Honokiol Prevents Intestinal Barrier Dysfunction in Mice with Severe Acute Pancreatitis and Inhibits JAK/STAT1 Pathway and Acetylation of HMGB1.

OBJECTIVE: To investigate the effect of honokiol (HON) and the role of high-mobility group protein B1 (HMGB1) on the pathogenesis of severe acute pancreatitis (SAP). METHODS: Thirty mice were numbered according to weight, and randomly divided into 5 groups using a random number table, including control, SAP, SAP and normal saline (SAP+NS), SAP and ethyl pyruvate (SAP+EP), or SAP+HON groups, 6 mice in each group. Samples of pancreas, intestine, and blood were collected 12 h after SAP model induction for examination of pathologic changes, immune function alterations by enzyme linked immunosorbent assay (ELISA), and Western blot. In vitro experiments, macrophages were divided into 5 groups, the control, lipopolysaccharide (LPS), LPS+DMSO (DMSO), LPS+anti-HMGB1 monoclonal antibody (mAb), and LPS+ HON groups. The tight connection level was determined by transmission electron microscopy and fluorescein isothiocyanate-labeled. The location and acetylation of HMGB1 were measured by Western blot. Finally, pyridone 6 and silencing signal transducer and activator of the transcription 1 (siSTAT1) combined with honokiol were added to determine whether the Janus kinase (JAK)/ STAT1 participated in the regulation of honokiol on HMGB1. The protein expression levels of HMGB1, JAK, and STAT1 were detected using Western blot. RESULTS: Mice with SAP had inflammatory injury in the pancreas, bleeding of intestinal tissues, and cells with disrupted histology. Mice in the SAP+HON group had significantly fewer pathological changes. Mice with SAP also had significant increases in the serum levels of amylase, lipase, HMGB1, tumor necrosis factor- α, interleukin-6, diamine oxidase, endotoxin-1, and procalcitonin. Mice in the SAP+HON group did not show these abnormalities (P<0.01). Studies of Caco-2 cells indicated that LPS increased the levels of occludin and claudin-1 as well as tight junction permeability, decreased the levels of junctional adhesion molecule C, and elevated intercellular permeability (P<0.01). HON treatment blocked these effects. Studies of macrophages indicated that LPS led to low nuclear levels of HMGB1, however, HON treatment increased the nuclear level of HMGB1 (P<0.01). HON treatment also inhibited the expressions of JAK1, JAK2, and STAT1 (P<0.01) and increased the acetylation of HMGB1 (P<0.05). CONCLUSION: HON prevented intestinal barrier dysfunction in SAP by inhibiting HMGB1 acetylation and JAK/STAT1 pathway.

SnoN residue (1-366) attenuates hypertrophic scars through resistance to transforming growth factor-ß1-induced degradation.

Hypertrophic scars (HSs) are characterized by fibroblast hyperproliferation and excessive matrix deposition. During wound healing, transforming growth factor (TGF)-ß1/Smad signaling acts as a key regulator. As a transcriptional corepressor of TGF-ß1/Smads, SnoN is expressed at low levels in many fibrotic diseases due to TGF-ß1/Smad-induced degradation. SnoN residue (1-366; SR) is resistant to TGF-ß1-induced degradation. However, the expression and role of SR in HSs are unknown. Here, we inhibited TGF-ß1/Smad signaling via overexpression of SR to block fibroblast transdifferentiation, proliferation, and collagen deposition during HS formation. Our results showed that SnoN was downregulated in HS fibroblasts (HSFs) owing to TGF-ß1/Smad-induced degradation. Overexpression of SR in normal human dermal fibroblasts (NHDFs) and HSFs successfully blocked phosphorylation of Smad2 and Smad3, thereby inhibiting NHDF transdifferentiation and HSF proliferation and reducing type I collagen (ColI) and type III collagen (ColIII) production and secretion. In addition, we applied overexpressed full-length SnoN (SF) and SR to wound granulation tissue in a rabbit model of HSs. SR reduced wound scarring, improved collagen deposition and arrangement of scar tissue, and decreased mRNA and protein expression of ColI, ColIII, and α-smooth muscle actin (α-SMA) more effectively than SF in vivo. These results suggest that SR could be a promising therapy for the prevention of HS.

