Glucagon-like peptide-1 receptor agonist exendin 4 ameliorates diabetes-associated vascular calcification by regulating mitophagy through the AMPK signaling pathway.

BACKGROUND: Vascular calcification (VC) is a complication in diabetes mellitus (DM) patients. Osteogenic phenotype switching of vascular smooth muscle cells (VSMCs) plays a critical role in diabetes-related VC. Mitophagy can inhibit phenotype switching in VSMCs. This study aimed to investigate the role of the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin 4 (EX4) in mitophagy-induced phenotype switching. MATERIALS AND METHODS: The status of VC in T2DM mice was monitored using Von Kossa and Alizarin Red S (ARS) staining in mouse aortic tissue. Human aortic smooth muscle cells were cultured in high glucose (HG) and ß-glycerophosphate (ß-GP) conditioned medium. Accumulation of LC3B and p62 was detected in the mitochondrial fraction. The effect of EX4 in vitro and in vivo was investigated by knocking down AMPKα1. RESULTS: In diabetic VC mice, EX4 decreased the percentage of von Kossa/ARS positive area. EX4 inhibited osteogenic differentiation of HG/ß-GP-induced VSMCs. In HG/ß-GP-induced VSMCs, the number of mitophagosomes was increased, whereas the addition of EX4 restored mitochondrial function, increased the number of mitophagosome-lysosome fusions, and reduced p62 in mitochondrial frictions. EX4 increased the phosphorylation of AMPKα (Thr172) and ULK1 (Ser555) in HG/ß-GP-induced VSMCs. After knockdown of AMPKα1, ULK1 could not be activated by EX4. The accumulation of LC3B and p62 could not be reduced after AMPKα1 knockdown. Knockdown of AMPKα1 negated the therapeutic effects of EX4 on VC of diabetic mice. CONCLUSION: EX4 could promote mitophagy by activating the AMPK signaling pathway, attenuate insufficient mitophagy, and thus inhibit the osteogenic phenotype switching of VSMCs.

microRNA-33a prevents epithelial-mesenchymal transition, invasion, and metastasis of gastric cancer cells through the Snail/Slug pathway.

Invasion and metastasis are responsible for the majority of deaths in gastric cancer (GC). microRNA-33a (miR-33a) might function as a tumor suppressor in multiple cancers. Here, we describe the regulation and function of miR-33a in GC and mechanisms involved in epithelial-mesenchymal transition (EMT) and metastasis. First, GC tissues and adjacent normal tissues were collected. miR-33a upregulation or SNAI2 depletion on GC cells were introduced to assess the detailed regulatory mechanism of them. We assessed the expression of miR-33a, SNAI2, Snail/Slug signaling pathway-related genes, and EMT-related markers in GC tissues and cells. miR-33a distribution in GC tissues and adjacent normal tissues was measured. Cell proliferation, migration and invasion, and cell cycle distribution were assessed. In nude mice, GC tumor growth and lymph node metastasis were observed. Furthermore, the predicative value of miR-33a in the prognosis of GC patients was evaluated. The obtained results indicated that lowly expressed miR-33a, highly expressed SNAI2, activated Snail/Slug, and increased EMT were identified in GC tissues. miR-33a was located mainly in the cytoplasm. miR-33a targeted and negatively regulated SNAI2. MKN-45 and MKN-28 cell lines were selected for in vitro experiments. Upregulated miR-33a expression or siRNA-mediated silencing of SNAI2 suppressed the activation of Snail/Slug, whereby GC cell proliferation, invasion and migration, EMT, tumor growth, and lymph node metastasis were inhibited. High expression of miR-33a was a protective factor influencing the prognosis of GC. This study suggests that miR-33a inhibited EMT, invasion, and metastasis of GC through the Snail/Slug signaling pathway by modulating SNAI2 expression.NEW & NOTEWORTHY miR-33a targets and inhibits the expression of SNAI2, overexpression of SNAI2 activates the Snail/Slug signaling pathway, the Snail/Slug signaling pathway promotes GC cell proliferation, invasion, and metastasis, and overexpression of miR-33a inhibits cell proliferation, invasion, and metastasis. This study provides a new therapeutic target for the treatment of GC.

