SDH, a novel diarylheptane compound, alleviates dextran sulfate sodium (DSS)-induced colitis by reducing Th1/Th2/Th17 induction and regulating the gut microbiota in mice.

Ulcerative colitis, a chronic inflammatory condition affecting the rectum and colon to varying degrees, is linked to a dysregulated immune response and the microbiota. Sodium (aS,9R)-3-hydroxy-16,17-dimethoxy-15-oxidotricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6(19),14,16-hexene-9-yl sulfate hydrate (SDH) emerges as a novel diarylheptane compound aimed at treating inflammatory bowel diseases. However, the mechanisms by which SDH modulates these conditions remain largely unknown. In this study, we assessed SDH's impact on the clinical progression of dextran sodium sulfate (DSS)-induced ulcerative colitis. Our results demonstrated that SDH significantly mitigated the symptoms of DSS-induced colitis, reflected in reduced disease activity index scores, alleviation of weight loss, shortening of the colorectum, and reduction in spleen swelling. Notably, SDH decreased the proportion of Th1/Th2/Th17 cells and normalized inflammatory cytokine levels in the colon. Furthermore, SDH treatment modified the gut microbial composition in mice with colitis, notably decreasing Bacteroidetes and Proteobacteria populations while substantially increasing Firmicutes, Actinobacteria, and Patescibacteria. In conclusion, our findings suggest that SDH may protect the colon from DSS-induced colitis through the regulation of Th1/Th2/Th17 cells and gut microbiota, offering novel insights into SDH's therapeutic potential.

Important molecular mechanisms in ferroptosis.

Ferroptosis is a type of cell death that is caused by the oxidation of lipids and is dependent on the presence of iron. It was first characterized by Brent R. Stockwell in 2012, and since then, research in the field of ferroptosis has rapidly expanded. The process of ferroptosis-induced cell death is genetically, biochemically, and morphologically distinct from other forms of cellular death, such as apoptosis, necroptosis, and non-programmed cell death. Extensive research has been devoted to comprehending the intricate process of ferroptosis and the various factors that contribute to it. While the majority of these studies have focused on examining the effects of lipid metabolism and mitochondria on ferroptosis, recent findings have highlighted the significant involvement of signaling pathways and associated proteins, including Nrf2, P53, and YAP/TAZ, in this process. This review provides a concise summary of the crucial signaling pathways associated with ferroptosis based on relevant studies. It also elaborates on the drugs that have been employed in recent years to treat ferroptosis-related diseases by targeting the relevant signaling pathways. The established and potential therapeutic targets for ferroptosis-related diseases, such as cancer and ischemic heart disease, are systematically addressed.

Modulating glycosphingolipid metabolism and autophagy improves outcomes in pre-clinical models of myeloma bone disease.

Patients with multiple myeloma, an incurable malignancy of plasma cells, frequently develop osteolytic bone lesions that severely impact quality of life and clinical outcomes. Eliglustat, a U.S. Food and Drug Administration-approved glucosylceramide synthase inhibitor, reduced osteoclast-driven bone loss in preclinical in vivo models of myeloma. In combination with zoledronic acid, a bisphosphonate that treats myeloma bone disease, eliglustat provided further protection from bone loss. Autophagic degradation of TRAF3, a key step for osteoclast differentiation, was inhibited by eliglustat as evidenced by TRAF3 lysosomal and cytoplasmic accumulation. Eliglustat blocked autophagy by altering glycosphingolipid composition whilst restoration of missing glycosphingolipids rescued autophagy markers and TRAF3 degradation thus restoring osteoclastogenesis in bone marrow cells from myeloma patients. This work delineates both the mechanism by which glucosylceramide synthase inhibition prevents autophagic degradation of TRAF3 to reduce osteoclastogenesis as well as highlighting the clinical translational potential of eliglustat for the treatment of myeloma bone disease.

Diterpenoid DGA induces apoptosis via endoplasmic reticulum stress caused by changes in glycosphingolipid composition and inhibition of STAT3 in glioma cells.

