Peraphan Pothacharoen

Diagnostic role of serum glypican-3 in differentiating hepatocellular carcinoma from non-malignant chronic liver disease and other liver cancers

Background and Aims:  The role of glypican‐3 (GPC3), a novel serum marker, in differentiating hepatocellular carcinoma (HCC) from non‐malignant chronic liver disease and other malignant space‐occupying lesions in the liver is largely unknown. The aims of this study were to evaluate its diagnostic role and clinical correlations in patients with HCC.

Chondroprotective and anti-inflammatory effects of sesamin

Osteoarthritis (OA) is a major disability of elderly people. Sesamin is the main compound in Sesamun indicum Linn., and it has an anti-inflammatory effect by specifically inhibiting Δ5-desaturase in polyunsaturated fatty acid biosynthesis. The chondroprotective effects of sesamin were thus studied in a porcine cartilage explant induced with interleukin-1beta (IL-1β) and in a papain-induced osteoarthritis rat model. With the porcine cartilage explant, IL-1β induced release of sulfated-glycosaminoglycan (s-GAG) and hydroxyproline release, and this induction was significantly inhibited by sesamin. This ability to inhibit these processes might be due to its ability to decrease expression of MMP-1, -3 and -13, which can degrade both PGs and type II collagen, both at the mRNA and protein levels. Interestingly, activation of MMP-3 might also be inhibited by sesamin. Moreover, in human articular chondrocytes (HACs), some pathways of IL-1β signal transduction were inhibited by sesamin: p38 and JNK. In the papain-induced OA rat model, sesamin treatment reversed the following pathological changes in OA cartilage: reduced disorganization of chondrocytes in cartilage, increased cartilage thickness, and decreased type II collagen and PGs loss. Sesamin alone might increase formation of type II collagen and PGs in the cartilage tissue of control rats. These results demonstrate that sesamin efficiently suppressed the pathological processes in an OA model. Thus, sesamin could be a potential therapeutic strategy for treatment of OA.

Sesamin stimulates osteoblast differentiation through p38 and ERK1/2 MAPK signaling pathways

BackgroundOsteoporosis is a worldwide health problem predominantly affecting post-menopausal women. Therapies aimed at increasing bone mass in osteoporetic patients lag behind comparable investigation of therapeutic strategies focusing on the bone resorption process. Sesamin, a major lignan compound found in Sesamun indicum Linn., has a variety of pharmacological effects, though its activity on bone cell function is unclear. Herein we examine the effect of this lignan on osteoblast differentiation and function.MethodCell cytotoxicity and proliferative in hFOB1.19 were examined by MTT and alamar blue assay up to 96 h of treatment. Gene expression of COL1, ALP, BMP-2, Runx2, OC, RANKL and OPG were detected after 24 h of sesamin treatment. ALP activity was measured at day 7, 14 and 21 of cultured. For mineralized assay, ADSCs were cultured in the presence of osteogenic media supplement with or without sesamin for 21 days and then stained with Alizarin Red S. MAPK signaling pathway activation was observed by using western blotting.ResultsSesamin promoted the gene expression of COL1, ALP, OCN, BMP-2 and Runx2 in hFOB1.19. On the other hand, sesamin was able to up-regulate OPG and down-regulate RANKL gene expression. ALP activity also significantly increased after sesamin treatment. Interestingly, sesamin induced formation of mineralized nodules in adipose derived stem cells (ADSCs) as observed by Alizarin Red S staining; this implies that sesamin has anabolic effects both on progenitor and committed cell stages of osteoblasts. Western blotting data showed that sesamin activated phosphorylation of p38 and ERK1/2 in hFOB1.19.ConclusionsThe data suggest that sesamin has the ability to trigger osteoblast differentiation by activation of the p38 and ERK MAPK signaling pathway and possibly indirectly regulate osteoclast development via the expression of OPG and RANKL in osteoblasts. Therefore, sesamin may be a promising phytochemical that could be developed for supplementation of osteoporotic therapy.

Two Related but Distinct Chondroitin Sulfate Mimetope Octasaccharide Sequences Recognized by Monoclonal Antibody WF6

