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Previous studies have shown that secreted protein acidic and rich in cysteine (SPARC) proteins can inhibit the development of cancer cells in various ways, such as by inhibiting angiogenesis and inhibiting cell proliferation. In fact, SPARC proteins may have an effect on the chemoresistance of gastric cancer cells to 5-Fluorouracil (5-FU), which needs further research in the future. Therefore, the purpose of this study was to explore the relationship between SPARC proteins and the chemosensitivity of gastric cancer cells to 5-FU. In vitro, after SPARC protein levels were regulated by plasmid, siRNA and human recombinant SPARC protein transfection in MGC-803, SGC-7901 and BGC-823 cells, we detected epithelial-mesenchymal transition (EMT), apoptosis markers and cell viability after 5-FU treatment. In vivo, we implanted BGC-823 cells with stable SPARC overexpression into nude mice. Tumour size was measured to assess the effect of SPARC protein on tumour formation and 5-FU chemosensitivity. In SGC-7901 and BGC-823 cells, both endogenous and exogenous upregulation of SPARC protein levels decreased cell viability, destroyed cytoskeletal F-actin, inhibited cell migration, and downregulated a series of transcription factors to inhibit cell EMT; it also upregulated cell apoptosis-related proteins to promote cell apoptosis. However, we obtained opposite results in SPARC knockdown MGC-803 cells. In vivo, compared with the control group, the group engrafted with BGC-823 cells stably overexpressing SPARC had a significant smaller tumour size. After 5-FU treatment, the new tumour gradually decreased in size. Our results show that the SPARC protein could enhance 5-FU chemosensitivity in gastric cancer cell lines by inhibiting EMT and promoting cell apoptosis.

Background: Surgery is the optimal choice for treatments of gastric cancers, and multivisceral resection may be the exclusive procedure for patients with apparent invasion to adjacent organs. However, for locally advanced gastric cancers, most surgeons refuse to select surgery as the primary treatment because of the theoretical higher mortality, morbidity and poorer prognosis. Briefly, multivisceral resection for locally advanced gastric cancer remains controversial. we aim to analyze the role of multivisceral resection for locally advanced gastric cancer with attention to the short, long-term results, prognostic factors and the selection standards for surgery. Methods: We retrospectively reviewed the medical records from January 1,1997 to December 31,2018 at Gastroenterology Department of Peking University First Hospital, 235 patients suffered from gastric cancer staged cT4b who underwent surgery were included for the study. And the suspected direct invasion to adjacent organs was determined by abdominal computed tomography and/or ultrasonography from medical records. Patients with distant metastasis, neoadjuvant treatments and metastatic gastric tumors were excluded. The primary endpoint was overall survival. Short-term results such as mortality, incidence of major complications and prognostic value of clinical and pathologic factors were also analyzed. Findings: Patients underwent multivisceral resection had an acceptable mortality and morbidity, for 1.3% and 13.1%, respectively. The overall 1,3,5-year survival rate of multivisceral resection was 56.1%, 26.2% and 15.4%, which statistically higher than that of palliative surgery ( P 15), vascular cancer emboli, differentiation grade, peritoneal metastasis and postoperative chemotherapy. What’s more, patients presented with pN3b did not benefit from multivisceral resection compared with palliative surgery. And D2 lymph node dissection but not extended D3 lymphadenectomy was enough for multivisceral resection ( P =0.42). Patients will benefit from ≥6 cycles chemotherapy after multivisceral resection. Interpretations: Patients presented with cT4b gastric cancers will benefit from multivisceral resection and experience an acceptable mortality and morbidity. Cases presented with obviously N3b status or completeness of resection can not be obtained should be excluded for multivisceral resection. Besides, ≥6 cycles chemotherapy should be performed after surgery. Funding Statement: Department of general surgery, Peking University First Hospital Declaration of Interests: All authors declare no competing interests. Ethics Approval Statement: Institutional review board approval had been obtained for the use of data.