Glioma is one of the most common malignant primary brain tumors, with poor prognosis and high recurrence. There are currently few drugs approved for brain tumors; thus, it is necessary to develop new effective drugs. Natural diterpenoids have important biological activities, including antiinflammatory, antioxidative, and antitumor effects. In this study, 7α,14ß-dihydroxy-ent-kaur-17-dimethylamino-3,15-dione (DGA), a diterpenoid compound modified from glaucocalyxin A, inhibited the proliferation of many tumor cells, especially glioma. Flow cytometry analysis showed that DGA induced apoptosis in glioma cells. DGA also inhibited xenograft tumors in nude mice. It affected the expression of ceramide synthases (CerS) in glioma cells; CerS1 decreased, and CerS2 and CerS5 increased, resulting in a change in the composition of glycosphingolipids containing varying acyl chain lengths. In glioma cells treated with DGA, the gene transcription of activating transcription factor 4 (ATF4), X-box binding protein-1 (XBP1), and C/EBP-homologous protein (CHOP) in unfolded protein response pathways was upregulated. Meanwhile, the ratio of proapoptotic protein Bcl-2-associated X protein (BAX) to antiapoptotic protein B-cell lymphoma 2 (Bcl-2) also increased. This suggested that an imbalance of glycosphingolipids caused by DGA induced severe endoplasmic reticulum stress and triggered cell apoptosis. Moreover, Western blotting showed DGA inhibited the signal transducers and activators of transcription 3 (STAT3) signaling pathway by reducing the phosphorylation of STAT3 and its upstream kinases, which also promoted the apoptosis of glioma cells. Together, these results explored the anticancer activities of DGA and highlighted it as a potential candidate for treating glioma.

SDH, a novel diarylheptane compound, is a potential treatment for inflammatory bowel disease by restoring epithelial barrier function.

BACKGROUND: The global prevalence of inflammatory bowel disease (IBD) is increasing, and mucosal healing is the preferred treatment target of IBD. Sodium (aS,9 R)- 3-hydroxy-16,17-dimethoxy-15-oxidotricyclo[12.3.1.12,6]nonadeca-1(18),2,4,6(19),14,16-hexene-9-yl sulfate hydrate (SDH) is a novel diarylheptane compound, which is designed to treat IBD. Hence, we investigated the potent therapeutic activity of SDH against IBD and explored the underlying mechanisms, and determined if SDH is a safe and well-tolerated oral therapeutic for IBD treatment. METHODS: We characterized its therapeutic properties in vitro and in vivo using Caco-2 cell monolayer and dextran sodium sulfate (DSS)- or 2,4,6-trinitro-benzene sulfonic acid (TNBS)-induced colitis models. We conducted nonclinical toxicology and safety pharmacology research, including general toxicity, toxicokinetics, pharmacokinetics, metabolism and plasma protein binding, cardiovascular safety pharmacology, central nervous system safety pharmacology, respiratory safety pharmacology, fertility and early embryonic development toxicity, reverse mutation assay, chromosomal aberration assay and micronucleus test. RESULTS: The results showed that SDH promoted expression of tight junction proteins, and protected the integrity and permeability of the epithelial barrier in both cell and animal models. Moreover, lower doses of SDH showed the similar or better efficacy than cyclosporine A (CsA) and mesalazine in DSS- or TNBS-induced colitis animals. Furthermore, our results identified that SDH has satisfactory safety in these studies we tested. In summary, SDH restored the epithelial barrier through tight junction proteins and was expected to be a novel therapeutic agent for the treatment of IBD.

DGT, a novel heterocyclic diterpenoid, effectively suppresses psoriasis via inhibition of STAT3 phosphorylation.

BACKGROUND AND PURPOSE: Psoriasis is a chronic immune-mediated inflammatory skin disease that easily recurs and is difficult to cure. DGT is a novel synthetic heterocyclic diterpenoid, whose structure has not been previously reported. We have investigated the action of DGT against psoriasis, specifically the hyperproliferation of epidermal keratinocytes, angiogenesis and pathogenic inflammatory responses. EXPERIMENTAL APPROACH: We investigated its pharmacokinetics in skin after topical administration. We characterized its pharmacological actions in vitro and in vivo using cell proliferation assay, cell apoptosis assay, diethylstilbestrol-induced mouse vaginal epithelial cell mitosis model, tube formation assay, cell migration assay, chick embryonic chorioallantoic membrane (CAM) assay, histological, flow cytometric analysis and imiquimod (IMQ)-induced psoriasis-like model. KEY RESULTS: DGT was found to be mainly distributed in the epidermis and dermis, which indicated that DGT was suitable as a topical treatment. DGT inhibited cell proliferation and induced apoptotic cell death of keratinocytes in vitro and in vivo. Moreover, DGT inhibited endothelial cell proliferation, tube formation and migration of in vitro angiogenesis, as well as in vivo CAM angiogenesis. In an IMQ-induced psoriasis-like skin inflammation murine model, topical application of DGT ameliorated keratinocyte proliferation and inflammatory response, especially in IL-17-related psoriasiform dermatitis. Furthermore, our results demonstrated that DGT prevented these pathological processes of psoriasis through suppression of STAT3 phosphorylation. CONCLUSION AND IMPLICATIONS: DGT has great potential as a novel therapeutic agent for the treatment of psoriatic skin disease.

