Abstract
Background: This research was undertaken in order to investigate the inhibitory potential of the Jamaican ball moss, Tillandsia recurvata against several kinases. The inhibition of these kinases has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Materials and Methods: Kinase inhibition was investigated using competition binding (to the ATP sites) assays, which have been previously established and authenticated. Results: Four hundred and fifty one kinases were tested against the Jamaican ball moss extract and a dose-response was tested on 40 kinases, which were inhibited by more than 35% compared to the control. Out of the 40 kinases, the Jamaican ball moss selectively inhibited 5 (CSNK2A2, MEK5, GAK, FLT and DRAK1) and obtained Kd50s were below 20 μg/ml. Conclusion: Since MEK5 and GAK kinases have been associated with aggressive prostate cancer, the inhibitory properties of the ball moss against them, coupled with its previously found bioactivity towards the PC-3 cell line, makes it promising in the arena of drug discovery towards prostate cancer.
Being the third leading cause of death worldwide (1-3), cancer is anticipated to claim more lives as it is projected that by 2030, there will be approximately 26 million new cancer cases and 17 million cancer deaths per year (4). Cancer is the leading cause of death in Jamaica and latest cancer statistics showed that breast cancer is the leading cause of cancer mortality amongst Jamaican women, while Jamaican men have arguably the highest reported cases of prostate cancer in the world (5).
Normal cell growth is tightly regulated and this tight regulation is facilitated by various checkpoint systems of whom selected kinases are the most noted. This regulation allows for controlled growth, differentiation along with identification and problem solving of genomic alterations (6, 7) within the cell. When this phenomenon is out of balance, which can be as a result of carcinogenesis or hereditary predisposition, then there is a classic case of de-regulated cell proliferation resulting in what we know as cancer. It has, therefore, become a recent and therapeutically relevant direction to locate molecular entities, be them inducers or inhibitors that can regulate particular kinases in hope of restoring the normal balance to cell proliferation (8). This is believed to provide a solution to uncontrollable growth evidenced in malignant tumours. With this in mind this research was undertaken in order to investigate the inhibitory properties of the Jamaican ball moss, Tillandsia recurvata L. against 451 kinases. We report, for the first time, findings on those kinases that were most significantly inhibited in the presence of the Jamaican ball moss.
Jamaica is known for its rich biodiversity and its abundant usage of medicinal plants, with approximately 30% of its terrestrial plants being endemic (9). Medicinal plants continue to play a role in drug discovery and development because of vast structural diversity of molecules, some of which become new drugs or leads for the development of new drugs (10). Tillandsia recurvata L. (Bromeliaceae) which is commonly called the “Jamaican Ball Moss” or the “Old Man's beard” is one of the several important plants found in Jamaica. Previous work done in our lab showed that the Jamaican ball moss exhibits both anti-cancer and anti-inflammatory activities (11, 12). Mechanistic studies further showed that its anticancer activity was expressed through the induction of apoptosis in five cancer cell lines (namely; PC-3 prostate cancer; Kaposi Sarcoma, B-cell lymphoma, breast cancer, and B-16 melanoma (11), and recent research showed that extracts from this plant also reducs angiogenesis (accepted for publication by Lowe et al., 2012). The ability of this plant to exhibit cytototoxic properties against several cancer cell lines, coupled with its ability to activate programmed cell death, reduce angiogenesis and further be able to inhibit several kinases, is indicative that it may be an effective therapeutic solution to several cancers.
Materials and Methods
Kinase inhibition assay. Competition binding assays were established, authenticated and executed as described previously (13, 14). For most assays, kinases were fused to T7 phage strains (13) and for the other assays, kinases were produced in HEK-293 cells after which they were tagged with DNA for quantitative PCR detection (data not shown). In general, full-length constructs were used for small-, single-domain kinases, and catalytic domain constructs for large multi-domain kinases. The binding assays utilized streptavidin-coated magnetic beads treated with biotinylated small-molecule ligands for 30 min at room temperature which generated affinity resins for the kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05 % Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17× PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 40x stocks in 100% DMSO and were diluted directly into the assay (Final DMSO concentration=2.5%). All reactions were performed in polypropylene 384-well plates in a final volume of 0.04 ml. The assay plates were incubated at room temperature with shaking for 1 h and the affinity beads were washed with washing buffer (1 × PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1 × PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Figure 1 presents a graphical illustration of the kinase interaction process. Kd50s were determined using a standard dose-response curve using the hill equation. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.
