On Osteopontin Expression Research Articles

1,2,3-Triazole tethered 1,2,4-trioxanes: Studies on their synthesis and effect on osteopontin expression in MDA-MB-435 breast cancer cells

Artemisinin and its analogs have shown potent anticancer activity in primary cancer cultures and cell lines by inhibiting cancer proliferation, metastasis, and angiogenesis. Despite its apparent compatibility to normal cells and low IC50 values in comparison to the commonly used anticancer drugs, the underlying mechanisms behind their cytotoxic effects are not yet fully understood. Surprisingly, the efficacy of synthetic 1,2,4-trioxanes against cancer has not been explored yet. Given the high antitumor activity of artemisinin dimers in comparison to their monomers, we report here the synthesis of simple 1,2,3-triazole conjugated 1,2,4-trioxanes and their potential antitumor activity by studying their inhibitory effect on osteopontin (OPN) expression in MDA-MB-435 breast cancer cells. It may be noted that despite being a strong marker to identify human tumor metastasis, no study on effect of artemisinin and its synthetic and semisynthetic derivatives on OPN expression has ever been studied. Although our initial studies did not notice any straight-line relationship between the number of trioxane units in a molecule to the extent of inhibition of OPN protein expression, we could observe better results in some cases in comparison to artemisinin. We have observed that artemisinin did not show appreciable OPN downregulation in MDA-MB-435 cancer cells, but dihydroartemisinin (DHA) and some synthetic 1,2,4-trioxane monomers and dimers showed downregulation of OPN expression. Therefore, these compounds may act as an anti-metastatic agent in controlling breast cancer cells metastasis.

Therapeutic effect of combined hyperbaric oxygen and radiation therapy for single brain metastasis and its influence on osteopontin and MMP-9.

The present study aimed to investigate the therapeutic effect of combined hyperbaric oxygen and radiation therapy for the treatment of single brain metastasis (SBM), as well as its influence on osteopontin (OPN) and matrix metalloproteinase-9 (MMP-9). A total of 86 patients with SBM were admitted to Hongqi Hospital from January 2013 to January 2016 and those included within the study were randomly divided into two groups. The control group was only treated with whole brain radiotherapy, while the observation group was treated with hyperbaric oxygenation combined with whole brain radiotherapy. OPN and MMP-9 expression was measured in each group by ELISA and the results prior to and following treatment were compared. The total effective rate (patients with complete remission, partial remission or stabilized lesions) in the observation group (95.3%) was significantly increased compared with the control group (67.4%). However, the OPN and MMP-9 protein levels observed in the observation group were significantly reduced compared with the control group (P<0.05). In addition, the quality of life and the incidence of adverse reactions in the observation group were significantly improved compared with the control group (P<0.05). For patients with SBM, hyperbaric oxygenation combined with radiotherapy may improve the efficiency of treatment and should be considered for further investigation and use within a clinical setting.

Abstract 4188: Preoperative supplementation of branched-chain amino acids suppresses Osteopontin expression in patients with hepatocellular carcinoma

Abstract Background: Recently, the new effect of branched-chain amino acid (BCAA) on Osteopontin (OPN), which correlates with cancer progression, has been reported. The aim of this study was that preoperative administration of BCAA could improve prognosis via degradation of OPN expression in hepatocellular carcinoma (HCC). Methods: Human hepatocellular carcinoma cell lines were treated with BCAA (2nM), and investigated OPN expression, cell proliferation and invasion in vitro. Next, patients undergoing elective liver resections for HCC (n=72) were randomized to receive or not receive preoperative oral supplementation with BCAA. OPN protein levels in HCC patients were examined using immunohistochemistry and compared with the clinicopathologic features of the tumors. Results: Experimentally, the expression levels of OPN were significantly decreased in HCC cells treated with BCAA. BCAA treatment significantly decreased cell invasion. In the clinical setting, OPN expression correlated with HBV or HCV infection (p = 0.03), venous invasion (p = 0.02) and liver fibrosis (p = 0.07). There was no difference in both DFS and OS between OPN-positive and OPN-negative group, and preoperative BCAA administration did not improve prognosis, either. For the patients without late recurrence, OPN-positive group showed significantly poor outcome in DFS (p = 0.047), and preoperative BCAA administration improved DFS (p = 0.062). Conclusions: OPN expression was associated with cancer cell invasion and intrahepatic metastasis. BCAA treatment suppressed HCC cell invasion and preoperative BCAA administration prevented early intrahepatic recurrence by decreasing the expression level of OPN in HCC. Citation Format: Yukihiko Hiroshima, Yasuhiro Yabushita, Yutaro Kikuchi, Daisuke Kawagushi, Takashi Murakami, Yasushi Ichikawa, Itaru Endo, Kuniya Tanaka. Preoperative supplementation of branched-chain amino acids suppresses Osteopontin expression in patients with hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4188.