Depletion of telomerase RNA inhibits growth of gastrointestinal tumors transplanted in mice.

AIM: To explore effects of telomerase RNA-targeting phosphorothioate antisense oligodeoxynucleotides (PS-ASODN) on growth of human gastrointestinal stromal tumors transplanted in mice. METHODS: A SCID mouse model for transplantation of human gastrointestinal stromal tumors (GISTs) was established using tumor cells from a patient who was diagnosed with GIST and consequently had been treated with imatinib. GIST cells cultured for 10 passages were used for inoculation into mice. Transfection of PS-ASODN was carried out with Lipotap Liposomal Transfection Reagent. GISTs that subsequently developed in SCID mice were subjected to intra-tumoral injection once daily from day 7 to day 28 post-inoculation, and mice were divided into the following four groups according to treatment: PS-ASODN group (5.00 µmoL/L of oligonucleotide, each mouse received 0.2 mL once daily); imatinib group (0.1 mg/g body weight); liposome negative control group (0.01 mL/g); and saline group (0.01 mL/g). On day 28, the mice were sacrificed, and tumor attributes including weight and longest and shortest diameters were measured. Tumor growth was compared between treatment groups, and telomerase activity was measured by enzyme-linked immunosorbent assay. Apoptosis was examined by flow cytometry. Real-time polymerase chain reaction was used to detect expression of the mRNA encoding the apoptosis inhibition B-cell leukemia/lymphoma 2 (bcl-2) gene. RESULTS: In the PS-ASODN group, tumor growth was inhibited by 59.437%, which was markedly higher than in the imatinib group (11.071%) and liposome negative control group (2.759%) [tumor inhibition = (mean tumor weight of control group--mean tumor weight of treatment group)/(mean tumor weight of control group) × 100%]. Telomerase activity was significantly lower (P < 0.01) in the PS-ASODN group (0.689 ± 0.158) compared with the imatinib group (1.838 ± 0.241), liposome negative control group (2.013 ± 0.273), and saline group (2.004 ± 0.163). Flow cytometry revealed that the apoptosis rate of tumor cells treated with PS-ASODN was 20.751% ± 0.789%, which was higher (P < 0.01) than that of the imatinib group (1.163% ± 0.469%), liposome negative control group (1.212% ± 0.310%), and saline group (1.172% ± 0.403%). Expression of bcl-2 mRNA in the transplanted GISTs was markedly downregulated (P < 0.01) in the PS-ASODN group (7.245 ± 0.739) compared with the imatinib group (14.153 ± 1.618) and liposome negative control group (16.396 ± 1.351). CONCLUSION: PS-ASODN can repress GIST growth, mediated perhaps by inhibition of telomerase activity and downregulation of bcl-2 expression.

Potentially predictive microRNAs of gastric cancer with metastasis to lymph node.

AIM: To detect the expression of 60 microRNAs (miRNAs) in gastric cancer tissues and find new predictive biomarkers of gastric cancer with metastasis. METHODS: The expressions of 60 candidate miRNAs in 30 gastric cancer tissues and paired normal tissues were detected by stem-loop real-time reverse transcription-polymerase chain reaction. After primary screening of miRNAs expression, 5 selected miRNAs were further testified in another 22 paired gastric tissues. Based on the expression level of miRNAs and the status of metastasis to lymph node (LN), receiver-operating-characteristic (ROC) curve were used to evaluate their ability in predicting the status of metastasis to LN. RESULTS: Thirty-eight miRNAs expressions in gastric cancer tissues were significantly different from those in paired normal tissues (P < 0.01). Among them, 31 miRNAs were found to be up-expressed in cancer tissues and 1 miRNAs were down-expressed ≥ 1.5 fold vs paired normal gastric tissue. Five microRNAs (miR-125a-3p, miR-133b, miR-143, miR-195 and miR-212) were differently expressed between different metastatic groups in 30 gastric cancer biopsies (P < 0.05). Partial correlation analysis showed that hsa-mir-212 and hsa-mir-195 were correlated with the status of metastasis to LN in spite of age, gender, tumor location, tumor size, depth of invasion and cell differentiation. ROC analysis indicated that miR-212 and miR-195 have better sensitivities (84.6% and 69.2%, respectively) and specificities (both 100%) in distinguishing biopsies with metastasis to LN from biopsies without metastasis to LN. CONCLUSION: miR-212 and miR-195 could be independent biomarkers in predicting the gastric cancer with metastasis to LN.

