Compound 3significantly sensitized all cell lines to each chemotherapeutic except for cisplatin, whose activity was decreased.In order to overcome solubility and stability issues for the future in vivo administration of compound 3,liposomal formulations were developed. By means of their combination index values obtained via checkerboard microplate method,a formulation showed superior activity to that of compound 3 alone. Because ecdysteroids act also on non-ABCB1 expressing (sensitive) cell lines, our results demonstrate that they do not or not exclusively exert their adjuvant anticancer activity as ABCB1 inhibitors, but other mechanisms must be involved, and they opened the way towards their in vivo bioactivity testing against various cancer xenografts.Ecdysteroids, analogs of the insect molting hormone, are known for their various mild,nonhormonal bioactivities in mammals. Previously, we reported that less-polar ecdysteroids can modulate the doxorubicinresistance of a multidrug resistant (MDR) mouse lymphoma cell line expressing the human ABCB1 transporter. Here,we describe the ability of 20-hydroxyecdysone ( Ecdysteroids represent a particularly interesting group of natural compounds from several aspects, with functions in all kingdoms of nature: in insects, they play a crucial hormonal role controlling molting and development ; in plan in vitro antiapoptotic effect of muristerone A was observed in RKO human colon carcinoma cells   as PEG-3000 PE were purchased from Avanti Polar Lipids Inc. (US). Cholesterol, polyoxyethylene-24-cholesterly ether, beta-D-glucopyranoside, anhydrous ethanol, sodium chloride, and sodium phosphate buffer salt (Na2HPO4·2H2O) were purchased from VWR Int. Ltd. (Austria). PES syringes and membrane syringe filters with pore sizes of 100, 220, and 900 nm and a diameter of 25 mm were purchased from Phenomenex Inc. .20E  phosphate buffer saline solution (pH 7.4) having the osmolality of 279 mOsm/L. The liposome dispersion was kept in a refrigerator at 4°C for 2 h prior to the droplet size reduction. The process was achieved by sonicating the liposome dispersion for 15 min at 100% amplitude . Liposomes of uniform size were prepared by filtration  once through 900 and 220 nm pore size filters. Sorbic acid (0.05% w/v) was added to the buffer solution in order to ensure microbiological stability.Liposomes were prepared by the hydration of thin dry lipid film method. The compositions used were selected according to a preliminary preformulation study (in preparation). Briefly, L-alpha phosphatidylcholine (L-PC), cholesterol, lecithin, polyoxyethylene-24-cholesterly ether (C-24), and beta-D-glucopyranoside or PEG-3000 PE were solubilized together with compoundn = 3).The liposome dispersion was characterized for average droplet size, polydispersity index , and surface electric charge by DLS (dynamic laser scattering) method with a Zetasizer Nano ZS . The optical parameters  and the conductivity were measured before the DLS measurement. The liposomes were stored at 25°C for 1 h prior to analysis and then diluted with ultrapure water. Liposomes were measured after 1 week storage time, which is enough to observe the possible phase separation  that could be caused by any undesirable alteration of the preparation process  at a flow rate of 1.5 mL/min at λ = 243 nm. Each measurement was performed in triplicates, as well as the calibration that was performed by injecting 0.2, 0.5, 1.0, 3.0, and 5.0 μg of compound 3.0.5 mL of LIP-1, LIP-2, or LIP-3 was measured into a 5.0 mL volumetric flask, diluted to 1.5 mL with HPLC-gradient water and adjusted to 5.0 mL with HPLC grade methanol. HPLC analysis was performed on a system of two Jasco PU-2080 pumps connected to a Jasco MD-2010 Plus photodiode-array detector, in isocratic mode by 70% aqueous methanol on a Zorbax XDB-C8 column (5 dox [dox was cultured in the presence of 1 μM of doxorubicin each third passage); prostate cancer cells PC3 and LNCaP cultured in RPMI 1640 media supplemented with 10% inactivated fetal bovine serum; in case of LNCaP medium also contained 1 mM Na-pyruvate, HEPES, and glucose; epidermal carcinoma cell line KB-3-1 and its subline KB-C-1 obtained by stepwise adaption to colchicine  cultured in RPMI 1640 media supplemented with 10% inactivated fetal bovine serum. All cells were cultured at 37°C and 5% CO2; all media contained nystatin, 2 mM L-glutamine, 100 U penicillin, and 0.1 mg streptomycin.Six human derived cancer cell lines were used in this study: breast cancer MCF7 cells and their subcell line obtained by adaptation to doxorubicin, MCF7dox  culturedMDR) cell line derived from L5178 by transfection with pHa MDR1/A retrovirus [2. MDR cell line was selected by culturing the infected cells with 60 μg/L colchicine (Sigma).Two mouse lymphoma cell lines were also used: a parental (L5178) cell line, L5178 mouse T-cell lymphoma cells , and a multidrug resistant . Fifty per cent inhibitory concentrations (IC50) were calculated using nonlinear regression curve fitting of log(inhibitor) versus normalized response and variable slope with a least squares (ordinary) fit of GraphPad Prism 5 software. 1–3 were found to exert very low cytotoxicity activity on each cell line (see below), the activity of 50 μM of compound on the IC50 of doxorubicin (Teva), paclitaxel (Mayen) cisplatine (Teva), or vincristine was tested using the same protocol as described above to the cytotoxicity testing for the respective cell lines. In each case, statistical analysis was carried out by one-way ANOVA with Bonferroni's post hoc test, and differences were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001. In order to prevent any possible false positive results and strengthen the relevance of our data for possible in vivo studies, we also set up a stricter criterion: statistically significant potentiation was considered relevant only when at least a two-time decrease in the IC50 of the chemotherapeutic agent was observed. Such measures were not applied in case of an antagonistic effect (see results for cisplatin).As compoundsMDR mouse lymphoma cells was determined using the checkerboard microplate method in 96-well flat-bottom microtiter plates as published before [4 cells/well were incubated with different concentrations of doxorubicin and liposomes containing compound 3 for 48 h at 37°C under 5% CO2. Cell viability rate was determined through MTT staining, and the interaction was evaluated by using the CompuSyn software  for the constant liposome versus doxorubicin ratios. Combination index (CI) values are presented for 50%, 75%, and 90% of growth inhibition. In each case, the amounts of liposomes were applied so that they represent the same amount of compound 3 within, based on the results of the quantitative determination by HPLC  and formulations LIP-1, LIP-2, and LIP-3 on the L5178 by Chou . 3, a less-polar ecdysteroid, against the doxorubicin resistance of ABCB1 expressing MDR murine lymphoma cells [MDR (mouse lymphoma), MCF7 and its ABCB1 expressing subcell line MCF7dox adapted to doxorubicin (mammary gland), and KB-3-1 and its ABCB1 expressing subcell line KB-C-1 adapted to colchicine (cervix). Two prostate cancer cell lines were also used in our experiments, the steroid dependent LNCaP and the nonsteroid dependent PC3. When tested alone on these cell lines, compounds 1, 2, and 3 exerted very low cytotoxic activities with IC50 values of typically >100 μM, except for compound 3 on the L5178 (81.12 ± 5.5 μM) and compound 2 on the MCF7 cell line (89.67 ± 4.1 μM).Our previous observations on the strong activity of compoundma cells  led to tμM of compounds 1–3 was tested on the cytotoxic activity of chemotherapeutics with distinct mechanisms of action, such as doxorubicin , paclitaxel , vincristine , and cisplatin ; results are shown in The effect of 50  1–3 exert negligible intrinsic cytotoxic activity at the applied concentration of 50 μM, this approach is still a simplification as compared to an appropriate calculation of synergism, for example, by using the checkerboard plate method [50 patterns in combination studies on checkerboard plates [50 value of the chemotherapeutic could be observed.It is important to mention that even though compoundse method , and, asd plates , we deci 3 exerted a significant sensitization effect on all cell lines when applied together with doxorubicin, vincristine, or paclitaxel. Results with doxorubicin could not be determined on the highly resistant MCF7dox cell line since that chemotherapeutic agent had to be applied in such high doses that the measurements were disturbed by its own color (data not shown); paclitaxel resistance of this cell line could also not be reverted. Altogether, the results that are in agreement with those observed by us previously and strongly support our previous assumption [ 3 could effectively sensitize non-MDR human cell lines with no detectable , in the presence of 50 μM of compound 3, while the IC50 values for doxorubicin alone on the L5178 and the L5178MDR cell lines were 228.3 and 3537 nM, respectively. Compound 3 was also able to reduce the paclitaxel resistance of PC3 cells to the same level of those of untreated LNCaP cells , while the treatment of LNCaP cells with compound 3 led to an even stronger cytotoxic activity of paclitaxel (IC50 = 16.5 nM). Moreover, KB-3-1 cells were sensitized by compound 3 to vincristine in a way that it reached much lower IC50 values than observed in KB-C-1 cells treated with the combination: the IC50 value was 0.67 nM (without compound 3: 2.84 nM) in case of KB-3-1, while it was ca. 10 times higher, 6.78 (without compound 3: >50 nM) in case of the MDR subline KB-C-1. A comparison of these values also reveals that resistance of KB-C-1 cells to vincristine could not completely be reverted by compound 3: even though a strong sensitizing activity was observed, the cytotoxicity of vincristine was still significantly weaker than in the parental KB-3-1 cell line (P < 0.001 by unpaired t-test).Compoundle  or  or  3 exle (MCF7 ) express2) showed tendencies for an activity pattern similar to the diacetonide 3, but with much weaker and in several cases irrelevant activities. Interestingly and somewhat unexpectedly, compound 1, 20-hydroxyecdysone, was also found to show significant and relevant sensitizing activity in case of one cell line, MCF7, when coadministered with paclitaxel.The 20,22-acetonide compound (dox) where all compounds significantly elevated its IC50 values. In case of the two prostate carcinoma cell lines, the compounds showed different behaviors: only compound 3 could significantly protect the steroid dependent LNCaP cells from the effect of cisplatin, while compounds 1 and 2 exerted such an activity in the PC3 cells where compound 3 did not influence the efficacy of cisplatin. Although androgen hormone dependency is a major difference between these two cell lines, it should be noted that ecdysteroids do not exert androgenic activity [ 1) available on the market, the possibility of unwanted interactions cannot be excluded. On the other hand, Konovalova et al. have previously found that 10 mg/kg of compound 1 could potentiate the activity and decrease the toxicity of cisplatin in P388 leukemia or B16 melanoma bearing mice, and the authors suggested that beneficial metabolic and immune system modulatory effects of this compound might be the reason for this phenomenon [ in vitro setup. Nevertheless, the strong potentiating activity of compound 3 on the activity of doxorubicin, vincristine, and paclitaxel on various cell lines is highly promising.On the other hand, the tested ecdysteroids showed an obvious general tendency to decrease the activity of cisplatin in all cell lines, especially in the two breast cancer cells lines  parameters of liposome samples of various lipid compositions are shown in  3 into LIP-1, LIP-2, and LIP-3 resulted in entrapment efficiencies of 43, 8, and 50%, respectively  is apparently not enough to hydrolyze the acetonide groups, which could have been a serious pitfall for these formulations if compound 3 would decompose to the much less active compound 2 [Based on our results, the formulations not only keep the strong synergistic activity of compoundysosomes  (that compound 2 .2) and in particularly the 2,3;20,22-diacetonide (3) derivatives of 20-hydroxyecdysone (1) are able to exert sensitizing activity to doxorubicin, paclitaxel, and vincristine in MDR cell lines, which express the ABCB1 membrane transporter, as well as in their susceptible counterparts. Even though, in this work, no conclusions can be made about the mechanism(s) of action of these compounds, these results strongly support our previous assumptions [We demonstrated that the 20,22-acetonide  showed stronger synergistic activity in combination with doxorubicin on L5178MDR cells as compared to the case when it was applied in solution. Based on these results, LIP-3 represents a highly prospective formulation for in vivo studies. Such studies have most recently started within our research program; results are going to be presented in the near future.Compound


