Probing the impact of quercetin-7-O-glucoside on influenza virus replication influence

Gansukh, E.,Kazibwe, Z.,Pandurangan, M.,Judy, G.,Kim, D.H. G. Fischer 2016 Phytomedicine Vol.23 No.9

<P>Background: Influenza virus is still at large and seriously affects social welfare and health. Dianthus superbus is a well-known medicinal plant widely used in Mongolian and Chinese traditional medicine for anti-inflammatory purposes. Purpose: To investigate the influence of this novel herbal medicinal product over virus infection and virus-induced symptoms Method: Quercetin-7-O-glucoside was isolated by bioassay (anti-influenza)-guided fractionation. The structural elucidation was made with 1H-NMR and 13C-NMR. Influenza A/Vic/3/75 (H3N2), A/PR/8/34 (H1N1), B/Maryland/1/59 and B/Lee/40 viruses were used for the evaluation of the antiviral activity. Virus-induced reactive oxygen species and autophagy formation levels were studied. The antiviral mechanism was elucidated via time-dependent, pre-, post-incubation assay methods. The viral RNA replication inhibition of Q7G was analyzed using quantitative RT-PCR method. The blocking of polymerase basic protein subunits of influenza viral RNA polymerase by Q7G was detected by in silico molecular docking assays using AutoDock Vina program with m(7)GTP. Additionally, Q7G was tested against M-MuLV RNA polymerase. Results: Q7G was not cytotoxic (CC50 > 100 mu g/ml) in MDCK cells and it showed 3.1 mu g/ml, 6.61 mu g/ml, 8.19 mu g/ml and 5.17 mu g/ml IC50 values against influenza A/PR/8/34, A/Vic/3/75, B/Lee/40 and B/Maryland/1/59 virus strains, respectively. Treatment of Q7G highly reduced ROS and autophagy formation induced by influenza virus infection. Q7G did not reduce NA activity and did not directly interact with the virus particles. Since viral RNA synthesis was blocked by treatment of Q7G. We targeted viral RNA polymerase for further probing. Interestingly, the binding energy of Q7G on viral PB2 protein was -9.1 kcal/mol and was higher than m(7)GTP recorded as -7.5 kcal/mol. It also was observe to block M-MuLV RNA polymerase. Conclusion: Isolated compound Q7G showed strong inhibition activity against influenza A and B viruses. It also reduced virus-induced ROS and autophagy formation. Q7G does not directly bind to the virus particles and did not affect NA activity. These results indicated that Q7G inhibits viral RNA polymerase, and that it occupies the binding site of m(7)GTP on viral PB2 protein. (C) 2016 Elsevier GmbH. All rights reserved.</P>

Daidzein causes cell cycle arrest at the G1 and G2/M phases in human breast cancer MCF-7 and MDA-MB-453 cells

Choi, E.J.,Kim, G.-H. G. Fischer 2008 Phytomedicine Vol.15 No.9

We examined the mechanisms by which daidzein inhibits the growth of breast cancer cells. First, we investigated its antiproliferative effects in MCF-7 and MDA-MB-453 cells exposed to 1-100μM daidzein for 24, 48, or 72h. Daidzein significantly inhibited cell proliferation in a dose- and time-dependent manner (p<0.05) and resulted in significant cell cycle arrest in the G1 and G2/M phases after 72h of treatment at concentrations over 5 and 10μM in MCF-7 and MDA-MB-453 cells, respectively (p<0.05). In addition, daidzein caused the accumulation of cells in sub-G0 phase in a dose-dependent manner in MDA-MB-453 (p<0.05), but not MCF-7, cells. As another biomarker of apoptosis induction, caspase-9 activity was significantly increased by daidzein in both cells. To investigate the effects of daidzein on the proteins regulating cell cycle arrest, cells were treated with 100μM daidzein for 72h. Similar changes in the expression of regulatory proteins were detected in both cells. Daidzein treatment resulted in decreases in cyclin D, CDK2, and CDK4, whereas the expression of CDK6 and cyclin E was unchanged. The protein expression of CDK1 related to the G2/M phase decreased markedly with daidzein treatment, whereas slight expression of cyclins A and B occurred. Daidzein treatment increased the expression of the CDK inhibitors p21<SUP>Cip1</SUP> and p57<SUP>Kip2</SUP>, but not that of p27<SUP>Kip1</SUP>. Thus, daidzein exerts its anticancer effects in human breast cancer cells via cell cycle arrest at the G1 and G2/M phases.