Paxillin (PXN) is a cytoplasmic protein that plays an important role in regulating focal adhesion, cytoskeletal rearrangements and cell motility. The present study aimed to investigate the role of PXN in the metastasis of human colorectal cancer (CRC) and its possible mechanisms. Immunohistochemical staining of tissues from 102 surgical CRC patients revealed that high PXN expression was positively correlated with tumour-node-metastasis (TNM) stage, lymph node metastasis, distant metastasis, and recurrence at distant sites after radical surgery. In 24 cases of stage IV CRC, PXN expression in liver metastasis was higher than that in the matched primary tumour. The knockdown of PXN inhibited the proliferation, migration and invasion potential of SW480 cells in vitro and in vivo. Transmission electron microscopy revealed the effect of PXN on ultrastructural characteristics, observed mainly in microvilli and desmosomes. The downregulation of PXN decreased the activation of extracellular regulated protein kinase (ERK) and suppressed the epithelial-mesenchymal transition (EMT) process. Following the downregulation of PXN, the addition of an ERK activator or inhibitor restored or further suppressed EMT, respectively, accompanied by corresponding changes in cell migration and invasion. Collectively, the present results confirmed the important role of PXN in CRC metastasis and revealed that PXN regulated EMT progression via the ERK signalling pathway. PXN may represent a future therapeutic strategy to prevent the EMT-associated progression and invasion of CRC.

Since the theory of seed and soil was put forward, people have increasingly recognized that the tumour microenvironment is an important regulator of tumour progression and therapeutic response. Among them, M2-type macrophages (M2, as the major macrophage subtype in the tumour foci) have important promoting effects on various biological behaviours. Secreted protein acidic and rich in cysteine (SPARC) is an important anti-tumour component in the microenvironment of gastric cancer. This study shows that macrophages are an important source of the SPARC and that SPARC overexpression in M2 can reduce M2-mediated promoting proliferation, migration and anti-apoptotic effects in gastric cancer. Additionally, the AKT/mTOR signalling pathways may participate in the malignant process.

Background: As the third confirmed gaseous transmitter, the role of hydrogen sulfide (H2S) in the pathogenesis of multiple types of cancer has been attracting increasing attention. Increased expression of cystathionine β-synthase (CBS) and H2S in colon cancer tissue samples has been validated and tumor-derived H2S, mainly produced by CBS, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer. Recently, the therapeutic manipulation of H2S has been proposed as a promising anticancer approach. However, the effect of aminooxyacetic acid (AOAA), which has been widely used as an inhibitor of CBS dependent synthesis of H2S, on the chemotherapeutic effect of oxaliplatin (OXA) and the underlying mechanisms remain to be illustrated. Methods: We examined the expression of CBS in human colorectal cancer specimens and matched normal mucosa by immunohistochemistry. The effect of AOAA on the sensitivity of colon cancer cells to OXA and the level of apoptosis induced by caspase cascade was investigated in both HCT116 and HT29 cell lines utilizing CCK-8 assays, flow cytometry analysis and western blot analysis. The endogenous levels of reactive oxygen species (ROS) were detected fluorescently by DCF-DA, and glutathione (GSH) levels were measured by a Total GSH Detection Kit. Tumor bearing xenograft mouse models and in vivo imaging systems were further used to investigate the effect of AOAA in vivo and immunohistochemistry (IHC) and TUNEL analysis were performed. Results: In the current study, we confirmed CBS, the main target of AOAA, is overexpressed in human colorectal cancer by immunohistochemistry. The inhibitory effect of AOAA on the synthesis of H2S was validated utilizing fluorescent probe and specific electrode. AOAA significantly reduced the IC50 values of OXA in both colon cancer cell lines. Co-incubation with AOAA elicited increased apoptosis induced by OXA, featured by increased activation of caspase cascade. Besides, AOAA further increased the levels of ROS induced by OXA and attenuated the synthesis of glutathione (GSH), which is a vital antioxidant. Besides, the results of in vivo imaging and following IHC and TUNEL analysis were in accordance with cellular experiments, indicating that AOAA sensitizes colon cancer cells to OXA via exaggerating intrinsic apoptosis. Conclusion: The results suggested that CBS is overexpressed in colorectal cancer tissues and AOAA sensitizes colon cancer cells to OXA via exaggerating apoptosis both in vitro and in vivo. Decreasing the endogenous level of GSH and consequently impaired detoxification of ROS might be one of the mechanisms underlying the effect of AOAA.

