Significance of NOTCH1 Expression in the Progression of Human Lung and Colorectal Cancers.

Lung and colorectal cancers are the most common types of cancer characterized by a poor prognosis and a high mortality rate. Mutations in the genes encoding components of the main intra- and extracellular signaling pathways, in particular the NOTCH1 gene (Notch1, a member of the Notch family of receptors), play one of the key roles in progression of these malignancies. Notch signaling is involved in maintaining homeostasis of the intestinal epithelium and structural and functional lung elements. Therefore, it is not surprising that the constitutive activity and hyperactivity of Notch signaling due to somatic mutations in genes coding for the products directly involved into its activation, could lead to the progression of these cancer types. The aim of our study was to investigate how the NOTCH1 downregulation via RNA interference (RNAi) affects the phenotype, characteristics, and Notch-dependent signaling of human A549 lung and HCT116 colorectal carcinoma cells. Several small harpin RNAs (shRNAs) were selected using the bioinformatic analysis and tested for their ability to suppress the NOTCH1 expression. The most efficient one was used to produce the A549 and HCT116 cells with NOTCH1 knockdown. The obtained cell lines demonstrated decreased proliferation rates, reduced colony-forming capacity under adhesive conditions, and decreased migration activity in a Boyden chamber. The NOTCH1 knockdown also significantly decreased expression of some Notch signaling target genes potentially involved in the acquisition and maintenance of more invasive and malignant cell phenotype. In vivo experiments in immunodeficient athymic female Balb/c nu/nu mice confirmed the results obtained in vitro: the NOTCH1 inhibition decreased the growth rates of the subcutaneous xenografts formed by A549 and HCT116 tumor cells. Therefore, downregulation of the gene encoding the Notch1 receptor potentially reduces malignant characteristics of human lung and colorectal carcinoma cells.

The Cytoplasmic Actins in the Regulation of Endothelial Cell Function.

The primary function of the endothelial cells (EC) lining the inner surface of all vessels is to regulate permeability of vascular walls and to control exchange between circulating blood and tissue fluids of organs. The EC actin cytoskeleton plays a crucial role in maintaining endothelial barrier function. Actin cytoskeleton reorganization result in EC contraction and provides a structural basis for the increase in vascular permeability, which is typical for many diseases. Actin cytoskeleton in non-muscle cells presented two actin isoforms: non-muscle ß-cytoplasmic and γ-cytoplasmic actins (ß-actins and γ-actins), which are encoded by ACTB and ACTG1 genes, respectively. They are ubiquitously expressed in the different cells in vivo and in vitro and the ß/γ-actin ratio depends on the cell type. Both cytoplasmic actins are essential for cell survival, but they perform various functions in the interphase and cell division and play different roles in neoplastic transformation. In this review, we briefly summarize the research results of recent years and consider the features of the cytoplasmic actins: The spatial organization in close connection with their functional activity in different cell types by focusing on endothelial cells.

Drug Targeting the Actin Cytoskeleton Potentiates the Cytotoxicity of Low Dose Vincristine by Abrogating Actin-Mediated Repair of Spindle Defects.

Antimicrotubule vinca alkaloids are widely used in the clinic but their toxicity is often dose limiting. Strategies that enhance their effectiveness at lower doses are needed. We show that combining vinca alkaloids with compounds that target a specific population of actin filaments containing the cancer-associated tropomyosin Tpm3.1 result in synergy against a broad range of tumor cell types. We discovered that low concentrations of vincristine alone induce supernumerary microtubule asters that form transient multi-polar spindles in early mitosis. Over time these asters can be reconstructed into functional bipolar spindles resulting in cell division and survival. These microtubule asters are organized by the nuclear mitotic apparatus protein (NuMA)-dynein-dynactin complex without involvement of centrosomes. However, anti-Tpm3.1 compounds at nontoxic concentrations inhibit this rescue mechanism resulting in delayed onset of anaphase, formation of multi-polar spindles, and apoptosis during mitosis. These findings indicate that drug targeting actin filaments containing Tpm3.1 potentiates the anticancer activity of low-dose vincristine treatment. IMPLICATIONS: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.

Identification of Cancer-Targeted Tropomyosin Inhibitors and Their Synergy with Microtubule Drugs.

Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2-M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo This combination warrants further study. Mol Cancer Ther; 16(8); 1555-65. ©2017 AACR.

In search of novel highly active mitochondria-targeted antioxidants: thymoquinone and its cationic derivatives.

Since the times of the Bible, an extract of black cumin seeds was used as a medicine to treat many human pathologies. Thymoquinone (2-demethylplastoquinone derivative) was identified as an active antioxidant component of this extract. Recently, it was shown that conjugates of plastoquinone and penetrating cations are potent mitochondria-targeted antioxidants effective in treating a large number of age-related pathologies. This review summarizes new data on the antioxidant and some other properties of membrane-penetrating cationic compounds where 2-demethylplastoquinone substitutes for plastoquinone. It was found that such a substitution significantly increases a window between anti- and prooxidant concentrations of the conjugates. Like the original plastoquinone derivatives, the novel compounds are easily reduced by the respiratory chain, penetrate through model and natural membranes, specifically accumulate in mitochondria in an electrophoretic fashion, and strongly inhibit H2O2-induced apoptosis at pico- and nanomolar concentrations in cell cultures. At present, cationic demethylplastoquinone derivatives appear to be the most promising mitochondria-targeted drugs of the quinone series.

Scavenging of reactive oxygen species in mitochondria induces myofibroblast differentiation.

The goal of this study was to investigate the possible role of reactive oxygen species (ROS) in signaling, in modulation of the cytoskeleton, and in differentiation of fibroblasts. For this purpose, we have applied a novel mitochondria-targeted antioxidant: plastoquinone conjugated with decyltriphenylphosphonium (SkQ1). This antioxidant at nanomolar concentration prevented ROS accumulation and cell death induced by H(2)O(2) in fibroblasts. We found that scavenging of ROS produced by mitochondria activated the Rho/ROCK/LIMK signaling pathway that was followed by phosphorylation of cofilin and stabilization of actin stress fibers. The mitochondria-targeted antioxidant induced differentiation of human subcutaneous fibroblasts to myofibroblasts as revealed by expression of fibronectin isoform (EDA-FN) and smooth muscle actin (α-SMA). This effect was shown to be mediated by transforming growth factor ß1 (TGFß1), which was activated by matrix metalloprotease 9 (MMP9) in the culture medium. Scavenging of ROS stimulated secretion of MMP9 rather than its processing. The same effect was achieved by the nontargeted antioxidant Trolox at higher concentration, but the thiol antioxidant N-acetylcysteine (NAC) inhibited MMP activity and was not able to induce myofibroblast differentiation. The myofibroblast phenotype was supported due to autocrine TGFß1-dependent stimulation after removal of SkQ1. It is concluded that ROS scavenging in mitochondria induces TGFß1-dependent myofibroblast differentiation.