Protein Phosphatase Inhibitor Cantharidin Research Articles

On Otis tarda and Marquis de Sade: what motivates male Great Bustards to consume Blister Beetles (Meloidae)?

Blister Beetles (Meloidae), invertebrates that can synthesize high doses of the protein phosphatase inhibitor cantharidin, are ingested preferentially by male Great Bustards (Otis tarda) during their mating season. Previous authors hypothesized that the bustards benefit from antihelminthic and antimicrobial properties of cantharidin. However, given that the ingestion of Blister Beetles is performed particularly in mating season, it may be that the cantharidin has more important direct effects on male behavior, possibly by increasing sexual arousal of males. Thus, we propose that cantharidin consumption by male Great Bustards is a form of self-medication to increase courtship display, willingness to participate in pre-breeding male–male competitions at leks, display intensity, and mating success.

A systems biology approach reveals the role of a novel methyltransferase in response to chemical stress and lipid homeostasis.

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors.

Flavin-containing polyamine oxidase is a hydrogen peroxide source in the oxidative response to the protein phosphatase inhibitor cantharidin in Zea mays L.

In this study, the specific contribution of polyamine oxidase (PAO), a hydrogen peroxide (H2O2)-producing enzyme, to the oxidative burst induced in maize mesocotyl by the phosphatase inhibitor cantharidin was examined. For this purpose, a pharmacological approach was applied using, either in vitro or in vivo, two strong inhibitors of maize PAO (MPAO), N-prenylagmatine (G3) and its structural analogue Ro5, as well as diphenyleneiodonium (DPI), an inhibitor of the phagocyte NAD(P)H oxidase. DPI was shown to be a good MPAO inhibitor in vitro. G3, Ro5, and DPI were very effective in inhibiting in vivo the extracellular accumulation of H2O2 that is released by mesocotyl segments upon spermidine supply. G3 and Ro5 did not show any inhibition in vitro of either horseradish peroxidase or barley oxalate oxidase. Moreover, G3 and Ro5 did not inhibit the extracellular accumulation of superoxide radical that is released in vivo upon NADH supply. G3, Ro5, and DPI strongly affected H2O2 production induced in maize mesocotyl by cantharidin. Histochemical localization of H2O2 in cantharidin-treated mesocotyl cross-sections revealed an increase of H2O2-specific staining in the epidermal and subepidermal tissues. The effect was also inhibited by G3 and DPI. Moreover, an increase in MPAO activity was observed in the same tissues upon cantharidin treatment. All these data suggest that G3 and Ro5 behave as powerful and selective inhibitors of MPAO activity either in vitro or in vivo and that MPAO activity contributes to a major part of the cantharidin-induced H2O2 synthesis in the apoplastic milieu of maize mesocotyl.

Indirect phosphorylation‐dependent modulation of postsynaptic nicotinic acetylcholine responses by 5‐hydroxytryptamine

Ionotropic nicotinic acetylcholine (ACh) receptors have been shown to be modulated by protein kinase-mediated phosphorylation in vitro. Here we demonstrate that 5-hydroxytryptamine (5-HT) can downregulate postsynaptic nicotinic ACh responses, elicited in an identified arthropod motoneuron in situ, by a mechanism dependent on protein kinase activity. Serotonergic modulation can be mimicked by perfusion with membrane-permeable analogues of either adenine (cAMP) or guanine (cGMP) cyclic nucleotides, and is prolonged in the presence of phosphodiesterase inhibitors. Furthermore, suppression of the ACh response by 5-HT is blocked by specific competitive inhibitors of protein kinase A and G, as well as the broad specificity protein kinase inhibitor staurosporine. The protein phosphatase inhibitor cantharidin similarly blocks recovery of the ACh response from suppression mediated by 5-HT. Thus, it appears that the nicotinic ACh response is modulated by a cAMP-mediated phosphorylation-dependent intracellular signalling pathway that is distinct from the direct block of mammalian nicotinic ACh receptors by 5-HT previously reported in vitro.

