Comparative effects of hepatocyte growth factor and tacrolimus on acute liver allograft early tolerance.

Allostimulated CD8+ T cells (aCD8+ T cells), as the main mediators of acute liver rejection (ARJ), are hyposensitive to apoptosis due to the inactivation of death receptor FAS-mediated pathways and fail to allow tolerance induction, eventually leading to acute graft rejection. Although tacrolimus (FK506), the most commonly used immunosuppressant (IS) in the clinic, allows tolerance induction, its use is limited because its target immune cells are unknown and it is associated with increased incidences of malignancy, infection, and nephrotoxicity, which substantially impact long-term liver transplantation (LTx) outcomes. The dark agouti (DA)-to-Lewis rat LTx model is a well-known ARJ model and was hence chosen for the present study. We show that both hepatocyte growth factor (HGF) (cHGF, containing the main form of promoting HGF production) and recombinant HGF (h-rHGF) exert immunoregulatory effects mainly on allogeneic aCD8+ T cell suppression through FAS-mediated apoptotic pathways by inhibiting cMet to FAS antagonism and Fas trimerization, leading to acute tolerance induction. We also showed that such inhibition can be abrogated by treatment with neutralizing antibodies against cMet (HGF-only receptor). In contrast, we did not observe these effects in rats treated with FK506. However, we observed that the effect of anti-rejection by FK506 was mainly on allostimulated CD4+ T cell (aCD4+ T cell) suppression and regulatory T cell (Treg) promotion, in contrast to the mechanism of HGF. In addition, the protective mechanism of HGF in FK506-mediated nephrotoxicity was addressed. Therefore, HGF as a tolerance inducer, whether used in combination with FK506 or as monotherapy, may have good clinical value. Additional roles of these T-cell subpopulations in other biological systems and studies in these fields will also be meaningful.

GINS2 was regulated by lncRNA XIST/miR-23a-3p to mediate proliferation and apoptosis in A375 cells.

Melanoma ranks second in aggressive tumors, and the occurrence of metastasis in melanoma results in a persistent drop in the survival rate of patients. Therefore, it is very necessary to find a novel therapeutic method for treating melanoma. It has been reported that lncRNA XIST could promote the tumorigenesis of melanoma. However, the mechanism by which lncRNA XIST regulates the progression of melanoma remains unclear. The proliferation of A375 cells was measured by clonal formation. Cell viability was detected by MTT assay. Flow cytometry was performed to detect cell apoptosis and cycle. The level of GINS2, miR-23a-3p, and lncRNA XIST was investigated by qRT-PCR. Protein level was detected by Western blot, and the correctness of prediction results was confirmed by Dual luciferase. In present study, GINS2 and lncRNA XIST were overexpressed in melanoma, while miR-23a-3p was downregulated. Silencing of GINS2 or overexpression of miR-23a-3p reversed cell growth and promoted apoptosis in A375 cells. Mechanically, miR-23a-3p directly targeted GINS2, and XIST regulated GINS2 level though mediated miR-23a-3p. Moreover, XIST exerted its function on cell proliferation, cell viability, and promoted the cell apoptosis of A375 cells though miR-23a-3p/GINS2 axis. LncRNA XIST significantly promoted the tumorigenesis of melanoma via sponging miR-23a-3p and indirectly targeting GINS2, which can be a potential new target for treating melanoma.

MiR-291a-3p regulates the BMSCs differentiation via targeting DKK1 in dexamethasone-induced osteoporosis.

