The iron-chelating peptides from soybean protein hydrolysates (SPH) were investigated using immobilized metal affinity chromatography (IMAC). The results demonstrated that SPH could absorb on the IMAC-Fe(3+) column, while the capability of the binding iron was different in SPH (10-30 kDa), SPH (3-10 kDa), and SPH (1-3 kDa). The highest binding amount on the column occurred with SPH (10-30 kDa). With the IMAC method, the iron-chelating peptides were shown to be formed at pH lower than 5.5, and they were not affected by NaCL with the concentration between 0.1 mol/L and 1 mol/L, while the iron-chelating peptides could be partially disrupted by 0.02 mol/L Na(2)HPO(4) at pH 8.0. Furthermore, the iron-chelating peptides were identified with reversed phase (RP)-HPLC, SDS-PAGE, and MALDI-TOF MS/MS. The binding characteristics of the SPH on IMAC-Fe(3+) and the sequences of the iron-chelating peptides revealed that binding sites between SPH and iron might be the carboxyl groups of Glu and Asp residues.
Toll‐like receptors (TLRs) play critical roles in host defense after recognition of conserved microbial‐ and host‐derived components, and their dysregulation is a common feature of various inflammation‐associated cancers, including gastric cancer (GC). Despite the recent recognition that metabolic reprogramming is a hallmark of cancer, the molecular effectors of altered metabolism during tumorigenesis remain unclear. Here, using bioenergetics function assays on human GC cells, we reveal that ligand‐induced activation of TLR2, predominantly through TLR1/2 heterodimer, augments both oxidative phosphorylation (OXPHOS) and glycolysis, with a bias toward glycolytic activity. Notably, DNA microarray‐based expression profiling of human cancer cells stimulated with TLR2 ligands demonstrated significant enrichment of gene‐sets for oncogenic pathways previously implicated in metabolic regulation, including reactive oxygen species (ROS), p53 and Myc. Moreover, the redox gene encoding the manganese‐dependent mitochondrial enzyme, superoxide dismutase (SOD)2, was strongly induced at the mRNA and protein levels by multiple signaling pathways downstream of TLR2, namely JAK‐STAT3, JNK MAPK and NF‐κB. Furthermore, siRNA‐mediated suppression of SOD2 ameliorated the TLR2‐induced metabolic shift in human GC cancer cells. Importantly, patient‐derived tissue microarrays and bioinformatics interrogation of clinical datasets indicated that upregulated expression of TLR2 and SOD2 were significantly correlated in human GC, and the TLR2‐SOD2 axis was associated with multiple clinical parameters of advanced stage disease, including distant metastasis, microvascular invasion and stage, as well as poor survival. Collectively, our findings reveal a novel TLR2‐SOD2 axis as a potential biomarker for therapy and prognosis in cancer.
Soybean protein hydrolysates (SPHs) bind with calcium, forming soluble SPH-calcium complexes via the carboxyl groups of glutamic and aspartic acid residues. However, their effect on calcium uptake is still unclear. In this study, Caco-2 cells were used to estimate the effect of SPH-calcium complexes with different molecular weights on calcium uptake in vitro. The changes in intracellular calcium ion concentration were measured by Fura-2 loading and expressed in fluorescence intensity. SPH-calcium complexes could promote calcium uptake. Improved fluorescence intensity was significantly different in SPH-calcium complexes (10 to 30 kDa), SPH-calcium complexes (3 to 10 kDa), and SPH-calcium complexes (1 to 3 kDa). The maximum levels of relative fluorescence intensity (18.3) occurred with SPH-calcium complexes (10 to 30 kDa). The effect of SPH-calcium complexes (10 to 30 kDa) on Ca(2+) increase was determined to be concentration dependent in the range of 0.5 to 4 mg/mL. Our results indicate that soybean protein itself might be responsible for promoting calcium absorption.
