Ambra1 is an essential regulator of autophagy and apoptosis in SW620 cells: Pro-survival role of Ambra1

Recent research has revealed a role for Ambra1, an autophagy-related gene-related (ATG) protein, in the autophagic pro-survival response, and Ambra1 has been shown to regulate Beclin1 and Beclin1-dependent autophagy in embryonic stem cells. However, whether Ambra1 plays an important role in the autophagy pathway in colorectal cancer cells is unknown. In this study, we hypothesized that Ambra1 is an important regulator of autophagy and apoptosis in CRC cell lines. To test this hypothesis, we confirmed autophagic activity in serum-starved SW620 CRC cells by assessing endogenous microtubule-associated protein 1 light chain 3 (LC3) localization, the presence of autophagosomes (transmission electron microscopy) and LC3 protein levels (Western blotting). Ambra1 expression was detected by Western blot in SW620 cells treated with staurosporine or etoposide. Calpain and caspase inhibitors were employed to verify whether calpains and caspases were responsible for Ambra1 cleavage. To examine the role of Ambra1 in apoptosis, Ambra1 knockdown cells were treated with staurosporine and etoposide. Cell apoptosis and viability were measured by annexin-V and PI staining and MTT assays. We determined that serum deprivation-induced autophagy was associated with Ambra1 upregulation in colorectal cancer cell lines. Ambra1 expression decreased during staurosporine- or etoposide-induced apoptosis. Calpains and caspases may be responsible for Ambra1 degradation. When Ambra1 expression was reduced by siRNA, SW620 cells were more sensitive to staurosporine- or etoposide-induced apoptosis. In addition, starvation-induced autophagy decreased. Finally, Co-immunoprecipitation of Ambra1 and Beclin1 demonstrated that Ambra1 and Beclin1 interact in serum-starved or rapamycin-treated SW620 cells, suggesting that Ambra1 regulates autophagy in CRC cells by interacting with Beclin1. In conclusion, Ambra1 is a crucial regulator of autophagy and apoptosis in CRC cells that maintains the balance between autophagy and apoptosis

Distribution and Clinical Significance of Th17 Cells in the Tumor Microenvironment and Peripheral Blood of Pancreatic Cancer Patients

This study was designed to investigate the distribution of Th17 cells in the tumor microenvironment and peripheral blood of pancreatic cancer patients, its clinical significance, and the expression profile of Th17 cell-associated cytokines. The percentage of Th17 cells detected by flow cytometry analysis (FACS) was significantly higher in 46 pancreatic tumor tissues (5.28 ± 1.65%) compared with corresponding adjacent normal tissues (2.57 ± 0.83%) (P = 0.031). In addition, the percentage of Th17 cells was significantly higher in stage III-IV tumors than stage I-II tumors (P = 0.039). The percentage of Th17 cells in peripheral blood of 20 pancreatic cancer patients (3.99 ± 1.15%) was significantly higher than 15 healthy volunteers (1.98 ± 0.57%) (P = 0.027). Immunohistochemistry (IHC) was performed to detect IL-17+ cells in 46 pancreatic tumor tissues, as well as expression of CD34 in 24 tumor tissues. IL-17 was shown to mainly locate in cytoplasm, and the frequency of IL-17+ cells in tumor tissues (39/46) was higher than control (29/46). The presence of IL-17+ cells in tumor tissues was associated with tumor, node, and metastasis (TNM) stage, and lymph node metastasis (P = 0.012 and P = 0.009) but not with patient sex, age, tumor size, and histological grade (P > 0.05). Interestingly, distribution of Th17 cells in tumor tissues was positively correlated with microvessel density (MVD) (r = 0.86, P = 0.018). Furthermore, the median survival time of patients with high and low level of IL-17+ cells frequency was 14.5 and 18.5 months respectively (P = 0.023). The serum levels of Th17 cell-associated cytokines, IL-17 and IL-23 in 20 pancreatic patients detected by enzyme-linked immunosorbent assay (ELISA) were 69.2 ± 28.5 pg/mL and 266.5 ± 98.1 pg/mL, respectively, which were significantly higher than 15 healthy volunteers (P = 0.015 and P = 0.02). Moreover, levels of IL-17 and IL-23 were significantly higher in stage III-IV tumors than stage I-II tumors (P = 0.04 and P = 0.036). This study suggests that increase in Th17 cells frequency and its related cytokines levels in pancreatic tumor tissues may indicate involvement in the invasion and metastasis of pancreatic cancer, which may thereby affect patient prognosis. Therefore, Th17 cells and related cytokines may be served as important immune indicators for predicting the prognosis of pancreatic cancer patients

