Uniform thin ice on ultraflat graphene for high-resolution cryo-EM.

Cryo-electron microscopy (cryo-EM) visualizes the atomic structure of macromolecules that are embedded in vitrified thin ice at their close-to-native state. However, the homogeneity of ice thickness, a key factor to ensure high image quality, is poorly controlled during specimen preparation and has become one of the main challenges for high-resolution cryo-EM. Here we found that the uniformity of thin ice relies on the surface flatness of the supporting film, and developed a method to use ultraflat graphene (UFG) as the support for cryo-EM specimen preparation to achieve better control of vitreous ice thickness. We show that the uniform thin ice on UFG improves the image quality of vitrified specimens. Using such a method we successfully determined the three-dimensional structures of hemoglobin (64 kDa), α-fetoprotein (67 kDa) with no symmetry, and streptavidin (52 kDa) at a resolution of 3.5 Å, 2.6 Å and 2.2 Å, respectively. Furthermore, our results demonstrate the potential of UFG for the fields of cryo-electron tomography and structure-based drug discovery.

α-Conotoxin recombinant protein ImI-AFP3 efficiently inhibits the growth and migration of lung cancer cells.

α-Conotoxin ImI is a selective antagonist of alpha7 nicotinic acetylcholine receptor (α7 nAChR) that is involved in cancer development. Human alpha fetoprotein domain 3 (AFP3) is a prototype of anticancer agents. In an effort to design drugs for anticancer treatments, we fused the ImI peptide to AFP3 as a fusion protein for testing. The fusion protein (ImI-AFP3) was highly expressed in the insect Bac-to-Bac system. The purified fusion protein was found to have improved anticancer activity and synergized with the drug gefitinib to inhibit the growth and migration of A549 and NCI-H1299 lung cancer cells. Our data have demonstrated that the recombinant protein ImI-AFP3 is a promising candidate for drug development to suppress lung cancer cell growth, especially to suppress hepatoid adenocarcinoma of the lung (HAL) cell growth.

α-Fetoprotein fragment synergizes with sorafenib to inhibit hepatoma cell growth and migration and promote the apoptosis.

Alpha fetoprotein (AFP) is associated with hepatocellular carcinoma (HCC) by stimulating the proliferation, metastasis and drug resistance. The application of AFP fragments to inhibit the malignant behaviours induced by AFP is a new strategy for the treatment of HCC. In an effort to design, screen and discover drugs, we attempted to express different human AFP fragments (AFP220-609 , AFP390-609 and AFP460-609 ) in a Bac-to-Bac system. We found that the AFP390-609 fragment was highly expressed in the system. Then, we assessed the bioactivity of the fragment in the human liver cancer cell line Bel7402, and the results indicated that the AFP fragment synergized with sorafenib to inhibit the hepatoma cell growth and migration and promote the apoptosis. This study provides a method to produce significant AFP fragments to screen AFP inhibitors for use in HCC therapy.

GATA5 inhibits hepatocellular carcinoma cells malignant behaviours by blocking expression of reprogramming genes.

Evidence indicated that GATA5 may suppress hepatocellular carcinoma (HCC) cell malignant transformation, but the mechanism of how GATA5 affects cancer cell reprogramming to inhibit HCC malignant behaviour is still unclear. In this study, we report that the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM was significantly higher in HCC tissues compared to normal liver tissues. In contrast, the expression of GATA5 was significantly lower in HCC tissues compared to normal liver tissues. Transfection of CDH-GATA5 vectors into HCC cells (HLE, Bel 7402 and PLC/PRF/5 cells) increased the GATA5 expression and decreased the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM. Increased GATA5 expression by transfection with its expression vectors was also able to inhibit the cell growth, colony formation and capability of migration, invasion, while promoting apoptosis in HCC cells. Results revealed that GATA5 co-localization with ß-catenin in the cytoplasm, preventing ß-catenin from entering the nucleus. Treatment with the specific Wnt/ß-catenin pathway inhibitor salinomycin was able to reduce the expression of ß-catenin and reprogramming genes. Salinomycin exerted a similar influence as GATA5, and siRNA-GATA5 restored ß-catenin and reprogramming gene expression. This study demonstrates that an increase in the expression of GATA5 inhibits the expression of ß-catenin and reprogramming genes and suppresses tumour growth, colony formation, metastasis and invasion, while promoting apoptosis in HCC cells. The mechanism of GATA5 inhibiting the malignant behaviours of HCC cells may involve in the disruption of the Wnt/ß-catenin pathway and the reduction of reprogramming gene expression.

