Proteome Landscapes of Human Hepatocellular Carcinoma and Intrahepatic Cholangiocarcinoma.

Liver cancer is among the top leading causes of cancer mortality worldwide. Particularly, hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA) have been extensively investigated from the aspect of tumor biology. However, a comprehensive and systematic understanding of the molecular characteristics of HCC and CCA remains absent. Here, we characterized the proteome landscapes of HCC and CCA using the data-independent acquisition (DIA) mass spectrometry (MS) method. By comparing the quantitative proteomes of HCC and CCA, we found several differences between the two cancer types. In particular, we found an abnormal lipid metabolism in HCC and activated extracellular matrix-related pathways in CCA. We next developed a three-protein classifier to distinguish CCA from HCC, achieving an area under the curve (AUC) of 0.92, and an accuracy of 90% in an independent validation cohort of 51 patients. The distinct molecular characteristics of HCC and CCA presented in this study provide new insights into the tumor biology of these two major important primary liver cancers. Our findings may help develop more efficient diagnostic approaches and new targeted drug treatments.

Identification of Protein Abundance Changes in Hepatocellular Carcinoma Tissues Using PCT-SWATH.

PURPOSE: To rapidly identify protein abundance changes in biopsy-level fresh-frozen hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: The pressure-cycling technology (PCT) is applied and sequential window acquisition of all theoretical mass spectra (SWATH-MS) workflow is optimized to analyze 38 biopsy-level tissue samples from 19 HCC patients. Each proteome is analyzed with 45 min LC gradient. MCM7 is validated using immunohistochemistry (IHC). RESULTS: A total of 11 787 proteotypic peptides from 2579 SwissProt proteins are quantified with high confidence. The coefficient of variation (CV) of peptide yield using PCT is 32.9%, and the R2 of peptide quantification is 0.9729. Five hundred forty-one proteins showed significant abundance change between the tumor area and its adjacent benign area. From 24 upregulated pathways and 13 suppressed ones, enhanced biomolecule synthesis and suppressed small molecular metabolism in liver tumor tissues are observed. Protein changes based on α-fetoprotein expression and hepatitis B virus infection are further analyzed. The data altogether highlight 16 promising tumor marker candidates. The upregulation of minichromosome maintenance complex component 7 (MCM7) is further observed in multiple HCC tumor tissues by IHC. CONCLUSIONS AND CLINICAL RELEVANCE: The practicality of rapid proteomic analysis of biopsy-level fresh-frozen HCC tissue samples with PCT-SWATH has been demonstrated and promising tumor marker candidates including MCM7 are identified.

Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils.

Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference.

Comparison of microRNA expression profiles in HCC-derived microvesicles and the parental cells and evaluation of their roles in HCC.

To determine whether the microRNAs (miRNAs) contained in cancer-derived microvesicles (MVs) mirror those of the parental tumor cells, we compared the miRNA expression profiles of MVs derived from their parental hepatocellular carcinoma (HCC) cells. The presence and levels of 888 miRNAs from SMMC-7721 cells and MVs were detected by Agilent miRNA microarray analysis. Four selected miRNAs were verified by real time qRT-PCR. Furthermore, the genes of the miRNAs were bioinformatically identified to explore potential roles of the miRNAs in HCC microenvironment. Our results showed that miRNAs expression profiles of MVs derived from HCC were significantly changed. Of all the miRNAs tested, 148 miRNAs were co-expressed in MVs and SMMC-7721 cells, only 121 and 15 miRNAs were detected in MVs and SMMC-7721 cells, respectively. Among the 148 co-expressing miRNAs, 48 miRNAs had the similar expression level and 6 of them were supposed to be oncogenic or suppressive miRNAs. According to the target prediction by Quantile Algorithm method, these miRNAs may regulate 3831 genes which were closely related to cell cycle, apoptosis and oncogenesis, and 78 were known tumor suppressors or oncogenes. Gene ontology (GO) analysis indicated that 3831 genes were mainly associated with nucleic acid binding, cell death, cell adhesion. MVs containing miRNAs, released into the HCC microenvironment, bear the characteristic miRNAs of the original cells and might participate in cancer progression.