Osteoporosis is a skeleton disease affecting 55% of people over age 60, and the number is still increasing due to an ageing population. One method to prevent osteoporosis is to increase the formation of new bone while preventing the resorption of older bone. Thus, osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is of great importance in improving the treatment of osteoporosis. On the other hand, glucocorticoids (GCs) are widely used to treat the chronic inflammatory disorders, but long-term exposure to GCs can induce osteoporosis. In present study, we treated BMSCs with dexamethasone (DEX) to simulate GC-induced osteoporosis. MTT assay, ALP activity, and Alizarin Red were used to evaluate the role miRNA-291a-3p in the DEX-induced osteogenic differentiation suppression. Further, we used qPCR and western blot to investigate the mechanisms of miRNA-291a-3p affecting BMSCs differentiation. The results showed that miRNA-291a-3p could improve the cell viability, osteogenic differentiation, and ALP activity, which are suppressed by DEX in BMSCs. Furthermore, we found that the osteogenesis genes Runx2, DMP1, and ALP were upregulated whereas the lipogenic genes C/EBPα and PPARγ were downregulated when miRNA-291a-3p mimics were transfected. Additionally, we demonstrated that miRNA-291a-3p promoted BMSCs' osteogenic differentiation by directly suppressing DKK1 mRNA and protein expression and subsequently activating Wnt/ß-catenin signaling pathway. Our study suggests that miR-291a-3p plays an important role in preventing osteoporosis and may serve as a potential miRNA osteoporosis biomarker.

Notch1-ADAM8 positive feed-back loop regulates the degradation of chondrogenic extracellular matrix and osteoarthritis progression.

BACKGROUND: Osteoarthritis (OA) is one of the most prevalent joint disease, and there are still no effective therapeutic agents or clinical methods for the cure of this disease to date. The degradation of cartilage extracellular matrix (ECM) is a major cause of OA. METHOD: IL-1ß was used to induce chondrogenic degradation. Q-PCR and Western blotting were used to detect mRNA and protein level, respectively. ELISA was used to detect the secreted TNF-α and IL-6 level. Immunofluorescence was used to detect the protein level of Aggrecan, Collagen II and ki67. TUNEL and flow cytometry were used to examine cell apoptosis of chondrocytes. ChIP and luciferase assay were used to study molecular gene regulation. Osteoarthritic animal model and Safranin-O staining were used to determine the in vivo OA phenotype. RESULTS: The expression of ADAM8 was up-regulated in osteoarthritic chondrocytes. Knockdown of ADAM8 suppressed the OA phenotype in the in vitro OA cell model. ADAM8 regulated OA progression through the activation of EGFR/ERK/NF-κB signaling pathway. Inhibition of Notch signaling suppressed OA phenotype in the in vitro OA cell model. Notch signaling regulated the gene expression of ADAM8 directly via Hes1. Notch1-ADAM8 positive feedback loop promoted the progression of OA in vivo. CONCLUSION: Notch1-ADAM8 feed-back loop regulates the degradation of chondrogenic extracellular matrix and osteoarthritis progression.

ADAM8 promotes chondrosarcoma cell migration and invasion by activating the NF-κB/MMP-13 signaling axis.

ADAM8 is reported to promote extracellular matrix degradation to provide conditions for tumor metastasis. However, the underlying mechanism of ADAM8 in modulating chondrosarcoma (CHS) metastasis remains unclear. We used two human CHS cell lines SW1353 and HCS-2/8 to analyze the expression profiles of ADAM8 in CHS cells compared with the normal chondrocytes. An important proteolytic enzyme MMP-13 was detected as a marker for extracellular matrix degradation in chondrocytes. Then, by silencing or overexpressing ADAM8, the effects on cell migration and invasion in SW1353 and HCS-2/8, and the downstream signal transduction pathways were evaluated. ADAM8 and MMP-13 were highly expressed, and the NF-κB pathway was activated in SW1353 and HCS-2/8 cells. Silencing ADAM8 significantly reduced the ability of cell migration and invasion, and blocked the NF-κB signaling pathway through IκBα and p65 dephosphorylation, leading to reduced NF-κB transcription activity and decreased MMP-13 expression. ADAM8 overexpression promoted these processes, which, however, were reversed by an inhibitor Bay 11-7085. Our data showed a novel regulation mechanism for ADAM8 in promoting CHS migration and invasion by activating the NF-κB/MMP-13 signaling axis. Modulation of their levels may serve as potential targets in the treatment of CHS and even other cartilage diseases.

Downregulation of survivin by adenovirus-mediated shRNA promotes apoptosis in skin cancer cells.