However, efficacy in the later stages of the disease occurs at concentrations difficult to achieve through dietary modification. We have therefore investigated what concentration(s) of enterolactone can restrict proliferation in multiple stages of prostate cancer using an in vitro model system of prostate disease. We determined that enterolactone at 20 μM significantly restricted the proliferation of mid and late stage models of prostate disease. These effects were strongly associated with changes in the expression of the DNA licencing genes , in reduced expression of the miR-106b cluster , and in increased expression of the PTEN tumour suppressor gene. We have shown anti-proliferative effects of enterolactone in earlier stages of prostate disease than previously reported and that these effects are mediated, in part, by microRNA-mediated regulation.The mammalian lignan, enterolactone, has been shown to reduce the proliferation of the earlier stages of prostate cancer at physiological concentrations  Prostate cancer is the second most common cancer in men worldwide with seventy percent of annual diagnoses occurring in Westernised societies . The incEnterolactone (ENL) is a weakly-oestrogenic  mammalian metabolite that is produced by the metabolism of plant lignans by intestinal bacteria, but may also be present in low amounts in dairy foods and meat as a consequence of ruminant intestinal metabolism , in vitro studies showing that purified ENL, or foods rich in ENL, can inhibit the development and progression of prostate cancer for example by reducing proliferation [in vitro and that these effects are associated with alterations in the expression of DNA replication licencing genes [Serum or urinary ENL levels, a biomarker of exposure, vary considerably by population and dietary preference, and typically ranges from 0.1 to 10 μM ,10,11. Tferation ,19,20,21feration , it is nThe correct initiation of DNA replication requires the licencing of origin of replications by the minichromosome maintenance complex (MCM) . The loain vitro model system of six prostate cell lines representing the early (RWPE-1 and WPE1-NA22), mid (WPE1-NB14 and WPE1-NB11) and later (WPE1-NB26 and LNCaP) stages of prostate tumourigenesis [in vitro model system was used to assess how the metabolic activity, growth rate, cell cycle progression changes with ENL exposure over 24 and 48 h. Based on these data we explored potential mechanisms for the anti-proliferative activity by measuring the expression of the GMMN, CDT1, MCM2 and MCM7, miR-106b cluster, and PTEN genes.Cancer is not composed of abnormal cells at the same stage of disease; rather it is a series of abnormal cells at differing stages of disease that collectively compromise the appropriate function of the tissue. Previous research has shown anti-proliferative effects of ENL in one or in limited stages of disease, rather than efficacy in a range of disease states ,19,20,21igenesis ,37. The Authenticated RWPE-1 (P52), WPE1-NA22 (P20), WPE1-NB14 (P16), WPE1-NB11 (P24), WPE1-NB26 (P15), and LNCaP (P22) cell lines were purchased from the American Type Culture Collection  to test the effects of ENL. All cell culture reagents were obtained from Life Technologies  unless otherwise stated. The cell lines were cultured and maintained as described previously , with alA stock solution of ENL  at 16.76 mM (100% DMSO) was prepared and used to prepare test concentrations of ENL in cell-line specific medium. Etoposide  was used as a positive control for proliferation as it blocks DNA synthesis resulting in apoptosis. The negative control for all experiments was cell-line specific medium adjusted to contain 0.36% v/v of DMSO.The effect of 10 to 100 μM ENL on the metabolic activity of the six cell lines over 48 h was measured using the water soluble tetrazolium cytotoxicity assay  as described previously . The neg5 cells. From these data the effect of ENL on the doubling time of the cell lines was calculated.The effect of 10 to 60 μM ENL on the viability of each cell line over 48 h was measured using trypan blue staining and the number of non-blue (viable) cells counted as described previously . For eac® 488 & Propidium Iodide from Life Technologies . This kit is a flow cytometry kit used to measure early apoptosis by detecting phosphatidyl serine expression and membrane permeability [The effect of 20 μM ENL on the cell cycle profile of six cell lines over 48 h was measured with the Dead Cell Apoptosis Kit with Annexin V Alexa Fluoreability ,38.5 cells. The fluorescence intensities of Alexa Fluor 488 and Propidium Iodide in each sample, at 585 nm, were measured using a FACSCalibur flow cytometer with CELLQuest Pro Software , and analysed using FlowJo V7.6.3 .Each assay was completed according to the manufacturer’s instructions. For each of three separate assays for each time point, each concentration was tested on three technical replicates with an initial seeding density of 5 × 105 cells were used.The expression of GMNN, CDT1, MCMs 2 and 7, PTEN, hsa-miR-106b, hsa-miR-93, and hsa-miR-25 by the six cell lines treated with 20 μM ENL over 48 h was quantified using probe-based real-time PCR. All reagents were obtained from Life Technologies  unless otherwise stated. For each gene and time point, two biological replicates (with triplicate qPCR measurements) of 5 × 10® miRNA kit  was used to extract large and small RNA in separate fractions from each of the samples according to the manufacturer’s instructions. RNA quantity and integrity was determined based on A260:280 and A260:230 nm ratios using a NanoDrop 1000 spectrophotometer  and a Agilent 2100 bioanalyser . Only RNA with both absorbance ratios of 1.8 to 2.1 and with a RIN value of 9 or greater were considered to be of sufficient quality and integrity.The NucleoSpinFor the large RNA fractions, 500 ng was reverse transcribed into cDNA using a high capacity RNA-to-cDNA kit according to the manufacturer’s instructions. The expression levels of the mRNA transcripts of the GMNN, CDT1, MCMs 2 and 7, and PTEN genes were quantified using pre-validated PrimeTime Nuclease assays  . The HPRT1 (Hs.PT.39a.22214821) reference gene was used to normalise for RNA content.For the small RNA fractions, 10 ng was reverse transcribed into cDNA with gene specific primers using the TaqMan microRNA RT kit according to the manufacturer’s instructions. The expression levels of the mature hsa-miR-106, hsa-miR-93, and hsa-miR-25 genes were quantified using pre-validated TaqMan assays . The RNU6B reference gene (001093) was used to normalise for RNA content.All real-time PCR assays were prepared as triplicate 10 μL reactions comprising a 9.0 μL aliquot of master mix , 0.5 μL of 20x mRNA or miRNA gene assay, 3.5 μL of nuclease-free water, and 1 μL of cDNA (10-fold dilution in nuclease-free water). The thermal profile used was: 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. The experiment was completed using a RotorGene 6000 qPCR instrument . Data were normalised to the appropriate reference gene and analysed for expression level changes (ratio compared to untreated) using the ΔCq method with efficiency correction. The efficiencies for all PCRs ranged between 1.91 and 2.03, where 2.0 represents 100% efficiency.p) value of less than 0.05 was considered to show a significant difference.All data were analysed for statistical significance using a one-way ANOVA with SigmaStat 12.3 . The normality of the data was tested using the Shapiro-Wilk method and the equality of variance using the Leven Median test. Non-normally distributed data was ranked and analysed using the Kruskal-Wallis ANOVA method. Following ANOVA, significantly different means were identified using the Dunnett’s post-hoc test. A probability , WPE1-NB14 (10 and 20 μM), WPE1-NB11 (10 and 20 μM), WPE1-NB26 (10 and 20 μM), and LNCaP (20 μM) cell lines was reduced at this time point. The RWPE-1 cell line tolerated up 40 μM ENL without significant alterations in metabolic activity. The metabolic activity of the WPE1-NB44, WPE1-NB11, WPE1-NB26, and LNCaP cell lines were significantly reduced in a dose dependent manner at concentrations of 20 μM of greater at both time points. The WPE1-NB14 and WPE1-NB11 cells were particularly sensitive to ENL at 24 and 48 h.i.e., a change in the time taken for a population of cells to double in number, we measured the doubling times of the cell lines in response to 10 and 20 μM ENL over 48 h.These data indicate that the lowest concentration of ENL that affects the metabolic activity in the “diseased” cell lines, but does not affect the “normal” cell line is 20 μM. As 40 to 100 μM ENL clearly affected all cell lines, these concentrations were excluded from further analysis. As changes in metabolic activity may result in altered growth rates, p < 0.018) decreased the metabolic activity and increased in the doubling time of the cell lines over 48 h. The WPE1-NB14, WPE1-NB11, and WPE1-NB26 cell lines were the most sensitive to the ENL-induced increased doubling time  of these cell lines. The doubling time of the LNCaP cell line was only affected by 20 μM ENL.The growth kinetics, based on the time for the population to double in number, of the RWPE-1 and WPE1-NA22 cell lines were unaltered by ENL. The positive control, 20 μM etoposide, significantly  this concentration was selected for further study.p < 0.014).The restriction of cell cycle progression in the cell lines with 20 μM ENL over 48 h is shown in 0/G1 phase of the cell cycle for the RWPE-1, WPE1-NB14, WPE1-NB11, WPE1-NB26, and LNCaP cell lines in response to 20 μM ENL. For the LNCaP cell line there was also decrease in the percentage of cells in the G2/M phase. At 48 h, the cell cycle of the WPE1-NB14 and WPE1-NB11 cell lines remained altered  by ENL. The G0/G1 and G2/M phases of the LNCaP cell line were also restricted, both reduced, after 48 h.At 24 h, there was an increase in the percentage of cells in the GThe data in These data indicate that the disrupted viability (metabolic activity and doubling times), shown in 0/G1 and S phases), reduced proliferation, and/or increased cell death.The expression of the DNA licencing genes in response to 20 μM ENL by the six cell lines are shown in The reduced expression of MCM7 suggests that the miR-106b cluster (located in one of the introns of MCM7) may also be influenced by ENL and if so this may affect the expression of the PTEN gene.The expression of the miR-106b cluster and PTEN genes in response to 20 μM ENL by the six cell lines are shown in These data suggest that the repression of the miR-106b cluster leads, in part, to increased PTEN expression. However, the expression of PTEN was increased by ENL in the WPE1-NA22 cell line despite no substantial change in the expression of the miR-106b cluster.in vitro proliferation of prostate cell lines. To our knowledge, this is the first study to examine the correlation between the expression of genes associated with DNA licencing and miR-106b cluster mediated PTEN and biological end-points of proliferation in vitro.The present study provides further evidence that a pure mammalian lignan inhibits the in vitro studies examining how the prostate cell lines respond to ENL have reported anti-proliferative effects for concentrations ranging from 25 to 100 μM and our data are in agreement with these findings [Previous findings ,20. In c1/S cell cycle block [The ENL-induced changes in genes required for DNA replication initiation may explain the effects of ENL on cell cycle control and consequently proliferation in the prostate cell lines. However, the effect of altering the GMNN/CDT1 balance in tumourigenic cells  is unclle block ,43; (2) le block , approprle block ,46; and le block  which tale block ,48,49.Reduced PTEN expression can contribute to cancer development due to decreased negative regulation of the PI3K/AKT pathway (known as quasi-sufficiency) . Unlike in vivo either through dietary or pharmacological intervention. We also have only demonstrated a link between gene expression and proliferation markers—further work is needed to establish whether the expression changes result in functional changes at the post-transcriptional level. We have shown that there appears to be a relationship between ENL-mediated expression of the PTEN gene, perhaps via suppression of the miR-106b cluster, and proliferation in prostate cancer cell lines. If these are confirmed at the proteomic and functional level, it may represent a novel mechanism for the anti-proliferative activity of ENL.The concentrations used in this study have yet to be shown to be achievable in the prostate In conclusion we have provided evidence for the anti-proliferative effects of ENL in mid and late prostate cell lines, and have shown that changes in the transcription of DNA licencing, miR-106b cluster, and PTEN genes may be involved in these effects. This is important as we have shown that ENL is effective in earlier stages of prostate cancer than previously reported and two important pathways in prostate tumourigenesis are linked through miRNA effects.


