ADP-ribosylation factor-like GTPase 15 enhances insulin-induced AKT phosphorylation in the IR/IRS1/AKT pathway by interacting with ASAP2 and regulating PDPK1 activity.

Decreased phosphorylation in the insulin signalling pathway is a hallmark of insulin resistance. The causes of this phenomenon are complicated and multifactorial. Recently, genomic analyses have identified ARL15 as a new candidate gene related to diabetes. However, the ARL15 protein function remains unclear. Here, we show that ARL15 is upregulated by insulin stimulation. This effect was impaired in insulin-resistant pathophysiology in TNF-α-treated C2C12 myotubes and in the skeletal muscles of leptin knockout mice. In addition, ARL15 localized to the cytoplasm in the resting state and accumulated in the Golgi apparatus around the nucleus upon insulin stimulation. ARL15 overexpression can enhance the phosphorylation of the key insulin signalling pathway molecules IR, IRS1 and AKT in C2C12 myotubes. Moreover, ARL15 knockdown can also specifically inhibit the phosphorylation of PDPK1 Ser241, thereby reducing PDPK1 activity and its downstream phosphorylation of AKT Thr308. Co-immunoprecipitation assays identified ASAP2 as an ARL15-interacting protein. In conclusion, we have identified that ARL15 acts as an insulin-sensitizing effector molecule to upregulate the phosphorylation of members of the canonical IR/IRS1/PDPK1/AKT insulin pathway by interacting with its GAP ASAP2 and activating PDPK1. This research may provide new insights into GTPase-mediated insulin signalling regulation and facilitate the development of new pharmacotherapeutic targets for insulin sensitization.

High-mobility group box 1 protein activating nuclear factor-κB to upregulate vascular endothelial growth factor C is involved in lymphangiogenesis and lymphatic node metastasis in colon cancer.

OBJECTIVES: To investigate the roles of high-mobility group box 1 (HMGB1) protein in lymphangiogenesis and lymphatic node metastasis in colon cancer. METHODS: Archival tumour specimens from patients with colon cancer were analysed in this retrospective immunohistochemical study. HMGB1, vascular endothelial growth factor C (VEGF-C) and podoplanin protein levels were analysed immunohistochemically. In vitro studies using the colon cancer cell line HCT116 were also undertaken to investigate the relationship between HMGB1, VEGF-C and nuclear factor (NF)-κB. RESULTS: Specimens from 70 patients with colon cancer were reviewed. The presence of positive HMGB1 immunohistochemical staining significantly correlated with lymphatic microvessel density, lymph node metastasis and VEGF-C immunohistochemical staining in colon cancer specimens. The presence of positive VEGF-C immunohistochemical staining significantly correlated with lymph node metastasis. The in vitro studies demonstrated that HMGB1 upregulated VEGF-C mRNA and protein in a dose-dependent manner in HCT116 cells, and that this was mediated via NF-κB. CONCLUSIONS: HMGB1 immunohistochemical staining was significantly associated with lymphangiogenesis and lymphatic node metastasis in colon cancer. There was evidence that HMGB1 upregulates VEGF-C by activating NF-κB in a colon cancer cell line.

Silencing hexokinase II gene sensitizes human colon cancer cells to 5-fluorouracil.

BACKGROUND/AIMS: Extensive trials have indicated that cancer cells with high glycolytic activity exhibit decreased sensitivity to anticancer agents. Moreover, recent research has proved that a specific inhibitor of hexokinase II, which is a key glycolytic enzyme, may enhance the activity of anticancer drugs. The purpose of this study is to further investigate the effect and mechanisms of hexokinase II on chemosensitivity of colon cancer cells to 5-fluorouracil. METHODOLOGY: The expression of hexokinase II gene was down regulated by RNA interference in colon cancer cell line LoVo, then the IC50 value of 5-fluorouracil to LoVo cells was carried out by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the protein expression of hexokinase II and thymidylate synthas by Western blot analysis Meanwhile, cell apoptosis and mitochondrial membrane potential were assessed by flow cytometry. Caspase-3 activity was also determined by its substrate color reaction. RESULTS: Down-regulation of the hexokinase II gene of LoVo cells resulted in decreased IC50 value of 5-fluorouracil and increased apoptosis rate, respectively. Furthermore, silencing hexokinase II of LoVo cells induced loss of mitochondrial membrane potential, activation of caspase-3, and inhibition of thymidylate synthase expression. CONCLUSIONS: Our findings suggest targeting hexokinase II has a potential role in the combination 5-fluorouracil treatments of colon cancer.

