Screening for natural anticancer agents using a fission yeast bioassay

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This is an Accepted Manuscript of an article published by Elsevier in Phytochemistry Letters on May 2014, available at: https://doi.org/10.1016/j.phytol.2013.09.011 .” 1 Screening for new natural anticancer drugs using a fission yeast bioassay Ángeles Sánchez-Picó a, Antonio J. León-González b, Carmen Martín-Cordero b, Rafael R. Daga a* a Centro Andaluz de Biologídel Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas, Junta de Andalucía, Sevilla, Spain bDepartamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, Spain (*) Corresponding author contact information: email: [email protected] Address: Universidad Pablo de Olavide Carretera de Utrera, Km1, 41013 Seville, Spain Phone: +34 954 977551 Fax: +34 954349376 Abstract A bio-guided screening to search for new anticancer drugs was carried out with extracts from several plant species from Andalusia (Spain), using the fission yeast Schizosaccharomyces pombe as model. The fission yeast is a valuable tool for screening of natural products. Since the major signaling pathways and cellular processes are conserved between fission yeast and mammalian cells, this simple eukaryotic organism is an excellent model for the identification of molecular mechanism of sensitivity to natural products from plants. Here, we show that an ethyl acetate extract from the leaves of Corema album (L.) D. Don (Ericaceae) inhibits proliferation of fission yeast cells. By fractionation of this extract, we identified 2´,4´- dihydroxychalcone (DHC) as the compound responsible for inhibiting cell proliferation in the biologically active fraction. We show that antiproliferative activity of this flavonoid is likely due to its ability to induce DNA damage which blocks cell cycle progression. 2 Keywords: Corema album; Ericaceae; fission yeast; Schizosaccharomyces pombe; antiproliferative; flavonoid; 2´,4´-dihydroxychalcone (DHC) 3 1. Introduction Cancer is the second leading cause of death in the developed countries and its incidence has been on the rise in the recent decades (Siegel et al., 2012). Cancer results from uncontrolled cell proliferation which is usually a consequence of deregulation of several signaling pathways related to cell cycle control and/or cell growth (Croce, 2008; Hanahan and Weinberg, 2000; Rao et al., 2009; Vogelstein and Kinzler, 2004). Despite the significant progress of tumor therapy in the last five decades, the multifactorial etiology of cell transformation makes it difficult to develop tumor-specific treatments. In addition, the emergence of drug resistance (or multidrug resistant phenotype, MDR) in certain patients and the considerable side effects of drugs currently in use, highlight the need to discover and develop new alternative drugs to make cancer treatment more effective (Gottesman, 2002; Szakacs et al., 2006; Wink et al., 2012). Development of new anticancer drugs would allow using drug combinations with synergy in their effects and would also enable tumor-specific treatments. The screening-based discovery of new anticancer molecules requires a source of compounds and a functional assay to test them (Srivastava et al., 2005; Suffness, and Pezzuto, 1991; Tohme et al., 2011). Search for of natural products suitable for the treatment of human cancer has resulted in the discovery of potent anti-proliferative chemicals that are widely used in the current clinical practice. Identification new such molecules remains a key area of anti-cancer drug development (Cragg et al., 2009; Bailly, 2009). Screening plant extracts for the presence of anti-proliferative compounds can be efficiently performed by using a cell-based assay. To explore the biodiversity in the search of anticancer drugs we have used a bioassay based on the fission yeast Schizosaccharomyces pombe, a simple model for such screenings. Fission yeast is a genetically tractable unicellular organism that allows for genetic, biochemical and cell biological analysis. Using fission yeast for drug screening facilitates identification of intracellular drug targets and helps uncover molecular mechanisms of drug action (Kim et al., 4 2010; Takeda et al., 2011). Conservation of many basic mechanisms regulating DNA metabolism and cell proliferation between yeast and human cells allows us to use yeast cell growth as a readout in such screening. In this work, using a fission yeast bioassay we have screened a collection of plant extracts from Andalusia (Spain). We show that the flavonoid 2’,4’-dihydroxychalcone (DHC) isolated from Corema album leaves produces a DNA damagedependent block of the cell cycle and inhibits cell proliferation. 