Ropivacaine promotes apoptosis of hepatocellular carcinoma cells through damaging mitochondria and activating caspase-3 activity.

BACKGROUND: Recent evidences indicated that some local anaesthetic agents played a role in inhibiting the proliferation of cancer cells; Whether ropivacaine is able to promote apoptosis of hepatocellular carcinoma (HCC) cells is still unclear. The aim of this study was to investigate the effect of ropivacaine on the apoptosis of HCC cells. METHODS: In the present study, we treated the HCC cell lines, Bel7402 and HLE with ropivacaine. MTT, DAPI stain, trypan blue exclusion dye assay, flow cytometry, electron microscopy, computational simulation, laser confocal microscope, Western blotting, and enzyme activity analysis of caspase-3 were applied to detect the growth and apoptosis of HCC cells and to explore the role mechanism of ropivacaine. RESULTS: Ropivacaine was able to inhibit proliferation and promote apoptosis of HCC cells in a dose- and time-dependent manner. Ropivacaine also has a trait to inhibit the migration of HCC cells; ropivacaine damaged the mitochondria of HCC cells. The results also indicated that ropivacaine was able to interact with caspase-3, promote cytoplasmic caspase-3 migration into the nucleus, stimulate cleavage of caspase-3 and PARP-1, caspase-9 proteins, inhibit the expression of Bcl-2, promote expression of Apaf-1 and mitochondria release cytochrome C, and activate the activity of caspase-3. CONCLUSIONS: Ropivacaine has a novel role in promoting apoptosis of HCC cells; The role mechanism of ropivacaine maybe involve in damaging the function of mitochondria and activating the caspase-3 signalling pathway in HCC cells. Our findings provide novel insights into the local anaesthetic agents in the therapy of HCC patients.

Structural basis for alpha fetoprotein-mediated inhibition of caspase-3 activity in hepatocellular carcinoma cells.

Alpha-fetoprotein (AFP) is an early serum growth factor in the foetal liver development and hepatic carcinogenesis; However, the precise biological role of cytoplasmic AFP remains elusive. Although we recently demonstrated that cytoplasmic AFP might interact with caspase-3 and inhibit the signal transduction of apoptosis in human hepatocellular carcinoma (HCC) cells, the details of this interaction are not clear. To reveal the molecular relationship between AFP and caspase-3, we performed molecular docking, co-immunoprecipitation (Co-IP), laser confocal microscopy, site-directed mutagenesis and functional experiments to analyse the key amino acid residues in the binding site of caspase-3. The results of Co-IP, laser confocal microscopy and functional analyses were consistent with the computational model. We also used the model to explain why AFP cannot bind to caspase-8. These results provide the molecular basis for the AFP-mediated inhibition of caspase-3 activity in HCC cells. Altogether, we found that AFP interacts with caspase-3 through precise amino acids, namely loop-4 residues Glu-248, Asp-253 and His-257. The results further demonstrated that AFP plays a critical role in the inhibition of the apoptotic signal transduction that mediated by caspase-3. Thus, AFP might represent a novel biotarget for the therapy of HCC patients.

HBx drives alpha fetoprotein expression to promote initiation of liver cancer stem cells through activating PI3K/AKT signal pathway.

