Anti-proliferation of Melanoma Cells and Immune Stimulation by the Cyanobacterial Indole-alkaloid Scytonemin

Under the stress of ultraviolet radiation some cyanobacteria synthesize scytonemin, a protective pigment against DNA photodamage. In addition to photoprotection, scytonemin has been shown to have an anti-proliferative effect on various types of malignant cells. In this study the effect of scytonemin on melanoma and spleen cells was assessed both in vitro using tissue cultures and in vivo in mice models. Melanoma and spleen cells were exposed to 0.08 to 10 μM of scytonemin, and cell proliferation was measured using tritiated thymidine uptake. The data suggest that scytonemin acts as an inhibitor for melanoma cells in a concentration-dependent manner while enhancing the proliferation of spleen cells, suggesting that it can potentially augment the immune response. Furthermore, mice injected with melanoma cells and scytonemin produced fewer tumors than mice that did not receive scytonemin, although the data were not significant. This study adds to the growing body of research that scytonemin may be beneficial as a future anticancer agent to prevent tumor cell growth.

Effects of Species Differences on Oocyte Regulation of Granulosa Cell Proliferation

<p>The role of oocytes in regulating ovulation quota between species is not fully understood. In humans, sheep, and rodents the oocyte-derived growth factors, bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9) have profound effects on ovarian follicular development and ovulation quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled ³H-thymidine uptake by granulosa cells (GC) both within and between the two species, and to assess species differences of GDF9 and BMP15 in co-incubations. For these experiments, oocytes denuded of cumulus-cells (DO) were co-incubated with a fixed number of GC from either species. Rat or sheep DO stimulated ³H-thymidine uptake by GC from the same species (P<0.005). Sheep oocytes also stimulated ³H-thymidine uptake by rat GC (P<0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with monoclonal antibodies specific to GDF9 or BMP15 or to a hydatids antigen (control). Both sheep and rat oocyte stimulation of ³H-thymidine uptake by GC was inhibited with the GDF9 antibody (P<0.05) but not the control antibody, irrespective of the species of GC. Sheep DO stimulation of rat GC was also inhibited using an antibody to BMP15 (P<0.05). However, when using the BMP15 antibody to block the effects of rat DO on rat GC, no inhibition of ³H-thymidine uptake was observed (P=0.988). The molecular forms of GDF9 and BMP15 protein in oocyte lysates and spent media were examined by Western blotting under reducing conditions. For both species, GDF9 protein was present in the mature form in both the lysate and the spend media. For sheep oocytes, BMP15 protein was present as pro-mature and monomeric mature forms in the lysate and in the spent media, whereas from rats pro-mature forms were detected in the oocyte lysate but there was minimal evidence for the mature form in the spent media. The mRNA levels of GDF9, BMP15 and several cumulus cell (CC) genes were measured at 8h intervals over a 24h incubation period. For both sheep and the rat, GDF9 and BMP15 mRNA expression levels were highly correlated (R²=0.99). In the rat, the relative mean expression level of Gdf9 mRNA was four times higher than Bmp15 mRNA, whereas in sheep the relative mean expression ratio of GDF9 to BMP15 was approximately one. For these studies, two types of incubations were performed, namely COC alone or co-incubations of DO with GC. In co-incubations of DO with GC, the relative GDF9 and BMP15 mRNA levels did not change significantly over time, whereas incubations of COC showed that the relative levels of GDF9 and BMP15 mRNA declined significantly during the incubation period. The CC genes (FSHR, LHR, KITL, CX43, AROM and CYCD2) were measured during the 24h incubation period for the COC but not the DO experiment. In rats, none of the CC genes showed any changes in mRNA levels over time except Kitl, where levels at 8h and 16h mRNA levels were significantly lower compared to those at 0h and 24h. In sheep, there were no significant mRNA changes in any of the CC genes except AROM. The level of AROM mRNA reduced to near zero by 8h and remained at low levels at both 16h and 24h. These findings suggest that, under the incubation conditions used, that the gene expression levels for most CC genes were maintained but that the oocyte growth factors alone were unable to maintain AROM gene expression in sheep. In conclusion, major differences were observed in GDF9 and BMP15 expression and regulation between rats and sheep. The lower expression levels of Bmp15 mRNA and protein are likely the reason why rat DO failed to stimulate ³H-thymidine uptake by sheep GC. The evidence suggests that oocyte-derived GDF9 is sufficient to stimulate ³H-thymidine incorporation and presumably DNA synthesis in rat GC whereas in sheep both BMP15 and GDF9 are required. These findings raise the possibility that in species with a low ovulation rate phenotype there is a higher level of GDF9 mRNA and protein synthesis and that BMP15 is a major factor in restricting ovulation quota in mammals.</p>

