MMP12 is a Potential Predictive and Prognostic Biomarker of Various Cancers Including Lung Adenocarcinoma.

OBJECTIVE: This study sought to explore the clinical value of matrix metalloproteinases 12 (MMP12) in multiple cancers, including lung adenocarcinoma (LUAD). METHODS: Using >10,000 samples, this retrospective study demonstrated the first pan-cancer analysis of MMP12. The expression of MMP12 between cancer groups and their control groups was analyzed using Wilcoxon rank-sum tests. The clinical significance of MMP12 expression in multiple cancers was assessed using receiver operating characteristic curves, Kaplan-Meier curves, and univariate Cox analysis. A further LUAD-related analysis based on 4565 multi-center and in-house samples was performed to verify the findings regarding MMP12 in pan-cancer analysis partly. RESULTS: MMP12 mRNA is highly expressed in 13 cancers compared to their controls, and the MMP12 protein level is elevated in some of these cancers (e.g., colon adenocarcinoma) (P < .05). MMP12 expression makes it feasible to distinguish 21 cancer tissues from normal tissues (AUC = 0.86). A high MMP12 expression is a prognosis risk factor in eight cancers, such as adrenocortical carcinoma (hazard ratio >1, P < .05). The elevated MMP12 expression is also a prognosis protective factor in breast-invasive carcinoma and colon adenocarcinoma (hazard ratio <1, P < .05). Some pan-cancer findings regarding MMP12 are verified in LUAD-MMP12 expression is upregulated in LUAD at both the mRNA and protein levels (P < .05), has the potential to distinguish LUAD with considerable accuracy (AUC = .91), and plays a risk prognosis factor for patients with the disease (P < .05). CONCLUSIONS: MMP12 is highly expressed in most cancers and may serve as a novel biomarker for the prediction and prognosis of numerous cancers.

LPCAT1 enhances the invasion and migration in gastric cancer: Based on computational biology methods and in vitro experiments.

BACKGROUND AND AIM: The biological functions and clinical implications of lysophosphatidylcholine acyltransferase 1 (LPCAT1) remain unclarified in gastric cancer (GC). The aim of the current study was to explore the possible clinicopathological significance of LPCAT1 and its perspective mechanism in GC tissues. MATERIALS AND METHODS: The protein expression and mRNA levels of LPCAT1 were detected from in-house immunohistochemistry and public high-throughput RNA arrays and RNA sequencing. To have a comprehensive understanding of the clinical value of LPCAT1 in GC, all enrolled data were integrated to calculate the expression difference and standard mean difference (SMD). The biological mechanism of LPCAT1 in GC was confirmed by computational biology and in vitro experiments. Migration and invasion assays were also conducted to confirm the effect of LPCAT1 in GC. RESULTS: Both protein and mRNA expression levels of LPCAT1 in GC were remarkably higher than those in noncancerous controls. Comprehensively, the SMD of LPCAT1 mRNA was 1.11 (95% CI = 0.86-1.36) in GC, and the summarized AUC was 0.85 based on 15 datasets containing 1727 cases of GC and 940 cases of non-GC controls. Moreover, LPCAT1 could accelerate the invasion and migration of GC by boosting the neutrophil degranulation pathway and disturbing the immune microenvironment. CONCLUSION: An increased level of LPCAT1 may promote the progression of GC.

AZGP1 Up-Regulation is a Potential Target for Andrographolide Reversing Radioresistance of Colorectal Cancer.