Hepatitis B virus (HBV)-X protein (HBx) plays critical role in inducing the malignant transformation of liver cells. Alpha fetoprotein (AFP) expression is closely related to hepatocarcinogenesis. We report that Oct4, Klf4, Sox2 and c-myc expression positively associated with AFP(+)/HBV(+) hepatocellular carcinoma(HCC) tissues, and the expression of the stemness markers CD44, CD133 and EpCAM was significantly higher in AFP(+)/HBV(+) HCC tissues compared to normal liver tissues or AFP (-)/HBV(-) HCC tissues. AFP expression turned on prior to expression of Oct4, Klf4, Sox2 and c-myc, and the stemness markers CD44, CD133 and EpCAM in the normal human liver L-02 cell line or CHL cell lines upon transfection with MCV-HBx vectors. Stem-like cells generated more tumour colonies compared to primary cells, and xenografts induced tumourigenesis in nude mice. Expression of reprogramming-related proteins was significantly enhanced in HLE cells while transfected with pcDNA3.1-afp vectors. The specific PI3K inhibitor Ly294002 inhibited the effects of pcDNA3.1-afp vectors. AFP-siRNA vectors were able to inhibit tumour colony formation and reprogramming-related gene expression. Altogether, HBx stimulates AFP expression to induce natural reprogramming of liver cells, and AFP plays a critical role in promoting the initiation of HCC progenitor/stem cells. AFP may be a potential novel biotarget for combating HBV-induced hepatocarcinogenesis.

Purification and characterization of a bioactive alpha-fetoprotein produced by HEK-293 cells.

Alpha-fetoprotein (AFP) is a biomarker that is used to diagnose hepatocellular carcinoma (HCC) and can promote malignancy in HCC. AFP is an important target in the treatment of liver cancer. To obtain enough AFP to screen for AFP inhibitors, we expressed and purified AFP in HEK-293 cells. In the present study, we produced AFP in the cells and harvested highly pure rAFP (or recombinant expression AFP in HEK-293 cells). We also analysed the bioactivity of rAFP and found that rAFP promoted growth of the human HCC cells, antagonize paclitaxel inhibition of HCC cell proliferation, suppress expression of active caspase-3, and promote expression of Ras and survivin. This study provides a method to produce significant amounts of AFP for use in biochemical assays and functional studies and to screen AFP inhibitors for use in HCC therapy.

Alpha fetoprotein plays a critical role in promoting metastasis of hepatocellular carcinoma cells.

A high level of serum alpha fetoprotein (AFP) is positively associated with human hepatocellular carcinoma (HCC) carcinogenesis and metastasis; however, the function of AFP in HCC metastasis is unknown. This study has explored the effects of AFP on regulating metastatic and invasive capacity of human HCC cells. Forty-seven clinical patients' liver samples were collected and diagnosed; HCC cells line, Bel 7402 cells (AFP-producing) and liver cancer cell line cells (non-AFP-producing) were selected to analyse the role of AFP in the metastasis of HCC cells. The results indicated that high serum concentration of AFP was positively correlated with HCC intrahepatic, lymph nodes and lung metastasis. Repressed expression of AFP significantly inhibited the capability of migration and invasion of Bel 7402 cells, expression of keratin 19 (K19), epithelial cell adhesion molecule (EpCAM), matrix metalloproteinase 2/9 (MMP2/9) and CXC chemokine receptor 4 (CXCR4) were also down-regulated in Bel 7402 cells; migration and invasion, expression of K19, EpCAM, MMP2/9 and CXCR4 were significantly enhanced when HLE cells were transfected with AFP-expressed vector. The results demonstrated that AFP plays a critical role in promoting metastasis of HCC; AFP promoted HCC cell invasion and metastasis via up-regulating expression of metastasis-related proteins. Thus, AFP may be used as a novel therapeutic target for treating HCC patients.

Residues Responsible for the Selectivity of α-Conotoxins for Ac-AChBP or nAChRs.

Nicotinic acetylcholine receptors (nAChRs) are targets for developing new drugs to treat severe pain, nicotine addiction, Alzheimer disease, epilepsy, etc. α-Conotoxins are biologically and chemically diverse. With 12-19 residues and two disulfides, they can be specifically selected for different nAChRs. Acetylcholine-binding proteins from Aplysia californica (Ac-AChBP) are homologous to the ligand-binding domains of nAChRs and pharmacologically similar. X-ray structures of the α-conotoxin in complex with Ac-AChBP in addition to computer modeling have helped to determine the binding site of the important residues of α-conotoxin and its affinity for nAChR subtypes. Here, we present the various α-conotoxin residues that are selective for Ac-AChBP or nAChRs by comparing the structures of α-conotoxins in complex with Ac-AChBP and by modeling α-conotoxins in complex with nAChRs. The knowledge of these binding sites will assist in the discovery and design of more potent and selective α-conotoxins as drug leads.