Effects of Species Differences on Oocyte Regulation of Granulosa Cell Proliferation

<p>The role of oocytes in regulating ovulation quota between species is not fully understood. In humans, sheep, and rodents the oocyte-derived growth factors, bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9) have profound effects on ovarian follicular development and ovulation quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled ³H-thymidine uptake by granulosa cells (GC) both within and between the two species, and to assess species differences of GDF9 and BMP15 in co-incubations. For these experiments, oocytes denuded of cumulus-cells (DO) were co-incubated with a fixed number of GC from either species. Rat or sheep DO stimulated ³H-thymidine uptake by GC from the same species (P<0.005). Sheep oocytes also stimulated ³H-thymidine uptake by rat GC (P<0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with monoclonal antibodies specific to GDF9 or BMP15 or to a hydatids antigen (control). Both sheep and rat oocyte stimulation of ³H-thymidine uptake by GC was inhibited with the GDF9 antibody (P<0.05) but not the control antibody, irrespective of the species of GC. Sheep DO stimulation of rat GC was also inhibited using an antibody to BMP15 (P<0.05). However, when using the BMP15 antibody to block the effects of rat DO on rat GC, no inhibition of ³H-thymidine uptake was observed (P=0.988). The molecular forms of GDF9 and BMP15 protein in oocyte lysates and spent media were examined by Western blotting under reducing conditions. For both species, GDF9 protein was present in the mature form in both the lysate and the spend media. For sheep oocytes, BMP15 protein was present as pro-mature and monomeric mature forms in the lysate and in the spent media, whereas from rats pro-mature forms were detected in the oocyte lysate but there was minimal evidence for the mature form in the spent media. The mRNA levels of GDF9, BMP15 and several cumulus cell (CC) genes were measured at 8h intervals over a 24h incubation period. For both sheep and the rat, GDF9 and BMP15 mRNA expression levels were highly correlated (R²=0.99). In the rat, the relative mean expression level of Gdf9 mRNA was four times higher than Bmp15 mRNA, whereas in sheep the relative mean expression ratio of GDF9 to BMP15 was approximately one. For these studies, two types of incubations were performed, namely COC alone or co-incubations of DO with GC. In co-incubations of DO with GC, the relative GDF9 and BMP15 mRNA levels did not change significantly over time, whereas incubations of COC showed that the relative levels of GDF9 and BMP15 mRNA declined significantly during the incubation period. The CC genes (FSHR, LHR, KITL, CX43, AROM and CYCD2) were measured during the 24h incubation period for the COC but not the DO experiment. In rats, none of the CC genes showed any changes in mRNA levels over time except Kitl, where levels at 8h and 16h mRNA levels were significantly lower compared to those at 0h and 24h. In sheep, there were no significant mRNA changes in any of the CC genes except AROM. The level of AROM mRNA reduced to near zero by 8h and remained at low levels at both 16h and 24h. These findings suggest that, under the incubation conditions used, that the gene expression levels for most CC genes were maintained but that the oocyte growth factors alone were unable to maintain AROM gene expression in sheep. In conclusion, major differences were observed in GDF9 and BMP15 expression and regulation between rats and sheep. The lower expression levels of Bmp15 mRNA and protein are likely the reason why rat DO failed to stimulate ³H-thymidine uptake by sheep GC. The evidence suggests that oocyte-derived GDF9 is sufficient to stimulate ³H-thymidine incorporation and presumably DNA synthesis in rat GC whereas in sheep both BMP15 and GDF9 are required. These findings raise the possibility that in species with a low ovulation rate phenotype there is a higher level of GDF9 mRNA and protein synthesis and that BMP15 is a major factor in restricting ovulation quota in mammals.</p>