BACKGROUND: Survivin, a member of the inhibitor of apoptosis protein family, is highly expressed in many cancers and has important roles in inhibiting apoptosis by blocking caspase activation. However, its antitumor effects remain largely unknown. Here we explore the function of survivin in skin cancer. METHODS: We used qPCR and Western blot to examine survivin expression in skin cancer patients and cell line. We generated several survivin shRNA constructs and tested the effects of survivin shRNA on cancer cell viability using MTT assay, flow cytometry, and TUNEL assay. RESULTS: We found that survivin was upregulated in both skin cancer patients and skin cancer cell line A431. Knockdown survivin via shRNA inhibited cancer cell proliferation and promoted apoptosis in both A431 cell and in vivo xenograft tumor mouse model. The antitumor effect is comparable to resveratrol, a drug known to inhibit cancer progression. Moreover, we showed that inhibition of survivin was able to increase the expression of cleaved caspase 7/caspase 9 and activate the ataxia-telangiectasia mutated-NF-κB pathway in A431 cells. CONCLUSION: Survivin-shRNA possesses antitumor abilities in vitro and in vivo by inhibiting the proliferation and promoting apoptosis of A431 cells. It may serve as a potential anticancer target for skin cancer therapy in the future.

miR-26b modulates OA induced BMSC osteogenesis through regulating GSK3ß/ß-catenin pathway.

BACKGROUNDS: Osteoactivin (OA) is a key regulator promoting bone marrow stromal cells osteogenesis progress, while Dexamethasone (Dex) could inhibit OA induced osteogenesis and lead to osteoporosis. miR-26b increased during BMSC osteogenesis but whether it participates in this progress is enigma. Osteogenesis is under regulation of canonical Wnt signaling pathway which could serve as potential target for miR-26b. It bears therapeutic potential if miR-26b could regulate osteogenesis and antagonize Dex induced Osteoporosis (OP). METHODS: BMSC were isolated from bone marrow of rats and induced for osteogenesis by OA administration. We detected miR-26b mRNA level together with osteogenesis related genes or Wnt signal pathway related genes by qRT-PCR. BMSC cells with miR-26b inhibitor or mimics revealed the effect of miR-26b on osteogenesis. The osteogenesis efficiency was detected by Alizarin Red staining and ALP activity. Protein level of canonical Wnt signal pathway and other proteins were detected by Western blot. The interaction between miR-26b and GSK3ß was detected by dual luciferase reporter assay. RESULTS: We found that miR-26b was increased during OA induced BMSC osteogenesis. Inhibiting miR-26b could lead to osteogenesis inhibition while miR-26b mimics could promote this progress. The key regulator of Wnt signal pathway GSK3ß is down-regulated when miR-26b was overexpressed, resulting in ß-catenin activation. Since Dex could promote GSK3ß expression and inhibit Wnt signal, miR-26b could also alleviate Dex induced osteogenesis inhibition. CONCLUSION: Our findings indicate that miR-26b promoted BMSC osteogenesis by directly targeting GSK3ß and activating canonical Wnt signal pathway, suggesting miR-26b might be serve as potential therapeutic candidate of osteoporosis.

Osteoactivin inhibits dexamethasone-induced osteoporosis through up-regulating integrin ß1 and activate ERK pathway.

BACKGROUNDS: Dexamethasone (Dex) is widely used in autoimmune diseases and inflammation treatment. A sever side effect of prolonged exposure to Dex is increased risk of osteoporosis (OP) or even femoral head necrosis, which would cause much suffer to patients. To reveal the mechanism behind this phenomenon, provide therapeutic guidance and potential target, we analyzed the inhibitory mechanism of Dex on osteogenesis of rat-BMSC. METHODS: Rat BMSC were obtained and characterized with FACS analysis. Osteogenesis and adipogenesis abilities were detected with Oil-O-Red staining, Alizarin Red staining and ALP activity analysis. These BMSC were then treated with Dex in combination with recombinant OA or not and detected for osteogenesis related gene expression with qRT-PCR. Protein interaction and expression were detected by Co-Immunoprecipitation and western blot. RESULTS: Osteoactivin (OA) could promote integrin ß 1 expression and interact with this protein physically, leading to ERK activation and promoting osteogenesis related genes' expression including Runx2, Col1a and OCN in BMSC. Dex, however, could block expression of several upstream genes of OA and decrease OA mRNA and protein level, and eventually suppress integrin ß1-ERK activation and lead to decreased osteogenesis, which could finally develop into OP. CONCLUSION: Recombinant OA treated BMSC exerted better osteogenesis potency even with Dex administration. This is because additional OA in medium counter-acts with Dex's influence and rescued osteoblast differentiation via up-regulating integrin ß1 and activate ERK/MAPK pathway which promotes osteogenesis. Hence, OA/integrin ß1 could serve as potential therapeutic target for OP.