Several studies in the past decades have reported anti-tumor activity of the bioactive compounds extracted from tea leaves, with a focus on the compound epigallocatechin-3-gallate (EGCG). However, further investigations are required to unravel the underlying mechanisms behind the anti-tumor activity of EGCG. In this study, we demonstrate that EGCG significantly inhibits the growth of 4T1 breast cancer cells in vitro and in vivo. EGCG ameliorated immunosuppression by significantly decreasing the accumulation of myeloid-derived suppressor cells (MDSCs) and increasing the proportions of CD4+ and CD8+ T cells in spleen and tumor sites in 4T1 breast tumor-bearing mice. Surprisingly, a low dose of EGCG (0.5–5 μg/mL) effectively reduced the cell viability and increased the apoptosis rate of MDSCs in vitro. EGCG down-regulated the canonical pathways in MDSCs, mainly through the Arg-1/iNOS/Nox2/NF-κB/STAT3 signaling pathway. Moreover, transcriptomic analysis suggested that EGCG also affected the non-canonical pathways in MDSCs, such as ECM–receptor interaction and focal adhesion. qRT-PCR further validated that EGCG restored nine key genes in MDSCs, including Cxcl3, Vcan, Col4a1, Col8a1, Oasl2, Mmp12, Met, Itsnl and Acot1. Our results provide new insight into the mechanism of EGCG-associated key pathways/genes in MDSCs in the murine breast tumor model.
Current studies aiming at identifying single immune markers with prognostic value have limitations in the context of complex antitumor immunity and cancer immune evasion. Here, we show how the integration of several immune markers influences the predictions of prognosis of gastric cancer (GC) patients. We analyzed Tissue Microarray (TMA) by multiplex immunohistochemistry and measured the expression of immune checkpoint molecule PD-L1 together with antitumor CD8 T cells and immune suppressive FOXP3 Treg cells in a cohort of GC patients. Unsupervised hierarchical clustering analysis of these markers was used to define correlations between CD8 T, FOXP3 Treg and PD-L1 cell densities. We found that FOXP3 and PD-L1 densities were elevated while CD8 T cells were decreased in tumor tissues compared to their adjacent normal tissues. However, patient stratification based on each one of these markers individually did not show significant prognostic value on patient survival. Conversely, combination of the ratios of CD8/FOXP3 and CD8/PD-L1 enabled the identification of patient subgroups with different survival outcomes. As such, high densities of PD-L1 in patients with high CD8/FOXP3 and low CD8/PD-L1 ratios correlated with increased survival. Collectively, this work demonstrates the need for the integration of several immune markers to obtain more meaningful survival prognosis and patient stratification. In addition, our work provides insights into the complex tumor immune evasion and immune regulation by the tumor-infiltrating effector and suppressor cells, informing on the best use of immunotherapy options for treating patients.
Cervical cancer is the leading cause of death with gynecological malignancies. We aimed to explore the molecular mechanism of carcinogenesis and biomarkers for cervical cancer by integrated bioinformatic analysis. We employed RNA-sequencing details of 254 cervical squamous cell carcinomas and 3 normal samples from The Cancer Genome Atlas. To explore the distinct pathways, messenger RNA expression was submitted to a Gene Set Enrichment Analysis. Kyoto Encyclopedia of Genes and Genomes and protein–protein interaction network analysis of differentially expressed genes were performed. Then, we conducted pathway enrichment analysis for modules acquired in protein–protein interaction analysis and obtained a list of pathways in every module. After intersecting the results from the 3 approaches, we evaluated the survival rates of both mutual pathways and genes in the pathway, and 5 survival-related genes were obtained. Finally, Cox hazards ratio analysis of these 5 genes was performed. DNA replication pathway (P < .001; 12 genes included) was suggested to have the strongest association with the prognosis of cervical squamous cancer. In total, 5 of the 12 genes, namely, minichromosome maintenance 2, minichromosome maintenance 4, minichromosome maintenance 5, proliferating cell nuclear antigen, and ribonuclease H2 subunit A were significantly correlated with survival. Minichromosome maintenance 5 was shown as an independent prognostic biomarker for patients with cervical cancer. This study identified a distinct pathway (DNA replication). Five genes which may be prognostic biomarkers and minichromosome maintenance 5 were identified as independent prognostic biomarkers for patients with cervical cancer.
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