Ursodeoxycholic Acid Modulates the Interaction of miR-21 and Farnesoid X Receptor and NF-κB Signaling.

(1) Background: This study investigates the effects of Ursodeoxycholic acid (UDCA) on NF-κB signaling, farnesoid X receptor (FXR) singling, and microRNA-21 in HepG2 cells. (2) Methods: HepG2 cells were treated with lipopolysaccharide (LPS) to simulate hepatic inflammation. The investigation focused on the expression of NF-κB activation, which was analyzed using Western blot, confocal microscopy, and Electrophoretic Mobility-shift Assays (EMSA). Additionally, NF-κB and farnesoid X receptor (FXR) singling expressions of micro-RNA-21, COX-2, TNF-α, IL-6, cyp7A1, and shp were assessed by RT-PCR. (3) Results: UDCA effectively downregulated LPS-induced expressions of NF-κB/65, p65 phosphorylation, and also downregulated FXR activity by Western blot. Confocal microscopy and EMSA results confirmed UDCA's role in modulating NF-κB signaling. UDCA reduced the expressions of LPS-induced COX-2, TNF-α, and IL-6, which were related to NF-κB signaling. UDCA downregulated LPS-induced cyp7A1 gene expression and upregulated shp gene expression, demonstrating selective gene regulation via FXR. UDCA also significantly decreased micro-RNA 21 levels. (4) Conclusions: This study demonstrates UDCA's potent anti-inflammatory effects on NF-κB and FXR signaling pathways, and thus its potential to modulate hepatic inflammation and carcinogenesis through interactions with NF-κB and FXR. The decrease in micro-RNA 21 expression further underscores its therapeutic potential.

Cytoplasmic CK1ε Protein Expression Is Correlated With Distant Metastasis and Survival in Patients With Melanoma.

BACKGROUND/AIM: Casein kinase 1 epsilon (CK1ε) is a member of the casein kinase 1 family, which includes highly conserved and ubiquitous serine/threonine protein kinases. Recent research has revealed that CK1ε plays an important role in a variety of human cancer types; however, its role in human melanoma remains unclear. The aim of this study was to elucidate the clinical role of CK1ε in patients with melanoma. PATIENTS AND METHODS: Samples from 34 patients with melanoma were analyzed by immunohistochemical staining. Formalin-fixed paraffin-embedded tissue microarrays were also examined by two histopathologists to assess CK1ε protein expression in humans. RESULTS: Cytoplasmic CK1ε protein expression was significantly lower in tumor tissue than in normal tissue. Lack of cytoplasmic CK1ε protein was significantly correlated with distant metastasis (p=0.022) and poorer survival (p=0.030). However, Kaplan-Meier survival analysis revealed that elevated expression of cytoplasmic CK1ε protein was not significantly associated with the overall survival of patients with melanoma. Univariate and multivariate analyses demonstrated that lack of cytoplasmic CK1ε protein expression was related to distant metastasis (p<0.001 and p=0.004), showing that CK1ε was a prognostic factor. CONCLUSION: CK1ε protein expression might serve as a prognostic indicator in the treatment of patients with melanoma.

UNC13C Suppress Tumor Progression via Inhibiting EMT Pathway and Improves Survival in Oral Squamous Cell Carcinoma.

Potential function of UNC13C in variety of cancers including, oral squamous cell carcinoma (OSCC) remains obscure. In the present study, immunohistochemical staining in tissue microarrays containing 268 OSCC samples showed that UNC13C protein levels were inversely correlated with AJCC Stage III and IV (P = 0.002) and death (P = 0.0134). Patients with lower UNC13C expression had a significantly shorter survival (P = 0.0231) than those with higher UNC13C expression. We also identified decreased overall UNC13C expression in oral cancer cell lines. In addition, our functional analysis of UNC13C shows that overexpression of UNC13C inhibited migration and invasion capacities of SCC-9 and SAS cells compared with the empty plasmid transfected cells. Further experiments suggested that transcription factors (Slug, Snail, Twist, and ZEB1) and mesenchymal marker (Vimentin) were down regulated and Tight Junction Protein (Claudin1) was up regulated after UNC13C overexpression in SCC9 and SAS cells. The novel role of UNC13C is revealed for the first time in OSCC. In summary, these results suggest that UNC13C as a novel tumor suppressor and an essential regulator of EMT signaling pathway during OSCC progression, and thus it could be used as a target for preventing oral cancer metastasis.

c-MYC expression in T (III/IV) stage oral squamous cell carcinoma (OSCC) patients.

