Vascular-homing peptides for targeted drug delivery and molecular imaging: Meeting the clinical challenges

The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.     

Limits of application of initiated chemiluminescence in monitoring of oncological process of mucous membrane of mouth and larynx

Investigation into the limits of application of chemiluminescence (CL) methods in oncology still attracts the attention of researchers. In the present work we analyze the screening and monitoring of oncological processes (OP) in the mucous membrane of the mouth and larynx by initiated CL (ICL). Chemiluminescence has already been used by stomatologists to define the start of OP, but methods that reflect the metabolic changes in organism under cancer diagnostics still have not found their place. This work presents results of ICL on blood serum (BS) of patients with oncological diseases at different stages of medical treatment compared with those of healthy people. We found an essential metabolic difference only in types of OP that are characterized by two maxima on chemiluminograms. These OP represent only 12.81% of groups of patients with oncological diseases. The possibility to apply ICL methods to monitor operation quality and control medical treatment at different stages when the two ICL maxima are present is established. At present, the chemiluminograms with the two maxima are mostly informative, but this does not exclude the quantitative analysis of other ICL kinetic methods and is encouraging for their investigation. Any OP introduces changes in organism function and these should be reflected in the ICL. Copyright © 2016 John Wiley & Sons, Ltd.     

Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-α-positive and -negative breast cancer cells

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-α (ER-α)-positive MCF-7 cells and ER-α-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-α transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3α, 17β-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin αvβ3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin αvβ3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-α-positive and ER-α-negative breast cancer cells, but by discrete mechanisms.     

Helicobacter pylori Infection and Family History of Gastric Cancer Decrease Expression of FHIT Tumor Suppressor Gene in Gastric Mucosa of Dyspeptic Patients

Background:  The expression of a fragile histidine triad (FHIT) protein is lost in stomach tumors. The study aimed at determining whether FHIT expression is affected by Helicobacter pylori infection, strain virulence ( vacA and cagA genes) and histopathological changes in the gastric mucosa of patients with functional dyspepsia having first-degree relatives with gastric cancer.
Materials and Methods:  Eighty-eight never-smoking patients with functional dyspepsia were selected for the study, and 48 of them had first-degree relatives with gastric cancer. Bacterial DNA amplification was used to identify H. pylori colonization. The level of FHIT gene expression was determined by qRT-PCR (mRNA) and Western blot (FHIT protein) analyses.
Results:  For patients having first-degree relatives with gastric cancer FHIT expression was lower (mRNA by ca. 40–45% and protein by 30%) compared with the control patients ( p  < .05). H. pylori infection decreased the FHIT mRNA level by 10–35% and the protein level by 10–20%. Bacterial strain vacA (+) cagA (+) lowered FHIT mRNA by ca. 30–35% in the antrum samples of both groups and in corpus samples of patients with first-degree relatives with gastric cancer ( p  < .05). The FHIT mRNA level was twice as high in control H. pylori- negative patients with intestinal metaplasia, compared with those with non-atrophic gastritis.
Conclusions:  The decreased FHIT gene expression associated with hereditary factors and with H. pylori infection, especially with vacA (+) cagA (+)-positive strains, may be related to gastric carcinoma development.     

Purification,characterization and kinetics of thiol protease inhibitor from goat (<Emphasis Type="Italic">Capra hircus</Emphasis>) lung

In the present study, two molecular forms of goat lung cystatin (GLC), I and II, were purified to homogeneity by a two-step procedure including ammonium sulfate precipitation (40–60%) and ion exchange chromatography. The inhibitor forms migrated as single bands under native and SDS-PAGE with and without reducing agent giving molecular mass of 66.4 and 76.4 kDa, respectively. GLC-I possesses 0.07% and GLC-II 2.3% carbohydrate content and no -SH groups. GLC-I showed greater affinity for papain than for ficin and bromelain. Immunological studies showed that the inhibitor was pure and there was cross reactivity between anti-GLC-I serum and goat brain cystatin. Both inhibitor forms were stable in the pH range of 3–10 and up to 75°C. GLC-I was found to possess 49% α-helical structure by CD spectroscopy. The inhibitor-papain complexes showed conformational changes as invoked by UV and fluorescence spectroscopic studies. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 963–971.     

The importin-alpha/nucleophosmin switch controls taspase1 protease function

Taspase1 is a threonine protease suspected to process (patho)biologically relevant nuclear and cytoplasmic substrates, such as the mixed lineage leukemia protein. However, neither the mechanisms regulating Taspase1's intracellular localization nor their functional consequences are known. Analysis of endogenous and ectopically expressed Taspase1 detected the protease predominantly in the nucleus accumulating at the nucleolus. Microinjection and ectopic expression studies identified an evolutionarily conserved bipartite nuclear import signal (NLS) (amino acids (197) KRNKRKLELA ERVDTDFMQLKKRR(220) ) interacting with importin-α. Notably, an NLS-mutated, import-deficient Taspase1 was biologically inactive. Although the NLS conferred nuclear transport already of the proenzyme, Taspase1's nucleolar localization required its autoproteolytic processing, triggering its interaction with the nucleolar shuttle protein nucleophosmin. In contrast, (auto)catalytically inactive Taspase1 mutants neither accumulated at the nucleolus nor bound nucleophosmin. Active nuclear import and interaction with nucleophosmin was found to be required for the formation of proteolytically active Taspase1 ensuring to efficiently process its nuclear targets. Intriguingly, coexpression of pathological nucleophosmin variants increased the amount of cytoplasmic Taspase1. Hence, Taspase1 appears to exploit the nuclear export activity of nucleophosmin to gain transient access to the cytoplasm required to also cleave its cytoplasmic substrates. Collectively, we here describe a hitherto unknown mechanism regulating the biological activity of this protease.     

Heterogenous induction of carcinoma-associated fibroblast-like differentiation in normal human prostatic fibroblasts by co-culturing with prostate cancer cells

In the tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are considered to play a critical role in the promotion of tumorigenesis. However, the mechanisms that generate CAFs are not well elucidated. To understand how CAFs are generated during primary cancer progression, we investigated the biochemical characteristics of normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer (PCa) cells in vitro. In primary cultures of human PCa-derived stromal cells (PCaSC-8 and PCaSC-9), expression of TNC, ACTA2, EGF, FGF7, and IGF1 mRNA was generally higher than PrSC but gene expression patterns were not uniform between PCaSC-8 and PCaSC-9 cells. Transforming growth factor β (TGFβ) and vascular endothelial growth factor (VEGF) protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than PrSC but levels of both secreted proteins were not same. When PrSCs were co-cultured with androgen-sensitive LNCaP cells or its sublines, androgen-low-sensitive E9 cells and androgen-insensitive AIDL cells, mRNA expression of IGF1 was significantly increased in all combinations. In contrast, expression of COL1A1, TNC, and ACTA2 mRNA was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Protein production of VEGF was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Increase of TGFβ protein was observed only in E9 + PrSC co-cultures. These biochemical characteristics of PrSC were partially recapitulated in TGFβ-treated PrSC. We have demonstrated that normal fibroblasts co-cultured with cancer cells become activated and exhibit biochemical characteristics of CAFs in a heterogenous manner. Our results suggest that heterogenous induction of CAF-like differentiation might be strongly dependent on biochemical characteristics of adjacent cancer cells.