Chondroitin sulfate (CS) proteoglycans are major components of cartilage and other connective tissues. The monoclonal antibody WF6, developed against embryonic shark cartilage CS, recognizes an epitope in CS chains, which is expressed in ovarian cancer and variably in joint diseases. To elucidate the structure of the epitope, we isolated oligosaccharide fractions from a partial chondroitinase ABC digest of shark cartilage CS-C and established their chain length, disaccharide composition, sulfate content, and sulfation pattern. These structurally defined oligosaccharide fractions were characterized for binding to WF6 by enzyme-linked immunosorbent assay using an oligosaccharide microarray prepared with CS oligosaccharides derivatized with a fluorescent aminolipid. The lowest molecular weight fraction recognized by WF6 contained octasaccharides, which were split into five subfractions. The most reactive subfraction contained several distinct octasaccharide sequences. Two octasaccharides, ΔD-C-C-C and ΔC-C-A-D (where A represents GlcUAβ1-3GalNAc(4-O-sulfate), C is GlcUAβ1-3Gal-NAc(6-O-sulfate), D is GlcUA(2-O-sulfate)β1-3GalNAc(6-O-sulfate), ΔCis Δ4,5HexUAα1-3GalNAc(6-O-sulfate), and ΔDis Δ4,5HexUA(2-O-sulfate)α1-3GalNAc(6-O-sulfate)), were recognized by WF6, but other related octasaccharides, ΔC-A-D-C and ΔC-C-C-C, were not. The structure and sequences of both the binding and nonbinding octasaccharides were compared by computer modeling, which revealed a remarkable similarity between the shape and distribution of the electrostatic potential in the two different octasaccharide sequences that bound to WF6 and that differed from the nonbinding octasaccharides. The strong similarity in structure predicted for the two binding CS octasaccharides (ΔD-C-C-C and ΔC-C-A-D) provided a possible explanation for their similar affinity for WF6, although they differed in sequence and thus form two specific mimetopes for the antibody.

The effects of p-hydroxycinnamaldehyde from Alpinia galanga extracts on human chondrocytes.

Osteoarthritis (OA) is the most common form of arthritis and affects millions of people worldwide. Patients have traditionally been treated with non-steroidal anti-inflammatory drugs (NSAIDs), but these are associated with significant side effects. Purification of the acetone extract of Alpinia galanga afforded p-hydroxycinnamaldehyde, as identified by nuclear magnetic resonance and mass spectrometry analyses. By exploiting the cartilage explant culture, p-hydroxycinnamaldehyde suppressed loss of uronic acid, resulting in release of hyaluronan (HA), sulfated glycosaminoglycans (s-GAGs) and matrix metalloproteinases (MMPs). p-Hydroxycinnamaldehyde and interleukin-1beta (IL-1beta), when incubated in primary human chondrocytes, also reduced release of HA, s-GAG and MMP-2. The results demonstrated: (a) that expression levels of the catabolic genes MMP-3 and MMP-13 were suppressed and (b) mRNA expression levels of anabolic genes of collagen II, SOX9 and aggrecan were increased. This study shows that p-hydroxycinnaldehyde from A. galanga Linn. is a potential therapeutic agent for treatment of OA.

SOX9 transduction increases chondroitin sulfate synthesis in cultured human articular chondrocytes without altering glycosyltransferase and sulfotransferase transcription

The transcription factor SOX9 (Sry-type high-mobility-group box 9) is expressed in all chondrocytes and is essential for the expression of aggrecan, which during biosynthesis is substituted with more than 10 times its weight of CS (chondroitin sulfate) and is secreted by chondrocytes to form the characteristic GAG (glycosaminoglycan)-rich ECM (extracellular matrix) of cartilage. SOX9 expression rapidly falls during monolayer culture of isolated chondrocytes and this turns off aggrecan and associated CS synthesis. We therefore investigated whether SOX9 transduction of cultured human articular chondrocytes had any effect on the gene expression of the glycosyltransferases and sulfotransferases necessary for GAG biosynthesis. Retroviral SOX9 transduction of passaged chondrocytes increased the endogenous rate of GAG synthesis and the total capacity for GAG synthesis assessed in monolayer culture with beta-xyloside. Both the endogenous rate and the total capacity of GAG biosynthesis were increased further in chondrogenic cell aggregate cultures. The GAG synthesized was predominantly CS and the hydrodynamic size of the newly synthesized chains was unchanged by SOX9 transduction. Aggrecan gene expression was increased in the SOX9-transduced chondrocytes and increased further in chondrogenic culture, but no comparable effects were found in SOX9 transduced dermal fibroblasts. However, the expression of CS glycosyltransferase and sulfotransferase genes in chondrocytes was unaffected by SOX9 transduction. Therefore SOX9 transduction in chondrocytes increased their CS synthetic capacity, but this was not accompanied by changes in the transcription of the CS biosynthetic enzymes and must occur by indirect regulation of enzyme activity through control of enzyme protein translation or enzyme organization.

Chondroitin sulfate epitope (WF6) and hyaluronic acid as serum markers of cartilage degeneration in patients following anterior cruciate ligament injury

Serum chondroitin sulfate epitope (WF6) and hyaluronic acid (HA) levels were determined to be of clinical relevance to an anterior cruciate ligament (ACL) injury. This cross-sectional study recruited participants from two distinct groups. Group A was comprised of 74 healthy controls, and group B consisted of 33 ACL injury patients. Serum samples were taken and assayed by a competitive immunoassay with monoclonal antibody WF6. Serum HA was also determined by an ELISA-based assay using biotinylated HA-binding proteins. Both groups A and B shared similar values of age, body mass index, white blood cell count and percentage of polymorphonuclear cells. ESR levels were also shown to be within normal limits. The serum WF6 epitope levels of group B were significantly higher than those of group A, whereas serum HA levels were not different between the two groups. The serum WF6 epitope level is more sensitive to changes in articular cartilage due to a non-inflammatory instability condition than the serum HA level, and should prove to be one of the most promising assays for early post-traumatic arthritis detection.

