Cadmium promotes the binding and centrosomal translocation of CCDC85C and PLK4 via ROS-GCLM pathway to trigger centrosome amplification in colon cancer cells.

Cadmium (Cd) is a prevalent heavy metal contaminant that can cause centrosome amplification (CA) and cancer. Since CA can initiate tumorigenesis, it is plausible that cadmium initiates tumorigenesis via CA. The present study investigated the signaling pathways underlying CA by Cd. Our findings confirmed that sub-toxic concentrations of Cd could induce CA in the HCT116 colon cancer cells, and revealed that reactive oxygen species (ROS), GCLM, CCDC85C and PLK4 were the signaling molecules that formed a pathway of ROS-GCLM-CCDC85C-PLK4. Cd not only increased the protein levels of CCDC85C and PLK4, but also promoted their distribution to the centrosomes. Molecular docking analysis revealed that CCDC85C and PLK4 had the binding potential. Indeed, antibodies against CCDC85C and PLK4 were able to pull down PLK4 and CCDC85C, respectively. Knockdown of CCDC85C decreased the Cd-promoted centrosomal distribution of PLK4. Similarly, knockdown of PLK4 reduced the centrosomal distribution of CCDC85C. Our results suggest that Cd activates ROS-GCLM pathway that triggers the expression of and binding between CCDC85C and PLK4, and promotes the translocation of CCDC85C-PLK4 complex to the centrosomes, which eventually leads to CA.

Synergistic effects and antityrosinase mechanism of four plant polyphenols from Morus and Hulless Barley.

Sanggenone C, oxyresveratrol, catechin and l-epicatechin exist in Morus and Hulless Barley as natural polyphenols with antityrosinase activity. Little research on their synergistic and structure-function relationships of them has been reported in recent years. In this paper, the inhibition mechanisms of these four plant polyphenols were investigated by enzyme kinetics, HPLC, fluorescence spectra, and molecular docking methods. The results showed that oxyresveratrol (IC50 = 1.096 ± 0.048 µg/mL), sanggenone C (IC50 = 13.360 ± 1.029 µg/mL), l-epicatechin (IC50 = 55.730 ± 1.762 µg/mL), and catechin (IC50 = 148.500 ± 3.355 µg/mL) exhibited tyrosinase inhibition activity. When sangenone C (14 µg/mL) was mixed with l-epicatechin (56 µg/mL) at 4:1 (40 µL + 10 µL), the highest tyrosinase inhibition was achieved. Molecular docking showed that the number and position of phenolic hydroxyls of polyphenols were the key for tyrosinase inhibition activity. This study provided new ideas for the application of these four plant polyphenols from Hulless Barley and Morus as tyrosinase inhibitors in food preservation.

Metagenomic Analysis Identifies Sex-Related Cecal Microbial Gene Functions and Bacterial Taxa in the Quail.

Background: Japanese quail (Coturnix japonica) are important and widely distributed poultry in China. Researchers continue to pursue genetic selection for heavier quail. The intestinal microbiota plays a substantial role in growth promotion; however, the mechanisms involved in growth promotion remain unclear. Results: We generated 107.3 Gb of cecal microbiome data from ten Japanese quail, providing a series of quail gut microbial gene catalogs (1.25 million genes). We identified a total of 606 main microbial species from 1,033,311 annotated genes distributed among the ten quail. Seventeen microbial species from the genera Anaerobiospirillum, Alistipes, Barnesiella, and Butyricimonas differed significantly in their abundances between the female and male gut microbiotas. Most of the functional gut microbial genes were involved in metabolism, primarily in carbohydrate transport and metabolism, as well as some active carbohydrate-degrading enzymes. We also identified 308 antibiotic-resistance genes (ARGs) from the phyla Bacteroidetes, Firmicutes and Euryarchaeota. Studies of the differential gene functions between sexes indicated that abundances of the gut microbes that produce carbohydrate-active enzymes varied between female and male quail. Bacteroidetes was the predominant ARG-containing phylum in female quail; Euryarchaeota was the predominant ARG-containing phylum in male quail. Conclusion: This article provides the first description of the gene catalog of the cecal bacteria in Japanese quail as well as insights into the bacterial taxa and predictive metagenomic functions between male and female quail to provide a better understanding of the microbial genes in the quail ceca.

