Increasing evidence suggests that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. Additionally, it is becoming apparent that cyclooxygenase (COX) signaling is associated with canine mammary tumor development. The goals of the present study were to investigate COX-2 expression patterns and their effect on CSC-mediated tumor initiation in primary canine mammary tissues and tumorsphere models using immunohistochemistry. Patterns of COX-2, CD44, octamer-binding transcription factor (Oct)-3/4, and epidermal growth factor receptor (EGFR) expression were examined in malignant mammary tumor (MMT) samples and analyzed in terms of clinicopathological characteristics. COX-2 and Oct-3/4 expression was higher in MMTs compared to other histological samples with heterogeneous patterns. In MMTs, COX-2 expression correlated with tumor malignancy features. Significant associations between COX-2, CD44, and EGFR were observed in low-differentiated MMTs. Comparative analysis showed that the levels of COX-2, CD44, and Oct-3/4 expression varied significantly among TSs of three histological grades. Enhanced COX-2 staining was consistently observed in TSs. Similar levels of staining intensity were found for CD44 and Oct-3/4, but EGFR expression was weak. Our findings indicate the potential role of COX-2 in CSC-mediated tumor initiation, and suggest that COX-2 inhibition may help treat canine mammary tumors by targeting CSCs.
Animals ; Antigens, CD44/genetics/metabolism ; Biomarkers, Tumor/genetics/metabolism ; Cell Transformation, Neoplastic/*genetics/metabolism ; Cyclooxygenase 2/*genetics/metabolism ; Dog Diseases/*genetics/metabolism ; Dogs ; Female ; Immunohistochemistry/veterinary ; Mammary Neoplasms, Animal/*genetics/metabolism ; Mammary Neoplasms, Experimental/*genetics/metabolism ; Neoplastic Stem Cells/*metabolism ; Octamer Transcription Factor-3/genetics/metabolism ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Retrospective Studies

Glycosaminoglycans are important structural components in the skin and exist as various proteoglycan forms, except hyaluronic acid. Heparan sulfate (HS), one of the glycosaminoglycans, is composed of repeated disaccharide units, which are glucuronic acids linked to an N-acetyl-glucosamine or its sulfated forms. To investigate acute ultraviolet (UV)-induced changes of HS and HS proteoglycans (HSPGs), changes in levels of HS and several HSPGs in male human buttock skin were examined by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR) after 2 minimal erythema doses (MED) of UV irradiation (each n = 4-7). HS staining revealed that 2 MED of UV irradiation increased its expression, and staining for perlecan, syndecan-1, syndecan-4, CD44v3, and CD44 showed that UV irradiation increased their protein levels. However, analysis by real-time qPCR showed that UV irradiation did not change mRNA levels of CD44 and agrin, and decreased perlecan and syndecan-4 mRNA levels, while increased syndecan-1 mRNA level. As HS-synthesizing or -degrading enzymes, exostosin-1 and heparanase mRNA levels were increased, but exostosin-2 was decreased by UV irradiation. UV-induced matrix metalloproteinase-1 expression was confirmed for proper experimental conditions. Acute UV irradiation increases HS and HSPG levels in human skin, but their increase may not be mediated through their transcriptional regulation.
Adult ; Agrin/genetics ; Antigens, CD44/genetics ; Base Sequence ; DNA Primers/genetics ; Gene Expression/radiation effects ; Glucuronidase/genetics ; Heparan Sulfate Proteoglycans/genetics/*metabolism ; Heparitin Sulfate/metabolism ; Humans ; Male ; Matrix Metalloproteinase 1/genetics ; N-Acetylglucosaminyltransferases/genetics ; RNA, Messenger/genetics/metabolism ; Skin/*metabolism/*radiation effects ; Skin Aging/genetics/physiology ; Syndecan-1/genetics ; Syndecan-4/genetics ; Ultraviolet Rays/*adverse effects ; Young Adult