Infusions of recipient-derived cytokine-induced killer cells of donor origin eradicated residual disease in a relapsed leukemia patient after allo-hematopoietic stem cell transplantation.

A female patient diagnosed with acute myelocytic leukemia M5a (AML-M5a) relapsed 986 days after her allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from an unrelated male donor with matched human leukocyte antigen (HLA). Three re-induction chemotherapies were administered, and partial remission was achieved. The patient was given repetitive infusion of cytokine-induced killer (CIK) cells expanded from recipient peripheral mononuclear cells of full donor chimerism due to loss of contact of quondam donor for donor lymphocyte infusion (DLI) and rejection of second transplantation. The patient achieved complete cytogenetical remission. This strategy might overcome the obstacle of donor unavailability and present an appealing new therapeutic alternative to donor-recruited adoptive immunotherapy for relapsed disease at post-transplantation.

[Influence of BCR-ABL inhibitor STI571 on SARI expression in K562 cells].

In order to investigate the molecular mechanisms of SARI expression regulation in chronic myeloid leukemia (CML), 46 patients with CML and 40 healthy volunteers were recruited in this study. SARI expression in the peripheral blood mononuclear cells (PBMNC) of CML patients and healthy volunteers was assayed by using real-time quantitative PCR. K562 cells were in vitro incubated with the BCR-ABL inhibitor STI571 (imatinib) at 37°C and 5% CO2 for 24 hours, then SARI expression was detected by using real-time quantitative PCR. All experiments were repeated three times. The results showed that as compared with healthy volunteers, the expression of SARI mRNA in PBMNC of CML patients presented a lower level (p < 0.001). After exposure of K562 cells to STI571 (2.5 µmol/L) for 24 hours, the SARI expression was higher than that in K562 cells treated without STI571 (p < 0.001). It is concluded that the suppression of SARI expression is involved in CML pathogenesis, and BCR-ABL mediates the down-regulation of SARI mRNA expression in K562 cells. These findings suggest a new orientation for gene therapy in CML patients.

[Influence of PP2A activator on proliferation of HL-60 cells and analysis of PP2A activity changes in patients with acute myeloid leukemia].

In order to investigate the effect of PP2A activator and PP2A inhibitor on proliferation of HL-60 cells and analyze the changes of PP2A activity in patients with acute myeloid leukemia (AML), HL-60 cells were treated with FTY720 alone or in combination with okadaic acid (OA) for 24 hours in culture. Cell proliferation was assayed with CCK8 kit. In addition, 20 AML patients including de novo AML and relapsed AML were enrolled in this study. The activity of PP2A in the peripheral blood mononuclear cells of patients was assayed with a PP2A Immunoprecipitation Phosphatase Assay Kit, the data were analyzed by software SPSS 16.0. The results indicated that as compared with control group, the proliferation of cells in FTY720 group was obviously inhibited (p < 0.05). The proliferation of cells in FTY720 + OA group was slightly inhibited as compared with the control group, there was no statistical difference (p > 0.05), but there was significant difference between the FTY720 + OA and FTY720 groups (p < 0.05). The activity of PP2A in AML patients (453.67 ± 102.52 pmol phosphate) was obviously lower than that in the normal controls (673.29 ± 96.32 pmol phosphate), there was significant difference between them (p < 0.01). It is concluded that the activation or inhibition of PP2A can affect the proliferation of HL-60 cells in vitro. Compared with healthy individuals, the activity of PP2A in AML patients is obviously lower. PP2A protein playing a key role in the occurrence and development of AML may be valuable for the diagnosis and treatment of AML.

[Mechanism of cinnamic aldehyde-inducing apoptosis of chronic myeloid leukemic cells in vitro].

