透明质酸合酶-2在癌相关成纤维细胞促进舌癌细胞侵袭中的作用

目的:检测透明质酸合酶2 (hyaluronan synthase 2,HAS2) 在正常成纤维细胞(normal zone fibroblasts,NFs)及癌相关成纤维细胞(cancer-associated fibroblasts,CAFs)中的表达,并探讨其在CAFs介导的舌鳞癌细胞系Cal27侵袭行为中的作用?方法:分别从同一舌癌患者手术切除标本的癌组织及癌旁组织获取CAFs和NFs?免疫细胞化学?Western blot法鉴定细胞表型?实时定量RT-PCR及Western blot检测透明质酸合酶在两种细胞中的表达?利用Transwell小室共培养模型,观察CAFs及NFs对舌癌细胞系Cal27侵袭能力的影响?利用siRNA抑制CAFs 中的HAS2,并分析其对Cal27侵袭能力的影响?结果:α-SMA表达于CAFs,NFs中几乎无表达?Cal27在CAFs组中的侵袭力显著高于NFs组及空白对照组(P < 0.01)?实时定量RT-PCR结果显示HAS2在CAFs中的表达是NFs的7倍(P < 0.01),蛋白水平则为NFs的3倍(P < 0.01);Cal27在HAS2干扰组的侵袭力显著低于CAFs组及无关序列组(P < 0.01)?结论:CAFs具有促进舌癌细胞侵袭能力的作用,高表达HAS2可能是其作用机制之一,抑制肿瘤微环境中CAFs的HAS2功能可能是一条新的抗肿瘤侵袭转移的途径?     

弗式不完全佐剂诱导小鼠腹腔淋巴管瘤模型方法的改进

目的改进弗式不完全佐剂诱导小鼠腹腔淋巴管瘤模型的建立方法。方法 20只C57BL/6小鼠中的16只按照小鼠体重随机分为两组,分别采取d1、d15(实验Ⅰ组)和d1、d8、d15(实验Ⅱ组)模式每次腹腔注射弗氏不完全佐剂与PBS以1:1体积比配制的乳悬液0.2ml。首次注射后约3个月,脱颈处死小鼠,观察小鼠腹腔内诱导形成的白色肿瘤样组织的生长部位,用游标卡尺记录大小,随后对其进行病理组织学观察,并通过免疫组化表达淋巴管内皮细胞透明质酸受体1(LYVE-1)来进一步证实为淋巴管瘤。另外4只小鼠分别作为两组的对照组(对照Ⅰ组和对照Ⅱ组)按照与实验组对应的注射模式腹腔注射0.2 ml PBS缓冲液。结果实验Ⅰ组、实验Ⅱ组小鼠腹腔内成瘤率均为100%。实验Ⅰ组每只小鼠腹腔成瘤总体积(227.82±124.87)mm3,明显小于实验Ⅱ组的(365.38±78.74)mm3(P<0.05)。实验Ⅰ组、实验Ⅱ组的肿瘤样组织均经HE染色后光学显微镜下观察,及免疫组化均强阳性的表达LYVE-1,证实为淋巴管瘤。对照Ⅰ组、对照Ⅱ组4只小鼠腹腔均未见任何新生物生成。结论利用C57BL/6小鼠腹腔注射弗氏不完全佐剂,以d1、d8、d1...     

Inhibition of hyaluronan retention by 4-methylumbelliferone suppresses osteosarcoma cells in vitro and lung metastasis in vivo

Background:

Hyaluronan (HA) plays crucial roles in the tumourigenicity of many types of malignant tumours. 4-Methylumbelliferone (MU) is an inhibitor of HA synthesis. Several studies have shown its inhibitory effects on malignant tumours; however, none have focused on its effects on osteosarcoma.

Methods:

We investigated the effects of MU on HA accumulation and tumourigenicity of highly metastatic murine osteosarcoma cells (LM8) that have HA-rich cell-associated matrix, and human osteosarcoma cell lines (MG-63 and HOS).

Results:

In vitro, MU inhibited HA retention, thereby reducing the formation of functional cell-associated matrices, and also inhibited cell proliferation, migration, and invasion. Akt phosphorylation was suppressed by MU (1.0 m). In vivo, although MU showed only a mild inhibitory effect on the growth of the primary tumour, it markedly inhibited (75% reduction) the development of lung metastasis. Hyaluronan retention in the periphery of the primary tumour was markedly suppressed by MU.