Binding between ROCK1 and DCTN2 triggers diabetes­associated centrosome amplification in colon cancer cells.

Type 2 diabetes increases the risk various types of cancer and is associated with a poor prognosis therein. There is also evidence that the disease is associated with cancer metastasis. Centrosome amplification can initiate tumorigenesis with metastasis in vivo and increase the invasiveness of cancer cells in vitro. Our previous study reported that type 2 diabetes promotes centrosome amplification via the upregulation and centrosomal translocation of Rho­associated protein kinase 1 (ROCK1), which suggests that centrosome amplification is a candidate biological link between type 2 diabetes and cancer development. In the present study, functional proteomics analysis was used to further investigate the molecular pathways underlying centrosome amplification by targeting ROCK1 binding partners. High glucose, insulin and palmitic acid were used to induce centrosome amplification, and immunofluorescent staining was employed to visualize centrosomal alterations. Combined with immunoprecipitation, mass spectrometry­based proteomics analysis was used to identify ROCK1 binding proteins, and protein complex disruption was achieved by siRNA­knockdown. In total, 1,148 ROCK1 binding proteins were identified, among which 106 proteins were exclusively associated with the treated samples, 193 were only associated with the control samples, and 849 were found in both the control and treated samples. Of the proteins with evidence of centrosomal localization, Dynactin subunit 2 (DCTN2) was confirmed to be localized to the centrosomes. Treating the cells with high glucose, insulin and palmitic acid increased the protein levels of ROCK1 and DCTN2, promoted their binding with each other, and triggered centrosome amplification. Disruption of the protein complex by knocking down ROCK1 or DCTN2 expression partially attenuated centrosome amplification, while simultaneous knockdown of both proteins completely inhibited centrosome amplification. These results suggested ROCK1­DCTN2 binding as a signal for the regulation of centrosome homeostasis, which is key for diabetes­associated centrosome amplification, and enriches our knowledge of centrosome biology. Therefore, the ROCK1­DCTN2 complex may serve as a target for inhibiting centrosome amplification both in research or future therapeutic development.

Secreted Wnt6 mediates diabetes-associated centrosome amplification via its receptor FZD4.

We recently published that type 2 diabetes promotes cell centrosome amplification via upregulation of Rho-associated protein kinase 1 (ROCK1) and 14-3-3 protein-σ (14-3-3σ). This study further investigates the molecular mechanisms underlying diabetes-associated centrosome amplification. We found that treatment of cells with high glucose, insulin, and palmitic acid levels increased the intracellular and extracellular protein levels of Wingless-type MMTV integration site family member 6 (Wnt6) as well as the cellular level of ß-catenin. The treatment also activated ß-catenin and promoted its nuclear translocation. Treatment of cells with siRNA species for Wnt6, Frizzled-4 (FZD4), or ß-catenin as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture medium could all attenuate the treatment-triggered centrosome amplification. Moreover, we showed that secreted Wnt6-FZD4-ß-catenin was the signaling pathway that was upstream of ROCK1 and 14-3-3σ. We found that advanced glycation end products (AGEs) were also able to increase the cellular and extracellular levels of Wnt6, the cellular protein level of ß-catenin, and centrosome amplification. Treatment of the cells with siRNA species for Wnt6 or FZD4 as well as introduction of antibodies against Wnt6 or FZD4 to the cell culture could all inhibit the AGEs-elicited centrosome amplification. In colon tissues from a diabetic mouse model, the protein levels of Wnt6 and 14-3-3σ were increased. In conclusion, our results showed that the pathophysiological factors in type 2 diabetes, including AGEs, were able to induce centrosome amplification. It is suggested that secreted Wnt6 binds to FZD4 to activate the canonical Wnt6 signaling pathway, which is upstream of ROCK1 and 14-3-3σ, and that this is the cell signaling pathway underlying diabetes-associated centrosome amplification.

