Scutellarin Suppresses RPMI7951 Melanoma Cell Proliferation by Targeting TOPK.

BACKGROUND: T-LAK cell-Originated Protein Kinase (TOPK) belongs to the serine/threonine protein kinase family. It is highly expressed in RPMI7951 melanoma cells. Scutellarin (SCU) is an active ingredient extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. Its main physiological functions are related to its anti-inflammatory and antitumour activities. METHODS: The relationship between SCU and TOPK was assessed by molecular docking, an in vitro binding assay and an in vitro kinase assay. The effect of SCU on RPMI7951 cells was detected by MTS and soft agar assays. TOPK knockdown was induced by lentiviral infection. The TOPK downstream signalling pathway was detected by western blot and immunohistochemical analyses in vitro and in vivo. RESULTS: SCU was found to directly bind with TOPK and inhibit TOPK activity in vitro. SCU inhibited the proliferation and colony formation of RPMI7951 cells in a dose-dependent manner. Silencing TOPK decreased the sensitivity of colon cancer cells to SCU. SCU inhibited the phosphorylation levels of Extracellular Regulated protein Kinases 1/2 (ERK1/2) and histone H3 in a time- and dose-dependent manner in RPMI7951 cells. In addition, SCU inhibited the growth of xenograft tumours of RPMI7951 cells and decreased the phosphorylation levels of extracellular regulated protein kinases 1/2 and histone H3 in vivo. CONCLUSION: The results showed that SCU exerts promising antitumour effects on human RPMI7951 cells by inhibiting the activity of TOPK.

Gossypetin Inhibits Solar-UV Induced Cutaneous Basal Cell Carcinoma Through Direct Inhibiting PBK/TOPK Protein Kinase.

BACKGROUND: Skin photoaging, skin inflammation and skin cancer are related with excessive exposure to solar UV. PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK), a member of the serine/threonine protein kinase, which regulates the signaling cascades of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal regulated kinase 1/2 (ERK1/2). PBK/TOPK plays a significant role in solar-UV-induced cutaneous basal cell carcinoma (BCC), and targeting PBK/TOPK can be supposed to treat and prevent cutaneous BCC. METHODS: The pathological feature and the expression level of PBK/TOPK in cutaneous BCC tissues of human were studied in clinical samples. SUV-induced the phosphorylation of p38 MAPK and ERK1/2 were demonstrated ex vivo. Moreover, the interaction between Gossypetin and PBK/TOPK were detected by in vitro kinase assay and Microscale thermophoresis (MST) assay. Furthermore, the effect of Gossypetin to solar UV-induced the activity of PBK/TOPK were detected ex vivo and in vivo. RESULTS: The clinical samples showed that the expression levels of PBK/TOPK, phosphor-p38 MAPK and phosphor- ERK1/2 were up-regulated in cutaneous BCC tissues of human. The expression of phosphor-p38 MAPK or phosphor-ERK1/2 increased in a dose and time dependent manner after solar UV treatment in HaCaT cells. MTT cytotoxicity assay results showed that Gossypetin has no effect on HaCaT cells. In vitro kinase assay and MST assay results showed that Gossypetin bound with PBK/TOPK and suppressed PBK/TOPK activity. Ex vivo results showed Gossypetin inhibited solar UV-induced phosphorylation of PBK/TOPK, p38 MAPK, ERK1/2 and H2AX by suppressing PBK/TOPK activity. In vivo test results indicated that Gossypetin suppressed solar UV-induced increase of PBK/TOPK, phosphor-p38 MAPK, phosphor-ERK1/2 and phosphor- H2AX in SKH-1 hairless mice. CONCLUSION: Our data demonstrated that Gossypetin can alleviate solar-UV-induced cutaneous BCC by blocking PBK/TOPK, and Gossypetin could be a remarkable agent for treating solar-UV induced cutaneous basal cell carcinoma.

The CD25+/CD4+ T cell ratio and levels of CII, CIX and CXI antibodies in serum may serve as biomarkers of pristane-induced arthritis in rats and Rheumatoid Arthritis in humans.