Background: Radiation resistance is a challenge that limits the therapeutic benefit of colorectal cancer (CRC) treatment, but the mechanism underlying CRC radiation resistance remains unclear. Andrographolide shows a broad-spectrum anti-tumor effect in various malignancies, including CRC, its effect and how it functions in CRC initiation, and radiation have not been established. This study aimed to explore the mechanism of CRC radiation resistance and the potential mechanisms of andrographolide on CRC radiation. Methods: Two acquired radioresistant cell lines were established and high throughput sequencing was employed to screen out the differentially expressed genes. The expression of AZGP1, which was upregulated in the acquired radioresistant tissues, was verified by microarray data recomputing. The common targets of andrographolide, CRC initiation, and radiation resistance were obtained, and the corresponding functional enrichment and pathway analysis were performed. The interaction between AZGP1 and andrographolide was investigated using molecular docking. Results: AZGP1 was upregulated in both the radioresistant cell model and microarray data. Moreover, AZGP1 was upregulated in cancerous colorectal tissue and displayed a tendency toward elevated expression in patients with an unfavorable prognosis. AZGP1 was identified as the common target of andrographolide, colorectal cancer initiation, and radiotherapy resistance. Ultimately, the protein structure of AZGP1 proved to be closely intertwined with the crystal texture of andrographolide. Conclusion: AZGP1 is recognized as a crucial factor for both CRC initiation and radioresistance. Andrographolide may affect the radioresistance of CRC via the targeting of AZGP1. Thus, the combination of andrographolide and AZGP1 intervention might be a promising strategy for improving the treatment benefit of CRC radiotherapy.

Upregulation of the transmembrane protease serine 3 mRNA level in radioresistant colorectal cancer tissues.

Aim: To investigate the clinical role of transmembrane protease serine 3 (TMPRSS3) in radioresistance and prognosis of colorectal cancer (CRC). Methods: Standardized mean difference (SMD) and summary area under the curve (AUC) of TMPRSS3 were calculated by combining all available high-throughput data globally. The prognostic significance of TMPRSS3 was determined by Kaplan-Meier and Cox regression analyses. Results:TMPRSS3 was remarkably upregulated in 198 CRC radioresistant cases compared with nonradioresistance (SMD = 0.38, AUC = 0.71). Overexpression of TMPRSS3 was observed in 1601 CRC patients compared with control subjects without CRC. TMPRSS3 was a risk factor for disease-free survival of CRC with the summarized hazard ratio 1.28. Conclusion: TMPRSS3 contributes to the radioresistance and unfavorable prognosis of CRC.

Downregulation of MicroRNA-1 and Its Potential Molecular Mechanism in Nasopharyngeal Cancer: An Investigation Combined with In Silico and In-House Immunohistochemistry Validation.

BACKGROUND: This study was aimed at elucidating the molecular biological mechanisms of microRNA-1 (miR-1) in nasopharyngeal carcinoma (NPC). METHOD: In this study, we performed a pooled analysis of miR-1 expression data derived from public databases, such as GEO, ArrayExpress, TCGA, and GTEx. The miRWalk 2.0 database, combined with the mRNA microarray datasets, was used to screen the target genes, and the genes were then subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis using the DAVID 6.8 database. We then used the STRING 11.0 database and Cytoscape 3.80 software to construct a protein-protein interaction (PPI) network for screening hub genes. Immunohistochemistry (IHC) was further used to validate the expression of hub genes. Finally, potential therapeutic agents for NPC were screened by the Connectivity Map (cMap) database. RESULTS: Pooled analysis showed that miR-1 expression was significantly decreased in NPC (SMD = -0.57; P < 0.05). The summary receiver operating characteristic curve suggested that miR-1 had a good ability to distinguish cancerous tissues from noncancerous tissues (AUC = 0.78). The results of GO analysis focused on mitotic nuclear division, DNA replication, cell division, cell adhesion, extracellular space, kinesin complex, and extracellular matrix (ECM) structural constituent. The KEGG analysis suggested that the target genes played a role in key signaling pathways, such as cell cycle, focal adhesion, cytokine-cytokine receptor interaction, ECM-receptor interaction, and PI3K/Akt signaling pathway. The PPI network suggested that cyclin-dependent kinase 1 (CDK1) was the hub gene, and the CDK1 protein was subsequently confirmed to be significantly upregulated in NPC tissues by IHC. Finally, potential therapeutic drugs, such as masitinib, were obtained by the cMap database. CONCLUSION: miR-1 may play a vital part in NPC tumorigenesis and progression by regulating focal adhesion kinase to participate in cell mitosis, regulating ECM degradation, and affecting the PI3K/Akt signaling pathway. miR-1 has the potential to be a therapeutic target for NPC.

