TLR5 agonist entolimod reduces the adverse toxicity of TNF while preserving its antitumor effects.

Tumor necrosis factor alpha (TNF) is capable of inducing regression of solid tumors. However, TNF released in response to Toll-like receptor 4 (TLR4) activation by bacterial lipopolysaccharide (LPS) is the key mediator of cytokine storm and septic shock that can cause severe tissue damage limiting anticancer applications of this cytokine. In our previous studies, we demonstrated that activation of another Toll-like receptor, TLR5, could protect from tissue damage caused by a variety of stresses including radiation, chemotherapy, Fas-activating antibody and ischemia-reperfusion. In this study, we tested whether entolimod could counteract TNF-induced toxicity in mouse models. We found that entolimod pretreatment effectively protects livers and lungs from LPS- and TNF-induced toxicity and prevents mortality caused by combining either of these agents with the sensitizer, D-galactosamine. While LPS and TNF induced significant activation of apoptotic caspase 3/7, lipid tissue peroxidation and serum ALT accumulation in mice without entolimod treatment, these indicators of toxicity were reduced by entolimod pretreatment to the levels of untreated control mice. Entolimod was effective when injected 0.5-48 hours prior to, but not when injected simultaneously or after LPS or TNF. Using chimeric mice with hematopoiesis differing in its TLR5 status from the rest of tissues, we showed that this protective activity was dependent on TLR5 expression by non-hematopoietic cells. Gene expression analysis identified multiple genes upregulated by entolimod in the liver and cultured hepatocytes as possible mediators of its protective activity. Entolimod did not interfere with the antitumor activity of TNF in mouse hepatocellular and colorectal tumor models. These results support further development of TLR5 agonists to increase tissue resistance to cytotoxic cytokines, reduce the risk of septic shock and enable safe systemic application of TNF as an anticancer therapy.

Increased human buccal cell autofluorescence is a candidate biomarker of tobacco smoking.

Human buccal cells display diverse changes that are associated with smoked and smokeless tobacco, and clinicopathologic studies have correlated human buccal cell changes with oral cancer. Reported herein are the results of studies that were undertaken to identify a high-throughput technology that would advance efforts to use human buccal cells. We report that (a) a relatively large (mean +/- SD, 2.1 +/- 1.4 x 10(5) cells) population of human buccal cells can be collected in a noninvasive manner with a toothbrush and purified (>98% human buccal cells; n = 138 samples of the oral mucosa; n = 69 donors); (b) despite their large size (diameter, approximately 65 microm), the human buccal cells were analyzed successfully with a single laser cytometer (FACScan) and an advanced multispectral cytometer (FACSAria) having three lasers (excitation = 488, 633, and 407 nm wavelengths) and nine distinct emission channels; (c) cytometry revealed that the buccal cells expressed a high level of autofluorescence that was displayed over a broad spectrum (450-780 nm wavelength); (d) autofluorescence of human buccal cells collected from the left and right cheek was consistent, illustrating the reproducibility of the sample collection and assay procedure; (e) human buccal cell autofluorescence differed significantly among 69 adult subjects; and (f) a statistical difference (P = 0.018) between current, former, and never smokers. Summarily, this report is thought to be the first to show the application of flow cytometry for assaying human buccal cells and identifies buccal cell autofluorescence as a candidate biomarker of tobacco smoking.

Level of FACT defines the transcriptional landscape and aggressive phenotype of breast cancer cells.

