Putrescine differently influences the effect of salt stress on polyamine metabolism and ethylene synthesis in rice cultivars differing in salt resistance

Effects of salt stress on polyamine metabolism and ethylene production were examined in two rice (Oryza sativa L.) cultivars [I Kong Pao (IKP), salt sensitive; and Pokkali, salt resistant] grown for 5 d and 12 d in nutrient solution in the presence or absence of putrescine (1 mM) and 0, 50, and 100 mM NaCl. The salt-sensitive (IKP) and salt-resistant (Pokkali) cultivars differ not only in their mean levels of putrescine, but also in the physiological functions assumed by this molecule in stressed tissues. Salt stress increased the proportion of conjugated putrescine in salt-resistant Pokkali and decreased it in the salt-sensitive IKP, suggesting a possible protective function in response to NaCl. Activities of the enzymes ornithine decarboxylase (ODC; EC 4.1.1.17) and arginine decarboxylase (ADC; EC 4.1.1.19) involved in putrescine synthesis were higher in salt-resistant Pokkali than in salt-sensitive IKP. Both enzymes were involved in the response to salt stress. Salt stress also increased diamine oxidase (DAO; 1.4.3.6) and polyamine oxidase (PAO EC 1.5.3.11) activities in the roots of salt-resistant Pokkali and in the shoots of salt-sensitive IKP. Gene expression followed by reverse transcription-PCR suggested that putrescine could have a post-translational impact on genes coding for ADC (ADCa) and ODC (ODCa and ODCb) but could induce a transcriptional activation of genes coding for PAO (PAOb) mainly in the shoot of salt-stressed plants. The salt-resistant cultivar Pokkali produced higher amounts of ethylene than the salt-sensitive cultivar IKP, and exogenous putrescine increased ethylene synthesis in both cultivars, suggesting no direct antagonism between polyamine and ethylene pathways in rice

Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

INTRODUCTION: Rapamycin, an inhibitor of the serine/threonine kinase target of rapamycin, induces G(1 )arrest and/or apoptosis. Although rapamycin and its analogues are attractive candidates for cancer therapy, their sensitivities with respect to growth inhibition differ markedly among various cancer cells. Using human breast carcinoma cell line MCF-7 as an experimental model system, we examined the growth-inhibitory effects of combinations of various agents and rapamycin to find the agent that most potently enhances the growth-inhibitory effect of rapamycin. METHOD: We evaluated the growth-inhibitory effect of rapamycin plus various agents, including cotylenin A (a novel inducer of differentiation of myeloid leukaemia cells) to MCF-7 cells, using either MTT assay or trypan blue dye exclusion test. The cell cycle was analyzed using propidium iodide-stained nuclei. Expressions of several genes in MCF-7 cells with rapamycin plus cotylenin A were studied using cDNA microarray analysis and RT-PCR. The in vitro results of MCF-7 cells treated with rapamycin plus cotylenin A were further confirmed in vivo in a mouse xenograft model. RESULTS: We found that the sensitivity of rapamycin to MCF-7 cells was markedly affected by cotylenin A. This treatment induced growth arrest of the cells at the G(1 )phase, rather than apoptosis, and induced senescence-associated β-galactosidase activity. We examined the gene expression profiles associated with exposure to rapamycin and cotylenin A using cDNA microarrays. We found that expressions of cyclin G(2), transforming growth factor-β-induced 68 kDa protein, BCL2-interacting killer, and growth factor receptor-bound 7 were markedly induced in MCF-7 cells treated with rapamycin plus cotylenin A. Furthermore, combined treatment with rapamycin and cotylenin A significantly inhibited the growth of MCF-7 cells as xenografts, without apparent adverse effects. CONCLUSION: Rapamycin and cotylenin A cooperatively induced growth arrest in breast carcinoma MCF-7 cells in vitro, and treatment with rapamycin and cotylenin A combined more strongly inhibited the growth of MCF-7 cells as xenografts in vivo than treatment with rapamycin or cotylenin A alone, suggesting that this combination may have therapeutic value in treating breast cancer. We also identified several genes that were markedly modulated in MCF-7 cells treated with rapamycin plus cotylenin A