2. Results and discussion 2.1. In vivo cell-based assay for testing plant extracts with antiproliferative properties To search for new antiproliferative natural compounds, we tested plant extracts from different Andalusian plants (listed in Experimental 4.1. subsection) using a disc assay. In this assay, 5 l of crude plant extracts containing 40 mg/ml of total extracted compounds were applied onto sterile paper discs which were placed on the surface of agar plates containing a lawn of growing fission yeast cells. Inhibition of yeast growth caused by the diffusion of plant active compounds from the disc into the agar plate was detected by the appearance of a growth inhibitory halo on the yeast lawn around the disc after 3 days of incubation at 30 ºC. Based on our previous experience in the search for natural cytotoxic compounds, we selected the intermediate polarity solvent, ethyl acetate (EtOAc) to obtain plant extracts (listed in experimental), of which, only the extract from leaves of Corema album inhibited fission yeast growth. Bio-guided fractionation of this extract was performed by silica gel column chromatography. Fraction three reproduced the inhibition of cell proliferation phenotype observed with the EtOAc extract (See Fig. 1A). By using HPLC / MS) we identified two flavonoids in this biologically active fraction, as pinocembrin and 2’,4’-dihydroxychalcone (DHC), comparing their retention time and multiple reaction monitoring (MRM) transitions 5 with commercial standards. By testing these two compounds individually, we found that DHC was the one responsible for inhibiting cell proliferation (See Fig. 1B). 2.2. DHC inhibits cell growth in a dose-dependent manner To better characterize the effect of DHC on cell growth, we analyzed the phenotype of cells treated with increasing concentrations of DHC using either a disc assay or liquid medium. Both assays showed that DHC impaired S. pombe growth in a dose-dependent manner (Fig. 1C, D). The microscopic analysis of cells treated with increasing concentrations of DHC revealed the presence of elongated cells (cdc-, cell cycle defective) at low or intermediate concentrations (8-20 µM), indicative of a cell cycle block and also showed cell lysis at higher DHC concentrations (>20 µM) (Fig. 1E). Given the toxicity observed at high DHC doses, in subsequent experiments we used a range of DHC concentrations where the cell cycle block was the most prominent phenotype (8-20 µM range). Thus, low doses of DHC impair cell cycle progression in fission yeast. 2.3. DHC induces cell cycle delay at the G2/M transition followed by aberrant DNA segregation during mitosis The appearance of elongated cells after DHC treatment can be a consequence of a cell cycle block at G1/S or G2/M transitions or, alternatively, result from a defective cytokinesis (Nurse et al., 1976). To distinguish between these possibilities, we analyzed the number of nuclei per cell in cells treated low doses of DHC (8-20 µM). To this end, S. pombe cells were treated with DHC, fixed and stained 4’-6’-Diamido-2-phenylindole (DAPI) to visualize nuclear DNA. Nuclear staining revealed that most elongated cells induced by DHC (42/45 cells) contained a 6 single nucleus, which is indicative of a cell cycle block (Fig. 2A, B). Analysis of DNA content by flow cytometry (FACS) further revealed that this cell cycle block was in the G2/M transition (data not shown). Importantly, we also observed around 10% of cells undergoing defective mitosis in which the DNA was cut by the division septum or asymmetrically segregated between daughter cells (Fig. 2C). Taken together, our data suggest that DHC induces a cell cycle block at the G2/M transition and a defective mitosis. Whether these phenotypes are related to each other will require further investigation. 