Objectives: To analyze the heterogeneous functions of secreted protein acidic and rich in cysteine (SPARC) from different origins and in different tumor microenvironments with the purpose of determining its clinical significance. Methods : The PubMed, CINAHL, Cochrane, Web of Science and Embase databases were utilized. Studies that focused on the effects of SPARC expression on solid tumor progression and clinical implications were used. The different outcomes including overall survival and disease-free survival were analyzed to evaluate their relations with tumor- and stroma-derived SPARC expression. Results : A total of 26 studies including 5,939 patients were enrolled in the present meta-analysis. Tumor-derived SPARC overexpression was significantly related with poor overall survival (hazard ratio: 1.478; 95% CI: 1.143-1.910; p =0.003), and a similar tendency was also observed in disease-free survival (hazard ratio: 1.476; 95% CI: 0.993-2.195; p =0.054). However, the hazard ratios for overall survival and disease-free survival did not present a statistical trend in stromal SPARC overexpression. Tumor type subgroup analysis revealed marked heterogeneity among outcomes. In pancreatic cancer, SPARC overexpression in the stroma was significantly associated with poorer overall survival and disease-free survival. In colorectal cancer, SPARC overexpression in the stroma was associated with better disease-free survival. Conclusion : For the majority of solid tumors, SPARC in cancer cells may be an unfavorable indicator for long-term survival for patients. As for stromal expression, SPARC indicates a poorer prognosis in pancreatic cancer, but a better disease-free survival in colorectal cancer. Secreted protein acidic and rich in cysteine might be a potential biomarker for solid tumor prognosis.

Background Long noncoding RNAs (lncRNAs) have been shown to participate in multiple biological processes and confer drug resistance. However, it remains unclear whether lncRNAs are involved in conferring cetuximab resistance in colorectal cancer (CRC) cells. Methods Cell Counting Kit-8 (CCK-8) assays were performed to assess the sensitivity of CRC cell lines to cetuximab treatment. We incubated Caco-2 cells, which are partially responsive to cetuximab, with increasing concentrations of cetuximab for approximately 6 months to generate Caco-2 cetuximab-resistant (Caco-2 CR) cells. Microarray analysis comparing Caco-2 CR with Caco-2 cells was used to identify lncRNAs that were potentially related to cetuximab resistance. Caco-2 cells were stably transduced with cetuximab resistance-associated RNA transcript 16 (CRART16) or an empty vector using lentiviral infection; the cells were designated Caco-2-CRART16 and Caco-2-NC, respectively, and were analyzed with RNA sequencing (RNA-seq). Quantitative real-time PCR (qRT-PCR) was performed to investigate RNA expression. Flow cytometry and TUNEL assays were used to assess apoptosis levels induced by cetuximab. The cell cycle, stemness biomarkers and membrane proteins of CRC cells were assessed via flow cytometry. RNA fluorescence in situ hybridization (FISH) was used to examine CRART16 localization and expression. Bioinformatics analysis was performed to predict the potential mechanism of CRART16, which was further validated by a dual-luciferase reporter assay. Differences in measurement data were compared using Student’s t test, one-way ANOVA followed by Dunnett’s test and two-way ANOVA. Results The novel lncRNA CRART16 was upregulated in Caco-2 CR cells. CRART16 overexpression reversed the effects of cetuximab on cell viability and reduced cetuximab-induced apoptosis. Meanwhile, CRART16 overexpression led to increases in the proportion of CD44+/CD133+ cells. In addition, CRART16 acts as a competing endogenous RNA (ceRNA) for miR-371a-5p to regulate V-Erb-B2 Erythroblastic Leukemia Viral Oncogene Homolog 3 (ERBB3) expression. MiR-371a-5p mimics counteracted the cetuximab resistance induced by CRART16 overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that after CRART16 was overexpressed, the resulting differentially expressed mRNAs were mainly enriched in the MAPK signaling pathway. Conclusions CRART16 overexpression may contribute to cetuximab resistance through the miR-371a-5p/ERBB3/MAPK pathway. Additionally, CRART16 contributes to the acquisition of stemness properties.

Background The aim of the present study was to clarify the correlations between SPARC expression in gastric cancer-associated fibroblasts (GCAFs) and the prognosis of patients with gastric cancer and to elucidate the role of GCAF-derived SPARC in stemness transformation and 5-fluorouracil resistance in gastric cancer. Methods One hundred ninety-two patients were enrolled in the present study. SPARC expression levels were evaluated by immunohistochemical staining. Primary GCAFs were obtained and cultured from cancer patients for in vitro study, and a lentivirus infection method was employed to knock down SPARC expression in GCAFs. The stemness phenotype and 5-fluorouracil (5-FU) response of gastric cancer cells were assessed via a 3D co-culture model. The apoptotic status and stemness alterations were monitored by flow cytometry and western blotting. Additionally, label-free quantification proteomics was used to identify the differentially expressed proteins and potential pathways in gastric cancer cells treated with GCAF-derived SPARC. Results Low expression of GCAF-derived SPARC was associated with decreased differentiation and reduced 5-year overall survival and was an independent predictive factor for prognosis in gastric cancer. The 3D tumour growth and 5-FU resistance abilities of gastric cancer cells were elevated after treatment with GCAFs with SPARC knockdown relative to these abilities in negative control cells. Additionally, suppressing SPARC expression in GCAFs facilitated the phenotypic alteration of gastric cancer cells towards CD44+/CD24− cancer stem cell (CSC)-like cells. Quantification proteomics analysis revealed that the differentially expressed proteins in gastric cancer cells were mainly involved in the AKT/mTOR and MEK/ERK signalling pathways. Conclusions SPARC expression in GCAFs is a useful prognostic factor in patients with gastric cancer. Low expression of GCAF-derived SPARC can lead to CSC transformation and 5-FU resistance. Additionally, the AKT/mTOR and MEK/ERK signalling pathways may participate in the malignant process. Electronic supplementary material The online version of this article (10.1186/s12935-019-0844-8) contains supplementary material, which is available to authorized users.