Aqueous and polyphenol-rich Larrea divaricata Cav. extracts as potential skin care agents

Skin cells are affected by UV-induced oxidative stress resulting in photoaging and diseases. The objective of this work was to assess the effect of an aqueous extract of Larrea divaricata(Aq) and a flavonoid rich fraction (EA), obtained by liquid/liquid fractionation, on the proliferation of keratinocytes and fibroblasts by the tritiated thymidine uptake assay and on cell viability by the trypan blue exclusion assay. The in vitro sun protection factor (SPF), peroxidase (Px)-like and superoxide dismutase (SOD)-like activities were determined by kinetic spectrophotometric assays, and the phytochemical composition was determined by HPLC-UV and HPLC MS/MS.Aq and EA induced keratinocytes and fibroblast proliferation, protected fibroblasts from H2O2-induced apoptosis and exerted SOD-like and Px-like activities. Aq and EA had a high sun UVB protection factor. So, L. divaricatacould be used in a future for the development of new dermocosmetic or phytotherapy adjuvant in skin oxidative damage.

Staphylococcal Enterotoxin C Subtypes Are Differentially Associated with Human Infections and Immunobiological Activities

ABSTRACT Staphylococcus aureus causes significant infections, responsible for toxic shock syndrome (TSS), hemorrhagic pneumonia, and many other infections. S. aureus secretes virulence factors, which include superantigens such as staphylococcal enterotoxins (SEs). We examined differences in immunobiological activities and disease associations among the four human SEC subtypes. We sequenced the sec gene from 35 human isolates to determine SEC subtypes. Upon finding differences in disease association, we used a [3H]thymidine uptake assay to examine SEC-induced superantigenicity. We also employed a rabbit model of SEC-induced TSS. SEC-2 and SEC-3 were associated with menstrual TSS and vaginal isolates from healthy women, whereas SEC-4 was produced by USA400 isolates causing purpura fulminans and hemorrhagic pneumonia. SEC subtypes differed in potency in a TSS rabbit model and in superantigenicity. There was no difference in superantigenicity when tested on human peripheral blood mononuclear cells. Despite differences, all SECs reacted with polyclonal antibodies raised against the other SEC subtypes. The associations of SEC subtypes with different infections suggest that S. aureus produces virulence factors according to host niches. IMPORTANCE Staphylococcal enterotoxin C has four subtypes that cause human diseases, designated SEC-1 to -4. This study shows that SEC-2 and SEC-3 are the most toxic subtypes in a rabbit model and are associated with human vaginal infections or colonization in association with another superantigen, toxic shock syndrome toxin 1. SEC-4 is associated with purpura fulminans and hemorrhagic pneumonia. SEC-1 is uncommon. The data suggest that there is some selective pressure for the SEC subtypes to be associated with certain human niches.

Cooperativity between Flt3/Syk and Proteasome Inhibitors in Flt3mutant/Wildtype AML, By Acting on the Oxphos and Wnt Endpoints of Syk and p62SQSTM1 Pathways Prohibits Protective Autophagy; While Downregulation of NRF2, NQO1, Jun, and b-Catenin Bypasses Interfering RAS or WT1 Co-Mutations

