Non-conventional rottlerin anticancer properties.

In the past few years, we focused the interest on rottlerin, an old/new natural substance that, over the time, has revealed a number of cellular and molecular targets, all potentially implicated in the fight against cancer. Past and recent literature well demonstrated that rottlerin is an inhibitor of enzymes, transcription factors and signaling molecules that control cancer cell life and death. Although the rottlerin anticancer activity has been mainly ascribed to apoptosis and/or autophagy induction, recent findings unveiled the existence of additional mechanisms of toxicity. The major novelties highlighted in this mini review are the ability to bind and inhibit key molecules, such as ERK and mTOR, directly, thus independently of upstream signaling cascades, and to cause a profound dysregulation of cap-dependent protein translation through the mTORC1/4EBP1/eIF4E axis and by inhibition of eIF2, an initiation factor of translation that is negatively regulated by endoplasmic reticulum (ER) stress. These last mechanisms, proved to be lethal in cancer cell lines derived from breast and skin, strongly enforce the potential of rottlerin as a promising natural lead compound for the development of novel therapeutic approaches.

Inhibitions of mTORC1 and 4EBP-1 are key events orchestrated by Rottlerin in SK-Mel-28 cell killing.

Earlier studies demonstrated that Rottlerin exerts a time- and dose-dependent antiproliferative effect on SK-Mel-28 melanoma cells during 24 h of treatment, but cytotoxicity due to cell death began only after a 48 h exposure. In the current study, in order to identify the type of cell death in this cell line, which is notoriously refractory to most anticancer therapies, and to clarify the underlying mechanisms of this delayed outcome, we searched for apoptotic, necrotic/necroptotic and autophagic traits in Rottlerin-exposed cells. Although SK-Mel-28 cells are both apoptosis and autophagy competent, Western blotting analysis, caspase activity assay, nuclear imaging and the effects of autophagy, apoptosis and necroptosis inhibitors, indicated that Rottlerin cytotoxicity was due to none of the aforementioned death mechanisms. Nevertheless, in growth arrested cells, the death did occur after a prolonged treatment and most likely ensued from the observed blockage of protein synthesis that reached levels expected to be incompatible with cell survival. From a mechanistic point of view, we ascribed this effect to the documented inhibition of mTORC1 activity; mTORC1 inhibition on the one hand led to a not deadly, rather protective autophagic response but, on the other hand caused a near complete arrest of protein synthesis. Interestingly, no cytotoxicity was found towards normal skin fibroblasts, which only resulted mildly growth arrested by the drug.

Phosphorylation-independent mTORC1 inhibition by the autophagy inducer Rottlerin.

We recently found that Rottlerin not only inhibits proliferation but also causes Bcl-2- and Beclin 1-independent autophagic death in apoptosis-resistant breast adenocarcinoma MCF-7 cells. Having excluded a role for canonical signaling pathways, the current study was aimed to investigate the contribution of the AMPK/mTOR axis in autophagy induction and to search for the upstream signaling molecules potentially targeted by Rottlerin. Using several enzyme inhibitors, Western blotting analysis, mTOR siRNA and pull down assay, we demonstrate that the Rottlerin-triggered autophagy is mediated by inhibition of mTORC1 activity through a novel AMPK and mTORC1 phosphorylation-independent mechanism, likely mediated by the direct interaction between Rottlerin and mTOR.

Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells.

Information about the harmful effects of vaping is sparse and inconsistent, therefore, since the use of electronic cigarettes (e-CIGs) has become increasingly popular as a tool to limit tobacco smoking, it is urgent to establish the toxicity of the commercial e-CIGs. Skin (HaCaT) and lung (A549) cells, the main targets of cigarette smoke (CS), were exposed to e-CIG vapor and CS using an in vitro system. The cytotoxic effect of the exposure was analyzed in both cell types by ultrastructural morphology, Trypan Blue exclusion test and LDH assay. In addition, pro-inflammatory cytokines were measured by the Bio-Plex assay. The cytotoxic components of e-CIG were restrained to the flavoring compound and, to a lesser extent, to nicotine although their effects were less harmful to that of CS. Humectants alone exhibited no cytotoxicity but induced the release of cytokines and pro-inflammatory mediators. Based on our results, we can state that exposure to e-CIG vapors results in far less toxic than exposure to CS. In fact, besides the deleterious effect of flavor and nicotine, even the humectants alone are able to evocate cytokines release. This study will hopefully promote the development of safer e-CIGs to help people quit smoking.

Rottlerin and cancer: novel evidence and mechanisms.

