Exploring the Different Stages of Bat Plant Growth

†Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229

‡Cancer Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229Find articles by

†Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229

‡Cancer Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229Find articles by

†Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229Find articles by

§Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX 78249Find articles by

‡Cancer Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229

§Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX 78249Find articles by

†Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229

‡Cancer Therapy and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229Find articles by

The biosynthesis of secondary metabolites provides higher plants mechanisms of defense against microbes, insects, and herbivores. One common cellular target of these molecules is the highly conserved microtubule cytoskeleton and microtubule targeting compounds with insecticidal, antifungal, nematicidal and anticancer activities have been identified from plants. A new retro-dihydrochalcone, taccabulin A, with microtubule destabilizing activity has been identified from the roots and rhizomes of Tacca species. This finding is notable because the microtubule stabilizing taccalonolides are also isolated from these sources. This is the first report of an organism producing compounds with both microtubule stabilizing and destabilizing activities. A two-step chemical synthesis of taccabulin A was performed. Mechanistic studies showed that taccabulin A binds within the colchicine site on tubulin and has synergistic antiproliferative effects against cancer cells when combined with a taccalonolide, which binds to a different site on tubulin. Taccabulin A is effective in cells that are resistant to many other plant-derived compounds. The discovery of a natural source that contains both microtubule stabilizing and destabilizing small molecules is unprecedented and suggests that the synergistic action of these compounds was exploited by nature long before it was discovered in the laboratory.

Plants as sessile organisms have evolved within their ecosystems to overcome biotic and abiotic stresses. Higher plants have developed sophisticated mechanisms of defense from insects, fungi, nematodes, and mammalian herbivores.1,2 Chemical defenses include the production of plant secondary metabolites that can be toxic, targeting fundamental biological processes, including mitochondrial respiration, proteases, ATPases, neuronal receptors, and cytoskeletal plasticity, which are thought to protect plants from a range of threats from infection to consumption.3,4 A common target for plant-derived secondary metabolites is the cytoskeleton. Plant-derived compounds that disrupt each of the major cytoskeletal components, intermediate filaments, microfilaments, and microtubules, have been discovered.5–8 We hypothesize that the ability of these compounds to interact directly with highly evolutionarily conserved cytoskeletal proteins may provide defense against a broad genera of predatory organisms.

Compounds that bind to tubulin/microtubules are divided into two categories, microtubule stabilizers and destabilizers, which increase or decrease cellular microtubule mass, respectively.7 Although they are named for their opposing effects on tubulin polymer equilibrium, at the lowest effective concentrations both stabilizers and destabilizers suppress microtubule dynamics, impairing cellular processes from vesicular transport to mitosis. The combination of a microtubule stabilizer and destabilizer that occupy different binding sites on tubulin can act synergistically to reduce microtubule function leading to cytotoxicity.9,10 Many microtubule destabilizing secondary metabolites have been isolated from plants, including colchicine, vinblastine, combretastatin A-4 (CA-4), and podophyllotoxin. Additionally, the first microtubule stabilizer identified, taxol, isolated from Taxus brevifolia, the Pacific yew11 is one of the most successful anticancer agents ever discovered. While microtubule stabilizers and destabilizers have been isolated from a wide range of plant species, no species has ever demonstrated the ability to produce compounds with each of these activities.

Here we show that the roots and rhizomes of Tacca sp, the bat flower, produce both a microtubule destabilizing retro-dihydrochalcone in addition to the known taccalonolide microtubule stabilizing compounds.12–14 These structurally diverse compounds have opposite effects on microtubule polymer mass, but at the lowest antiproliferative concentrations they cause super-additive effects against cancer cells. It is interesting to speculate that together these compounds could provide superior chemical defenses for the plant.

Taccabulin A ( Inhibits Tubulin Polymerization and Binds Within the Colchicine Site on Tubulin

All known microtubule destabilizers bind directly to tubulin, which allows the effects of these drugs to be observed in biochemical preparations containing only purified tubulin and guanosine triphosphate (GTP) in a glycerol buffer. Under these conditions, tubulin can assemble into microtubules, as observed by an increase in turbidity over time ( ). Taccabulin A (2) caused a dose dependent decrease in the rate and extent of microtubule polymerization. A 25 μM concentration of 2 caused approximately 50% inhibition of polymerization ( ) indicating that it is less potent than CA-4 which caused 50% inhibition of polymerization at 2 – 3 μM.21

Two sites for microtubule destabilizers have been identified on tubulin: the vinca site and the colchicine site. While the large complex natural products of the vinca family bind within the vinca site, most small molecule microtubule destabilizers bind to the colchicine site. Owing to the strong structural similarities between 2 and colchicine-site agents, including CA-4,21 we hypothesized that 2 also binds to the colchicine site. When colchicine is bound to tubulin, a conformational change causes the drug to fluoresce, a property that unbound colchicine does not possess.22 This property of colchicine-tubulin binding was utilized to determine whether 2 could compete for binding to this site on tubulin. A dose dependent inhibition of colchicine fluorescence was observed when 2 was added to biochemical preparations containing purified tubulin, colchicine, and GTP ( ). Interestingly, the concentrations of 2 that caused a 50% inhibition of colchicine binding or a 50% inhibition of tubulin polymerization in biochemical assays were equivalent (compare ).

Taccabulin A ( Disrupts Microtubule-dependent Processes

Microtubule stabilizers and destabilizers have seemingly opposite cellular effects based on their respective ability to increase or decrease interphase microtubule mass ( ). However, the prevailing view of the mechanism by which both classes of drugs inhibit the proliferation of cells in culture is by inhibiting microtubule dynamics. During mitosis, the mitotic spindle and kinetochore microtubules rapidly grow and shrink to properly align and separate sister chromatids and this requires highly dynamic microtubules. Mitotic microtubules are 3.6-fold more dynamic than interphase microtubules.23

Microtubule stabilizers and destabilizers disrupt highly dynamic mitotic spindles leading to mitotic arrest at concentrations lower than those that cause gross changes in interphase microtubule mass. Indeed, 2, like the taccalonolides, arrested cells in the G2/M phase of the cell cycle as determined by flow cytometry ( ). Consistent with the flow cytometry results, when the microtubules of 2-treated cells were visualized, abnormal mitotic spindles were observed. Normal bipolar mitotic spindles were present in vehicle-treated cells ( ) while the taccabulin A (2)-treated cells contained multiple aberrant spindle asters ( ). The formation of aberrant spindles during mitosis contributes to the inability of both taccalonolide A (1) and taccabulin A (2)-treated cells to progress through the cell cycle, even though the morphologies of the spindles formed by these two drugs are distinct (compare ).

Growing the Black Bat Flower (Tacca chantrieri)