PYK2 mediates BzATP-induced extracellular matrix proteins synthesis

Mechanical stimuli such as fluid shear and cyclic tension force induced extracellular adenosine triphosphate (ATP) release in osteoblasts. In particular, cyclic tension force-induced ATP enhances bone formation through P2X7 activation. Proline-rich tyrosine kinase 2 (PYK2) mediate osteoblasts differentiation is induced by mechanical stimuli. Furthermore, activation of PYK2 also was a response to integrin by mechanical stimuli. Extracellular matrix protein (ECMP)s, which are important factors for bone formation are expressed by osteoblasts. However, the effect of the interaction of 2’(3)-Ο-(4-Benzoylbenzoyl) adenosine-5’-triphosphate (BzATP), which is the agonist of the mechanosensitive receptor P2X7, with PYK2 on ECMP production is poorly understood. Thus, our purpose was to investigate the effects of PYK2 on BzATP-induced ECMP production in osteoblasts.BzATP increased phospho-PYK2 protein expression on days 3 and 7 of culture. Furthermore, the PYK2 inhibitor PF431394 inhibited the stimulatory effect of BzATP on the expression of type I collagen, bone sialoprotein and osteocalcin expression. PF431396 did not inhibit the stimulatory effect of BzATP on osteopontin (OPN) mRNA expression.These results suggest that mechanical stimuli activate P2X7 might induce ECMPs expression through PYK2 except in the case of OPN expression. Altogether, mechanical stimuli-induced ECMPs production might be implicated by extracellular ATP secretion or integrin via PYK2 activation.

Higher Matrix Stiffness Upregulates Osteopontin Expression in Hepatocellular Carcinoma Cells Mediated by Integrin β1/GSK3β/β-Catenin Signaling Pathway.

Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3β and the expression of nuclear β-catenin were also elevated, indicating that GSK3β/β-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin β1 showed that OPN expression and p-GSK3β level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3β led to accumulation of β-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin β1/GSK-3β/β-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin β1/GSK-3β/β-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.

Wogonin suppresses osteopontin expression in adipocytes by activating PPARα.

Wogonin (5,7-dihydroxy-8-methoxyflavone), a major bioactive compound of the flavonoid family, is commonly extracted from the traditional Chinese medicine Scutellaria baicalensis and possesses antioxidant and anti-inflammatory activities and is assumed to have anti-diabetes function. Indeed, a current study has shown that it can possibly treat metabolic disorders such as those found in db/db mice. However, the underlying molecular mechanism remains largely unclear. The aim of this study was to investigate the impact of wogonin on osteopontin (OPN) expression in adipose tissue from type 1 diabetic mice and in 3T3-L1 adipocytes. Type 1 diabetes was induced by streptozotocin (STZ) injection. 3T3-L1 preadipocytes were converted to 3T3-L1 adipocytes through treatment with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine (IBMX). Western blot analysis and RT-PCR were performed to detect protein expression and mRNA levels, respectively. Wogonin treatment suppressed the increase in serum OPN levels and reduced OPN expression in adipose tissue from STZ-induced type 1 diabetic mice. Administration of wogonin enhanced PPARα expression and activity. Silencing of PPARα diminished the inhibitory effects of wogonin on OPN expression in 3T3-L1 adipocytes. Furthermore, the levels of c-Fos and phosphorylated c-Jun were reduced in wogonin-treated adipose tissue and 3T3-L1 adipocytes. In addition, wogonin treatment dramatically mitigated p38 MAPK phosphorylation. Pharmacological inhibition of p38 MAPK by its specific inhibitor SB203580 increased PPARα activity and decreased OPN expression. Our results suggest that wogonin downregulated OPN expression in adipocytes through the inhibition of p38 MAPK and the sequential activation of the PPARα pathway. Given the adverse effects of high OPN levels on metabolism, our results provide evidence for the potential administration of wogonin as a treatment for diabetes.

