Selective Growth Inhibition Research Articles

Anti–microbial principles of selected remedial plants from Southern India

Objective To examine the anti–bacterial activity of leaf extracts of Morus alba L. ( Moraceae) and Piper betel L. ( Piperaceae), and seed extracts of Bombax ceiba L. ( Borabacaceae). Methods We have partially purified plant extracts by solvent extraction method, and evaluated the effect of individual fractions on bacterial growth using Escherichia coli ( E. coli), Pseudomonas aeruginosa ( P. aeruginosa) and Staphylococcus aureus ( S. aureus) bacterial strains. Results Compared with Morus and Bombax fractions, Piper fractions showed significant growth inhibition on all the three types of bacteria studied. The EtOAc-hexane fractions of Piper leaves exhibited significant anti–bacterial activity with minimum inhibitory concentrations (MIC) of 50 μg/ml culture against both gram-positive and gram-negative bacteria. The EtOAc-fractions I, II, and IV inhibited bacterial colony formation on soft agar in addition to growth inhibition. A combination treatment of piper fractions with ampicillin resulted in significant growth inhibition in E. coli and P. aeruginosa, and combination with anticancer drug geldanamycin (2μg/ml) showed selective growth inhibition against P. aeruginosa and S. aureus. Three major compounds, i.e., eugenol, 3-hexene-ol and stigmasterol, were primarily identified from Piper betel leaf extractions. Among the individual compounds, eugenol treatment showed improved growth inhibition compared with stigmasterol and 3-hexene-ol. Conclusions We are reporting potential anti–bacterial compounds from Piper betel against both gram-positive and gram-negative bacteria either alone or in combination drug treatment.

Selective Growth Inhibition of Human Breast Cancer Cells by Graviola Fruit Extract In Vitro and In Vivo Involving Downregulation of EGFR Expression

The epidermal growth factor receptor (EGFR) is an oncogene frequently overexpressed in breast cancer (BC), and its overexpression has been associated with poor prognosis and drug resistance. EGFR is therefore a rational target for BC therapy development. This study demonstrated that a graviola fruit extract (GFE) significantly downregulated EGFR gene expression and inhibited the growth of BC cells and xenografts. GFE selectively inhibited the growth of EGFR-overexpressing human BC (MDA-MB-468) cells (IC50 = 4.8 μg/ml) but had no effect on nontumorigenic human breast epithelial cells (MCF-10A). GFE significantly downregulated EGFR mRNA expression, arrested cell cycle in the G0/G1 phase, and induced apoptosis in MDA-MB-468 cells. In the mouse xenograft model, a 5-wk dietary treatment of GFE (200 mg/kg diet) significantly reduced the protein expression of EGFR, p-EGFR, and p-ERK in MDA-MB-468 tumors by 56%, 54%, and 32.5%, respectively. Overall, dietary GFE inhibited tumor growth, as measured by wet weight, by 32% (P < 0.01). These data showed that dietary GFE induced significant growth inhibition of MDA-MB-468 cells in vitro and in vivo through a mechanism involving the EGFR/ERK signaling pathway, suggesting that GFE may have a protective effect for women against EGFR-overexpressing BC.

Abstract 3580: Discovery and SAR study of a novel and potent Itk inhibitor as a targeted therapeutic agent for T-cell lymphoma and leukemia

Abstract Itk (IL2-inducible T-cell kinase) is a non-receptor protein tyrosine kinase. It is selectively expressed in T cells, Mast cells, and NK cells. It plays an important role in T-cell maturation, proliferation, and malignancies. Using Western blotting, Itk was only detected in three T-cell lymphoma and leukemia cell lines (Jurkat, Molt-4, and Hut78) out of 41 different cancer cell lines. A small molecule named CTA056 showed potent and selective inhibition of Itk kinase activity and growth in each of the three T-cell cancer cell lines. After treatment of Jurkat cells with CTA056, Itk phosphorylation was inhibited in a dose-dependent manner. In addition, apoptosis was induced in Jurkat cells following treatment with CTA056. In order to better understand structure-activity relationship (SAR) on CTA056, a series of analogs of CTA056 were designed, synthesized, and tested for their inhibition of both Itk phosphorylation and cancer cell growth. Several analogs showed better activity and selectivity. The results will be presented. Success in this endeavor could lead to the development of novel targeted therapy, rather than traditional chemotherapy, for T-cell lymphomas and leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3580. doi:10.1158/1538-7445.AM2011-3580