Protective effects of HY1702 on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome is an inflammatory disease with no effective pharmacological treatment. We investigated the therapeutic effect of HY1702, a new small molecule diterpene obtained from the processing and modification of Glaucocalyxin A and may exhibit anti-inflammatory activity. Specifically, we studied the anti-inflammatory effects of HY1702 on lipopolysaccharide-induced inflammatory responses in RAW264.7 and THP-1 cells in vitro and its protective efficacy on lipopolysaccharide-induced mild acute respiratory distress syndrome in mice. Our results showed that HY1702 significantly decreased lipopolysaccharide-induced inflammatory cytokine expression in RAW264.7 and THP-1 cells and attenuated the secretion of nitric oxide and prostaglandin E2 by down-regulating the expression of inducible nitric oxide synthase and cyclooxygenase 2 in RAW264.7 cells. In mice with lipopolysaccharide-induced mild acute respiratory distress syndrome, HY1702 alleviated histological alterations in the lungs and reduced the alveolar cavity protein leakage and inflammatory cytokine expression in murine bronchial alveolar lavage fluid. HY1702 decreased the myeloperoxidase activity and lung wet to dry weight ratio. In our mechanism studies in lipopolysaccharide-exposed RAW264.7 cells, HY1702 suppressed the inflammation stimulated by lipopolysaccharide through inhibiting phosphorylation of inhibitor of nuclear factor κB kinase subunit α/ß (IKKα/ß) and inhibitor of nuclear factor κB subunit α (IκBα), further affecting the nuclear transfer of phosphorylated p65. Meanwhile, phosphorylation of p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) was inhibited. These data suggest that HY1702 can reduce inflammation on lipopolysaccharide-stimulated macrophages and attenuate the symptoms of mild acute respiratory distress syndrome in a murine model by regulating the nuclear factor κB and MAP kinase signalling pathways.

Agl22 and Agl23 are involved in the synthesis and utilization of the lipid-linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica.

Like both eukaryotes and bacteria, archaea can decorate proteins with N- and O-linked glycans. Whereas pathways and roles of N-glycosylation have been studied in several model archaeal organisms, little is known of O-glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S-layer glycoproteins are modified by an N-linked glucose-α-(1, 2)-[sulfoquinovosamine-ß-(1, 6)-]galactose trisaccharide and an O-linked glucose-α-(1, 4)-galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose-DolP onto galactose-DolP to form the glucose-α-(1, 4)-galactose-DolP precursor of the N-glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase-associated protein responsible for flipping of hexose-DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N- and O-linked glycans onto target protein occurs on the outer surface of the cell using hexose-DolPs as sugar donors. Deletion mutant showed that N- and O-glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.

Abberant Immunoglobulin G Glycosylation in Rheumatoid Arthritis by LTQ-ESI-MS.

Aberrant glycosylation has been observed in many autoimmune diseases. For example, aberrant glycosylation of immunoglobulin G (IgG) has been implicated in rheumatoid arthritis (RA) pathogenesis. The aim of this study is to investigate IgG glycosylation and whether there is an association with rheumatoid factor levels in the serum of RA patients. We detected permethylated N-glycans of the IgG obtained in serum from 44 RA patients and 30 healthy controls using linear ion-trap electrospray ionization mass spectrometry (LTQ-ESI-MS), a highly sensitive and efficient approach in the detection and identification of N-glycans profiles. IgG N-glycosylation and rheumatoid factor levels were compared in healthy controls and RA patients. Our results suggested that total IgG purified from serum of RA patients shows significantly lower galactosylation (p = 0.0012), lower sialylation (p < 0.0001) and higher fucosylation (p = 0.0063) levels compared with healthy controls. We observed a positive correlation between aberrant N-glycosylation and rheumatoid factor level in the RA patients. In conclusion, we identified aberrant glycosylation of IgG in the serum of RA patients and its association with elevated levels of rheumatoid factor.

