The association of circulating amylin with β-amyloid in familial Alzheimer's disease.

Introduction: This study assessed the hypothesis that circulating human amylin (amyloid-forming) cross-seeds with amyloid beta (Aβ) in early Alzheimer's disease (AD). Methods: Evidence of amylin-AD pathology interaction was tested in brains of 31 familial AD mutation carriers and 20 cognitively unaffected individuals, in cerebrospinal fluid (CSF) (98 diseased and 117 control samples) and in genetic databases. For functional testing, we genetically manipulated amylin secretion in APP/PS1 and non-APP/PS1 rats. Results: Amylin-Aβ cross-seeding was identified in AD brains. High CSF amylin levels were associated with decreased CSF Aβ42 concentrations. AD risk and amylin gene are not correlated. Suppressed amylin secretion protected APP/PS1 rats against AD-associated effects. In contrast, hypersecretion or intravenous injection of human amylin in APP/PS1 rats exacerbated AD-like pathology through disruption of CSF-brain Aβ exchange and amylin-Aβ cross-seeding. Discussion: These findings strengthened the hypothesis of circulating amylin-AD interaction and suggest that modulation of blood amylin levels may alter Aβ-related pathology/symptoms

Advancements in Oncoproteomics Technologies: Treading toward Translation into Clinical Practice

Proteomics continues to forge significant strides in the discovery of essential biological processes, uncovering valuable information on the identity, global protein abundance, protein modifications, proteoform levels, and signal transduction pathways. Cancer is a complicated and heterogeneous disease, and the onset and progression involve multiple dysregulated proteoforms and their downstream signaling pathways. These are modulated by various factors such as molecular, genetic, tissue, cellular, ethnic/racial, socioeconomic status, environmental, and demographic differences that vary with time. The knowledge of cancer has improved the treatment and clinical management; however, the survival rates have not increased significantly, and cancer remains a major cause of mortality. Oncoproteomics studies help to develop and validate proteomics technologies for routine application in clinical laboratories for (1) diagnostic and prognostic categorization of cancer, (2) real-time monitoring of treatment, (3) assessing drug efficacy and toxicity, (4) therapeutic modulations based on the changes with prognosis and drug resistance, and (5) personalized medication. Investigation of tumor-specific proteomic profiles in conjunction with healthy controls provides crucial information in mechanistic studies on tumorigenesis, metastasis, and drug resistance. This review provides an overview of proteomics technologies that assist the discovery of novel drug targets, biomarkers for early detection, surveillance, prognosis, drug monitoring, and tailoring therapy to the cancer patient. The information gained from such technologies has drastically improved cancer research. We further provide exemplars from recent oncoproteomics applications in the discovery of biomarkers in various cancers, drug discovery, and clinical treatment. Overall, the future of oncoproteomics holds enormous potential for translating technologies from the bench to the bedside

Role of PXR in Hepatic Cancer: Its Influences on Liver Detoxification Capacity and Cancer Progression

<div><p>The role of nuclear receptor PXR in detoxification and clearance of xenobiotics and endobiotics is well-established. However, its projected role in hepatic cancer is rather illusive where its expression is reported altered in different cancers depending on the tissue-type and microenvironment. The expression of PXR, its target genes and their biological or clinical significance have not been examined in hepatic cancer. In the present study, by generating DEN-induced hepatic cancer in mice, we report that the expression of PXR and its target genes CYP3A11 and GSTa2 are down-regulated implying impairment of hepatic detoxification capacity. A higher state of inflammation was observed in liver cancer tissues as evident from upregulation of inflammatory cytokines IL-6 and TNF-α along with NF-κB and STAT3. Our data in mouse model suggested a negative correlation between down-regulation of PXR and its target genes with that of higher expression of inflammatory proteins (like IL-6, TNF-α, NF-κB). In conjunction, our findings with relevant cell culture based assays showed that higher expression of PXR is involved in reduction of tumorigenic potential in hepatic cancer. Overall, the findings suggest that inflammation influences the expression of hepatic proteins important in drug metabolism while higher PXR level reduces tumorigenic potential in hepatic cancer.</p></div

Correlations between the reduced levels of PXR and PXR-regulated CYP3A11 DME with enhanced levels of inflammatory proteins in hepatic cancer.