Due to the upcoming enforcement of the tobacco product directive for e-cigarettes and e-liquids in May 2016, attempts have been made to first establish testing protocols to obtain toxicological data. However, these efforts are limited to chemical data, which might not be sufficient to ensure complete consumer protection in the future. The complexity of the problem cannot be solved by a simple toxicological screening method and should be based on different assays addressing the cytotoxic spectrum of e-liquids and/or their aerosols. In our opinion, it is also necessary to analyze the effects of e-liquid aerosols in vitro, since the generated inhalable vapor represents the actual hazardous compounds interacting directly with the epithelium of the respiratory tract. In general, we are in agreement with Polosa in vitro testing of e-liquid aerosols in general. However, due to the fact that the primary impact site of e-liquid aerosol is the respiratory tract, cells from this anatomical region are the most suitable ones. In our opinion, primary cells from healthy human lung tissue would be the most relevant cell model, but due to donor-dependent variations, limited lifespan and limited availability, those cells have their limitations for standard routine testing. Here, immortalized cell lines offer an alternative, because they have unlimited availability and allow testing procedures with comparable cell populations. In our opinion, cytotoxic studies should also not be limited to acute toxicity testing with undifferentiated cells of the respiratory tract, but should also include long-term (chronic) studies on differentiated 3D constructs with all characteristic cell types to address cell-specific cytotoxic effects relevant for the in vivo situation. In this context, it is of great importance to have one cell line, which can be used to perform both acute and long-term toxicity studies, in order to obtain a broad spectrum of toxicological data. In our publication , we did et al. [Polosa et al.  mentioneet al.  and becoet al. . The alset al. . In our We compared the cellular effects (viability and the production of reactive oxygen species) after e-liquid aerosol and mainstream smoke exposure on freshly isolated primary bronchial epithelial cells, the immortalized cell line CL-1548 and the alveolar cell line A549 . Our expet al.’s [Regarding Polosa et al.’s  notes abin vitro, because testing extracts under submersed culture conditions does not reflect the situation after vaping/smoking in vivo. In the lung, the cells are not covered with a liquid layer as found during submersed cultivation, but are exposed directly to the surrounding atmosphere. Furthermore, water-soluble and volatile vapor components cannot be trapped in the extracts and are therefore not analyzed during the testing. In our case, we did not work according to the standard protocol (ISO 10993-5) for testing substance extracts In summary, we are convinced that direct exposure studies with normal human bronchial epithelial cells or relevant immortalized cell lines in an undifferentiated as well as differentiated stage will contribute to the evaluation of the cytotoxic potency of e-liquid vapor. Such investigations should be included in a validated research protocol accepted by international regulatory authorities.


There is merit in considering a simple toxicological screening method that evaluates the total cytotoxic potential of e-liquids or electronic cigarettes (ECs) aerosol emissions in one single testing. However, there is growing confusion, with several researchers endorsing their personal solution to the problem. Here, we discuss as an example the recent paper by Scheffler and colleagues, in which the authors suggest that more relevant and well differentiated cell lines from human airways could be the most suitable candidates for toxicological evaluation of ECs aerosol emissions. We advance recommendations for validated protocols and advocate for an international coordinated effort aimed at establishing consensus on methodology. According to the tobacco product directive (TPD) of the European Union, electronic cigarettes (ECs) will be regulated from May 2016 [in vitro cellular cytotoxicity system may also detect toxicological potential attributable to unknown contaminants/by-products in the ECs emissions.In relation to this, it is worth noting that there is merit in considering a simple toxicological screening method that evaluates the total cytotoxic potential of e-liquids or ECs aerosol emissions in one single testing, rather than presenting a long list of toxicological risk assessment of several dozens of chemicals tested in isolation. An additional advantage is that toxicological findings from such i.e., CL-1548) would be the most suitable candidate for toxicological evaluation.Although it should not be too difficult to put this in practice, there is growing confusion with several researchers endorsing their personal solution to the problem. As an example, we discuss the recent paper by Scheffler and colleagues . The auti.e., the conducting zone of the respiratory tract) in order to establish a more relevant cell culture model for a toxicological evaluation of ECs emissions. Based on the assumption that the most of these emissions impact on the respiratory tract , they conclude that the human bronchial epithelial cell culture is the most suitable model and propose their in-house immortalized human bronchial cell line as a candidate . The main problem with this approach is that fully characterized human bronchial epithelial cell lines are already available from ATCC  and generally used for regulatory purposes by FDA [Their working hypothesis is that it is important to consider the anatomical site of primary impact of aerosols .The authors compared cell viability of ECs aerosol emission to that of cigarette smoke (positive control) and clean air (negative control) 24-h post exposure of normal human bronchial epithelial (NHBE) cells, immortalized human bronchial epithelial (CL-1548) cells, and adenocarcinoma human alveolar basal epithelial (A549) cells. After 24-h incubation with aerosol emission, cell viability was reduced in CL-1548 much more than A549 and less than NHBE and based on this observation the authors conclude that it is best to use CL-1548 for testing of ECs aerosol emissions by virtue of its heightened cytotoxic sensitivity. The sensitivity of a cell line is a relative concept and it is not surprising that different cell types expressing different grade of differentiation may exhibit different sensitivity. Therefore, conclusions about the appropriateness of a cell line compared to another, as the most “suitable” candidate for toxicological evaluation, requires justification beyond simplistic considerations about the anatomical site of primary impact of aerosols. What if after exposure ECs aerosol emissions, cell viability was not reduced in alternative bronchial epithelial cell lines ? These cell lines should be included for cross checking purposes and to support these authors working hypothesis. Nonetheless, cell differentiation is not an essential requirement for regulatory cytotoxicity studies, it may be a valid scientific approach to when addressing other aspect concerning bronchial epithelial health , defines the level of exposure (extract of 1% concentration) and is used for approval of medical devices or products. It is critical that future evaluation of the harm potential of ECs aerosol emissions gets away from the controversial toxicological debate that has been generated in recent laboratories studies because of experimental protocols that do not mimic realistic condition of use [In conclusion, when assessing potential cytotoxicity effects of ECs aerosol emissions, it is mandatory to compare them with those resulting from the exposure of cigarette smoke. In the absence of clearly defined ECs aerosol generation methods and exposure protocols, it is recommended to perform an ISO 10993-5 [n of use . Last bu


Malignant astrocytomas are highly invasive neoplasms infiltrating diffusely into regions of normal brain. Migration or invasion of tumor cells into normal tissue is a principal cause of mortality and is thought to be a multi-factorial process, consisting of cell interaction with ECM and adjacent cells, as well as accompanying biochemical processes supportive of active cell movement. Rho family members are known to regulate malignant transformation motility and invasion of cancer cells, but the clinicopathological significance of Rnd3 remains unclear. We evaluated the protein expression level of RhoE in gliomas tissues, cell lines and brain tumor initiating stem cells (BTSCs). Results showed that the expression of RhoE was significantly higher in patients with highly invasive gliomas. Next, we constructed a mammalian expression plasmid containing human RhoE and ectopically expressed it in U87. Ectopic expression of RhoE was found to induce membrane ruffle formation, increased migration and invasion both in vitro and in vivo and altered the relative levels of GTP-bound Rac1 and RhoA. Finally RhoE siRNA in the glioma cell line U251 inhibited cell invasion in vitro. In conclusion, the expression of RhoE appears to have a role in the pathogenesis of gliobstoma multiforme.