Spatial localization of the JAG1/Notch1/osteopontin cascade modulates extrahepatic metastasis in hepatocellular carcinoma.

The model of Notch-driven carcinogenesis and development of hepatocellular carcinoma remains controversial and is based on observations of developmental stage- and dose-dependent Notch activation. In this study, the relevance of the spatial distribution of Notch cascade members to the promotion of hepatocellular carcinoma metastasis was evaluated. The spatial expression patterns of the members of the Jagged1 (JAG1)/Notch1 cascade in HCC were evaluated in a tissue microarray of 112 tumors and 46 peri-tumors. Regulation of JAG1/Notch1 on osteopontin (OPN) was evaluated by RNA interference. Tumor cells with JAG1 expressed on the membrane (JAG1(Mem)) were more likely to undergo extrahepatic metastasis [p<0.001; hazard ratio (HR), 0.166; 95% CI, 0.068-0.402], and JAG1(Mem) was a strong independent prognostic factor for metastasis (HR, 0.467; 95% CI, 0.271-0.806; p=0.006). JAG1(Mem) also showed a strong positive correlation with Notch1(Mem). In addition, tumors with JAG1(Mem) expression had more poorly encapsulated membranes (p=0.014). Furthermore, Notch1(Mem) expression correlated with HCC metastasis and was the strongest predictive factor for metastasis. However, in peri-tumoral tissues, most JAG1 (45/46) and Notch1 (41/46) was localized to the cytoplasm. The expression of OPN, one of the main targets of JAG1/Notch1 signaling and a crucial metastasis-related gene in HCC, correlated significantly with JAG1(Mem) expression. Knockdown of JAG1 expression or Notch1 expression induced the downregulation of OPN in HCC cells. Taken together, protein localization is a critical factor affecting the activity of the Notch cascade in the development of hepatocellular carcinoma. Furthermore, our results suggest that the JAG1/Notch1/OPN cascade represents a potential therapeutic target for hepatocellular carcinoma metastasis.

Transmembrane receptor CXCR7 increases the risk of extrahepatic metastasis of relatively well-differentiated hepatocellular carcinoma through upregulation of osteopontin

Recurrence and metastasis are the main obstacles to improving the survival of patients with post-resective hepato-cellular carcinoma (HCC). Our previous study suggests a critical role of CXCR7 in the metastasis of HCC. In the present study, the effect of CXCR7 as a risk factor for metastasis of HCC was evaluated. Immunohistochemical assay was performed on tissue microarrays based on HCC with extrahepatic metastases after hepatectomy. Two categories based on staining scores were used to evaluate the risk effect of CXCR7, respectively. The effect of CXCR7 on osteopontin (OPN) was explored by RNA interference. Based on the results, in both categories, highly expressed CXCR7 was a dependent risk factor for extrahepatic metastasis because of the potential association with relatively good cell differentiation. Stratification analyses indicated that CXCR7 was a strong independent risk factor (OR, 3.40; 95% CI, 1.07-18.84; P=0.038 in category 1 and OR, 6.40; 95% CI, 1.64-24.92; P=0.007 in category 2, respectively) in patients with Edmondson grade 1/2. Furthermore, CXCR7 correlated well and positively with expression of OPN (P=0.019 and P<0.001 in two categories, respectively) in HCC cases with Edmondson grade 1/2. Immunocytochemistry and RT-PCR demonstrated downregulation of OPN in a highly metastatic HCC cell line following knockdown of CXCR7. Taken together, these findings suggest that high expression of CXCR7 increases the risk of metastasis in post-resective HCC patients with relatively good differentiated tumors, potentially through upregulation of OPN. This group of patients may acquire a survival benefit from early detection and treatment of recurrence and metastasis.