BACKGROUND: CD44 protein is known as a homing cellular adhesion molecule that is linked to diverse cellular functions such as adhesion, migration and invasion, which are all important in cancer progression and metastasis. The expression of CD44 standard and variant isoforms (CD44 standard isoform [CD44s] and CD44 splice variants containing exon v6 [CD44v6], respectively) is associated with an unfavorable clinical outcome in various neoplasms. METHODS: Forty patients who were diagnosed with diffuse large B-cell lymphoma (DLBCL) through biopsy at Hanyang University Hospital between 1996 and 2003 were included in this study. CD44 proteins expression was analyzed by immunohistochemical staining on a tissue microarray and the correlation of CD44 with the types of DLBCL and clinical parameters, including the factors defined by the International Prognostic Index, was evaluated. RESULTS: A high CD44s and intermediate to strong CD44v6 expression, including cytoplasmic membranous staining patterns, was present in 35% (14/40) and 25% (10/40) of DLBCL patients, respectively. High CD44s expression was correlated significantly with non-germinal center B-cell-like types (non-GCB, p=0.004) and patients with old age (p=0.041). CONCLUSIONS: High CD44s expression may be significantly associated with the non-GCB type compared to the GCB type and may be essential to the prediction of disease outcome in tumor stage III in DLBCL patients.
Antigens, CD44 ; B-Lymphocytes ; Biopsy ; Cytoplasm ; Exons ; Humans ; Lymphoma, B-Cell ; Lymphoma, Large B-Cell, Diffuse ; Neoplasm Metastasis ; Protein Isoforms ; Proteins

BACKGROUND/AIMS: Cluster differentiation 44 standard isoform (CD44s) is a transmembrane glycoprotein. CD44s is a known prognostic factor in various cancers, due to its involvement in tumor cell growth, invasion and metastasis. Its prognostic role, however, is debated because it can be a positive or negative prognostic factor depending on tumor type and is still an ambiguous prognostic indicator in other cancers, especially hepatocellular carcinoma (HCC). We investigated the relationship between CD44s expression and survival in HCC patients. METHODS: A total of 260 HCC samples were collected to generate a tissue microarray. Staining of the arrays with a primary mouse CD44s monoclonal antibody was followed by evaluation of the relationship between CD44s expression and tumor differentiation. The effect of CD44s expression on patient survival was analyzed. RESULTS: CD44s protein expression correlated with histological grade (most and worst Edmondson grade) of the HCC (p=0.029 and p=0.039, respectively) and adversely affected the disease free survival period based on univariate and multivariate analyses (p=0.038 and p=0.077, respectively). CONCLUSIONS: High CD44s protein expression correlates with shorter disease free survival and poorly differentiated HCC. CD44s-targeted therapy may be efficacious for HCC treatment in the future.
Animals ; Antigens, CD44 ; Carcinoma, Hepatocellular ; Disease-Free Survival ; Glycoproteins ; Humans ; Mice ; Multivariate Analysis ; Neoplasm Metastasis ; Protein Array Analysis ; Recurrence

PURPOSE: This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs). MATERIALS AND METHODS: Aliquots (10 g) of the lipoaspirates were stored at 4degrees C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed. RESULTS: When the lipoaspirates were stored at 4degrees C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1x1014 fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells. CONCLUSION: ASCs isolated from lipoaspirates and stored for 24 hours at 4degrees C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4degrees C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.
5'-Nucleotidase/metabolism ; Adipose Tissue/*cytology ; Adult ; Antigens, CD/metabolism ; Antigens, CD44/metabolism ; Antigens, Thy-1/metabolism ; Cell Differentiation/physiology ; Cells, Cultured ; Female ; Humans ; Immunoblotting ; Immunohistochemistry ; Immunophenotyping ; Mesenchymal Stem Cells/metabolism ; Muscle Development/genetics/physiology ; Osteogenesis/genetics/physiology ; Receptors, Cell Surface/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells/*cytology/metabolism ; Young Adult