In vitro Assessment of Renal Toxicity and Inflammatory Events of Two Protein Phosphatase Inhibitors Cantharidin and Nor‐Cantharidin*

In China, cantharidin has been reported to be active against various human cancers, but with severe side effects such as nephrotoxicity. In order to reduce this toxicity, its demethylated analogue nor-cantharidin has been synthesized and used in cancer therapy, but with only few data regarding safety assessment. The aim of this study was to compare the in vitro effects of cantharidin and nor-cantharidin on renal toxicity and on inflammatory events associated with tumoural process where protein phosphatases could be involved (energy status, prostanoid production, glutathione and nitrite contents) on RAW 264.7 and LLC-PK1 cells. In macrophages, both cantharidin and nor-cantharidin decreased cell viability, in a concentration- and time-dependent manner. However, IC50 was lower with cantharidin than with nor-cantharidin. These two drugs significantly decreased the ATP level after 24 hr incubation. However, ATP decreased much more with cantharidin (up to 4 times) than with nor-cantharidin. When control macrophages were activated with lipopolysaccharide+interferon-gamma for 24 hr a significant increase in nitrite content and in prostanoids were observed. Addition of either drug decreased nitrite generation and prostanoids, however these decreases were greater with cantharidin than with nor-cantharidin. In LLC-PK1 cells, incubated with either cantharidin or nor-cantharidin, our results show significant differences between the two drugs, similar to those observed in peritoneal macrophages, except for GSH content with opposite variations in both cells. We provide a better understanding of the various mechanisms of cantharidin side effects, allowing an easier comparison with nor-cantharidin which could be an attractive therapeutic potential in cancer chemotherapy in western countries.

Hermaphroditism, gonochorism, and asexual reproduction in Cassiopea sp.—an immigrant in the islands of Hawai'i

Summary Two populations of the conspicuous, semi-sessile medusae of the rhizostome genus Cassiopea, both morphologically similar to Cassiopea andromeda from the Red Sea and Indopacific, were recently studied on Oahu (Hawai'i), one from a sheltered saltwater pond (the Hilton Lagoon) next to Ala Wai Harbor, Honolulu, and the second from the canals of a fish farm near Kahuku on NE Oahu. Gonad differentiation, checked in 1998, and again in 1999 and 2000, was unexpectedly different in medusae from these two locations. Contrary to all earlier reports and personal observations of strict gonochorism in C. andromeda, the majority of the Hilton Lagoon medusae collected in 1998 were hermaphroditic with female and male parts intermingling in the gonads. This is an exceptional case in scyphozoans. However, this hermaphroditic condition, which was observed again in 1999, was not stable over time. All six medusae taken from the same lagoon in 2000 were distinctly gonochoristic; four were males, and two were females brooding egg masses on the oral disk. Planula larvae hatching in the laboratory were successfully reared to the scyphopolyp stage. By contrast, medusae sampled from the population were all males in 1998, 1999, and 2000. Polyps were found abundantly on dark, deteriorating leaves of the Red Mangrove, occasionally on other plant remnants, and on discarded plastic materials in the canals. The polyps propagated asexually both by strobilation of ephyrae and by production of motile, larva-like buds that settled to form additional polyps. We speculate on a clonal origin of this male population. Asexually formed propagules were induced to form polyps by exposure to either the peptide Z-GPGGPA or biogenic substrata as has been shown before in other Cassiopea species. The serine/threonine protein phosphatase inhibitor cantharidin stimulated bud development too, but interfered strongly with pattern formation, polarity, and the sequence and timing of morphogenetic events. It did not induce typical bud-to-polyp metamorphosis.

Alterations in the distribution of histone H3 phosphorylation in mitotic plant chromosomes in response to cold treatment and the protein phosphatase inhibitor cantharidin.

The function of the phosphorylation of histone H3 at Ser 10 in plant cell division is uncertain. The timing correlates with chromosome condensation, and studies in plant meiosis suggest that it is involved in sister chromatid cohesion. In mitosis, plant chromosomes are highly phosphorylated in the pericentromeric region only. In order to modulate H3 phosphorylation, root meristems of different plant species were treated with the protein phosphatase inhibitor cantharidin or with ice-water. Immunostaining using an antibody specific to phosphorylated H3 at Ser 10 revealed a high level of H3 phosphorylation along the whole mitotic chromosome after cantharidin treatment, which resembles the distribution seen exclusively in first meiotic division. In chromosomes that were isolated from meristems treated with ice-water, the heterochromatic regions and nucleolar organizer regions, in addition to the pericentromeric region, were highly phosphorylated at H3. Cantharidin and ice-water also affected spindle assembly and chromosome length, but these effects did not seem to be directly linked to changes in H3 phosphorylation.