BCAT1: A risk factor in multiple cancers based on a pan-cancer analysis.

BACKGROUND: Although branched chain amino acid transaminase 1 (BCAT1) has been identified to play an essential role in multiple tumors, no studies on its role in pan-cancer have been consulted before. METHODS: The study comprehensively analyzes the expression, potential mechanisms, and clinical significance of BCAT1 in pan-cancer through utilizing 16,847 samples, providing novel clues for the treatment of cancers. A Kruskal-Wallis test and the Wilcoxon rank-sum and signed-rank tests were applied to investigate diverse BCAT1 expression between various groups (e.g., cancer tissues versus normal tissues). Spearman's rank correlation coefficient was used in all correlation analyses in the study. Cox analyses and Kaplan-Meier curves were utilized to identify the prognosis significance of BCAT1 expression in cancers. The significance of BCAT1 expression in differentiating cancer and non-cancer tissues was explored via the area under the receiver operating characteristic curves (AUC). RESULTS: The differential expression of BCAT1 was detected in various cancers (p < 0.05), which is relevant to some DNA methyltransferases expression. BCAT1 expression was associated with mismatch repair gene expression, immune checkpoint inhibitors expression, microsatellite instability, and tumor mutational burden in some cancers, indicating its potential in immunotherapy. BCAT1 expression showed prognosis significance and played a risk role in multiple cancers (hazard ratio > 0, p < 0.05). BCAT1 expression also demonstrated conspicuous ability to distinguish some cancers tissues from their normal tissues (AUC > 0.7), indicating its potential to detect cancers. Further analyses on head and neck squamous cell carcinoma certified upregulated BCAT1 expression at both mRNA and protein levels in this disease based on in-house tissue microarrays and multicenter datasets. CONCLUSIONS: For the first time, the research comprehensively demonstrates the overexpression of BCAT1 in pan-cancer, which improves the understanding of the pathogenesis of BCAT1 in pan-cancer. Upregulated BCAT1 expression represented a poor prognosis for cancers patients, and it serves as a potential marker for cancer immunotherapy.

Down-regulation of microRNA-125b-2-3p is a risk factor for a poor prognosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of mortality in cancer patients, but the association between miR-125b-2-3p and the onset and prognosis of HCC has not been reported in previous studies; thus, the clinicopathological implications of miR-125b-2-3p in HCC require elaboration. To examine the expression of miR-125b-2-3p in HCC, both in-house RT-qPCR and public datasets were used to calculate the standard mean difference (SMD) and the summary receiver operating characteristic (sROC). MiR-125b-2-3p was markedly lower in HCC than in non-tumor tissue as assessed by the in-house RT-qPCR which was confirmed by the integrative analysis showing the SMD being -0.69 and the area under the curve (AUC) being 0.84 based on 1,233 cases of HCC and 630 cases of non-HCC controls. To gain a overview of the clinical value of miR-125b-2-3p in HCC, all possible datasets were integrated, and lower miR-125b-2-3p levels could lead to poorer differentiation and a more advanced clinical stage of HCC. The hazard ratio (HR) of miR-125b-2-3p was also calculated using a Cox proportional hazards model, and the miR-125b-2-3p level could act as an protective indication for the survival with the HR being 0.74 based on 586 cases of HCC. Furthermore, the effect of nitidine chloride (NC), a natural bioactive phytochemical alkaloid, on the regulation of miR-125b-2-3p and its potential targets was also investigated. The miR-125b-2-3p level was increased after NC treatment, while the expression of its potential target PRKCA was reduced. Above all, a low-expressed level of miR-125b-2-3p plays a tumor suppressive role in HCC.

