Lea-Yea Chuang

Role of receptor for advanced glycation end-product (RAGE) and the JAK/STAT-signaling pathway in AGE-induced collagen production in NRK-49F cells

Advanced glycation end‐product (AGE) is important in the pathogenesis of diabetic nephropathy (DN), and captopril (an angiotensin converting enzyme inhibitor) is effective in treating this disorder. We have shown that the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) cascade is responsible for AGE‐induced mitogenesis in NRK‐49F (normal rat kidney fibroblast) cells, but its role in renal fibrosis in DN remains unknown. Therefore, we have sought to determine whether JAK/STAT is involved in AGE‐regulated collagen production in NRK‐49F cells. We found that AGE time (1–7 days) and dose (10–200 μg/ml)‐dependently increased collagen production in these cells. Additionally, AGE increased RAGE (receptor for AGE) protein expression. AGE‐induced RAGE expression was dose‐dependently inhibited by antisense RAGE oligodeoxynucleotide (ODN) and captopril. AGE‐induced type I collagen production and JAK2‐STAT1/STAT3 activation were decreased by AG‐490 (a specific JAK2 inhibitor), antisense RAGE ODN and captopril. Meanwhile, STAT1 and STAT3 decoy ODNs also suppressed the induction of collagen by AGE. We concluded that RAGE and the JAK2‐STAT1/STAT3 pathway were involved in AGE‐induced collagen production in NRK‐49F cells. Furthermore, captopril was found to reverse AGE‐induced collagen production, probably by attenuating RAGE expression and JAK2‐STAT1/STAT3 activities. J. Cell. Biochem. 81:102–113, 2001.

Non-steroidal anti-inflammatory drugs inhibit matrix metalloproteinase-2 expression via repression of transcription in lung cancer cells.

Recent studies show that up‐regulation of cyclooxygenase‐2 (COX‐2) in human cancer cells induces activation of matrix metalloproteinases (MMPs) and increase of metastatic potential. In this study, we investigate the effect of a COX‐2 selective inhibitor, NS398, on the expression and enzymatic activity of MMPs in human lung cancer cells. We found that NS398 inhibited MMP‐2, not MMP‐9, mRNA expression. NS398 also reduced the amount of MMP‐2, not MMP‐9, released into the medium. Additionally, this COX‐2 inhibitor attenuated the degrading activity of MMP‐2 as demonstrated by gelatin zymography. Investigation of cellular MMP‐2 by Western blotting indicated that synthesis and processing of MMP‐2 was significantly suppressed by NS398. We performed promoter activity assay to address whether NS398 might affect MMP‐2 gene transcription. Our results indicated that NS398 directly inhibited MMP‐2 promoter activity. However, the inhibitory effect of NS398 is not fully dependent on inhibition of COX‐2 because a high concentration of NS398 was needed to suppress MMP‐2 expression and addition of prostaglandin E2 only partially reversed the action of NS398. Moreover, a non‐selective COX inhibitor indomethacin also suppressed the expression of MMP‐2. Taken together, these results indicate that non‐steroidal anti‐inflammatory drugs suppress MMP‐2 expression via repression of transcription and support the notion that COX inhibitors may be useful in inhibition and/or prevention of metastasis.

Bone Morphogenetic Protein-2 Antagonizes Renal Interstitial Fibrosis by Promoting Catabolism of Type I Transforming Growth Factor-β Receptors