OBJECTIVE: Collagen antibodies in serum are involved in the pathogenesis of Rheumatoid Arthritis (RA). The objective of this study was to identify the subtype of collagen antibodies and T cell subtype distribution in pristane-induced arthritis (PIA) and to clarify their roles in the initiation and maintenance of arthritis. METHODS: Arthritis was induced in Dark Agouti (DA) rats by injection of pristane. The severity was evaluated by macroscopic and microscopic score systems. The alteration of CD25+/CD4+ T cell ratio in rats was detected by flow cytometry. Collagen type II (CII), CIX, or CXI antibody in serum was determined by ELISA. The levels of Nitric oxide (NO) and tartrate-resistant acid phosphatase (TRAP) were measured by kits. RESULTS: The serum levels of CII, CIX, CXI antibodies were significantly increased in RA patients while slightly increased in PIA rats. The ratio of CD25+/CD4+ T cells was significantly higher in RA rats than that in the control group. The serum levels of NO and TRAP in PIA rats and RA patients were higher than that in the control groups, which suggested that the activity of osteoclast was increased in RA. CONCLUSION: The ratio of CD25+/CD4+ T cells plays a pivotal role in the development of PIA. The serum levels of NO and TRAP are inflammatory and osteoclast activity indicators. The serum levels of CII, CIX and CXI antibodies may serve as the clinical diagnostic indicators. These findings are important to our understanding of the pathogenesis of RA, and may provide biomarkers of RA diagnosis and therapeutic targets for the treatment of RA.

The Pathogenesis of Ossification of the Posterior Longitudinal Ligament.

Ossification of the posterior longitudinal ligament (OPLL) is a multi-factorial disease involving an ectopic bone formation of spinal ligaments. It affects 0.8-3.0% aging Asian and 0.1-1.7% aging European Caucasian. The ossified ligament compresses nerve roots in the spinal cord and causes serious neurological problems such as myelopathy and radiculopathy. Research in understanding pathogenesis of OPLL over the past several decades have revealed many genetic and non-genetic factors contributing to the development and progress of OPLL. The characterizations of aberrant signaling of bone morphogenetic protein (BMP) and mitogen-activated protein kinases (MAPK), and the pathological phenotypes of OPLL-derived mesenchymal stem cells (MSCs) have provided new insights on the molecular mechanisms underlying OPLL. This paper reviews the recent progress in understanding the pathophysiology of OPLL and proposes future research directions on OPLL.

Pre-miR-149 rs71428439 polymorphism is associated with increased cancer risk and AKT1/cyclinD1 signaling in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common lethal malignancies in the world, and the current knowledge on the molecular and genetic basis of HCC is still limited. Previous study has shown miR-149 plays a tumor suppressive role in HCC, here we aimed to investigate the association between rs71428439 polymorphism, which located in the pre-miR-149, and the risk of HCC in a Chinese Han population. A total of 177 HCC patients and 103 healthy controls were genotyped, by a multivariate logistic regression, we found that individuals with GG genotype have significantly higher risk of HCC (adjusted OR=3.397, 95% CI=1.565-7.375, P=0.002) compared with those with AA genotype, similar results were also observed in recessive model (adjusted OR=2.563, 95% CI=1.300-5.054, P=0.007) and dominant model (adjusted OR=2.074, 95% CI=1.147-3.752, P=0.016). We further observed that tumor tissues in patients with GG genotype expressed lower level of miR-149 compared with those with AA or AG genotype, and consequently, AKT1, a pre-validated miR-149 target in vitro, was found to have higher expression level in tumors with GG genotype. In summary, our data indicated that rs71428439 may be a genetic risk factor of HCC in the Chinese Han population, and its mechanism possibly involves downregulated miR-149 expression and upregulated AKT1 expression.

[Preparation and identification of the polyclonal antibody against extracellular domain (405T-573Q) of rat Toll-like receptor 2].