Plant material. Tillandsia recurvata extraction. The whole T. recurvata plant was collected from trees and electricity poles at Kingston, Jamaica. A voucher specimen of the plant was identified at the Institute of Jamaica Herbarium where it is deposited with Accession Number: IJ 3411. The collected plant material was air-dried, pulverized into powder. 2.3 kg of Ball Moss biomass was extracted twice with 5 L of chloroform. The filtrate was dried in a rotavapor to obtain a dark green residue (87.6 g).
Results and Discussion
Out of the 451 kinases tested, a dose-response was run on 40 which were inhibited by more than 35%, compared to the controls. Out of the 40 kinases, the Jamaican ball moss selectively inhibited 5 (MEK5, GAK, CSNK2A2, FLT and DRAK1) and obtained a Kd50<20 μg/ml, as shown in Table I.
Mitogen/extracellular signal-regulated kinase-5 (MEK5) belongs to a network of mitogen-activated protein kinase pathways while Cyclin G-associated kinase (GAK) is identified as an androgen receptor (AR) protein belonging to the steroid receptor family. Both of these kinases have been associated with the process of carcinogenesis (15, 16) and their further overexpression has been shown to promote cellular proliferation, motility and invasion (17), linking them to aggressive prostate (15, 18) and breast cancers (19). Since previous work performed in our laboratory demonstrated that the ball moss extract reduced the viability of a human prostate cancer cell line (PC-3) via apoptosis (11), its further bioactivity towards the inhibition of MEK5 (Kd50=12 μg/ml) and GAK (Kd50=8 μg/ml), kinases associated with aggressive prostate cancer makes this plant extract even more relevant in the arena of drug discovery against this cancer.
Present in all eukaryotic cells, casein kinase II (CSNKA2) is a highly conserved (20, 21) multifunctional protein kinase that catalyzes the phosphorylation of amino acids, serine and threonine (22), and is implicated in a variety of cellular processes and functions including mitosis and cellular transformation. It has been shown to be expressed in several cancers (23, 24), as well as in the pathogenesis of a number of infectious and neurodegenerative diseases and cardiovascular disorders (8, 25). Inhibition of this kinase, as demonstrated by the Jamaican ball moss (Kd50=14 μg/ml), implicates induction of the p53-dependent cell-cycle arrest and consequently apoptosis, as demonstrated by Dixit et al., (26). Also, since CSNKA2 is implicated in neurodegenerative diseases and cardiovascular disorders, then their inhibitors may show promise in the treatment of these diseases and disorders.
FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase (TK) structurally related to the receptors for platelet-derived growth factor (PDGF), expressed by immature hematopoietic cells and is important for the normal development of stem cells and the immune system (27). Mutations of this kinase have been associated with leukemia (28) and so the search for molecular drug entities that can inhibit these mutated kinases is ongoing in hopes of finding a solution to cancers of the blood and immune system. The Jamaican ball moss inhibited this kinase (12 μg/ml) making it an ideal candidate for future research against the mutant variant and also making it an ideal candidate for further development in the treatment of the above cancers.
Among the five kinases inhibited by the Jamaican ball moss, was the kinase-related apoptosis inducing protein kinase 1 (DRAPK1, Kd50=12 μg/ml) a kinase known to enhance the cell's sensitivity, as well as overcoming its resistance to gemcitabine in pancreatic cancer cells (29). Because of the role this kinase plays in the promotion of apoptosis, inducers rather than inhibitors are the preferred traits of plant extracts/molecular entities, making the activity of the ball moss towards this particular kinase not ideal. Notwithstanding, the search for key phytochemicals from the Jamaican ball moss such as isolated cycloartanes (submitted for publication, Lowe et al., 2012) and a novel glycoside (30) previously conducted in our lab can be explored for induction properties towards this particular kinase.
How the KinomeScan assay works (Courtesy of DiscoveRx).
Kinases showing good interaction with Ball Moss and their disease relevance.
Conclusion
Out of the 451 kinases, the Jamaican ball moss selectively inhibited 5, thus validating its potential anticancer activity. Our findings show that the bioactivity of the Jamaican ball moss inhibits two of the tested kinases, MEK5 and GAK, known to be associated with prostate cancer. Coupled with previous findings that demonstrate the bioactivity of the plant extract to apoptotically reduce the viability of a human prostate cancer cell line the present results are promising in the arena of drug discovery towards prostate cancer.
Acknowledgements
The Authors are grateful to Dr. Rena Lapidus of the Translational Core of the Greenebaum Cancer Center, University of Maryland School of Medicine for validating the results of the anticancer activity of Cycloartane-3,24,25-triol and to the KINOMEscan group (San Diego) for carrying out the kinase inhibition assays.
- Received July 31, 2012.
- Accepted August 31, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