Anti-H1N1 viral activity of three main active ingredients from zedoary oil.

Influenza virus is one of the most widespread infectious diseases in the world. It poses a serious public health threat to humans. With the emergence of drug-resistant virus strains, antiviral drugs are urgently needed to control virus transmission and disease progression. In this study, three main active substances-curcumol, curdione and germacrone-were isolated from the traditional Chinese medicine zedoary. They inhibited the replication of influenza A (H1N1) virus in a dose-dependent manner. After treatment with these compounds, the expression of viral protein and RNA synthesis were inhibited. In vivo, these compounds also reduced H1N1-induced lung damage and the load of virus in serum as well as whole blood cells. In a proteomic analysis, after treatment with germacrone, the expression of antiviral protein and the amount of intracellular virus were significantly reduced, further proving that germacrone can inhibit viral replication. Our experiments have shown that curcumol, curdione and germacrone can inhibit the replication of H1N1 virus; in particular, germacrone shows potential both in vitro and in vivo as a therapeutic drug.

Possible role of ß-galactosidase in rheumatoid arthritis.

Objective: To investigate the potential role of ß-galactosidase in altering immunoglobulin G (IgG) galactosylation in serum of rheumatoid arthritis (RA).Methods: The expression level and activity of ß-galactosidase in serum and CD 19+ B cells were measured by enzyme-linked immune sorbent assay (ELISA). The effect of ß-galactosidase on the N-glycan changes in serum from mice intravenously treated with ß-galactosidase was observed by linear ion-trap quadrupole-electrospray ionization mass spectrometry (LTQ-ESI-MS). We established a collagen-induced arthritis (CIA) rat model to explore the biological function of ß-galactosidase in RA.Results: The expression level of ß-galactosidase in serum of 32 patients was elevated when compared with those of 30 healthy controls. The activity and expression level of ß-galactosidase in CD19+ B cells from RA patients was higher than those from healthy controls. The ratio of m/z 1142/937 was reduced in mice treated with ß-galactosidase when compared with normal mice. We found that ß-galactosidase was implicated in the development of inflammation by affecting body weight and elevating the expression level of interleukin-6, tumor necrosis factor-α, and rheumatoid factor in the serum.Conclusions: Our results suggested the high level of ß-galactosidase in B cells and serum of RA patients and revealed that altered ß-galactosidase may be implicated in the progression of inflammation.

Mass spectrometry analysis reveals aberrant N-glycans in colorectal cancer tissues.

Aberrant glycosylation is strongly correlated with the development of various cancers. Tumor-associated carbohydrate antigens, including N-glycans, are predominantly expressed on the tumor cell surface. Because the incidence of colorectal cancer is high in China, we investigated aberrant N-glycans from colorectal cancer tissues (CRC) in Chinese patients. By Linear ion trap quadrupole-electrospray ionization mass spectrometry, we performed glycomic assays on N-glycans obtained from solid CRC tissues and paired peritumoral tissues. In total, aberrant N-glycans were expressed in the colorectal tumor tissues. Specifically, seven bisecting structures (M/Z 9732+, 10602+, 10752+, 11622+, 11772+, 12642+, 13522+) decreased, M/Z 10552+ (two-antennae complex N-glycan) and M/Z 12792+ (three-antennae complex N-glycan) decreased, M/Z 10132+ and M/Z 11162+ (high-mannose N-glycan) increased, and M/Z 12282+ (bifucosylated N-glycan) increased. To evaluate the MS profile data, several statistical tools were applied, including student's t test, orthogonal partial least squares discriminant analysis and receiver operating characteristic curve. The measurement of the degree of bisecting N-glycans had an area under the curve value of 0.823. Interestingly, we observed that the bisecting N-glycans decreased with the tumor stages. This phenomenon was not found in esophageal squamous cell carcinoma, in which the bisecting N-glycans had no change. Thus, the expression of bisecting N-glycans may be an interesting point in the study of colorectal cancer.