Purpose: c-MYC has been noted in many tumor types, but its functional significance and clinical utility in oral squamous cell carcinoma (OSCC) are not well known. Here we studied the expression of c-MYC in correlation to clinical outcome in patients with oral squamous cell carcinoma. Methods: The current study, using immunohistochemical staining, first examined c-MYC expression in OSCC patients and further correlated its expression with clinicopathological parameters. Results: c-MYC was expressed in the majority of OSCC patients (n=133). The c-MYC expression is associated with histological grade (P=0.0205) of patients with oral squamous cell carcinoma. Multivariate Cox regression analysis revealed that TN stage (P<0.001), American Joint Committee on Cancer (AJCC) stage (P<0.0001), and tumor differentiation (P=0.0025) were independent factors for overall survival in patients with OSCC except for c-MYC expression (P>0.05). Multiplicative-scale interaction between T stage (III/IV) and low c-MYC expression on mortality risk was identified (P=0.0233). Kaplan-Meier survival analysis demonstrated that oral cancer patients (T III/IV stage) with high c-MYC expression had better survival than those with low and medium c-MYC expression (P=0.0270). Conclusion: Our data indicate that c-MYC is a potential biomarker that can be used as a therapeutic target for treating OSCC patients with T stage (III/IV).

CIP2A overexpression in Taiwanese oral cancer patients.

Introduction: Oral cancer is a prevalent form of cancer worldwide, particularly in Taiwan, and mechanisms involved in oral squamous cell carcinoma (OSCC) progression remain relatively unknown. Cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncoprotein, is aberrantly expressed in many human malignant tumors including oral cancer. However, the expression and role played by CIP2A in oral cancer pathogenesis remain obscure. Methods: In this study, immunohistochemistry was used to analyze CIP2A expression between OSCC tissues and their adjacent noncancerous tissues. Furthermore, associations between CIP2A expression and histopathological parameters were investigated. Results: In this study, we showed that CIP2A was overexpressed in most of the OSCC tissues. High CIP2A expression was significantly associated with moderate/poor tumor differentiation (P=0.02). No significant association was found between CIP2A expression and other clinical parameters. Kaplan-Meier analysis revealed that high CIP2A expression showed poorer survival rates than those with low CIP2A expression (P=0.047). Multivariate Cox regression analysis indicated that CIP2A expression, N stage, American Joint Committee on Cancer stage and clinical therapy were independent prognostic factors for survival. Conclusion: Thus, our study suggests that CIP2A is an independent prognostic marker for OSCC and a novel target for OSCC treatment.

The effect of proton pump inhibitors on the gastric mucosal microenvironment.

BACKGROUND: Proton pump inhibitors (PPIs) are widely applied for acid related disorders, and possess pleiotropic biological functions. The effect of PPIs on the gastric mucosa, neutrophil and Helicobacter pylori (H. pylori) infiltration and glandular atrophy has not been well investigated, particularly the duration of the effects of PPIs. OBJECTIVES: To investigate the effects of PPIs on neutrophil infiltration, H. pylori infiltration and the gastric mucosa. MATERIAL AND METHODS: A total of 76 adult patients with gastrointestinal symptoms who had undergone upper gastrointestinal endoscopy were enrolled in the study. Each patient's history was recorded, including smoking, alcohol consumption and the duration of PPI use prior to gastric biopsy. Endoscopic biopsies of gastric antral mucosa were performed and evaluated by histology. Neutrophil and H. pylori infiltration were graded by H & E staining in accordance with the updated Sydney system. RESULTS: Among the 76 patients, 44 patients had H. pylori infection and 19 patients had taken PPIs for varying durations prior to gastric biopsy. Neutrophil infiltration was significantly inhibited by PPIs (p = 0.005). The duration of PPI use was correlated with inhibition of neutrophil and H. pylori infiltration. A logistical regression analysis demonstrated that PPIs significantly inhibited neutrophil infiltration in the gastric mucosa and were associated with atrophy of the mucosa. CONCLUSIONS: PPIs attenuated neutrophil infiltration of gastric mucosa, and may be related to atrophy of the mucosa.

