An Essential Role of cAMP Response Element Binding Protein in Ginsenoside Rg1-Mediated Inhibition of Na+/Glucose Cotransporter 1 Gene Expression.

The Na(+)/glucose cotransporter 1 (SGLT1) is responsible for glucose uptake in intestinal epithelial cells. It has been shown that the intestinal SGLT1 level is significantly increased in diabetic individuals and positively correlated with the pathogenesis of diabetes. The development of targeted therapeutics that can reduce the intestinal SGLT1 expression level is, therefore, important. In this study, we showed that ginsenoside Rg1 effectively decreased intestinal glucose uptake through inhibition of SGLT1 gene expression in vivo and in vitro. Transient transfection analysis of the SGLT1 promoter revealed an essential cAMP response element (CRE) that confers the Rg1-mediated inhibition of SGLT1 gene expression. Chromatin immunoprecipitation assay and targeted CRE-binding protein (CREB) silencing demonstrated that Rg1 reduced the promoter binding of CREB and CREB binding protein associated with an inactivated chromatin status. In addition, further studies showed that the epidermal growth factor receptor (EGFR) signaling pathway also plays an essential role in the inhibitory effect of Rg1; taken together, our study demonstrates the involvement of the EGFR-CREB signaling pathway in the Rg1-mediated downregulation of SGLT1 expression, which offers a potential strategy in the development of antihyperglycemic and antidiabetic treatments.

A gut microbial metabolite of ginsenosides, compound K, induces intestinal glucose absorption and Na(+) /glucose cotransporter 1 gene expression through activation of cAMP response element binding protein.

SCOPE: The Na(+) /glucose cotransporter 1 (SGLT1) plays a crucial role in glucose uptake in intestinal epithelial cells (IECs), which has been shown essential in ameliorating intestinal inflammation. Ginseng has historically been used to treat inflammatory disorders. Understanding the regulatory mechanism of ginseng-mediated induction of SGLT1 gene expression in human intestinal cells is therefore important. METHODS AND RESULTS: We demonstrate that ginsenoside compound K (CK) enhances SGLT1-mediated glucose uptake in mice and human intestinal Caco-2 cells. Transient transfection analysis using SGLT1 promoter-luciferase reporters demonstrated that the presence of an essential cAMP response element (CRE) is required for CK-mediated induction of SGLT1 gene expression. The ChIP assays indicated that increased CRE-binding protein (CREB) and CREB-binding protein (CBP) binding to the SGLT1 promoter in CK-treated cells is associated with an activated chromatin state. Our result showed that the increased CREB phosphorylation is directly correlated with SGLT1 expression in IECs. Further studies indicated that the epidermal growth factor receptor (EGFR) signaling pathway is involved in the CK-mediated effect. CONCLUSION: These findings provide a novel mechanism for the CK-mediated upregulation of SGLT1 expression through EGFR-CREB signaling activation, which could contribute to reducing gut inflammation.

Caffeic acid phenethyl ester induces E2F-1-mediated growth inhibition and cell-cycle arrest in human cervical cancer cells.

Caffeic acid phenyl ester (CAPE) has been identified as an active component of propolis, a substance that confers diverse activities in cells of various origins. However, the molecular basis of CAPE-mediated cellular activity remains to be clarified. Here, we show that CAPE preferentially induced S- and G2 /M-phase cell-cycle arrests and initiated apoptosis in human cervical cancer lines. The effect was found to be associated with increased expression of E2F-1, as there is no CAPE-mediated induction of E2F-1 in the pre-cancerous cervical Z172 cells. CAPE also up-regulated the E2F-1 target genes cyclin A, cyclin E and apoptotic protease activating of factor 1 (Apaf-1) but down-regulated cyclin B and induced myeloid leukemia cell differentiation protein (Mcl-1). These results suggest the involvement of E2F-1 in CAPE-mediated growth inhibition and cell-cycle arrest. Transient transfection studies with luciferase reporters revealed that CAPE altered the transcriptional activity of the apaf-1 and mcl-1 promoters. Further studies using chromatin immunoprecipitation assays demonstrated that E2F-1 binding to the apaf-1 and cyclin B promoters was increased and decreased, respectively, in CAPE-treated cells. Furthermore, E2F-1 silencing abolished CAPE-mediated effects on cell-cycle arrest, apoptosis and related gene expression. Taken together, these results indicate a crucial role for E2F-1 in CAPE-mediated cellular activities in cervical cancer cells.

Radioimmunotherapy and apoptotic induction on CK19-overexpressing human cervical carcinoma cells with Re-188-mAbCx-99.

BACKGROUND: The overexpression of Ck19 antigen occurs frequently in human carcinomas. The strategy and mechanism of radioimmunotherapy using Re-188-mAbCx-99 to Ck19 on human cervical carcinoma cells was investigated in this study. MATERIALS AND METHODS: Using mAbCx-99, the overexpression of Ck19 protein in lysates of cell lines and tissues from various patients' cervixes were verified by immunobinding and immunoblot analysis. The therapeutic effect of Re-188-mAbCx-99 on ME180 cells was examined in vitro by cell proliferation, apoptosis, DNA fragmentation and intemucleosomal levels. RESULTS: A relatively high expression of Ck19 was found in all human cervical carcinoma cell lines (4- to 44-fold) and in tissue lysates (26.8- to 79-fold) from patients (31 out of 34) with cervical, endometrial or ovarian carcinomas compared with that of benign or normal control samples. The growth inhibition of ME180, CC7T and Hela cells were significantly higher (p < 0.001) in the Re-188-mAbCx-99-treated (60-80%) than in the Re-188-MOPCIgG1-treated lines (8-18%) after 72-h treatment. After 48 h of treatment with Re-188-mAbCx-99, ME180 cells significantly exhibited DNA fragmentation and morphological features of apoptosis. This effect markedly elevated the expression of p21, p53 and Bcl-xS protein, while the Mcl-1 and Caspase-8 proteins were down-regulated. CONCLUSION: We suggest that an elevated Ck19 level is associated with disease stage in most patients with cervical cancer. The therapeutic effect of Re-188-mAbCx-99 was verified through apoptosis on targeting the enriched Ck19 protein of carcinoma cells.