Glycolipid iGb3 feedback amplifies innate immune responses via CD1d reverse signaling.

The cross-talk between cellular lipid metabolism and the innate immune responses remains obscure. In addition to presenting lipid antigens to Natural Killer T-cells (NKT cells), the Cluster of Differentiation 1D Glycoprotein (CD1d) might mediate reverse signaling in antigen-presenting cells (APCs). Here we found CD1d deficiency attenuated Toll-like receptor (TLR)-triggered inflammatory innate responses in macrophages and dendritic cells, protecting mice from endotoxin shock. TLR activation in macrophages induced metabolic changes of glycosphingolipids (GSLs), among which glycolipid isoglobotrihexosylceramide (iGb3) was rapidly produced. The endogenously generated iGb3 bound CD1d in endosomal compartments and then synergized with the initially activated TLR signal to induce Tyr332 phosphorylation of CD1d intracellular domain. This led to the recruitment and activation of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 interacted with IκB kinase ß (IKKß) and TANK-binding kinase 1 (TBK1), and enhanced tyrosine phosphorylation of Tyr188/199 of IKKß and Tyr179 of TBK1 and thus, their activation to promote full activation of TLR signaling. Thus, intracellular CD1d reverse signaling, triggered by endogenous iGb3, amplifies inflammatory innate responses in APCs. Our findings identify a non-canonical function of CD1d reverse signaling activated by lipid metabolite in the innate immune response.

Antineoplastic activity of isoliquiritigenin, a chalcone compound, in androgen-independent human prostate cancer cells linked to G2/M cell cycle arrest and cell apoptosis.

Isoliquiritigenin is a natural chalcone derived from Glycyrrhiza, which has been reported to have anti-tumor activity in recent years. Here, we investigate the anticancer efficacy and associated mechanisms of isoliquiritigenin in human prostate cancer PC-3 and 22RV1 cells. Isoliquiritigenin (25-50µM) inhibited cell proliferation, induced cell apoptosis, and caused G2/M cell cycle arrest in vitro. This agent also repressed the growth of PC-3 xenograft tumors in vivo with the results of hematoxylin/eosin staining and immunohistochemistry staining showing differences between isoliquiritigenin-treated groups and control group. Next, we used microarray transcriptional profiling to identify isoliquiritigenin-regulated genes on PC-3 prostate cancer cells. Multiple genes involved in cell cycle, DNA damage, and apoptosis signaling pathways were changed remarkably with the treatment of isoliquiritigenin. Molecular studies revealed that G2/M arrest was associated with a decrease in cyclin B1, cyclin-dependent kinase 1 (CDK1), and phosphorylated CDK1 (Thr14, Tyr15, and Thr161), whereas the expression of 14-3-3σ and growth arrest and DNA damage-inducible 45 alpha (GADD45A) was increased. The complexes of cyclin B1-CDK1 were also examined to show a decrease in the binding of CDK1 with cyclin B1. In addition, treatment with relatively high concentrations of isoliquiritigenin induced apoptosis, mainly associated with enhancing apoptosis regulator (Bax/Bcl-2) ratio. Collectively, these findings indicate that isoliquiritigenin modulates cyclin B1-CDK1 for G2/M arrest, together with an alteration of cell cycle regulators and apoptotic factors in human prostate cancer cells. However, we observed pleiotropic effects for isoliquiritigenin in microarray results, suggesting that other biological mechanisms also contribute to its efficacy, which could be of interest for future investigations.

Overexpression of ceramide synthase 1 increases C18-ceramide and leads to lethal autophagy in human glioma.