Hsp74/14-3-3σ Complex Mediates Centrosome Amplification by High Glucose, Insulin, and Palmitic Acid.

It has been reported recently that type 2 diabetes promotes centrosome amplification via 14-3-3σ/ROCK1 complex. In the present study, 14-3-3σ interacting proteins are characterized and their roles in the centrosome amplification by high glucose, insulin, and palmitic acid are investigated. Co-immunoprecipitation in combination with MS analysis identified 134 proteins that interact with 14-3-3σ, which include heat shock 70 kDa protein 4 (Hsp74). Gene ontology analyses reveal that many of them are enriched in binding activity. Kyoto Encyclopedia of Genes and Genomes analysis shows that the top three enriched pathways are ribosome, carbon metabolism, and biosynthesis of amino acids. Molecular and functional investigations show that the high glucose, insulin, and palmitic acid increase the expression and binding of 14-3-3σ and Hsp74 as well as centrosome amplification, all of which are inhibited by knockdown of 14-3-3σ or Hsp74. Moreover, molecular docking analysis shows that the interaction between the 14-3-3σ and the Hsp74 is mainly through hydrophobic contacts and a lesser degree ionic interactions and hydrogen bond by different amino acids residues. In conclusion, the results suggest that the experimental treatment triggers centrosome amplification via upregulations of expression and binding of 14-3-3σ and Hsp74.

Advanced Glycation End Products Increase MDM2 Expression via Transcription Factor KLF5.

Type 2 diabetes increases the risk for all-site cancers including colon cancer. Diabetic patients present typical pathophysiological features including an increased level of advanced glycation end products (AGEs), which comes from a series of nonenzymatic reactions between sugars and biological macromolecules, positively associated with the occurrence of diabetic complications. MDM2 is an oncogene implicated in cancer development. The present study investigated whether diabetes promoted MDM2 expression in colon cells and the underlying mechanisms. Our results showed that AGE increased the protein level of MDM2 in a cell model and promoted binding between MDM2 and Rb as well as p53, which led to degradation of Rb and p53. KLF5 was able to bind to the regulatory sequence of the MDM2 gene, and knockdown of the KLF5 protein level inhibited the AGE-triggered MDM2 overexpression, which indicated that KLF5 was the transcription factor for MDM2. In a mouse model of diabetes, we found that AGE level was increased in serum. The protein levels of both KLF5 and MDM2 were increased. KLF5 was able to bind to the regulatory sequence of the MDM2 gene. In conclusion, our results suggest that diabetes increases the level of AGE which enhances the expression of MDM2 via transcription factor KLF5 in colon cells. MDM2 overexpression is a candidate biological link between type 2 diabetes and colon cancer development.

Type 2 Diabetes Promotes Cell Centrosome Amplification via AKT-ROS-Dependent Signalling of ROCK1 and 14-3-3σ.