<p>Relative protein levels of TNF-α, P65, and IL-6 were correlated with PXR and CYP3A11 protein levels using Pearson’s correlation coefficient (r) in scattered plot. Respective p-value (p) represents the significance between the correlations.</p

Levels of inflammatory proteins are enhanced in DEN-induced hepatic cancer.

<p>A. Real-time PCR analysis was performed for <i>Tnf-α</i>, <i>p65</i>, and <i>Stat3</i> with total RNA extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. GAPDH served as an internal control. B. Cell extracts of control and DEN-induced hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse TNF-α, anti-mouse P65, and anti-mouse IL-6 antibody. Bands of human and mouse P65 were detected at 65 KDa and 60 KDa respectively. β-Actin antibody served as control; C. The relative endogenous TNF-α, P65 and IL-6 protein expressions in control and DEN-induced hepatic cancer were quantified by densitometry. The experiments were performed with seven samples of each control and DEN-induced cancerous mice, and the values are represented as the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. P-value <0.05 represented by a single asterisk (*), P-value <0.005 represented by a double asterisk (**), P-value <0.0005 represented by a triple asterisk (***).</p

PXR enhances expression of apoptotic genes while reduces expression of cell-cycle regulatory genes in hepatic cancer cells A.

<p>Real-time PCR analysis was performed for <i>Bcl-xl</i>, <i>Bcl-2</i>, <i>Cdk2</i> and <i>Cdk4</i> with total RNA extracted from HepG2, human HepXR and mouse HepXR cell lines for various cell cycle and apoptosis regulatory genes. β-Actin served as an internal control. B. Higher cellular expression of PXR increased the doubling time in human HepXR and mouse HepXR cells in comparison to HepG2 cells. The experiments were performed three times with three samples of each cell lines and the values are represented as the mean ±SE. The P-value represents the significance in human HepXR and mouse HepXR cell lines as compared to the control HepG2 cell line. P-value <0.05 represented by a single asterisk (*), P-value <0.005 represented by a double asterisk (**), P-value <0.0005 represented by a triple asterisk (***).</p

Altered expression of PXR-regulated genes in DEN-induced hepatic cancer.

<p>A. Total RNA was extracted from mouse liver tissues of saline-treated control and DEN-induced hepatic cancer mice. Real-time PCR analysis was performed for <i>Cyp3A11</i>, <i>Gsta2</i> and <i>Mrp3</i>. For <i>Mdr1</i> semi-quantitative PCR was performed. GAPDH served as an internal control. B. Cell lysates of control and hepatic cancerous mice liver tissues were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-mouse Cyp3a11 antibody (upper panel). β-Actin antibody served as control (lower panel); C. The relative endogenous Cyp3a11 protein expression in control and DEN-induced hepatic cancer was quantified by densitometry. The experiments were performed with six samples of each control and DEN-induced cancerous mice, and the values are represented as the mean ±SE. The P-value represents the significance in DEN-induced hepatic cancer mice as compared to the control mice. The P-value <0.05 represented by a single asterisk (*), P-value <0.005 represented by a double asterisk (**), P-value <0.0005 represented by a triple asterisk (***).</p

Overexpression of FLAG-tagged mPXR gene at protein level and its effect on tissue histology.

<p>A. Liver tissue extracts of Control and transgenic mice were electrophoresed (50 μg per sample) on a 10% SDS-PAGE. The proteins were transferred onto the methanol-activated PVDF membrane and probed with anti-FLAG antibody and anti-mouse PXR antibody (upper panels). β-Actin antibody served as control (lower panel). B. Control and transgenic mice were sacrificed and their livers were removed. Paraffin sections were prepared from control and transgenic mice livers harvested in the experiment and immunodetection were performed with anti-FLAG antibody (indicated by arrows). The immunodetected tissues were viewed and recorded at 20X magnification using a light microscope. The experiments were performed with three samples of each of the controls and the transgenic mice.</p

PXR reduces cell-ECM interactions in hepatic cancer cells.

<p>Total RNA was extracted from HepG2, human HepXR and HepR21 cell lines and used for real-time PCR analysis of A. human HABP1; B. human PXR; and C. human MDR1. β-Actin served as an internal control. The values are represented as the mean ±SE. The P-value represents the significance in human HepXR and HepR21 cell lines as compared to the control HepG2 cell line. P-value <0.05 represented by a single asterisk (*), P-value <0.005 represented by a double asterisk (**), P-value <0.0005 represented by a triple asterisk (***).</p