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Background: The Hepatitis B Virus X protein (HBx) plays a major role in hepatocellular carcinoma (HCC) development, however, its contribution to tumor invasion and metastasis has not been established so far. Heat shock protein 90 alpha (HSP90alpha) isoform is an ATP-dependent molecular chaperone that maintains the active conformation of client oncoproteins in cancer cells, which is abundantly expressed in HCC, especially in hepatitis B virus (HBV)-related tumors, might be involved in tumor progression., Methods: The levels of HSP90alpha, extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2) and c-Myc in HBx-transfected HepG2 cells were determined by western blots analysis. The endogenous ERKs activity was demonstrated by ELISA assay. The regulation of c-Myc-mediated HSP90 alpha promoter transactivation by HBx was evaluated through electrophoretic mobility shift analysis (EMSA). The c-Myc-mediated HSP90alpha transcription was analysed by promoter assay. The HBx-expressing cells were transfected with specific small interference RNA (siRNA) against c-Myc. The in vitro invasion potentials of cells were evaluated by Transwell cell invasion assay., Results: HBx induces HSP90alpha expression at the transcription level. The induction effect of HBx was inhibited after treatment with c-Myc inhibitor, 10058-F4. In addition, the luciferase activity of the HSP90alpha promoter analysis revealed that the HBx is directly involved in the c-Myc-mediated transcriptional activation of HSP90alpha. Furthermore, HBx induces c-Myc expression by activation of Ras/Raf/ERK1/2 cascades, which in turn results in activation of the c-Myc-mediated HSP90alpha promoter and subsequently up-regulation of the HSP90alpha expression. Overexpression of HSP90alpha in HBx-transfected cells enhances tumor cells invasion. siRNA-mediated c-Myc knockdown in HBx-transfected cells significantly suppressed HSP90alpha expression and cells invasion in vitro., Conclusion: These results demonstrate the ability of HBx to promote tumor cells invasion by a mechanism involving the up-regulation of HSP90alpha and provide new insights into the mechanism of action of HBx and its involvement in tumor metastasis and recurrence of HCC.

Introduction: Cannabis-based medicines have a number of therapeutic indications, including anti-inflammatory and analgesic effects. The endocannabinoid receptor system, including the cannabinoid receptor 1 (CB1) and receptor 2 (CB2) and the endocannabinoids, are implicated in a wide range of physiological and pathophysiological processes. Pre-clinical and clinical studies have demonstrated that cannabis-based drugs have therapeutic potential in inflammatory diseases, including rheumatoid arthritis (RA) and multiple sclerosis. The aim of this study was to determine whether the key elements of the endocannabinoid signalling system, which produces immunosuppression and analgesia, are expressed in the synovia of patients with osteoarthritis (OA) or RA., Methods: Thirty-two OA and 13 RA patients undergoing total knee arthroplasty were included in this study. Clinical staging was conducted from x-rays scored according to Kellgren-Lawrence and Larsen scales, and synovitis of synovial biopsies was graded. Endocannabinoid levels were quantified in synovial fluid by liquid chromatography-mass spectrometry. The expression of CB1 and CB2 protein and RNA in synovial biopsies was investigated. Functional activity of these receptors was determined with mitogen-activated protein kinase assays. To assess the impact of OA and RA on this receptor system, levels of endocannabinoids in the synovial fluid of patients and non-inflamed healthy volunteers were compared. The activity of fatty acid amide hydrolase (FAAH), the predominant catabolic endocannabinoid enzyme, was measured in synovium., Results: CB1 and CB2 protein and RNA were present in the synovia of OA and RA patients. Cannabinoid receptor stimulation of fibroblast-like cells from OA and RA patients produced a time-dependent phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 which was significantly blocked by the CB1 antagonist SR141716A. The endocannabinoids anandamide (AEA) and 2-arachidonyl glycerol (2-AG) were identified in the synovial fluid of OA and RA patients. However, neither AEA nor 2-AG was detected in synovial fluid from normal volunteers. FAAH was active in the synovia of OA and RA patients and was sensitive to inhibition by URB597 (3'-(aminocarbonyl) [1,1'-biphenyl]-3-yl)-cyclohexylcarbamate)., Conclusion: Our data predict that the cannabinoid receptor system present in the synovium may be an important therapeutic target for the treatment of pain and inflammation associated with OA and RA.

The efficacy of an oily calcium hydroxide suspension (Osteoinductal) as an adjunct to periodontal regenerative therapy has been demonstrated in recent clinical and histological studies. However, little is known about the molecular mechanisms in vitro, particularly, about the effect of oily calcium hydroxide paste on periodontal ligament (PDL) cells. Therefore the aim of the present study was to analyze the mitogenic response of cultured PDL cells to Osteoinductal in comparison to calcium hydroxide and enamel matrix derivative (EMD) in vitro. Human periodontal ligament cells were derived from a third molar extracted for orthodontic reasons and incubated in the presence of Osteoinductal, calcium hydroxide, EMD, phosphate-buffered saline plus 10% glycerol (PBS) or standard culture medium for 15 and 60 minutes. The mitogenic response of the PDL cells was determined by Western Blot with antibodies specific for extracellular signal-related kinase (ERK)1/2 as well as the activated, tyrosine-phosphorylated form of ERK1/2 (p-ERK). Relative phosphorylation of ERK1/2 was normalized to total ERK1/2 levels by densitometry. Cell proliferation was measured after 1, 3 and 8 days using a Neubauer haemocytometer to determine the total cell number. Results demonstrated that the mitogenic response to Osteoinductal, calcium hydroxide and enamel matrix derivative was associated with the activation of ERK1/2 and was more pronounced as compared to PBS and standard culture medium treated cells. Although Osteoinductal and calcium hydroxide activated mitosis and caused phosphorylation of ERK1/2 in PDL cells, its effects were less pronounced as compared to EMD. Furthermore EMD exhibited the highest proliferative response in comparison to Osteoinductal, calcium hydroxide and the negative control after one, three and eight days. In conclusion, our data indicate that Osteoinductal enhances the mitogenic response of human PDL cells by activation of ERK1/2 and increases cell proliferation, however, it is inferior in comparison to EMD.