HBx induced AFP receptor expressed to activate PI3K/AKT signal to promote expression of Src in liver cells and hepatoma cells.

BACKGROUND: Hepatitis B virus (HBV)-X protein(HBx) is a transactivator of host several cellular genes including alpha-fetoprotein(AFP) and AFP receptor(AFPR) which contributes to HBV-associated tumor development. The expression of AFP/AFPR are correlated with hepatocellular carcinoma(HCC)-initial cells. But the role of AFP and AFPR in promoting occurrence of HBV-related HCC were still unclear. METHODS: A total of 71 clinical patients' liver specimens, normal human liver cells L-02 and HCC cell lines, PLC/PRF/5 were selected for analyzing the effects of HBx on expression of AFP, AFPR and Src. The expression of goal proteins were detected by Immunohistochemical stained and Western blotting; HBx-expressed vectors were constructed and transfected into L-02 cells, laser confocal microscopy was applied to observe expression and location of AFP, AFPR and Src in the normal liver cells and HCC cells, soft agar colony formation assay was used to observe colonies formed of the cells. RESULTS: We confirmed HBx gives preference to promote the expression of AFP and AFPR; HBx priors to up-regulate the expression of AFPR and AFP in L-02 cells and in normal liver specimens; AFPR signal been able to stimulate Src expression. The results also indicated that phosphatidylinositol 3-kinase(PI3K) inhibitors Ly294002 and GDC0941 effectively suppress AFPR mediated up-regulation expression of Src in AFPR positive HCC lines. CONCLUSIONS: HBx priors to drive the expression of AFP and AFPR to promote expression of Src in normal liver cells and hepatoma cells; AFP and AFPR maybe play pivotal role in HBV-related hepatocarcinogenesis; Targeting AFPR is an available therapeutic strategy of HCC.

The role of alpha-fetoprotein in the tumor microenvironment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent malignant cancer worldwide, characterized by high morbidity and mortality rates. Alpha-fetoprotein (AFP) is a glycoprotein synthesized by the liver and yolk sac during fetal development. However, the serum levels of AFP exhibit a significant correlation with the onset and progression of HCC in adults. Extensive research has demonstrated that the tumor microenvironment (TME) plays a crucial role in the malignant transformation of HCC, and AFP is a key factor in the TME, promoting HCC development. The objective of this review was to analyze the existing knowledge regarding the role of AFP in the TME. Specifically, this review focused on the effect of AFP on various cells in the TME, tumor immune evasion, and clinical application of AFP in the diagnosis and treatment of HCC. These findings offer valuable insights into the clinical treatment of HCC.

The diagnostic, prognostic, drug sensitivity and ceRNA internet of PPP4R1 in liver hepatocellular carcinoma (LIHC).