Disparate Impact of Butyroyloxymethyl Diethylphosphate (AN-7), a Histone Deacetylase Inhibitor, and Doxorubicin in Mice Bearing a Mammary Tumor

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Dox- toxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of γH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in γH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in γH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection

Nitric Oxide Controls Constitutive Freezing Tolerance in Arabidopsis by Attenuating the Levels of Osmoprotectants, Stress-Related Hormones and Anthocyanins

[EN] Plant tolerance to freezing temperatures is governed by endogenous constitutive components and environmental inducing factors. Nitric oxide (NO) is one of the endogenous components that participate in freezing tolerance regulation. A combined metabolomic and transcriptomic characterization of NO-deficient nia1,2noa1¿2 mutant plants suggests that NO acts attenuating the production and accumulation of osmoprotective and regulatory metabolites, such as sugars and polyamines, stress-related hormones, such as ABA and jasmonates, and antioxidants, such as anthocyanins and flavonoids. Accordingly, NO-deficient plants are constitutively more freezing tolerant than wild type plants.This work was supported by grants BIO2011-27526 and BIO2014-56067-P from the Spanish Ministry of Economy and Competitiveness and FEDER funds. We thank support and comments from Janice Jones and Danny Alexander (Metabolon Inc., USA) on metabolomic analyses.Costa-Broseta, Á.; Perea-Resa, C.; Castillo López Del Toro, MC.; Ruíz, MF.; Salinas, J.; Leon Ramos, J. (2018). Nitric Oxide Controls Constitutive Freezing Tolerance in Arabidopsis by Attenuating the Levels of Osmoprotectants, Stress-Related Hormones and Anthocyanins. Scientific Reports. 8. https://doi.org/10.1038/s41598-018-27668-8S8Janská, A., Marsík, P., Zelenková, S. & Ovesná, J. Cold stress and acclimation - what is important for metabolic adjustment? Plant Biol (Stuttg) 12, 395–405 (2010).Eremina, M., Rozhon, W. & Poppenberger, B. Hormonal control of cold stress responses in plants. Cell Mol Life Sci 73, 797–810 (2016).Winkel-Shirley, B. Biosynthesis of flavonoids and effects of stress. Curr Opin Plant Biol 5, 218–223 (2002).Cuevas, J. C. et al. Putrescine is involved in Arabidopsis freezing tolerance and cold acclimation by regulating abscisic acid levels in response to low temperature. Plant Physiol 148, 1094–105 (2008).Chen, M. & Thelen, J. J. Acyl-lipid desaturase 1 primes cold acclimation response in Arabidopsis. Physiol Plant 158, 11–22 (2016).Takahashi, D., Kawamura, Y. & Uemura, M. Cold acclimation is accompanied by complex responses of glycosylphosphatidylinositol (GPI)-anchored proteins in Arabidopsis. J Exp Bot 67, 5203–5215 (2016).van Buer, J., Cvetkovic, J. & Baier, M. Cold regulation of plastid ascorbate peroxidases serves as a priming hub controlling ROS signaling in Arabidopsis thaliana. BMC Plant Biol 16(1), 163 (2016).Zhao, M. G., Chen, L., Zhang, L. L. & Zhang, W. H. Nitric reductase dependent nitric oxide production is involved in cold acclimation and freezing tolerance in Arabidopsis. Plant Physiol 151, 755–767 (2009).Puyaubert, J. & Baudouin, E. New clues for a cold case: nitric oxide response to low temperature. Plant Cell & Environ 37, 2623–2630 (2014).Siddiqui, M. H., Al-Whaibi, M. H. & Basalah, M. O. Role of nitric oxide in tolerance of plants to abiotic stress. Protoplasma 248, 447–455 (2011).Arasimowicz-Jelonek, M. & Floryszak-Wieczorek, J. Nitric oxide: an effective weapon of the plant or the pathogen? Mol. Plant Pathol. 