Infrared Spectroscopic Studies of Cells and Tissues: Triple Helix Proteins as a Potential Biomarker for Tumors

In this work, the infrared (IR) spectra of living neural cells in suspension, native brain tissue, and native brain tumor tissue were investigated. Methods were developed to overcome the strong IR signal of liquid water so that the signal from the cellular biochemicals could be seen. Measurements could be performed during surgeries, within minutes after resection. Comparison between normal tissue, different cell lineages in suspension, and tumors allowed preliminary assignments of IR bands to be made. The most dramatic difference between tissues and cells was found to be in weaker IR absorbances usually assigned to the triple helix of collagens. Triple helix domains are common in larger structural proteins, and are typically found in the extracellular matrix (ECM) of tissues. An algorithm to correct offsets and calculate the band heights and positions of these bands was developed, so the variance between identical measurements could be assessed. The initial results indicate the triple helix signal is surprisingly consistent between different individuals, and is altered in tumor tissues. Taken together, these preliminary investigations indicate this triple helix signal may be a reliable biomarker for a tumor-like microenvironment. Thus, this signal has potential to aid in the intra-operational delineation of brain tumor borders. © 2013 Stelling et al

Recombinant Lysyl Oxidase Propeptide Protein Inhibits Growth and Promotes Apoptosis of Pre-Existing Murine Breast Cancer Xenografts

Lysyl oxidase propeptide (LOX-PP) ectopic overexpression inhibits the growth of cancer xenografts. Here the ability and mode of action of purified recombinant LOX-PP (rLOX-PP) protein to inhibit the growth of pre-existing xenografts was determined. Experimental approaches employed were direct intratumoral injection (i.t.) of rLOX-PP protein into murine breast cancer NF639 xenografts, and application of a slow release formulation of rLOX-PP implanted adjacent to tumors in NCR nu/nu mice (n = 10). Tumors were monitored for growth, and after sacrifice were subjected to immunohistochemical and Western blot analyses for several markers of proliferation, apoptosis, and for rLOX-PP itself. Direct i.t. injection of rLOX-PP significantly reduced tumor volume on days 20, 22 and 25 and tumor weight at harvest on day 25 by 30% compared to control. Implantation of beads preloaded with 35 micrograms rLOX-PP (n = 10) in vivo reduced tumor volume and weight at sacrifice when compared to empty beads (p<0.05). A 30% reduction of tumor volume on days 22 and 25 (p<0.05) and final tumor weight on day 25 (p<0.05) were observed with a reduced tumor growth rate of 60% after implantation. rLOX-PP significantly reduced the expression of proliferation markers and Erk1/2 MAP kinase activation, while prominent increases in apoptosis markers were observed. rLOX-PP was detected by immunohistochemistry in harvested rLOX-PP tumors, but not in controls. Data provide pre-clinical findings that support proof of principle for the therapeutic anti-cancer potential of rLOX-PP protein formulations

Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

B lymphocyte-induced maturation protein 1 (Blimp1) is a master regulator of B cell differentiation, and controls migration of primordial germ cells. Recently we observed aberrant Blimp1 expression in breast cancer cells resulting from an NF-κB RelB to Ras signaling pathway. In order to address the question of whether the unexpected expression of Blimp1 is seen in other epithelial-derived tumors, we selected lung cancers as they are frequently driven by Ras signaling. Blimp1 was detected in all five lung cancer cell lines examined and shown to promote lung cancer cell migration and invasion. Interrogation of microarray datasets demonstrated elevated BLIMP1 RNA expression in lung adenocarcinoma, pancreatic ductal carcinomas, head and neck tumors as well as in glioblastomas. Involvement of Ras and its downstream kinase c-Raf was confirmed using mutant and siRNA strategies. We next addressed the issue of mechanism of Blimp1 activation in lung cancer. Using knockdown and ectopic expression, the role of the Activator Protein (AP)-1 family of transcription factors was demonstrated. Further, chromatin immunoprecipitation assays confirmed binding to identified AP-1 elements in the BLIMP1 promoter of ectopically expressed c-Jun and of endogenous AP-1 subunits following serum stimulation. The propeptide domain of lysyl oxidase (LOX-PP) was identified as a tumor suppressor, with ability to reduce Ras signaling in lung cancer cells. LOX-PP reduced expression of Blimp1 by binding to c-Raf and inhibiting activation of AP-1, thereby attenuating the migratory phenotype of lung cancer cells. Thus, Blimp1 is a mediator of Ras/Raf/AP-1 signaling that promotes cell migration, and is repressed by LOX-PP in lung cancer

Single-cell microRNA sequencing method comparison and application to cell lines and circulating lung tumor cells

Molecular single cell analyses provide insights into physiological and pathological processes. Here, in a stepwise approach, we first evaluate 19 protocols for single cell small RNA sequencing on MCF7 cells spiked with 1pg of 1,006 miRNAs. Second, we analyze MCF7 single cell equivalents of the eight best protocols. Third, we sequence single cells from eight different cell lines and 67 circulating tumor cells (CTCs) from seven SCLC patients. Altogether, we analyze 244 different samples. We observe high reproducibility within protocols and reads covered a broad spectrum of RNAs. For the 67 CTCs, we detect a median of 68 miRNAs, with 10 miRNAs being expressed in 90% of tested cells. Enrichment analysis suggested the lung as the most likely organ of origin and enrichment of cancer-related categories. Even the identification of non-annotated candidate miRNAs was feasible, underlining the potential of single cell small RNA sequencing. Technologies for small non-coding RNA sequencing at the single-cell level are less mature than for sequencing mRNAs. Here the authors evaluate available protocols for analysis of circulating lung cancer tumour cells

Lipocalin-2 expressed in innate immune cells is an endogenous inhibitor of inflammation in murine nephrotoxic serum nephritis.