ASCL2 expression contributes to gastric tumor migration and invasion by downregulating miR223 and inducing EMT.

Achaete­scute homolog 2 (ASCL2), a basic helix­loop­helix transcription factor, serves an essential role in the maintenance of adult intestinal stem cells and the growth of gastric cancer (GC). However, the function of ASCL2 in the metastasis of GC is poorly understood. The present study aimed to evaluate the effect of ASCL2 expression on gastric tumor metastasis. ASCL2 protein expression was detected in 32 cases of gastric metastasis and its relevant primary tumors using western blotting and immunohistochemistry. The data suggested that the expression of ASCL2 was highest in metastatic tumors, among adjacent normal tissues, primary gastric tumors and gastric metastatic tumors. Furthermore, ASCL2­overexpressing GC cell lines MKN1­ASCL2 and SNU16­ASCL2 were established. An in vitro assay suggested that microRNA 223 (miR223) expression was downregulated following ASCL2 overexpression, and that the expression of the epithelium­associated protein E­cadherin was significantly decreased, while a series of mesenchyme­associated proteins, including zinc finger E­box­binding homeobox 1 (Zeb­1), twist­related protein 1, integrin, vimentin, 72 kDa type IV collagenase and matrix metalloproteinase­9 were upregulated in ASCL2­overexpressing cells. Overexpression of miR223 attenuated the epithelial­mesenchymal transition (EMT)­promoting effect induced by ASCL2 expression. In addition, the results of the chromatin immunoprecipitation and luciferase reporter gene assays indicated that ASCL2 was able to interact with the promoter of pre­miR223, and to inhibit the maturation of miR223, which may interact with the 3' untranslated region of Zeb­1 and inhibit EMT in tumor cells. The results of the present study demonstrated that ASCL2 was able to downregulate the expression level of miR223, contribute to EMT and promote gastric tumor metastasis, which indicated that ASCL2 may serve as a therapeutic target in the treatment of GC.

Cantharidin Inhibits the Growth of Triple-Negative Breast Cancer Cells by Suppressing Autophagy and Inducing Apoptosis in Vitro and in Vivo.

BACKGROUND/AIMS: Cantharidin, a type of terpenoid secreted by the blister beetle Mylabris phalerata (Pallas), has attracted great attention in cancer therapy because of its potential anti-cancer activities. Here, we report the effects on apoptosis and autophagy in human triple-negative breast cancer (TNBC) cell lines after treatment with cantharidin and attempt to elucidate the underlying mechanisms. METHODS: MDA-MB-231 and MDA-MB-468 cells were treated with cantharidin and cell proliferation was examined using CCK-8 and clone formation assays. The expression of apoptosis- and autophagy-associated proteins was detected by western blotting. Cells were infected with lentivirus carrying the Beclin-1 gene, and MDA-MB-231-beclin1 (MB231-Bec) and MDA-MB-468-beclin-1(MB468-Bec) cells stably expressing Beclin-1 were established. Autophagic vacuoles in cells were observed with LC3 staining using fluorescence microscopy, and apoptotic cells were detected via flow cytometry. Tumor growth was assessed by subcutaneous inoculation of TNBC cells into BALB/c nude mice. RESULTS: Cantharidin inhibited the proliferation of MDA-MB-231 and MDA-MB-468 cells, and induced cell apoptosis. Cantharidin additionally inhibited the conversion of LC3 I to LC3 II and autophagosome formation by suppressing the expression of Beclin-1. Furthermore, overexpression of Beclin-1 in TNBC cells attenuated the cytotoxicity of cantharidin. In vivo, cantharidin inhibited the growth of MDA-MB-231 and MDA-MB-468 xenografts in nude mice by suppressing autophagy and inducing apoptosis, and Beclin-1 overexpression in TNBC cells reduced the efficacy of cantharidin. CONCLUSIONS: Cantharidin inhibits autophagy by suppressing Beclin-1 expression and inducing apoptosis of TNBC cells in vitro and in vivo, thereby representing a potential strategy for the treatment of TNBC.