A new form of diffraction lines similar to Rutherford, Kikuchi and Kossel lines has been identified. They can be used to eliminate the need for sample/source matching in Lonsdale’s triple convergent line method in lattice-parameter determination. A new form of diffraction lines has been identified, similar to Rutherford, Kikuchi and Kossel lines. This paper highlights some of the properties of these lines and shows how they can be used to eliminate the need for sample/source matching in Lonsdale’s triple convergent line method in lattice-parameter determination. Lines similar to Rutherford, Kikuchi and Kossel lines arising from incoherent X-ray multiple-scattering processes have been observed for numerous materials. We will present data from Cu and Au samples.aufhellung and was first reported by Wagner (1920umweganregung (umweg for short) and was first reported by Renninger . DMS lines exhibit several exploitable differences from the above processes which will be explored in the current paper. In particular, we will extend the capabilities of Lonsdale’s method in the context of tunable synchrotron radiation. Finally, we will provide a convenient algorithm for calculating the geometry of the DMS features.2.2.1.a). At its most basic, multiple scattering occurs when two or more reciprocal-lattice vectors simultaneously intersect the Ewald sphere. This will result in a secondary reflection emitted in a direction away from the primary scattered beam vector (L) and their corresponding Ewald slices. This is done by converting hkl’s to reciprocal-lattice vectors using the B matrix  and their axes parallel to G. The radius of the circles is given by the norm of the projection of L on the plane perpendicular to G. The radius of the Ewald slice is given by r is the component of the reciprocal-lattice vectors projected onto G and θ is the Bragg angle of the primary reflection.In multiple scattering, the geometry at which multiple scattering occurs is determined by several parameters, namely, the energy of the incident beam, which fixes the size of the Ewald sphere, the direction of the primary scattering vector, the reciprocal-space origin, which is set by the primary Bragg angle, and an azimuthal reference with respect to which the azimuthal angles are defined . This is analogous to having a range of incident angles providing a continuous set of reciprocal-space origins. Calculating the azimuthal angles at which the crystal reciprocal-lattice vectors intercept the Ewald sphere for each of these origins results in lines across the sphere’s surface. Figs. 1c) and 1d) show DMS is almost identical to normal multiple scattering in terms of its geometry. If we extend the Ewald construction so we are no longer considering the integer case but have an extended source within the crystal arising from the diffuse scatter of a nearby Bragg peak or crystal truncation rod (CTR), we have the scheme depicted in Fig. 1Ignoring polarization, this is geometrically equivalent to defining an internal spherical wave which then produces conics where the Bragg condition is met. This equivalence is demonstrated in Fig. 22.3.a) and 3c) show experimental data collected for a Au(111) crystal measured at the I16 beamline  and (c) consist of several discrete images taken for different azimuths, which is why the diffuse spots are separated according to the resolution of the stitching or azimuthal step size. The lines, however, flow continuously from image to image for the reasons explained in §2.2Figs. 3et al., 1996In contrast to Kossel lines, which are isotropic and unpolarized  crystal. The only significant difference between the two crystals is the direction of the surface cut and, therefore, the direction of the truncation rod.So far we have looked at multiple scattering where the primary reflection is diffuse and is filtered by secondary and tertiary reflections resulting in relatively clean lines. A further component that can make a contribution to the observed signal is the CTR. We measured the DMS lines on two Cu crystals with differing surface cuts, (111) and (311), see Fig. 4Presenting the data for both crystals as a function of azimuth about the 2.05 2.05 2.05 primary reflection reveals the complex interaction between the DMS and the CTR. The CTR and nearby 222 are reinforcing each other and in turn reinforcing the DMS lines Fig. 5. While f3.Strain measurements. The divergent-beam method was used by Lonsdale to study diamond, which cannot produce Kossel lines because of the low energy of the C et al., 2004The precision is limited by the energy bandwidth of the source X-rays, but Lonsdale reported a precision of 4.in situ manipulation-type measurements. Finally, because no coating or external divergent source is required, domain mapping could also be achieved using DMS with microfocusing.We have demonstrated, for the first time, a new form of diffraction lines. We have highlighted some of the properties of these lines and shown the potential for DMS lines as a strain-measurement technique that truly exploits the tunable capabilities of synchrotron radiation. Furthermore, because we are using X-rays, the measurements can be made in the presence of electric or magnetic fields, thus extending the scope of


Uveal melanoma (UM) is recognized as the most common intraocular malignancy and the second most common form of melanoma. Nearly 50% of UM patients develop untreatable and fatal metastases. The 48-member nuclear receptor (NR) superfamily represents a therapeutically targetable group of transcription factors known for their regulation of key cancer pathways in numerous tumor types. Here, we profiled the expression of the 48 human NRs by qRT-PCR across a melanoma cell line panel including 5 UM lines, 9 cutaneous melanoma (CM) lines, and normal primary melanocytes. NR expression patterns identified a few key features. First, in agreement with our past studies identifying RXRg as a CM-specific marker, we found that UM cells also exhibit high levels of RXRg expression, making it a universal biomarker for melanoma tumors. Second, we found that LXRb is highly expressed in both UM and CM lines, suggesting that it may be a therapeutic target in a UM metastatic setting as it has been in CM models. Third, we found that RARg, PPARd, EAR2, RXRa, and TRa expressions could subdivide UM from CM. Previous studies of UM cancers identified key mutations in three genes: GNAQ, GNA11, and BRAF. We found unique NR expression profiles associated with each of these UM mutations. We then performed NR-to-NR and NR-to-genome expression correlation analyses to find potential NR-driven transcriptional programs activated in UM and CM. Specifically, RXRg controlled gene networks were identified that may drive melanoma-specific signaling and metabolism. ERRa was identified as a UM-defining NR and genes correlated with its expression confirm the role of ERRa in metabolic control. Given the plethora of available NR agonists, antagonists, and selective receptor modulators, pharmacologic manipulation of these NRs and their transcriptional outputs may lead to a more comprehensive understanding of key UM pathways and how we can leverage them for better therapeutic alternatives. Uveal melanoma (UM) is the most common form of intraocular cancer in adults and comprises about 5% of all malignant melanoma diagnoses . UM tumoV600E or NRAS mutations, only ~5% of UMs  exhibit BRAFV600E mutations, and NRAS mutations are typically not observed in UM tumors  to transform immortalized melanocytes . Some suThe nuclear receptor (NR) superfamily of transcription factors includes 48 members, most of which activate complex transcriptional programs via ligand binding . NRs regNuclear receptors have also been noted as having tumor suppressive functions including VDR’s protective function in colon cancer ; PPARg’sThe expression and activity of NRs in UMs have been, to date, completely unstudied. To address this issue, we have used high-throughput qRT-PCR to profile the expression of the 48 members of the NR superfamily in various UM cell lines derived from both primary and metastatic lesions and in a normal melanocyte cell line. Based on these results, we report UM-specific NR expression patterns as well as pharmacologically targetable NR-regulated gene networks that could be driving proliferative or oncogenic signaling in UM.All UM cell lines were a kind gift from Dr. Jerry Niederkorn at UT Southwestern and were grown as originally described . CM cellwww.nursa.org under the rapid release tab. Universal cDNA standards generated from human adult RNA  were used for analysis of all receptors except CAR, FXRb, PXR, SHP, DAX-1, ERb, LRH-1, PNR, SF-1, and TLX, which were too limited in expression to use the universal RNA set. For these receptors, commercially available tissue-specific total RNA standards derived from cell lines or adult organ donors were used from liver, ovary, eye, adrenal, and brain, as appropriate. qRT-PCR data were analyzed using ABI instrument software SDS2.1. Baseline values of amplification plots were set automatically, and threshold values were kept constant to obtain normalized cycle times and linear regression data. Because PCR efficiencies for each receptor primer set vary, individual receptor PCR efficiencies were determined to permit receptor-to-receptor comparisons. PCR efficiencies were calculated from the slope of the resulting standard curves as reported previously  was performed in triplicate using the TaqMan-based efficiency-corrected cycle threshold method with 12.5 ng cDNA per reaction for 50 cycles in an ABI 7900HT sequence detection system  as previously described . NR mRNAeviously . Normalihttp://www.ncbi.nlm.nih.gov/gds/) (All microarray data were obtained from published datasets available at the GEO Database (ov/gds/) –25. The p-Values for comparisons between groups of measurements were performed using Student’s t-tests. Bonferroni Corrections were used to account for multiple-hypothesis testing as appropriate.Dendrograms were generated using R Statistical Software. Analysis parameters included distance calculations using a Manhattan methodology and Ward’s method for cluster aggregation. Correlation coefficients for comparisons between data were calculated using Pearson Correlation. r > 0.6 was used). Culled lists were input into gene ontology (GO) analysis tool GOrilla and GO analyses were performed as described (Correlation coefficients (Pearson) were calculated for each gene and the NR in question (either ERRa or RXRg). Lists were then culled to retain the most significantly positively correlated genes (cutoff of escribed .n = 48) in UM, we performed high-throughput qRT-PCR expression analysis across a cell line panel consisting of five UM cell lines, nine CM cell lines from the NCI-60 , suggesting that the data presented here for UM lines are representative of findings in clinical UM samples.Data quality was assessed by comparison to 63 previously published microarray profiles generated from patient UM samples  . In thisTo visualize relationships within the dataset, unsupervised hierarchical clustering analyses were performed on the qRT-PCR dataset for both the 13 cell lines and the 48 NRs  were particularly distinct from the other NRs in the dataset due to their exceptionally high expression. Previously published work from our group noted that RXRg expression is very high in CM cell lines while essentially unexpressed in every other tissue type in the NCI-60 cancer cell line panel . High leWe next examined NRs differentially expressed between UM and CM Figure . First, To better understand the relationships between the NRs themselves within the melanoma panel, we calculated correlation coefficients (performed as before) for all possible pairwise combinations of the 48 NRs. The results of this unsupervised clustering analysis are shown as a heat map