TGF-beta is a therapeutic target for renal fibrosis. Scientists have long sought ways to antagonize TGF-beta to ameliorate diabetic nephropathy. Bone morphogenetic protein (BMP-2) is a member of the TGF-beta superfamily and is highly regulated in the kidney. Thus, the role of BMP-2 was investigated in NRK-49F cells (rat fibroblasts). We showed that TGF-beta1 induces an increase in fibronectin. Treatment with exogenous BMP-2 or pCMV-BMP-2 significantly reversed the TGF-beta1-induced increase in fibronectin concomitant with a significant decrease in type I TGF-beta receptors (TGF-beta RI). Moreover, BMP-2 significantly shortened the half-life of TGF-beta RI. These results are related to proteosomal activation because MG132, a proteasome inhibitor, abolished BMP-2-mediated degradation of TGF-beta RI. This was confirmed because BMP-2 time course dependently enhanced the ubiquitination level of TGF-beta RI. In addition, Smads would seem to be involved in the interaction of BMP-2 and TGF-beta. We demonstrated that BMP-2 significantly reversed the TGF-beta1-induced increase in pSmad2/3 and reversed the TGF-beta1-induced decrease in inhibitory Smad7. Most importantly, Smad7 small interfering RNA abolished the BMP-2-induced decrease in TGF-beta RI. We evaluated the clinical efficacy of BMP-2 using unilateral ureteral obstruction rats. BMP-2 was administered ip for 7 d. In the unilateral ureteral obstruction kidneys, interstitial fibrosis was prominent. However, treatment with BMP-2 dramatically reduced Massons trichrome staining (collagen) in the interstitial and tubular areas of the kidneys concomitantly with a reduction in TGF-beta RI. These results suggest that BMP-2 acts as a novel fibrosis antagonizing cytokine partly by down-regulating TGF-beta RI and Smads.

Expression of cyclin D1 and c-Ki-ras gene product in human epithelial ovarian tumors☆

The expression of cyclin D1 gene product in human ovarian tumors was studied. We found that cyclin D1 is expressed at high levels in several ovarian cancer cell lines. Immunohistochemical study also showed that a significant proportion of primary ovarian tumor tissues overexpressed cyclin D1 gene product. Clear nuclear staining of cyclin D1 protein was detected in 28% of the cases. We also characterized the expression of c-Ki-ras gene product in ovarian cancer cell lines and tumor tissues. Amplification or overexpression of this proto-oncogene has been reported in ovarian tumors from Taiwan. These results show that c-Ki-ras is strongly expressed in PA-1 and NIH:OVCAR-3 cells in which cyclin D1 also expressed at high levels. Specific cytoplasmic staining of c-Ki-ras protein was detected in 11 tumors (52%). Statistical analyses show a strong positive correlation between cyclin D1 and c-Ki-ras immunoexpression. Thus, these data support the ideas that cyclin D1 may be involved in the pathogenesis of ovarian cancer, and coactivation of cyclin D1 and c-Ki-ras gene expression may represent one of the major pathways that lead to the development of ovarian cancer in Taiwan.

Role of AKT kinase in sphingosine‐induced apoptosis in human hepatoma cells

Our previous work has shown that a number of sphingolipid metabolites including sphingosine, sphinganine, and other long‐chain bases potently induced apoptosis in human hepatoma cells. In this study, we examined the possibility that sphingosine may trigger apoptosis in human hepatoma cells via inhibition of anti‐apoptotic pathways. We investigated the effect of sphingosine on AKT kinase, a serine/threonine kinase which was found to protect cells from apoptosis induced by a variety of extracellular stresses. Our results indicated that sphingosine inhibited basal and serum‐stimulated AKT kinase activity in a dose‐dependent manner in hepatoma cells. Additionally, sphingosine‐induced inhibition of AKT kinase was correlated with induction of apoptosis in these cells. Pretreatment of insulin, a potent stimulator of AKT kinase, partially reversed the inhibition of AKT kinase by sphingosine and counteracted the apoptotic action of this sphingolipid. Expression of activated AKT kinase partially protected cells from sphingosine‐induced apoptosis, whereas expression of kinase‐dead AKT kinase had no effect. The molecular mechanism by which AKT kinase suppressed the apoptotic action of sphingosine was investigated. Our results showed that increased release of cytochrome C from mitochondria and subsequent activation of caspase‐3 were detected in sphingosine‐treated hepatoma cells. On the contrary, expression of activated AKT kinase in Hep3B cells attenuated cytochrome C release and caspase‐3 activation induced by sphingosine. Taken together, these findings suggest that suppression of AKT kinase is one of the mechanisms by which sphingosine induces apoptosis in hepatoma cells and activation of AKT kinase may inhibit sphingosine‐induced apoptosis by blocking a step upstream of cytochrome C release and caspase‐3 activation.

