Protein Signatures for Distinguishing Colorectal Cancer Liver Metastases from Primary Liver Cancer Using Tissue Slide Proteomics.

BACKGROUND: Colorectal cancer liver metastasis (CRLM) and hepatocellular carcinoma (HCC) are both high incidence tumors in China. In certain poorly differentiated cases they can exhibit comparable imaging and pathological characteristics, which impedes accurate clinical diagnosis. The use of protein-based techniques with tissue slides offers a more precise means to assess pathological changes and has the potential to assist with tumor diagnosis. METHODS: A simple in situ protein digestion protocol was established for protein fingerprint analysis of paraffin-embedded tissue slide samples. Additionally, machine learning techniques were employed to construct predictive models for CRLM and HCC. The accuracy of these models was validated using tissue slides and a clinical database. RESULTS: Analysis of differential protein expression between CRLM and HCC groups reliably identified 977 proteins. Among these, 53 were highly abundant in CRLM samples and 57 were highly abundant in HCC samples. A prediction model based on the expression of six proteins (CD9, GSTA1, KRT20, COL1A2, AKR1C3, and HIST2H2BD) had an area under curve (AUC) of 0.9667. This was further refined to three proteins (CD9, ALDH1A1, and GSTA1) with an AUC of 0.9333. CONCLUSIONS: Tissue slide proteomics can facilitate accurate differentiation between CRLM and HCC. This methodology holds great promise for improving clinical tumor diagnosis and for identifying novel markers for challenging pathological specimens.

DRR1 promotes glioblastoma cell invasion and epithelial-mesenchymal transition via regulating AKT activation.

Metastatic invasion is the primary cause of treatment failure for GBM. EMT is one of the most important events in the invasion of GBM; therefore, understanding the molecular mechanisms of EMT is crucial for the treatment of GBM. In this study, high expression of DRR1 was identified to correlate with a shorter median overall and relapse-free survival. Loss-of-function assays using shDRR1 weakened the invasive potential of the GBM cell lines through regulation of EMT-markers. The expressions of p-AKT were significantly decreased after DRR-depletion in SHG44 and U373 cells. Moreover, the invasion was inhibited by the AKT inhibitor, MK-2206. The expression of Vimentin, N-cadherin, MMP-7, snail and slug was significantly inhibited by MK-2206, while the expression of E-cadherin was upregulated. Our results provide the first evidence that DRR1 is involved in GBM invasion and progression possibly through the induction of EMT activation by phosphorylation of AKT.

Application of acellular intima from porcine thoracic aorta in full-thickness skin wound healing in a rat model.

The tunica intima of aorta is made up of one layer of smooth endothelium and basement membrane. The basement membrane is rich in extracellular matrix (ECM) molecules, including collagen, glycosaminoglycans (e.g., heparan sulfate), proteoglycans (e.g., perlecan), and glycoproteins. All or most of these components are involved in wound healing process. In this work, we determined whether the acellular intima from porcine thoracic aorta can be a new kind of xenograft to repair the skin-wound surface in a rat model. Acellular intima xenografts (AIX) were prepared from tunica intima, and then the swelling ratio, moisture content ratio, water retention ratio, degradation rate, and water vapor transmission rate of the materials were measured. Prothrombin time test was applied to assess its hemostatic property in vitro, in vitro cell experiment was used to test its cellular biocompatibility, and animal experiment was used to evaluate its effect on wound healing. Results showed that AIX, with some reasonable treatments, has good hemostatic property, cell biocompatibility, and histocompatibility. AIX can also promote angiogenesis in the healing process and thus accelerate comprehensive healing, thereby confirming supporting its functionality and excellent application potential in wound healing.

Prokineticin 2 Plays a Pivotal Role in Psoriasis.

Psoriasis is histologically characterized by keratinocytes (KC) hyperproliferation, inflammation, and increased angiogenesis, but the pathological factor responsible for these symptoms is unknown. Here, a neuroendocrine peptide (prokineticin 2, PK2), is highly expressed in human and mouse psoriatic skins but no significant change in other autoimmune diseases, suggesting that PK2 is a psoriasis-specific factor. Bacterial products significantly up-regulated PK2, implying that infection induces PK2 over-expression. PK2 promoted KC and macrophage to produce interleukin-1 (IL-1), the central player of inflammation and psoriasis, which acts on adjacent fibroblast to induce inflammatory cascades and KC hyperproliferation. IL-1 feeds back on macrophages to induce PK2 production to perpetuate PK2-IL-1 positive feedback loop. PK2 also promoted angiogenesis, another psoriatic symptom. In mouse models, PK2 over-expression aggravated psoriasis while its knock-down inhibited pathological development. The results indicate that PK2 over-production perpetuates psoriatic symptoms by creating PK-2-IL-1 vicious loop. PK2 is a central player in psoriasis and a promising psoriasis-specific target.