Follicle stimulating hormone (FSH) is a kind of glycoprotein gonadotropin, and plays an important role in the diagnosis and treatment of infertility. Follicle stimulating hormone receptor (FSHR) is a kind of G protein coupled receptor (GPCR), found in the ovary and testes, and its activation is required for the hormonal operation during the breeding period. In this study, an experimental model of FSHR mediated FSH into cell membrane, which exhibited a phenomenon of fluorescent localized on cell surfaces internalized into cell interior, was established to verify biological activity of FSH.

Follicle-stimulating hormone (FSH) plays a central role in mammals reproduction, with the actions of FSH mediated by follicle-stimulating hormone receptors (FSHRs) on the surface of target cells. The purposes of this study were to determine and evaluate the biological activities for the commercialization of recombinant follicle-stimulating hormone (rFSH) in vitro through the cellular internalization using cloned 293T-FSHR cell lines as target. Using imaging approaches we have found here that a little fluorescent signal from the surface of the cell transferred to the cytoplasm and accumulated around the nucleus by endocytosis. Compared with the control groups, the commercialization of rFSH have not the significant differences of internalization, but the rFSH have promoted the internalization of the fluorescent, suggested that this detection system might as a protocol for the bioactivity of recombinant therapeutic proteins in vitro.

Understanding molecular mechanisms underlying plant salinity tolerance provides valuable knowledgebase for effective crop improvement through genetic engineering. Current proteomic technologies, which support reliable and high-throughput analyses, have been broadly used for exploring sophisticated molecular networks in plants. In the current study, we compared phosphoproteomic and proteomic changes in roots of different soybean seedlings of a salt-tolerant cultivar (Wenfeng07) and a salt-sensitive cultivar (Union85140) induced by salt stress. The root samples of Wenfeng07 and Union85140 at three-trifoliate stage were collected at 0 h, 0.5 h, 1 h, 4 h, 12 h, 24 h, and 48 h after been treated with 150 mm NaCl. LC-MS/MS based phosphoproteomic analysis of these samples identified a total of 2692 phosphoproteins and 5509 phosphorylation sites. Of these, 2344 phosphoproteins containing 3744 phosphorylation sites were quantitatively analyzed. Our results showed that 1163 phosphorylation sites were differentially phosphorylated in the two compared cultivars. Among them, 10 MYB/MYB transcription factor like proteins were identified with fluctuating phosphorylation modifications at different time points, indicating that their crucial roles in regulating flavonol accumulation might be mediated by phosphorylated modifications. In addition, the protein expression profiles of these two cultivars were compared using LC MS/MS based shotgun proteomic analysis, and expression pattern of all the 89 differentially expressed proteins were independently confirmed by qRT-PCR. Interestingly, the enzymes involved in chalcone metabolic pathway exhibited positive correlations with salt tolerance. We confirmed the functional relevance of chalcone synthase, chalcone isomerase, and cytochrome P450 monooxygenase genes using soybean composites and Arabidopsis thaliana mutants, and found that their salt tolerance were positively regulated by chalcone synthase, but was negatively regulated by chalcone isomerase and cytochrome P450 monooxygenase. A novel salt tolerance pathway involving chalcone metabolism, mostly mediated by phosphorylated MYB transcription factors, was proposed based on our findings. (The mass spectrometry raw data are available via ProteomeXchange with identifier PXD002856).