Interaction of omeprazole and Helicobacter pylori-induced nuclear factor-κB activation and mediators in gastric epithelial cells.

BACKGROUND: Omeprazole (OMP), a proton pump inhibitor, is a highly effective drug for the management of acid-related disorders. Infections resulting from cytotoxin antigen A (CagA) positive Helicobacter pylori strains have been associated with higher grades of gastric mucosal inflammation. Nuclear factor (NF)-κB activation has been reported to participate in H. pylori-induced gastritis in humans. The complex interaction of OMP on the H. pylori and NF-κB related molecular mechanisms within the gastric mucosa remains unclear. In the present study, we investigated OMP, specifically its effects on NF-κB activation, and COX-2, IL-6, and IL-8 production in gastric cells (Kato-III cells) treated with CagA positive (CagA(+)) and negative (CagA(-)) H. pylori strains. METHODS: Kato-III cells were stimulated with H. pylori water extracts (HPE) containing ATCC 43504 (CagA(+)) and ATCC 51932 (CagA(-)) strains. NF-κB activation, inhibitory IκB expression and phosphorylation, and cyclooxygenase (COX)-2, interleukin (IL)-6, and IL-8 expression were assessed in the absence and presence of OMP. RESULTS: Both CagA(+) and CagA(-) HPE induced NF-κB activation, whereas OMP suppressed NF-κB activation in the CagA(-) strain. HPE demonstrated a similar effect on IκB protein expression in the absence and presence of OMP. OMP alone decreased IκB phosphorylation without promoting NF-κB and IκB expression. Additionally, both CagA(+) and CagA(-) HPE induced COX-2 expression, but no significant effect on IL-6 and IL-8. However, OMP downregulated the transcription of COX-2, IL-6, and IL-8 in CagA(-) HPE treated cells. CONCLUSION: Using the Kato-III cells model, H. pylori induces NF-κB activation in a CagA-independent manner. Both CagA(+) HPE and CagA(-) HPE induced COX-2 gene expression, but not for IL-6 and IL-8 expression. However, OMP suppressed NF-κB activation via a downregulation of IκB phosphorylation in CagA(-) HPE treated condition. OMP also suppressed CagA(-)H. pylori induced-transcription of proinflammatory COX-2, IL-6, and IL-8. OMP may provide different effects on CagA(+) and CagA(-)H. pylori infection conditions.

Clarithromycin modulates Helicobacter pylori-induced activation of nuclear factor-κB through classical and alternative pathways in gastric epithelial cells.

Infection of gastric epithelial cells by Helicobacter pylori stimulates the activation of nuclear factor-κB (NF-κB) and the upregulation of interleukin-8 (IL-8) expression. Activation of NF-κB can occur through classical (p50/p65) and alternative (p52/RelB) pathways. The role of the bacterial cag pathogenicity island (PAI) in these events is controversial. This study aimed to evaluate the hypothesis that the CagA protein is required for H. pylori-induced activation of NF-κB and upregulation of IL-8 expression, and for clarithromycin (CAM) to exert its molecular effects. Cultured KATO-III human gastric cancer cells were treated with extracts of H. pylori strains ATCC43504 (cag PAI(+)) and ATCC51932 (cag PAI(-)) for 24 h. NF-κB and phospho-IκB protein expression was then evaluated using western blotting. IL-8 mRNA expression was evaluated using the reverse transcription polymerase chain reaction. Following the separation of the proteins using two-dimensional gel electrophoresis, proteomes of the two bacterial extracts were compared using nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis. Although the protein profiles of the two extracts differed, both extracts induced IκBα phosphorylation, upregulation of IL-8 expression, and NF-κB activation through classical and alternative pathways. In cells treated with either of the bacterial extracts, CAM inhibited H. pylori-induced activation of NF-κB and upregulation of IL-8 expression. These results suggested that CagA is not required for H. pylori-induced activation of NF-κB and upregulation of IL-8 expression in gastric epithelial cells. H. pylori-induced NF-κB signaling can occur through classical and alternative activation pathways, and that CAM inhibits these two pathways.