A sulfated glycosaminoglycan array for molecular interactions between glycosaminoglycans and growth factors or anti-glycosaminoglycan antibodies

Glycosaminoglycans (GAGs) take part in numerous biological processes by binding to protein molecules and functionally regulating protein-ligand interactions; therefore, molecular interactions of GAGs have been studied by several methods, including surface plasmon resonance, enzyme-linked immunosorbent assays (ELISAs), and GAG microarrays. To achieve rapid, sensitive, and high-throughput screening of GAG interactions, we have developed a novel microarray in which GAGs, including chondroitin sulfate, heparan sulfate, and heparin, were immobilized. The microarray is made from cyclic polyolefin substrate coated with metacrylate polymers, which have phospholipid groups as side chains. The polymer also has aminooxy groups that react specifically with aldehyde groups at the reducing termini of GAG chains, whereas the phospholipid groups prevent nonspecific adsorption of proteins. Thus, minute amounts of GAGs can be chemically immobilized on the surface with low nonspecific binding of proteins. Using this array, interactions between GAGs and antibodies against chondroitin or heparan sulfate and heparin-binding growth factors were examined. The results were in agreement with previously reported specificities, suggesting that the GAG array is useful for high-throughput interaction analyses between GAGs and functional proteins in miniscule amounts and can be applied to both basic studies of GAGs and the development of diagnostic methods for metabolic diseases involving GAGs.

Comparison of glucose derivatives effects on cartilage degradation

BackgroundGlucosamine (GlcN) is a well-recognized candidate for treatment of osteoarthritis. However, it is currently used in derivative forms, such as glucosamine-hydrochloride (GlcN-HCl) or glucosamine sulfate (GlcN-S). However, the molecular mode of action remains unclear. In this study, we compared the effects of Glucose (Glc), Glucuronic acid (GlcA), Glucosamine hydrochloride (GlcN-HCl) and Glucosamine sulfate (GlcN-S) on cartilage degradation.MethodsPorcine cartilage explants were co-cultured with recombinant human IL-1β and each tested substance for 3 days. HA, s-GAG and MMP-2 releases to media were measured using ELISA, dye-binding assay and gelatin zymography, respectively. Similar studies were performed in a human articular chondrocytes (HAC) monolayer culture, where cells were co-treated with IL-1β and each reagent for 24 hours. Subsequently, cells were harvested and gene expression measured using RT-PCR. All experiments were carried out in triplicate. Students t-tests were used for statistical analysis.ResultsIn cartilage explants treated with IL-1β, GlcN-S had the highest chondroprotective activity of all four chemicals as shown by the inhibition of HA, s-GAG and MMP-2 released from cartilage. The anabolic (aggrecan core protein; AGG, SOX9) and catabolic (MMP-3, -13) genes in HACs treated with IL-1β and with/without chemicals were studied using RT-PCR. It was found that, GlcN-HCl and GlcN-S could reduce the expression of both MMP-3 and -13 genes. The IL-1β induced-MMP-13 gene expression was decreased maximally by GlcN-S, while the reduction of induced-MMP-3 gene expression was greatest with GlcN-HCl. Glc and GlcA reversed the effect of IL-1β on the expression of AGG and SOX9, but other substances had no effect.ConclusionThis study shows that glucosamine derivatives can alter anabolic and catabolic processes in HACs induced by IL-1β. GlcN-S and GluN-HCl decreased induced MMP-3 and -13 expressions, while Glc and GlcA increased reduced-AGG and SOX9 expression. The chondroprotective study using porcine cartilage explant showed that GlcN-S had the strongest effect.

The effect of doxycycline on canine hip osteoarthritis: design of a 6-months clinical trial

Twenty-five dogs were included in a randomized, double-blind trial to assess the efficacy of doxycycline (DOX) orally administered twice a day at 4 mg/kg/day (n = 12) for the treatment of osteoarthritis of the hip. Chondroitin sulfate (CS; 525 mg/day) was used as a positive control (n = 13). Dogs were re-examined monthly for 6 months after initiation of treatment. The assessment protocol included clinical score, radiographic findings and serum osteoarthritis biomarkers. Dogs treated with DOX showed statistically significant improvements (p < 0.05) in lameness, joint mobility, pain on palpation, weight-bearing and overall score at 2, 6, 4, 4 and 4 months, respectively, after treatment. Biomarker levels of CS-WF6 epitope and hyaluronan were significantly increased and decreased (p < 0.05) at 2 and 3 months after treatment compared to pretreatment. These results showed that DOX had a positive therapeutic effect in dogs with osteoarthritis.