OBJECTIVE: To prepare the polyclonal antibody of the extracellular domain (405T-573Q) of rat Toll-like receptor 2 (TLR2) and identify its activity. METHODS: Firstly, the extracellular domain (507 bp) of rat TLR2 was amplified from rat hepatocytes by reverse transcription PCR, and the recombinant plasmid pET28a-TLR2ex was constructed. Secondly, the fusion protein TLR2ex-6×His was expressed in E.coli BL21(DE3) and purified by affinity chromatography. And then, the polyclonal antibody was prepared by immunizing the BALB/c mice with the purified fusion protein. Finally, the titer and specificity of the polyclonal antibody were analyzed by ELISA and Western blotting, respectively. RESULTS: The recombinant plasmid pET28a-TLR2ex was successfully constructed, and the TLR2ex-6×His fusion protein was successfully expressed and purified. Western blotting demonstrated that the antibody from the TLR2ex-6×His-immunized BALB/c mice specifically and selectively bound with rat TLR2. ELISA also indicated that the titer of polyclonal antibody reached 1:76 800. CONCLUSION: The polyclonal antibody against the extracellular domain (405T-573Q) of rat TLR2 has been successfully prepared.

Antitumor and immunomodulatory activity of a polysaccharide from fungus Coprinus comatus (Mull.:Fr.) Gray.

In our study, a water-soluble polysaccharide (CCPa-1) was successfully purified from the fruiting bodies of Coprinus comatus by DEAE-cellulose and Sepharose CL-6B column chromatography. The molecular weight was evaluated to be 53.6 kDa as determined by high performance size exclusion chromatography (HPSEC). Sugar composition analysis revealed that CCPa-1 consisted primarily of galactose, glucose and arabinose in a molar ratio of 6.6:1.2:2.2. CCPa-1 could not only inhibit the growth of sarcoma 180 (S180) tumor transplanted in mice, but also increase the relative spleen/thymus indexes and body weight of tumor bearing mice. Moreover, Con A- or LPS-induced splenocyte proliferation was also enhanced after CCPa-1 administration in tumor-bearing mice. Furthermore, CCPa-1 significantly enhanced the Con A- or LPS-induced splenocyte proliferation and increased the production of TNF-α and IL-2. All the data demonstrated that CCPa-1 had a potential application as natural antitumor agent with immunomodulatory activity.

Levels of HTNV-specific CD8+ T lymphocytes in PBMC from the patients with hemorrhagic fever with renal syndrome.

The pathogenesis of hemorrhagic fever with renal syndrome (HFRS) has not been fully clarified. Cell-mediated immunity appears to play a crucial role in the immune pathogenesis of HFRS. To explore the relationship between Hantaan (HTNV)-specific CD8(+) T lymphocytes and the immune pathogenesis of HFRS, the levels of interferon γ (IFN-γ) secreted by HTNV-specific CD8(+) T lymphocytes were detected by flow cytometry in peripheral blood mononuclear cells. Levels of HTNV-specific CD8(+) T lymphocytes in patients with HFRS were associated with different phases of HFRS. In fever phase, it was significantly elevated. The levels of HTNV-specific CD8(+) T lymphocytes in PBMC of patients with HFRS were negatively correlated with the levels of blood urea nitrogen and creatinine in plasma. The results show that the HTNV-specific CD8(+) T lymphocyte levels correlate with disease phases. Therefore, dynamic observation of these levels in patients with HFRS can help to judge the status of HFRS disease and to clarify the immune pathogenesis of HFRS.

[Construction and identification of human PRMT1 gene eukaryotic vector].

