Interrogation of ethnomedicinal plants for synthetic lethality effects in combination with deficiency in the DNA repair endonuclease RAD1 using a yeast cell-based assay
Graphical abstract
Introduction
Colorectal cancer is the third most common cancer in both sexes worldwide with over 700,000 deaths annually, accounting for 8% of all deaths from cancer (Ferlay et al., 2013, Siegel et al., 2017). More than 1.4 million new cases of colorectal cancer are reported each year and the incidence is increasing, even in countries where risk has been traditionally low (Ferlay et al., 2013, Watson and Collins, 2011). Treatments for colorectal cancer include surgery, radiotherapy, chemotherapy, or a combination of these strategies (Watanabe et al., 2012). Surgery is the main treatment for colorectal cancer; although, the effectiveness of surgery may be limited in advanced stages and in these cases, adjuvant chemotherapy is often essential to prevent recurrence (de Gramont et al., 2003, Watanabe et al., 2012)
Adjuvant chemotherapy for colorectal cancer patients primarily utilizes 5-fluorouracil, capecitabine (metabolized to 5-fluorouracil), and oxaliplatin (de Gramont et al., 2003, Ling et al., 2011, Longley et al., 2003). However, these drugs do not specifically target cancer and instead cause generalized toxicity to cells that rapidly proliferate (Longley et al., 2003, Raymond et al., 1998). Many patients cannot tolerate the side effects of 5-fluorouracil, capecitabine, and oxaliplatin, which include nausea, vomiting and diarrhea, leukopenia, cardiotoxicity, and neurological damage (de Gramont et al., 2003, Han et al., 2008, McQuade et al., 2014, Ng et al., 2005, Teixeira et al., 2004). Targeted therapy based on the understanding of the molecular changes between normal and cancer cells has the potential to limit adverse side effects by making drugs more selective. DNA damage is typically increased in cancer cells compared to adjacent normal tissues due to mutations in DNA damage response genes (Bartkova et al., 2005). Mutation and epigenetic silencing of genes involved in the DNA damage response is common in cancer and this altered DNA damage repair ability can be used to differentiated cancer cells from normal cells (Curtin, 2012, Helleday et al., 2008). Mutation or repression of one DNA repair pathway in cancer cells can be partially compensated by increased activity of other pathways that exhibit partially overlapping functions. The activity of these compensating DNA repair pathways enhanced genome integrity of cancer cells and allow them to maintain viability. In addition, the DNA repair pathways capable of compensating for the primary mutation can promote resistance to chemotherapy and radiotherapy, making them interesting targets for intervention (Curtin, 2012).
Strategies that take advantage of the genetic concept of synthetic lethality can be used to identify inhibitors for the compensating DNA repair systems in cancer cells (Canaani, 2009, Ferrari et al., 2010). Synthetic lethality or synthetic sick interactions occur when the deletion of two genes or inhibition of their gene products leads to a dramatic decrease in survival or death, even though inhibition of each gene alone does not cause substantial loss of viability (Canaani, 2009, Ferrari et al., 2010, Garber, 2002). Screens for synthetic lethal genetic and chemical genetic interactions have been used to identify genes with compensating functions in essential pathways as well as inhibitors of these pathways (Ferrari et al., 2010, Forsburg, 2001). Exploiting the dependence of cancer cells on secondary DNA repair pathways based on the principle of synthetic lethality using inhibitors of DNA repair enzymes has proven successful (Bryant et al., 2005, Farmer et al., 2005). An interesting target for synthetic lethal interactions in colorectal cancer is ERCC4 (excision repair cross-complementation group 4), encoding the DNA repair endonuclease XPF (Brookman et al., 1996). Approximately 55% of colorectal cancers exhibit reduced expression of ERCC4, (Facista et al., 2012) which functions in both nucleotide excision repair (NER) and double-strand break repair (DSBR) pathways.
Identification of compounds with synthetic lethal/sick properties can be facilitated through the use of model organisms such as the baker's yeast Saccharomyces cerevisiae as a surrogate for more complex experimental systems (Holbeck and Simon, 2007, Matuo et al., 2012, Ross-Macdonald, 2003). The DNA repair systems of S. cerevisiae are well described and are remarkably similar to those in human cells (Dudas and Chovanec, 2004, Hsieh, 2001, Iyer et al., 2006) and S. cerevisiae RAD1 is equivalent to human ERCC4 (Brookman et al., 1996, Friedberg, 1985). Comparing the sensitivities of a wild-type yeast and an isogenic mutant, with a defect in a DNA repair pathway, to an array of compounds or other bioactive materials can aid in the identification of drug candidates that selectively target mutant cells (Simon and Yen, 2003). This can be especially important in anticancer drug development as many drugs are cytotoxic to both normal and cancer cells (Longley et al., 2003).