Proapoptotic and TRAIL-sensitizing constituents isolated from Salvia militiorrhiza (Danshen).

Natural compounds isolated from medicinal plants are invaluable resources for drug discovery. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent unique by its cancer cell-specific proapoptotic action, but its potential is heavily curbed by acquired resistance. We herein reported for the first time the identification of cytotoxic and TRAIL-sensitizing components of Salvia miltiorrhiza (Danshen), a traditional medicinal plant effective for treating cardiovascular disorders. Specifically, we found that the ethanol extract and its group 5 fraction of S. miltiorrhiza showed evident cytotoxicity against the human lung adenocarcinoma cell line A549 and ovarian adenocarcinoma cell line TOV-21G in a concentration-dependent manner. Likewise, a dose-dependent cytotoxicity was exerted by the standard solutions of cryptotanshinone, tanshinone I and tanshinone IIA, the major components of the group 5 fraction, where tanshinone IIA were most potent and displayed an IC50 of 2.00 ± 0.36 µM and 2.75 ± 0.23 µM for A549 and TOV-21G, respectively. Induction of apoptosis represents an essential mechanism underlying tanshinone IIA-mediated cytotoxic action, as evidenced by the proteolytic processing of PARP upon tanshinone IIA stimulation and, importantly, a marked rescue of the viability of tanshinone IIA-treated cells when co-treatment with the pan-caspase inhibitor z-VAD-fmk. Noteworthy, stimulation with cryptotanshinone, tanshinone I or tanshinone IIA all effectively potentiated TRAIL to reduce viability and inhibit the colony formation capacity of TRAIL-resistant TOV-21G and SKOV3. Collectively, we revealed the proapoptotic and TRAIL-sensitizing components of S. miltiorrhiza and further implicated the potential of developing these active compounds as monotherapeutic agent or TRAIL-based therapy for cancer chemoprevention or chemotherapy.

Matrix metalloproteinases 2 and 9 correlate with alanine aminotransferase and hepatitis C virus in blood donors.

BACKGROUND/AIMS: Matrix metalloproteinases 2 and 9 (MMP-2 and -9) have been suggested to play an important role in hepatitis and hepatic fibrosis. The aims of this study were to determine the plasma levels of enzyme activities of matrix metalloproteinases 2 and 9 in healthy and deferred blood donors. The relationships between activities of matrix metalloproteinases 2 and 9 and the level of alanine aminotransferase and the status of hepatitis B virus and hepatitis C virus infection were investigated. METHODOLOGY: This study included 51 healthy volunteer blood donors and 175 deferred blood donors. Of the deferred donors, 54 donors had levels of alanine aminotransferase > or = 45 IU/L, 99 donors were positive for hepatitis B virus surface antigen, and 32 donors were positive for antibodies against hepatitis C virus. Activities of matrix metalloproteinases 2 and 9 were measured by zymography using impregnated gel. RESULTS: Activity of matrix metalloproteinase 2 in deferred blood donors with elevated ALT was higher than that in healthy donors (331 +/- 139 vs. 266 +/- 122, p < 0.001). Further, activity of matrix metalloproteinase 2 was higher in HCV-infected donors than in healthy donors (319 +/- 97 vs. 275 +/- 133, p < 0.05). Matrix metalloproteinase 9 activity was also higher in deferred donors with HCV infection than in healthy donors (153 +/- 45 vs. 108 +/- 48, p < 0.001). CONCLUSION: These findings suggest that hepatitis C virus infection may upregulate the activities of matrix metalloproteinases 2 and 9 and that these matrix metalloproteinases may be linked to liver dysfunction.