Conclusion:

These findings suggested that MU suppressed HA retention and cell-associated matrix formation in osteosarcoma cells, resulting in a reduction of tumourigenicity, including lung metastasis. 4-Methylumbelliferone is a promising therapeutic agent targeting both primary tumours and distant metastasis of osteosarcoma, possibly via suppression of HA retention.     

Activation of p38 mitogen-activated protein kinase is inhibited by hyaluronan via intercellular adhesion molecule-1 in articular chondrocytes stimulated with type II collagen peptide

This study examined the activation of p38 mitogen-activated protein kinase with matrix metalloproteinase-13 (MMP-13) production by a synthetic peptide derived from type II collagen (CB12-II) and its inhibition by high molecular weight hyaluronan (HA) in chondrocytes. When cartilage explants or isolated chondrocytes in monolayer were incubated with CB12-II, the peptide (50 μM, 72 h) activated p38 in association with enhanced MMP-13 production. Inhibition studies with SB203580 (0.1 - 1 μM) indicated the requirement of p38 for CB12-II-induced MMP-13 production. Pretreatment with 2700 kDa HA (1 mg/ml, 1 h) resulted in significant suppression of CB12-II-stimulated MMP-13 production in cartilage as well as in chondrocyte monolayer cultures. HA (1 mg/ml) suppressed p38 activation by CB12-II, leading to a decrease in MMP-13 production. The antibody (20 μg/ml) to intercellular adhesion molecule-1 (ICAM-1), which has been recognized as a receptor of HA on chondrocytes, reversed the HA effect on CB12-II action. Thus, the present study clearly demonstrated that high molecular weight HA suppressed CB12-II-activated p38 via ICAM-1 in articular chondrocytes. HA could down-regulate the catabolic action of type II collagen fragments in osteoarthritic joints through the mechanism demonstrated in this study.     

Upregulation of hyaluronan and its binding receptors in an experimental model of chronic cyclosporine nephropathy

Aim: Hyaluronan (HA) is an important extracellular matrix (ECM) proteoglycan. The localization of HA and its binding receptors, CD44 and LYVE‐1, was evaluated in an experimental model of chronic cyclosporine A (CsA)‐induced nephropathy. Methods: Sprague–Dawley rats maintained on a low‐salt diet (0.05% sodium) received an s.c. injection of vehicle (1 mL/kg per day olive oil; VH groups) or CsA (15 mg/kg per day; CsA groups) for 1 or 4 weeks. Induction of chronic CsA nephropathy was evaluated according to renal function and pathology and expression of HA, CD44, LYVE‐1, ED‐1 and α‐smooth muscle actin (α‐SMA). Results: CsA treatment for 4 weeks caused renal dysfunction, which was accompanied by typical striped interstitial fibrosis. In the VH group, HA immunoreactivity was observed only in the inner medulla. However, the area of HA immunoreactivity increased with the duration of CsA treatment: CsA treatment for 1 week extended HA immunoreactivity to the outer medulla, and CsA treatment for 4 weeks caused a further extension of HA immunoreactivity to the cortex, which was vulnerable to CsA‐induced renal injury. HA binding receptor, CD44 and LYVE‐1 expression were also upregulated in the CsA groups, and were localized to the area of fibrosis and the peritubular capillaries of the cortex. In the CsA groups, ED‐1 and α‐SMA were predominantly expressed in fibrotic areas in which HA had accumulated. Conclusion: These findings suggest that upregulation of HA and its binding receptors are involved in interstitial fibrosis in chronic CsA‐induced renal injury.     

Bioactive tetrapeptide GEKG boosts extracellular matrix formation: in vitro and in vivo molecular and clinical proof

The 'matrikine' concept claims that processing of the precursors for collagen results in the formation of peptides such as KTTKS which in turn augments extracellular matrix (ECM) production. In the present study, we show the development of an anti-ageing active from an in silico approach by molecular design resulting in the tetrapeptide GEKG derived from ECM proteins. The efficacy of the peptide to significantly induce collagen production of the protein level and mRNA level has been demonstrated in vitro in human dermal fibroblasts and in vivo in a double-blind, randomized, placebo-controlled study enroling 10 volunteers with an average age of 48.2 years. The effect of GEKG on facial wrinkles was studied in 30 volunteers using state of the art fringe projection, which allows determination of surface roughness in three-dimensions. Here, only GEKG but not the placebo was able to significantly decrease skin roughness as a measure for wrinkles.     