Roles of Signaling Pathways in the Epithelial-Mesenchymal Transition in Cancer.

The epithelial-mesenchymal transition (EMT) is a cellular process though which an epithelial phenotype can be converted into a phenotype of mesenchymal cells. Under physiological conditions EMT is important for embryogenesis, organ development, wound repair and tissue remodeling. However, EMT may also be activated under pathologic conditions, especially in carcinogenesis and metastatic progression. Major signaling pathways involved in EMT include transforming growth factor ß(TGF-ß), Wnt, Notch, Hedgehog and other signaling pathways. These pathways are related to several transcription factors, including Twist, Smads and zinc finger proteins snail and slug. These interact with each other to provide crosstalk between the relevant signaling pathways. This review lays emphasis on studying the relationship between EMT and signaling pathways in carcinogenesis and metastatic progression.

[Study of apoptosis related factors regulatory mechanism of resveratrol to human skin squamous cell carcinoma A431 xenograft in nude mice].

OBJECTIVE: To explore the anti-tumor effects of resveratrol (Res) upon human skin squamous cell carcinoma A431 xenograft in nude mice and elucidate the regulatory mechanisms of survivin and caspase-3. METHODS: The model of human skin squamous cell carcinoma (A431) xenograft in nude mice was established. And the animals were randomly divided into saline-negative control, cyclophosphamide (CTX) positive control, Res high-, medium- and low-dosage and blank control groups (n = 10 each). After drug intervention, tumor-bearing mice were sacrificed. The tumor growth curve was plotted and the Res inhibition rate calculated by terminal tumor weight. The morphological changes of tumor cell among groups were observed by hematoxylin and eosin staining; cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL; the impact of Res upon the protein expressions of survivin and caspase-3 in tumor issues was observed by Western blot. Analysis of variance and Pearson's correlation were employed for statistical analyses. RESULTS: (1) By the end of treatment, the tumor volume of CTX, Res high-, medium-, low-dosage, saline-negative control and blank control groups were (1154 ± 255), (1002 ± 115), (1207 ± 176), (1342 ± 211), (1642 ± 226), (1564 ± 156) mm(3) respectively, and tumor weight of CTX, Res high-, medium-, low-dosage, saline-negative control and blank control groups (1.84 ± 0.30), (1.72 ± 0.39), (1.96 ± 0.40), (2.67 ± 0.73), (3.16 ± 0.52), (3.33 ± 0.59) g respectively. Through analysis of variance, the tumor volume and weight of Res high-, medium-, low-dosage groups were smaller than those of saline-negative control and blank control groups (all P < 0.05). The inhibition rate of Res high-, medium- and low-dosage groups were 45.57%, 37.97% and 15.51% respectively. (2) The apoptosis index of the above groups were 36.79% ± 8.86%, 33.15% ± 6.00%, 18.09% ± 3.92%, 10.53% ± 4.20%, 3.87% ± 1.63%, 2.73% ± 1.61%. Through analysis of variance, the apoptosis index of Res groups were higher than those of saline-negative control and blank control groups (all P < 0.05). (3) The protein expression of survivin/ß-actin of each group were 0.48 ± 0.20, 0.19 ± 0.11, 0.22 ± 0.12, 0.28 ± 0.24, 0.98 ± 0.41, 0.85 ± 0.34. The protein expression of caspase-3/ß-actin of each group were 0.42 ± 0.09, 0.31 ± 0.10, 0.31 ± 0.07, 0.22 ± 0.08, 0.14 ± 0.04, 0.13 ± 0.05 respectively. Through analysis of variance, the protein expression of survivin of Res groups was lower than those of the saline-negative control and blank control groups (all P < 0.05). And the protein expression of caspase-3 of Res groups were higher than those of the saline-negative control and blank control group (all P < 0.05). Through Pearson's analysis, the protein expression of survivin and caspase-3 had no correlation (r = -0.279, P > 0.05). CONCLUSIONS: Res inhibits the growth of human skin squamous cell carcinoma A431 xenograft in nude mice. And its mechanism may be associated with the apoptosis of tumor cell through the depression of survivin and the activation of caspase-3.