Active Enhancer Assessment by H3K27ac ChIP-seq Reveals Claudin-1 as a Biomarker for Radiation Resistance in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) represents the third most common malignant tumor in the worldwide. Radiotherapy is the common therapeutic treatment for CRC, but radiation resistance is often encountered. ChIP-seq of Histone H3K27 acetylation (H3K27ac) has revealed enhancers that play an important role in CRC. This study examined the relationship between an active CRC enhancer and claudin-1 (CLDN1), and its effect on CRC radiation resistance. METHODS: The target CRC genes of active enhancers were obtained from public H3K27ac ChIP-seq, and the genes highly expressed in radio-resistant CRC were screened and intersected with enhancer-driven genes. The clinical roles of CLDN1 in radiation resistance were examined using the t-test, standard mean deviation (SMD), summary receiver operating characteristic curve and Kaplan-Meier curves. The co-expressed genes of CLDN1 were calculated using Pearson Correlation analysis, and Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes and Gene Set Variation Analysis (GSVA) analyses were used to examine the molecular mechanisms of CLDN1. RESULTS: Total 13 703 CRC genes were regulated by enhancers using 58 H3K27ac ChIP-seq. Claudin-1 (CLDN1) was enhancer-driven and notably up-regulated in CRC tissues compared to non-CRC controls, with a SMD of 3.45 (95 CI % = .56-4.35). CLDN1 expression was increased in radiation-resistant CRC with a SMD of .42 (95% CI = .16-.68) and an area under the curve of .74 (95% CI = .70-.77). The cell cycle and immune macrophage levels were the most significant pathways associated with CLDN1. CONCLUSION: CLDN1 as an enhancer-regulated gene that can boost radiation resistance in patients with CRC.

Ulinastatin attenuates LPS-induced inflammation in mouse macrophage RAW264.7 cells by inhibiting the JNK/NF-κB signaling pathway and activating the PI3K/Akt/Nrf2 pathway.

Ulinastatin (UTI) is a broad-spectrum serine protease inhibitor isolated and purified from human urine with strong anti-inflammatory and cytoprotective actions, which is widely used for the treatment of various diseases, such as pancreatitis and sepsis. Although the therapeutic effects of UTI are reported to be associated with a variety of mechanisms, the signaling pathways mediating the anti-inflammatory action of UTI remain to be elucidated. In the present study we carried out a systematic study on the anti-inflammatory and anti-oxidative mechanisms of UTI and their relationships in LPS-treated RAW264.7 cells. Pretreatment with UTI (1000 and 5000 U/mL) dose-dependently decreased the mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, iNOS) and upregulated anti-inflammatory cytokines (IL-10 and TGF-ß1) in LPS-treated RAW264.7 cells. UTI pretreatment significantly inhibited the nuclear translocation of NF-κB by preventing the degradation of IκB-α. UTI pretreatment only markedly inhibited the phosphorylation of JNK at Thr183, but it did not affect the phosphorylation of JNK at Tyr185, ERK-1/2 and p38 MAPK; JNK was found to function upstream of the IκB-α/NF-κB signaling pathway. Furthermore, UTI pretreatment significantly suppressed LPS-induced ROS production by activating PI3K/Akt pathways and the nuclear translocation of Nrf2 via promotion of p62-associated Keap1 degradation. However, JNK was not involved in mediating the anti-oxidative stress effects of UTI. In summary, this study shows that UTI exerts both anti-inflammatory and anti-oxidative effects by targeting the JNK/NF-κB and PI3K/Akt/Nrf2 pathways.

Cerebral ganglion ultrastructure and differential proteins revealed using proteomics in the aplysiid (Notarcus leachii cirrosus Stimpson) under cadmium and lead stress.

Cadmium (Cd) and lead (Pb) are both highly toxic metals in environments. However the toxicological mechanism is not clear. In this study, the aplysiid, Notarcus leachii cirrosus Stimpson (NLCS) was subjected to Cd (NLCS-Cd) or Pb (NLCS-Pb). The cerebral ganglion of NLCS was investigated with a transmission electron microscope. Next the differential proteins were separated and identified using proteomic approaches. Eighteen protein spots in NLCS-Cd and seventeen protein spots in NLCS-Pb were observed to be significantly changed. These protein spots were further excised in gels and identified. A hypothetical pathway was drawn to show the correlation between the partially identified proteins. The results indicated that damage to the cerebral ganglion was follows: cell apoptosis, lysosomes proliferation, cytoskeleton disruption, and oxidative stress. These phenomena and data indicated potential biomarkers for evaluating the contamination levels of Cd and Pb. This study provided positive insights into the mechanisms of Cd and Pb toxicity.

