Implication of integrin α5ß1 in senescence of SK-Mel-147 human melanoma cells.

Downregulation of α5ß1 integrin in the SK-Mel-147 human melanoma culture model sharply inhibits the phenotypic manifestations of tumor progression: cell proliferation and clonal activity. This was accompanied by a 2-3-fold increase in the content of SA-ß-Gal positive cells thus indicating an increase in the cellular senescence phenotype. These changes were accompanied by a significant increase in the activity of p53 and p21 tumor suppressors and components of the PI3K/Akt/mTOR/p70 signaling pathway. Pharmacological inhibition of mTORC1 reduced the content of SA-ß-Gal positive cells in the population of α5ß1-deficient SK-Mel-147 cells. A similar effect was observed with pharmacological and genetic inhibition of the activity of Akt1, one of the three Akt protein kinase isoenzymes; suppression of other Akt isozymes did not affect melanoma cell senescence. The results presented in this work and previously obtained indicate that α5ß1 shares with other integrins of the ß1 family the function of cell protection from senescence. This function is realized via regulation of the PI3K/Akt1/mTOR signaling pathway, in which Akt1 exhibits a non-canonical activity.

[Integrin α3ß1 signaling in regulation of the SK-Mel-147 melanoma cell senescence]. / Signaling integrina α3ß1 v regulirovanii stareniia kletok SK-MEL-147 melanomy cheloveka.

Using a model of the human SK-Mel-147 melanoma cell line, it was shown that blocking the expression of integrin α3ß1 by transduction of cells with α3-specific shRNA did not affect their proliferation, but sharply increased the proportion of SA-ß-Gal-positive cells, a phenotypic feature of cell senescence. These findings were accompanied by a significant increase in the activity of the Akt and mTOR protein kinases and the expression of p53 and p21 oncosupressors. Pharmacological inhibition of mTORC1 reduced the number SA-ß-Gal-positive cells in the SK-Mel-147 cell population depleted of α3ß1. Based on our recent data on a non-canonical function of Akt isomers in the regulation of SK-Mel-147 cell senescence caused by deficiency of α2ß1 receptor, we investigated the role of Akt isomers in senescence induced by the α3ß1 knockdown. It appeared that in the cell population with downregulated α3ß1, inhibition of Akt1 reduced the number SA-ß-Gal positive cells to the level of control cell population, while inhibition of Akt2 had no visible effect. Our results demonstrate that the laminin-specific integrin α3ß1, like the collagen-specific receptor α2ß1, is involved in tumor cell protection from senescence, and senescence induced by α3ß1 depletion, like that caused by α2ß1 deficiency, is based on a signaling mechanism employing a non-canonical function of the Akt1 isoform.

Forecasting the general surgery physician workforce deficit in the state of Georgia: a public health perspective.

OBJECTIVES: The aim of the study was to quantify the current shortage of general surgeons in the state of Georgia and to estimate the shortage in 2040. STUDY DESIGN: This is a population-based longitudinal study. METHODS: Data were collected from the US Census Bureau, the Georgia Board for Physician Workforce, and the Accreditation Council for Graduate Medical Education to evaluate changes in the supply of general surgeons from 2000 to 2017 and estimate an expected shortage through 2040. RESULTS: The state of Georgia experienced a net loss of 120 surgeons from 2000 to 2017 and currently faces a shortage of 139 general surgeons. We project a deficit of between 285 and 725 general surgeons in the state of Georgia by 2040. CONCLUSION: Georgia will face a substantial general surgical workforce shortage by 2040. Enhanced efforts at boosting both the output of surgical training programs as well as recruitment and retention of surgeons may help alleviate this deficit.

Implication of Integrin α2ß1 in Proliferation and Invasion of Human Breast Carcinoma and Melanoma Cells: Noncanonical Function of Akt Protein Kinase.