2.4. The G2/M delay induced by DHC is dependent on the DNA damage checkpoint proteins A cell cycle block at the G2/M transition might be a direct consequence of DHC acting on a cell cycle regulator that control this transition. Alternatively, DHC could induce DNA damage or interfere with DNA replication, which would eventually result in the activation of the cell cycle checkpoints, thus blocking cell cycle in G2. To test whether mutants in the DNA damage checkpoint were able to suppress the cell cycle block produced by DHC, we analyzed the effect of DHC on deletions of rad3, chk1 and cds1, three key kinases required for DNA checkpoint operating at G2/M (Canman, 2001; Rhind and Russell, 2000). Rad3 is required to signal downstream of either DNA replication problems or DNA damage, whereas Chk1 transmits DNA damage induced signal and Cds1 signals in response to defective DNA replication (Bentley et al., 1996; Boddy et al., 1998; Brondello et al., 1999; Jimenez et al., 1992; Latif et al., 2004). Interestingly, both rad3 and chk1 deletions suppressed the cell cycle block produced by DHC (Fig. 3A), while cds1 deletion showed partial suppression, suggesting that the G2/M block observed in DHC is mostly caused by the activation of DNA damage checkpoint response. Consistent with these observations, analysis of cell viability after treatment of these mutants with DHC revealed that rad3  presented increased sensitivity to DHC, measured as an 7 increase in the growth inhibitory halo produced by DHC in the disc assay performed using these strains (Fig. 3B, C). We also found that deletion of rhp51, a gene essential for DNA homologous recombination in the mitotic cell cycle, was hypersensitive to DHC (Fig. 3B, C). In this assay, methyl methanesulfonate (MMS), an alkylant compound that induces DNA damage (Wyatt and Pittman, 2006), was used as control (Fig. 3B). Thus, our results suggest that treatment of fission yeast cells with low concentrations of DHC (8-20 µM) produces, directly or indirectly, damage to the DNA that results in a Rad3 and Chk1-dependent cell cycle block at the G2/M transition. 2.5. Cells blocked at G2/M transition contain DNA repair factories The strong sensitivity of rhp51  cells to DHC and the Rad3 and Chk1-dependent cell cycle block at G2/M produced by DHC prompted us to check whether DHC was inducing double strand breaks, a damage that requires Rhp51 to be repaired and the Rad3 and Chk1 branch of the DNA damage checkpoint to block cell cycle progression during DNA repair. Upon DNA double strand breaks, many proteins implicated in DNA repair cluster to form nuclear structures called DNA repair factories or foci. These factories promote DNA double strand break repair by homologous recombination (Thorpe et al., 2011). When assembled, repair factories are indicative of ongoing DNA repair and can be followed in vivo by the Rad22-GFP marker, a component of these factories (Meister et al., 2003; Tohme et al., 2011). To determine whether DHC was inducing double strand breaks, we analyzed the presence of DNA repair factories in cells treated with increasing concentrations of this compound. It is important to note that DNA repair factories are normally assembled after every S-phase to repair DNA breaks produced during replication (Fig. 4A, arrowheads) (Meister et al., 2003). Interestingly, 8 all cells blocked in the cell cycle (30/30) contained assembled Rad22-YFP foci (Fig. 4A and B). Thus, accumulation of DNA repair factories in cells treated with DHC suggest that this flavonoid is inducing DNA double strand breaks that might activate the DNA damage response and block cell cycle progression at G2/M. Alternatively, our data would also be consistent with DHC inhibiting replication or post-replication DNA repair. 3. Conclusions In this work, we use fission yeast as a model for a bio-guided screening of plant extracts to search for new antiproliferative agents. We have identified in Corema album leaves the bioactive compound 2’,4’-dihydroxychalcone (DHC) that inhibits cell proliferation. Similar to other plant secondary metabolites, DHC has lipophilic properties that allow it to readily pass biomembranes and interfere with molecular targets in mamals and microbes acting as a defence mechanism (Wink, 2008; Wink and Schimmer, 2010; Wink et al., 2012). It is known that DHC has in vitro antiproliferative activity in different cancer cell lines, thus validating our bioassay, but the mechanism of DHC action is still unknown (Calliste et al., 2001; Iwata et al., 1995; Lou et al., 2009; Middleton et al., 2000). We have shown that DHC blocks fission yeast cell cycle progression at G2/M and produces aberrant mitosis. The G2/M block is dependent on DNA