BACKGROUND/AIMS: Type 2 diabetes is associated with oxidative stress and DNA damage which can cause centrosome amplification. Thus, the study investigated centrosome amplification in type 2 diabetes and the underlying mechanisms. METHODS: Centrosome numbers in human peripheral blood mononuclear blood cells (PBMC) from healthy subjects and patients with type 2 diabetes were compared to access the association between type 2 diabetes and centrosome amplification. Colon cancer cells were used to investigate the molecular mechanisms underlying the centrosome amplification triggered by high glucose, insulin and palmitic acid. Western blot analysis was used to quantify the level of protein and protein phosphorylation. Immunofluorescent staining was performed to detect centrosomes. ROS was quantified using flow cytometry technique. Transcriptpmic profiling was performed using Illumina HiSeqTM500 platform. RESULTS: We found that centrosome amplification was increased PBMC from the type 2 diabetic patients, which correlated with the levels of fasting blood glucose and HbA1c. High glucose, insulin and palmitic acid, alone or in combinations, induced ROS production and centrosome amplification. Together, they increased AKT activation as well as the expression, binding and centrosome translation of ROCK1 and 14-3-3σ. Results from further analyses showed that AKT-ROS-dependent upregulations of expression, binding and centrosome translocation of ROCK1 and 14-3-3σ was the molecular pathway underlying the centrosome amplification in vitro triggered by high glucose, insulin and palmitic acid. Moreover, the key in vitro molecular signalling events activated by high glucose, insulin and palmitic acid were verified in PBMC from the patients with type 2 diabetes. CONCLUSION: Our results show that type 2 diabetes promotes cell centrosome amplification, and suggest that the diabetic pathophysiological factors-activated AKT-ROS-dependent signalling of ROCK1 and 14-3-3σ is the underlying molecular mechanism.

CacyBP/SIP nuclear translocation induced by gastrin promotes gastric cancer cell proliferation.

AIM: To investigate the role of nuclear translocation of calcyclin binding protein, also called Siah-1 interacting protein (CacyBP/SIP), in gastric carcinogenesis. METHODS: The expression of CacyBP/SIP protein in gastric cancer cell lines was detected by Western blot. Immunofluorescence experiments were performed on gastric cancer cell lines that had been either unstimulated or stimulated with gastrin. To confirm the immunofluorescence findings, the relative abundance of CacyBP/SIP in nuclear and cytoplasmic compartments was assessed by Western blot. The effect of nuclear translocation of CacyBP/SIP on cell proliferation was examined using MTT assay. The colony formation assay was used to measure clonogenic cell survival. The effect of CacyBP/SIP nuclear translocation on cell cycle progression was investigated. Two CacyBP/SIP-specific siRNA vectors were designed and constructed to inhibit CacyBP/SIP expression in order to reduce the nuclear translocation of CacyBP/SIP, and the expression of CacyBP/SIP in stably transfected cells was determined by Western blot. The effect of inhibiting CacyBP/SIP nuclear translocation on cell proliferation was then assessed. RESULTS: CacyBP/SIP protein was present in most of gastric cancer cell lines. In unstimulated cells, CacyBP/SIP was distributed throughout the cytoplasm; while in stimulated cells, CacyBP/SIP was found mainly in the perinuclear region. CacyBP/SIP nuclear translocation generated a growth-stimulatory effect on cells. The number of colonies in the CacyBP/SIP nuclear translocation group was significantly higher than that in the control group. The percentage of stimulated cells in G1 phase was significantly lower than that of control cells (69.70% ± 0.46% and 65.80% ± 0.60%, control cells and gastrin-treated SGC7901 cells, P = 0.008; 72.99% ± 0.46% and 69.36% ± 0.51%, control cells and gastrin-treated MKN45 cells, P = 0.022). CacyBP/SIPsi1 effectively down-regulated the expression of CacyBP/SIP, and cells stably transfected by CacyBP/SIPsi1 were then chosen for further cellular assays. In CacyBP/SIPsi1 stably transfected cells, CacyBP/SIP was shown to be distributed throughout the cytoplasm, irregardless of whether they were stimulated or not. After CacyBP/SIP nuclear translocation was reduced, there had no major effect on cell proliferation, as shown by MTT assay. There had no enhanced anchorage-dependent growth upon stimulation, as indicated by colony formation in flat plates. No changes appeared in the percentage of cells in G0-G1 phase in either cell line (71.09% ± 0.16% and 70.86% ± 0.25%, control cells and gastrin-treated SGC7901-CacyBP/SIPsi1 cells, P = 0.101; 74.17% ± 1.04% and 73.07% ± 1.00%, control cells and gastrin-treated MKN45-CacyBP/SIPsi1 cells, P = 0.225). CONCLUSION: CacyBP/SIP nuclear translocation promotes the proliferation and cell cycle progression of gastric cancer cells.