Chemiluminescence assay of mucosal reactive oxygen species in gastric cancer, ulcer and antral mucosa.

BACKGROUND/AIMS: Reactive oxygen species (ROS) have been implicated in inflammatory and cancerous illness, including that of the gastrointestinal tract. The oxidative damage incurred during human gastric ulcer or cancer mucosa may be related to acumination of ROS. In this study, we aimed to demonstrate oxidative stress of gastric ulcer and cancer mucosa compared to gastric antral mucosa. PATIENTS: Thirty-four patients with gastric ulcer and gastric cancer were enrolled in this study. Gastric mucosa specimens, taken from upper GI endoscopic biopsy, from the lesion (ulcer or cancer) and antrum were sent for the activity of O2- or H2O2 determined by chemiluminescence assay. Protein concentrations in the tissue homogenates were determined by Bio-Red protein assay. The production of O2- or H2O2 per unit of protein was calculated by dividing the tissue CL level by the protein content of a tissue. RESULTS: The oxidative stress metabolites O2- and H2O2 of mucosa were evaluated by chemiluminescence assay in gastric lesions (27 ulcers and 7 cancers) and gastric antrum. Gastric lesion showed significantly increased O2- than antral mucosa (18.77 +/- 45.18 (counts/sec x microg), 95% CI 3.01, 34.53 vs. 3.58 +/- 6.89 (counts/sec x microg), 95% CI 1.18, 5.98, p < 0.05). There was also significantly greater expression of H2O2 in gastric lesion than gastric antral mucosa (76.06 +/- 148.36 (counts/sec x microg), 95% CI 24.30, 127.83 vs. 912.41 +/- 20.22 (counts/sec x microg), 95% CI 5.35, 19.46, p = 0.008). Differences of mucosal O2- and H2O2 between gastric ulcer and cancer were not significant. There was significant correlation of O2- and H2O2 generation in gastric lesion mucosa. CONCLUSIONS: Oxidative stress is now thought to make a significant contribution to inflammatory disease and malignancy. The reason that overproduction of free radicals is a feature of such a broad spectrum of diseases derived from the fact that oxidative metabolism is a necessary part of every cell's metabolism. In this study, we demonstrated increased ROS production in gastric ulceration and cancer compared with gastric antral mucosa.

The prevalence of SEN virus infection in blood donors in Taiwan.

OBJECTIVES: To determine the prevalence of SENV infections among blood donors in central Taiwan and to clarify the relationship between these infections and elevated alanine aminotransaminase (ALT) values. METHODS: DNA was extracted from plasma of 200 blood donors and amplified by seminested PCR. RESULTS: For all donors, the prevalence of SENV-D was 32%, and of SENV-H was 30.5%. Prevalence of mixed SENV-D/H infection was 11.5% and of SENV-D and/or SENV-H (SENV-D/H) was 51%. Infections were not associated with age, gender, or raised ALT values. CONCLUSIONS: SENV-D and SENV-H infections are common among blood donors in central Taiwan but are unlikely to contribute to abnormal ALT values.

Down-regulation of dehydroepiandrosterone sulfotransferase gene in human hepatocellular carcinoma.

Differential display (DD) PCR cloning of differentially expressed genes in hepatocellular carcinoma (HCC) and adjacent unaffected tissue demonstrated preferential down-regulation of a vital sex steroid precursor (dehydroepiandrosterone sulfotransferase; DHEA-ST; SULT2A1) in HCC. SULT2A1 mRNA and/or protein expression in HCC were markedly reduced in 61 of 120 (50.8%) primary unicentric HCCs. The down-regulation was more frequent in grade III versus grade I HCC (68.1% versus 32.1%, P = 0.0025), and in stage 3 versus stage 1 HCC (62.7% versus 29.2%, P = 0.007). The lowered expression in tumor cells of SULT2A1 in HCC tissues involved in metabolism and/or inactivation of sex steroids is consistent with a regulatory role of the SULT2A1 gene product in the development and/or tumor cell differentiation and progression of human HCC. This suggestion is partly supported by our observations that the down-regulated SULT2A1 gene expression correlated with a higher grade and stage of HCC.