Neutrophil adhesion and migration are critical in hepatic ischemia/reperfusion (I/R) injury. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy in reperfusion injury. Therefore, the aim of this study was to assess the role of CD44 in neutrophil infiltration and liver injury from hepatic I/R. In this study, using a partial hepatic ischemic model in vivo, we determined the potential role of CD44 in neutrophil infiltration and liver injury from I/R. Reperfusion caused significant hepatocellular injury as it was determined by plasma ALT levels and liver histopathology. The injury was associated with a marked neutrophil recruitment and CD44 expression into the ischemic livers. Administration of anti-CD44 antibody to mice reduced the infiltration of neutrophil into the ischemic tissue, associated with liver function preservation. These results support crucial roles of CD44 in neutrophil recruitment and infiltration leading to liver damage in hepatic I/R injury. Moreover, they provide the rationale for targeting to CD44 as a potential therapeutic approach in liver I/R injury.
Alanine Transaminase/blood ; Animals ; Antibodies/immunology/pharmacology ; Antigens, CD44/immunology/metabolism/*physiology ; Cytokines/metabolism ; Disease Models, Animal ; Liver/*metabolism/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Neutrophils/immunology/physiology ; Reperfusion Injury/metabolism/pathology/*prevention & control

BACKGROUND: Epithelial tumor cells with a CD44(+)/CD24(-/low) immunoprofile may have the ability to cause breast cancer. We studied these cells and their clinicopathological significance. METHODS: The clinicopathologic findings of 100 invasive ductal carcinoma (IDC) cases and 45 ductal carcinoma in situ (DCIS) cases were reviewed. CD44(+)/CD24(-/low) tumor cells were identified by immunohistochemistry, and their clinicopathological implications in IDC and DCIS were analyzed. RESULTS: IDC with a high prevalence of CD44(+)/CD24(-/low) tumor cells was significantly associated with larger mass, higher grade, estrogen receptor (ER) negativity, and tumor cells with a higher frequency of metastasis. The proportion of CD44(+)/CD24(-/low) tumor cells in IDC, and its DCIS components was not significantly different, whereas the proportion of CD44(+)/CD24(-/low) tumor cells was higher in DCIS than in the DCIS component of IDC (p < 0.001). CONCLUSIONS: IDC with a high prevalence of CD44(+)/CD24(-/low) tumor cells might correlate with aggressive features, such as ER and higher grades. Moreover, the proportion of CD44(+)/CD24(-/low) tumor cells in the DCIS components of IDC and DCIS might harbor different biology, which may lead to differences in cancer progression and early carcinogenesis.
Antigens, CD24 ; Antigens, CD44 ; Biology ; Breast ; Breast Neoplasms ; Carcinoma, Ductal ; Carcinoma, Intraductal, Noninfiltrating ; Estrogens ; Immunohistochemistry ; Neoplasm Metastasis ; Neoplastic Stem Cells ; Prevalence

BACKGROUND: CD44 is a cell surface receptor that has been implicated in tumor cell invasion and metastasis in a range of tumors of various organs, including breast, ovary, colon, lung, and brain. CD44 stimulates the invasive ability by interacting with matrix metalloproteinase 14 (MMP14). The expression of MMP14 on the cell surface is thought to trigger multiple proteinase cascades and to stimulate cell migration. METHODS: A total 54 astrocytoma patients were eligible for this study. We performed a retrospective clinicopathological review and CD44 and MMP14 immunohistochemistry. RESULTS: The expressions of CD44 and MMP14 were significantly correlated with the World Health Organization (WHO) grade. On univariate analysis, the WHO grade and the expression of CD44 were the significant prognostic factors affecting overall survival (OS) and disease progression free survival (DPFS). On the multivariate analysis by the Cox regression model, the only WHO grade was shown to be a significant independent prognostic factor for predicting the DPFS and OS. CONCLUSIONS: In this study, the CD44 and MMP14 expressions were related to the WHO grade of astrocytoma. The CD44 expression status was a prognostic factor for DPFS and OS on univariate analysis, but it was not an independent prognostic factor on the multivariate analysis.
Antigens, CD44 ; Astrocytoma ; Brain ; Breast ; Colon ; Disease Progression ; Female ; Humans ; Lung ; Matrix Metalloproteinase 14 ; Multivariate Analysis ; Neoplasm Metastasis ; Ovary ; Prognosis ; Retrospective Studies ; World Health Organization