An association between parvovirus B19 and Kikuchi-Fujimoto disease.

Although Kikuchi-Fujimoto disease (KFD) has a higher prevalence among Asian countries, it is a well-defined entity throughout the world. However, its etiology and pathogenesis remain undetermined. To study whether B19 infection is associated with idiopathic KFD (iKFD), we examined the presence of the viral genome and proteins in paraffin-embedded tissues of lymph nodes retrospectively from 33 iKFD patients and 16 age- and sex-matched control subjects by nested PCR (nPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). B19 was detected in 87.1, 69.7, and 57.6% of iKFD specimens by nPCR, ISH, and IHC, respectively, whereas the virus was positive in only 56.3, 31.3, and 25.0% of control tissues by the respective methods (nPCR: p = 0.029; ISH: p = 0.011; IHC: p = 0.032). The IHC-ISH double-staining assay demonstrated that B19-infected cells were mainly lymphocytes and a small number of histiocytes. These results showed for the first time a high frequency of localized persistence of B19 in lymph nodes from iKFD patients, suggesting that B19 might play an important role in the pathogenesis of iKFD.

Gossypol-Induced Differentiation in Human Leukemia HL-60 Cells.

The main treatment of leukemia is traditional radiochemotherapy, which is associated with serious side effects. In the past twenty years, differentiation was found as an important effective measure to treat leukemia with fewer side effects. Gossypol, a natural compound which has been used as an effective contraceptive drug, has been proposed to be a potent drug to treat leukemia, but the differentiation effect has not been studied. In the present study, we investigated the pro-differentiated effects, in vitro, of gossypol on the classic human myeloid leukemia HL-60 cell line. The effects of gossypol were investigated by using morphological changes, nitroblue tetrazolium (NBT) reduction, surface markers, cell-cycle analysis and Western blot analysis, etc. When HL-60 cells were incubated with low concentrations of gossypol (2-5µM) for 48hr, a prominent G0/G1 arrest was observed. At 96 hr of treatment, 90% of HL-60 cells differentiated, as evidenced by morphological changes, NBT reduction, and increase in cell surface expression of some molecules were detected. This study is the first to identify gossypol's pro-differentiated effects on the leukemia cell line, and it induced differentiation through the PBK (PDZ-binding kinase)/TOPK (T-LAKcell-originated protein kinase) (PBK/TOPK) pathway. It is concluded that gossypol could induce differentiation in the leukemia HL-60 cells, and it may be a potential therapeutic agent, chemoprevention or chemotherapeutic adjuvant especially in combination drug therapy for leukemia.

[Experimental study on dog's bone marrow stem cells transfected by pIRES2-EGFP-IGF-1 gene].

OBJECTIVE: To establish the bone marrow stem cells (MSC) model which could highly express the insulin-like growth factor 1 (IGF-1) transfected by dog's IGF-1 gene. METHODS: pIRES2-EGFP-IGF-1 was transfected into MSC by lipofectamine. Positive clones were selected with G418. The expression of IGF-1 protein in the MSC was determined by immunohistochemistry and Western blot analysis. The IGF-1 in the supernatant of the transfected MSC was detected by sandwich-in ELISA. The periodontal ligament cells (PDLC) were cultured in the supernatant of the transfected MSC. The changes of PDLC' proliferation were observed by MTT. RESULTS: IGF-1-transfected MSC could apparently express IGF-1. The IGF-1 protein in the supernatant of the transfected MSC was confirmed by sandwich-in ELISA. IGF-1 could promote the PDLC' proliferation. CONCLUSIONS: The MSC transfected by dog's IGF-1 gene can highly express IGF-1, which may lay the foundation for further study on periodontal regeneration.