Formononetin sensitizes glioma cells to doxorubicin through preventing EMT via inhibition of histone deacetylase 5.

Chemoresistance is a major obstacle to successful chemotherapy for glioma. Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human glioma cells, and further elucidated the molecular mechanism underlying the anti-tumor property. We found that formononetin enhanced doxorubicin cytotoxicity in glioma cells. Combined treatment with formononetin reversed the doxorubicin-induced epithelial-mesenchymal transition (EMT) in tumor cells. Moreover, we found that formononetin treatment significantly decreased the expression of HDAC5. Overexpression of HDAC5 diminished the suppressive effects of formononetin on glioma cell viability. Furthermore, knockdown of HDAC5 by siRNA inhibited the doxorubicin-induced EMT in glioma cells. Taken together, these results demonstrated that formononetin-combined therapy may enhance the therapeutic efficacy of doxorubicin in glioma cells by preventing EMT through inhibition of HDAC5.

Toxicological responses of the hard clam Meretrix meretrix exposed to excess dissolved iron or challenged by Vibrio parahaemolyticus.

The responses of genes encoding defense components such as ferritin, the lipopolysaccharide-induced tumor necrosis factor-alpha factor (LITAF), the inhibitor of nuclear factor-κB (IκB), metallothionein, and glutathione peroxidase were assessed at the transcriptional level in order to investigate the toxicological and immune mechanism of the hard clam Meretrix meretrix (HCMM) following challenge with iron or a bacterium (Vibrio parahaemolyticus). Fe dissolved in natural seawater led to an increase of Fe content in both the hepatopancreas and gill tissue of HCMM between 4 and 15 days of exposure. The ferritin gene responded both transcriptionally as indicated by real-time quantitative PCR and translationally as shown by western blotting results to iron exposure and both transcriptional and translational ferritin expression in the hepatopancreas had a positive correlation with the concentration of dissolved iron in seawater. Both iron and V. parahaemolyticus exposure triggered immune responses with similar trends in clam tissues. There was a significant post-challenge mRNA expression of LITAF and IκB at 3h, ferritin at 24h, and metallothionein and glutathione peroxidase at 48h. This behavior might be linked to their specific functions in physiological processes. These results suggested that similar signaling pathways were triggered during both iron and V. parahaemolyticus challenges. Here, we indicated that the ferritin of Meretrix meretrix was an intermediate in the pathway of iron homeostasis and in its innate immune defense mechanism.

Histone deacetylase 5 promotes the proliferation of glioma cells by upregulation of Notch 1.

Histone deacetylases (HDACs) constitute a family of enzymes that play important roles in the epigenetic regulation of gene expression and contribute to the growth, differentiation and apoptosis of cancer cells. However, the biological function of HDAC5 in glioma cells has not been fully understood. In the present study, we found that the mRNA and protein levels of HDAC5 are increased in human glioma tissues and cell lines. In addition, overexpression of HDAC5 promoted proliferation of glioma cells, as measured by the MTT assay. By contrast, HDAC5 gene silencing using small interfering RNA (siRNA) inhibited cell proliferation. Furthermore, we demonstrated that HDAC5 enhances Notch 1 expression at both the mRNA and the protein level in glioma cell lines. Taken together, these results demonstrated, for the first time to the best of our knowledge, that HDAC5 promotes glioma cell proliferation, and suggest that this effect involves the upregulation of Notch 1. Therefore, our study may provide a novel therapeutic target for treatment of gliomas.

The evidence of HeLa cell apoptosis induced with tetraethylammonium using proteomics and various analytical methods.