Adam17, a Target of Mir-326, Promotes Emt-Induced Cells Invasion in Lung Adenocarcinoma.

BACKGROUND/AIMS: A disintegrin and metalloprotease (ADAM) 17 has been reported to be implicated in cancer cells invasion. Nevertheless, its potential role in lung adenocarcinoma has not been addressed clearly. METHODS: RT-PCR and Western blot were used to detect the expression of miR-326 and ADAM17 in lung adenocarcinoma samples (n=73). miR-326 mimics and inhibitor were tansfected in human A549 and SPCA1 cell lines. The transwell assay was used to detect the cell invasive ability. The regulation mechanism was evaluated by luciferase reporter assay. The markers of (epithelial-to-mesenchymal transition) EMT were detected by using Western blot assay. RESULTS: We found increased expression of ADAM17 in lung adenocarcinoma and cell lines. In vitro, up-regulation of ADAM17 promoted cells invasion, while silencing of ADAM17 inhibited cells invasion. Meanwhile, ADAM17 could affect the markers of EMT. Furthermore, we confirmed that ADAM17 is a target of miR-326, which is involved in EMT and cells invasion. CONCLUSIONS: These findings revealed that ADAM17, a target of miR-326, promoted EMT-induced cells invasion in lung adenocarcinoma.

B7-H6 expression in non-small cell lung cancers.

B7 family has been known to be a negative regulator of immunity response in patients with lung cancer. B7-H6 as a novel identified member of B7 family is found to trigger natural killer (NK) cell cytotoxicity and cytokine secretion by binding natural cytotoxicity receptor NKp30. Up until now, no investigations have been made about B7-H6 expression in lung cancer. We present the result of the B7-H6 prognostic value in 65 non-small cell lung cancer (NSCLC) tissues and 65 matched adjacent non-tumor tissues by Immunohistochemistry (IHC). Meanwhile, fluorescence activated cell sorter (FACS) analysis was used to detect B7-H6 receptor NKp30 expression in 7 non-small cell lung cancer tissues and 7 adjacent non-tumor tissues. Here, the result showed B7-H6 immunoreactivity in 6/65 (9.23%) lung cancer patients and 4/65 (6.15%) in adjacent non-tumor tissues. No relationship was found between B7-H6 expression and clinic pathological features. Similarly, no relevance was found for NKp30 expression in lung cancer tissues and non-tumor tissues. However, B7-H6 positive carcinomas were significantly correlated with degree of differentiation (P = 0.044). Three year survival rate after operation did not show the prognostic value for B7-H6 expression. Our study suggests that B7-H6 has a limited value as a prognostic marker in the patients of lung cancer.

Bacterial surface display of endoglin by antigen 43 induces antitumor effectiveness via bypassing immunotolerance and inhibition of angiogenesis.

Various angiogenesis-related self-molecules have been considered to be therapeutic targets. However, the direct use of self-molecules as vaccines is not recommended because of the inherent ability of the host to develop immune tolerance. Antigen 43 (Ag43) is a surface protein found in E. coli and contains an α and a ß subunits, which contains multiple T epitopes in α subunit. Here we construct a novel Ag43 surface display system (Ag43 system) to express Ag43 chimeric proteins to disrupt immune tolerance against self-molecules. The Ag43 system was constructed from an Escherichia coli strain Tan109, derived from JM109, in which the Ag43 gene was deleted and a recombinant plasmid (pETAg43') expressing a partial Ag43 gene was introduced. The extracellular domain of angiogenesis-related endoglin gene was then subcloned into plasmid pETAg43', resulting in a recombinant plasmid pETAg43'/END(e) which was then used to transform Tan109 for protein expression. We found that Ag43 and endoglin chimeric protein (Ag43'/END(e) ) was expressed on the bacterial surface. The chimeric protein could be separated from the bacterial surface by heating to 60°C and yet retain activity. We used Ag43'/END(e) as a protein vaccine and found that it could disrupt immune tolerance against endoglin by inducing significant antitumor activities and inhibit angiogenesis in several tumor models without significant side effects. These data suggest that Ag43'/END(e) chimeric protein is a potential model vaccine for active tumor immunotherapy, and that Ag43 system could be an effective tool for novel vaccine preparation to break immune tolerance to other angiogenesis-related self-molecules for cancer therapy.