Dual IGF-1R/SRC inhibitors based on a N′-aroyl-2-(1 H-indol-3-yl)-2-oxoacetohydrazide structure

The N′-aroyl-2-(1 H-indol-3-yl)-2-oxoacetohydrazide motif was identified as a novel scaffold for the development of kinase inhibitors. Derivatives with a biphenyl element attached to the hydrazide structure proved to be submicromolar dual inhibitors of the cancer-related kinases IGF-1R and SRC. One of the most potent kinase inhibitors of the series produced a selective growth inhibition in a panel of cultivated cancer cell lines.

The selective growth inhibition of oral cancer by iron core-gold shell nanoparticles through mitochondria-mediated autophagy

Nanoparticles with an iron core and gold shell (denoted “Fe@AuÓ”) have been reported to limit cancer-cell proliferation and therefore have been proposed as a potential anti-cancer agent. However, the underlying mechanisms are still unknown. In this study, we used flow cytometry, confocal fluorescence microscopy, and transmission electron microscopy to analyse the morphological and functional alterations of mitochondria in cancerous cells and healthy cells when treated with Fe@Au. It was found that Fe@Au caused an irreversible membrane-potential loss in the mitochondria of cancer cells, but only a transitory decrease in membrane potential in healthy control cells. Production of reactive oxygen species (ROS) was observed; however, additions of common ROS scavengers were unable to protect cancerous cells from the Fe@Au-induced cytotoxicity. Furthermore, iron elements, before oxidation, triggered mitochondria-mediated autophagy was shown to be the key factor responsible for the differential cytotoxicity observed between cancerous and healthy cells.

Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in HPV 16-positive cells

Background"High risk" Human Papillomavirus strains are the causative agents of the vast majority of carcinomas of the uterine cervix. In these tumors, the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins.MethodsIn this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" HPV 16 E7 oncoprotein, scFv 43M2 and scFv 51. These scFvs were expressed by retroviral system in different cell compartments of the HPV16-positive SiHa cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the tumor suppressor pRb protein, were then investigated by immunoassays, PepSet™technology and Surface Plasmon Resonance.ResultsThe expression of the two scFvs in the nucleus and the endoplasmic reticulum of SiHa cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the pRb binding site. Nevertheless, the binding of scFv 43M2 to E7 was inhibited by pRb in a non-competitive manner.ConclusionsBased on the overall results, the observed inhibition of HPV-positive SiHa cells proliferation could be ascribed to an interaction between scFv and E7, involving non-pRb targets. The study paves the way for the employment of specific scFvs in immunotherapeutic approaches against the HPV-associated lesions.

NF kappa B inhibitors and antitrypanosomal metabolites from endophytic fungus Penicillium sp. isolated from Limonium tubiflorum

Chemical investigation of the endophytic fungus Penicillium sp. isolated from Limonium tubiflorum growing in Egypt afforded four new compounds of polyketide origin, including two macrolides, penilactone (1) and 10,11-epoxycurvularin (2), a dianthrone, neobulgarone G (7), and a sulfinylcoumarin, sulfimarin (14), along with twelve known metabolites (3-6, 8-13, 15 and 16). The structures of all compounds were assigned by comprehensive spectral analysis (1D and 2D NMR) and mass spectrometry. Compounds 3, 4, 13 and 16 showed pronounced antitrypanosomal activity with mean MIC values ranging from 4.96 to 9.75μM. Moreover, when tested against a panel of three human tumor cell lines compounds 3, 4, 6 and 12 showed selective growth inhibition against Jurkat and U937 cell lines with IC(50) values ranging from 1.8 to 13.3μM. The latter compounds also inhibited TNFα-induced NF-κB activity in K562 cells with IC(50) values ranging from 1.6 to 10.1μM, respectively.