BACKGROUND: The overexpression of Cox-2 in tumors is important for tumor invasion, angiogenesis, resistance to apoptosis and the suppression of host immunity. Moreover, a tumor's CD44 expression plays an important role in tumor invasion and metastasis. We examined the expression of COX-2 and also CD44 and its variants as well as the biological implications and relationship between Cox-2 and the CD44 variants in non-small cell lung carcinoma. METHODS: The expressions of Cox-2 and also CD44s and its variants (CD44v3 and CD44v6) were examined by performing immunohistochemistry on 98 surgical specimens. RESULTS: The expressions of CD44s, CD44v3 and CD44v6 were significantly more frequent in squamous cell carcinoma specimens than in the adenocarcinoma (CD44s, p=0.033; CD44v3, p=0.007; CD44v6, p=0.022). The loss of CD44s and CD44v3 were significantly correlated with poor tumor differentiation (CD44s, p=0.03; CD44v3, p=0.011). Patients with Cox-2 positive-adenocarcinoma tumors had a significantly worse cumulative survival than did those adenocarcinoma patients without the Cox-2 (p=0.048). The expression of Cox-2 was significantly associated with the CD44s expression in non-small cell lung cancer, and especially in squamous cell carcinoma. CONCLUSIONS: These findings suggest that expression of CD44s is associated with the expression of Cox-2 in NSCLC, and especially squamous cell carcinoma.
Adenocarcinoma ; Antigens, CD44 ; Apoptosis ; Carcinoma, Non-Small-Cell Lung ; Carcinoma, Squamous Cell ; Cyclooxygenase 2* ; Humans ; Immunohistochemistry ; Lung Neoplasms* ; Lung* ; Neoplasm Metastasis

BACKGROUND: Assay of glycosaminoglycans or proteoglycans from skin is complicated due to individual methods where the measurements are highly specialized. The results from these different methods are not able to be compared and there is a large variance. OBJECTIVE: It seems reasonable that a major glycosaminoglycan in the skin, hyaluronic acid, might be ideal as a representative instead of the whole components of glycosaminoglycan. To develop a simple and reliable assay method, the in vitro cell culture system was selected to reduce time and variety of data. The usefulness of the ELISA method, using hyaluronic acid binding protein (HA-ELISA), was evaluated. METHODS: The amount of hyaluronic acid synthesis was measured under a standardized protocol for cultured human skin fibroblasts from the elderly and neonates, as well as the NIH 3T3 mouse fibroblast cell line. To see whether this screening method (HA-ELISA) could be time-saving and reliable under in vitro conditions, some well-known stimulants for glycosaminoglycan synthesis such as retinol, retinyl palmitate, polyethoxyretinide retinamide and hydroxyproline were treated. RESULTS: The production of hyaluronic acid was influenced by both culture condition and source of fibroblasts. The level of quantity showed different patterns due to factors such as culture period, serum in the medium and cell proliferation rate. We found that stable levels of hyaluronic acid assay from culture supernatant were obtained by delaying the sampling time after 24 hours of treatment with stimulants. CONCLUSION: For a reliable quantitative assay, either NIH 3T3 mouse fibroblasts or neonate fibroblasts were suitable. The culture condition and time of harvest should be determined first to estimate the stable kinetics of hyaluronic acid synthesis. This in vitro test protocol can be used as an additional evaluation system towards a potential agent for dermal connective tissue, while further efforts are still mandatory to correlate the confounding factors of in vitro and in vivo.
Aged ; Animals ; Antigens, CD44 ; Cell Culture Techniques ; Cell Line ; Cell Proliferation ; Connective Tissue ; Enzyme-Linked Immunosorbent Assay* ; Fibroblasts* ; Glycosaminoglycans ; Humans* ; Hyaluronic Acid* ; Hydroxyproline ; Infant, Newborn ; Kinetics ; Mass Screening ; Mice ; Proteoglycans ; Skin* ; Vitamin A