Complete Inhibition Of Colony Formation Research Articles

Abstract 5083: BSCHL-2046, a lead chalcone derivative from a newly synthesized library, is a potent neddylation inhibitor against the growth of prostate cancer cell lines

Abstract Members of the chalcone family of molecules and their derivatives have been shown to inhibit various forms of cancer cells. In particular, Flavokawain A has been shown to inhibit prostate cancer cell lines. In order to capitalize on the bioactive nature of chalcone derivatives, we created a library of chalcone derivatives based on the scaffold of Flavokawain A but with an additional anti-cancer moiety. Here we have demonstrated that BSCHL-2046, the most potent member of the library, inhibits cell growth of prostate cancer cell lines including LNCaP, C4-2B, DU145, 22Rv1, and PC3 with IC50’s ranging from 0.44µM-7.5µM. BSCHL-2046 inhibited Ubc12 neddylation in an in vitro assay at an IC50 of 1.5µM and demonstrated complete inhibition at a concentration of 6µM. BSCHL-2046 also reduced Nedd-8 conjugations to both Cullin-1 and Ubc12 in vivo in DU145 cells. The inhibition of neddylation by BSCHL-2046 resulted in an increased expression of p27 and CDT-1. Using molecular modeling, BSCHL-2046 was predicted to bind directly to the Cullin-RING E3 ligase complex. The Cellular Thermal Shift Assay (CETSA, an in vivo assay) demonstrated that BSCHL-2046 significantly decreased heat-induced protein degradation of E3 subunits, supporting BSCHL-2046 directly binding to the complex. Furthermore, BSCHL-2046 at a concentration of 500nM and 1.25µM displayed a 75% to complete inhibition of colony formation of C4-2B cells in soft agar. Taken together, these results demonstrate BSCHL-2046 and its structural analogs are potent neddylation inhibitors that deserve further investigation for the prevention and treatment of prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Matthew A. Tippin, Dong Jun Fu, Liankun Song, Victor Pham, Xaolin Zi. BSCHL-2046, a lead chalcone derivative from a newly synthesized library, is a potent neddylation inhibitor against the growth of prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5083.

Characterization of the Maize Stalk Rot Pathogens Fusarium subglutinans and F. temperatum and the Effect of Fungicides on Their Mycelial Growth and Colony Formation.

Maize is a socioeconomically important crop in many countries. Recently, a high incidence of stalk rot disease has been reported in several maize fields in Gangwon province. In this report, we show that maize stalk rot is associated with the fungal pathogens Fusarium subglutinans and F. temperatum. Since no fungicides are available to control these pathogens on maize plants, we selected six fungicides (tebuconazole, difenoconazole, fluquinconazole, azoxystrobin, prochloraz and kresoxim-methyl) and examined their effectiveness against the two pathogens. The in vitro antifungal effects of the six fungicides on mycelial growth and colony formation were investigated. Based on the inhibition of mycelial growth, the most toxic fungicide was tebuconazole with 50% effective concentrations (EC50) of <0.1 μg/ml and EC90 values of 0.9 μg/ml for both pathogens, while the least toxic fungicide was azoxystrobin with EC50 values of 0.7 and 0.5 μg/ml for F. subglutinans and F. temperatum, respectively, and EC90 values of >3,000 μg/ml for both pathogens. Based on the inhibition of colony formation by the two pathogens, kresoxim-methyl was the most toxic fungicide with complete inhibition of colony formation at concentrations of 0.1 and 0.01 μg/ml for F. subglutinans and F. temperatum, respectively, whereas azoxystrobin was the least toxic fungicide with complete inhibition of colony formation at concentrations >3,000 μg/ml for both pathogens.

Potential biological role of laccase from the sponge Suberites domuncula as an antibacterial defense component

BackgroundLaccases are copper-containing enzymes that catalyze the oxidation of a wide variety of phenolic substrates. MethodsWe describe the first poriferan laccase from the marine demosponge Suberites domuncula. ResultsThis enzyme comprises three characteristic multicopper oxidase homologous domains. Immunohistological studies revealed that the highest expression of the laccase is in the surface zone of the animals. The expression level of the laccase gene is strongly upregulated after exposure of the animals to the bacterial endotoxin lipopolysaccharide. To allow the binding of the recombinant enzyme to ferromagnetic nanoparticles, a recombinant laccase was prepared which contained in addition to the His-tag, a Glu-tag at the N-terminus of the enzyme. The recombinant laccase was enzymatically active. The apparent Michaelis constant of the enzyme is 114μM, using syringaldazine as substrate. Exposure of E. coli to the nanoparticles, coated with Glu-tagged laccase, and to the mediator 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the presence of lignin, as the oxidizable substrate, resulted in an almost complete inhibition of colony formation. Quantitative studies of the effect of the laccase-coated iron oxide nanoparticles were performed using E. coli grown in suspension in reaction tubes within a magnetic nanoparticle separator. ConclusionsThis newly designed magnetic nanoparticle separator allowed a removal of the nanoparticles after terminating the reaction. Using this system, a strong dose-dependent inhibition of the growth of E. coli by the laccase iron oxide nanoparticles was determined. General significanceFrom our data we conclude that the sponge laccase is involved in the anti-bacterial defense of the sponge organism.