Differential effects of high-fat-diet rich in lard oil or soybean oil on osteopontin expression and inflammation of adipose tissue in diet-induced obese rats

To examine the effect of different dietary fat types on osteopontin (OPN) expressions and inflammation of adipose tissues in diet-induced obese rats. Male Sprague-Dawley rats were randomly assigned to one control group fed standard diet (LF, n = 10) and two high-fat diet groups fed isoenergy diet rich in lard or soybean oil (HL or HS, n = 45 each). Diet-induced obese rats in HL and HS group were then subdivided into two groups either continuously fed high-fat diet or switched to low-fat diet for 8 more weeks. Fasting serum glucose, insulin, and OPN concentrations were assayed and QUICKI was calculated; the expression of OPN, IL-6, IL-10, TNF-α, NF-κB, and F4/80 in adipose tissue was determined. Both high-fat diets lead to comparable development of obesity characterized by insulin resistance and adipose tissue inflammation. Obese rats continuously fed high-fat diet rich in lard oil exhibited the highest fasting serum insulin level and adipose tissue OPN, F4/80, TNF-α, and NF-κB expression level. In both high-fat diet groups, switching to low-fat diet resulted in less intra-abdominal fat mass, decreased expression of F4/80, TNF-α, and NF-κB, while decreased OPN expression was only observed in lard oil fed rats after switching to low-fat diet. Reducing diet fat or replacing lard oil with soybean oil in high-fat diet alleviates obesity-related inflammation and insulin resistance by attenuating the upregulation of OPN and macrophage infiltration into adipose tissue induced by high-fat diet.

Expression and clinicopathological significance of OPN and CD44v6 in lung cancer

There have been a lot of studies about surface adhesion molecule (CD44) in recent years, however study on osteopontin (OPN) is still few. The aim of this study is to investigate the levels of OPN and CD44v6 in lung cancer and their correlation. OPN and CD44v6 expression were detected in 78 lung cancer tissues by immunohistochemistry. The positive rate of OPN and CD44v6 expression in non-small cell lung cancer (NSCLC) was 58.46% and 66.15% respectively, but no positive case in small cell lung cancer. The expression of OPN and CD44v6 in NSCLC was closely related to TNM stages (P < 0.01), but not to cell differentiation (P > 0.05). There was a remarkably positive correlation between the expression of OPN and CD44v6 (r=0.255, P < 0.05). The co-expression of OPN and CD44v6 may be involved in progression and metastasis of lung cancer. The interrelationship of OPN and CD44v6 was coordinative with development and metastasis of lung cancer. It might be helpful to predict the metastasis and prognosis of NSCLC.

Expression and clinicopathologic significance of OPN, CD44v6 and MMP-2 in squamous cell carcinoma and adenocarcinoma of the lung

There are a lot of studies on relationship of surface adhesion molecule (CD44) and matrix metalloproteinase (MMP) with tumors in recent years, however study on osteopontin (OPN) is still few. The aim of this study is to investigate the levels of OPN, CD44v6 and MMP-2 in squamous cell carcinoma and adenocarcinoma of the lung, and to clearly understand their roles in growth, invasion and metastasis of squamous cell carcinoma and adenocarcinoma. OPN, CD44v6 and MMP-2 were detected in 69 patients with squamous cell carcinoma and adenocarcinoma by immunohistochemical method. The expression rate of OPN, CD44v6 and MMP-2 was significantly related to histological classification, TNM stages and lymph node metastasis (P < 0.05), but not to cell differentiation (P > 0.05). There was a positive correlation between OPN and CD44v6 expression (P < 0.001), as well as between OPN and MMP-2 expression (P < 0.001). There was no correlation between CD44v6 and MMP-2 expression. OPN, CD44v6 and MMP-2 expression is related to the histology, TNM stages and lymph node metastasis of lung cancer. They might be used as clinical indicators to predict the progress and metastatic potential for squamous cell carcinoma and adenocarcinoma of the lung.

Insulin-like growth factor-I attenuates the inhibitory effect of type I collagen through β1 integrin receptor

Extracellular matrix and growth factors are the crucial factors that regulate healing and regenerating processes in human periodontal ligament cells. The purpose of this study was to examine the effects of type I collagen and insulin-like growth factor-I (IGF-I) on osteopontin (OPN) expression. The data showed that OPN expression was significantly decreased when cells were cultured on collagen-coated plates. Addition of IGF-I obviously induced OPN expression only in a collagen-coated condition, suggesting an attenuating effect of IGF-I on the decrease of OPN expression. Cells treated with a combination of inhibitory antibody to β1 integrin and IGF-I showed the same level of OPN expression as those treated with either inhibitory antibody to β1 integrin or IGF-I alone. These results indicate that IGF-I counteracts with the inhibitory signal from type I collagen through β1 integrin receptor.