Although breast cancer (BrCa) may be detected at an early stage, there is a shortage of markers that predict tumor aggressiveness and a lack of targeted therapies. Histone chaperone FACT, expressed in a limited number of normal cells, is overexpressed in different types of cancer, including BrCa. Recently, we found that FACT expression in BrCa correlates with markers of aggressive BrCa, which prompted us to explore the consequences of FACT inhibition in BrCa cells with varying levels of FACT.FACT inhibition using a small molecule or shRNA caused reduced growth and viability of all BrCa cells tested. Phenotypic changes were more severe in "high- FACT" cells (death or growth arrest) than in "low-FACT" cells (decreased proliferation). Though inhibition had no effect on the rate of general transcription, expression of individual genes was changed in a cell-specific manner. Initially distinct transcriptional profiles of BrCa cells became similar upon equalizing FACT expression. In "high-FACT" cells, FACT supports expression of genes involved in the regulation of cell cycle, DNA replication, maintenance of an undifferentiated cell state and regulated by the activity of several proto-oncogenes. In "low-FACT" cells, the presence of FACT reduces expression of genes encoding enzymes of steroid metabolism that are characteristic of differentiated mammary epithelia.Thus, we propose that FACT is both a marker and a target of aggressive BrCa cells, whose inhibition results in the death of BrCa or convertion of them to a less aggressive subtype.

Prognostic value of histone chaperone FACT subunits expression in breast cancer.

Understanding the underlying reasons for tumor aggressiveness, such as why some tumors grow slowly and locally, while others rapidly progress to a lethal metastatic disease, is still limited. This is especially critical in breast cancer (BrCa) due to its high prevalence and also due to the possibility that it can be detected early. Several oncogenes and tumor suppressors have been identified and are used in the prognosis and treatment of BrCa. However, even with these markers, the outcome within BrCa subtypes is highly variable. Chromatin organization has long been acknowledged as a factor that plays an important role in tumor progression, but molecular mechanisms defining chromatin dynamics are largely missing. We have recently found that histone chaperone FACT (facilitates chromatin transcription) is overexpressed in ~18-20% of BrCa cases. FACT is elevated upon transformation of mammary epithelial cells and is essential for viability of tumor cells. BrCa cells with high FACT have a more aggressive transcriptional program than those with low FACT cells. Based on this we propose that FACT may be a marker of aggressive BrCa. In this study, we aimed to comprehensively characterize the pattern of FACT expression in BrCa in relation to other molecular and clinical prognostic markers. We developed and tested an assay for the detection and quantitation of protein levels of both FACT subunits, SSRP1, and SPT16, in clinical samples. We compared the value of mRNA and protein as potential markers of disease aggressiveness using a large cohort of patients (n=1092). We demonstrated that only SSRP1 immunohistochemical staining is a reliable indicator of FACT levels in tumor samples. High SSRP1 correlated with known markers of poor prognosis, such as negative hormone receptor status, presence of Her2, high-grade tumors, and tumors of later clinical stage. At the same time, no strong correlation between SSRP1 expression and survival was detected when all samples were analyzed together. Clear trend toward longer survival of patients with low or no SSRP1 expression in tumor samples was seen in several subgroups of patients, and most importantly significant association of high SSRP1 expression with shorter disease-free survival was detected in patients with early-stage and low-grade BrCa, the category of patients with the highest demand in predictive marker of disease progression.

Smoking and smokeless tobacco-associated human buccal cell mutations and their association with oral cancer--a review.

Reported herein are the results of a structured literature review that was undertaken to (a) determine if human buccal (mouth) cell changes are associated with smoking and smokeless ("chewing") tobacco, (b) tabulate different buccal cell alterations that have been reported, (c) delineate buccal cell assays that have been used successfully, (d) determine whether buccal cell changes correlate with oral cancer as defined in clinicopathologic investigations, and (e) assess the feasibility of developing a high-throughput buccal cell assay for screening smokers for the early detection of oral cancer. The results of the studies reported herein have established that diverse buccal cell changes are associated with smoking and smokeless tobacco. This review documents also that buccal cells have been collected in a noninvasive manner, and repetitively for serial studies, from different sites of the mouth (e.g., cheek, gum, and tongue) and from normal tissue, preneoplastic lesions (leukoplakia), and malignant tumors. Tobacco-associated genetic mutations and nongenetic changes have been reported; a partial listing includes (a) micronuclei, (b) bacterial adherence, (c) genetic mutations, (d) DNA polymorphisms, (d) carcinogen-DNA adducts, and (e) chromosomal abnormalities. Clinical studies have correlated buccal cell changes with malignant tumors, and some oral oncologists have reported that the buccal cell changes are practical biomarkers. Summarily, the literature has established that buccal cells are useful not only for characterizing the molecular mechanisms underlying tobacco-associated oral cancers but also as exfoliative cells that express diverse changes that offer promise as candidate biomarkers for the early detection of oral cancer.

Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry.

Numerous highly fluorescent macrophages (MPhi), designated "smoker cells," exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MPhi, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MPhi is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., "tar"). Studies reported herein were undertaken to test the hypothesis that the origin of the MPhi fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MPhi isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MPhi from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MPhi is due to tobacco tar.

Weekly administration of rapamycin improves survival and biomarkers in obese male mice on high-fat diet.

Recent discoveries have revealed the key role of mTOR (target of rapamycin) in aging. Furthermore, rapamycin extends lifespan in mice, especially in female mice. Here, we treated obese male mice on high-fat diet with rapamycin given intermittently: either weekly (once a week) or alternating bi-weekly (three injections every other week). While only marginally reducing obesity, intermittent administration of rapamycin significantly extended lifespan. Significance was achieved for weekly treated group and for the three rapamycin-received groups combined. In weekly treatment group, 100% mice were alive by the age of 2 years, whereas 60% of mice died in untreated group by this age. The effect of weekly treatment on survival was highly significant and cannot be fully explained by partial reduction in obesity. Alternating bi-weekly treatments seem to be less effective than weekly treatment, although effects of additional factors (see ) may not be excluded. After one year of treatment, all survived mice were sacrificed 8 days after the last administration of rapamycin to avoid its direct interference with parameters examined. Fasting levels of cardiac and hepatic p-S6, a marker of mTORC1 activity, were lower in weekly treatment group compared with control mice. In contrast, levels of p-Akt (S473), glucose, triglycerides and insulin were unchanged, whereas leptin and IGF-1 tended to be lower. Thus, weekly treatment with rapamycin may slow down aging in obese male mice on high-fat diet.

Comparison of rapamycin schedules in mice on high-fat diet.

At a wide range of doses, rapamycin extends life span in mice. It was shown that intraperitoneal injections (i.p.) of rapamycin prevent weight gain in mice on high-fat diet (HFD). We further investigated the effect of rapamycin on weight gain in female C57BL/6 mice on HFD started at the age of 7.5 months. By the age of 16 and 23 months, mice on HFD weighed significantly more (52 vs 33 g; p = 0.0001 and 70 vs 38 g; p < 0.0001, respectively) than mice on low fat diet (LFD). The i.p. administration of 1.5 mg/kg rapamycin, 3 times a week every other week, completely prevented weight gain, whereas administration of rapamycin by oral gavash did not. Rapamycin given in the drinking water slightly decreased weight gain by the age of 23 months. In addition, metabolic parameters were evaluated at the age of 16 and 23 months, 6 and 13 days after last rapamycin administration, respectively. Plasma leptin levels strongly correlated with body weight, (P < 0.0001, r=0.86), suggesting that the difference in weight was due to fat tissue mass. Levels of insulin, glucose, triglycerides and IGF1 were not statistically different in all groups, indicating that these courses of rapamycin treatment did not impair metabolic parameters at least after rapamycin discontinuation. Despite rapamycin discontinuation, cardiac levels of phospho-S6 and pAKT(S473) were low in the i.p.-treated group. This continuous effect of rapamycin can be explained by prevention of obesity in the i.p. group. We conclude that intermittent i.p. administration of rapamycin prevents weight gain without causing gross metabolic abnormalities. Intermittent gavash administration minimally affected weight gain. Potential clinical applications are discussed.

Fasting levels of hepatic p-S6 are increased in old mice.