Abstract 1743: A dual FLT-3 and MER tyrosine kinase small molecule inhibitor in acute myeloid leukemia cell lines and patient samples

Abstract FLT-3 and MER tyrosine kinases have been previously identified as potential targets in the treatment of acute myeloid leukemia (AML). Expression of FLT-3 internal tandem duplication (ITD) occurs in ∼30-40% of AML patient samples and MER overexpression has been detected in ∼80-100%. Here we describe a novel first-in-class small molecule inhibitor that has potent activity against both of these kinases and mediates growth inhibition or apoptosis of cell lines and patient myeloblasts. UNC1666 is an ATP-competitive reversible small molecule inhibitor that potently inhibits FLT-3 and MER, preventing phosphorylation of these kinases and resultant downstream signaling. In these studies, the effects of treatment with UNC1666 were analyzed in FLT3-ITD-positive (Molm-13 and MV4;11) and MER-positive (Kasumi-1 and U937) AML cell lines and in primary AML patient samples with variable expression of FLT3-ITD and MER. Short term exposure to UNC1666 in cell lines that express either a FLT3-ITD or MER resulted in a dose-dependent decrease in AKT and STAT6 activation compared to cells treated with vehicle, confirming that UNC1666 inhibits both targets in cell-based assays. AML cell lines were also stained with Yo-Pro-1 iodide and propidium iodide and analyzed by flow cytometry to determine induction of apoptosis in response to treatment with UNC1666. Treatment of MER-positive cell lines with UNC1666 resulted in a two to five-fold induction of apoptosis relative to vehicle-treated cells (66±10% and 20±10% apoptotic cells respectively; p&amp;lt;0.01). Treatment of FLT3-ITD cell lines with UNC1666 resulted in an even more dramatic nine-fold induction of apoptosis (90±6% verses10±2% in vehicle-treated cultures, p&amp;lt;0.001). When AML cell lines were cultured in soft agar, treatment with the dual inhibitor resulted in decreased colony formation compared to cells treated with vehicle (relative colony counts were 100 for vehicle-treated cultures versus, 34±15 for MER-positive cell lines and 15±12 for FLT3-ITD cell lines treated with UNC1666, p&amp;lt;0.01). Six primary patient samples that were MER and/or FLT3-ITD positive were analyzed in similar assays and exhibited dose-dependent induction of apoptosis and near complete inhibition of colony formation in methylcellulose after treatment with UNC1666. In summary, UNC1666 is a novel first-in-class small molecule with ability to inhibit activation of both FLT-3 and MER tyrosine kinases. Treatment with UNC1666 blocked activation of pro-survival signaling, including AKT and STAT6, induced apoptosis, and decreased myeloblast colony-forming potential in FLT3-ITD positive and MER-expressing AML cell lines. Moreover, similar anti-leukemia effects were observed in primary samples from patients with AML. These data validate dual FLT-3/MER inhibition as a new and attractive approach for treatment of AML with potential for clinical application. Citation Format: Alisa Lee Sherick, Kelly Menachof, Amanda Hill, Sean Rinella, Deborah DeRyckere, Jing Liu, Xiaodong Wang, Stephen Frye, H. Shelton Earp, Douglas Graham. A dual FLT-3 and MER tyrosine kinase small molecule inhibitor in acute myeloid leukemia cell lines and patient samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1743. doi:10.1158/1538-7445.AM2014-1743

Abstract 1796: SAR302503: A Jak2 inhibitor with antitumor activity in solid tumor models