Background Cancer-associated fibroblasts (CAFs) play important roles in tumor progression. However, the behaviors of activated CAFs in gastric cancer remain to be determined. The aim of the present study was to investigate the correlations between activated gastric CAFs and the prognosis of patients with gastric cancer, and to determine the effects of activated CAFs on the malignant phenotype and 5-fluorouracil resistance in this cancer. Methods Ninety-five patients with primary gastric cancer were enrolled in this study. Activation states of gastric CAFs were evaluated by immunohistochemistry. A modified method for the primary culture of gastric CAFs was employed. Types of CAFs and activation states were identified by immunocytochemical and immunofluorescent staining. Cell co-culture and gastric CAF conditioned medium transfer models were established to investigate the paracrine effects of activated CAFs on the migration and invasion of gastric cell lines. The half maximal inhibitory concentration of 5-fluorouracil and levels of cell apoptosis were examined using cell viability assay and flow cytometry, respectively. Protein expression levels of associated molecules were measured by Western blotting. Results Kaplan–Meier survival curves showed that activated gastric CAFs identified via fibroblast activation protein were significantly related to poorer cumulative survival in gastric cancer patients. Five strains of CAFs were successfully cultured via the modified culture method, and three gastric CAFs strains were identified as activated gastric CAFs. The migration and invasion abilities of gastric cells were significantly enhanced in both the co-culture group and the conditioned medium group. The half maximal inhibitory concentration for 5-fluorouracil in BGC-823 cells was elevated after treatment with conditioned medium, and early apoptosis was inhibited. Additionally, an obvious elevation of epithelial–mesenchymal transition level was observed in the conditioned medium group. Conclusions Activated gastric CAFs correlate with a poor prognosis of cancer patients and may contribute to the malignant phenotype and the development of resistance to 5-fluorouracil via paracrine action in gastric cancer. Gastric CAFs with a specific activation state might be used as a tumor biomarker within the microenvironment for prognosis and as a new therapeutic target for chemoresistant gastric cancer. Electronic supplementary material The online version of this article (10.1186/s12935-018-0599-7) contains supplementary material, which is available to authorized users.

Mammalian cells, e.g., CHO, BHK, HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering. They are widely used for the production of recombinant therapeutic proteins, vaccines, anticancer agents, and other clinically relevant drugs. HEK293 (human embryonic kidney 293) cells and their derived cell lines provide an attractive heterologous system for the development of recombinant proteins or adenovirus productions, not least due to their human-like posttranslational modification of protein molecules to provide the desired biological activity. Secondly, they also exhibit high transfection efficiency yielding high-quality recombinant proteins. They are easy to maintain and express with high fidelity membrane proteins, such as ion channels and transporters, and thus are attractive for structural biology and electrophysiology studies. In this article, we review the literature on HEK293 cells regarding their origins but also stress their advancements into the different cell lines engineered and discuss some significant aspects which make them versatile systems for biopharmaceutical manufacturing, drug screening, structural biology research, and electrophysiology applications.

Background Several proteins have been identified as potential diagnostic biomarkers in imaging, genetic, or proteomic studies in Alzheimer disease (AD) patients and mouse models. However, biomarkers for presymptom diagnosis of AD are still under investigation, as are the presymptom molecular changes in AD pathogenesis. Objective In this study, we aim to analyzed the early proteomic changes in APPSw,Ind mice and to conduct further functional studies on interesting proteins. Methods We used the isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with mass spectrometry to examine the early proteomic changes in hippocampi of APPSw,Ind mice. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immuno-blotting were performed for further validation. Finally, the functions of interesting proteins β-spectrin and Rab3a in APP trafficking and processing were tested by shRNA knockdown, in N2A cells stably expressing β-amyloid precursor protein (APP). Results The iTRAQ and RT-PCR results revealed the detailed molecular changes in oxidative stress, myelination, astrocyte activation, mTOR signaling and Rab3-dependent APP trafficking in the early stage of AD progression. Knock down of β -spectrin and Rab3a finally led to increased APP fragment production, indicating key roles of β-spectrin and Rab3a in regulating APP processing. Conclusion Our study provides the first insights into the proteomic changes that occur in the hippocampus in the early stages of the AD mouse model. In addition to improving the understanding of molecular alterations and functional cascades involved in early AD pathogenesis, our findings raise the possibility of developing potential biomarkers and therapeutic targets for early AD.

A full-length cDNA encoding a candidate Oxysterol-binding protein(OSBP) from Aspergillus oryzae (AoOSBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoOSBP protein from the importantly industrial fungus A. oryzae was purified by amylose resin and chromatography column. SDS-PAGE showed that MBP-AoOSBP has an estimated molecular weight of 182 kDa. OSBP and its homologues (ORPs) own the affinity for oxysterols, cholesterol and glycerophospholipids. According to the superiority of A. oryzae in the fermented foods and also in food-grade productions pharmaceutical enzyme manufacture, it is meaningful to identify the biochemical properties of OSBP in A. oryzae .