Phyllanthus spp. Induces Selective Growth Inhibition of PC-3 and MeWo Human Cancer Cells through Modulation of Cell Cycle and Induction of Apoptosis

Background Phyllanthus is a traditional medicinal plant that has been used in the treatment of many diseases including hepatitis and diabetes. The main aim of the present work was to investigate the potential cytotoxic effects of aqueous and methanolic extracts of four Phyllanthus species (P.amarus, P.niruri, P.urinaria and P.watsonii) against skin melanoma and prostate cancer cells.Methodology/Principal Findings Phyllanthus plant appears to possess cytotoxic properties with half-maximal inhibitory concentration (IC50) values of 150–300 µg/ml for aqueous extract and 50–150 µg/ml for methanolic extract that were determined using the MTS reduction assay. In comparison, the plant extracts did not show any significant cytotoxicity on normal human skin (CCD-1127Sk) and prostate (RWPE-1) cells. The extracts appeared to act by causing the formation of a clear “ladder” fragmentation of apoptotic DNA on agarose gel, displayed TUNEL-positive cells with an elevation of caspase-3 and -7 activities. The Lactate Dehydrogenase (LDH) level was lower than 15% in Phyllanthus treated-cancer cells. These indicate that Phyllanthus extracts have the ability to induce apoptosis with minimal necrotic effects. Furthermore, cell cycle analysis revealed that Phyllanthus induced a Go/G1-phase arrest on PC-3 cells and a S-phase arrest on MeWo cells and these were accompanied by accumulation of cells in the Sub-G1 (apoptosis) phase. The cytotoxic properties may be due to the presence of polyphenol compounds such as ellagitannins, gallotannins, flavonoids and phenolic acids found both in the water and methanol extract of the plants.Conclusions/Significance Phyllanthus plant exerts its growth inhibition effect in a selective manner towards cancer cells through the modulation of cell cycle and induction of apoptosis via caspases activation in melanoma and prostate cancer cells. Hence, Phyllanthus may be sourced for the development of a potent apoptosis-inducing anticancer agent.

Anti-Cancer Effect of HIV-1 Viral Protein R on Doxorubicin Resistant Neuroblastoma

Several unique biological features of HIV-1 Vpr make it a potentially powerful agent for anti-cancer therapy. First, Vpr inhibits cell proliferation by induction of cell cycle G2 arrest. Second, it induces apoptosis through multiple mechanisms, which could be significant as it may be able to overcome apoptotic resistance exhibited by many cancerous cells, and, finally, Vpr selectively kills fast growing cells in a p53-independent manner. To demonstrate the potential utility of Vpr as an anti-cancer agent, we carried out proof-of-concept studies in vitro and in vivo. Results of our preliminary studies demonstrated that Vpr induces cell cycle G2 arrest and apoptosis in a variety of cancer types. Moreover, the same Vpr effects could also be detected in some cancer cells that are resistant to anti-cancer drugs such as doxorubicin (DOX). To further illustrate the potential value of Vpr in tumor growth inhibition, we adopted a DOX-resistant neuroblastoma model by injecting SK-N-SH cells into C57BL/6N and C57BL/6J-scid/scid mice. We hypothesized that Vpr is able to block cell proliferation and induce apoptosis regardless of the drug resistance status of the tumors. Indeed, production of Vpr via adenoviral delivery to neuroblastoma cells caused G2 arrest and apoptosis in both drug naïve and DOX-resistant cells. In addition, pre-infection or intratumoral injection of vpr-expressing adenoviral particles into neuroblastoma tumors in SCID mice markedly inhibited tumor growth. Therefore, Vpr could possibly be used as a supplemental viral therapeutic agent for selective inhibition of tumor growth in anti-cancer therapy especially when other therapies stop working.

Enhanced anti-tumor activity of a new curcumin-related compound against melanoma and neuroblastoma cells