Inhibitory effect of mimosine on proliferation of human lung cancer cells is mediated by multiple mechanisms.

The plant amino acid mimosine has been reported to block cell cycle progression in the late G1 phase. A recent study showed that mimosine might induce growth arrest by activating the expression of p21CIP1, a cyclin-dependent kinase inhibitor (CDKI), and by inhibiting the activity of cyclin E-associated kinases in human breast cancer cells. However, mimosine at higher concentrations also blocked proliferation of p21-/- cells by unknown mechanisms. In this study, we investigated the effect of mimosine on the expression of cyclins and CDKIs in human lung cancer cells. We found that mimosine specifically inhibited cyclin D1 expression in H226 cells. The expression of another G1 cyclin, cyclin E, was not regulated by mimosine in all lung cancer cell lines examined. Moreover, mimosine induced p21CIP1 expression in H226 and H358 cells, while it activated p27KIP1 expression in H322 cells. However, mimosine does not affect transcription of these genes directly because significant changes in cyclin D1 or CDKI expression were observed at 12-24 h after drug addition. Our results indicate that mimosine may block cell proliferation by multiple mechanisms and this amino acid is a useful agent for the study of cell cycle control.

Serum insulin-like growth factor-II as a serologic marker of small hepatocellular carcinoma

Objective Alpha-fetoprotein (AFP) is not a useful tumor marker for diagnosis of small hepatocellular carcinoma (HCC). There is over-expression of insulin-like growth factor (IGF)-II in HCC tissue. This study investigates the diagnostic application of IGF-II in small HCC. Material and methods Serum levels of IGF-II and AFP were determined in 41 patients with small cirrhotic HCC (≤3 cm), 41 sex- and age-matched patients with cirrhosis alone (LC), and 41 healthy adults. The optimal cut-off values for diagnosing HCC were determined with receiver operating characteristics (ROC) curve. Results Both IGF-II and AFP levels in HCC were higher than those in LC patients or controls (each p=0.0001). The IGF-II levels in LC patients were lower than those in controls (p=0.001). In HCC patients, multivariate analysis indicated that that both IGF-II (odds ratio, 4.54; 95% confidence interval, 2.15–9.55; p=0.0001) and AFP (odds ratio, 1.05; 95% confidence interval, 1.01–1.08; p=0.003) were found to be associated with an increased risk of presence of HCC. The optimal cut-off values of IGF-II (4.1 mg/g prealbumin) and AFP (50 ng/ml) were determined with ROC curves. The sensitivity, specificity, and diagnostic accuracy values for IGF-II were 63%, 90%, and 70%, respectively. Those for AFP were 44%, 95%, and 70%, respectively. Determination of both markers in parallel significantly increase the diagnostic accuracy (88%) and sensitivity (80%), with a high specificity (90%). Conclusions Serum IGF-II level can be used as an independent serologic marker or a complementary tumor marker to AFP for diagnosis of small HCC.

Thrombospondin-1 mediates distal tubule hypertrophy induced by glycated albumin.

