentally secreted proteins have the same function and bind to the same receptor. Furthermore, the requirement of post‐translational modifications for the activity of these highly glycosylated proteins remains undetermined.

Effect of homeopathic treatment on gene expression in Copenhagen rat tumor tissues.

Background: Increasing evidence suggests that the inability to undergo apoptosis is an important factor in the development and progression of prostate cancer. Agents that induce apoptosis may inhibit tumor growth and provide therapeutic benefit. In a recent study, the authors found that certain homeopathic treatments produced anticancer effects in an animal model. In this study, the authors examined the immunomodulating and apoptotic effects of these remedies. Materials and Methods: The authors investigated the effect of a homeopathic treatment regimen containing Conium maculatum, Sabal serrulata, Thuja occidentalis, and a MAT-LyLu Carcinosin nosode on the expression of cytokines and genes that regulate apoptosis. This was assessed in prostate cancer tissues, extracted from animals responsive to these drugs, using ribonuclease protection assay or reverse transcription polymerase chain reaction. Results: There were no significant changes in mRNA levels of the apoptotic genes bax, bcl-2, bcl-x, caspase-1, caspase-2, caspase-3, Fas, FasL, or the cytokines interleukin (IL)–1α, IL-1β, tumor necrosis factor (TNF)–β, IL-3, IL-4, IL-5, IL-6, IL-10, TNF-α, IL-2, and interferon-γ in prostate tumor and lung metastasis after treatment with homeopathic medicines. Conclusions: This study indicates that treatment with the highly diluted homeopathic remedies does not alter the gene expression in primary prostate tumors or in lung metastasis. The therapeutic effect of homeopathic treatments observed in the in vivo experiments cannot be explained by mechanisms based on distinct alterations in gene expression related to apoptosis or cytokines. Future research should explore subtle modulations in the expression of multiple genes in different biological pathways.

Characterization of receptors for murine pregnancy specific glycoproteins 17 and 23

In primates and rodents, trophoblast cells synthesize and secrete into the maternal circulation a family of proteins known as pregnancy specific glycoproteins (PSG). The current study was undertaken to characterize the receptor for two members of the murine PSG family, PSG17 and PSG23. Binding of recombinant PSG17 and PSG23 to CHO-K1 and L929 cells and their derived mutants was performed to determine whether these proteins bound to cell surface proteoglycans. We also examined binding of these proteins to cells transfected with syndecans and glypican-1 by flow cytometry. The interaction with glycosaminoglycans was confirmed in solid phase assays. Our results show that PSG17 binds to CD9 and to cell surface proteoglycans while PSG23 binds only to the latter. We found that the amino acids involved in CD9 binding reside in the region of highest divergence between the N1-domains of murine PSGs. For both proteins, the N-terminal domain (designated as N1) is sufficient for binding to cells and the ability to bind cell surface proteoglycans is affected by the cell line employed to generate the recombinant proteins. We conclude that while substantially different at the amino acid level, some murine PSGs share with human PSG1 the ability to bind to cell surface proteoglycans and that at least one PSG binds to more than one type of molecule on the cell surface.

ORIGINAL ARTICLE: N-glycosylation is Required for Binding of Murine Pregnancy-Specific Glycoproteins 17 and 19 to the Receptor CD9

Murine pregnancy‐specific glycoproteins (PSGs) are encoded by 17 different genes. Different family members have different expression levels at different stages of embryonic development. It is currently unknown whether all members of this family of placentally secreted proteins have the same function and bind to the same receptor. Furthermore, the requirement of post‐translational modifications for the activity of these highly glycosylated proteins remains undetermined.

Early Expression of Pregnancy-Specific Glycoprotein 22 (PSG22) by Trophoblast Cells Modulates Angiogenesis in Mice

Mouse and human pregnancy-specific glycoproteins (PSG) are known to exert immunomodulatory functions during pregnancy by inducing maternal leukocytes to secrete anti-inflammatory cytokines that promote a tolerogenic decidual microenvironment. Many such anti-inflammatory mediators also function as proangiogenic factors, which, along with the reported association of murine PSG with the uterine vasculature, suggest that PSG may contribute to the vascular adaptations necessary for successful implantation and placental development. We observed that PSG22 is strongly expressed around the embryonic crypt on Gestation Day 5.5, indicating that trophoblast giant cells are the main source of PSG22 during the early stages of pregnancy. PSG22 treatment up-regulated the secretion of transforming growth factor beta 1 and vascular endothelial growth factor A (VEGFA) in murine macrophages, uterine dendritic cells, and natural killer cells. A possible role of PSGs in uteroplacental angiogenesis is further supported by the finding that incubation of endothelial cells with PSG22 resulted in the formation of tubes in the presence and absence of VEGFA. We determined that PSG22, like human PSG1 and murine PSG17 and PSG23, binds to the heparan sulfate chains in syndecans. Therefore, our findings indicate that despite the independent evolution and expansion of human and rodent PSG, members in both families have conserved functions that include their ability to induce anti-inflammatory cytokines and proangiogenic factors as well as to induce the formation of capillary structures by endothelial cells. In summary, our results indicate that PSG22, the most abundant PSG expressed during mouse early pregnancy, is likely a major contributor to the establishment of a successful pregnancy.