Antiviral activity of raoulic acid from Raoulia australis against Picornaviruses

Choi, H.J.,Lim, C.H.,Song, J.H.,Baek, S.H.,Kwon, D.H. G. Fischer 2009 Phytomedicine Vol.16 No.1

RNA viruses are a major source of respiratory diseases worldwide. The lack of effective therapeutical treatment underlines the importance of research for new antiviral compounds. Raoulic acid is a principal ingredient of the plant Raoulia australis Hook. F. Antiviral assay using cytopathic effect (CPE) reduction method showed that raoulic acid possessed strong antiviral activity against human rhinovirus 2 (HRV2) with a 50% inhibition concentration (IC<SUB>50</SUB>) value of less than 0.1μg/ml, human rhinovirus 3 (HRV3) with a IC<SUB>50</SUB> value of 0.19μg/ml, coxsackie B3 (CB3) virus with IC<SUB>50</SUB> values of 0.33μg/ml, coxsackie B4 (CB4) virus with IC<SUB>50</SUB> values of 0.40μg/ml, and enterovirus 71 (EV71) virus with IC<SUB>50</SUB> values of less than 0.1μg/ml. However, the compound did not possess antiviral activity against influenza A (Flu A/PR, Flu A/WS, H1N1) and B viruses at four concentrations ranging from 0.1 to 100μg/ml.

The D-type cyclin gene (Nicta;CycD3;4) controls cell cycle progression in response to sugar availability in tobacco

Kwon, H.K.,Wang, M.H. G. Fischer 2011 Journal of plant physiology Vol.168 No.2

D-type cyclins play key roles in the G1-to-S phase transition that occurs in response to nutrient and hormonal signals. In higher plants, sucrose is the major transported carbon source, and is likely to be a major determinant of cell division. To elucidate how sugar affects on the regulation of cell cycle machinery and plant development, we examined the role of carbon sources on the expression of cell-cycle-related genes in transgenic tobacco plants overexpressing Nicta;CycD3;4. The Nicta;CycD3;4 overexpressed transgenic plants showed accelerated growth and remarkable increase in the number of cells in the S and G2 phases in response to sucrose concentrations. Increased expressions level of Nicta;CycD3;4 gene was observed in transgenic tobacco plants grown on ½ strength MS medium supplemented with a high concentration of sugar. Moreover, the expression of sugar-sensing-related gene, invertase, was also maintained at a high level in transgenic tobacco plants with elevated sugar availabiliy. These findings indicate that sugar availability plays a role during the G1 phase and the transition of the G1-to-S phase of cell cycle by controlling the expression of Nicta;CycD3;4.

Kurarinol, tyrosinase inhibitor isolated from the root of Sophora flavescens

Ryu, Y.B.,Westwood, I.M.,Kang, N.S.,Kim, H.Y.,Kim, J.H.,Moon, Y.H.,Park, K.H. G. Fischer 2008 Phytomedicine Vol.15 No.8

It is well known that flavanones, sophoraflavanone G 1, kurarinone 2, and kurarinol 3, from the root of Sophora flavescens, have extremely strong tyrosinase inhibitory activity. This study delineates the principal pharmacological features of kurarinol 3 that lead to inhibition of the oxidation of l-tyrosine to melanin by mushroom tyrosinase (IC<SUB>50</SUB> of 100nM). The inhibition kinetics analyses unveil that compounds 1 and 2 are noncompetitive inhibitors. However similar analysis shows kurarinol 3 to be a competitive inhibitor. Compounds 1 and 2 exhibited potent antibacterial activity with 10μg/disk against Gram-positive bacteria, whereas kurarinol 3 did not ostend any antibacterial activity. Interestingly, kurarinol 3 inhibits production of melanin in S. bikiniensis without affecting the growth of microorganism. It is thus distinctly different from the other tyrosinase inhibitors 1 and 2. In addition, kurarinol 3 manifests relatively low cytotoxic activity (EC<SUB>50</SUB>>30μM) compared to 1 and 2. To account for these observations, we conducted molecular modeling studies. These suggested that the lavandulyl group within 3 is instrumental in the interaction with the enzyme. More specifically, the terminal hydroxy function within the lavandulyl group is most important for optimal binding.