The inhibition of resveratrol to human skin squamous cell carcinoma A431 xenografts in nude mice.

Squamous cell carcinoma (SCC) is one of the commonest dermatological malignancies. Resveratrol (Res) is one type of polyphenolic compound which was first identified from the roots of Veratrum grandinorum in 1940. The previous studies found that Res can promote apoptosis of a variety of tumor cell, especially SCC cells. However it is rare to study the inhibition mechanism of Res in the animal model. In this study, through the establishment of human cutaneous SCC A431 xenografts in nude mice, we observed Res inhibition effect and investigated the inhibition mechanism by checking the expression of apoptosis-related factors, p53, ERK and survivin. The results showed that the xenograft volume and weight of Res groups were less than those of the control groups (P<0.05), but the net body mass of nude mice of Res groups was not significantly different from the control groups (P>0.05). The apoptotic index of Res groups were significantly higher than the control groups (P<0.05). The protein and mRNA expression of p53 and ERK were statistically positively correlated (P<0.05) and significantly increased in Res high- and medium-dose groups compared with the control groups (P<0.05). Moreover, the protein and mRNA expression of SVV were negatively correlated with p53 (P<0.05) and lower than the control groups (P<0.05). The results demonstrate Res inhibitory effect and indicate that the inhibition mechanism of Res is to upgrade the protein and mRNA expression of p53 and to downgrade the protein and mRNA expression of SVV, thus inducing the apoptosis of tumor cells.

Reduced expression of Raf kinase inhibitor protein correlates with poor prognosis in pancreatic cancer.

AIM: Raf kinase inhibitory protein (RKIP) is an inhibitor of the Raf/MEK/MAP kinase signaling cascade and a suppressor of cancer metastasis. But its function in pancreatic cancer was not yet clarified completely. The aim of this study was to investigate the involvement of RKIP in pancreatic cancer. METHODS: RKIP expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumor tissue samples from a series (n = 99) of consecutive patients with pancreatic cancer. Survival was calculated using Kaplan-Meier curves. Parameters found to be of prognostic significance in univariate analysis were verified in a multivariate Cox regression model. RESULTS: RKIP expression was high in normal pancreatic epithelium and retained to varying degrees in pancreatic cancer tissues. However, in tumor tissues with lymph node metastasis (P = 0.008) and high UICC stage (P = 0.006), RKIP expression was highly significantly reduced or lost. Furthermore, the reduced expression of RKIP significantly correlated with both poor overall and disease-free survival (P = 0.008 and 0.01, respectively). Multivariate analyses revealed RKIP to be an independent prognosticator. CONCLUSION: These findings suggest that RKIP could be a promising marker for predicting a better prognosis in pancreatic cancer.

Upregulation of a disintegrin and metalloprotease 8 influences tumor metastasis and prognosis in patients with osteosarcoma.

To investigate the clinicopathological and prognostic value of a disintegrin and metalloprotease 8 (ADAM8) in osteosarcoma. ADAM8 expression in osteosarcoma tissues was examined by immunohistochemistry in 69 patients. ADAM8 was positively expressed in 61 of 69 (88.4%) osteosarcoma specimens with cytoplasmic staining, and also increased in the specimens with recurrence (P = 0.008) and metastasis (P = 0.002). Patients with strong ADAM8 expression had significantly poorer overall survival (OS) and disease-free survival (DFS) (both P < 0.001) when compared with the patients with the weak expression of ADAM8. On multivariate analysis, ADAM8 expression was found to be an independent prognostic factor for both OS (P < 0.001) and DFS (P < 0.001). Our results suggest for the first time that ADAM8 might be applied as a novel marker for the prediction of recurrence and metastasis potency and a significant indicator of poor prognosis for patients with osteosarcoma.