15, 406–416 (2014).Gupta, K. J., Fernie, A. R., Kaiser, W. M. & van Dongen, J. T. On the origins of nitric oxide. Trends Plant Sci. 16, 160–168 (2011).Mur, L. A. et al. Nitric oxide in plants: an assessment of the current state of knowledge. AoB Plants 5, pls052 (2013).Thomas, D. D. Breathing new life into nitric oxide signaling: A brief overview of the interplay between oxygen and nitric oxide. Redox Biol. 5, 225–33 (2015).Correa-Aragunde, N., Foresi, N. & Lamattina, L. Nitric oxide is a ubiquitous signal for maintaining redox balance in plant cells: regulation of ascorbate peroxidase as a case study. J. Exp. Bot. 66, 2913–2921 (2015).Groβ, F., Durner, J. & Gaupels, F. Nitric oxide, antioxidants and prooxidants in plant defence responses. Front. Plant Sci. 4, 419 (2013).Astier, J. & Lindermayr, C. Nitric oxide-dependent posttranslational modification in plants: an update. Int. J. Mol. Sci. 13, 15193–15208 (2012).Hess, D. T. & Stamler, J. S. Regulation by S-nitrosylation of protein post-translational modification. J. Biol. Chem. 287, 4411–4418 (2012).Guerra, D. D. & Callis, J. Ubiquitin on the move: the ubiquitin modification system plays diverse roles in the regulation of endoplasmic reticulum- and plasma membrane-localized proteins. Plant Physiol. 160, 56–64 (2012).Cantrel, C. et al. Nitric oxide participates in cold-responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana. New Phytol. 189, 415–427 (2011).Lozano-Juste, J. & León, J. Enhanced abscisic acid-mediated responses innia1,2noa1-2 triple mutant impaired in NIA/NR- and AtNOA1-dependent nitric oxide biosynthesis in Arabidopsis. Plant Physiol. 152, 891–903 (2010).Gibbs, D. J. et al. Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors. Mol. Cell 53, 369–379 (2014).Lee, B. H., Henderson, D. A. & Zhu, J. K. The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. Plant Cell 17, 3155–3175 (2005).Kilian, J. et al. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J. 50, 347–363 (2007).Hu, Y., Jiang, L., Wang, F. & Yu, D. Jasmonate regulates the inducer of cbf expression-C-repeat binding factor/DRE binding factor1 cascade and freezing tolerance in Arabidopsis. Plant Cell 25, 2907–2924 (2013).Lee, H. G. & Seo, P. J. The MYB96-HHP module integrates cold and abscisic acid signaling to activate the CBF-COR pathway in Arabidopsis. Plant J. 82, 962–977 (2015).Kasukabe, Y. et al. Overexpression of spermidine synthase enhances tolerance to multiple environmental stresses and up-regulates the expression of various stress-regulated genes in transgenic Arabidopsis thaliana. Plant & Cell Physiol 45, 712–722 (2004).Korn, M., Peterek, S., Mock, H. P., Heyer, A. G. & Hincha, D. K. Heterosis in the freezing tolerance, and sugar and flavonoid contents of crosses between Arabidopsis thaliana accessions of widely varying freezing tolerance. Plant Cell & Environ. 31, 813–827 (2008).Guy, C., Kaplan, F., Kopka, J., Selbig, J. & Hincha, D. K. Metabolomics of temperature stress. Physiol. Plant. 