Induction and Activation of Latent Transforming Growth Factor-β1 Are Carried out by Two Distinct Domains of Pregnancy-specific Glycoprotein 1 (PSG1)

Background: PSG1 is secreted by placental cells and regulates TGF-β1. Results: The B2 domain of PSG1 activates latent TGF-β1, whereas macrophage induction of latent TGF-β1 relies on the LYHY amino acid sequence within the N-terminal domain. Conclusion: PSG1 exerts bifunctional activity through two distinct domains, resulting in an increase in active TGF-β1. Significance: These data describe a mechanism by which PSG1 modulates TGF-β1 during pregnancy. Pregnancy-specific glycoproteins (PSGs) are a family of Ig-like proteins secreted by specialized placental cells. The PSG1 structure is composed of a single Ig variable region-like N-terminal domain and three Ig constant region-like domains termed A1, A2, and B2. Members of the human and murine PSG family have been shown to induce anti-inflammatory cytokines from monocytes and macrophages and to stimulate angiogenesis. We recently showed that recombinant forms of PSG1 (PSG1-Fc and PSG1-His) and PSG1 purified from the serum of pregnant women are associated with the immunoregulatory cytokine TGF-β1 and activated latent TGF-β1. Here, we sought to examine the requirement of specific PSG1 domains in the activation of latent TGF-β1. Plasmon surface resonance studies showed that PSG1 directly bound to the small latent complex and to the latency-associated peptide of TGF-β1 and that this binding was mediated through the B2 domain. Furthermore, the B2 domain alone was sufficient for activating the small latent complex. In separate experiments, we found that the PSG1-mediated induction of TGF-β1 secretion in macrophages was dependent on the N-terminal domain. Mutagenesis analysis revealed that four amino acids (LYHY) of the CC′ loop of the N-terminal domain were required for induction of latent TGF-β1 secretion. Together, our results show that two distinct domains of PSG1 are involved in the regulation of TGF-β1 and provide a mechanistic framework for how PSGs modulate the immunoregulatory environment at the maternal-fetal interface for successful pregnancy outcome.

Activation of latent transforming growth factor-β1, a conserved function for pregnancy-specific beta 1-glycoproteins

STUDY QUESTION Do all 10 human pregnancy-specific beta 1-glycoproteins (PSGs) and murine PSG23 activate latent transforming growth factor-β1 (TGF-β1)? SUMMARY ANSWER All human PSGs and murine PSG23 activated latent TGF-β1. WHAT IS KNOWN ALREADY Two of the 10 members of the PSG1 family, PSG1 and PSG9, were previously shown to activate the soluble small latent complex of TGF-β1, a cytokine with potent immune suppressive functions. STUDY DESIGN, SIZE, DURATION Recombinant PSGs were generated and tested for their ability to activate the small latent complex of TGF-β1 in a cell-free ELISA-based assay and in a bioassay. In addition, we tested the ability of PSG1 and PSG4 to activate latent TGF-β bound to the extracellular matrix (ECM) or on the membranes of the Jurkat human T-cell line. PARTICIPANTS/MATERIALS, SETTING, METHODS Recombinant PSGs were generated by transient transfection and purified with a His-Trap column followed by gel filtration chromatography. The purified PSGs were compared to vehicle (PBS) used as control for their ability to activate the small latent complex of TGF-β1. The concentration of active TGF-β was measured in an ELISA using the TGF-β receptor II as capture and a bioassay using transformed mink epithelial cells that express luciferase in response to active TGF-β. The specificity of the signal was confirmed using a TGF-β receptor inhibitor. We also measured the binding kinetics of some human PSGs for the latent-associated peptide (LAP) of TGF-β using surface plasmon resonance and determined whether PSG1 and PSG4 could activate the large latent complex of TGF-β1 bound to the ECM and latent TGF-β1 bound to the cell membrane. All experiments were performed in triplicate wells and repeated three times. MAIN RESULTS AND THE ROLE OF CHANCE All human PSGs activated the small latent complex of TGF-β1 (P < 0.05 vs. control) and showed similar affinities (KD) for LAP. Despite the lack of sequence conservation with its human counterparts, the ability to activate latent TGF-β1 was shared by a member of the murine PSG family. We found that PSG1 and PSG4 activated the latent TGF-β stored in the ECM (P < 0.01) but did not activate latent TGF-β1 bound to glycoprotein A repetitions predominant (GARP) on the surface of Jurkat T cells. LIMITATIONS, REASONS FOR CAUTION The affinity of the interaction of LAP and PSGs was calculated using recombinant proteins, which may differ from the native proteins in their post-translational modifications. We also utilized a truncated form of murine PSG23 rather than the full-length protein. For the studies testing the ability of PSGs to activate membrane-bound TGF-β1, we utilized the T-cell line Jurkat and Jurkat cells expressing GARP rather than primary T regulatory cells. All the studies were performed in vitro. WIDER IMPLICATIONS OF THE FINDINGS Here, we show that all human PSGs activate TGF-β1 and that this function is conserved in at least one member of the rodent PSG family. In vivo PSGs could potentially increase the availability of active TGF-β1 from the soluble and matrix-bound latent forms of the cytokine contributing to the establishment of a tolerogenic environment during pregnancy. LARGE-SCALE DATA None. STUDY FUNDING/COMPETING INTEREST(S) The research was supported by a grant from the Collaborative Health Initiative Research Program (CHIRP). No conflicts of interests are declared by the authors.

ORIGINAL ARTICLE: N-glycosylation is Required for Binding of Murine Pregnancy-Specific Glycoproteins 17 and 19 to the Receptor CD9: MURINE PSGS-CD9 INTERACTION

Murine pregnancy‐specific glycoproteins (PSGs) are encoded by 17 different genes. Different family members have different expression levels at different stages of embryonic development. It is currently unknown whether all members of this family of placentally secreted proteins have the same function and bind to the same receptor. Furthermore, the requirement of post‐translational modifications for the activity of these highly glycosylated proteins remains undetermined.