BackgroundSharing the common neuroectodermal origin, melanoma and neuroblastoma are tumors widely diffused among adult and children, respectively. Clinical prognosis of aggressive neuroectodermal cancers remains dismal, therefore the search for novel therapies against such tumors is warranted. Curcumin is a phytochemical compound widely studied for its antioxidant, anti-inflammatory and anti-cancer properties. Recently, we have synthesized and tested in vitro various curcumin-related compounds in order to select new anti-tumor agents displaying stronger and selective growth inhibition activity on neuroectodermal tumors.ResultsIn this work, we have demonstrated that the new α,β-unsaturated ketone D6 was more effective in inhibiting tumor cells growth when compared to curcumin. Normal fibroblasts proliferation was not affected by this treatment. Clonogenic assay showed a significant dose-dependent reduction in both melanoma and neuroblastoma colony formation only after D6 treatment. TUNEL assay, Annexin-V staining, caspases activation and PARP cleavage unveiled the ability of D6 to cause tumor cell death by triggering apoptosis, similarly to curcumin, but with a stronger and quicker extent. These apoptotic features appear to be associated with loss of mitochondrial membrane potential and cytochrome c release. In vivo anti-tumor activity of curcumin and D6 was surveyed using sub-cutaneous melanoma and orthotopic neuroblastoma xenograft models. D6 treated mice exhibited significantly reduced tumor growth compared to both control and curcumin treated ones (Melanoma: D6 vs control: P < 0.001 and D6 vs curcumin P < 0.01; Neuroblastoma: D6 vs both control and curcumin: P < 0.001).ConclusionsOur data indicate D6 as a good candidate to develop new therapies against neural crest-derived tumors.

Total Synthesis of Englerin A

Total synthesis of englerin A, a recently reported sesquiterpenoid exhibiting potent and selective growth inhibition against renal cancer cell lines, has been accomplished. The successful strategy featured a [5 + 2] cycloaddition reaction to cast the seven-membered oxabicyclic key intermediate in both racemic and optically active forms. Synthetic (+/-)-englerin A, (+/-)-englerin B, (+/-)-englerin B acetate, a hydroxy acetate, a tert-butyldimethylsilyl ether, and hydrogenated (+/-)-englerin A (31) were tested for their cytotoxicity against a selected panel of cancer cell lines, and the results are path-pointing to more focused structure-activity relationship studies.

Abstract 1024: Selective inhibition of bcl-2 gene expression and leukemic cell growth by the bcl-2 quadruplex-forming sequence

Abstract Bcl-2 (B-cell CLL/lymphoma 2), a mitochondrial membrane oncoprotein functioning as an inhibitor of apoptosis, is commonly overexpressed in a variety of hematological malignancies promoting apoptotic resistance and leading to poor patient survival. Inhibition of Bcl-2 may result in sustained regression of leukemia/lymphoma. Recently, the promoters of several cancer-related oncogenes, including Bcl-2, were found to contain sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The Bcl-2 quadruplex-forming sequence (Bcl-2 q, 23 bp) is located upstream of the P1 promoter of the Bcl-2 gene and is implicated in the regulation of Bcl-2 transcription, however, the biological role of this sequence remains unclear. We hypothesize that treatment of leukemia cells with the single-stranded, quadruplex-forming sequence (Bcl-2 q) induces cell death by inhibiting gene expression. To determine the biological role of the Bcl-2 q on leukemic cell proliferation, U937 and HL60 were treated with Bcl-2 q or the corresponding mutant sequence (MutBcl-2), which lacks runs of two or more guanines. Changes in cell proliferation were compared to levels of Bcl-2 protein expression. Quadruplex formation was confirmed by circular dichroism spectroscopy. Our results demonstrate that Bcl-2 q formed a stable parallel quadruplex structure, while the MutBcl-2 sequence did not form a quadruplex. Treatment of leukemia cells with Bcl-2 q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days as determined by MTT assay. No change in the growth of nontransformed stromal cells occurred in response to Bcl-2 q indicating the effect is specific for malignant cells. Two additional leukemia cell lines, Raji and K562 cells demonstrated dramatic growth inhibition while three solid tumor cell lines, DU145 (prostate), A549 (lung), and SK-BR-3 (breast) showed very little growth inhibition at concentrations up to10µM. Interestingly, the degree of growth inhibition induced by Bcl-2 q correlated with baseline Bcl-2 gene expression. Confocal and flow cytometry analysis of cells treated with FITC-labeled Bcl-2 q or MutBcl-2 showed prominent uptake and nuclear localization of Bcl-2 q not MutBcl-2 after 72 h. Inhibition of cell proliferation corresponded with decreased Bcl-2 protein expression. No significant change in the cell cycle was noted after 72 h, however, induction of cell death corresponded with a decrease in mitochondrial transmembrane potential. These results demonstrate striking growth inhibition in response to the Bcl-2 quadruplex-forming sequence related to inhibition of Bcl-2 expression. This oligonucleotide shows considerable therapeutic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1024.