132, 220–235 (2008).Berger, S. et al. Enzymatic and non enzymatic lipid peroxidation in leaf development. Biochem. Biophys. Acta 1533, 266–276 (2001).Yoshida, Y., Umeno, A. & Shichiri, M. Lipid peroxidation biomarkers for evaluating oxidative stress and assessing antioxidant capacity in vivo. J Clin. Biochem. Nutr. 52, 9–16 (2013).Catalá, R. et al. The Arabidopsis 14-3-3 protein RARE COLD INDUCIBLE 1A links low-temperature response and ethylene biosynthesis to regulate freezing tolerance and cold acclimation. Plant Cell 26, 3326–3342 (2014).Tähtiharju, S. & Palva, T. Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana. Plant J. 26, 461–470 (2001).Kawamura, Y. & Uemura, M. Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation. Plant J. 36, 141–154 (2003).Xin, Z. & Browse, J. Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proc. Natl. Acad. Sci. USA 95, 7799–7804 (1998).Nanjo, T. et al. Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett. 461, 205–210 (1999).Zuther, E., Schulz, E., Childs, L. H. & Hincha, D. K. Clinal variation in the non-acclimated and cold-acclimated freezing tolerance of Arabidopsis thaliana accessions. Plant Cell & Environ. 35, 1860–1878 (2012).Alcázar, R., García-Martínez, J. L., Cuevas, J. C., Tiburcio, A. F. & Altabella, T. Overexpression of ADC2 in Arabidopsis induces dwarfism and late-flowering through GA deficiency. Plant J. 43, 425–436 (2005).Alet, A. I. et al. Putrescine accumulation in Arabidopsis thaliana transgenic lines enhances tolerance to dehydration and freezing stress. Plant Signal. & Behav. 6, 278–286 (2011).Nägele, T., Stutz, S., Hörmiller, I. I. & Heyer, A. G. Identification of a metabolic bottleneck for cold acclimation in Arabidopsis thaliana. Plant J. 72, 102–114 (2012).Krol, M. et al. Low-temperature stress and photoperiod affect an increased tolerance to photoinhibition in Pinus banksiana seedlings. Canadian Journal of Botany 73, 1119–1127 (1995).Harvaux, M. & Kloppstech, K. The protective functions of carotenoid and flavonoid pigments against excess visible radiation at chilling temperature investigated in Arabidopsis npq and tt mutants. Planta 213, 953–966 (2001).Schulz, E., Tohge, T., Zuther, E., Fernie, A. R. & Hincha, D. K. Flavonoids are determinants of freezing tolerance and cold acclimation in Arabidopsis thaliana. Sci. Rep. 6, 34027 (2016).Llorente, F., Oliveros, J. C., Martínez-Zapater, J. M. & Salinas, J. A freezing-sensitive mutant of Arabidopsis, frs1, is a new aba3 allele. Planta 211, 648–655 (2000).Lozano-Juste, J., Colom-Moreno, R. & León, J. In vivo protein tyrosine nitration in Arabidopsis thaliana. J. Exp. Bot. 62, 3501–3517.Gill, S. S. & Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem. 48, 909–930 (2010).Begara-Morales, J. C. et al. Antioxidant Systems are Regulated by Nitric Oxide-Mediated Post-translational Modifications (NO-PTMs). Front. Plant Sci. 7, 152 (2016).Castillo, M. C. & León, J. Expression of the beta-oxidation gene 3-ketoacyl-CoA thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf senescence in Arabidopsis. J. Exp. Bot. 59, 2171–2179 (2008).Guo, F. Q., Okamoto, M. & Crawford, N. M. Identification of a plant nitric oxide synthase gene involved in hormonal signaling. Science 302, 100–103 (2003).Solfanelli, C., Poggi, A., Loreti, E., Alpi, A. & Perata, P. Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis. Plant Physiol. 140, 637–646 (2006).Seo, M., Jikumaru, Y. & Kamiya, Y. Profiling of Hormones and Related Metabolites in Seed Dormancy and Germination Studies. Meth. Mol. Biol. 773, 99–111 (2011)