Mannitol induces the conversion of conidia to chlamydospore-like structures that confer enhanced tolerance to heat, drought, and UV in Gibberella zeae

Son, H.,Lee, J.,Lee, Y.W. G. Fischer 2012 MICROBIOLOGICAL RESEARCH Vol.167 No.10

Fungi use mannitol to store carbon, balance redox, and mannitol serves as an antioxidant. Several fungi also increase stress tolerance by accumulating mannitol. The results of this study showed that conidia of the cereal head blight fungus Gibberella zeae were readily changed to chlamydospore-like structures (CLS) in cultures supplemented with high amounts of mannitol. CLS cellular features were atypical of chlamydospores, but accumulated high levels of glycogen, lipids, and chitin in the cytoplasm. In addition, CLS exhibited increased tolerance to environmental stresses, including UV, heat, and drought compared to normal conidia. Molecular approaches revealed that several genes associated with lipid metabolism, signal transduction, acetyl-CoA production, and chitin synthesis were involved in CLS formation. This is the first report to characterize conidia modifications similar to chlamydospores in G. zeae applying histological and molecular approaches. The results suggest CLS serve a role in G. zeae survival strategies under hot and dry field conditions.

In vitro and in vivo antifungal efficacy of plant based lawsone against Fusarium oxysporum species complex

Dananjaya, S.H.S.,Udayangani, R.M.C.,Shin, S.Y.,Edussuriya, M.,Nikapitiya, C.,Lee, J.,De Zoysa, M. G. Fischer 2017 MICROBIOLOGICAL RESEARCH Vol.201 No.-

<P>Fusarium oxysporum is an ascomycete facultative fungus which generally affects to plants. However, it is recently known as a serious emerging opportunistic pathogen of human and other animals. F. oxysporum shows broad resistance to commonly used antifungal agents and therefore development of alternative therapeutic agents is required. In this study, we investigated the antifungal efficacy of plant based natural lawsone against pathogenic F. oxysporum. Antifungal susceptibility test determined the concentration dependent growth inhibition of lawsone against F. oxysporum with minimum inhibitory concentration (MIC) at 100 mu g/mL. Ultra-structural analysis indicates the prominent damage on cell wall of the mycelium after lawsone treatment, and suggests that it could increase the membrane permeability and disintegration of cells leading to cellular death. Propidium iodide (PI) uptake assay results showed the higher level of cell death in lawsone treated F. oxysporum which further confirms the loss of plasma membrane integrity. Also, detection of reactive oxygen species (ROS) using DCFH-DA has clearly indicated that lawsone (100 mu g/mL) can induce the ROS level in the filaments of F. oxysporum. MTT assay results showed the loss of viability and germination capacity of F. oxysporum spores by lawsone in concentration dependent manner. Moreover, lawsone treatment induced the mRNA expression of two autophagy related genes (ATG1 and ATG8) indicating that lawsone may activate the autophagy related pathways in F. oxysporum due to the oxidative stress generated by ROS. F. oxysporum infected zebrafish has recovered after lawsone therapy as a topical treatment suggesting that lawsone is a potential natural antifusariosis agent.</P>

12-O-Tetradecanoyl phorbol-13-acetate (TPA)-induced growth arrest is increased by silibinin by the down-regulation of cyclin B1 and cdc2 and the up-regulation of p21 expression in MDA-MB231 human breast cancer cells

Kim, S.,Lee, H.S.,Lee, S.K.,Kim, S.H.,Hur, S.M.,Kim, J.S.,Kim, J.H.,Choe, J.H.,Shin, I.,Yang, J.H.,Lee, J.E.,Nam, S.J. G. Fischer 2010 Phytomedicine Vol.17 No.14

TPA is a potent regulator of cell growth, including cell proliferation and differentiation. In this study, we determined the effect of silibinin on TPA-induced growth arrest in breast cancer cells. Silibinin increased growth arrest of the G2/M phase in a dose-dependent fashion. Silibinin decreased the basal level of cyclin B1 and cdc2 expression, which is involved in S phase and G2/M transition. In addition, TPA-induced G2/M phase arrest was increased by silibinin. Under the same conditions, TPA-induced down-regulation of cyclin B1 and cdc2 was decreased by silibinin. In contrast, TPA-induced p21 expression was further increased by silibinin. To determine the regulatory mechanism of TPA-induced growth arrest, we pretreated cells with various inhibitors, such as UO126, SB203580, and LY294002. Interestingly, TPA-induced growth arrest was significantly increased by LY294002, but not by UO126 and SB203580. In addition, TPA-induced down-regulation of cyclin B1 was inhibited by LY294002; however, the basal level of p21 was increased by TPA and TPA-induced p21 expression was further increased by LY294002. Finally, adenoviral constitutively active-Akt (Ad-CA-Akt) overexpression regulated the up-regulation of cyclin B1 and the down-regulation of p21. Therefore, we have demonstrated that silibinin has an additive effect on TPA-induced growth arrest through the PI-3-kinase/Akt-dependent pathway.