Compounds present in ethnomedicinal plants previously implicated as having utility in cancer therapy are good candidates for potential therapeutic agents (Balandrin et al., 1985, Clark, 1996, Lee and Houghton, 2005, Sangmalee et al., 2012). In this study, we utilized a library containing extracts from 32 ethnomedicinal species to search for synthetic lethal/sick chemical-genetic interactions in rad1∆ yeast. We report here the identification of Bacopa monnieri and Colubrina asiatica extracts as potential sources of compounds with specificity toward inhibiting growth of S. cerevisiae deficient for the ERCC4 homologue RAD1.
Section snippets
Yeast strains and plasmids
S. cerevisiae strains used in this study were derived from BY4742 (Mat α, leu2∆0, lys2∆0, ura3∆0, his3∆1) (Brachmann et al., 1998) and single deletion strains were obtained from Open Biosystems (Huntsville, AL, USA). Yeast strains are described in Table 1. Gene deletions were verified by in vivo PCR with a BioRad MJ Mini thermocycler (Hercules, CA, USA) using flanking primers (Longtine et al., 1998). Plasmids were generated using standard procedures and the DNA sequence integrity was verified
Results
A library of extracts from 32 ethnomedicinal plant species, containing samples for multiple plant parts in some cases giving a total of 37 extracts, was assayed for specific toxicity to yeast cells deleted for RAD1. The plant family, names for each plant, as well as the parts used are listed in Table 3. Plant extracts at a concentration of 2000 µg/mL were used to evaluate toxicity in the erg6∆ strain. Growth of treated cells was compared to samples containing vehicle control (2% DMSO, 0.2%
Discussion
A potential disadvantage of using yeast in drug screens is the barrier provided by the cell wall and plasma membrane that acts to limit the uptake of small molecules (Simon et al., 2000). This permeability barrier can be weakened using yeast mutants defective for synthesis of membrane components, such as sterols. ERG6 is a nonessential gene that encodes a methyltransferase required for the synthesis of ergosterol, equivalent to cholesterol in mammalian cells (McCammon et al., 1984). Yeast
Conclusion
The use of the yeast system is a powerful tool for analysis of synthetic lethal interactions since cells that are isogenic except for a single gene mutation can be readily evaluated. This method for selecting synthetic lethal interactions has validated the ethnomedicinal literature regarding use of B. monnieri extract as an anti-cancer medicine. Our screen did not give positive responses for all plants with reported anti-cancer properties, highlighting the specific nature of the type of
Acknowledgments
This research project was supported by grants from the Thailand Research Fund RSA5780047 and IRG5980008, the Mahidol-Norway Capacity Building Initiative for ASEAN, and the Mahidol University Central Instrument Facility. We thank T. Phadungcharoen for identification of plant materials, the Olympus Biomaging Center, Mahidol University for assistance with fluorescence microscopy, and P. Jarulakkhana for the ERG6 deletion plasmid and strains.
Details of the contributions of individual authors
Hsu Mon Aung: Performed experiments, analyzed and interpreted data,
Chananya Huangteerakul: Analyzed and interpreted data
Wittaya Panvongsa: Performed experiments.
Amornrat N. Jensen: Involved in the conception and design of the study, analyzed and interpreted data, and revised the article
Arthit Chairoungdua: Involved in design of the study.
Suchada Sukrong: Performed experiments. Involved in the conception and design of the study.
Laran T. Jensen: Involved in the conception and design of the study,
References (148)
- et al.
New insights into the mechanism of homologous recombination in yeast
Mutat. Res.
(2004) - et al.
Triterpenes from the stem bark of Mitragyna diversifolia and their cytotoxic activity
Chin. J. Nat. Med.
(2014) - et al.
Chemopreventive and anti-cancer properties of the aqueous extract of flowers of Butea monosperma
J. Ethnopharmacol.
(2010) - et al.
Antiproliferative and antioxidant activities of a tricin acylated glycoside from sugarcane (Saccharum officinarum) juice
Phytochemistry
(2007) - et al.
DNA double-strand break repair by homologous recombination
Mutat. Res.
(2004) - et al.
A lethal combination for cancer cells: synthetic lethality screenings for drug discovery
Eur. J. Cancer
(2010) - et al.
Treatment of fibroids: the use of beets (Beta vulgaris) and molasses (Saccharum officinarum) as an herbal therapy by Dominican healers in New York City
J. Ethnopharmacol.
(2004) - et al.
Plants used for treatment of dysentery and diarrhoea by the Bhoxa community of district Dehradun, Uttarakhand, India
J. Ethnopharmacol.
(2013) - et al.
Dammarane-type triterpenoid saponins from Bacopa monniera
Phytochemistry
(1996) - et al.
Nucleotide excision repair in yeast is mediated by sequential assembly of repair factors and not by a pre-assembled repairosome
J. Biol. Chem.
(1996)