Colchicine Suppresses Osteopontin Expression and Inflammatory Cell Infiltration in Chronic Cyclosporine Nephrotoxicity

Background: Colchicine (Col) is beneficial to renal injury because of its anti-inflammatory effect, but its mechanism has yet to be elucidated. The present study was designed to evaluate the inhibitory effects of colchicine on osteopontin (OPN) expression and the macrophage accumulation in chronic cyclosporine (CsA) nephrotoxicity in rats. Methods: Male adult Sprague-Dawley rats on a low salt diet (LSD, 0.05% sodium) were treated daily with Col (30 µg/kg), CsA (15 mg/kg), and both CsA and colchicine or vehicle (olive oil 1 ml/kg) for 4 weeks. The effects of colchicine on chronic CsA nephrotoxicity were evaluated by examining renal function, histopathology, and ED-1 positive cells. The expressions of OPN mRNA and protein were estimated respectively by Northern blot and immunohistochemistry. Results: Compared with vehicle-treated rats, CsA-treated rats showed an increase in serum creatinine, a decline in creatinine clearance rate, and tubulointerstitial fibrosis (all p < 0.01). Concomitant administration of colchicine reversed all of the above parameters (all p < 0.01). Of note, the upregulated expression of osteopontin mRNA and protein seen in CsA-treated rats was significantly decreased after colchicine treatment. Furthermore, the expression of osteopontin mRNA was strongly correlated with the number of ED-1 positive cells (r = 0.712, p < 0.001) and the tubulointerstitial fibrosis score (r = 0.586, p = 0.007). Conclusion: Colchicine is capable of abrogating the upregulation of chemotactic OPN expression and macrophage influx, and this is associated with improved renal tubulointerstitial fibrosis in chronic CsA nephrotoxicity.

Effects of citrate on renal stone formation and osteopontin expression in a rat urolithiasis model.

Previous studies have described the inhibitory effects of citrate on calcium oxalate crystallization in place of crystal growth, but the effects of citrate on matrix proteins of stones has not been studied in vivo. To examine the effect of citrate on the matrix, we investigated the effect of citrate on osteopontin (OPN) expression, which we had previously identified as an important stone matrix protein. Control rats were treated with saline while rats of the stone group were treated with ethylene glycol (EG) and vitamin D3, and the citrate groups (low-dose and high-dose groups) were treated with a citrate reagent compound of sodium citrate and potassium citrate, in addition to EG and vitamin D3. The rate of renal stone formation was lower in the citrate groups than in the stone group. This was associated with a low expression of OPN mRNA in citrate-treated rats relative to that in the stone group. Citrate was effective in preventing calcium oxalate stone formation and reduced OPN expression in rats. Our results suggest that citrate prevents renal stone formation by acting against not only the crystal aggregation and growth of calcium oxalate but also OPN expression.

The effect of takusha, a kampo medicine, on renal stone formation and osteopontin expression in a rat urolithiasis model.

Kampo medicine is a traditional Japanese therapeutic system which originated in China and was used to treat various diseases for hundreds of years. Kampo medicine had been also used for the cure and the prevention of urinary calculi for many years, but the effect and the mechanism of this use of kampo medicine are unclear. We examined the inhibitory effect of the kampo medicine takusha on the formation of calcium oxalate renal stones induced by ethylene glycol (EG) and vitamin D3 in rats. We also investigated the effect of takusha on osteopontin (OPN) expression, which we previously identified as an important stone matrix protein. The control group rats were non-treated; the stone group rats were administered EG and vitamin D3, and the takusha group was administered takusha in addition to EG and vitamin D3. The rate of renal stone formation was lower in the takusha group than in the stone group; thus, the OPN expression in the takusha group was smaller than in the stone group. Takusha was effective in preventing oxalate calculi formation and OPN expression in rats. These findings suggest that takusha prevents stone formation including not only calcium oxalate aggregation but also proliferation.