Pinoresinol-4,4'-di-O-β-d-glucoside from Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse embryo fibroblasts

Do, K.H.,Choi, Y.W.,Kim, E.K.,Yun, S.J.,Kim, M.S.,Lee, S.Y.,Ha, J.M.,Kim, J.H.,Kim, C.D.,Son, B.G.,Kang, J.S.,Khan, I.A.,Bae, S.S. G. Fischer 2009 Phytomedicine Vol.16 No.6

Lignans are major constituents of plant extracts and have important pharmacological effects on mammalian cells. Here we showed that pinoresinol-4,4'-di-O-β-d-glucoside (PDG) from Valeriana officinalis induced calcium mobilization and cell migration through the activation of lysophosphatidic acid (LPA) receptor subtypes. Stimulation of mouse embryo fibroblast (MEF) cells with 10μM PDG resulted in strong stimulation of MEF cell migration and the EC<SUB>50</SUB> was about 2μM. Pretreatment with pertussis toxin (PTX), an inhibitor of G<SUB>i</SUB> protein, completely blocked PDG-induced cell migration demonstrating that PDG evokes MEF cell migration through the activation of the G<SUB>i</SUB>-coupled receptor. Furthermore, pretreatment of MEF cells with Ki16425 (10μM), which is a selective antagonist for LPA<SUB>1</SUB> and LPA<SUB>3</SUB> receptors, completely blocked PDG-induced cell migration. Likewise, PDG strongly induced calcium mobilization, which was also blocked by Ki16425 in a dose-dependent manner. Prior occupation of the LPA receptor with LPA itself completely blocked PDG-induced calcium mobilization. Finally, PDG-induced MEF cell migration was attenuated by pretreatment with a phosphatidylinositol 3-kinase (PI3K) inhibitor such as LY294002. Cells lacking downstream mediator of PI3K such as Akt1 and Akt2 (DKO cells) showed loss of PDG-induced migration. Re-expression of Akt1 (but not Akt2) completely restored PDG-induced DKO cell migration. Given these results, we conclude that PDG is a strong inducer of cell migration. We suggest that the pharmacological action of PDG may occur through the activation of an LPA receptor whereby activation of PI3K/Akt signaling pathway mediates PDG-induced MEF cell migration.

The alkaloid Berberine inhibits the growth of Anoikis-resistant MCF-7 and MDA-MB-231 breast cancer cell lines by inducing cell cycle arrest

Kim, J.B.,Yu, J.H.,Ko, E.,Lee, K.W.,Song, A.K.,Park, S.Y.,Shin, I.,Han, W.,Noh, D.Y. G. Fischer 2010 Phytomedicine Vol.17 No.6

Berberine is a pure phenanthren alkaloid isolated from the roots and bark of herbal plants such as Berberis, Hydrastis canadensis and Coptis chinensis. Berberine has been established to inhibit the growth of breast cancer cells, but its effects on the drug resistance and anoikis-resistance of breast cancer cells have yet to be elucidated. Anoikis, or detachment-induced apoptosis, may prevent cancer progression and metastasis by blocking signals necessary for survival of localized cancer cells. Resistance to anoikis is regarded as a prerequisite for metastasis; however, little is known about the role of berberine in anoikis-resistance. We established anoikis-resistant cells from the breast cancer cell lines MCF-7 and MDA-MB-231 by culturing them on a Poly-Hema substratum. We then investigated the effects of berberine on the growth of these cells. The anoikis-resistant cells had a reduced growth rate and were more invasive than their respective adherent cell lines. The effect of berberine on growth was compared to that of doxorubicine, which is a drug commonly used to treat breast cancer, in both the adherent and anoikis-resistant cell lines. Berberine promoted the growth inhibition of anoikis-resistant cells to a greater extent than doxorubicine treatment. Treatment with berberine-induced cell cycle arrest at G0/G1 in the anoikis-resistant MCF-7 and MDA-MB-231 cells as compared to untreated control cells. In summary, these results revealed that berberine can efficiently inhibit growth by inducing cell cycle arrest in anoikis-resistant MCF-7 and MDA-MB-231 cells. Further analysis of these phenotypes is essential for understanding the effect of berberine on anoikis-resistant breast cancer cells, which would be relevant for the therapeutic targeting of breast cancer metastasis.