The aim of this study was to investigate the apoptosis-inducing effect of cinnamic aldehyde (CA) on chronic myeloid leukemic (CML) cells and its mechanism. K562 cells and primary bone marrow mononuclear cells (MNC) from patients with CML were treated by various concentrations of CA. Flow cytometry was employed to measure the apoptosis of K562 cells and primary CML bone marrow MNC. Western blot was used to determine the expression of C-MYC and the phosphorylation of CrkL in K562 cells, and real-time polymerase chain reaction (real-time PCR) was used to quantify the expression of BCR-ABL mRNA in K562 cells. The results indicated that CA induced the apoptosis of K562 cells in a time- and dose-dependent manner. CA induced apoptosis of CML MNC dose-dependently. CA inhibited the expression of BCR-ABL mRNA and C-MYC, reduced CrkL phosphorylation levels in K562 cells. It is concluded that CA induces apoptosis of CML cells in vitro. Down-regulation of the expression and function of BCR-ABL may be one of its most important anti-leukemia mechanisms.

[Dyskeratosis congenital: clinical features and genotype analysis in two Chinese patients].

OBJECTIVE: To analysis the clinic and genotype in two Chinese patients with Dyskeratosis congenita (DC). METHODS: The two patients were characterized by mucocutaneous abnormalities (abnormal nails, lacey reticular pigmentation, and oral leukoplakia), bone marrow failure. They were diagnosed with DC. DC genes were amplified by polymerase chain reaction (PCR), including DKC1, TERT, TERC, TINF2, NOP10, NHP2, then DNA sequencing was performed for abnormal exons. RESULTS: An abnormal peak was found in exon 6 of TINF2 gene of the two patients. DNA sequencing showed a 845G→A transition in TINF2 gene in the two patients. CONCLUSION: We should think about DC if the young patients with mucocutaneous abnormalities and marrow failure. TINF2 c.845G→A(R282H) does exist in the two patients. It is reported in China for the first time.

Cytotoxic effect of trans-cinnamaldehyde on human leukemia K562 cells.

AIM: To investigate the effects of trans-cinnamaldehyde (TCA) on the human leukemia K562 cell line and the cytotoxicity of cytokine-induced killer (CIK) cells against K562 cells. METHODS: Apoptosis, Fas expression, and mitochondrial transmembrane potential in K652 cells were analyzed using flow cytometry. K562 cells were labeled with CFSE. The cytotoxic effect of expanded CIK cells on CFSE-labeled K562 cells was determined by FACS flow cytometry. RESULTS: Treatment with TCA 180 micromol/L for 9 h induced apoptosis in 8.9%+/-1.23% of K562 cells. Treatment with 120 or 180 micromol/L TCA for 24 h significantly increased the apoptotic cells to 18.63%+/-1.42 % and 38.98%+/-2.74%, respectively. TCA significantly upregulates Fas expression and decreases mitochondrial transmembrane potential in K562 cells. TCA treatment at 120 and 180 micromol/L for 9 h enhanced the percentage of lysis of K562 cells by expanded CIK cells from 34.84%+/-2.13% to 48.21%+/-2.22 % and 64.81%+/-3.22% at the E:F ratio of 25:1 and from 49.26%+/-3.22% to 57.81%+/-5.13% and 73.36%+/-5.98% at E:F ratio of 50:1. CONCLUSION: TCA exerts cytotoxic effects on human leukemia K562 cells by inducing apoptosis and synergizing the cytotoxicity of CIK cells against K562 cells. These properties of TCA are beneficial to the treatment of leukemia, even in the patients who have received hematopoietic stem cells transplantation (HSCT).

Comprehensive flow cytometry phenotype in acute leukemia at diagnosis and at relapse.