Evidence of nigericin as a potential therapeutic candidate for cancers: A review

Emerging studies have shown that nigericin, an H+, K+ and Pb2+ ionophore, has exhibited a promising anti-cancer activity in various cancers. However, its anti-cancer mechanisms have not been fully elucidated. In this review, the recent progresses on the use of nigericin in human cancers have been summarized. By exchanging H+ and K+ across cell membranes, nigericin shows promising anti-cancer activities in in vitro and in vivo as a single agent or in combination with other anti-cancer drugs through decreasing intracellular pH (pHi). The underlying mechanisms of nigericin also include the inactivation of Wnt/β-catenin signals, blockade of Androgen Receptor (AR) signaling, and activation of Stress-Activated Protein Kinase/c-Jun N-terminal Kinase (SAPK/JNK) signaling pathways. In many cancers, nigericin is proved to specifically target putative Cancer Stem Cells (CSCs), and its synergistic effects on photodynamic therapy are also reported. Other mechanisms of nigericin including influencing the mitochondrial membrane potentials, inducing an increase in drug accumulation and autophagy, controlling insulin accumulation in nuclei, and increasing the cytotoxic activity of liposome-entrapped drugs, are also discussed. Notably, the potential adverse effects such as teratogenic effects, insulin resistance and eryptosis shall not be ignored. Taken together, these reports suggest that treatment of cancer cells with nigericin may offer a novel therapeutic strategy and future potential of translation to clinics

Deuterium Inverse Isotopic Effect in Ammonia Synthesis over Ru-Based and Fe-Based Catalysts

K2MoO4-NiO/SiO2 catalyst samples prepared by the co-impregnation method were calcined at different temperatures and atmosphere. X-ray diffraction, thermal gravimetric-differential scanning calorimetry, hydrogen temperature-programmed reduction, Raman spectroscopy, and electron spin resonance techniques were used to characterize the catalyst samples. The catalytic performance of the catalyst for one-step synthesis of methanethiol from high H2S-containing syngas was evaluated. The results showed that the catalyst calcined in air sintered seriously because of the heat release from citric acid oxidation. With the increase of calcination temperature, octahedral coordination Mo(O-h) gradually changed into tetrahedral coordination Mo(T-d), which made the reduction of Mo6+ more difficult, decreased the number of CO adsorption sites (coordinatively unsaturated sites) of Mo, and at last led to the decrease of CO conversion. Methanethiol synthesis was closely related to the Mo-S-K phase, and hydrocarbon synthesis was related to the MoS2 phase. Compared with the catalyst calcined in N-2, there were more MoS2 phase and less Mo-S-K phase on the surface of the catalyst calcined in air, resulting in the higher selectivity for hydrocarbon and lower selectivity for CH3SH

Autophagy is induced by starvation in SW620 cells.

<p>A: Western blot analysis of LC3 expression in SW620 cells. B: Quantitation of the optical density of LC3-II/LC3-I at various time points. C: Western blot of Ambra1, Beclin1 and cathepsin D expression after serum starvation at various time points. D: Quantitation of the optical density of Ambra1, cathepsin D and Beclin1. E: The relative Ambra1 expression after serum starvation for the indicated times as determined by real-time PCR. F: Immunofluorescent images of SW620 cells treated with or without 100 nM BafA1 for the indicated times. G: Quantification of autophagy in SW620 cells after serum starvation. The data are presented as the mean ± SD of three independent experiments (p<0.05).</p

The pro-survival function of Ambra1 in SW620 cells.

<p>A: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control or Ambra1-siRNA oligonucleotides. These cells were analyzed by MTT assay. B: SW620 cells cultured in serum-free medium for 12 or 24 h. Cell viability was determined by MTT assay. C: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control and Ambra1-siRNA oligonucleotides. These cells were analyzed by flow cytometry. D: SW620 cells serum starved for 24 h. Cell death was measured by flow cytometry. E: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control or Ambra1-siRNA oligonucleotides. These cells were assayed for staurosporine-induced cell death. F: SW620 cells treated with staurosporine (2 µM for 6 h). Cell death was measured by flow cytometry. G: Western blot analysis of Ambra1 expression in SW620 cells after transient transfection with negative control or Ambra1-siRNA oligonucleotides. These cells were assayed for etoposide-induced cell death. H: SW620 cells treated with etoposide (5 µg/ml for 24 h). Cell death was measured by flow cytometry. The data are presented as the mean ± SD of three independent experiments (p<0.05).</p

Ambra1 interacts with Beclin1 in SW620 cells.

<p>A and B: Co-immunoprecipitation assays with SW620 cell lysates. SW620 cells were serum-starved or not (control) for 12 h. Next, the cells were incubated with anti-Ambra1 antisera or control sera (A) or anti-Beclin1 antisera or control sera (B) covalently bound to protein A-sepharose beads. After extensive washes, the eluted proteins were separated and subjected to Beclin1 and Ambra1 immunoblotting. The procedure for (C) and (D) is similar to that for (A) and (B) with the exception that the cells were treated or not with rapamycin (100 nM for 12 h) instead of being serum-starved.</p

Ambra1 degradation during etoposide-induced apoptosis.

<p>A: SW620 cells treated with 1 µM staurosporine. Ambra1, Beclin1, Vps34 and ATG5 expression was measured by Western blot at various time points. B: Normalized quantitation of the Ambra1 optical density in SW620 cells treated with etoposide for the indicated times. C: SW620 cells treated with 1 µM staurosporine for the indicated times and analyzed by Western blot for pro-capsase-3 and cleaved caspase-3. D: Normalized quantitation of the cleaved caspase-3 optical density in SW620 cells treated with etoposide for the indicated times. E: SW620 cells treated with various doses of etoposide for 6 h. Ambra1 levels were measured by Western blot. F: Normalized quantitation of the Ambra1 optical density in SW620 cells treated with the indicated doses of staurosporine. The data are presented as the mean ± SD of three independent experiments (p<0.05).</p