Probing the circadian control of phosphoenolpyruvate carboxylase kinase expression in Kalanchoë fedtschenkoi.

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. In crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) kinase is expressed at night under the control of a circadian oscillator. We have proposed that this is an indirect effect, secondary to circadian fluctuations in the level of a metabolite, possibly cytosolic malate, resulting from a primary effect on the permeability of the tonoplast (Nimmo 2000, Trends in Plant Science 5, 75-80). Here we show that the nocturnal accumulation of PEPC kinase translatable mRNA and phosphorylation of PEPC in Kalanchoë fedtschenkoi is blocked by the protein phosphatase inhibitor cantharidin. This implicates protein dephosphorylation in the circadian pathway that regulates expression of PEPC kinase. We also show that the effect of reducing the temperature from 30 to 15 °C on CO2 fixation by detached leaves held in constant darkness and normal air is 'gated' by the circadian clock. This strongly supports the view that the effect of the clock on the expression of PEPC kinase is secondary rather than direct. We have developed a non-aqueous fractionation protocol that separates the cytosolic material in mature leaves from vacuolar material. The cytosolic malate in mature leaves represents a very small part of the total malate, and its concentration cannot be measured precisely by this method.

Protein phosphatase inhibitor cantharidin inhibits steroidogenesis and steroidogenic acute regulatory protein expression in cultured rat preovulatory follicles

The effect of cantharidin, a natural toxicant of blister beetles and a strong inhibitor of protein phosphatases types 1 and 2A, on luteinizing hormone (LH)-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450scc) enzyme. Treatment of preovulatory follicles dissected from ovaries of immature rats primed with pregnant mares' serum gonadotropin (10 IU) with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 100 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of follicles with increasing concentrations (10 – 1000 ng/ml) of cantharidin suppresssed LH (100 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450scc protein and the conversion of 22R-hydroxycholesterol to progesterone were not affected by cantharidin. This indicates that cantharidin did not interfere with the activity of P450scc. Cantharidin also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (10 −5 M) or a cAMP analog 8-Br-cAMP (0.5 mM). These results demonstrate that cantharidin inhibits the LH-induced StAR protein levels, and, thus, suggest that phosphoprotein phosphatase activity is required for the cAMP-protein kinase A–stimulated steroidogenic activity of the preovulatory follicle.

Protein phosphatase inhibitors activate defense responses in rice (Oryza sativa) leaves

The protein phosphatase inhibitor cantharidin activates defense responses in rice leaves when applied exogenously at concentrations ranging from 100 to 500 μM. Responses include the accumulation of the major rice phenolic phytoalexin sakuranetin and the lactone phytoalexin momilactone A. Accumulation of sakuranetin was preceded by an induction of phenylalanine ammonia lyase (PAL) activity and an increase in the activity of naringenin 7‐O‐methyltransferase (NOMT), the key enzyme in sakuranetin biosynthesis. Cantharidin also strongly induced accumulation of the probenazole (PBZ)‐inducible protein (PBZ1) and two novel, related proteins named PBZ2 and PBZ3. Endothall, a herbicide and potent protein phosphatase inhibitor, but not its inactive analog (1,4‐dimethylendothall) also induced sakuranetin accumulation, increased activity of NOMT and accumulation of the 3 PBZ proteins. In contrast, two other protein phosphatase inhibitors, calyculin A and microcystin LR, did not activate these defense responses. Induction of NOMT and PAL activity, and sakuranetin accumulation, was completely blocked by cycloheximide. Leaf segments treated with cantharidin and endothall showed brownish and orange colored lesions, respectively, similar to the lesion mimic mutants of rice. These results indicate a direct role for protein phosphorylation/dephosphorylation events in the activation of defense responses in rice, in particular on the accumulation of antifungal phytoalexins and the PBZ proteins.