Because cancers are caused by deregulation of hundreds of genes, an ideal anticancer agent should target multiple gene products or signaling pathways simultaneously. Recently, extensive research has addressed the chemotherapeutic potential of plant-derived compounds. Among the ever-increasing list of naturally occurring anticancer agents, Rottlerin appears to have great potentiality for being used in chemotherapy because it affects several cell machineries involved in survival, apoptosis, autophagy, and invasion. The underlying mechanisms that have been described are diverse, and the final, cell-specific, Rottlerin outcome appears to result from a combination of signaling pathways at multiple levels. This paper seeks to summarize the multifocal signal modulatory properties of Rottlerin, which merit to be further exploited for successful prevention and treatment of cancer.

Beta-carotene prevents ozone-induced proinflammatory markers in murine skin.

Beta-carotene has been thought to protect against oxidative stress generated by ultraviolet radiation and thus prevents skin cancer and skin aging (Biesalski and Obermueller-Jevic, 2001). However, nothing is known about its potential effects against other environmental sources of oxidative stress such as ozone (O3) in skin. Intake of oral beta-carotene supplements before exposure to sunlight (and thus inevitably also to (O3) has been recommended on a population-wide basis. However, although some studies have shown beta-carotene as providing skin protection as an antioxidant, other studies using skin cells in culture have shown that beta-carotene may have unexpected prooxidant properties (Obermüller-Jevic, et al., 2001). Given this, there is an ongoing debate regarding the protective or potentially harmful role(s) of beta-carotene in human skin. In this study, the effect of beta-carotene on ozone's effects on the skin of hairless mice was assessed. After feeding a diet supplemented with 0.5% beta-carotene for 1 month, mice were subjected to O3 exposure (0.8 ppm 6 h/day; 7 days) and the induction of proinflammatory markers such as tumor necrosis factor-alpha (TNFalpha), macrophage inflammatory protein 2 (MIP2), and inducible nitric oxide synthase (iNOS), and markers of oxidative stress, heme-oxygenase-1 (HO-1), were quantitated. The data showed that beta-carotene downregulated the induction of TNFalpha, MIP2, iNOS, and HO-1 in response to O3. We conclude that beta-carotene provides protection against O3-induced skin oxidative stress in vivo, which is consistent with a protective role for beta-carotene in the skin.

Effects of rifaximin on bacterial virulence mechanisms at supra- and sub-inhibitory concentrations.

Rifaximin, a poorly absorbed rifamycin derivative, exhibited time-dependent bactericidal activity and at concentrations as low as 1/32 of the minimum inhibitory concentration (MIC) caused morphological alterations in both susceptible and resistant bacterial strains. Spontaneous rifaximin-resistant clones appeared with an incidence of 2.6 x 10(-7). The percentage of Escherichia coli cells cured of various plasmids ranged from: 4.5-70% (Flac), 0-18% (pBP507), 7.7-43.8% (plasmid carrying ESBL genes) and 22.4-41.6% (plasmid encoding toxin from ETEC mex264). 8.4-18.2 and <0.1-18% of Staphylococcus aureus cells were cured (plasmid-mediated penicillinase), 9.5-58.6% of Morganella morganii (ESBL), 10.6-47.1% Citrobacter freundii (ESBL), 2.3-38.7% of Proteus mirabilis (ESBL) and 14.3-66.6% of Klebsiella pneumoniae (ESBL). Rifaximin reduced plasmid transfer from donor to recipient strains by >99%. The MIC of ceftazidime was reduced (2-4 dilutions) in the presence of rifaximin (0.5 x MIC) in ESBL producing strains. Rifaximin lowered the viability and virulence of the bacteria even though they developed resistance to the compound. In conclusion, the present findings add new features to the microbiological characteristics of rifaximin and suggest that if in vivo pathogens are exposed to sub-MICs of the drug, not only are their physiological functions compromised, but gene virulence and antibiotic resistance are not fully expressed.

Rottlerin inhibits the nuclear factor kappaB/cyclin-D1 cascade in MCF-7 breast cancer cells.

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.

Antiproliferative and survival properties of PMA in MCF-7 breast cancer cell.

Although PKCs are assumed to be the main targets of phorbol esters (PMA), additional PMA effectors, such as chimaerins (a family of RacGTPase activating proteins) and RasGRP (exchange factor for Ras/Rap1), can counteract or strengthen the PKC pathways. In this study, we evaluated the proliferative behavior of PMA-treated MCF-7 breast cancer cell and found that: PMA induced growth arrest and inhibited cell death; PMA activated ERKs, which, in turn, induced p21; and inhibitors of ERK (PD98059) and PKC (GF109203X) prevented p21 induction and abolished the PMA survival effect. We conclude that PMA inhibits MCF-7 cell growth and simultaneously stimulates cell survival; both responses are linked to ERK-dependent and p53-independent p21 induction.

In vitro activity of ceftibuten at sub-inhibitory concentrations in comparison with other antibiotics against respiratory and urinary tract pathogens.