Abstract Numerous studies have recognized the critical role of STAT3 in malignant transformation and tumor progression. Constitutive STAT3 activation is frequently found in cancer cell lines and tumor samples and it is usually linked to the presence of IL-6. Autocrine or paracrine IL-6 loops have been described to provide tumor cells with the ability to proliferate, survive, migrate and metastasize. At the molecular level, IL-6 binding to its receptor results in activation of Jak/STAT3 signaling and other signaling cascades, namely PI3K/Akt, MEK/ERK1-2, with a well established role in cancer. We have evaluated the ability of SAR302503, a selective Jak2 inhibitor entering a PhIII clinical trial in myelofibrosis patients, to block these pathways in a panel of ∼20 tumor cell lines representing different cancer types (prostate, breast, lung, colorectal, pancreas, hepatocellular, etc). A 45 minutes treatment with different concentrations of SAR302503 (0.1-10 µM) was sufficient to block both basal or IL-6 induced STAT3 phosphorylation in a dose dependent manner. Compound concentrations equal or greater than 0.1 µM were able to reduce the phosphorylation levels of STAT3 by an extent greater than 50% in all the cell lines included in the study. The impact of SAR302503 in tumor cell proliferation and survival was evaluated using different assays including clonogenic assays. Complete inhibition of colony formation was achieved at concentrations of SAR302503 below 1 µM in most of the cell lines. The antitumor activity of SAR302503 was evaluated in mice xenotransplanted subcutaneously with DU145 human prostate cancer cells. Oral administration of SAR302503 for 10 days resulted in significant dose dependent tumor growth inhibition, near to stasis at the highest dose (T/C=19% at 100 mg/kg, bid). In summary, we demonstrate that SAR302503 negatively impacts the proliferation and survival of different solid tumor cells and our data supports a role for a selective Jak2 inhibition in treating solid tumors with activated Jak/STAT signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1796. doi:1538-7445.AM2012-1796

Antimicrobial Properties of Nanostructured TiO&lt;SUB&gt;2&lt;/SUB&gt; Plus Fe Additive Thin Films Synthesized by a Cost-Effective Sol–Gel Process

We prepared thin films composed of pure TiO2 or TiO2 with an Fe additive (at concentrations of 0.2-0.8 wt%) via a simple and cost effective sol gel process, and tested their antifungal properties (against Candida albicans (MTCC-1637), Candida tropicalis (MTCC-184), Candida parapsilosis (MTCC-2509), and Candida glabrata (MTCC-3019) and antibacterial properties (against Staphylococcus faecalis (NCIM-2604) Staphylococcus epidermidis (NCIM-2493), Staphylococcus aureus (NCIL-2122), and Bacillus subtilis (NCIM-2549)). The films were deposited on glass and Si substrates and subjected to annealing at 400 degrees C for 3 h in ambient air. The film structural and morphological properties were investigated by X-ray photoelectron spectroscopy profilometry and scanning electron microscopy, respectively. Antifungal and antibacterial tests were conducted using the drop test method. Among the species examined, Candida albicans (MTCC-1637), and Staphylococcus aureus (NCIL-2122) showed complete colony formation inhibition after exposure for 4 h for the TiO2 loaded with 0.8 wt% Fe thin films. These results indicate that increasing the Fe concentration increased the antimicrobial activity, with complete inhibition of colony formation after 4 h exposure.

Inhibition of the AKT pathway with genistein combined polysaccharide (GCP) plus external beam radiation therapy (EBRT) in a prostate cancer (CaP) xenograft

3135 Background: While EBRT is an effective treatment for primary CaP, many patients will fail biochemically within 5 years. Activation of the AKT pathway of survival has been identified in response to EBRT. Genistein, a primary component of GCP, is an isoflavone aglycone that acts as a protein kinase inhibitor. Single agent GCP inhibits CaP cell growth in vitro and in xenografts models. GCP-treated CaP cells have shown inhibition of the PI3-K/AKT pathway and our previous work showed near complete inhibition of colony formation after GCP + EBRT treatment. We hypothesized that combining a tyrosine kinase inhibitor, such as genistein, with EBRT would result in decreased activation of the AKT survival pathway and greater inhibition of tumor growth than EBRT alone. Methods: We examined the combination of GCP with EBRT in PC-3 xenografts. A clinically relevant dose of radiation (2 Gy) was used to determine either increased combined activity or antagonism by addition of GCP. PC-3 (5x106) cells were injected subcutaneously into Balb/c nu/nu mice. Tumors were treated with GCP alone, EBRT alone, GCP + EBRT, or were untreated. Animals receiving either GCP or GCP + EBRT therapy received daily oral doses of GCP (10% GCP in 0.1ml/10 kg body weight). Within 2 weeks after initial GCP treatment, animals in the EBRT and GCP + EBRT groups were irradiated locally at the tumor site (a single dose of 2Gy). Tumor volume was measured thrice weekly. Results: A reduction in tumor growth was seen in each of the single treatment groups GCP and EBRT (40%) versus untreated tumors up to 40 days. The combination of EBRT and GCP inhibited tumor growth (80%) versus untreated over the same time period. By day 70, 80% of GCP + EBRT-treated tumors showed a reduction in tumor volume with some tumors no longer measurable. Conclusion: 1) GCP + EBRT inhibited CaP tumor growth which was followed by tumor regressions in most animals. 2) These data support additional studies combining GCP with EBRT as a potential treatment for primary CaP. (Support: Amino Up Chemical Co., Ltd.) Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amino Up Chemical Co., Ltd.