Blocking the expression of integrin α2ß1, which was accomplished by transduction of α2-specific shRNA, resulted in significant inhibition of proliferation and clonal activity in human MCF-7 breast carcinoma and SK-Mel-147 melanoma cells. Along with these changes, deprivation of α2ß1 caused a sharp decrease in melanoma cell invasion in vitro. Analysis of integrin-mediating signal pathways that control cell behavior revealed a significant increase in activity of Akt protein kinase in response to depletion of α2ß1. The increase in Akt activity that accompanies a suppressive effect on cell invasion contradicts well-known Akt function aimed at stimulation of tumor progression. This contradiction could be explained by the "reversed" (noncanonical) role played by Akt in some cells that consists in suppression rather than promotion of invasive phenotype. To test this suggestion, the effects of Akt inhibitors on invasive activity of SK-Mel-147 cells were investigated. If the above suggestion is true, then inhibition of Akt in cells depleted of α2ß1 should result in the restoration of their invasive activity. It appeared that treatment with LY294002, which inhibits all Akt isoforms (Akt1, Akt2, Akt3), not only failed to restore the invasive phenotype of melanoma cells but further attenuated their invasive activity. However, treatment of the cells with an Akt1-specific inhibitor significantly increased their invasion. Thus, the stimulating effect of α2ß1 integrin on invasion of melanoma cells is realized through a mechanism based on inhibition of one of the Akt isoforms, which in these cells exhibits a noncanonical function consisting in suppression of invasion.

Hyperexpression of Integrin α5ß1 Promotes Resistance of MCF-7 Human Breast Carcinoma Cells to Doxorubicin via ERK Protein Kinase Down-regulation.

In MCF-7 human breast carcinoma cells, α5ß1 integrin hyperexpression, which was accomplished by transduction of a full-length α5 integrin cDNA, increased by about 50-70% the number of cells, survived during 48-72 h cell treatment with doxorubicin. Up-regulation of α5ß1 reduced the level of the apoptogenic p53 protein and p21 cell cycle inhibitor, but enhanced the activity of Akt and mTOR protein kinases. In addition to these findings, we observed a significant decrease in the activity of both isoforms of phosphokinase Erk1/2, which is known to play a key role in cell viability pathways, including pathways alleviating stress damages caused by distinct antitumor drugs. Diminished Erk activity accompanying the rise of drug resistance can be explained by an "atypical" function of this kinase, which, in the cells studied, promotes an enhanced rather than reduced sensitivity to doxorubicin. To verify this suggestion, the effect of a specific Erk inhibitor, PD98059, on the resistance to doxorubicin of control and α5 cDNA-transduced MCF-7 cells was investigated. The data showed that suppression of Erk activity increased the resistance of control cells (transduced with an "empty" vector) to a level higher than that demonstrated by the α5 cDNA-transduced cells. The highest level of resistance was observed in α5ß1-trancduced cells treated with PD98059. Akt and mTOR kinase inhibitors had little if any effect on doxorubicin resistance of α5 cDNA-transduced MCF-7 cells. The data show for the first time that integrin α5ß1 can stimulate drug resistance of tumor cells through a mechanism based on the inhibition of protein kinase Erk. From a more general view, the results of this investigation suggest that signal protein kinases can perform in tumor cells "non-canonical" functions, opposite to those, which are the basis for using kinase inhibitors in targeted cancer therapy. It follows that if a protein kinase is supposed to be used as a target for such therapy, it is important to explore its features in the particular tumor prior to the onset of treatment.

[Protective action of fish muscle extracts against cellular senescence induced by oxidative stress]. / Zashchitnoe deistvie ékstraktov iz myshts ryb ot indutsirovannogo okislitel'nym stressom kletochnogo stareniia.

Muscle extracts of some fish species, i.e. pike (Esox lucius), sterlet (Acipenser ruthenus), pink salmon (Oncorhynchus gorbuscha) and, to a lesser extent, perch (Perca fluviatilis) and Russian sturgeon, (Acipenser gueldenstaedtii) prevent the development of premature senescence of the human embryonic fibroblasts induced by the sublethal concentration of H2O2. Muscle extracts of other fish species tested, i.e. coho salmon (Oncorhynchus kisutch) and zander (Sander lucioperca), have not demonstrated this feature. Cell proliferation increased after the action of the senescence-inhibiting muscle extracts. Possible mechanisms of the action of nature biologically active compounds that interfere with the development of stress-induced cell senescence are discussed.

Microphthalmia-associated transcription factor suppresses invasion by reducing intracellular GTP pools.

Melanoma progression is associated with increased invasion and, often, decreased levels of microphthalmia-associated transcription factor (MITF). Accordingly, downregulation of MITF induces invasion in melanoma cells; however, little is known about the underlying mechanisms. Here, we report for the first time that depletion of MITF results in elevation of intracellular GTP levels and increased amounts of active (GTP-bound) RAC1, RHO-A and RHO-C. Concomitantly, MITF-depleted cells display larger number of invadopodia and increased invasion. We further demonstrate that the gene for guanosine monophosphate reductase (GMPR) is a direct MITF target, and that the partial repression of GMPR accounts mostly for the above phenotypes in MITF-depleted cells. Reciprocally, transactivation of GMPR is required for MITF-dependent suppression of melanoma cell invasion, tumorigenicity and lung colonization. Moreover, loss of GMPR accompanies downregulation of MITF in vemurafenib-resistant BRAFV600E-melanoma cells and underlies the increased invasion in these cells. Our data uncover novel mechanisms linking MITF-dependent inhibition of invasion to suppression of guanylate metabolism.