Tetraethylammonium (TEA) is a potassium channel (KCh) blocker applied in the functional and pharmacological studies of the KChs. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, a colorimetric assay to quantitatively measure living cells, demonstrated that TEA reduced the HeLa cell viability dose-dependently. Flow cytometry analysis indicated an increased apoptosis rate of the HeLa cell after exposing to TEA. The patch clamp technique revealed that the K(+) current of the HeLa cell was inhibited up to 80% when exposed to TEA. In addition, quantitative real-time PCR approach set up cross-talk among the cytotoxicity of TEA, 4-aminopyridine, and anti-cancer drug such as cisplatin. Using comparative proteomics combined with MALDI-TOF MS/MS, 33 significantly changed proteins were found from TEA treatment group; among these proteins, 12 were up-regulated, and 21 were down-regulated. Here we indicated that these proteins were closely connected with many biological functions such as oxidative stress response, signal transduction, metabolism, protein synthesis, and degradation. Both Western blotting and quantitative real-time PCR approaches further verified these differential proteins. Ingenuity Pathways Analysis software, a tool to analyze "omics" data and model biological system, was applied to analyze the interaction pathways of these proteins. The subcellular locations of the differential proteins are also predicted from Uniprot. All results above can help in our understanding of the mechanism of TEA-induced cytotoxicity and provide potential cancer biomarkers. Various experimental results in this study (like those for cisplatin) indicated that TEA is not only a KCh blocker but also a potential anti-cancer drug.

Proteomic approach for identifying gonad differential proteins in the oyster (Crassostrea angulata) following food-chain contamination with HgCl2.

Hg discharged into the environmental waters can generally be bioaccumulated, transformed and transmited by living organisms, thus resulting in the formation of Hg-toxicity food chains. The pathway and toxicology of food chain contaminated with environmental Hg are rarely revealed by proteomics. Here, we showed that differential proteomics had the potential to understand reproduction toxicity mechanism in marine molluscs through the Hg-contaminated food chain. Hg bioaccumulation was found in every link of the HgCl2-Chlorella vulgaris-oyster-mice food chain. Morphological observations identified the lesions in both the oyster gonad and the mice ovary. Differential proteomics was used to study the mechanisms of Hg toxicity in the oyster gonad and to find some biomarkers of Hg contamination in food chain. Using 2-DE and MALDI-TOF/TOF MS, we identified 13 differential protein spots, of which six were up-regulated, six were down-regulated, while one was undecided. A portion of these differential proteins was further confirmed using real-time PCR and western blotting methods. Their major functions involved binding, protein translocation, catalysis, regulation of energy metabolism, reproductive functioning and structural molecular activity. Among these proteins, 14-3-3 protein, GTP binding protein, arginine kinase (AK) and 71kDa heat shock connate protein (HSCP 71) are considered to be suitable biomarkers of environmental Hg contamination. Furthermore, we established the gene correspondence, responding to Hg reproductive toxicity, between mouse and oyster, and then used real-time PCR to analyze mRNA differential expression of the corresponding genes in mice. The results indicated that the mechanism of Hg reproductive toxicity in mouse was similar to that in oyster. We suggest that the proteomics would be further developed in application research of food safety including toxicological mechanism. BIOLOGICAL SIGNIFICANCE: It is well known that mercury (Hg) is one of the best toxic metal elements in nature. The research reports as previously described indicated that multiple mercury compounds can directly contaminate the aquatic animals by flowing of water body and through the diffusion of air. The pollution sources of the mercury compounds in marine water were mainly found from the pathways such as steam power plant and mineral exploitation which are located on the inshore. Of note, after being released into environmental waters, mercury compounds undergo the processes of bioaccumulation, transformation and transmission in living organisms, thus resulting in the multiple forms of Hg found in Hg-toxicity food chains, and among them, methyl mercury (MeHg) showing the high toxic characteristics is the main form of Hg. The abundant reports indicated that the metal salts were easily found within the various organs of the animals, but it is difficult to judge the level of its perniciousness according to its content only in vivo. Here, the algae to have been contaminated by the mercury compounds have the ability for contaminating both the fish and shellfish as food pathway quickly. If these fish and shellfish edible as food will be taken by human, they will further affect the human health badly. However, studies about their perniciousness are rarely reported, especially in using proteomics. The oysters as normal food are largely consumed in Southern China, especially in Xiamen City. Similarly, a pathway question that the contaminated oysters can effect on the human health such as cancer is unclear or poorly understood. Here, we showed that an analytical technology such as differential proteomics has potential to understand toxicity mechanism induced by Hg-contamination through the food pathway. It is for reason that the oyster proteomics including relative analytical methods have been used to reveal the contaminant level and to determine its perniciousness using toxic algae as food. Here, we also indicated that the research here shows great significance for both analysis of food safety and toxicology of the metal compounds. In addition, a few biomarkers have shown their strong potential for monitoring the level of Hg pollution in sea in the manuscript and gene correspondence between mouse and oyster, the two contiguous links of the Hg-contaminated food chain, was further investigated to better illustrate our finding in the analysis of food chain proteomics. Moreover, similar research work is rarely reported compared to the current proteomic development, showing that a lot of novel results by proteomic methods in the manuscript have strong potential for developing the new area of food chain proteomics.