In vitro studies on the immunosuppressive effect of 2′,2′-difluorodeoxycytidine (dFdC) and its metabolite 2′,2′-difluorodeoxyuridine (dFdU)

The effect of 2',2'-difluorodeoxycytidine (dFdC) on in vitro human lymphocyte response was assessed in comparison with that of its major metabolite 2',2'-difluorodeoxyuridine (dFdU). Peripheral blood mononuclear cells (PBMNC) from healthy human volunteers were used for assay of mixed lymphocyte reaction (MLR), blastogenesis and colony forming by PHA. Both substances inhibited mitogen and alloantigen responses of PBMNC in a dose-dependent manner, but dFdU was up to 10,000-fold less potent than its parent compound dFdC. The data indicate that activation by alloantigen is more sensitive to the action of dFdU and dFdC than the response to PHA. Thus, dFdU inhibits MLR-induced response at significantly lower doses than PHA-induced proliferation (IC50 +/- SD, 23.55 +/- 8 microM versus 133.2 +/- 12 microM) (p = 0.0003). dFdC also proved to be about 12.3-fold more potent against alloantigen response compared to PHA-induced proliferation of PBMNC (IC50 +/- SD, 2.28 +/- 0.5 nM versus 28.1 +/- 0.5 nM) (p = 0.0001). To get an insight into the toxic profile of dFdU and dFdC, both substances were additionally tested on the in vitro clonal growth of CD34+ cells. Cells were cultured in methylcellulose in the continuous presence of dFdU and dFdC in doses up to 640 microM and 16 nM, respectively. The results show a marked inhibition of erythroid (BFU-E) and myeloid progenitors (CFU-GM) in a dose-dependent manner, but BFU-E was more sensitive to the action of dFdU and dFdC than CFU-GM (p=0.0001). Compared to T-lymphocytes, however, similar or even higher doses of dFdU and dFdC were required for complete inhibition of colony formation obtained from CD34+ cells. To test the role of deoxycytidine kinase (dCK) in the metabolism of dFdU in comparison to that of dFdC, reversal studies with deoxycytidine (dCyd), the natural substrate of dCK, were performed on dFdU- and dFdC-treated HL-60 cells. The data show that relatively low concentrations of dCyd (10 microM) were sufficient to protect HL-60 cells from cytotoxicity of lethal doses of dFdU (160 microM), whereas 100-fold higher concentrations of deoxycytidine (dCyd) (1 mM) were required for a complete reversal of dFdC-mediated toxicity. This suggests that activation of dFdU is due to its phosphorylation by dCK, but dFdU has low affinity to dCK. These effects of dFdU and dFdC in relation to T-lymphocytes and CD34+ cells suggest their possible use as immunosuppressive agents.

Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR)

Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpinPss, a 34.7kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpinPssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6h. There were similarities in the harpinPss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100U ml−1catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpinPss-mediated cell death.

PROTECTIVE EFFECTS OF HUMAN INTERLEUKIN-1 ON HEMATOPOIETIC PROGENITOR CELLS FROM L-PHENYLALANINE MUSTARD

The effects of interleukin-1 (IL-1) on protecting both human and murine bone marrow cells were studied using in vitro clonogenic assays, long-term bone marrow cultures and in vivo mouse studies. Incubation with 100 ng/ml human recombinant IL-1 beta for 20 hours prior to a one hour exposure to L-phenylalanine mustard (L-PAM) provided significant protection of bone marrow colony forming cells when compared to bone marrow cells not exposed to IL-1. Complete inhibition of colony formation was observed above 40 mu M L-PAM in the absence of IL-1 preincubation; whereas, colonies were still detectable in cultures which were initiated with IL-1-treated bone marrow cells. Similar results demonstrating greater protection with IL-1 incubation from L-PAM were seen when murine bone marrow cells were assayed for long-term culture-initiating cells. Furthermore, IL-1 protects long-term repopulating hematopoietic stem cells from L-PAM when studied using an in vivo irradiated mouse assay. In contrast, incubation with IL-1 does not protect colony formation by K562, KG-1 or HL-60 leukemic cell lines implying that protection by IL-1 may be selective. Finally, the protection observed by IL-1 preincubation could be abrogated by incubation with 50 mu M L-buthionine sulfoximine (BSO). This result indicates that IL-1 may increase the amount of glutathione in hematopoietic cells and be responsible for the observed protection from L-PAM.