Recent attempts at single agent targeted therapy of AMLs described by mutation of Flt3 or nuclear epigenetic effectors, has led to the conclusion that combination targeted approaches will be required (CM McMahon et al. Cancer Discov 2019). The simplest combination therapy would involve inhibitors directed at mutant drivers at each level (receptor, nuclear). However, the number of those inhibitors is limited. A broader strategy would target common endpoints for converging pathways such as Wnt/beta-catenin activation elicited by mutation of IDH1/2, TET2, DNMT3A. We found marked cytoplasmic accumulation of ubiquitinated protein (especially inactive b-catenin excluded from the nucleus) by treatment with proteasome inhibitor(PI) to be an efficient, dose-dependent inducer of endoplasmic reticulum (ER) stress apoptosis in mutant Flt3/Wnt effector AML's, requiring concentrations =/>100nM Ixazomib, or =/>20nM Bortezomib, when used alone on cultured blasts. Indeed, a compensatory pathway to protective autophagic escape from PI in poor-risk AML is linked to the levels of NRF2, a major transcriptional activator of NADPH quinone reductase1 (NQO1) that buffers oxidative stress. However, the Flt3/Syk inhibitor (FSI) in clinical trial, TAK-659, at =/<250 nM, significantly broadened the ED30-66 concentrations for these PIs when acting on AML blasts annotated for the presence of mutations Flt3, DNMT3A, IDH1/2, NPM1, where inhibitory activity has been reported for all other Flt3 inhibitors (JW Tyner et al, 2018). We identified that activity involved reduction by the combination of PI with TAK-659, of p62SQSTM1, the scaffolding adaptor protein linked to both sequestration/disposal of ubiquitinated and damaged proteins, as well as to mitochondrial remodeling for OXPHOS. P62, jun, NRF2, NQO1, and b-catenin were reduced by the combination, to levels that equaled or exceeded effects by TAK-659 alone, with heightened impact on blast cell extinction by visible apoptosis and thymidine uptake. However, the combination of PI Ixazomib/Bortezomib with TAK-659 demonstrated unexpected heightened activity also for Flt3mutant and Flt3WT AML blasts with RAS or WT1 mutations, resulting in significant shifts in the ED concentrations for blasts in response to PI alone or the combination with TAK-659 at the clinically relevant dose of 250nM. In addition, blasts annotated for RAS mutation, without or with WT1 mutation (which abrogates expression of the nuclear chaperone for b-catenin, TBL1X) had heightened nuclear content for RAC1, which can participate in b-catenin chaperoning. TAK/PI combinations reduced nuclear RAC content as a measure of the antileukemic cooperativity, while also diminishing levels of nuclear b-catenin and its activating (PAK-dependent) phosphorylation on S675. The combination was also active for AML blasts with MLL-PTD, without or with additional RAS mutation. Further, genetic epistasis experiments were performed with siRNA transduced into MV-411 cells to establish the functional relationship of the individual effectors. P62 knockdown had a dominant effect on reducing NRF2 and NQO1 content, as well reducing nuclear/active expressions of c-jun, and b-catenin, thus establishing its importance in both OXPHOS and Wnt/b-catenin pathways. On the other hand, siRNA knockdown of Syk supported its role as a signaling substation for b-catenin abundance, also dependent on c-jun. Whether the consistent ability for TAK-659 to inhibit p62 expression in primary blasts depends upon it's inhibition of Syk vs. Flt3 is under further investigation. However, both NRF2 and c-jun are known to transactivate p62 gene, and p62 product can serve as scaffolding for JNK1. Indeed, an OXPHOS phenotype is dependent upon jun as well as NRF2, and jun also affects b-catenin en route to HOXA9/10, where overlapping expression of HOXA's and OXPHOS species is described. On the other hand, RAS mutations have been found to occupy an AML signaling space that is OXPHOS independent (I Baccelli, et al. Cancer Cell, 2019). In summary, we have identified common intracellular effectors for OXPHOS as well as Wnt/b-catenin-> HOXA in AML, as compared to the RAS and WT1 pathways, and have established a combination therapy (TAK-659 plus PI) that affects the inhibitory effectors elicited by these co-mutational states, which are responsible for negating activity for most Flt3 selective targeted agents, so as to allow antileukemic response. Disclosures Roodman: Amgen: Membership on an entity's Board of Directors or advisory committees. Konig:Agios: Consultancy; Amgen: Honoraria.

Loss-of-function phenotype of a PKCθT219A knockin mouse strain

Background Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line. Methods The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry. Results Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression. Conclusion This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.