Abstract LB-308: Protein kinase C beta selective inhibition induces apoptosis and arrests cell cycle in acute lymphoblastic leukemia cell lines

Abstract Acute lymphoblastic leukemia (ALL) is the most common leukemia in children and accounts for 20% of acute leukemia in adults. The intensive induction-consolidation-maintenance therapeutic regimens used currently have improved the 5-year disease free survival to around 80% in children and to 25%-40% in adults. The poorer response in adults is basically due to the inability to tolerate the intensive chemotherapy, and to the biology of adult disease which is associated with poor-risk prognostic factors. In the present era of target-specific therapy, PKCß targeting arose as a new, promising, and well tolerated treatment strategy in a variety of neoplasms, especially in B-cell malignancies. PKCß plays a major role in B-cell receptor signaling, but studies describing the role of PKCß in B-cell ALL are lacking. In the present study, we measured the sensitivity of a variety of B-cell ALL cell lines to PKC selective inhibition. Five cell lines were studied: RS4;11 and SEM-K2 [both Pro-B ALL with t(4;11)(q21;q23)], HB-1119 [Pre-B ALL with t(11;19)(q23;p13)] TOM-1 [Ph-positive Pro-B ALL with t(9;22)], and Reh [Pre-B ALL with t(12;21)(p13;q22)]. Cells were tested for PKCß1 and PKCß2 expression by immunoblot. Cell viability was measured in the presence of PKCß-selective inhibitor at concentrations of 1, 2.5, 5, 10, 20 and 30 μM for 48 hours with 10% fetal bovine serum (FBS). MTS assay was performed to quantify cell viability, and TUNEL assay with propidium iodide staining was used to detect apoptotic induction and effect on cell cycle. Results showed that all five cell lines express PKCß1 and PKCß2. Treatment with PKCß-selective inhibitor resulted in a dose-dependent inhibition of cell proliferation. RS4;11 and SEM-K2 cell lines showed the greatest sensitivity with statistically significant cell growth inhibition at inhibitor concentrations as low as 1 μM and an IC50 of 5 μM. Other cell lines required relatively higher inhibitor concentrations, with cell growth inhibition starting at 5 μM and an IC50 of 14 μM. The mechanism of PKC -selective cell growth inhibition was shown by flow cytometric TUNEL assay to involve apoptotic induction. No effect on cell cycle progression was observed, except in the Reh cell line. Apoptotic induction in RS4;11 and SEM-K2 cell lines was linked to the inhibition of GSK-3ß phosphorylation; flow cytometric analysis showed a 25% decrease in phosphorylated GSK-3ß after 24 hours incubation with the PKCß-selective inhibitor. These results indicate that PKCß plays an important role in the malignant process in B-cell ALL, and suggest that PKCß targeting should be considered as a potential treatment, whether in combination with the current regimens used or as a single agent monotherapy. Ongoing studies in our lab will detail the mechanism of PKCß inhibition and uncover possible relationships between PKCß signaling and t(4;11) (q21;q23). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-308.

Synthesis, structure and cytotoxicity studies of four-coordinate bis ( cis–bis(diphenylphosphino)ethene) gold(I) complexes, [Au(dppey) 2

Four-coordinate 1:2 gold(I) complex salts with cis–bis(diphenylphosphino)ethene, [Au(dppey) 2]X have been synthesized for X = PF 6 −, CF 3SO 3 −, BF 4 −, Cl −, Br − and BPh 4 − and characterized by NMR spectroscopy and electrospray mass spectrometry. Single crystal X-ray structure determinations show the BF 4 −, Cl − and Br − complexes to be isostructural, although with different degrees of hydration, while the BPh 4 − complex crystallizes as an acetone solvate with two molecules in the asymmetric unit. The Au(P–P) 2 core for the BF 4 −, Cl − and Br − complexes adopts D 2 symmetry with Au–P bond lengths 2.3980(7)–2.4009(7) Å and inter-ligand P–Au–P angles 114.78(2)–127.82(2))°. The Au(P–P) 2 core in the BPh 4 − complex is unsymmetrical with Au–P bond lengths 2.364(1)–2.420(1) Å and inter-ligand P–Au–P angles 104.76(5)–137.50(4)°. In vitro cytotoxicity studies show the PF 6 −, CF 3SO 3 −, BF 4 −, Cl −, Br − and I − complexes to be potent and selective growth inhibitors of the human cell lines MCF7 (hormone-dependent breast cancer), MDA-MB-231 (hormone-independent breast cancer), MM96L (melanoma), CI80-13S (cisplatin resistant ovarian cancer) and a normal cell line NFF (neonatal foreskin fibroblasts), achieving IC 50 values between 13 and 196 nM. The halogen and triflate salts were approximately twice as potent towards the MCF7 and MDA-MB-231 cell lines compared to the PF 6 − and BF 4 − derivatives; while the cytotoxicity of all complexes towards the sensitive CI80-13S and MM96L cancer cell lines was approximately 10-fold greater than that displayed towards the normal human cell line (NFF).