Transferrin-cisplatin specifically deliver cisplatin to HepG2 cells in vitro and enhance cisplatin cytotoxicity.

Cisplatin is a major broad-spectrum chemotherapeutic agent, however, its dose-dependent side effects limit the administration of large doses. Presently, developing a drug targeted delivery system is suggested as one of the most promising approaches to minimize the side effects of cisplatin. Here, we found that each human serum transferrin (HTf) has the potential to bind with over 22 cisplatins, and the complex of apo-HTf-cisplatin can specifically deliver cisplatin to HepG2 cells (human hepatocellular liver carcinoma cell line) in vitro, and facilitate HepG2 cells to apoptosis. Moreover, proteomics methods revealed that the abundances of 23 proteins in HepG2 cells were remarkably altered in response to cisplatin/apo-HTf-cisplatin exposure, and Realtime-PCR revealed that a number of important genes related to chemotherapeutic cytotoxicity and chemotherapeutic resistance are differentially transcribed between the HepG2 cells of cisplatin exposed and HTf-cisplatin exposed. The pathway analysis of the differentially expressed proteins and gene transcriptions indicated that those regulated proteins and gene transcriptions are involved in apoptosis regulation, transcription, cell cycle control, protein biosynthesis, energy metabolism, signal transduction, protein binding and other functions. It indicated that the cisplatin toxicity in HepG2 cell is diverse, the transport process has an effect on the cisplatin cytotoxicity, and the mechanism of the apoptosis of HepG2 cells induced by apo-HTf-cisplatin is different from that of cisplatin.

Differential protein profile in zebrafish (Danio rerio) brain under the joint exposure of methyl parathion and cadmium.

As different chemicals, methyl parathion (MP) and cadmium (Cd) can induce neurotoxicity on the brain of aquatic ecosystems. This study aims to explore the differential expression proteins in the brain induced by their joint stress and their joint effects, which are poorly reported, and devotes finding novel biomarkers for monitoring their contamination in water and assessing their neurological effects. The bioaccumulation of MP and Cd in tissues after 96 h of exposure was first analyzed by GC and inductively coupled plasma-MS to provide insights into the interaction. Protein profile changes in the brains of the zebrafish (Danio rerio) exposed to MP and Cd were further investigated using the proteomic approach. The correlation of gene expression on the transcription level of mRNA and the translation level of protein was examined by real-time quantitative PCR and Western blotting analysis. It showed that Cd and MP have an interaction on their bioaccumulation, which suggests that their joint effect over 96 h might be antagonistic. Proteomics revealed that 22 protein spots changed their expression levels under stress, of which 16 proteins were identified using MS. These proteins were involved in oxidation/reduction, metabolism, energy production, receptor activity, and cytoskeleton assembly. Among them, five proteins with a remarkable abundance change are significantly suggested to play important roles in the joint effect. This work demonstrates that there exists an interaction between MP and Cd toxicities, which may aid in our understanding of the mechanism of neurotoxicity induced by joint stress. The results may also provide the possibility of the establishment of candidate biomarkers for monitoring MP and Cd contamination in water.