Some new features of the in vitro activity of ceftibuten, an oral third generation cephalosporin, have been studied in reference to respiratory and urinary tract pathogens included in its antibacterial spectrum. At 0.25XMIC (minimum inhibitory concentration) and 0.5XMIC levels, ceftibuten was able to affect the biofilm production in 2/3 of both Escherichia coli and Proteus mirabilis strains, and reduced the number of strains capable of adhering to epithelial cells by about 35% in comparison to the control. Surface hydrophobicity was also influenced by ceftibuten and the other drugs at 0.25-0.5XMIC. In general, no marked variation in the virulence traits of the pathogens studied were found by exposing bacteria to sub-MICs of ceftibuten. Plasmid loss (from 1.8 to 37.2%), and Flac transfer inhibition (about 30-50% reduction in the number of recombinants) were detected under the experimental conditions used. This study confirms the excellent antibacterial properties of ceftibuten by adding new information about the effects of this antibiotic against pathogens often involved in respiratory and urinary tract infections that may be treated with this compound, supporting the appropriate use of this cephalosporin.

Functional interactions of protein kinase A and C in signalling networks: a recapitulation.

On the basis of evidence collected from the literature, we propose a general model by which protein kinase (PK) A and the different PKC isoforms can inversely affect cell growth. Molecular switches, which are able to direct the signal towards antiproliferative or mitogenic pathways, are the different isoforms of Raf and PKC. Conflicting data are also reported and discussed in an attempt to reconcile them.

Multiple plasma proteins control atrial natriuretic peptide (ANP) aggregation.

We have recently demonstrated that human alpha-atrial natriuretic peptide (alpha-hANP), an amyloidogenic peptide responsible for isolated atrial amyloidosis, binds to a dimeric form of apo A-I belonging to small high-density lipoproteins (HDL). This binding phenomenon is considered a protective mechanism since it inhibits or strongly reduces the ANP aggregation process. The observation that plasma exhibits at least four times greater amyloid inhibitory activity than HDL prompted us to determine whether small HDL are the only ANP plasma-binding factors. After incubation of whole plasma with labelled ANP, the macromolecular complexes were subjected to two-dimensional gel electrophoresis followed by autoradiography. The results presented here provide novel evidence of additional binding proteins, in addition to apo A-I dimer, able to bind ANP in vitro and to prevent its aggregation. The mass spectrometry analysis of the radioactive spots identified them as albumin, alpha-1 antitrypsin, orosomucoid and apo A-IV-TTR complex. The putative impact of these findings in the amyloidogenic/antiamyloidogenic peptides network is discussed.

The complexity of parathyroid hormone-related protein signalling.

Our understanding of the mode of action of parathyroid hormone-related protein (PTHrP) has changed profoundly during the last decade. Most PTHrP activities are mediated by membrane receptors through autocrine/paracrine pathways. However, both endogenous and exogenous PTHrP also appear to have intracrine effects through translocation into the nucleus. The present review proposes unconventional PTHrP signalling, based on novel clues. First, PTHrP binding to its membrane receptor triggers internalization of the whole complex, mediated by beta-arrestin. There is growing evidence that the receptor and arrestin are the effectors of biological responses, rather than the ligand (or in addition to the ligand). Second, the existence of putative PTHrP targets within the cytoplasm is beginning to be supported. Recent findings of interactions between a COOH-terminus of PTHrP and beta-arrestin and between the PTHrP receptor and 14-3-3 proteins represent the starting point for identification of intracellular partners of both the hormone and its receptor.

ERKs are the point of divergence of PKA and PKC activation by PTHrP in human skin fibroblasts.

Parathyroid hormone-related peptide (PTHrP) receptors, coupled to trimeric G proteins, operate in most target cells through at least three different transduction routes: Galpha s-mediated stimulation of adenylylcyclase (AC), Galpha q-mediated activation of phospholipase Cbeta (PLC) and mitogen-activated protein kinase (MAPK) activation. In this study we investigated the relative role of different pathways in human skin fibroblast proliferation. Using chemical inhibitors and activators of signal transduction, we demonstrated that: (i) AC/cAMP and PLC/1,4,5 inositol triphosphate/diacylglycerol second-messenger systems are simultaneously activated following PTHrP binding to its receptors; (ii) the mitogenic response to PTHrP derives from a balance between two counteracting pathways--an activating route mediated by protein kinase C (PKC) and an inhibitory route mediated by protein kinase A (PKA); (iii) PTHrP mitogenic effects are largely dependent on MAPKs, whose activity can be modulated by both PKA and PKC. Our results indicate that MAPKs are common targets of both transduction routes and, at the same time, their point of divergence in mediating PTHrP dual and opposite mitogenic effects.

Plasma factors controlling atrial natriuretic peptide (ANP) aggregation: role of lipoproteins.