TOR is involved in aging in a wide range of species from yeast to mammals. Here we show that, after overnight fasting, mTOR activity is higher in the livers of 28 months old female mice compared with middle-aged mice. Taken together with previous reports, our data predict that the life-extending effect of calorie restriction (CR) may be diminished, if CR is started in very old age. In contrast, rapamycin is known to be effective, even when started late in life.

Dysregulation of the mTOR pathway in p53-deficient mice.

Mammalian or mechanistic target of rapamycin (mTOR) is involved in growth, aging, and age-related diseases including cancer. There is an extensive cross talk between p53 and mTOR. In cell culture, p53 inhibits the mTOR pathway in a cell type-dependent manner. p53-deficient mice develop pro-inflammation and cancer. We have shown that rapamycin delayed cancer and extended lifespan, thus partially substituting for p53. Here we show that a marker of mTOR activity, phosphorylated S6 (p-S6), is increased in the hearts of p53-deficient mice. Furthermore, cardiac p-S6 correlated with body weight. Also, p53(-/-) mice were slightly hyperinsulinemic with a tendency to elevated IGF-1. Radiation exacerbated the difference between IGF-1 levels in normal and p53(-/-) mice. Noteworthy, radiation induced Thr-308 Akt phosphorylation in the livers (but not in the hearts) of both p53(+/+) and p53(-/-) mice. Simultaneously, radiation decreased p-S6 in the livers of normal mice, consistent with the negative effect of p53 on mTOR. Our data indicate that the activity of mTOR is increased in some but not all tissues of p53(-/-) mice, associated with the tendency to increased insulin and IGF-1 levels. Therefore, the absence of p53 may create oncophilic microenvironment, favoring cancer.

Mechanistic or mammalian target of rapamycin (mTOR) may determine robustness in young male mice at the cost of accelerated aging.

Males, who are bigger and stronger than females, live shorter in most species from flies to mammals including humans. Cellular mass growth is driven in part by mTOR (Target of Rapamycin). When developmental growth is completed, then, instead of growth, mTOR drives aging, manifested by increased cellular functions, such as hyper-secretion by fibroblasts, thus altering homeostasis, leading to age-related diseases and death. We hypothesize that MTOR activity is elevated in male mice compared with females. Noteworthy, 6 months old males were 28 % heavier than females. Also levels of phosphorylated S6 (pS6) and phospho-AKT (p-AKT, Ser 473), markers of the mTOR activity, were higher in male organs tested. Levels of pS6 were highly variable among mice and correlated with body weight and p-AKT. With age, the difference between levels of pS6 between sexes tended to minimize, albeit males still had hyperactive mTOR. Unlike fasting, the intraperitoneal (i.p.) administration of rapamycin eliminated pS6 in all organs of all females measured by immunoblotting and immunohistochemistry without affecting p-AKT and blood insulin. Although i.p. rapamycin dramatically decreased levels of pS6 in males too, it was still detectable by immunoblotting upon longer exposure. Our study demonstrated that both tissue p-AKT and pS6 were higher in young male mice and were associated with increased body weight and insulin. These data can explain bigger body size and faster aging in males. Our data suggest higher efficacy of rapamycin compared to fasting. Higher sensitivity of females to rapamycin may explain more pronounced life extension by rapamycin observed in females compared to males in several studies.

Rapamycin extends lifespan and delays tumorigenesis in heterozygous p53+/- mice.

TOR (Target of Rapamycin) pathway accelerates cellular and organismal aging. Similar to rapamycin, p53 can inhibit the mTOR pathway in some mammalian cells. Mice lacking one copy of p53 (p53+/- mice) have an increased cancer incidence and a shorter lifespan. We hypothesize that rapamycin can delay cancer in heterozygous p53+/- mice. Here we show that rapamycin (given in a drinking water) extended the mean lifespan of p53+/- mice by 10% and when treatment started early in life (at the age less than 5 months) by 28%. In addition, rapamycin decreased the incidence of spontaneous tumors. This observation may have applications in management of Li-Fraumeni syndrome patients characterized by heterozygous mutations in the p53 gene.