in Figure Finally, we compared NR profiles across the different UM cell lines Figure . BecauseWithin the UM panel, three cell lines were derived from primary tumors  and two cell lines were derived from metastatic lesions (OMM1 and OMM2.3). Comparison between these two groups found that NGFIB was up-regulated in the metastatic lines versus the primary tumor lines and the melanocyte control line Figure B. There Examples of all three of the key UM mutations are represented in the cell line panel Table . Becauser = 0.82 for ERRa and 0.7 for RXRg). Lists of microarray probes positively correlated with either ERRa or RXRg expression patterns were generated, culled to the top 400 genes correlating with each receptor , and subjected to gene ontology (GO) analysis using publically available GO analysis tool Gorilla  and identified receptors  exhibiting mutation-specific expression patterns.In this study, we measured expression levels of the 48 human NRs by qRT-PCR across a panel of UM cell lines and a normal melanocyte control to expand on our original work across the NCI-60 panel. We demonstrated that UM (like CM) is distinguished from other cancer cell lines by high expression of RXRg, an NR that we previously reported separates melanomas from other cancers. Furthermore, UM and CM can be differentiated based solely on their NR expression profiles with several NRs differentially expressed between the two . Our results confirm the distinction of UM and CM as separate diseases in line with their differing mutational profiles. We also examined whether there are NRs preferentially expressed in the different mutational subtypes of UM  also showed generally higher expression in the metastasis-derived UM cell lines. The role of NR4 family members in cancer is controversial as it has been noted to be pro-proliferative in some contexts and tumor suppressive in others . One othFinally, we examined NR-to-NR correlation patterns as well as NR-to-genome correlation patterns to identify receptors that might be interacting with each other and to identify networks of genes that certain, key NRs might be controlling within the UM context. ERRa was one of the NRs that differentiated the UM cluster from the CM cluster, and is particularly important given its role as a therapeutic target in other cancers (particularly breast cancer) and the growing availability of ERRa-targeted therapeutics  includinAnalogous studies conducted previously by our group and others have successfully utilized NR expression profiles to subdivide between different cancer types. It has been demonstrated that expression profiles of the 48 NRs alone can properly distinguish between cancers of vastly different origins (within the NCI-60 panel) , betweenIt is of significant interest that several NRs are differentially expressed between UM and CM tumors. Particularly, the findings RARg, PPARd, EAR2, TRa, and LXRb that are retained in UM at levels comparable to the melanocyte while being reduced or lost in CM suggests that there may be opportunities for NR-directed therapeutic interventions in UM that are not available in CM due to CM-specific loss of these receptors. It is also worth noting that the UM-specific loss of RXRa expression. Because RXRa is a heterodimeric binding partner for many NRs, lower RXRa expression might suggest indirect downregulation of NR signaling in UM tumors.in vivo.It has recently been reported that metastatic CM can be inhibited by administration of LXRb agonists in pre-clinical models of CM . Here, wMelanoma is an aggressive, highly metastatic disease that is notoriously difficult to treat using standard cytotoxic agents . Mechaniin vitro growth of GNAQ/11 UM mutant cell lines  in more than 80% of UM cases. Although these mutations would seem obvious targets for therapeutic intervention, GNAQ/11 mutations have not been amenable to therapeutic development in UM and recent work has instead focused on inhibiting downstream events and gene networks driven by these mutations , 35. As ll lines , it is ill lines . Recent ll lines .As transcription factors, activated NRs are extremely effective in eliciting widespread physiologic changes in cells through alteration of the transcriptional output and architecture of the genome . One of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://journal.frontiersin.org/article/10.3389/fendo.2015.00093The Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.


Using gene expression data to enhance our knowledge of control networks relevant to cancer biology and therapy is a challenging but urgent task. Based on the premise that genes that are expressed together in a variety of cell types are likely to functions together, we derived mutually correlated genes that function together in various processes in epithelial-like tumor cells. Expression-correlated genes were derived from data for the NCI-60 human tumor cell lines, as well as data from the Broad Institute’s CCLE cell lines. NCI-60 cell lines that selectively expressed a mutually correlated subset of tight junction genes served as a signature for epithelial-like cancer cells. Those signature cell lines served as a seed to derive other correlated genes, many of which had various other epithelial-related functions. Literature survey yielded molecular interaction and function information about those genes, from which molecular interaction maps were assembled. Many of the genes had epithelial functions unrelated to tight junctions, demonstrating that new function categories were elicited. The most highly correlated genes were implicated in the following epithelial functions: interactions at tight junctions ; interactions at adherens junctions ; interactions at desmosomes ; transcription regulation of cell-cell junction complexes (GRHL1 and 2); epithelial RNA splicing regulators (ESRP1 and 2); epithelial vesicle traffic ; epithelial Ca(+2) signaling ; terminal differentiation of epithelial cells ; maintenance of apico-basal polarity . The findings provide a foundation for future studies to elucidate the functions of regulatory networks specific to epithelial-like cancer cells and to probe for anti-cancer drug targets. Progress in cancer biology and therapy depends in large part on comprehending the molecular interactions that govern key regulatory networks. The vast amount of data on gene expression in cancer cells should assist in reaching that goal, but effectively utilizing that information remains challenging. Most malignant solid tumors derive from epithelial tissues and retain epithelial characteristics to a variable degree that correlates inversely with malignant virulence. We aimed to utilize gene expression data for cell lines derived from various human tumors to elucidate molecular interaction networks controlling functions key to epithelial cell types, leading eventually to deeper understanding of the factors that govern transitions to mesenchymal character, a change that is thought to be central to acquisition of the ability of cancer cells to invade tissue and form distant metastases. The current work focuses on genes that are expressed selectively in epithelial cells, while a subsequent communication will focus on transitions between epithelial and mesenchymal cell states.Epithelia are arguably the best defined as well as the embryonically earliest multicellular phenotype. A prominent characteristic essential to epithelia is tight junctions, which help to hold adjacent cells together and regulate transport of molecules through the paracellular space between adjacent cells The relative expression levels of over 23,000 genes in the National Cancer Institute’s 60 human tumor cell lines (NCI-60) have been assembled into a freely and readily accessible database We now show that a subset of the NCI-60 cell lines that are selective in expression of certain tight junction-associated genes serve as a signature for epithelial character of tumor cells, and that genes positively correlated with that signature can be assembled into networks involved in the control of epithelial functions. We show that the expression patterns in the NCI-60 human cancer cell lines correlates well with expression in the CCLE/Broad cell lines.Although gene expression at the mRNA level is not the sole determinant of corresponding protein expression (for which we do not yet have adequate data), the function correlations are striking.The current work combines gene expression correlations with molecular interaction information directly from current scientific literature to assemble molecular interaction networks specific to epithelial-like cells. In addition to the bioinformatics analysis, an integral part of this study includes a comprehensive review of molecular interactions of genes (and gene products) having epithelial-related functions in human cancer cell lines.http://discover.nci.nih.gov/cellminer/). This tool provides relative quantitation for the cell lines from five microarray platforms Gene expression profiles and correlations for NCI-60 human tumor cell lines were obtained using the “Gene transcript level z score” web-based tool provided by CellMiner (http://www.broadinstitute.org/ccle/data/browseData?conversationPropagation=begin). The downloaded file was pre-processed using a combination of UNIX commands and R programs, e.g., to remove entries for which the gene name was missing. The expression values for each gene were converted to a z-score across all samples in the dataset , using the R program scale. The resulting matrix of gene expression profiles was saved as an R object. An in-house R package was used to compile and normalize the data from individual samples into a coherent dataset for each cancer type. The expression values for each gene were converted to a z-score across all samples in the dataset , using the R program scale. The resulting matrix of gene expression profiles was saved to hard drive as an R object.Gene expression data (CCLE_Expression_Entrez_2012-09-29.gct) for human tumor cell lines of the Cancer Cell Line Encyclopedia (CCLE) of the Broad Institute of MIT and Harvard were downloaded from (Clustered image maps for gene expression and correlations were generated using an in-house R package.http://discover.nci.nih.gov/mim/. The MIM symbols used in the current work are defined in Information on molecular interactions and functions was assembled from recent literature in PubMed. The number of cited references was limited by citing recent publications that contain citations to earlier literature. The molecular interaction maps (MIMs) were prepared using the notation described by Kohn et al Tight junctions are bands of specific structural proteins that seal cell-cell junctions and regulate passage of small ions or molecules through the intercellular space; they are an essential characteristic of epithelial cell types Using the CellMiner NCI-60 analysis tools Structurally associated with tight junctions are adherens junctions whose central component is the epithelial marker, CDH1/E-cadherin. Because of that functional relationship and the close similarity of its NCI-60 expression profile with that of the tight-junction genes displayed in The expression correlations of the NEC genes and cell lines in the context of all NCI-60 cell lines and all tight junction and cadherin gene family members is shown as a clustered image map (CIM) in In addition to the NEC genes listed in Many of the gene family members included in the CIM do not have epithelial-related functions or are expressed at plasma membrane regions other than tight junctions. CDH2/N-cadherin, for example, forms adherens junctions in mesenchymal cells, which do not have cell-cell junctions of the type that is unique to epithelial cells Since the NCI-60 contain a limited number of cell lines per tissue type, we asked whether the NEC subset of tight-junction and cadherin family would