Ceramide synthase 1 (CERS1) is the most highly expressed CERS in the central nervous system, and ceramide with an 18-carbon-containing fatty acid chain (C18-ceramide) in the brain plays important roles in signaling and sphingolipid development. However, the roles of CERS1 and C18-ceramide in glioma are largely unknown. In the present study, measured by electrospray ionization linear ion trap mass spectrometry, C18-ceramide was significantly lower in glioma tumor tissues compared with controls (P < 0.001), indicating that C18-ceramide might have a role in glioma. These roles were examined by reconstitution of C18-ceramide in U251 and A172 glioma cells via addition of exogenous C18-ceramide or overexpression of CERS1, which has been shown to specifically induce the generation of C18-ceramide. Overexpression of CERS1 or adding exogenous C18-ceramide inhibited cell viability and induced cell death by activating endoplasmic reticulum stress, which induced lethal autophagy and inhibited PI3K/AKT signal pathway in U251 and A172 glioma cells. Moreover, overexpression of CERS1 or adding exogenous C18-ceramide increased the sensitivity of U251 and A172 glioma cells to teniposide (VM-26). Thus, the combined therapy of CERS1/C18-ceramide and VM-26 may be a novel therapeutic strategy for the treatment of human glioma.

Glycosphingolipid GM3 is Indispensable for Dengue Virus Genome Replication.

Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease of humans worldwide. Glycosphingolipids (GSLs) are involved in virus infection by regulating various steps of viral-host interaction. However, the distinct role of GSLs during DENV infection remains unclear. In this study, we used mouse melanoma B16 cells and their GSL-deficient mutant counterpart GM95 cells to study the influence of GSLs on DENV infection. Surprisingly, GM95 cells were highly resistant to DENV infection compared with B16 cells. Pretreatment of B16 cells with synthetase inhibitor of GM3, the most abundant GSLs in B16 cells, or silencing GM3 synthetase T3GAL5, significantly inhibited DENV infection. DENV attachment and endocytosis were not impaired in GM95 cells, but DENV genome replication was obviously inhibited in GM95 cells compared to B16 cells. Furthermore, GM3 was colocalized with DENV viral replication complex on endoplasmic reticulum (ER) inside the B16 cells. Finally, GM3 synthetase inhibitor significantly reduced the mortality rate of suckling mice that challenged with DENV by impairing the viral replication in mouse brain. Taken together, these data indicated that GM3 was not required for DENV attachment and endocytosis, however, essential for viral genome replication. Targeting GM3 could be a novel strategy to inhibit DENV infection.

Liquid chromatography mass spectrometry-based O-glycomics to evaluate glycosylation alterations in gastric cancer.

PURPOSE: Gastric cancer is the fourth most common malignant cancer worldwide. Important for tumorigenesis and progression, aberrant glycosylation occurs frequently in cancers. We investigated the differences in O-glycosylation in the serum of 157 gastric cancer patients (GC) and 144 healthy donors. EXPERIMENTAL DESIGN: We used the method of labeling O-glycans (released from proteins) with 1-phenyl-3-methyl-5-pyrazolone followed by LC-MS analysis. Analyzing the LC-MS data by partial least squares discriminant and unpaired Student t test, combined with the structural information of O-glycans from LC-MS/MS in positive mode. RESULTS: The expression level of core1, core2, ST antigen, and core2 complex O-glycans (m/z 733.33, m/z 809.42) were increased significantly (p < 0.0001), whereas m/z 529.75 and diST-antigen were decreased in the serum of GC compared with controls (p < 0.001). In addition, there were significant correlations between the abundance of the O-glycans and glycoproteins (MUC1, CEA) in the serum of GC. CONCLUSION AND CLINICAL RELEVANCE: Glycomics approaches identified multiple candidate antigens for patients with GC. The O-glycan structures are increased in the serum of GC, they may be candidates for carbohydrate tumor markers.

Elevated level of serum glycoprotein bifucosylation and prognostic value in Chinese breast cancer.

Aberrant glycosylation is highly associated with cancer progression. The aim of this study was to compare bifucosylated N-glycans in sera obtained from healthy controls and breast cancer patients, with the goal of identifying a potential indicator for monitoring the recurrence and metastasis of breast cancer. A unique structural pattern of bifucosylated N-glycan, with both core and antennary fucosylation, was identified in breast cancer patients. The spectrum of antennary fucosylation was a composite of the standard spectra of Lewis X and H2, indicating a mixture of the two epitopes. Permethylated N-glycans of the glycoproteins extracted from 91 breast cancer patients and 43 healthy controls were detected using linear ion-trap quadrupole-electrospray ionization mass spectrometry, which appeared to be a highly sensitive and useful approach in the detection and identification of N-glycans. To evaluate MS profile data, several statistical tools were applied, including Student'st-test, partial least squares discriminant analysis and receiver-operating characteristic curve. The results showed that the measurement of bifucosylation degree and CEA levels had an improved diagnostic performance compared with that of CEA alone. We compared the potential of bifucosylated N-glycan as an indicator of breast cancer recurrence with the current clinical biomarkers, i.e., CEA, CA 15-3 and CA125. The result revealed that, compared with CEA, CA 15-3 and CA125, the bifucosylation degree of N-glycans could be a more reliable indicator of breast cancer recurrence.