[Implication of integrin alpha5beta1 signal pathways in proliferation and apoptosis of MCF-7/Dox human breast carcinoma cells]. / Razlichiia mezhdu integrinom al'fa5beta1i retseptorom EGRF v signal'nykh putiakh, kontroliruiushchikh proliferatsiiu i apoptoz kletokMCF-7/Dox kartsinomy molochnoi zhelezy cheloveka.

In MCF-7/Dox human breast carcinoma cells, down-regulation of integrin alpha5beta1 and inhibition of epidermal growth factor receptor (EGFR) markedly reduced rates of cell proliferation. Mitotic cycle analysis showed that alpha5beta1 down-regulation resulted in cell cycle arrest at the S phase, followed by a significant increase in the population of apoptotic cells (subG1 population). Inhibition of EGFR activity also caused cell cycle arrest at the S-phase but without any increase in the subG1 population. Down-regulation of alpha5beta1 and EGFR inhibition resulted in a significant decrease of cell content of the active (phosphorylated) forms of FAK and Erk protein kinases. The data obtained suggest that alpha5beta1 integrin is implicated in cell growth control via inhibition of apoptotic cell death and through EGFR activation.

Electrical stimulation to optimize cardioprotective exosomes from cardiac stem cells.

Injured or ischemic cardiac tissue has limited intrinsic capacity for regeneration. While stem cell transplantation is a promising approach to stimulating cardiac repair, its success in humans has thus far been limited. Harnessing the therapeutic benefits of stem cells requires a better understanding of their mechanisms of action and methods to optimize their function. Cardiac stem cells (CSC) represent a particularly effective cellular source for cardiac repair, and pre-conditioning CSC with electrical stimulation (EleS) was demonstrated to further enhance their function, although the mechanisms are unknown. Recent studies suggest that transplanted stem cells primarily exert their effects through communicating with endogenous tissues via the release of exosomes containing cardioprotective molecules such as miRNAs, which upon uptake by recipient cells may stimulate survival, proliferation, and angiogenesis. Exosomes are also effective therapeutic agents in isolation and may provide a feasible alternative to stem cell transplantation. We hypothesize that EleS enhances CSC-mediated cardiac repair through its beneficial effects on production of cardioprotective exosomes. Moreover, we hypothesize that the beneficial effects of biventricular pacing in patients with heart failure may in part result from EleS-induced preconditioning of endogenous CSC to promote cardiac repair. With future research, our hypothesis may provide applications to optimize stem cell therapy and augment current pacing protocols, which may significantly advance the treatment of patients with heart disease.

Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity.

Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF(V600E) or NRAS(Q61R) human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.

Implication of α2ß1 integrin in anoikis of MCF-7 human breast carcinoma cells.

Silencing of α2ß1 integrin expression significantly promoted anchorage-dependent apoptosis (anoikis) and drastically reduced clonal activity of MCF-7 human breast carcinoma cells. Depletion of α2ß1 enhanced the production of apoptotic protein p53 and of inhibitor of cyclin-dependent protein kinases, p27, while downregulating antiapoptotic protein Bcl-2 and multifunctional protein cMyc. Blocking the expression of α2ß1 had no effect on activity of protein kinase Akt, but it sharply increased the kinase activity of Erk1/2. Pharmacological inhibition of Erk1/2 had a minor effect on anoikis of control cells, while it reduced anoikis of cells with downregulated α2ß1 to the level of control cells. The data show for the first time that integrin α2ß1 is implicated in the protection of tumor cells from anoikis through a mechanism based on the inhibition of protein kinase Erk.

[Integrins as a potential target for targeted anticancer therapy].

The review briefly summarizes information of structure of integrins and their involvement in the development and malignant progression of tumors. Special attention is paid to approaches based on modification of functional properties of integrins that prevent/antagonize tumor growth and progression; these approaches developed in modem experimental biology have certain perspective in clinical application.

[Implication of integrin alpha5beta1 in human breast carcinoma apoptosis and drug resistance].

