Ruta Graveolens Extract Induces DNA Damage Pathways and Blocks Akt Activation to Inhibit Cancer Cell Proliferation and Survival
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ANTICANCER RESEARCH 31 : 233-242 (2011) Ruta graveolens Extract Induces DNA Damage Pathways and Blocks Akt Activation to Inhibit Cancer Cell Proliferation and Survival KHALDA FADLALLA 1, ANGELA WATSON 1, TESHOME YEHUALAESHET 1, TIMOTHY TURNER 2 and TEMESGEN SAMUEL 1 1Department of Pathobiology and 2Department of Biology and Tuskegee University Center for Cancer Research (TUCCR), Tuskegee University, Tuskegee, A L 36088, U.S.A. Abstract. Background: Ruta graveolens is a medicinal herb based on natural products (1, 2). Despite the decline of that has been used for centuries against various ailments. This interest by the pharmaceutical industry in research and study examined the anticancer properties of the herb using development of natural compounds, these unrefined cancer cell lines. Materials and Methods: Methanolic extract compounds from terrestrial and aquatic sources continue to of R. graveolens was tested on colon, breast and prostate serve as the chemical foundations from which modified or cancer cells. Viability, cell cycle profiles, clonogenicity and synthetic versions can be derived. capase activation were measured. Induction and subcellular Ruta graveolens is a medicinal and culinary plant that is localizations of p53, 53BP1 and γ-H2AX proteins were native to the Mediterranean region of southern Europe and examined. Results: The extract dose-dependently decreased the northern Africa. Widely grown in different parts of the world, viability and the clonogenicity of treated cells and induced this herb has historically been in use since the ancient times (3). G2/M arrest, aberrant mitoses, and caspase-3 activation. It Its documented therapeutic uses include the treatment of also induced the p53 pathway and focal concentration of the inflammatory conditions, eczema, ulcers, arthritis, fibromyalgia, DNA damage response proteins 53BP1 and γ-H2AX. antidote for venoms, insect repellent, and as an abortifacient (4- Moreover, the levels of phospho-Akt and cyclin B1 were 6). The plant has also been commonly used to season some food reduced by treatment, whereas only cyclin B1 was reduced in items such as soup, cheese, butter, coffee, and tea, and in normal dermal fibroblasts. Conclusion: R. graveolens extract medicinal preparations such as rue oil and infusions that are contains bioactive compounds which, independently of known used as antispasmodics and emmenagogues (7). photoactivatable mechanisms, potently inhibit cancer cell Chemical compounds so far known to be present in R. proliferation and survival through multiple targets. graveolens include furanocumarins, carotenoids, chlorophyll, and furanoquinolones (4, 8). Psoralens, a family of Plants are important sources of medicinal compounds furanocumarins in R. graveolens , have been widely studied worldwide. A few examples are: aspirin from willow tree, for their DNA interstrand cross-linking activity when digitalis from foxglove, artemisinin from wormwood, taxol exposed to ultra violet (UV) light. This photoactivation from the pacific yew tree, vinblastine and vincristine from property of psoralens has been utilized in the treatment of periwinkle, etoposide from mayapple, etc . More than 60% of various skin malignancies including psoriasis, vitiligo and cancer therapeutics on the market or in pre-clinical trials are cutaneous lymphoma as a psoralen plus UV-A or UV-B (PUVA or PUVB) regimen (9-11). Although some bioactive components in the herbal This article is freely accessible online. extracts and infusions made from R. graveolens have been suggested to be responsible for the plant’s beneficial or Correspondence to: Temesgen Samuel, College of Veterinary phototoxic effects, molecular studies that extensively Medicine, Nursing and allied Health, Department of Pathobiology decipher the activities of most of its bioactive ingredients are and TUCCR, Patterson Hall, Room A408, Tuskegee, AL 36088, scarce. While the phytophototoxicity caused by R. U.S.A. Tel: +1 3347244547, Fax: +1 3347244110, e-mail: [email protected] graveolens has been known for a long time, the bioactivities of R. graveolens extracts and its various preparations against Key Words: Medicinal herb, bioactivity, p53 pathway, apoptosis, tumor cells or pathogenic microbes have attracted attention Ruta graveolens , Akt . only recently (12-17). 