Molecular basis of antibody binding to mucin glycopeptides in lung cancer.

Glycopeptides bearing Tn epitopes are emerging targets for cancer diagnosis and immunotherapy. In this study, we analyzed membrane proteins containing O-glycosylated tandem repeat (TR) sequences in lung cancer patients of different types and stages, using gene microarray data in public domain. The expression of Tn and glycopeptide epitopes on the surface of lung cancer cell lines were studied by monoclonal IgG antibodies 14A, 16A, and B72.3. The binding of mAbs to synthetic glycopeptides were studied by surface plasmon resonance. Nine mucin mRNAs were found to be expressed in lung cancer patients but at similar level to healthy individuals. At protein level, a glycopeptide epitope on cancer cell surface is preferably recognized by mAb 16A, as compared to peptide-alone (14A) or sugar-alone epitopes (B72.3). 14A and 16A favor clustered TR containing more than three TR sequences, with 10-fold lower Kd than two consecutive TR. B72.3 preferrably recognized clustered sialyl-Tn displayed on MUC1 but not other O-glycoproteins, with 100-fold stronger binding when MUC1 is transfected as a sugar carrier, while the total sugar epitopes remain unchanged. These findings indicate that clusters of both TR backbones and sugars are essential for mAb binding to mucin glycopeptides. Three rules of antibody binding to mucin glycopeptides at molecular level are presented here: first, the peptide backbone of a glycopeptide is preferentially recognized by B cells through mutations in complementarity determining regions (CDRs) of B cell receptor, and the sugar-binding specificity is acquired through mutations in frame work of heavy chain; secondly, consecutive tandem repeats (TR) of peptides and glycopeptides are preferentially recognized by B cells, which favor clustered TR containing more than three TR sequences; thirdly, certain sugar-specific B cells recognize and accommodate clustered Tn and sialyl-Tn displayed on the surface of a mucin but not other membrane proteins.

[Effect of compound Chinese traditional medicine PC-SPES II in inhibiting proliferation of human prostate cancer cell LNCaP and on expressions of AR and PSA].

To investigate the effect of compound Chinese traditional medicine PC-SPES II I in inhibiting proliferation of human prostate cancer cell LNCaP based on the androgen receptor (AR) signaling pathway. The effect of PC-SPES II on LNCaP cell proliferation was detected by MTT assay. According to the findings, at the mass concentration of 180-1 440 mg x L(-1), PC-SPES II significantly inhibited the proliferation of LNCaP cells; the IC50 of PC-SPES II at 24 h and 48 h were 311.48, 199.01 mg x L(-1), respectively. The flow Cytometry detection showed 240 mg x L(-1) PC-SPES II arrested cells in G2/M phase, and an obvious apoptotic peak appeared before G0/G1 peak and rose over time. Meanwhile, Hoechst 33258 staining revealed apoptotic cellular morphology. Annexin V-FITC/PI staining manifested an increase in apoptotic cell ratio at the PC-SPES II concentration of 480 mg x L(-1) in a dose dependent manner. The prostate specific antigen (PSA) secretion of LNCaP cells was tested by PSA ELISA kit. Besides, compared with 25 mg x L(-1) Bic, 480 mg x L(-1) PC-SPES II significantly reduced the cell secretion of PSA. The AR and PSA mRNA and protein expressions were detected by qRT-PCR and Western blot. According to the results, after the induction of LNCaP cells with synthetic androgen 25 µg x L(-1) R1881, 240-480 mg x L(-1) PC-SPES II notably down-regulated the AR and PSA mRNA and protein expressions and inhibited the translocation of AR from cytoplasm to nucleus. In summary, PC-SPES II significantly can inhibit the in vitro proliferation of LNCaP cells and arrest cell cycle arrest in G2/M phase. Its mechanism may be associated with the down-regulation of the AR and PSA expressions and the inhibition of AR nuclear translocation.