[Surgical treatment and prognostic analysis of retroperitoneal paragangliomas: a study of 19 cases].

OBJECTIVE: To investigate the clinical characteristics, surgical treatment and prognostic analysis of retroperitoneal paragangliomas and to enhance the diagnostic and therapeutic levels of retroperitoneal paragangliomas. METHODS: The clinical data of all patients undergoing paraganglioma resection at our department from November 1999 to March 2009 were retrospectively analyzed. The parameters included clinical manifestations, tumor function, surgical findings, operative approach, tumor pathology, imaging study and post-operative survival time. RESULTS: (1) The ratio of male to female was 1.375:1 and the median age 50 years old. The most common presenting symptom was abdominal mass (9/19, 47%). And the preoperative CT misdiagnosis rate was high (89%). (2) The most common tumor location was periaortic and percival (9/19, 47%). The average maximal diameter of tumors was 8.6 cm. 58% (11/19) tumors had integral peplow, 42% (8/19) adhered to adjacent organs and 26% (5/19) required adjacent organ resection. (3) The rate of functional tumor was 63% (12/19). Preoperative and intra-operative hypertension occurred in 67% (8/12) and 33% (4/12) respectively. (4) Immunohistochemical staining was performed in 18 tumors of 16 patients. Among all tumors, 89% (16/18) showed positive immunoreactivity for chromogranin and 67% (12/18) for S-100. PCNA staining showed different proliferative activities (0%-48% positive). Only malignant tumors showed positive immunoreactivity for Ki-67 staining and P53 staining (20% & 34% respectively). (5) The overall 5-year survival was 77%. Survival was significantly worse after metastasis (χ2=6.604, P=0.01). But it was not dependent on tumor diameter (χ2=3.208, P=0.201), the secreting function of tumor (χ2=0.121, P=0.728) and the status of tumor margins (χ2=0.036, P=0.849). CONCLUSION: It is difficult to make an early diagnosis of retroperitoneal paragangliomas. Survival is significantly worse after metastasis. Lifelong follow-up for recurrence is important. And it is absolutely essential to perform immunohistochemical staining for tumors.

[Effect of adjuvant chemotherapy of ginsenoside Rg3 combined with mitomycin C and tegafur in advanced gastric cancer].

OBJECTIVE: To evaluate the enhancing effects of ginsenoside Rg3 combined with mitomycin C and tegafur (MF) on postoperative chemotherapy in advanced gastric cancer. METHODS: Seventy-one postoperative patients with advanced gastric cancer were randomly divided into two groups, the control group (n=33), which received treatment with only MF (Mitomycin C+Tegafur), and the trial group (n=38), which were treated with ginsenoside Rg3+MF. The serum VEGF levels in the control group and trial group were detected preoperatively and postoperatively, meanwhile, the serum VEGF levels in 30 healthy persons were detected as comparison. The relations between patients survival and serum VEGF levels were analyzed. RESULTS: The levels of serum VEGF in advanced gastric cancer were higher than those in healthy persons [(297.8+/-129.6) pg/ml vs (212.3+/-67.5) pg/ml] (P<0.01), and were correlated with the depth of tumor invasion, lymph node metastasis, tumor size > 4 cm and TNM stage (P<0.05). Fourteen weeks after operation, the levels of serum VEGF in trial group decreased below those of preoperation and approached to normal range, while in the control group, the levels of serum VEGF decreased near those of preoperation only. The median survival of patients in trial group and control group were 40 and 25 months respectively. The survival rate of patients in trial group was significantly higher than that in control group (P=0.047). CONCLUSION: The combined application of ginsenoside Rg3+MF chemotherapy can decrease the concentration of serum VEGF and improve the survival rate in advanced gastric cancer patients.