AIM: To construct the eukaryotic recombinant expression plasmid of pcDNA3.1(+)-PRMT1. METHODS: Human PRMT1 cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR). After digested by BamH I, Hind III and ligation, PRMT1 was inserted into pcDNA3.1(+)eukaryotic expression vector. The positive colonies were screened and identified by PCR and sequencing. pcDNA3.1(+)-PRMT1 plasmid was then transfected into the cultured A549 cell line with Lipofectamine(TM);2000. Realtime-PCR and Western blot were used to detect the mRNA and protein expression of PRMT1 respectively. RESULTS: The PRMT1 cDNA was successfully amplified, and pcDNA3.1(+)-PRMT1 were constructed. The inserted sequence in pcDNA3.1(+)-PRMT1 was the same as the sequence of PRMT1 cDNA published in NCBI GenBank. Further, Realtime PCR and Western blot results validated the recombinant plasmid expressed in A549 cell line efficiently. CONCLUSION: pcDNA3.1(+)-PRMT1 recombinant was successfully constructed.

Expression of VLA-4 molecule in PBMC from patients with hemorrhagic fever with renal syndrome.

OBJECTIVE AND DESIGN: The very late antigen-4 (VLA-4) bound to vascular cell adhesion molecule-1 could provide co-stimulatory signals for the activation of T lymphocytes, and these adhesion molecules play key roles in leukocyte adherence and propagation of inflammatory responses. We examined the levels of VLA-4 in the peripheral blood mononuclear cells (PBMC) of patients with hemorrhagic fever with renal syndrome (HFRS). MATERIALS AND METHODS: The levels of VLA-4 in PBMC samples collected from 53 patients by immunohistochemical staining were detected. RESULTS: The expression of VLA-4 in PBMC of HFRS patients at different stages were significantly higher than those in normal controls (P < 0.05), except recovery stage (P > 0.05). The expression of VLA-4 in PBMC of HFRS patients at different types were significantly higher than those in healthy controls (P < 0.05). The levels of VLA-4 in patients with HFRS were positively correlated with serum levels of blood urea nitrogen (BUN) and creatinine (Cr). CONCLUSIONS: VLA-4 might play an important role in the immunopathological lesions of HFRS. We found that VLA-4 levels were closely correlated to the severity of the HFRS and the degree of kidney damage.

Up-regulation of Tiam1 and Rac1 correlates with poor prognosis in hepatocellular carcinoma.

OBJECTIVE: T-cell lymphoma invasion and metastasis 1 (Tiam1) specifically activates Rho-like GTPases (e.g. Rac1) and Tiam1-Rac1 pathway affects the migration and invasion of many tumors, such as nasopharyngeal carcinoma, breast cancer and retinoblastoma. However, no studies have yet comprehensively examined the involvement of Tiam1-Rac1 pathway in hepatocellular carcinoma. In this study, we examined the relationship of the up-regulation of Tiam1 and Rac1 with clinicopathological features in patients with hepatocellular carcinoma. METHODS: Expression of Tiam1 and Rac1 was assessed in 242 hepatocellular carcinoma tissues and their adjacent normal hepatic tissues by performing immunohistochemistry and was gauged regarding stage, grade and survival. RESULTS: Immunohistochemistry showed that patients with a high clinical stage hepatocellular carcinoma (III-IV) and α-fetoprotein levels had a higher tendency to express Tiam1 and Rac1 on tumor cells than the patients with low pathologic grade hepatocellular carcinoma (I-II) (P = 0.008 and 0.01, respectively) and low α-fetoprotein levels (P = 0.006 and 0.002, respectively). In addition, Tiam1 and Rac1 up-regulation was also significantly associated with vascular invasion status (both P = 0.02), intrahepatic metastasis status (P = 0.009 and 0.01, respectively) and histological differentiation (P = 0.008 and 0.009, respectively) of patients with hepatocellular carcinoma. Moreover, post-operative survival analysis indicated that hepatocellular carcinoma patients with strong Tiam1 (P = 0.01) and Rac1 (P = 0.02) expression had shorter disease-specific survival than those with weak expression. Multivariate analysis also showed that Tiam1 and Rac1 overexpression could be two predictors of poor prognosis (P = 0.02 and 0.03, respectively). CONCLUSIONS: The current study demonstrated for the first time that the Tiam1-Rac1 pathway may play a critical role in tumor progression of hepatocellular carcinoma. The expression of Tiam1 and Rac1 can be considered as the two useful indicators for predicting the prognosis of hepatocellular carcinoma.