Doxorubicin-resistant MCF-7Dox line, which is a derivative of the drug-sensitive MCF-7 human breast carcinoma line, differs from the latter by a strongly reduced expression of the alpha2beta1 integrin and a highly increased expression of the alpha5beta1 receptor. Silencing of this integrin in the MCF-7Dox cells by transfection with alpha5-specific siRNA markedly stimulated anoikis and increased sensitivity of the cells to doxorubicin. Alpha5beta1 silencing also leads to significant inhibition of the activity of kinases Akt and Erk2 in MCF-7Dox cells. Our results suggest that integrins alpha5beta1-induced signals, controlling distinct aspects of cell behavior, are conducted through the common signal pathways.

Implication of alpha5beta1 integrin in invasion of drug-resistant MCF-7/ADR breast carcinoma cells: a role for MMP-2 collagenase.

Expression of alpha5beta1 integrin in the drug-resistant MCF-7/ADR breast carcinoma cells was inhibited by treatment of these cells with alpha5-specific siRNA. The decrease of alpha5beta1 expression resulted in a sharp decrease of expression of MMP-2 collagenase and inhibition of invasion activity of these cells in vitro. Similar decrease of invasion was also observed during inhibition of MMP-2 expression by treatment of these cells with MMP-2-specific siRNA. Inhibition of alpha5beta1 expression was also accompanied by significant decrease in cell content of active (phosphorylated) forms of signal protein kinases Akt and Erk1/2. Inhibition of activity of these kinases by treatment of cells with PI-3K/Akt-specific inhibitor LY294002 or Erk-specific inhibitor PD98059 resulted in inhibition of MMP-2 expression and the decrease of invasion in vitro. These data suggest that alpha5beta1 controls invasion ability of these cells by regulating expression of MMP-2, which involves PI-3K and Erk1/2 protein kinase signaling.

C-MYC overexpression is required for continuous suppression of oncogene-induced senescence in melanoma cells.

Malignant melanomas often harbor activating mutations in BRAF (V600E) or, less frequently, in NRAS (Q61R). Intriguingly, the same mutations have been detected at higher incidences in benign nevi, which are largely composed of senescent melanocytes. Overexpression of BRAF(V600E) or NRAS(Q61R) in human melanocytes in vitro has been shown to induce senescence, although via different mechanisms. How oncogene-induced senescence is overcome during melanoma progression remains unclear. Here, we report that in the majority of analysed BRAF(V600E)- or NRAS(Q61R)-expressing melanoma cells, C-MYC depletion induced different yet overlapping sets of senescence phenotypes that are characteristic of normal melanocytes undergoing senescence due to overexpression of BRAF(V600E) or NRAS(Q61R), respectively. These senescence phenotypes were p16(INK4A)- or p53-independent, however, several of them were suppressed by genetic or pharmacological inhibition of BRAF(V600E) or phosphoinositide 3-kinase pathways, including rapamycin-mediated inhibition of mTOR-raptor in NRAS(Q61R)-expressing melanoma cells. Reciprocally, overexpression of C-MYC in normal melanocytes suppressed BRAF(V600E)-induced senescence more efficiently than NRAS(Q61R)-induced senescence, which agrees with the generally higher rates of activating mutations in BRAF than NRAS gene in human cutaneous melanomas. Our data suggest that one of the major functions of C-MYC overexpression in melanoma progression is to continuous suppress BRAF(V600E)- or NRAS(Q61R)-dependent senescence programs.

Alcohol trait and transcriptional genomic analysis of C57BL/6 substrains.

C57BL/6 inbred mice have been widely used as research models; however, widespread demand has led to the creation of several B6 substrains with markedly different phenotypes. In this study, we report that two substrains of C57BL/6 mice, C57BL/6J (B6J) and C57BL/6NCrl (B6C), separated over 50 years ago at two different breeding facilities differ significantly in alcohol consumption and alcohol preference. The genomes of these two substrains are estimated to differ by only 1-2% of all gene loci, providing a unique opportunity to extract particular expression signatures between these substrains that are associated with quantifiable behavioral differences. Expression profiling of the cortex and striatum, hippocampus, cerebellum and the ventral brain region from alcohol-naïve B6C and B6J mice showed intervals on three chromosomes that are enriched in clusters of coregulated transcripts significantly divergent between the substrains. Additional analysis identified two genomic regions containing putative copy number differences between the substrains. One such region on chromosome 14 contained an estimated 3n copy number in the B6J genome compared with B6C. Within this interval, a gene of unknown function, D14Ertd449e, was found to be both associated with alcohol preference and vary in copy number across several inbred strain lineages. H2afz, Psen1, Wdfy1 and Clu were also identified as candidate genes that may be involved in influencing alcohol consumption.