Selective Growth Inhibitory Effect of Biochanin A Against Intestinal Tract Colonizing Bacteria

Both bifidobacteria and clostridia are part of the natural gut microflora and while clostridia may be responsible for severe intestinal infections, bifidobacteria are probiotic microorganisms belonging to the most important prospective bacteria in the bowel. The antimicrobial activity of biochanin A was tested in vitro against six Bifidobacterium spp., and eight Clostridium spp. using the broth microdilution method. Biochanin A showed an inhibition against all clostridia in the range of minimum inhibitory concentrations (MIC) from 64 μg/mL (for Cl. clostridioforme, strains DSM 933 and I3) to 1,024 μg/mL (for Cl. perfringens, DSM 11778). No bifidobacteria were suppressed at four-fold higher concentration (MICs > 4,096) than MIC of Cl. perfringens. These results indicate selective growth inhibition of biochanin A and its potential use in antimicrobial prevention and/or protection.

Investigating the mechanisms for the opposing pH relationships of fungal and bacterial growth in soil

Soil pH is one of the most influential variables in soil, and is a powerful factor in influencing the size, activity and community structure of the soil microbial community. It was previously shown in a century old artificial pH gradient in an arable soil (pH 4.0–8.3) that bacterial growth is positively related to pH, while fungal growth increases with decreasing pH. In an attempt to elucidate some of the mechanisms for this, plant material that especially promotes fungal growth (straw) or bacterial growth (alfalfa) was added to soil samples of the pH gradient in 5-day laboratory incubation experiments. Also, bacterial growth was specifically inhibited by applying a selective bacterial growth inhibitor (bronopol) along the entire pH gradient to investigate if competitive interaction caused the shift in the decomposer community along the gradient. Straw benefited fungal growth relatively more than bacterial, and vice versa for alfalfa. The general pattern of a shift in fungal:bacterial growth with pH was, however, unaffected by substrate additions, indicating that lack of a suitable substrate was not the cause of the pH effect on the microbial community. In response to the bacterial growth inhibition by bronopol, there was stimulation of fungal growth up to pH 7, but not beyond, both for alfalfa and straw addition. However, the accumulation of ergosterol (an indicator of fungal biomass) during the incubation period after adding alfalfa increased at all pHs, indicating that fungal growth had been high at some time during the 5-day incubation following joint addition of alfalfa and bronopol. This was corroborated in a time-series experiment. In conclusion, the low fungal growth at high pH in an arable soil was caused to a large extent by bacterial competition, and not substrate limitation.

Selective inhibition of human leukemia cell growth and induction of cell cycle arrest and apoptosis by pseudolaric acid B