Multiparameter flow cytometry (MFC) plays a vital role in the detection of minimal residual disease (MRD) and diagnosis of relapse in acute leukemia. However, application of a limited panel of antibodies in MFC leads to high rates of false-negative and false-positive results. Thirteen patients with acute lymphoblastic leukemia (ALL) and 12 patients with acute myeloid leukemia (AML) were immunophenotyped by MFC at diagnosis and at relapse using a comprehensive panel of monoclonal antibodies (McAbs) to 27 antigens and CD45/SSC gating. In 23 of 25 patients (92.3%), changes in at least one of progenitor-associated, myeloid and lymphoid antigens between diagnosis and relapse were observed. Antigen changes were observed in 92 of 239 antigens (38.5%) expressed in 25 patients, in 49 of 117 antigens (41.9%) expressed in 13 ALL patients, and in 43 of 122 antigens (35.2%) expressed in 12 AML patients. Phenotypic changes were characterized by the expression of cross-lineage antigens. The intralineage change was observed in the majority of patients. However, myeloid lineage shift was identified by MFC in two patients with T-ALL. Multiple panels of three or more McAbs are likely to be required in the monitoring of MRD and diagnosis of relapse in acute leukemia by MFC.

[Methylation status of JunB and CDH13 gene promoter in CD34(+)CD38(-) chronic myelogenous leukemia cells].

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. The expressions of JunB and CDH13 (cadherin-13) gene as tumor suppressor gene were inactivated by promoter methylation in CML patients. This study was purposed to investigate the methylation difference of JunB and CDH13 gene promoter and the expression levels of JunB and CDH13 gene in CD34(+)CD38(-) cells in CML patients vs normal individuals. CD34(+)CD38(-) cells from 8 cases of CML and 5 normal individuals were selected by flow cytometry. The methylation status of JunB and CDH13 genes were detected by MS-PCR in selected CD34(+)CD38(-) cells. The expression levels of JunB and CDH13 gene was detected with real time polymerase chain reaction (RT-PCR). The results showed that no methylation of JunB and CDH13 gene was detected in CD34(+)CD38(-) cells of 5 normal individuals. Methylations of JunB and CDH13 promoter were found in 87.5% (7/8) and 50% (4/8) CML CD34(+)CD38(-) cells, percentages of which were significantly higher than those in normal individuals. The difference was statistically significant (p < 0.05). The relative expression levels of JunB and CDH13 mRNA in CD34(+)CD38(-) cells of CML patients were significantly lower than those in normal individuals (2(-DeltaDeltaCT) were 1/5.21 and 1/10.63 respectively). It is concluded that the high methylation of JunB and CDH13 gene promoter occurs in CD34(+)CD38(-) cells of CML patients, their mRNA expression level is significantly lower, thus the methylation of JunB and CDH13 gene promoter probably plays a role in the pathogenesis of CML and may have clinical significance in predicting prognosis of CML.

Cross-lineage expression in 505 patients with acute lymphoblastic leukemia by multiparametric flow cytometry analysis.

The expression of immunological markers of one hematopoietic lineage on the abnormal cells of another lineage (cross-lineage expression) is a known feature of leukemia. The present study was aimed to investigate the cross-lineage expression in ALL cells. The cross-lineage expression in ALL cells from 505 patients was detected by flow cytometry using 23 monoclonal antibodies (McAbs) in triple staining combinations. The results showed that in whole ALL, the expression of myeloid antigens occurred in 56.4% of the cases, and CD13 was the most frequently expressed myeloid marker (32.7%) followed by CD33 (29.5%), CD15 (19.2%) and CD11b (7.7%). CD13/CD33 expressions were more frequent in CD34(+) cases than in CD34(-) cases. In B-ALL, T-cell antigen CD4, CD5, CD7 and CD2 were found in 27 (6.3%), 12 (2.8%), 8 (1.9%), and 6 (1.4%) cases respectively, and CD7(+), CD2(+) and CD4(+) cases commonly expressed CD13/CD33. In T-ALL, B-cell antigen cCD79a, CD19 and CD22 were found in 6 (8.1%), 5 (6.8%), and 2 (2.8%) cases respectively, and all of CD19(+) and CD22(+) cases were all accompanied with CD13/CD33. It is concluded that cross-lineage expression in ALL mostly exists in the immature stages, ALL cells more frequently express phenotypes B(+)M(+), T(+)M(+) and occasionally B(+)T(+)M(+), but B(+)T(+)M(-) phenotype is extremely rare.