also be evident in the data for the much larger number of human tumor cell lines of the Broad Institute’s Cancer Cell Line Encyclopedia (CCLE) Most of the colon cell lines uniquely also express CDH17; selective expression of CDH17 is seen in 35 of the 54 (65%) epithelial-like CCLE colon lines . Among tMany tight junction family genes also function elsewhere in the cell, and particular NEC cell lines may or may not have normal tight junctions. We ask however whether other genes selectively expressed in the NEC cell lines have additional epithelial-related functions, which would further test the inference that NEC gene expression provides a signature for epithelial character of tumor cells.Having defined an NEC cell line signature based on selective expression of a subset of tight junction and cadherin family genes, we used the pattern comparison tool of CellMiner to identify other genes selectively expressed (or selectively not expressed) by those NEC cell lines. We found 76 genes whose z-score correlations with respect to selective expression in the NEC cell lines was r>0.75. For each of those 76 genes, we assembled information about molecular interactions and functions from recent scientific literature. We found relevant information for 44 of the genes  for selective expression in the NEC lines, as given by CellMiner.Many of the most highly NEC-correlated genes were found to interact in a molecular interaction network related to cell-cell junctions: tight junctions, adherens junctions, and desmosomes; these genes are colored red in the molecular interaction map (MIM) in CLDN3, 4 and 7  (CLDN7 (r = 0.93) is a nearly perfect match to the NEC pattern. Closely associated with the claudins in tight junctions is OCLN/occludin (r = 0.58), although its precise role in tight junctions is not clear. When epithelial cells migrate during wound healing, OCLN in complex with INADL (r = 0.69) moves from cell-cell junctions to the leading edge of the migrating cells MARVELD3 (r = 0.95), a tetraspanning transmembrane protein MARVELD2/tricellulin (r = 0.77), which is localized at 3-cell junctions in the epithelial monolayer TJP3/ZO-3 (r = 0.87) correlated strongly with the NEC gene expression pattern  . Note th pattern .CGN/cingulin (r = 0.80), which can bind both TJP1–3 and actomyosin TJP proteins link tight junctions with the cortical actin cytoskeleton and are required for its structural integrity CRB3/Crum3 (r = 0.81) and INADL/Patj (r = 0.69) (LLGL2 (r = 0.80), which participates in the complex that maintains apical/basolateral polarity. LLGL2 was able to reverse an epithelial-mesenchymal transition Directly associated with tight junctions are  = 0.69) , which bEPCAM/TACSTD1/TROP1 (r = 0.84) and TACSTD2/TROP2 (r = 0.64), both of which bind CLDN7  codes for a PDZ domain-containing E3 ubiquitin ligase that targets CLDN3, as well as serine/threonine kinase PBK and other proteins The highly NEC-correlated gene CDH1/E-cadherin (r = 0.77) is the major structural component. Additional components of adherens junctions are CAMSAP3 (r = 0.76) and PLEKHA7 (r = 0.52), which bind to each other in a complex that could bring together several components: CAMSAP3 binds the minus ends of microtubules, and PLEKHA7 binds CTNND1/p120-catenin (r = 0.48), which in turn binds CDH1/E-cadherin ADAP1/CENTA1 (r = 0.82). Thus ADAP1, whose expression is highly NEC-correlated, maintains adherens junctions and preserves epithelial character Closely associated with tight junctions are adherens junctions, of which DSC2 (r = 0.60), and desmogleins, such as DSG3 (r = 0.38), are desmosomal cadherins that form calcium-dependent cell-cell junctions similar to those of the adherens junctions of CDH1/E-cadherin. Plakophilins, such as PKP3/plakophilin (r = 0.71), and JUP/plakoglobin (r = 0.70), constitute the outer dense plaque that connects to the desmosomal cadherins and to the cytoskeletal linker protein DSP/desmoplakin (r = 0.62) on the cytoplasmic side of the plasma membrane , thereby loosening epithelial cell-cell adhesion and promoting cell invasion and metastasis PPL/periplakin (r = 0.76), binds EVPL/envoplakin (r = 0.49) and the two bind intermediate filaments ANXA9/annexinA9 (r = 0.81); the two proteins, whose expression is highly correlated with the NEC genes, co-localize at cell-cell junctions of epithelial cells Desmosomes confer strong cell-cell adhesion in association with adherens junctions in epithelial cells and provide linkage to the cytoskeleton, particularly keratin intermediate filaments. The interactions of the desmosomal proteins are shown in membrane . PKP3 isJUP/plakoglobin (r = 0.70) is a structural component of both desmosomes and adherens junctions, where it binds the cytoplasmic tail of CDH1/E-cadherin DSP/desmoplakin (r = 0.62) binds the epithelia-specific keratin intermediate filaments KRT8 (r = 0.63), KRT19 (r = 0.63) and KRT18 (r = 0.59). In intestinal epithelium, these keratins function to maintain proper architecture of microvilli even without linkage to desmosomes GRHL1 and GRHL2 (r = 0.83 and 0.89), suggesting key rolls in the functions in the of epithelial cell-cell junctions (RAB25 (r = 0.94), whose expression is almost perfectly correlated with the NEC genes. RAB25 also enhances the expression of CLDN4, induces its localization in tight junctions, and activates the transcription of TACSTD2 (r = 0.62), a binder of CLDN7 at tight junctions Several NEC-correlated genes interact indirectly with tight or adherens junctions, regulate their functions, and link them to other cell structures . The larunctions . In partERBB3 (r = 0.54), which forms an oncogenic heterodimer with ERBB2 (r = 0.37) in breast cancer cells GRHL2 promotes transcription of LLGL2 (r = 0.80), which associates with tight junctions via the highly NEC-correlated CRB3 (r = 0.81) (The expression of CDH1/E-cadherin is further enhanced by the highly NEC-correlated  = 0.81) . LLGL2 tSeveral NEC genes were selectively and sometimes exclusively expressed in the NCI-60 colon cancer cell lines and have intestine-specific functions. Some of their interactions are included in the molecular interaction map in VIL1/villin-1 (r = 0.68) and PLS1/plastin-1/fimbrin (r = 0.64)  . VIL1 li = 0.64) . PLS1 waELMO3 (r = 0.88), which is activated by the transcription factors CDX2 and SP1 The normal progression of intestinal epithelial cells from crypt to villus involves the highly NEC-correlated gene CBLC/CBL3 (r = 0.86) and EPHA1 (ephrin receptor A1) (r = 0.82) are both expressed highly selectively in the NEC cell lines, including all of the NCI-60 colon cancer cell lines, except SW620, and both are expressed selectively in the epithelial-like CCLE colon and breast cancer cell lines (not shown). CBLC is expressed specifically in epithelia, most prominently in intestinal epithelia, where it is expressed mainly in post-proliferative cells in villi EFNA1 (ephrin-A1) (r = 0.53), is a membrane receptor tyrosine kinase that avidly binds its major ligand located on an adjacent cell and is expressed mainly in epithelial tissues. Of the 10 EPHA receptors, only EPHA1 was expressed highly selectively in the NEC cell lines. When activated by interaction with EFNA1, EPHA1 binds and inhibits integrin-linked kinase ILK, enhances adhesion to extracellular matrix, and inhibits cell migration and invasion FGF19 (r = 0.61) was expressed exclusively in 5 colon cell lines of the NCI-60; its receptor FGFR4 (r = 0.44) was expressed in most of those colon cancer cell lines but also in the breast cancer MCF7 and several other cell lines. In colon HCT116 and COLO205 (which were among the NCI-60 cell lines that expressed FGF19 and FGFR4), FGF19 promoted tyrosine phosphorylation of CTNNB1/beta-catenin and inhibited its binding to CDH1/E-cadherin MACC1 (metastasis associated colon cancer 1) (r = 0.80) was selectively expressed in the NCI-60 colon cancer cell lines, as well as most of the other NEC cell lines. It binds and activates the MET promoter and enhances cell proliferation, motility and scattering in a MET and HGF-dependent manner that induces nuclear translocation of MACC1. MACC1 and MET mRNA expression are directly correlated with metastasis in colon cancer, and MACC1 is a useful clinical marker for the ability of tumor cells derived from epithelial tissues, such as colon and stomach, to metastasize CDH17, as well as CLDN2 and CLDN15, in colon cancer cell lines has already been noted above in relation to The selective expression of Alterative RNA splicing at consensus nucleotide sequences is an efficient way a master controller protein can simultaneously regulate the function of multiple pathways. Although we do not have adequate data on the expression levels of alternative transcripts, we did find splice-regulators among the highly NEC-correlated (or negatively-correlated) genes.ESRP1 and 2   A central control of mesenchymal to epithelial transition by way of altered RNA splicing involves FGF2 (r = –0.44) and its receptor FGFR1 (r = –0.38) are implicated in repression of epithelial genes via ZEB1/2 Fibroblast growth factor QKI/Quaking, which exhibited a negative expression correlation with that of the NEC cell lines (r = –0.71) mRNA splicing is also modulated by the RNA-binding protein Vesicles such as endosomes move cell components along microtubules from one site to another, thereby regulating both cell structure and signaling functions at various locations in the cell, and inducing epithelial cell polarity. Internalization and recycling of cell surface components in endosomes regulates signaling and ion channels at the cell surface of polarized cells EPN3/epsin-3 (r = 0.83) was expressed selectively in the NEC cell lines. EPN3 is involved in the transport of endosomal cargo proteins to and from the apical surface of epithelial cells. These cargo proteins include sodium ion channels, NOTCH ligands, and epithelial growth factor (EGF) receptors, which are removed from the cell surface in endosomes and then may be recycled to the cell surface, thereby regulating the functions of these molecules Endocytosis is mediated in part by epsins NOTCH3 (r = 0.46) exhibited significant expression selectivity in the NEC cell lines.Epsins are required for intercellular communication mediated by NOTCH ligands and receptors Despite its highly selective expression in the epithelial-like cell lines, however, EPN3 may promote cell migration and invasion RAB25 (r = 0.94), whose expression in the NCI-60 cell lines was almost perfectly selective for the epithelial-like NEC cell lines, and whose expression is prominent in epithelial tissues VIM/vimentin (r = –0.86), but is activated by GRHL2 (grainyhead-like 2) (r = 0.89) Another factor involved in vesicle transport along microtubules is the small GTPase ADAP1/CENTA1/centaurin-alpha1 (r = 0.82). In its active GTP form, ARF6 promotes clathrin-dependent endocytosis of CDH1/E-cadherin with consequent disassembly of adherens junctions and conferring migration and invasion capabilities Vesicle transport is promoted by ARF6, which is inactivated by the GTPase activating protein (GAP) MYO5B (r = 0.79) and MYO5C (r = 0.73). MYO5B is ubiquitously expressed, whereas MYO5C is expressed mainly in glandular and epithelial cells RAB25 and/or RAB11A (r = 0.49) Motor function for vesicle transport is provided by PLEKHG6/MyoGEF (r = 0.85) is recruited by ezrin to the apical pole of epithelial cells, where it induces the formation of microvilli and membrane ruffles. It forms a complex with ezrin and RhoG or with ezrin and ELMO (an effector of RhoG), wherein PLEKHG6 can activate RhoG in response to EGF in EGFR-expressing cells SCNN1A/ENaC (r = 0.83) is a subunit of an epithelial sodium channel that facilitates electrogenic uptake of sodium from the luminal surface of epithelia. It is activated by cleavage of its extracellular domain by ST14/matriptase (r = 0.89) EPN3 (r = 0.83) and