Lipocalin-2 (Lcn-2) is involved in divergent processes such as acute kidney injury or bacterial host defence. Our study was designed to evaluate the functional role of Lcn-2 in nephrotoxic serum nephritis (NTS). Since Lcn-2 is expressed in tubular epithelial cells as well as in cells of innate immunity such as macrophages and polymorphonuclear neutrophils (PMN), we induced NTS in wild-type (WT), Lcn-2 knock-out (KO) mice and WT/Lcn-2 KO chimeras. Mice lacking Lcn-2 exhibited more glomerular damage with increased proteinuria and interstitial leukocyte accumulation compared to WT mice. Chimeras able to express Lcn-2 in macrophages and PMN but not in epithelial cells were found to develop NTS comparable to wild-type controls. In contrast, chimeras expressing Lcn-2 in tubular epithelial cells with no expression in innate immune cells developed increased NTS due to decreased concerted apoptosis but increased necrosis and formation of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB-1) in the kidney. In vivo blockade of HMGB-1, a toll-like receptor (TLR)-2 agonist, significantly reduced inflammation and NTS in Lcn-2 knock-out mice. In parallel, TLR-2 signalling was found to drive Lcn-2 transcription in vitro. Taken together, Lcn-2 expressed in innate immune cells is protective in NTS by inducing concerted apoptosis and inhibiting the formation of HMGB-1 thereby limiting cytokine production via TLR-2 signalling. In parallel, TLR-2 dependent transcription of Lcn-2 is an endogenous inhibitor of inflammation in NTS

Identification of CIN85 and CD2AP as LOX-PP interacting proteins in breast cancer cells.

<p>(<b>A</b>) Extracts from ZR-75 cells transfected with vectors expressing GST or LOX-PP-GST were precipitated with Glutathione-Sepharose 4B beads, resolved by SDS-PAGE and silver stained. The band(s) at ∼85 kDa was analyzed by LC-MS/MS mass spectrometry and identified as CIN85 and CD2AP. *, non-specific proteins. The positions of the co-precipitated CD2AP/CIN85 and LOX-PP proteins are indicated by the solid and large hatched arrows, respectively, and of GST by the dashed line. (<b>B–C</b>) GST or LOX-PP-GST (PP-GST) was co-expressed with GFP-CIN85 WT (B) or FLAG-CD2AP (C) in HEK293T cells, and LOX-PP associated proteins isolated by GST-pull down assays and subjected to WB for GFP (B) or FLAG (C) and GST. Input, 4% of lysates (4%). (<b>D</b>) Recombinant LOX-PP-myc-His (0.5 µM) was subjected to a GST-pull down assay using 0.5 µM of either GST or GST (G)-CIN85, and WB for the His or CIN85 (Calbiochem) antibody. Input, 5%. (<b>E</b>) Samples of whole cell extracts (10 µg) of the indicated human and mouse cells were subjected to WB for CIN85 (Upstate). (<b>F</b>) (Left) TX-100 extracts of Hs578T (Upper) or ZR-75 (Lower) cells were immunoprecipitated with mouse IgG or CIN85 (Upstate) antibody, and analyzed for CIN85 (Upstate) and LOX-PP. (Right) TX-100 extracts of Hs578T (upper) or ZR-75 (lower) cells were immunoprecipitated with rabbit IgG or LOX-PP antibodies, and subjected to WB.</p

Structural model of CIN85 SH3-B domain in complex with the LOX-PP peptide. <i>Upper part</i>:

<p>Front and rear views of the modeled SH3-B (pale green) and LOX-PP (yellow sticks) complex are depicted; selected, key electrostatic interactions are represented by blue dashed lines. The open arrow (upper right) indicates the approximate position of Arg(-2) in structures that can assemble the ternary complex: SH3–[RxP¹xxxxR/K peptide]–SH3. <i>Inset box</i>: Detail showing the structural dissimilarity in the n-Src loop between CIN85 SH3-B (our model) and SH3-A, superimposed (PDB code: 2BZ8; brown cartoon). In the SH3-A structure, the bound Cbl-b peptide backbone (light brown) is also seen to diverge more towards its C-terminus from the LOX-PP peptide position. <b><i>Lower part</i></b><b>:</b> Comparison of the electrostatic potential surfaces of SH3 domains A and B in equivalent orientations, illustrating their differential charge distributions. The Adaptive Poisson-Boltzmann Solver (APBS) software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077288#pone.0077288-Baker1" target="_blank">[55]</a> was used to generate the electrostatic potential map, contoured in varying colour intensity from -15 (red) through 0 (white) to +15 (blue) kT/e, and rendered within PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>).</p