Hyperglycaemia and aberrated insulin signalling stimulate tumour progression via induction of the extracellular matrix component hyaluronan

Epidemiological studies have detected a higher incidence of various tumour entities in diabetic patients. However, the underlying mechanisms remain insufficiently understood. Glucose‐derived pericellular and extracellular hyaluronan (HA) promotes tumour progression and development. In our study, we tested the hypothesis that a diabetic metabolic state, characterised by hyperglycaemia and concomitant aberrant insulin signalling, stimulates tumour progression via the induction of HA synthesis. In a streptozotocin‐induced diabetic nude mouse tumour xenograft model, hyperglycaemia and lack of insulin caused an increased formation of tumour‐associated HA‐matrix, which in turn accelerated tumour progression and neoangiogenesis. This process was effectively attenuated by treatment with 4‐methylumbelliferone, a pharmacological inhibitor of HA‐synthesis. To define the mechanisms behind these in vivo observations, we investigated the impact of hyperglycaemia and insulin on the glucose metabolism in oesophageal squamous cell cancer cells (ESCC). Hyperglycaemia induced HA synthesis while insulin diminished HA production by directing glucose metabolites to glycolysis. Vice versa, inhibition of glycolysis, either by knockdown of the glycolytic key enzyme phosphofructokinase or by an experimental abrogation of insulin signalling (knockdown of the insulin receptor and long‐term treatment with insulin) augmented HA synthesis. Consequently, these processes induced invasion, anchorage‐independent growth and adhesion of ESCC to endothelial cells in vitro. Thus, the cellular shift in glucose usage from catabolism of glucose to anabolism of HA driven by hyperglycaemia and insulin resistance may represent an important link between diabetes and cancer progression. Hence, therapeutical inhibition of HA synthesis may represent a promising approach for tumour treatment in diabetic patients.     

The efficacy of intra-articular sodium hyaluronate in patients with reducing displaced disc of the temporomandibular joint

In this clinical trial, we examined the efficacy of intra-articular hyaluronic acid (HA) treatment in 38 patients with reducing displaced disc of the temporomandibular joint (TMJ). Subjects received two unilateral upper space injections of HA or physiological saline solution with 1 week apart. Efficacy was based on the following measurements: pain and sound intensity of the joint measured by visual analogue scale (VAS), modified Helkimo's clinical dysfunction index and the intensity of joint vibration during opening and closing the mouth measured by accelerometers. These measurements were performed before the first injection and 1 and 6 months after the last injection. In the treatment group (n=19), all measurements improved significantly at month 1 and at month 6 compared with the baseline (P < 0.01). The same measurements, in the placebo group (n=19), did not show any change, except for the pain intensity which improved at month 1 and month 6 (P < 0.05). The change in baseline measurements of all of the efficacy criteria at month 1 and at month 6 in the treatment group was significantly better compared with the change obtained with placebo at the same time intervals. This study demonstrates that intra-articular sodium hyaluronate (Orthovisc) injection into the TMJ is an effective treatment for a reducing displaced disc.     

Topical hyaluronidase decreases hyaluronic acid and CD44 in human skin and in reconstituted human epidermis: evidence that hyaluronidase can permeate the stratum corneum

Hyaluronic acid (HA), a high molecular weight glycosaminoglycan of the extracellular matrix involved in growth, inflammation and wound healing, also contributes to the hydration and plastic properties of skin. Several drug and cosmetic formulations contain HA. We have initiated investigations that explore whether it is possible, by topical application, to modulate endogenous HA levels in skin. We developed a model epidermal culture system that exhibited a differentiated stratum corneum, and expressed HA and the HA receptor CD44, in a pattern similar to that observed in intact skin. Such in vitro skin equivalents are useful models for investigating the effect of topical drugs. HA and bacterial hyaluronidase were applied to the in vitro skin equivalent and to human skin. Their effects on endogenous HA and CD44 expression were examined using histochemical analysis. Topical HA treatment had no significant effect on HA or CD44 expression in either system. However, hyaluronidase decreased HA and CD44 expression in a dose-dependent manner in both the epidermal culture system and in skin. Apparently, HA is not able to permeate the epidermal culture system or human skin to a significant degree, but bacterial hyaluronidase does permeate both human skin and the culture system, depleting HA and decreasing CD44 expression. These effects were more prominent in the dermal than in the epidermal layers, suggesting that marked differences in HA metabolism exist in these two skin compartments. The ability of hyaluronidase to permeate the stratum corneum suggests that topical application may, additionally, be useful as a clinical modality.