may be recycled. Thus at least 3 genes involved in the regulation of these channels were expressed highly selectively in the NEC cell lines.EPN3/epsin-3 (r = 0.83), RAB25 (r = 0.94), GRHL2 (r = 0.89), ADAP1/CENTA1 (r = 0.82), MYO5B (r = 0.79), MYO5C (r = 0.73), AP1M2 (r = 0.75), and PLEKHG6/MyoGEF (r = 0.85).In summary, several of the epithelial-specific genes, identified by their selective expression in the NEC cell lines, are implicated in endocytosis, vesicle transport and cell polarity, including ATP2C2/SPCA2 (r = 0.90), S100A14 (r = 0.89), and BSPRY (B-box and SPRY domain containing) (r = 0.87). The extraordinary expression selectivity of these genes in the NEC cell lines suggest that their Ca(2+)-regulated gene products have specific roles in epithelial tumor cell lines, which however are still not well understood.Ca(2+) signaling is involved in coordinating the transport of cargo-containing vesicles from endoplasmic reticulum to Golgi to plasma membrane ATP2C2/SPCA2 is a Ca(2+)-ATPase that activates calcium channel ORAI1 and mediates uptake of Ca(2+) and Mn(2+) by Golgi membranes; deficiency leads to increased cytosolic Ca(2+) and Golgi disruption BHLHA15/MIST1 (r = 0.51) in serous exocrine epithelial cells, including acini of pancreas, salivary, and lactating mammary glands S100A14, an EF-hand Ca(2+)-binding protein, is the only S100A-family member whose expression was highly selective for the NEC cell lines. It is expressed in several normal epithelial tissues, and low expression is associated with poor prognosis in colorectal tumors and adenocarcinomas of the small intestine BSPRY co-localizes and negatively regulates TRPV5, which is a channel for active calcium uptake by epithelia. BSPRY inhibited the TRPV5-mediated influx of Ca(2+) in kidney cells. Thus BSPRY may be a negative regulator of Ca(2+) transport IRF6 (r = 0.93), OVOL1 (r = 0.83) and OVOL2 (r = 0.75) (ST14/matriptase (r = 0.89) and PRSS8/prostasin/CAP1 (r = 0.83), and their inhibitors SPINT1/Hai1 (r = 0.88) and SPINT2/Hai2 (r = 0.66). All 7 of these terminal differentiation regulators were expressed highly selectively in the NCI-60 epithelial consensus (NEC) cell lines. This is consistent with a continued function of terminal differentiation controls in epithelial cancer cell lines. The known interactions regulating epithelial cell proliferation and differentiation by these components is depicted as molecular interaction maps in The maintenance and cell renewal in epithelial tissues requires delicate controls on cell proliferation and terminal differentiation  = 0.75) . TerminaST14/matriptase and PRSS8/prostasin are required for terminal differentiation of epithelia IRF6 (r = 0.93) drives the differentiation of epithelial cells, although the mechanism has not been fully defined; down-regulation of IRF6 inhibits keratinocyte differentiation and promotes RAS-induced tumorigenesis MYB (r = 0.35) ST14/Matriptase (r = 0.89) is thought to be auto-activated by cleavage in trans after oligomerization SPINT1/Hai1 (r = 0.88), which inhibits ST14/matriptase enzyme activity. SPINT1/Hai1 is an unusually effective, albeit reversible, and rapidly acting inhibitor PRSS8/prostasin (r = 0.83), a glycosylphosphatidylinositol-anchored serine protease on the plasma membrane. Activation of prostasin and matriptase occurs by cleavage of these proteins, which are both rapidly inhibited by binding SPINT1/Hai1 PRSS8/prostasin (r = 0.83) is abundantly expressed in normal breast and prostate tissues, but down-regulated in cancers. Matriptase can cleave the extracellular domain of EGFR, and prostasin can enhance this action SCNN1A/ENaC (r = 0.83) is another protein that is cleavage-activated by ST14/matriptase and has already been discussed above.EHF/ESE3 (r = 0.80) regulates the self-renewal of stem-like epithelial cell and favors transition to a differentiation pathway, at least in prostate cells ELL3 (r = 0.75) promotes proliferation and cancer stem cell-like properties of breast cancer cells and may confer resistance to 5-fluorouracil; these actions are largely due to activation of MEK/ERK signaling IL17RE (r = 0.81), in complex with IL17RA, binds IL17C and engages in an autocrine mechanism to induce innate immune pathways in epithelial cells In addition to positively NEC-correlated genes  and 3, wThe large amount of accumulated information on cancer-related molecular interactions and gene expression patterns challenges us to use that information to comprehend cell functions. The current work used gene expression data to elucidate cancer cell regulatory functions, based on the premise that genes expressed together in a variety of cancer cell types are likely to function together. We showed how genes mutually co-expressed in human tumor cell lines comprised molecular interaction networks regulating coherent functions in epithelial-like cells. The focus was on genes that confer epithelial-related functions and serves as a foundation for subsequent investigation of the mechanisms that control transitions between epithelial and mesenchymal phenotype.A unique aspect of the current work is the assembly of detailed and comprehensive molecular interaction maps (MIMs) of networks regulating structure and function of epithelial cancer cells –16. ThatWe first identified a set of mutually co-expressed tight-junction and cadherin family genes in a subset of NCI-60 human tumor cell lines derived from epithelial tissues  . Based oWe systematically assembled the recently published molecular interaction data for the highly NEC-correlated genes in It is remarkable how many NEC-correlated genes were found to participate in a network of interactions at cell-cell junctions (NEC-correlated genes may link between cell-cell junctions and cytoskeleton: CGN/cingulin (r = 0.80) can bind both tight junctions and F-actin; CAMSAP3 (r = 0.76) links adherens junctions to microtubules; DSP/desmoplakin (r = 0.62) links desmosomes to the intermediate filament keratins KRT8, 18, and 19  .An unexpected finding was the disparity between expression of ZEB1 and ZEB2, which seems not to have been reported previously. In the NCI-60 melanoma cell lines, ZEB2 had relatively high expression, whereas expression of ZEB1 was relatively low . In the Many NEC-correlated genes have functions involved in maintaining apical-basal polarity, a key feature of epithelial cells An efficient mechanism of switching between epithelial and mesenchymal phenotypes may be by way of alternative RNA splicing induced by the highly NEC-correlated genes ESRP1 and ESRP2 . ESRP1 had the strongest and essentially perfect correlation with selective expression in NEC cell lines; this is striking because it was not one of the input genes from which the consensus cell lines were derived, nor is it related to any of those genes by sequence, function, or chromosome location. ESRP1 and 2 are not tight junction or adherens junction genes and do not interact with those structures. Instead they control mRNA splicing and are master regulators of an epithelial splicing network Cancers derived from epithelia are often defective in the normal transition of cells having sustained proliferative potential to cells undergoing terminal differentiation. Since defective regulation of terminal differentiation allows uncontrolled proliferation of the tumor cells, it is important to understand how terminal differentiation is normally regulated and how the regulation is defective in tumors. Indeed some investigated therapies attempt to induce terminal differentiation of epithelial, as well as other tumor types. We found that a major part of the network regulating epithelial terminal differentiation involves highly NEC-selective genes , which hEpithelial terminal differentiation requires a cleaved form of PRSS8/prostasin (r = 0.83), the production of which depends in turn on a cleaved form of ST14/matriptase (r = 0.89) . The proIn a second level of regulation, epithelial terminal differentiation is dependent on IRF6 (r = 0.93) and negatively regulated by OVOL1 and OVOL2 . IRF6, OInputs to the system via regulation of OVOL1 can occur via the TGF beta and beta-catenin pathways. Both pathways transcriptionally activate OVOL1, thereby restricting epithelial cell proliferation and differentiation. The TGF beta pathway couples that action with inhibition of cell proliferation via regulation of cyclin D. The transcriptional inhibition of OVOL1 via beta-catenin is dampened by binding to CDH1/E-cadherin.Inputs to the system via regulation of OVOL1 can occur via the TGF beta and beta-catenin pathways. Both pathways transcriptionally activate OVOL1, thereby restricting epithelial cell proliferation and differentiation. The TGF beta pathway couples that action with inhibition of cell proliferation via regulation of cyclin D. The transcriptional inhibition of OVOL1 via beta-catenin is dampened by binding to CDH1/E-cadherin.This model suggests how epithelial terminal differentiation may be highly integrated, both in time and over the cell population, and how some inputs regulate the system. These conjectures invite computer simulations and cell culture experiments and may provide deeper understanding of the regulation defects in cancer.The unusually high NEC correlations of IRF6 (r = 0.93), OVOL1 (r = 0.83), OVOL2 (r = 0.75), ST14/matriptase (r = 0.89), PRSS8/prostasin (r = 0.83), and SPINT1/Hai1 (r = 0.88) are striking and indicate that these proliferation/differentiation control genes work together specifically in epithelial tumor cell lines.Finally, it may be noted that expression of many of these NEC-correlated genes is associated with relatively low carcinoma aggressiveness. On the other hand, many of the NEC negatively correlated genes appear to be involved in functions associated with non-epithelial or mesenchymal cell types. These genes and their functions in transitions between epithelial and mesenchymal phenotypes are of great interest to cancer biology.In summary, gene expression correlations at the mRNA level were remarkably successful as a means to delineate network functions in epithelial-like human tumor cell lines. The expression of a subset of tight-junction genes provided a key whereby expression correlation identified genes functioning in a broad range of epithelia-related molecular interactions. The gene expression correlations, initially derived in the NCI-60 human tumor cell lines, were consistent with gene expression patterns in the much larger set of CCLE human tumor cell lines of epithelial origin, which provided additional information. The mutually correlated expression of a set of tight junction genes provided a signature for epithelial-like cancer cell lines, distinct from mesenchymal-like tumor cells. The retrieved tight-junction expression-correlated genes had a major role in molecular interaction maps depicting many functions at epithelial cell-cell junctions and depicting regulation of the balance between proliferative and terminally differentiating epithelial cells. Our studies implicated tight-junction expression-correlated genes in a variety of structural and functional characteristics of epithelial cells: (1) maintenance of junction complexes and desmosomes; (2) maintenance of apical-basal polarity by vesicle transport of appropriate components to apical or basal regions of the cell; (3) linkage of cell-cell junction components to actin, microtubule, and intermediate filament cytoskeletons; (4) control of endosomal recycling or degradation of cell-cell junction components; (5) formation of microvilli in intestinal epithelial cells; (6) regulation of RNA splicing in a manner promoting epithelial gene variants; (7) regulation of terminal differentiation of epithelial cells; (8) suppression of tumor growth and metastasis; and (9) inhibition of the epithelial-to-mesenchymal transition as well as promotion of the reverse transition.