NK Cell Induced T Cell Anergy Depends on GRAIL Expression

NK cells (natural killer cells) being a part of the innate immune system have been shown to be involved in immunoregulation of autoimmune diseases. Previously we have shown that HINT1/Hsp70 treatment induced regulatory NK cells ameliorating experimental autoimmune encephalomyelitis (EAE) course and CD4+ T cells proliferation. NK cells were isolated from mice treated with HINT1/Hsp70 and co-cultured with proteolipid protein (PLP)-stimulated CD4+ T cells isolated from EAE mice. Cell proliferation was assessed by thymidine uptake, cytotoxicity by lactate dehydrogenase (LDH) release assay and fluorescence activated cell sorting (FACS) analysis, protein expression by Western blot, mRNA by quantitative RT-PCR. Gene related to anergy in lymphocytes (GRAIL) expression was downregulated by specific siRNA and GRAIL overexpression was induced by pcDNA-GRAIL transfection. HINT1/Hsp70 pretreatment of EAE SJL/J mice ameliorated EAE course, suppressed PLP-induced T cell proliferation by enhancing T cell expression of GRAIL as GRAIL downregulation restored T cell proliferation. HINT1/Hsp70 treatment induced immunoregulatory NK cells which inhibited PLP-stimulated T cell proliferation not depending on T cell necrosis and apoptosis. This immunoregulatory NK cell function depended on NK cell expression of GRAIL as GRAIL downregulation diminished inhibition of NK cell suppression of T cell proliferation. Similarly GRAIL overexpression in NK cells induced their regulatory function. HINT1/Hsp70 treatment generated regulatory NK cells characterized by expression of GRAIL.

Targeting FGFR with BGJ398 in Breast Cancer: Effect on Tumor Growth and Metastasis

Background: Endocrine resistance and metastatic dissemination comprise major clinical challenges for breast cancer treatment. The fibroblast growth factor receptor family (FGFR) consists of four tyrosine kinase transmembrane receptors, involved in key biological processes. Genomic alterations in FGFR have been identified in advanced breast cancer and thus, FGFR are an attractive therapeutic target. However, the efficacy of FGFR inhibitors on in vivo tumor growth is still controversial. Objective: The purpose of this study was to evaluate the role of FGFR in tumor growth and breast cancer progression. Methods: Cell proliferation was assessed by 3H-thymidine uptake and cell counting in primary cultures of endocrine resistant mammary carcinomas and a human cell line, respectively. Tumor transplants and cell injections were used to determine in vivo growth and spontaneous metastasis. FGFR1-3 and αSMA expression were evaluated on primary tumors by immunohistochemistry. Results: Antiprogestin resistant murine transplants and a human xenograft express high levels of total FGFR1-3. In vitro treatment with the FGFR inhibitor, BGJ398, impaired cell proliferation of resistant variants versus vehicle. In vivo, versus control, BGJ398 treatment decreased one out of four resistant tumors, however all tumors showed a decreased epithelial/stromal ratio. Finally, in a model of hormone resistant mammary cancer that spontaneously metastasizes to the lung, BGJ398 decreased the number of mice with lung metastasis. Conclusion: FGFR inhibitors are promising tools that require further investigation to identify sensitive tumors. These studies suggest that targeting FGFR combined with other targeted therapies will be useful to impair breast cancer progression.

Effect of Hochuekkito (Buzhongyiqitang) on Nasal Cavity Colonization of Methicillin-Resistant Staphylococcus aureus in Murine Model

Background: Methicillin-resistant Staphylococcus aureus (MRSA) infections are largely preceded by colonization with MRSA. Hochuekkito is the formula composing 10 herbal medicines in traditional Kampo medicine to treat infirmity and to stimulate immune functions. We evaluated the efficacy of hochuekkito extract (HET) against MRSA colonization using a nasal infection murine model. Methods: We evaluated the effects of HET as follows: (1) the growth inhibition by measuring turbidity of bacterial culture in vitro, (2) the nasal colonization of MRSA by measuring bacterial counts, and (3) the splenocyte proliferation in mice orally treated with HET by the 3H-thymidine uptake assay. Results: HET significant inhibited the growth of MRSA. The colony forming unit (CFU) in the nasal fluid of HET-treated mice was significantly lower than that of HET-untreated mice. When each single crude drug—Astragali radix, Bupleuri radix, Zingiberis rhizoma, and Cimicifugae rhizome—was removed from hochuekkito formula, the effect of the formula significantly weakened. The uptake of 3H-thymidine into murine splenocytes treated with HET was significantly higher than that from untreated mice. The effects of the modified formula described above were also significantly weaker than those of the original formula. Conclusions: Hochuekkito is effective for the treatment of MRSA nasal colonization in the murine model. We suggest HET as the therapeutic candidate for effective therapy on nasal cavity colonization of MRSA in humans.