Toxicity of HPA-23 (ammonmm-21-tungsto-9-antimoniate) for normal human myeloid progenitor cells (GM-CFU) in vitro

HPA-23 is a competitive inhibitor of the RNA-dependent DNA polymerase (reverse transcriptase) of the human immunodeficiency virus (HIV). It may therefore potentially benefit patients with HIV infection. This study aimed at defining the haematopoietic toxicity of this drug and particula.iy its effects on the normal human granulocyte-macrophage progenitor cells (GM-CFU). Our in vitro studies, in semi-solid agar, have shown an inhibitory effect of increasing concentrations of HPA-23 on colony and cluster formation. This effect is probably dose-dependent. An almost complete inhibition of colony formation was observed at doses of more than 20 μg ml . Regarding cluster formation, a similar although much more progressive inhibitory effect was found. Our experimental data should be extrapolated with caution to clinical situations. However, they must be kept in mind for optimal design of HPA-23 therapy in HIV infected patients.

In vitro antiproliferative efficacy of Interferon-alpha,-gamma and Tumor Necrosis Factor on two human renal tumor xenografts

The in vitro antitumor activity of recombinant alpha- and gamma-Interferon as well as recombinant Tumor Necrosis Factor alpha was tested on renal cell carcinoma xenografts using the double layer soft agar method. Using this assay, the effect of the drugs on the clonogenic potential of tumor cells in soft agar was determined and used as an indication for the antiproliferative capacity of these drugs. There appeared to be a differential response of the tested xenografts towards these drugs in a dose dependent way. However, when used in combination in most cases an additive or synergistic effect was observed. Even though again the response was differential, the combination of alpha-Interferon (10 ng/dish) and Tumor Necrosis Factor (100 ng/dish) resulted in a complete inhibition of colony formation in soft agar for both tumor lines. We conclude that there is a direct effect of alpha-Interferon, gamma-Interferon and Tumor Necrosis Factor on renal cell carcinoma xenografts. In combination drug tests the effect of alpha-Interferon and Tumor Necrosis Factor was strongly synergistic. The implication of these studies for in vivo use of these drugs remain to be established.

Effectiveness of various food preservatives in controlling the outgrowth of Byssochlamys nivea ascospores.

Potassium sorbate, sodium benzoate, sulfur dioxide, and diethylpyrocarbonate (DEPC) were tested for their effectiveness in preventing the outgrowth of Byssochlamys nivea Westling ascospores. Sulfur dioxide was the most inhibitory of the test antimycotics, complete inhibition of colony formation occuring in acidified (pH 3.5) potato dextrose agar containing 50 ppm of the preservative. Complete inhibition of B. nivea ascospore outgrowth in grape juice stored for 60 days was noted in the presence of 300 ppm sulfur dioxide, 400 ppm potassium sorbate, and 600 ppm DEPC. Growth was observed in grape juice containing 1000 ppm sodium benzoate. The presence of up to 100 ppm potassium sorbate in grape juice during heat activation appears to have a stimulatory effect on breaking dormancy, while the other test preservatives at this concentration decrease the heat resistance of B. nivea ascospores. The time elapsed between heat shock and exposure to DEPC or sodium benzoate is critical with respect to the sensitivity of ascospores to these preservatives.

Protective effect of vitamin E on reduction in colony formation of cultured human cells by bisulfite

Bisulfite had a severe cytotoxic effect on HeLa S-3 cells at a concentration of 10 −4 M, causing complete inhibition of colony formation. A concentration of 10 −M bisulfite had a moderate cytotoxic effect on the cells, reducing their colony-forming activity to one-third of that of untreated control cultures. Addition of vitamin E at a concentration of 10 −7 M restored the colony-forming activity of 10 −5 M bisulfite-treated cells to 71% of that of control cultures. Addition of 10 −6 M vitamin E resulted in almost complete recovery of the colony-forming activity of bisulfite-treated cells to that of the control cultures. The counteraction of vitamin E to the cytotoxic effect of bisulfite on human cells was discussed.