Expression of Tiam1 and VEGF-C correlates with lymphangiogenesis in human colorectal carcinoma.

OBJECTIVE: To investigate the relationship between Tiam1 and lymphangiogenesis in human colorectal carcinoma (CRC) tissues, as well as the expression of VEGF-C in a CRC cell line (HCT116) after knockdown of the Tiam1 gene with RNA interference (RNAi). RESULTS: In the specimens of CRC tissue, the positivity rate of Tiam1 and VEGF-C was 84% and 58%, respectively. The positivity rate of VEGF-C in the Tiam1 positive group (64.3%) was significantly higher than that in the Tiam1 negative group (25.0%). The LMVD in the Tiam1 positive group (11.35 +/- 3.34) was significantly higher than that in the Tiam1 negative group (7.38 +/- 2.27). In addition, the expression of the Tiam1 gene was efficiently blocked by RNAi. Downregulation of Tiam1 gene expression significantly suppressed HCT116 cell growth in vitro. Compared with untransfected HCT116 cells, HCT116 cells transfected with pGenesil-1-Tiam1 plasmids showed a significant decrease in the expression of VEGF-C. METHODS: The expressions of Tiam1, Rac1, VEGF-C and Podoplanin in 50 samples of CRC were detected by immunohistochemical analysis. The lymph microvessel density (LMVD) in Podoplanin positive specimens was evaluated. The results were analyzed statistically to investigate the correlation of Tiam1, VEGF-C, lymph node metastasis and other clinicopathological parameters. An shRNA eukaryotic expression vector against Tiam1 gene was constructed and transfected into HCT116 cells. The expression of Tiam1 gene was assessed by RT-PCR and western blot analysis. CONCLUSIONS: We suggest that the Tiam1 gene may act as a crucial therapeutic target for Lymphangiogenesis in CRC.

Downregulation of the hexokinase II gene sensitizes human colon cancer cells to 5-fluorouracil.

BACKGROUND: Extensive trials have indicated that cancer cells with high glycolytic activity exhibit decreased sensitivity to anticancer agents. Moreover, recent research has proven that specific inhibitors of hexokinase (HK) II, a key glycolytic enzyme, may enhance the activity of anticancer drugs. The aim of this study was to investigate the effect and mechanisms of HK II on chemosensitivity of a colon cancer cell line (LoVo) to 5-fluorouracil (5-FU). METHODS: HK II gene expression was downregulated by RNA interference in the colon cancer cell line LoVo, which was detected by Western blot analysis. Then the IC(50) value of 5-FU was determined in LoVo cells via MTT assay. In addition, cell apoptosis and mitochondrial membrane potential (MMP) were assessed by flow cytometry and caspase-3 activity by its substrate color reaction. RESULTS: In LoVo cells, HK II downregulation resulted in a decreased IC(50) value of 5-FU and increased apoptosis. Furthermore, HK II downregulation resulted in a decreased MPP and activation of caspase-3. CONCLUSION: Our findings suggest that targeting HK II may be beneficial for patients with colon cancer treated with 5-FU.

Stable RNA interference of hexokinase II gene inhibits human colon cancer LoVo cell growth in vitro and in vivo.

The purpose of this study is to investigate the effect of silencing hexokinase II (HK II) gene with RNA interference (RNAi) technique on colon cancer LoVo cell proliferation in vitro and in vivo. A short hairpin RNA (shRNA) eukaryotic expression vector against HK II gene was constructed, named as plasmid pGenesil-1-HK II, and transfected into LoVo cells. The expression of HK II gene was detected by RT-PCR and Western blot analysis respectively. Then, tumor colony formation was observed, and cell cycle was also assessed by flow cytometry and the contents of intracellular adenosine triphosphate (ATP) by high performance liquid chromatography (HPLC). Furthermore, LoVo cells were injected subcutaneously into nude mice. After a 4-week follow-up period, the sizes and weights of tumors were measured. Moreover, the expression of Ki67 protein was observed by immunohistochemical technique, and cell apoptosis by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Consequently, the expression of HK II gene was efficiently blocked by RNAi. Downregulation of HK II gene expression significantly suppressed cloning efficiency and cell cycle of LoVo cell in vitro and tumor growth in vivo. Compared with untransfected LoVo cells, LoVo cells transfected with pGenesil-1-HK II plasmids showed significant decrease in the cellular ATP contents and Ki67 expression, and obvious increase in the apoptosis indexes. Our results suggest that HK II gene can act as a crucial therapeutic target for slowing colon cancer growth.