Diabetic nephropathy is characterized by early hypertrophy in both glomerular and tubuloepithelial elements. However, no studies to date have established a direct causal link between hyperglycaemia and renal hypertrophy. Our previous studies have found that high glucose does not induce cellular hypertrophy or expression of TGF-beta1 (transforming growth factor-beta1) in distal renal tubule cells [Yang, Guh, Yang, Lai, Tsai, Hung, Chang and Chuang (1998) J. Am. Soc. Nephrol. 9, 182-193]. In the present study, we used AGEs (advanced glycation end-products) to mimic long-term hyperglycaemia. Similar to glucose, AGEs did not induce TGF-beta1 mRNA in distal renal tubule cells [MDCK (Madin-Darby canine kidney) cells]; however, TGF-beta1 bioactivity was increased significantly. This result indicated post-translational regulation. Since TSP-1 (thrombospondin-1) has been demonstrated to activate latent TGF-beta1 in a variety of systems, the following experiments were performed. We found that AGEs dose-dependently increased both intracellular and extracellular levels of TSP-1. Purified TSP-1, like AGEs, increased the cellular protein content. Furthermore, anti-TSP-1 neutralizing antibodies attenuated the AGE-induced increase in TGF-beta1 bioactivity and hypertrophy. Thus TSP-1 might mediate AGE-induced distal renal tubule hypertrophy. In addition, we observed several putative transcription factor binding sites in the TSP-1 promoter, including those for AP-1 (activator protein-1), CREB (cAMP response element binding protein), NF-kappaB (nuclear factor-kappaB), SRF (serum response factor) and HSF (heat-shock factor), by sequence mapping. We used an enhancer assay to screen possible transcription factors involved. We showed that AP-1 and CREB were specifically induced by AGEs; furthermore, TFD (transcription factor decoy) for AP-1 could attenuate the AGE-induced increases in TSP-1 levels and cellular hypertrophy. Thus regulation of TSP-1 might be critical for hyperglycaemic distal tubule hypertrophy. Furthermore, TSP-1 TFD might be a potential approach to ameliorate diabetic renal hypertrophy.

Serum Insulin-Like Growth Factor-II and α-Fetoprotein as Tumor Markers of Hepatocellular Carcinoma

To evaluate the diagnostic application of serum insulin-like growth factor-II (IGF-II) and α-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC), IGF-II and AFP were determined in 100 cirrhotic patients with HCC, 100 sex- and age-matched patients with cirrhosis alone and 50 healthy controls. The results indicated that IGF-II and AFP levels in patients with HCC were higher than in those with cirrhosis alone (p = 0.0001). There is an inverse correlation between IGF-II and logAFP (r = –0.410, p = 0.0001) in patients with HCC. Multivariate analysis indicated that IGF-II and AFP were closely associated, in a dose-related fashion, with the presence of HCC. Receiver operating characteristic curves were used to determine the optimal cutoff values of IGF-II (4.5 mg/g prealbumin) and AFP (100 ng/ml), respectively. Both IGF-II and AFP show a high specificity and positive likelihood ratio. The sensitivity was 42.0% for IGF-II and 73.0% for AFP. Determination of both markers in parallel significantly increased the diagnostic accuracy (96.5%) and sensitivity (97.9%), with a high specificity (95.1%) and positive likelihood ratio (19.9). In conclusion, IGF-II and AFP may be used as complementary tumor markers to discriminate HCC from cirrhosis.

Clinical relevance of transforming growth factor-beta 1 in the urine of patients with hepatocellular carcinoma.

To assess the clinical relevance of transforming growth factor-beta 1 (TGF-beta 1) in the urine of patients with hepatocellular carcinoma (HCC), TGF-beta 1 was measured, by radioimmunoassay, in 140 patients with HCC, 50 cirrhotic patients, 30 patients with chronic active hepatitis, and 50 healthy controls. The results indicate that there were significantly increased urinary TGF-beta 1 levels in patients with HCC. Raised TGF-beta 1 levels were associated, in a dose-related fashion, with increased risk for development of HCC (odds ratio, 1.05, 95% confidence interval, 1.03-1.07). HCC patients with raised TGF-beta 1 levels had shorter survival than those with normal TGF-beta 1 levels (p = 0.038). TGF-beta 1 levels decreased after successful anticancer therapy (p < 0.0001). There was an inverse correlation between TGF-beta 1 and serum alpha-fetoprotein (AFP) (r = -0.199, p < 0.04). Receiver operating characteristics (ROC) curve analysis indicated that parallel determination of TGF-beta 1 and AFP significantly increased the sensitivity and diagnostic accuracy, with a high specificity. In conclusion, raised urinary TGF-beta 1 was associated with HCC development. It is a predictor of poor prognosis, and a tumor marker for diagnosis and therapeutic follow-up of HCC.