Background: Recent studies have reported a role of protein phosphatase 4 regulatory subunit 1 (PPP4R1) in cancer development. However, its expression, diagnostic significance, prognostic value and biological function in liver hepatocellular carcinoma (LIHC) are not known. Methods: The expression level of PPP4R1 in pan-cancer was evaluated by analyzing publicly accessible data from the University of California Santa Cruz (UCSC) Xena database. The diagnostic value of PPP4R1 for tumors was assessed using receiver operating characteristic (ROC) curves, whereas the impact of PPP4R1 on tumor prognosis was determined using Kaplan-Meier survival curves, and a prognostic model for LIHC was established using cox regression analysis. In addition, analysis of the correlation between PPP4R1 and anti-cancer drugs using Spearman's correlation coefficient was carried out. Four databases, miRWalk (mRNA-miRNA interactions), MicroT-CDS (mRNA-miRNA interactions), LncBase (miRNA-lncRNA interactions) and Encyclopedia of RNA Interactomes (ENCORI), were used to predict the competitive endogenous RNA (ceRNA) regulatory network of PPP4R1. Finally, the expression of PPP4R1 protein levels was verified using experiments. Results: The findings indicated that the PPP4R1 expression level in cancerous tissues was notably greater than in adjacent tissues (P<0.05). PPP4R1 showed diagnostic significance for 14 tumors based on the ROC curves results area under the curve >0.7. Furthermore, the Kaplan-Meier survival plots demonstrated that PPP4R1 exhibited prognostic significance for all five tumors (P<0.05). According to the cox regression analysis, LIHC patients' prognosis was independently influenced by pathological stage, M stage, and PPP4R1 (P<0.05). The drug sensitivity analysis revealed a positive correlation between the expression level of PPP4R1 and the half maximal inhibitory concentration (IC50) of fludarabine. Additionally, the ceRNA network prediction indicated that the FGD5 antisense RNA 1 (FGD5-AS1)-hsa-miR-22-3p-PPP4R1 ceRNA network could potentially contribute to the progression of LIHC. The experimental results showed that the expression level of PPP4R1 protein was higher in cancer tissues than in paracancerous tissues. Conclusions: PPP4R1 has diagnostic value in most cancers, and high expression of PPP4R1 is associated with poor prognosis, drug resistance and natural killer cell-mediated toxicity, particularly in LIHC. Therefore, PPP4R1 may be a prognostic biomarker and a potential target for immunotherapy in LIHC.

Structural characteristics of alpha-fetoprotein, including N-glycosylation, metal ion and fatty acid binding sites.

Alpha-fetoprotein (AFP), a serum glycoprotein, is expressed during embryonic development and the pathogenesis of liver cancer. It serves as a clinical tumor marker, function as a carcinogen, immune suppressor, and transport vehicle; but the detailed AFP structural information has not yet been reported. In this study, we used single-particle cryo-electron microscopy(cryo-EM) to analyze the structure of the recombinant AFP obtained a 3.31 Å cryo-EM structure and built an atomic model of AFP. We observed and identified certain structural features of AFP, including N-glycosylation at Asn251, four natural fatty acids bound to distinct domains, and the coordination of metal ions by residues His22, His264, His268, and Asp280. Furthermore, we compared the structural similarities and differences between AFP and human serum albumin. The elucidation of AFP's structural characteristics not only contributes to a deeper understanding of its functional mechanisms, but also provides a structural basis for developing AFP-based drug vehicles.

Tumor-associated macrophage polarization in the inflammatory tumor microenvironment.

The chronic inflammation of tumor continues to recruit TAMs (tumor-associated macrophages) to the TME (tumor microenvironment) and promote polarization. Pro-inflammatory signals polarize macrophages to the M1 phenotype to enhance inflammation against pathogens. Tumor inflammatory development changes the pro-inflammatory response to an anti-inflammatory response, resulting in the alteration of macrophages from M1 to M2 to promote tumor progression. Additionally, hypoxia activates HIF (hypoxia-inducible factors) in the TME, which reprograms macrophages to the M2 phenotype to support tumor development. Here, we discuss the factors that drive phenotypic changes in TAMs in the inflammatory TME, which will help in the development of cancer immunotherapy of macrophages.

Glucose metabolism reprogramming promotes immune escape of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a complex process that plays an important role in its progression. Abnormal glucose metabolism in HCC cells can meet the nutrients required for the occurrence and development of liver cancer, better adapt to changes in the surrounding microenvironment, and escape the attack of the immune system on the tumor. There is a close relationship between reprogramming of glucose metabolism and immune escape. This article reviews the current status and progress of glucose metabolism reprogramming in promoting immune escape in liver cancer, aiming to provide new strategies for clinical immunotherapy of liver cancer.

Alpha fetoprotein promotes polarization of macrophages towards M2-like phenotype and inhibits macrophages to phagocytize hepatoma cells.