Bmi1 functions as an oncogene independent of Ink4A/Arf repression in hepatic carcinogenesis.

Bmi1 is a polycomb group proto-oncogene that has been implicated in multiple tumor types. However, its role in hepatocellular carcinoma (HCC) development has not been well studied. In this article, we report that Bmi1 is overexpressed in human HCC samples. When Bmi1 expression is knocked down in human HCC cell lines, it significantly inhibits cell proliferation and perturbs cell cycle regulation. To investigate the role of Bmi1 in promoting liver cancer development in vivo, we stably expressed Bmi1 and/or an activated form of Ras (RasV12) in mouse liver. We found that while Bmi1 or RasV12 alone is not sufficient to promote liver cancer development, coexpression of Bmi1 and RasV12 promotes HCC formation in mice. Tumors induced by Bmi1/RasV12 resemble human HCC by deregulation of genes involved in cell proliferation, apoptosis, and angiogenesis. Intriguingly, we found no evidence that Bmi1 regulates Ink4A/Arf expression in both in vitro and in vivo systems of liver tumor development. In summary, our study shows that Bmi1 can cooperate with other oncogenic signals to promote hepatic carcinogenesis in vivo. Yet Bmi1 functions independent of Ink4A/Arf repression in liver cancer development.

[Correlation between FoxP3(+) regulatory T cells and chronic obstructive pulmonary disease].

OBJECTIVE: To investigate whether FoxP3(+) regulatory T cells (Treg) are present in the acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and the correlation between Treg and COPD. METHODS: Peripheral blood samples were collected from 21 patients of AECOPD, 20 males and 1 female, aged (70 +/- 9) (52-85). Lymphocytes were isolated by three-color labeled three colors monoclonal antibodies flow cytometry to examine the quantities and percentages of CD4(+)CD25(+), CD4(+)CD25(+)FoxP3(+) (CD4(+)Treg), CD8(+)CD25(+), and CD8(+)CD25(+)FoxP3(+) (CD8(+)Treg). ELISA was used to detect the expression of transforming growth factor beta1 (TGF-beta1), interleukin-10 (IL-10), and tumor necrosis factor alpha (TNF-alpha). Middle correlation coefficient r> or = 0.3 was analysed and discussed. RESULTS: The percentages of CD4(+)CD25(+), CD4(+)Treg, CD8(+)CD25(+), and CD8(+)Treg in AECOPD were (18 +/- 6)%, (19 +/- 13)%, (5 +/- 4)%, and (12 +/- 10)% respectively. Linear correlation analysis indicated that the quantity and percentage of Treg were significantly correlated with age, course of disease, smoking index, quantity of white cells, and blood pH, and there were complex causal relations between the immunity of patients and these factors. However, TGF-beta1 and IL-10 showed no correlation with Treg. CONCLUSION: CD4(+)Treg and CD8(+)Treg are expressed in the peripheral blood of AECOPD patients and contribute to the immune suppression of these patients, so they can be used as new markers of immunity of these patients.

[Differential expression profiles of MicroRNA during the development of human cord blood CD34(+)CD38(-) cells to CD34(+)CD38(+) cells].