0250-7005/2011 $2.00+.40 233 ANTICANCER RESEARCH 31 : 233-242 (2011) This study examined the potency of an extract from R. Corp.) and centrifuged. Pellets were resuspended in 300 μl phosphate- graveolens on cancer cell lines. This study shows that this buffered saline (PBS; Invitrogen Corp.), fixed by addition of 700 μl extract has potent anticancer activity, exhibited through strong 100% ethanol while vortexing, and stored at –20˚C for a minimum of 12 hours. Fixed cells were centrifuged, and stained in FACS staining anti-proliferative and ant i- survival effects on cancer cells. solution (320 mg/ml RNase A, 0.4 mg/ml propidium iodide) in PBS without calcium and magnesium for 15 minutes at 37˚C. Stained cells Materials and Methods were filtered through a 70 μm pore size filter and analyzed by flow cytometry on C6 Accuri ® flow cytometer (Accuri Cytometers, Ann Cell culture and treatments. The human colorectal cancer cell line Arbor, MI, USA). Data was analyzed and histograms were prepared HCT116 was a generous donation from Dr. Bert Vogelstein (Johns using CFlow™ software (Accuri Cytometers). Hopkins University, MD, USA). The cell line was maintained in McCoy’s medium (Lonza, Walkersville, MD, USA) supplemented Immunofluorescent staining and microscopy. Phase contrast images with 10% fetal bovine serum (FBS) and 10,000 U/ml of cells were taken at ×20 and ×40 magnification objectives (and ×10 penicillin/10mg/ml streptomycin. The MCF7 cell line was a gift from eyepiece) using an Olympus I×71 inverted microscope (Olympus Dr. Leslie Wilson (University of California at Santa Barbara, CA, America Inc, Melville, NY, USA) fitted with digital image capture USA). RKO (purchased from ATCC, Manassas, VA, USA) and MCF7 cameras (Digital Microscopy Lab, College of Veterinary Medicine, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Nursing and Allied Health, Tuskegee University). HCT116 cells for Invitrogen Corp, Carlsbad, CA, USA) supplemented with FBS and immunofluorescent staining were grown in 4-well chamber slides. penicillin/streptomycin. Prostate cancer cell lines PC3 and DU-145 Staining was performed as described previously (19). Confocal cells, purchased from ATCC were cultured in T-medium (Invitrogen images were taken using an Olympus DSU spinning disk confocal Corp, Carlsbad, CA, USA), supplemented with 10% FBS and microscope (Olympus America Inc) at the Research Center at penicillin/streptomycin. For experimental treatments, cells were seeded Minority Institutions (RCMI) core-facility using a ×40 dry objective. in dishes with 96 well s (for viability assays), 12 well s (for colony Images were captured using Metamorph Premium software (Strategic formation assays), 6 well s (for flow cytometry and clonogenicity Triangle Inc, Toronto, Canada), stored in TIFF format and further assays), 6 cm culture plates (for cell lysate preparations), or 4-well processed in Adobe Photoshop. chamber slides (for immunofluorescence staining). All cell cultures were incubated at 37˚C and 5% CO 2 in a humidified incubator. Immunoblotting. Cell lysates were prepared in NP-40 lysis buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 10% glycerol and 0.2% NP-40 Extract preparation. Fresh leaves of R. graveolens were minced plus protease inhibitor cocktail) and protein concentrations were finely in a food processor and extracted in 80% methanol for 24 determined using DC detergent compatible protein assay (BioRad hours. Particulate matters w as removed by two rounds of Laboratories Inc, Hercules, CA, USA). Samples containing equivalent centrifugations at 1000× g and 10,000× g for 5 and 10 minutes, protein concentrations were mixed with Laemmli buffer, and boiled respectively. The soluble fraction was desiccated in a rotary for 5 minutes. Proteins were resolved by SDS-PAGE, transferred to evaporator, and the evaporated solids were resuspended in DMSO PVDF membranes (GE Healthcare Life Sciences, Piscataway, NJ, at a final concentration of 60 mg/ml. USA) and blocked in 5% non-fat dry milk. Primary antibodies were used at 1:1000 dilutions. Peroxidase-conjugated anti-rabbit and anti- Antibodies. Anti-p53, -β-actin, -phospho-γ-H2AX ser139, -53BP1, -Akt, mouse IgG secondary antibodies were purchased from GE Healthcare -phospho-Akt, -cyclin B1 and - CDK1 antibodies were purchased from Life Sciences and used at a 1:5000 dilution. Chemiluminescent Cell Signaling Technology (Danvers, MA, USA). Anti-p21 WAF1 was detections were performed using Chemiluminescent HRP Substrate purchased from Invitrogen Corp.). Monoclonal antibody to α-tubulin (GE Healthcare Life Sciences). (clone DM1A) was purchased from Sigma (St Louis, MO, USA). Caspase-3 activation assay. The assay was performed using a Clonogenicity assays. Clonogenicity assay was performed as colorimetric caspase 3 activation assay kit ( caspase-3-C; Sigma). described elsewhere (18). The clonogenic potential of untreated and Briefly, treated and control cells were lysed in 1× lysis buffer. Thirty treated cells was determined by seeding approximately 150 cells per micrograms of total protein were used to perform the assay according well of a 6-well dish. Cells were allowed to adhere for approximately to the manufacturer’s recommendations. Recombinant