Targeting FACT complex suppresses mammary tumorigenesis in Her2/neu transgenic mice.

Development of safe and effective tumor-preventive treatments for high-risk patient populations and therapies for early-stage cancer remains a critical need in oncology. We have recently discovered compound with anticancer activity, Curaxin-137, which modulates several important signaling pathways involved in even the very early stages of cancer. In tumor cells, Curaxin-137 inhibits NF-κB- and HSF1-dependent transcription (prosurvival pathways) and activates p53 (a proapoptotic pathway) without inducing DNA damage. These effects result from chromatin trapping and inhibition of activity of the FACT (facilitates chromatin transcription) complex by Curaxin-137. FACT has not been previously implicated in cancer, but we found that its subunits are overexpressed in breast cancer. On the basis of this background, we tested whether Curaxin-137 could suppress tumorigenesis in MMTV-neu transgenic mice, which spontaneously develop mammary carcinoma due to steroid receptor-regulated expression of the Her2 proto-oncogene. We found that chronic administration of Curaxin-137 in a preventive regimen to MMTV-neu mice did not cause any detectable changes in normal organs and tissues, yet inhibited tumor onset, delayed tumor progression, and prolonged survival of mice in a dose-dependent manner. Curaxin-137 induced changes in FACT, altered NF-κB localization, and activated p53 in tumor cells as expected from its defined mechanism of action. These results support further investigation of Curaxin-137 as a potential preventive and/or early-stage therapeutic agent for breast cancer.

Cigarette smoke, bacteria, mold, microbial toxins, and chronic lung inflammation.

Chronic inflammation associated with cigarette smoke fosters malignant transformation and tumor cell proliferation and promotes certain nonneoplastic pulmonary diseases. The question arises as to whether chronic inflammation and/or colonization of the airway can be attributed, at least in part, to tobacco-associated microbes (bacteria, fungi, and spores) and/or microbial toxins (endotoxins and mycotoxins) in tobacco. To address this question, a literature search of documents in various databases was performed. The databases included PubMed, Legacy Tobacco Documents Library, and US Patents. This investigation documents that tobacco companies have identified and quantified bacteria, fungi, and microbial toxins at harvest, throughout fermentation, and during storage. Also characterized was the microbial flora of diverse smoking and smokeless tobacco articles. Evidence-based health concerns expressed in investigations of microbes and microbial toxins in cigarettes, cigarette smoke, and smokeless tobacco products are reasonable; they warrant review by regulatory authorities and, if necessary, additional investigation to address scientific gaps.

Expression of FACT in mammalian tissues suggests its role in maintaining of undifferentiated state of cells.

The Facilitates Chromatin Transcription (FACT) chromatin remodeling complex, comprised of two subunits, SSRP1 and SPT16, is involved in transcription, replication and DNA repair. We recently showed that curaxins, small molecules with anti-cancer activity, target FACT and kill tumor cells in a FACT-dependent manner. We also found that FACT is overexpressed in human and mouse tumors and that tumor cells are sensitive to FACT downregulation. To clarify the clinical potential of FACT inhibition, we were interested in physiological role(s) of FACT in multicellular organisms. We analyzed SSRP1 and SPT16 expression in different cells, tissues and conditions using Immunohistochemical (IHC) staining of mouse and human tissues and analysis of publically available high-content gene expression datasets. Both approaches demonstrated coordinated expression of the two FACT subunits, which was primarily associated with the stage of cellular differentiation. Most cells of adult tissues do not have detectable protein level of FACT. High FACT expression was associated with stem or less-differentiated cells, while low FACT levels were seen in more differentiated cells. Experimental manipulation of cell differentiation and proliferation in vitro, as well as tissue staining for the Ki67 proliferation marker, showed that FACT expression is related more to differentiation than to proliferation. Thus, FACT may be part of a stem cell-like gene expression signature and play a role in maintaining cells in an undifferentiated state, which is consistent with its potential role as an anti-cancer target.