We have previously shown that human plasma atrial alpha-natriuretic peptide (alpha-hANP) sequestering is a protective phenomenon against amyloid aggregation. In the present work, the possible role of lipoproteins as alpha-hANP binding factors has been investigated in vitro using an experimental model, developed in our laboratory, that allows to work at physiological concentrations. This approach consists of gel filtration on Sephacryl S-300 HR of big alpha-[(125)I]hANP generated in phosphate buffered saline or in human normal plasma supplemented or not with lipoproteins. The results of these experiments indicate that high density lipoproteins (HDL) are responsible for the ANP binding phenomenon observed in vitro, while low density lipoproteins and very low density lipoproteins do not directly interact with ANP. Moreover, the HDL remodeling process occurring in vitro has been analyzed during plasma incubation by monitoring the redistribution of lipids and apolipoproteins among the HDL subclasses. The changes in HDL size and composition observed in incubated plasma were compared with the redistribution of endogenous and labeled big ANP. The obtained results revealed that both tend to follow the molecular rearrangement in plasma of apolipoprotein A-I containing particles and suggested that, among HDL species, the small particles are mainly involved in the ANP binding phenomenon. This hypothesis was further demonstrated by ligand blotting experiments that confirmed the existence of differences in the ability of HDL particles to bind alpha-[(125)I]hANP.

Molecular assembly of endogenous and synthetic big atrial natriuretic peptide (ANP) and its amyloidogenic implications.

The aggregation process of alpha-hANP has been investigated in vitro at physiological concentrations by gel chromatographic procedures using a radiolabeled tracer incubated in PBS and in plasma. In PBS big forms of ANP are organized as a peak eluting from both Sephacryl S-100 and S-300 HR in the void volume of the columns; in plasma, besides this major peak, a second radioactive peak is evident, eluting from Sephacryl S-100 HR around the HSA region. After gel chromatography on Sephacryl S-300 HR the major peak appears to consist of three components of different molecular size. Some information about the nature of these peak materials comes from the result of parallel incubations of partially aggregated (seed or nucleus) and aggregate depleted tracer. The comparison between the two time courses of big ANP formation indicates that: (a) ANP aggregation is a nucleation-dependent process, with a lag time longer than 8 days, at picogram peptide levels and (b) the aggregated forms of peptide are those eluting in the void volume, the other plasma peaks being probably expression of a binding, neither saturable or reversible, to some plasma components. The principle of seeded polymerization, used to detect ANP aggregates present in the plasma, indicates that: (a) the endogenous big ANP cannot act as a nucleus for polymerization and it likely consists of non-fibrillar ANP aggregates and/or bound ANP, and (b) this experimental approach can be suitable to evidence ANP binding plasma factors for further characterization studies.

Responses of atrial natriuretic peptide and other fluid regulating hormones to long distance swimming in the sea.

The plasma hormonal response following a swimming competition in the sea (18 km) was evaluated in 12 top level male endurance swimmers. At the end of the effort, while plasma renin activity (PRA) and aldosterone concentration (ALDO) were unchanged, a significant increase in plasma atrial natriuretic peptide (ANP) and antidiuretic hormone (ADH) concentrations were recorded. These changes were associated with a decrease in haematocrit and an increase in Na+ and Cl- plasma concentrations. The individual variations of ANP (difference between the final and initial concentrations) were inversely correlated with the corresponding individual variations of PRA and ADH. The results suggest that, during prolonged swimming, ANP may exert an inhibitory effect on the PRA-ALDO axis and have a modulatory role with regard to ADH secretion.

Hypercalcemia in breast cancer.

Hypercalcemia is relatively frequent in malignancy with or without osteolytic bone metastases. It is thought that neoplastic cells may secrete substances which not only stimulate osteoclastic activity but are also capable of modifying the absorption, excretion, and resorption of calcium and phosphate ions. Since 1987, we have studied 24 breast cancer patients with hypercalcemia (22 with bone metastases and two without). The group of 22 patients with bone metastases were divided into two subgroups. The first consisted of 10 patients with high serum levels of humoral factors, such as parathyroid hormone-related protein (PTHrP), and/or prostaglandin E2 (PGE2) and/or interleukin 1 (IL-1), and high levels of bone markers, such as alkaline phosphatase, bone Gla protein and urinary hydroxyproline. The second subgroup consisted of 12 patients with high levels of bone markers alone. Bone histologic analysis showed an osteoclastic activation surrounding metastatic tumor tissue in six out of 10 patients of the first subgroup, while an evident osteolysis caused by the tumor cells was noted in seven out of 12 patients of the second subgroup. The two patients without bone metastases showed normal biochemistry and bone histologic examination. The authors, having tried to explain the pathogenesis of hypercalcemia, emphasize the importance of humoral factors secreted by tumor cells as a direct or indirect cause of hypercalcemia. The origin of hypercalcemia remains unclear in two patients without bone metastases.