[Expression of hexokinase-II gene in human colon cancer cells and the therapeutic significance of inhibition thereof].

OBJECTIVE: To investigate the expression of hexokinase (HK)-II gene in human colon cancer cells and the therapeutic significance of inhibition of HK-II gene. METHODS: Human colon cancer cells of the lines HCT-116, LOVO, HT-19, and SW480 were cultured. The mRNA expression and protein expression of HK-II in these cells were detected by RT-PCR and Western blotting respectively. 3-Bromopyruvic acid (3-BrPA), a HK-II specific inhibitor, of different concentrations was added into the culture fluid of the colon cancer cells for 48 h, then MTT method was used to examine the proliferation of the cells. 3-BrPA combined with adriamycin (ADM) of the concentrations of 0.05 and 0.1 microg/ml, or with oxaliplatin (L-OHP) of the concentration of 0.5 and 1.0 microg/ml was added into culture fluid for 48 h to observe the change of the cell proliferation rate. 3-BrPA of different concentrations was co-cultivated with LOVO cells for 24 h, and then enzyme labeling instrument was used to measure the activity of caspase-3, a cell apoptosis signal. 3-BrPA and CaCl2 were added into the culture fluid of LOVO cells and then ultra-violet spectrum was used to detect the mitochondrial permeability transition pore (PTP) opening degree. Flow cytometry (FCM), with addition of 10 microg/ml Rh123, was used to measure the mitochondrial membrane potential (DeltaPsim) of LOVO cells. RESULTS: Both HK-II mRNA and HK-II protein were expressed in the HCT-116, LOVO, HT-19, and SW480 cells. Treated by 3-BrPA combined with ADM of the concentrations of 0.05 and 0.1 microg/ml for 48 h, the cell proliferation inhibiting rates in the 4 lines were increased from 5.1% +/- 1.3% and 10.5% +/- 2.0% to 46.5% +/- 3.2% and 57.9% +/- 3.3% respectively (all P < 0.01). Treated by 3-BrPA combined with L-OHP of the concentration of 0.5 and 1.0 microg/ml for 48 h, the cell proliferation inhibiting rates were increased from 19.2% +/- 6.1% and 32.2% +/- 2.2% to 48.4% +/- 3.2% and 60.5% +/- 4.6% respectively (all P < 0.01). 24 h after the co-cultivation of 3-BrPA of different concentrations, the caspase-3 activity of the LOVO cells was increased along with the increase of the concentration of 3-BrPA (P = 0.000). The PTP opening degrees induced by 3-BrPA and CaCl(2) of the LOVO cells were 40.0% +/- 3.5% and 37.4% +/- 2.3% respectively (P = 0.348). After treated with 100 microml/L 3-BrPA for 1, 3, and 5 h, the DeltaPsim decrease rates of the LOVO cells were 12.7%, 15.4%, and 26.8% respectively. CONCLUSION: HK-II gene may be an effective therapeutic target for gene may be an effective therapeutic target for gene may be an effective therapeutic target for colon cancer.

Identification of leukemia-associated antigens in chronic myeloid leukemia by proteomic analysis.

The immune system plays an important role in the treatment of chronic myeloid leukemia (CML). Identification of leukemia-associated antigens (LAAs) eliciting an immune response in patients is a prerequisite for specific immunotherapy of CML. To identify new LAAs in CML, We utilized a novel approach based serology and proteomics technologies. LAAs were identified by comparing the reactivity of proteins resolved by 2-DE with sera from CML patients and healthy donors. Several new LAAs were identified including alpha enolase, aldolase A, HSP70 protein8, beta-tubulin and tropomyosin isoforms. Although, the functions of these identified proteins in CML need further investigation, the detection of autoantibodies in CML may have value on CML screening, diagnosis, or follow-up. Additionally, identification of LAAs in CML may also be of vital importance in antigen-based immunotherapy.