The role of beta1 integrin subfamily in anchorage-dependent apoptosis of breast carcinoma cells differing in multidrug resistance.

Integrin expression was investigated in MCF-7 human breast adenocarcinoma line and in the MCF-7Dox line, which was selected from MCF-7 by a resistance to multiple antitumor drugs (MDR). We have shown that acquisition of MDR was accompanied by a drastically reduced expression of some integrins of the beta1-subfamily (alpha2beta1, alpha3beta1, alpha6beta1) and of alpha vbeta5 intergin in the adenocarcinoma cells. In contrast, expression of alpha5beta1 integrin was markedly increased in the MDR cells. Along with multiple antitumor drug resistance, MCF-7Dox cells demonstrate elevated resistance to anchorage-dependent apoptosis (anoikis) and enhanced in vitro invasive activity. To elucidate the implication of beta1-integrins in the above phenotypic modifications, the effect of beta1-integrin signaling was assayed. Stimulation of beta1-mediated signaling was accomplished by treating of the cells with antibodies to the beta1-subunit common for members of the beta1-subfamily. These data show that activation of beta1-integrin signaling markedly upregulated anoikis of the adenocarcinoma cells.

Expression and role of integrins in invasive activity of oncotransformed fibroblasts differing in spontaneous metastasizing.

Four closely related lines of RSV-transformed Syrian hamster fibroblasts differing drastically in their spontaneous metastatic capacity were investigated for the surface expression of integrins, in vitro invasion, and production of MMP-2 collagenase. The highly metastasizing HET-SR-2SC-LNM cells differ from the lowly metastasizing parental HET-SR cells in a high level of the surface expression of the collagen-specific alpha1beta1, alpha2beta1, and alphavbeta3 integrins, a high invasive activity, and an increased production of MMP-2. The same properties are characteristic for the actively metastasizing cells of the independent HET-SR-1 line. The lowly metastasizing fibroblasts that are derived from HET-SR-2SC-LNM retain a high level of the expression of the alpha1beta1 and alpha2beta1 integrins, but, unlike the parental line, they exhibit a decreased expression of the alphavbeta3 integrin, invasion in Matrigel, and MMP-2 production. Substrate stimulation of the signal function of the collagen-specific integrins increases the production of MMP-2 by the metastatically active fibroblasts. Inhibition of the signal activity of the integrins by RGD-containing pentapeptide or by genistein reduces markedly in vitro invasion in Matrigel and MMP-2 production. The role of specific properties of the extracellular matrix surrounding tumor cells and of specific surface integrins expressed in these cells in developing of the malignant phenotype is discussed.

[Antioxidants in malignant neoplasms in humans and animals]. / Antioksidanty v zlokachestvennykh opukholiakh cheloveka i zhivotnogo.

This review is devoted to the role of low molecular weight antioxidants and antioxidant enzymes in cancerogenesis. The data on accumulation of lipid-soluble and water-soluble antioxidants in a number of malignant human and experimental tumors especially in the renal cell carcinoma tissues are analyzed. A possibility is discussed concerning the pro- and anticarcinogenic effects of the abundant tumor content of antioxidants. Implication of antioxidants in tumor resistance to chemotherapeutic drugs and ionizing radiation is discussed.

Role of integrin alphavbeta3 in substrate-dependent apoptosis of human intestinal carcinoma cells.

Incubation of human intestinal carcinoma Caco-2 cells in suspension (i.e., in the absence of substrate contacts) leads to massive cell death by apoptosis. Since this type of apoptosis has been referred to as anoikis, we designated these cells as anoikis-positive. However, a minor proportion of Caco-2 cells, designated as anoikis-negative, survived in suspension. Extended incubation of the cells in suspension resulted in the reduction of the number of viable cells. In comparison to the original Caco-2 cell population, the anoikis-negative cells demonstrated markedly decreased levels of expression of integrin alphavbeta3 on the cell surface and of transcription of the alphav subunit gene. Activation of the signaling function of alphavbeta3 in the original Caco-2 cells led to substantial stimulation of anoikis, while the inhibition of expression of this receptor resulted in better resistance of the cells to anoikis. The data provide the first evidence that alphavbeta3 integrin can generate apoptosis-stimulating signals.