Cloning analysis of ferritin and the cisplatin-subunit for cancer cell apoptosis in Aplysia juliana hepatopancreas.

Ferritin, an iron storage protein, plays a key role in iron metabolism in vivo. Here, we have cloned an inducible ferritin cDNA with 519 bp within the open reading frame fragment from the hepatopancreas of Aplysia juliana (AJ). The subunit sequence of the ferritin was predicted to be a polypeptide of 172 amino acids with a molecular mass of 19.8291kDa and an isoelectric point of 5.01. The cDNA sequence of hepatopancreas ferritin in AJ was constructed into a pET-32a system for expressing its relative protein efficiently in E. coli strain BL21, under isopropyl-ß-d-thiogalactoside induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately 20 kDa mass using both SDS-PAGE and mass spectrometry. The secondary structure and phosphorylation sites of the deduced amino acids were predicted using both ExPASy proteomic tools and the NetPhos 2.0 server, and the subunit space structure of the recombinant AJ ferritin (rAjFer) was built using a molecular operating environment software system. The result of in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was AjFer. ICP-MS results indicated that the rAjFer subunit could directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 17.6 CDDP/ferritin subunits and forming a novel CDDP-subunit. This suggests that a nanometer CDDP core-ferritin was constructed, which could be developed as a new anti-cancer drug. The flow cytometry results indicated that CDDP-rAjFer could induce Hela cell apoptosis. Results of the real-time PCR and Western blotting showed that the expression of AjFer mRNA was up-regulated in AJ under Cd(2+) stress. The recombinant AjFer protein should prove to be useful for further study of the structure and function of ferritin in Aplysia.

Construction of nanometer cisplatin core-ferritin (NCC-F) and proteomic analysis of gastric cancer cell apoptosis induced with cisplatin released from the NCC-F.

Both transmission electron microscopy (TEM) and fluorescence spectrometry were used to reveal the characteristics of both subunit disassociation and recombination in apo-pig pancreas ferritin (apoPPF) in an alkaline medium ranging reversibly from pH 7.0 to 13.0. The experimental results indicated that apoPPF could be completely disassociated into 24 free subunits at pH 13.0, and then these subunits could be quickly reassembled into the original apoPPF once the pH of the reactive medium was returned to pH7.0. This novel and simple method could be used to effectively construct a novel nanometer cisplatin core-PPF (NCC-PPF). The major characteristics of NCC-PPF were investigated using various analytical methods such as ultraviolet-spectrometry, circular dichroism spectrometry and TEM, which indicated that its molecular structure was still similar to that of the original apoPPF. Results from the inductively coupled plasma mass spectrometer (ICP-MS) method showed that 11.26 cisplatin (CDDP) molecules were successfully packaged within the NCC-PPF shell, indicating that each molecule of apoPPF had the ability to enwrap 11.26 CDDP molecules for constructing the NCC-PPF. Flow cytometry showed that NCC-PPF also had the ability to release CDDP for inducing the apoptosis of gastric cancer cells BGC823 (GCC), but this phenomenon could scarcely be observed using apoPPF. A differential proteomic technique using two-dimensional electrophoresis (2-DE) gels selected and identified the differential proteins from cell apoptosis in order to reveal the molecular pathway of GCC apoptosis by both NCC-PPF and free CDDP, giving 13 differential expression proteins. These differential proteins could be further classified into six groups, which were described as being involved in the regulation of apoptosis, RNA transcription, oxidative stress response, signal transduction, cell metabolism, and cytoskeleton changes. In addition, a real-time PCR method was used to prove the expression level of mRNA and to identify the reliability of the protein expression according to these differential proteins, which indicated that the mRNA level changes of six differential proteins corresponded to those of its differential protein expression in 2-DE gels. These studies played an important role in reasonably revealing the different pathways of GCC apoptosis induced with both the CDDP released by NCC-PPF and the free CDDP. We thus suggest that apoPPF has great potential for constructing a nanometer carrier filled with various drugs for application in clinical work.