Loss of the Opa interacting protein 5 inhibits breast cancer proliferation through miR-139-5p/NOTCH1 pathway.

Opa interacting protein 5 (OIP5) has been reported to be over-expressed in several kinds of human cancer. However, the biological function and clinical significance of OIP5 in human breast cancer remains unknown. In this study, we found that OIP5 was notably over-expressed in breast cancer tissues compared with their corresponding nontumorous tissues. Statistical analysis showed a significant correlation of OIP5 expression with advanced clinical stage. Ablation OIP5 inhibited the proliferation of breast cancer cells. OIP5 over-expression inhibited hsa-miR-139-5p expression, antagonized its functions and led to the de-repression of its endogenous target NOTCH1, which was a core oncogene in promoting breast cancer progression. Our results suggested that OIP5 is a potential diagnosis biomarker and therapeutic target for breast cancer.

SHCBP1 is over-expressed in breast cancer and is important in the proliferation and apoptosis of the human malignant breast cancer cell line.

BACKGROUND: SHC SH2-binding protein 1, a member of Src homolog and collagen homolog (Shc) family, has been recently identified in different contexts in unbiased screening assays. It has been reported to be over-expressed in several malignant cancers. METHODS: Immunohistochemistry of SHCBP1 on 128 breast cancer tissues and adjacent normal tissues were used to evaluate the prognostic significance of SHCBP1. Survival analyses were performed by Kaplan-Meier method. CRISPR/CAS9 method was used to knockout SHCBP1 expression. CRISPR/CAS9 technology was used to knockout SHCBP1 in 2 breast cancer cell lines. MTT assay, BrdU assay, colony formation assay, cell cycle assay and apoptosis analysis in MCF-7 and MDA-MB-231 cell lines were carried out to evaluate the effects of SHCBP1 on breast cancer in vitro. RESULTS: Immunohistochemical analysis revealed SHCBP1 was significantly up-regulated in breast cancer tissues compared with adjacent normal tissues (82 of 128, 64%). Over-expressed SHCBP1 was correlated with advanced clinical stage and poorer survival. Ablation of SHCBP1 inhibited the proliferation in vitro. SHCBP1 knockout increased cyclin-dependent kinase inhibitor p21, and decreased the Cyclin B1 and CDK1. CONCLUSION: Our study suggests SHCBP1 is dysregulated expressed in breast cancer and plays a critical role in cancer progression, which can be a potential prognosis predictor of breast cancer.

Cartilage Oligomeric Matrix Protein-Angiopoietin-1 Has a Protective Effect of Vascular Endothelial Barrier in Rat With Acute Necrotizing Pancreatitis.

OBJECTIVE: To investigate the protective effect of angiopoietin-1 (Ang-1) from capillary endothelial damage in rats with acute necrotizing pancreatitis (ANP). METHODS: 96 male Sprague-Dawley rats were randomly averaged and divided into control group, ANP group, Si-Ang-1 group, and COMP (cartilage oligomeric matrix protein)-Ang-1 group. Animals were killed at 6, 12, and 24 hours after molding. Levels of serum amylase, porcine endothelin 1, C-reactive protein, and Ang-1 were detected; histopathological changes in the pancreas were observed; capillary permeability and Ang-1 expression of the pancreatic tissue were detected by Evans Blue extravasation assay, immunohistochemistry, Western blot, and quantitative polymerase chain reaction. RESULTS: (1) Levels of serum amylase, C-reactive protein, and porcine endothelin-1 increased and level of Ang-1 decrease in the ANP group and Si-Ang-1 group compared with the control group, whereas COMP-Ang-1 group could improve the changes. (2) The order of pancreas pathological changes (mild to severe) is: control group, COMP-Ang-1 group, ANP group, and Si-Ang-1 group. (3) Capillary permeability of the pancreatic tissue in the COMP-Ang-1 group was lower than that in the ANP group. (4) Ang-1 mRNA and protein expression in the COMP-Ang-1 group was significantly higher than in the ANP group. CONCLUSIONS: COMP-Ang-1 can upregulate the expression of Ang-1 protein to promote angiogenesis and improve early inflammatory and pathological damage in ANP group.

