Alpha-2-Glycoprotein 1(AZGP1) Regulates Biological Behaviors of LoVo Cells by Down-Regulating mTOR Signaling Pathway and Endogenous Fatty Acid Synthesis

<div><p>AZGP1 is a multifaceted protein associated with lipid mobilization, a process that is regulated by FASN and other metabolic pathways such as mTOR signaling. The active mTOR signaling pathway has been found to be involved in a variety of tumors. However, it remains unclear whether it is involved in the regulation of AZGP1 and FASN. An AZGP1-expressing plasmid was transfected into a human colorectal cancer cell line (LoVo) with a low expression of AZGP1. The expression of AZGP1, FASN, eIF4E, p-mTOR, p-S6,and S6K1 were measured by Western blot analysis, and target genes were detected by RT-PCR. Cell proliferation was studied using the MTT and colony formation assays. The analysis of apoptosis and the cell cycle phase were assessed by flow cytometry. The capacity of cell migration was investigated using the transwell migration assay. We found that the expression of AZGP1 was up-regulated while the expression of FASN, eIF4E, p-mTOR, p-S6, and S6K1 were down-regulated in LoVo cells after AZGP1 was expressed. The proliferation of malignant cells was reduced in AZGP1-overexpression cells, which is consistent with an increased in the G₂-arrest and apoptosis rate. Furthermore, the migration of AZGP1-overexpression cells was decreased. The overexpression of AZGP1 suppressed the activation of the mTOR pathway and endogenous FASN-regulated fatty acid synthesis, mitigating the malignant phenotype of LoVo cells. Herein, we provide evidence that AZGP1 may constitute a novel tumor suppressor for LoVo colorectal cancer cells.</p></div

Primary antibodies used for the detection in the Western blot analysis.

<p>Primary antibodies used for the detection in the Western blot analysis.</p

Target cell selection.

<p>(a) AZGP1 and GAPDH protein expression were detected by Western bolt. (b) The relative expression of AZPG1 (compared with the GAPDH protein) *compared to other cell lines, LoVo cells exhibited lower AZGP1 protein expression <i>P</i><0.05. (c) AZGP1 gene expression was detected with agarose gel electrophoresis and measured using gel imaging and an analysis system. (d) The relative expression of AZPG1 (compared with the GAPDH gene) *compared to the other cell lines, LoVo cells exhibited lower AZGP1 expression <i>P</i><0.05. (e) The expression profile of the AZPG1 gene in three kinds of LoVo cells. (f) The relative expression of the AZPG1 gene in three groups. *compared to blank group and control group, the experimental group showed higher expression of the AZGP1 gene. <i>P</i><0.05, no significant difference was detected between blank group and control group.</p

AZGP1 blocks the proliferation and migration of LoVo cells.

<p>(a) Up-regulation of AZGP1 expression inhibits the proliferation of LoVo cells. *compared with the two control groups, <i>P</i><0.05. (b), (c) The colony formation of LoVo cells after transfection. *compared with the two control groups, <i>P</i><0.05. (d), (e) Migration of LoVo cells after transfection. **compared with blank group and control groups, the number of LoVo cells which pass through membrane of experimental group was less (<i>P</i><0.01).</p

AZGP1 overexpression induces apoptosis and cell cycle arrest.

<p>(a), (b) Cell cycle analysis for the three groups. *compared with blank group and control group, the rate of G₂ phase in LoVo cells which were transfected with the pGCMV/EGFP/AZGP1 plasmid was higher (<i>P</i><0.05). **compared to the blank group and control group, the experimental group had a higher rate of apoptosis (<i>P</i><0.01). (c), (d) Apoptosis rate. *compared with the other groups, the experimental group exhibited higher apoptosis rates (<i>P</i><0.05).</p

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Additional file 4. Effects of chronic treatment of Scu or Scu-loaded nanoparticles on body weight and blood-glucose in STZ-induced diabetic rats. (A) The changes of body weight of rats. (B) The changes of blood-glucose in rats. STZ was administrated to the DM group, DM +Chit-DC group, DM + Scu group, DM + Chit-DC-Scu group, and DM + Chit-DC-VB12-Scu group at week 8(n = 8)

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Additional file 3. FTIR spectra and photo of Chit-DC-FITC. (A) FTIR spectra of Chit-DC and Chit-DC-FITC. (B) Fluorescence spectra and a photo of FITC-labeled amphiphilic chitosan derivatives

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Additional file 2. Synthesis of FITC-labelled amphiphilic chitosan derivatives: (A) the Chit-DC-FITC derivative and (B) the Chit-DC-B12-FITC derivative

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Additional file 1. 1H NMR spectra of (a) deoxycholic acid (DMSO-d6), (b) Chit-DC (D2O), (c) Chit-DC-VB12 (D2O) and (d) vitamin B12 (D2O)