Alpha-fetoprotein(AFP) is a cancer biomarker for the diagnosis of hepatocellular carcinoma(HCC); however, its role in macrophage polarization and phagocytosis remains unclear. In the present study, we explored the correlation between AFP regulation of macrophage function and the possible regulatory mechanisms. Human mononuclear leukemia cells (THP-1) and monocytes from healthy donors were used to analyze the effect of AFP on the macrophages' phenotype and phagocytosis. THP-1 cells and healthy human donor-derived monocytes were polarized into M0 macrophages induced by phorbol ester (PMA), and M0 macrophages were polarized into M1 macrophages induced by lipopolysaccharide(LPS) and interferon-γ(IFN-γ). Interleukin-4(IL-4) and interleukin-13(IL-13) were used to induce M0 macrophage polarization into M2 macrophages. Tumor-derived AFP(tAFP) stimulated M0 macrophage polarization into M2 macrophages and inhibited M1 macrophages to phagocytize HCC cells. The role of AFP in promoting macrophage polarization into M2 macrophages and inhibiting the M1 macrophages to phagocytize HCC cells may be involved in activating the PI3K/Akt signaling pathway. AFP could also enhanced the migration ability of macrophages and inhibited the apoptosis of HCC cells when co-cultured with M1-like macrophages. AFP is a pivotal cytokine that inhibits macrophages to phagocytize HCC cells.

[Corrigendum] SCF increases cardiac stem cell migration through PI3K/AKT and MMP­2/­9 signaling.

Following the publication of this article, the authors have realized that they mistakenly used the total AKT blot featured in Fig. 4A for the GAPDH blot in Fig. 3B on p. 116. The corrected version of Fig. 3, featured the correct data for the GAPDH experiment, is shown opposite. The authors regret that this error was not picked up upon before the paper was sent to press, and thank the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish a corrigendum. The error did not affect either the results or the conclusions reported in the study, and all the authors agree to this corrigendum. Furthermore, they regret any inconvenience caused to the readership. [the original article was published in International Journal of Molecular Medicine 34: 112­118, 2014; DOI: 10.3892/ijmm.2014.1773].

Erratum: Hepatitis B Virus X Protein Driven Alpha Fetoprotein Expression to Promote Malignant Behaviors of Normal Liver Cells and Hepatoma Cells: Erratum.

[This corrects the article DOI: 10.7150/jca.13628.].

Dynamic contrast-enhanced MRI predicts PTEN protein expression which can function as a prognostic measure of progression-free survival in NPC patients.

OBJECTIVES: The objective of our study was to investigate whether a phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression was associated with dynamic contrast-enhanced MRI (DCE-MRI) parameters and prognosis in nasopharyngeal carcinoma (NPC). METHODS: Two-hundred-and-forty-five (245) patients with NPC who underwent pretreatment biopsy, expression of PTEN detected by immunohistochemistry of biopsy, and radical intensity-modulated radiation therapy (IMRT) with or without chemotherapy were included. Tumor segmentations were delineated on pretreatment MRI manually. The pharmacokinetic parameters (Ktrans, Kep, Ve, and Vp) derived from dynamic contrast-enhanced MRI (DCE-MRI) using the extended Toft's model within the tumor segmentations were estimated. The following demographics and clinical features were assessed and correlated against each other: gender, age, TNM stage, clinical-stage, Epstein-Barr virus (EBV), pathological type, progression-free survival (PFS), and prognosis status. DCE parameter evaluation and clinical feature comparison between the PTEN positive and negative groups were performed and correlation between PTEN expression with the PFS and prognosis status using Cox regression for survival analysis were assessed. RESULTS: A significantly lower Ktrans and Kep were found in NPC tumors in PTEN negative patients than in PTEN positive patients. Ktrans performed better than Kep in detecting PTEN expression with the ROC AUC of 0.752. PTEN negative was associated with later TNM stage, later clinical-stage, shorter PFS, and worse prognosis. Moreover, N stage, pathological type, Kep, and prognostic status can be considered as independent variables in discrimination of PTEN negative expression in NPCs. CONCLUSIONS: PTEN negative indicated a shorter PFS and worse prognosis than PTEN positive in NPC patients. Ktrans and Kep derived from DCE-MRI, which yielded reliable capability, may be considered as potential imaging markers that are correlated with PTEN expression and could be used to predict PTEN expression noninvasively. Combined radiological and clinical features can improve the performance of the classification of PTEN expression.