Identification and characterization of the retinoic acid response elements in the human RIG1 gene promoter.

The expression of retinoic acid-induced gene 1 (RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all-trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5'-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the -4910/-5509 fragment of the 5'-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between -5048 and -5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5'-TGACCTctattTGCCCT-3' (DR5, -5243/-5259), and an inverted repeat sequence with six nucleotide spacing, 5'-AGGCCAtggtaaTGGCCT-3' (IR6, -5323/-5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the atRA-mediated induction. Taken together, we have identified and characterized the functional atRA response element that is responsible for the atRA-mediated induction of RIG1 gene.

Comparison of different adjuvants of protein and DNA vaccination for the prophylaxis of IgE antibody formation.

A high-molecular-weight mite allergen Der f11 that was hardly purified for immunotherapy was used to develop the DNA vaccine pDf11. We have shown that vaccination of mice with pDf11 induces Th1 responses characterized by suppression of IgE responses. In the present study, effects of different adjuvants on pDf11 were first studied. Mice receiving pDf11 +/- CpG, bestatin, and bupivacaine had better suppression of IgE responses than those receiving pDf11 +/- lipofectin or alum. Bestatin could greatly boost IgG2a responses. Immunomodulating effects of different adjuvants between protein and DNA vaccines were further elucidated. CpG was the best for both protein and DNA vaccines to profoundly suppress IgE responses, but alum, bestatin and lipofectin were useless for rDf11 to induce IgE inhibition. Neither did the combination of rDf11 and pDf11 have further IgE suppression. In conclusion, CpG is the unique adjuvant for the protein vaccine rDf11 to inhibit IgE responses. In contrast, the DNA vaccine pDf11 +/- CpG, bestatin, or bupivacaine induces profound suppression of IgE responses.

Apoptotic effect of caffeic acid phenethyl ester and its ester and amide analogues in human cervical cancer ME180 cells.

BACKGROUND: Caffeic acid phenylether ester (CAPE) has potent antioxidant, anti-inflammatory, antiviral, anti-proliferative, immunomodulatory and pro-apoptotic activities. The activities of CAPE and its novel synthetic derivatives, caffeic acid octyl ester (CAO) and 1-octyl caffeamide (CAN-8), were investigated in this study. MATERIALS AND METHODS: Cultured human cells were incubated with or without these compounds. The effect of these compounds on cell apoptosis, intracellular level of hydrogen peroxide and mitochondrial potential were analyzed. Western blot analysis was used to study the effect of alterations in protein level of caspases, Bcl-2 family, p21, p53 and c-Jun upon drug treatment. RESULTS: These compounds arrested cell proliferation, triggered cell apoptosis and caused a marked scavenging effect of hydrogen peroxide. Apoptosis induced by CAPE or CAO is associated with increased expression of p53, p21 and c-Jun. While the levels of Bcl-2 and Bcl-xL were relatively unchanged, these compounds induced a marked reduction in Mcl-1 level. The CAPE- or CAO-induced apoptosis was also accompanied by a rapid loss of mitochondrial transmembrane potential and activation of caspase-3 and caspase-8, suggesting a mitochondrial-dependent mechanism. In causing these cellular actions, CAO was shown to be comparable or more potent than CAPE, whereas the amide analogue CAN-8 displayed much weaker activities than both CAPE and CAO. Since these three compounds contain similar antioxidant functionality, the difference in their potency suggests that the octyl moiety in CAO is an important determinant for the enhanced activities. CONCLUSION: We have characterized a novel CAPE structure analogue, CAO, which showed strong antioxidant and proapoptotic activities. In addition, we demonstrated that down-regulation of Mcl-1 gene expression and activation of caspase-8 are associated with CAPE-triggered cell apoptosis.

The concentration of serum transforming growth factor beta-1 (TGF-beta1) is decreased in cervical carcinoma patients.

Transforming growth factor beta-1 (TGF-beta1) is a multifunctional growth factor and known to inhibit the proliferation of epithelial cell. The relationship between serum TGF-beta1 level and the presence of cervical cancer was investigated in this study. The patients with cervical squamous cell carcinoma (SCC) had significantly lower level of serum TGF-beta1 (39.14 +/- 9.03 ng/mL; mean +/- SD) than those with myoma (49.17 +/- 9.38 ng/mL) and normal subjects (49.13 +/- 8.81 ng/mL), (p < 0.007 and p < 0.001, respectively). TGF-beta1 level was also lower in the patients with cervical intraepithelial neoplasia (CIN3) (42.07 +/- 9.38 ng/mL) than in normal controls (49.13 +/- 8.81 ng/mL) (p < 0.04). It suggested that diminution of the production of TGF-beta1 has close association with the neoplastic transformation of cervical epithelium.