Hirsutella thompsonii produces an insecticidal protein named hirsutellin A (HtA), which has been described to be toxic to several species of mites, insect larvae, and cells. On the other hand, on the basis of an extensive biochemical and structural characterization, HtA has been considered to be a member of the ribotoxins family. Ribotoxins are fungal extracellular ribonucleases, which inactivate ribosomes by specifically cleaving a single phosphodiester bond located at the large rRNA. Although ribotoxins were brought to light in the 1960s as antitumor agents, their biological function has remained elusive. Thus, the consideration of hirsutellin A, an insecticidal protein, as a singular ribotoxin recalled the idea of the biological activity of these toxins as insecticidal agents. Further studies have demonstrated that the most representative member of the ribotoxin family, α-sarcin, also shows strong toxic action against insect cells. The determination of high resolution structures, the characterization of a large number of mutants, and the toxicity assays against different cell lines have been the tools used for the study of the mechanism of action of ribotoxins at the molecular level. The aim of this review is to serve as a compilation of the facts that allow identification of HtA as a paradigmatic example of the insecticidal function of fungal ribotoxins.The fungal pathogen  Toxins are molecules affecting vital physiological systems. Many toxins have been explored and developed into drugs for the treatment of many different diseases and others are still under study with the same purpose. But one question to answer is why do organisms need to produce toxins?Aspergillus giganteus, isolated from a sample of farm soil. The protein responsible for these effects was named α-sarcin after its anti-sarcoma activity [All living beings develop several strategies to survive, including a variety of mechanisms for defense, preying, and feeding. Fungi have traditionally been a major source of toxins for study and biotechnological application. They are a rich source of nitrogen and phosphorous and suffer constant attacks by fungivorous animals such as mites, insects, and arthropods ,2,3. Thuactivity . A few yactivity ,7. This activity ,9,10. Thactivity ,11,12, lactivity .Aspergillus related diseases [Nowadays, four of these ribotoxins have been thoroughly studied and characterized: α-sarcin, the first one to be discovered ,9; restrdiseases ,15; and diseases  and causCarpophilus freemani species that are able to feed on Aspergillus restrictus, the restrictocin-producing organism, could not feed upon this fungus during conidia maturation. On the other hand, beetles were not able to feed on Aspergillus nidulans genetically modified to produce the ribotoxin by transforming the fungus with the cDNA of restrictocin placed under the control of the glucoamylase promoter [A. restrictus [The biological function of ribotoxins has been a matter of speculation since their discovery. Apart from the generalized idea of being involved in defense or predation, the assignment of an insecticidal function for fungal ribotoxins has been pointed out ,18. In tpromoter . Accordistrictus ,20.in vitro or in vivo conditions. These authors suggest that restrictocin could be sequestered in membrane systems and transported to certain locations or secreted outside. Immunofluorescence studies on the localization of restrictocin in A. restrictus support this idea. An alternative hypothesis to the active secretion is that an inactivating protein could bind restrictocin until its liberation, but no evidence exists to support such a system in A. restrictus.In relation to ribotoxin biosynthesis, little is known about the mechanisms by which the producing fungi might protect themselves from their own toxicity. Lamy and Davies  suggesteHirsutella has over 50 species that are able to colonize a wide variety of insects. Several studies have reported that crude filtrates of a particular species of this genus denominated Hirsutella thompsonii were toxic to various groups of arthropods as moth, fly and mosquito larvae, aphids, and mites [Acarina inhabiting citrus and other plants in most subtropical and tropical regions [Phyllocoptruta oleivora [The fungal genus nd mites ,24, caus regions  and had oleivora . The insoleivora ,28 Hirsutella thompsonii [in vivo to the adult citrus rust mite, Phyllocoptruta oleivora, the natural host to this parasitic fungus [Galleria mellonella larvae [Spodoptera frugiperda cells [in vitro translation system [The insecticidal protein HtA was originally detected in 1995 while looking for the effective toxic agents produced by ompsonii ,29. It hc fungus . Moreoveda cells . HtA alsn system .Recently, evidence has been found that HtA is able to reproduce the specific ribonucleolytic action and other abilities of ribotoxins, and so it has been included as a new member of the α-sarcin/restrictocin family. Although the question of whether HtA is just an exception within the ribotoxin family has been proposed, both its insecticidal and its ribotoxin activities have been clearly demonstrated ,29,30,31Ustilago sphaerogena, stands out as the unspecific fungal extracellular RNase most closely related to ribotoxins [vs. 101). Ribotoxins also share, with RNases of the T1 family, their main structural core, but they present a number of characteristics that make them unique within the whole superfamily. Ribotoxins are basic proteins, and they are around 40 residues longer (140–150 amino acids). The main structural differences between ribotoxins and RNases of the T1 family are the length and arrangement of the non-ordered protein loops and the N-terminal β-hairpin, which are positively charged in ribotoxins. Thus, these regions are supposed to be the determinants of the extra activities of ribotoxins, such as their specificity and cytotoxicity [Ribotoxins are an intriguing group of proteins regarding their evolution and structure-function relationships ,33. Theybotoxins ,37. It dtoxicity . The abiRibotoxins are considered to be naturally engineered proteins that evolved from nontoxic ribonucleases . They exRibotoxins and non-cytotoxic ribonucleases of the RNase T1 family show high structural homology but different specific activities. Although classic ribotoxins, such as α-sarcin and restrictocin (about 150 amino acids), are larger than non-toxic RNases (about 96–110 amino acids), they share a similar central structure region, including a conserved β-sheet core composed of five strands connected by loops of different lengths. Structural differences between both families are mainly concentrated at these loops and the N-terminal β-hairpin. Consequently, they have been proposed to be the structural determinants responsible for ribotoxin toxic properties . The thrHirsutella thompsonii, the cDNA of HtA was cloned and sequenced [After its discovery as the molecule responsible for the insecticidal activity of the entomopathogenic fungus equenced . The corequenced . These lequenced ,37 as isE. coli [m) determined by circular dichroism (CD), and differential scanning calorimetry (DSC) was 62 ºC. This value was 10 ºC higher than that reported for α-sarcin [m values for the ribotoxins AspF1 and restrictocin, respectively [From the clone of the cDNA of HtA, plasmid pTac-TacHtA was constructed for the expression of mature HtA in E. coli . HtA wasE. coli . The midα-sarcin  but closectively . In 2009ectively  confirmeAs far as the active site is concerned, that of α-sarcin is well known . Among tIt is well known that ribotoxins are cyclizing ribonucleases that exhibit a two-step enzymatic mechanism ,30,53. FTheir toxicity arises from their ability to cross cell membranes and, once inside the cell, impair ribosome function by cleaving a single phosphodiester bond, that between G4325 and A4326 in 28S rRNA (rat ribosome numbering) at the sarcin/ricin loop (SRL) . This clHtA produces the same cleavage as α-sarcin but shows two-fold higher specific activity when assayed against eukaryotic ribosomes. HtA is also able to cleave 35mer synthetic SRL-RNA, liberating 14mer and 21mer oligonucleotides, with identical activity values than those of α-sarcin . ResidueAlthough passage through the cell membrane is the first and limiting step for ribotoxin cytotoxicity , knowledin vivo cytotoxicity would only be manifested against cells targeted by the conjugated antibody.The ability to interact with lipid membranes has been associated with the non-ordered protein loops and the N-terminal β-hairpin of ribotoxins , where tHirsutella thompsonii is able to infect arthropods [in vivo conditions, conidia contact the host, attach to the cuticle, germinate, and penetrate through it [Hirsutella thompsonii as a biological control agent of the parasitic mite Varroa destructor, a honey bee parasite, has also been shown [The assignment of an insecticidal function to fungal ribotoxins has been suggested before ,18. Sevethropods ,24. Underough it . The poten shown ,87.Insect cells have a different plasma membrane composition from mammalian cells, with a higher content of phosphatidylethanolamine and phosphatidylinositol and a significant lower cholesterol/phospholipid ratio . Thus, iG. mellonella larvae  and Trichoplusia ni (High Five). Both α-sarcin and HtA cause a dramatic effect on both inhibition of in vivo protein biosynthesis and decreasing cell viability  upon degradation of the target ribosomes. In fact, the production of α-fragment was observed in treated insect cells. The results revealed the cleavage of 28S rRNA by both ribotoxins with an identical pattern as that described for isolated ribosomes . These dFinally, the long studied antitumoral activity of ribotoxins could be a side effect of their insecticidal action and ribotoxins could be considered host-defense proteins and thus included in the new generation of bioinsecticides ,91. Hirsutellin A (HtA) has been recently demonstrated to be the smallest ribotoxin known to date . It is 2Spodoptera frugiperda Sf9 cell cultures showed specific ribonucleolytic activity. Second, their insecticidal activity against two different insect cell lines (Spodoptera frugiperda Sf9 and Trichoplusia ni High five) has been proved and directly related to the catalytic production of the α-fragment. Finally, both ribotoxins exhibit high toxicity when injected at low doses into G. mellonella larvae. According to this, it seems reasonable to think that ribotoxins evolved from non-specific RNases driven by the necessity of fungal defense against insects. In this regard, it is very interesting to consider the fact that insect cell membranes show many of the characteristics of transformed cells and, thus, the insecticidal biological role of ribotoxins could also explain their intriguing antitumoral properties.In addition, HtA displays a well-characterized insecticidal biological action . InteresCarpophilus freemani, a fungus-feeding beetle, killed 38.5% of larvae in 48 h. Adult C. freemani were not affected, but they exhibited an aversion to restrictocin in their diet [In relation to the production of potential insecticidal agents based on ribotoxins, further work must be carried out. Pure restrictocin (1000 p.p.m.) added to the diet of eir diet . In thisAlthough the unspecific cytotoxic action of ribotoxins could limit their potential use as insecticidal agents, the present accumulation of data about their mechanism of action allow an optimistic view. In this regard, the development of immunotoxins based on these fungal ribonucleases stands out as an alternative in the mid-term future. Nowadays, second generation immunotoxins, based on the fusion of ribtoxins to a single chain containing only the variable domains needed for antigen recognition, have been already obtained . In this