Inflammatory response of lung macrophages and epithelial cells to tobacco smoke: a literature review of ex vivo investigations.

Chronic inflammation contributes to the initiation and progression of tumors and tobacco smoke-associated inflammation is associated with malignant and certain non-neoplastic lung diseases. Reported herein are the results of an interpretative synthesis review of the literature assessing the inflammatory response of lung macrophages (MPhi) and epithelial cells to tobacco smoke as measured ex vivo. Papers were retrieved using Boolean operations from PubMed and Scopus. Many writings reported the results of assays of human MPhi from fresh surgically excised human lung tissue, bronchoalveolar lavage, activated blood monocytes, long-term cell lines and MPhi from different laboratory animals. Some publications reported the findings of comparative studies of lung MPhi freshly isolated from the lungs of smokers and non-smokers. Other papers described the effect of tobacco smoke on lung epithelial cells. Most investigators quantified the response of the target cells to tobacco smoke by measuring the production of pro-inflammatory mediators; these included chemokines, cytokines, reactive oxygen species and enzymes. Investigators have reported conflicting observations of the response of human and animal MPhi and epithelial cells to tobacco smoke. The spectrum included papers describing robust production of various inflammatory mediators, significant reduction of a pro-inflammatory response to a known stimulant and overt cytotoxicity. This literature review documents that there exists no consensus, and no emerging trend line, of the reproducible effect(s) of cigarette smoke. This discrepancy reflects the absence of standardized protocols for collecting, processing and bioassaying the smoke, a highly complex aerosol, and identifies the need for establishing collaborative research schemes.

Review: Is lung inflammation associated with microbes and microbial toxins in cigarette tobacco smoke?

Chronic inflammation that has been observed for malignant and non-neoplastic lung diseases of smokers has been attributed to the numerous and diverse particulate ('tar')-phase and gas-phase chemicals in mainstream smoke, most of which arise from the burning of tobacco. The primary cell-mediator of lung inflammation is the macrophage. Most probably, inflammation is promoted also from some of the more than 50 other cell types of the lung. Cured tobacco in diverse types of cigarettes is known to harbor a plethora of bacteria (Gram-positive and Gram-negative), fungi (mold, yeast), spores, and is rich in endotoxin (lipopolysaccharide). Reviewed herein are recent observations of the authors' team and other investigators that support the hypothesis that lung inflammation of long-term smokers may be attributed in part to tobacco-associated bacterial and fungal components that have been identified in tobacco and tobacco smoke.

Cigarette filter-based assays as proxies for toxicant exposure and smoking behavior--a literature review.

BACKGROUND: Cigarettes are being marketed with filters that differ in composition and design. The filters have different toxicant trapping efficiencies, and smoking stains reflect variations in smoking behavior. Presented herein are the results of a structured literature review that was done to identify cigarette filter-based assays that may serve as proxies for mouth-level exposure and assessing smoking methods. METHODS: A search of the published scientific literature and internal tobacco company documents from 1954 to 2009 was carried out. RESULTS: The literature search identified diverse schemes for assessing cigarette filters, including visual inspection and digital imaging of smoked-stained spent filters, and quantitative determinations for total particulate matter (TPM), nicotine, and solanesol. The results also showed that: (a) there are sufficient data to link filter-based chemical measures to standardized smoking machine-measured yields of tar and nicotine; (b) TPM eluted from filters or in chemical digest of filters can be used to estimate the efficiency of the filter for trapping smoke solids; (c) visual and digital inspection of spent filters is useful in finding indicators of variations in smoking behaviors; and (d) there is a correlation between solanesol and nicotine measured in filters and exposure biomarkers in smokers. CONCLUSIONS: The cigarette filter may prove useful in estimating smoking behaviors such as filter vent blocking and puffing intensity, and may have utility as proxy measures of mouth-level smoke exposure in clinical trials. Additional investigations are needed to compare the different proposed assay schemes and the assay results with measurements of human biomarker assays of smoke exposure.