Zoledronic acid inhibits growth of hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND: Growing preclinical evidence shows that zoledronic acid (ZOL) exhibits direct antitumor activity in various cancer cell lines. However, the cytotoxic effects of ZOL on human hepatocellular carcinoma (HCC) cells have not been established. In the present study, we investigated the effect of ZOL on HCC both in vitro and in vivo. METHODS: Cytotoxicity and cell cycles were assessed with Sulforhodamine B colorimetric assay and flow cytometry. Expression levels of cell cycle phase-linked proteins were examined. The effect of ZOL on HCC in vivo was explored based on H22-subcutaneous injection (s.c.) and H22-intraperitoneal injection (i.p.) mice model. RESULTS: ZOL inhibited the growth of SK-HEP-1 and H22 cells and induced S-phase arrest through downregulating cdc2 protein and upregulating cyclin A. It inhibited the growth of s.c tumors, and increased the survival of both H22-s.c. and H22-i.p. mice in vivo. CONCLUSION: ZOL inhibits growth of HCC cells in vitro and in vivo.

Induction of tumor inhibition and apoptosis by a candidate tumor suppressor gene DRR1 on 3p21.1.

Down-regulated in renal cell carcinoma gene (DRR1) is one of the candidate tumor suppressor genes (TSGs) on human 3p21.1. This study was performed to validate the expression status of DRR1 gene in cancer cells and the expression pattern of the protein in clinical specimens of human lung cancer and to examine its potential as a molecular target for treatment of lung cancer in vivo. DRR1 expression was analyzed in 7 human lung cancer cell lines. DRR1 protein expression was also examined in clinical non-small cell lung cancer (NSCLC) specimens. Furthermore, effects of DRR1 re-expression on A549 cells in vitro and A549 xenograft tumors in nude mice were evaluated. Loss of DRR1 mRNA expression was detected in 6 of the 7 human cancer cell lines, the exception was the renal cancer cell line OS-RC-2. DRR1 protein expression was absent in 15 of 20 (75%) human NSCLC specimens by immunostaining. Transfection of DRR1 gene into DRR1-negative-expressing A549 cells resulted in significant cell growth suppression and apoptosis. Plasmids containing DRR1 cDNA complexed with DOTAP:Chol liposomes were administered intravenously via tail vein to nude mice bearing A549 xenograft tumors resulting in tumor growth inhibition and elevation of apoptosis compared with the controls. DRR1 is a potent growth suppressor of NSCLC, acting through apoptosis pathway in vivo and it may be a potential therapeutic gene for human lung cancer.

Anti-tumor effects of a human VEGFR-2-based DNA vaccine in mouse models.

BACKGROUND: Vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2 (Flk-1/KDR), play a key role in tumor angiogenesis. Blocking the VEGF-VEGFR-2 pathway may inhibit tumor growth. Here, we used human VEGFR-2 as a model antigen to explore the feasibility of immunotherapy with a plasmid DNA vaccine based on a xenogeneic homologue of this receptor. METHODS: The protective effects and therapeutic anti-tumor immunity mediated by the DNA vaccine were investigated in mouse models. Anti-angiogenesis effects were detected by immunohistochemical staining and the alginate-encapsulate tumor cell assay. The mechanism of action of the DNA vaccine was primarily explored by detection of auto-antibodies and CTL activity. RESULTS: The DNA vaccine elicited a strong, protective and therapeutic anti-tumor immunity through an anti-angiogenesis mechanism in mouse models, mediated by the stimulation of an antigen-specific response against mFlk-1. CONCLUSION: Our study shows that a DNA vaccine based on a xenogeneic homologue plasmid DNA induced autoimmunity against VEGFR-2, resulting in inhibition of tumor growth. Such vaccines may be clinically relevant for cancer immunotherapy.

Combination of recombinant xenogeneic endoglin DNA and protein vaccination enhances anti-tumor effects.

The immunization approaches with DNA vaccine priming and subsequent protein or peptide boosting has been widely tested in various models of infectious diseases. However, these approaches are seldom reported in the areas of cancer immunotherapy. In this study we combined endoglin plasmid DNA and recombinant protein as vaccines and used them to prime and boost, simultaneously, as a vaccine strategy. Our results showed that combination of endoglin DNA and protein vaccines could enhance both protective and therapeutic anti-tumor efficacy in both colon carcinoma and Lewis lung carcinoma models. Significant inhibition of tumor angiogenesis was found in the tumor tissues. The titers of autoantibodies against murine endoglin were significantly increased and the antibody levels lasted longer in the mice with combined endoglin DNA and recombinant protein vaccination. CTL response against endoglin-positive HUVECs, but not against endoglin-negative tumor cells was found in the mice combined DNA with protein vaccination. In addition, combination of endoglin DNA and recombinant protein vaccination significantly induced IFN-gamma secreting cells. These observations suggested that a combination of endoglin DNA and recombinant protein immunization as a vaccine strategy was superior to those using endoglin DNA or recombinant protein alone as vaccines.