NDRG2 down-regulation and CD24 up-regulation promote tumor aggravation and poor survival in patients with gallbladder carcinoma.

Recent studies have demonstrated that N-Myc downstream-regulated gene 2 (NDRG2) may reduce the metastatic potential of breast cancer and hepatocellular carcinoma cells by regulating the expression of CD24, which is expressed in a large variety of solid tumors. The aim of this study was to clarify the clinical value of NDRG2 and CD24 expression in primary gallbladder carcinoma (GBC). One hundred and thirty GBC tissues were evaluated by immunohistochemistry for NDRG2 and CD24 expression. The associations of NDRG2 and CD24 expression with the clinicopathological characteristics and the overall survival of patients with GBC were analyzed. NDRG2 and CD24 were positively expressed in 49/130 (37.69%) and 107/130 (82.31%) of GBC tissues, respectively. In addition, the tumors with the down-regulation of NDRG2 and the up-regulation of CD24 more frequently had lymph node metastasis and lymphovascular invasion. Moreover, the tumors with the down-regulation of NDRG2 and the up-regulation of CD24 tended to show deeper invasion depth and higher TNM stage. There was a negative correlation between NDRG2 expression and CD24 expression in GBC tissues (r = -0.86, P < 0.001). The patients with NDRG2 negative expression correlated with poor prognosis of GBC (P = 0.01), as opposed to CD24 (P = 0.01). The survival rate of the patients with NDRG2-/CD24+ expression was the lowest (P < 0.001), and conjoined expression of NDRG2-/CD24+ was an independent prognostic indicator of GBC (P = 0.003). Our data suggest that NDRG2 down-regulation or CD24 up-regulation is an important feature of GBC. A combined detection of NDRG2/CD24 co-expression may benefit us in prediction of the prognosis in GBC.

Prognostic value of matrix metalloproteinase-1/ proteinase-activated receptor-1 signaling axis in hepatocellular carcinoma.

Matrix metalloproteinase-1 (MMP-1) is proposed to be involved in both tumor cell invasion and metastasis. MMP-1 proteolytically activates protease activated receptor-1 (PAR-1), which also plays an important role in tumor development and progression. However, it is currently unknown whether MMP-1 activation of PAR-1 has relevance to the progression of hepatocellular carcinoma (HCC). To address this problem, we investigate the clinicopathological and prognostic value of MMP-1/PAR-1 signaling axis in HCC. Immunohistochemistry assay was used to determine the expression of MMP-1 and PAR-1 proteins in normal and HCC tissues. The correlations of MMP-1 and PAR-1 expression with clinicopathological parameters were assessed by Chi-squared test. Patient survival and their differences were determined by Kaplan-Meier method and log-rank test. Cox regression was adopted for multivariate analysis of prognostic factors. MMP-1 and PAR-1 immunoreactivities were negative or low in normal liver tissues, but high in HCC tissues. PAR-1 expression was significantly correlated with that of MMP-1 (r = 0.896, p < 0.0001). The overexpression of MMP-1 and PAR-1 was significantly associated with recurrence, TNM staging and portal vein invasion of HCC. Patients with high MMP-1 and PAR-1 expression had significantly poorer overall survival (OS) (both P < 0.001) and disease-free survival (DFS) (both P < 0.001) when compared with patients with the low expression of MMP-1 and PAR-1. On multivariate analysis, MMP-1 and PAR-1 expression patterns were found to be independent prognostic factors for OS (both P < 0.001) and DFS (both P < 0.001). Our results suggest for the first time that the MMP-1/PAR-1 signaling axis might be applied as a novel marker for the prediction of recurrence and metastasis potency and a significant indicator of poor prognosis for patients with HCC.