The leukemias account for the largest number of cases of childhood cancer and remain the primary cause of cancer-related mortality among children in the United States. There is a need for novel antileukemia agents due to toxicity and resistant to existing chemotherapeutic agents. In this study, the effects of pseudolaric acid B (PAB) on three human leukemia cell lines, acute promyelocytic leukemia HL-60 cells, acute lymphoblastic leukemia CCRF-CEM cells, and human chronic myeloid leukemia blast-phase K562 cells were investigated in vitro, compared to normal human peripheral blood mononuclear cells (PBMC). Cell viability was determined using CellTiter-Glo luminescent reagent. Colony formation was assessed by Microtitration cloning assay. Cell cycle analysis was carried out by flow cytometry. Tubulin polymerization was measured by recording the increase in absorbance. Inhibition of topoisomerase I (topo I) and topoisomerase II (topo II) enzyme activities was measured by DNA relaxation assay using topo I and II drug screening kit. Apoptosis was observed by DAPI staining assay and Caspase3/7 activities was measured using Caspase-Glo((R)) 3/7 assay kit. Pseudolaric acid B selectively inhibited the growth of human leukemia HL-60, CCRF-CEM and K562 cells, but not normal PBMC. PAB suppressed colony formation in HL-60 cells. Cell cycle analysis showed that PAB blocked the cell cycle at G(2)/M phase in HL-60 cells, suggesting that it suppresses mitosis. DNA topo I and topo II were not inhibited, but tubulin polymerization was inhibited. PAB-induced apoptosis and activated caspase-3/7 activity. This study indicates that PAB has a potential for use against leukemia and its effects might be mediated by inhibiting tubulin polymerization, preventing cell division and activating caspase-3, which leads to apoptosis.

Human telomeric G‐quadruplex: The current status of telomeric G‐quadruplexes as therapeutic targets in human cancer

The 3'-ends of human chromosomal DNA terminate in short single-stranded guanine-rich tandem-repeat sequences. In cancer cells, these are associated with the telomere-maintenance enzyme telomerase together with the end-binding protein hPOT1. Small molecules that can compete with these proteins and induce the single-stranded DNA to form quadruplex-ligand complexes are, in effect, able to expose these 3'-ends, which results in the activation of a DNA damage response and selective inhibition of cell growth. Several of these G-quadruplex binding molecules have shown promising anticancer activity in tumour xenograft models, which indicate that the approach may be applicable to the treatment of a wide range of human cancers. This minireview summarizes the available data on these compounds and the challenges posed for drug discovery.

Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo

BackgroundOncolytic virotherapy is an attractive drug platform of cancer gene therapy, but efficacy and specificity are important prerequisites for success of such strategies. Previous studies determined that Apoptin is a p53 independent, bcl-2 insensitive apoptotic protein with the ability to specifically induce apoptosis in tumor cells. Here, we generated a conditional replication-competent adenovirus (CRCA), designated Ad-hTERT-E1a-Apoptin, and investigated the effectiveness of the CRCA a gene therapy agent for further clinical trials.ResultsThe observation that infection with Ad-hTERT-E1a-Apoptin significantly inhibited growth of the melanoma cells, protecting normal human epidermal melanocytes from growth inhibition confirmed cancer cell selective adenoviral replication, growth inhibition, and apoptosis induction of this therapeutic approach. The in vivo assays performed by using C57BL/6 mice containing established primary or metastatic tumors expanded the in vitro studies. When treated with Ad-hTERT-E1a-Apoptin, the subcutaneous primary tumor volume reduction was not only observed in intratumoral injection group but in systemic delivery mice. In the lung metastasis model, Ad-hTERT-E1a-Apoptin effectively suppressed pulmonary metastatic lesions. Furthermore, treatment of primary and metastatic models with Ad-hTERT-E1a-Apoptin increased mice survival.ConclusionsThese data further reinforce the previously research showing that an adenovirus expressing Apoptin is more effective and advocate the potential applications of Ad-hTERT-E1a-Apoptin in the treatment of neoplastic diseases in future clinical trials.

海洋生物からの医薬シーズの探索

In search of new pharmaceutically valuable substances from marine organisms, we have been engaged in chemical studies 1] structural elucidation; 2] asymmetric total synthesis; 3] elucidation of pharmacophore by analysis of structure–activity relationship; 4] action mechanism and search for target protein on the bioactive substances, which were isolated from marine invertebrates and marine microorganisms at Japanese coasts and Indonesian coral reefs on the guidance of new practical bioassay. a) Anti–angiogenic substance: selective growth–inhibitor against human umbilical vein endothelial cells (HUVECs) (e.g. cortistatin A: 9(10–19)–abeo–androstane–type steroidal alkaloid having isoquinoline unit) b) Hypoxia–selective growth inhibitor: growth inhibitor against tumor cells under hypoxic environment selectively (e.g. furospinosulin–1: furanosesterterpene) c) Anti–mycobacterial substances, which are effective to both active and non–replicating persistent (NRP) states of tuberculosis (e.g. halicyclamine A: macrocyclic alkaloid)