To establish a basis for deep investigation of the role of microRNA (miRNA) in the regulation of hematopoiesis, differential expression profiles of miRNA between human cord blood CD34(+)CD38(-) and CD34(+)CD38(+) cells were analyzed. Mononuclear cells from cord blood (CB) of healthy donors were separated by Ficoll-Hypaque density gradients. CD34(+)CD38(-) and CD34(+)CD38(+) cells were sorted by using FACS Vantage SE. Their mRNA were then extracted and hybridized to miRNA microarray chip. The resulting data were analyzed with GeneSpring and informatics technique. The results showed that eleven miRNAs were found to be downregulated and 73 miRNAs to be upregulated by at least two-fold in the CD34(+)CD38(+) cells of CB, compared with the CD34(+)CD38(-) cells, which maintained CD34(+)CD38(-) cells' self-renewal and multiple lineage potential, that were defined as "stemness" miRNAs. 12 of the 84 genes (14.29%) were common to 33 hematopoietic-expressed miRNAs expressed by CD34(+) cells from both peripheral blood and bone marrow in Georgantas's study, which included 10 upregulated miRNAs (hsa-miR-23b, -26b, -92, -107, -130a, -181a, -197, -213, -222, -223) and 2 downregulated ones (hsa-miR-16a, -155). Some "stemness" miRNAs undergo CD34 antigen-like expression pattern during development and commmitted differeniation of hematopoietic stem cell/progenitors. Hematopoiesis-associated miRNA clusters and putative target genes could be found with informatics technique. It is concluded that the hematopoietic "stemness" miRNAs play important roles in normal hematopoiesis: miRNA expression profiles of hematopoietic stem cell/progenitors --> their gene expression profiles --> their self-renewal and lineage-commmitted differeniation.

CD4+CD25high regulatory cells in peripheral blood of cancer patients.

AIM: Regulatory T cells (Treg) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. Experimental tumor models in mice revealed that Tregs are potent inhibitors of an antitumor immune response. The purpose of the study was to identify a CD4+ population of regulatory T cells expressing high levels of CD25(CD4+CD25 high) in the peripheral blood of cancer patients and provide the opportunity to determine whether cancer patients exhibit an expanded CD4+CD25high pool. METHODS: The frequency of CD4+CD25high in the peripheral blood of 62 cancer patients and 15 healthy donors was determined by flow cytometry. RESULTS: Compared with healthy donors, cancer patients have an increasing prevalence of CD4+CD25high T cells in the peripheral blood with characteristics of Tregs, i.e. they are CD45-RA(), CD69(-). Among patients, those with higher percentages of CD4+CD25high T cells had a poor prognosis than did those with lower percentages. CONCLUSION: We provide evidence of an increased pool of CD4+CD25high in the peripheral blood of cancer patients, which may be related to immunosuppression and tumor progress in cancer patients. This finding suggests that the use of immunomodulatory therapy to treat cancer patients may be an effective strategy.

[Effect of WISp39 on proliferation, cell cycle and apoptosis of U937 cells].

To investigate the effect of a novel p21-modulating protein WISp39 on proliferation, apoptosis and cell cycle of leukemia cells, the plasmid pLenti6/V5-WISp39 was constructed and transfected into the human myelocytic leukemia cell line-U937 cells. The expression of WISp39 was detected by real-time PCR at 48 hours after transfection, proliferation of U937 cells assayed by CCK-8, apoptosis and cell cycle were determined by flow cytometry. The results showed that plasmid pLenti6/V5-WISp39 could readily enhance the expression of WISp39 in U937 cells. A significant growth inhibition (37.6%) was observed in cells tranfected with pLenti6/V5-WISp39, while the control plasmid pLenti6/V5-lacZ showed little effect on U937 growth. Further analysis revealed that pLenti6/V5-WISp39 did not show obvious apoptosis induction effect, but it could really regulate U937 proliferation via cell cycle modulation. Compared with pLenti6/V5-lacZ, pLenti6/V5-WISp39 resulted in increase of cells in G(0)/G(1) phase by 10% at 48 hours after transfection. It is concluded that the WISp39 gene has no significant apoptosis induction effect on leukemic cells, but it can increase cells at G(0)/G(1) phase via effect on cell cycle, thus inhibiting the U937 proliferation. This result means WISp39 gene can act as a negative modulator on tumour cells.