Loss of imprinting and abnormal expression of the insulin-like growth factor 2 gene in gastric cancer.

This study examined the frequency of loss of imprinting (LOI) and expression of the insulin-like growth factor 2 (IGF2) gene, and their relationship to selected clinical and pathological factors, in a well defined series of 90 Chinese patients with gastric cancer (GC) and 90 matched patients (controls) diagnosed with nonmalignant conditions. Using peripheral blood and gastric tissue samples, polymerase chain reaction-based assays and restriction endonuclease (Apa I) digestion revealed 33 GC patients and 21 controls to be Apa I informative. LOI of IGF2 was positive in 48.5% (16/33) of primary GC tumor tissues, in 21.2% (7/33) of histologically normal adjacent gastric mucosa (AM) and in 12.1% (4/33) of distant gastric mucosa (DM), and in 15.2% (5/33) of peripheral blood lymphocytes (PBLs). The prevalence of IGF2 LOI in PBL was not statistically different between GC patients (5/33, 15.2%) and control subjects (2/21, 9.5%), P = 0.69. Although patients who were found to have LOI of IGF2 were more likely to have advanced stage gastric tumors (P = 0.04), no statistically significant differences in survival were found based on imprinting status. IGF2 LOI was associated with an increased expression of IGF2 level in both tumors (P < 0.01) and blood (P < 0.01). The results of this study implicate IGF2 LOI in the molecular pathogenesis of GC, most likely through increased IGF2 expression. Although the precise molecular mechanisms by which LOI of IGF2 increases GC risk require further study, LOI of IGF2 may be a potentially important clinical epigenetic marker to identify individuals at increased risk for gastric malignancy.

[Update in the research of gene therapy for pancreatic carcinoma].

With the advances in immunology and molecular biology, new recognition in the pathogenesis, progression, and metastasis of carcinoma have been achieved. Studies on gene therapy for pancreatic carcinoma have been attempted in different ways, such as inhibiting oncogene, activating tumor suppressor gene, inducing apoptosis, applying gene directed enzyme prodrug therapy, and immune activation. New specific target genes and further development of gene technology may bring the break-through in this field.

[Significance of detection of K-ras gene mutations and CA19-9 in serum for diagnosis of pancreatic carcinoma].

BACKGROUND & OBJECTIVE: The early diagnosis of pancreatic carcinoma is difficult. The serum tumor markers such as CA19-9 have a relatively high sensitivity but with low specificity. The oncogene K-ras is frequently mutated in pancreatic carcinoma and with high specificity. The aim of this study was to assess the feasibility of detection of K-ras mutation combined with serum content of CA19-9 as an approach for diagnosis of pancreatic carcinoma. METHODS: Serum DNA was extracted from 39 patients with pancreatic carcinoma. Mutation enriched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine codon 12 mutations of K-ras. Serum content of CA19-9 was determined by radioimmunoassay. In addition, the sera from 17 patients with other pancreatic diseases and 21 healthy individuals were also analyzed as control. RESULTS: K-ras gene mutations at codon 12 were detected in the sera of 71.79%(28/39) patients with pancreatic carcinoma and 11.76%(2/17) of patients with benign pancreatic tumors. The positive rates of CA19-9 were 71.79% and 41.18%, respectively. Parallel combined test increased the diagnostic sensitivity to 94.87%; and serial combined test increased the diagnostic specificity to 94.12%. Negative results of K-ras gene and CA19-9 were obtained in all sera from healthy controls. CONCLUSION: Combined detection of K-ras mutation and CA19-9 could increase the sensitivity and specificity in diagnosing pancreatic carcinoma, and would seem to be merited in clinic.