A Novel Rice (Oryza sativa L.) Acidic PR1 Gene Highly Responsive to Cut, Phytohormones, and Protein Phosphatase Inhibitors

A novel rice acidic pathogenesis-related (PR) class 1 cDNA (OsPR1a) was isolated from jasmonic acid (JA)-treated rice seedling leaf. The OsPR1a cDNA is 830 bp long and contains an open reading frame of 507 nucleotides encoding 168 amino acid residues with a predicted molecular mass of 17,560 and pI of 4.4. The deduced amino acid sequence of OsPR1a has a high level of identity with acidic and basic PR1 proteins from plants. Southern analysis revealed that OsPR1a is a member of a multigene family. The OsPR1a gene was found to be cut-inducible, whereas the phytohormones JA, salicylic acid (SA), 3-indoleacetic acid, gibberellin, and ethylene (using ethylene generator ethephon, ET) enhanced accumulation of OsPR1a transcript, as well as the protein phosphatase inhibitors cantharidin (CN) and endothall (EN). Induced expression of OsPR1a gene by JA, CN or EN, and ET was light/dark- and dose-dependent and was almost completely inhibited by cycloheximide. Dark downregulated CN-, EN-, and ET-induced OsPR1a gene expression, whereas it was further enhanced with JA. SA and abscisic acid blocked JA-induced OsPR1a transcript. Simultaneous application of staurosporine (ST) enhances CH- or EN-induced OsPR1a transcript, but not with JA. This is the first report on cloning of a rice acidic PR1 gene (OsPR1a), which is regulated by phytohormones, phosphorylation/dephosphorylation event(s), and light.

Phosphatase inhibitor cantharidin blocks adenosine A(1) receptor anti-adrenergic effect in rat cardiac myocytes.

Experiments were performed to examine whether the protein phosphatase inhibitor cantharidin blocks the anti-adrenergic effect of adenosine A(1) receptor stimulation. In electrically stimulated adult rat ventricular myocytes loaded with the intracellular calcium concentration ([Ca(2+)](i)) indicator fluo-3, isoproterenol (10 nM) increased systolic [Ca(2+)](i) by 46%, increased twitch amplitude by 56%, and increased total cellular cAMP content by 140%. The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentlyadenosine (CCPA) reduced isoproterenol-stimulated [Ca(2+)](i) and contractility by 87 and 80%, respectively, but reduced cAMP content by only 18%. Cantharidin had no effects on myocyte [Ca(2+)](i), contractility, or cAMP in the absence or presence of isoproterenol but blocked the effects of CCPA on [Ca(2+)](i) and contractility by approximately 44%. Cantharidin had no effect on CCPA attenuation of isoproterenol-induced increases in cAMP. Pretreatment with CCPA also reduced the increase in contractile parameters produced by the direct cAMP-dependent protein kinase A (PKA) activator 8-bromocAMP. These results suggest that activation of protein phosphatases mediate, in part, the anti-adrenergic effect of adenosine A(1) receptor activation in ventricular myocardium.

Early and delayed tolerance to simulated ischemia in heat-preconditioned endothelial cells: a role for HSP27.

An ischemia-mimicking metabolic stress in cultured endothelial cells from the human aorta or umbilical vein caused ATP depletion, a rise in cytosolic free Ca2+, fragmentation and aggregation of actin microfilaments, retraction of the cytoplasm, and disintegration of cell monolayer. Simultaneously, the constitutive heat shock protein 27 (HSP27) underwent dephosphorylation and formed granules inside cell nuclei. Prior heat shock (45 degreesC, 10 min) in confluent cultures conferred two phases (early and delayed) of tolerance to simulated ischemia. Although heat preconditioning did not retard the ATP drop and the free Ca2+ overload within ischemia-stressed cells, each phase of the tolerance was manifested in longer preservation of normal cell morphology during the stress. Cells exhibiting the early tolerance within 3 h after heating altered the F-actin response to ischemic stress; no microfilament debris but, instead, translocation of F-actin to the tight submembranous layer was observed. In contrast, the delayed cytoprotection preserved the preexisting F-actin bundles under simulated ischemia; this happened only after 12- to 14-h post-heat shock recovery, elevating the intracellular HSP content, and was sensitive to blockers of HSP synthesis, cycloheximide and quercetin. The dephosphorylation and intranuclear granulation of HSP27 were markedly suppressed in both phases of the heat-induced tolerance. Without heat pretreatment, similar attenuation of the HSP27 dephosphorylation/granulation and the actin cytoskeleton stability during simulated ischemia were achieved by treating cells with the protein phosphatase inhibitors cantharidin or sodium orthovanadate. We suggest that prior heat shock ameliorates the F-actin response to ischemic stress by suppressing the HSP27 dephosphorylation/granulation; this prolongs a sojourn in the cytosol of phosphorylated HSP27, which protects microfilaments from the disruption and aggregation.