damage checkpoints, and blocked cells contain DNA repair factories, suggesting that inhibition of cell proliferation by DHC is likely due to its capability to induce DNA damage. Interestingly, it has been recently shown that DHC induces a cell cycle arrest in the G2/M phase followed by apoptosis in cancer cells (Lou et al., 2010). Importantly, in vitro treatment of cancer cells with another chalcone, isoliquiritigenin that contains an extra hydroxyl group in position 4 (4,2’,4’-trihydroxychalcone) produces a block in 15 Figure legends Fig. 1. Corema album leaf extract blocks cell proliferation. A. Disc assay using EtOAc extract and active fraction 3 B. Identification and assay of the active compounds infraction 3, pinocembrin and 2´,4´-dihydroxychalcone (DHC). C. Disc assay using increasing concentration of DHC (40, 80, 120 y 160 mM), c: denotes control, DMSO. D. Optical density changes over time in cells treated with the indicated concentrations of DHC. E. Differential interphase contrast (DIC) images of S. pombe cells treated with indicated concentrations of DHC. Scale bar, 5µm. Fig. 2. DHC produces cell cycle block and defective mitosis. A. DIC and DAPI staining of wild type cells treated with the indicated concentrations of DHC. B, C. Percentage of elongated cdccells and aberrant mitoses respectively at indicated concentrations of DHC. Scale bar, 5µm. Fig. 3. The G2/M block produced by DHC depends on the DNA damage checkpoints. A. DIC images of wildtype and DNA damage mutants at indicated DHC concentrations. B. Drop assay using indicated DNA damage mutants treated with methyl methanesulfonate (MMS, 0.004%), DHC (120 mM) or DMSO as a negative control. C. Measurements of halo diameter relative to the wildtype control. Scale bar, 5µM. Fig. 4. Cell blocked at G2/M contain assembled DNA foci. A. DIC and fluorescence (YFP) life-cell imaging of wild type cells treated with and without 12µM DHC. B. Plot of percentage of cdccells containing DNA repair foci. Scale bar, 5µm. 16 References Bailly, C., 2009. Ready for a comeback of natural products in oncology. Biochemical Pharmacology 77, 1447-1457. Bentley, N.J., Holtzman, D.A., Flaggs, G., Keegan, K.S., DeMaggio, A., Ford, J.C., Hoekstra, M., Carr, A.M., 1996. The Schizosaccharomyces pombe rad3 checkpoint gene. The EMBO Journal 15, 6641-6651. Boddy, M.N., Furnari, B., Mondesert, O., Russell, P., 1998. Replication checkpoint enforced by kinases Cds1 and Chk1. Science 280, 909-912. Brondello, J.M., Boddy, M.N., Furnari, B., Russell, P., 1999. Basis for the checkpoint signal specificity that regulates Chk1 and Cds1 protein kinases. Molecular and Cellular Biology 19, 4262-4269. Calliste, C.A., Le Bail, J.C., Trouillas, P., Pouget, C., Habrioux, G., Chulia, A.J., Duroux, J.L. ,2001. Chalcones: structural requirements for antioxidant, estrogenic and antiproliferative activities. Anticancer Research 21, 3949-3956. Canman, C.E., 2001. Replication checkpoint: preventing mitotic catastrophe. Current Biology 11, R121-124. Cragg, G.M., Grothaus, P.G., Newman, D.J., 2009. Impact of natural products on developing new anti-cancer agents. Chemical Reviews 109, 3012-3043. Croce, C.M., 2008. Oncogenes and cancer. The New England Journal of Medicine 358, 502511. Glavinas, H., Krajcsi, P., Cserepes, J., Sarkadi, B., 2004. The role of ABC transporters in drug resistance, metabolism and toxicity. Current Drug Delivery 1, 27-42. Gottesman, M.M., 2002. Mechanisms of cancer drug resistance. Annual Review of Medicine 53, 615-627. 17 Han, Y., Riwanto, M., Go, M.L., Ee, P.L., 2008. Modulation of breast cancer resistance protein (BCRP/ABCG2) by non-basic chalcone analogues. Eurpean Journal of Pharmacological Sciences 35, 30-41. Hanahan, D., Weinberg, R.A., 2000. The hallmarks of cancer. Cell 100, 57-70. Iwata, S., Nishino, T., Nagata, N., Satomi, Y., Nishino, H., Shibata, S., 1995. Antitumorigenic activities of chalcones. I. Inhibitory effects of chalcone derivatives on 32Pi-incorporation into phospholipids of HeLa cells promoted by 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Biological & Pharmaceutical Bulletin 18, 1710-1713. Jimenez, G., Yucel, J., Rowley, R., Subramani, S., 1992). The rad3+ gene of Schizosaccharomyces pombe is involved in multiple checkpoint functions and in DNA repair. Proceedings of the National Academy of Sciences of the United States of America 89, 4952-4956. Kim, D. U., Hayles, J., Kim, D., Wood, V., Park, H. O., Won, M., Yoo, H.S., Duhig, T., Nam, M., Palmer, G. et al., 2010. Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nature Biotechnology 28, 617-623. Latif, C., den Elzen, N., O'Connell, M., 2004. DNA damage checkpoint maintenance through sustained Chk1 activity. Journal of Cell Science 117, 3489-3498. Locher, K.P., 2009. Structure and mechanism of ATP-binding cassette transporters. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 364, 239-245. Lou, C., Wang, M., Yang, G., Cai, H., Li, Y., Zhao, F., Yang, H., Tong, L., Cai, B., 2009. Preliminary studies on anti-tumor activity of 2',4'-dihydroxychalcone isolated from Herba oxytropis in human gastric cancer MGC-803 cells. Toxicology in vitro 23, 906-910. 18 Lou, C., Yang, G., Cai, H., Zou, M., Xu, Z., Li, Y., Zhao, F., Li, W., Tong, L., Wang, M. et al., 2010. 2',4'-Dihydroxychalcone-induced apoptosis of human gastric cancer MGC-803 cells via down-regulation of survivin mRNA. Toxicology in vitro 24, 1333-1337. Meister, P., Poidevin, M., Francesconi, S., Tratner, I., Zarzov, P., Baldacci, G., 2003. Nuclear factories for signalling and repairing DNA double strand breaks in living fission yeast. Nucleic Acids Research 31, 5064-5073. Middleton, E., Jr., Kandaswami, C., Theoharides, T.C., 2000. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacological Reviews 52, 673-751. Nurse, P., Thuriaux, P., Nasmyth, K., 1976. Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Molecular & General Genetics 146, 167-178. Park, I., Park, K.K., Park, J.H., Chung, W.Y., 2009. Isoliquiritigenin induces G2 and M phase arrest by inducing DNA damage and by inhibiting the metaphase/anaphase transition. Cancer Letters 277, 174-181. Rao, C.V., Yamada, H.Y., Yao, Y., Dai, W., 2009. Enhanced genomic instabilities caused by deregulated microtubule dynamics and chromosome segregation: a perspective from genetic studies in mice. Carcinogenesis 30, 1469-1474. Rees, D.C., Johnson, E., Lewinson, O., 2009. ABC transporters: the power to change. Nature reviews. Molecular Cell Biology 10, 218-227. Rhind, N., Russell, P., 2000. Chk1 and Cds1: linchpins of the DNA damage and replication checkpoint pathways. Journal of Cell Science 113, 3889-3896. Suffness, M., Pezzuto, J.M., 1991. Assays related to cancer drug discovery, in: Hostettmann, K. (Ed.). Methods in Plant Biochemistry, Vol 6, Assays for Bioactivity. Academic press, London, pp 71-153. 19 Siegel, R., Ward, E., Brawley, O., Jemal, A., 2012. Cancer statistics, 2011: the impact of eliminating socioeconomic and racial disparities on premature cancer deaths. A Cancer Journal for Clinicians 61, 212-236. Srivastava, V., Negi, A.S., Kumar, J.K., Gupta, M.M., Khanuja, S.P., 2005. Plant-based anticancer molecules: a chemical and biological profile of some important leads. Bioorganic & Medicinal Chemistry 13, 5892-5908. Szakacs, G., Paterson, J.K., Ludwig, J.A., Booth-Genthe, C., Gottesman, M.M., 2006. Targeting multidrug resistance in cancer. Nature reviews. Drug discovery 5, 219-234. Takeda, K., Mori, A., Yanagida, M., 2011. Identification of genes affecting the toxicity of anticancer drug bortezomib by genome-wide screening in S. pombe. PloS one 6, e22021. Thorpe, P. H., Alvaro, D., Lisby, M., Rothstein, R., 2011. Bringing Rad52 foci into focus. The Journal of Cell Biology 194, 665-667. Tohme, R., Darwiche, N., Gali-Muhtasib, H., 2011. A journey under the sea: the quest for marine anti-cancer alkaloids. Molecules 16, 9665-9696. Vogelstein, B., Kinzler, K. W., 2004. Cancer genes and the pathways they control. Nature Medicine 10, 789-799. Wink, M., 2008. Evolutionary advantage and molecular modes of action of multi-component mixtures used in phytomedicine. Current Drug Metabolism 9, 996-1009. Wink, M., Ashour, M.L., El-Readi, M.Z., 2012. Secondary Metabolites from Plants Inhibiting ABC transporters and reversing resistance of cancer cells and microbes to cytotoxic and antimicrobial agents. Frontiers in Microbiology 3, 130. Wink, M., Schimmer, O., 2010. Molecular modes of action of defensive secondary metabolites, in: Wink, M. (Ed.) Functions and Biotechnology of Plant Secondary Metabolite. WileyBlackwell, Oxford, pp.21-161. 20 Wyatt, M.D., Pittman, D. L., 2006. Methylating agents and DNA repair responses: Methylated bases and sources of strand breaks. Chemical Research in Toxicology 19, 1580-1594. 21 Figure 1 HO O O HO OH O Pinocembrin 2’,4’-dihydroxychalcone 22 23