Cantharidin Research Articles

Molecular modes of action of cantharidin in tumor cells

Cancer chemotherapy is often limited by patient's toxicity and tumor drug resistance indicating that new drug development and modification of existing drugs is critical for improving the therapeutic response. Traditional Chinese medicine is a rich source of potential anticancer agents. In particular, cantharidin (CAN), the active principle ingredient from the blister beetle, Mylabris, has anti-tumor activity, but the cytotoxic mechanism is unknown. In leukemia cells, cantharidin induces apoptosis by a p53-dependent mechanism. Cantharidin causes both DNA single- and double-strand breaks. Colony-forming assays with knockout and transfectant cells lines showed that DNA polymerase β, but not ERCC1, conferred increased cell survival after cantharidin treatment, indicating that base excision repair (BER), rather than nucleotide excision repair (NER), is important for CAN-induced DNA lesions. Oxidative stress-resistant thymic lymphoma-derived WEHI7.2 variants are also more resistant to cantharidin. These data suggest that cantharidin treatment causes oxidative stress that provokes DNA damage and p53-dependent apoptosis.

Regulation of OsRac1 mRNA Against Defense-Related Stimuli in Wild-Type Rice Seedlings

A rice (Oryza sativa L.) homolog of mammalian Rac-GTPase, OsRac1, characterized only in transgenic rice, was proposed to involve in cell death/disease resistance. However, its role in wild-type rice remains uncharacterized and unclear. We examined expression of the OsRac1 mRNA in response to stress signalling components, jasmonic acid (JA), hydrogen peroxide (H2O2), and two protein phosphatase inhibitors, cantharidin (CN) and endothall (EN), using rice (cv. Nipponbare) seedlings. The OsRac1 transcript, whose accumulation required certain de novo synthesized protein factor(s), increased in the leaves upon CN/EN and H2O2 treatment, but not by JA. Using two pathogenesis-related (PR) protein gene markers, OsPR5 and OsPR10, our results also reveal OsRac1 mRNA accumulates later than that of these two important defense/stress-related OsPR genes.

Importance of ascorbate peroxidases OsAPX1 and OsAPX2 in the rice pathogen response pathways and growth and reproduction revealed by their transcriptional profiling

We have identified and characterized two rice ascorbate peroxidase ( OsAPX1 and OsAPX2) genes against diverse signals known to involve in defense/stress responses, using “2-week-old rice seedling in vitro model system”. Oryza sativa ascorbate peroxidase 1 and 2 ( OsAPX1/2) share 83% similarity to each other at amino acid level and belong to a small gene family in the rice genome. OsAPX1/2 show a weak constitutive expression in leaves. Their transcripts were up-regulated upon wounding (by cut), and diverse signals such as salicylic acid (SA), ethylene (using the ethylene generator, ethephon), abscisic acid (ABA), hydrogen peroxide, copper sulfate, protein phosphatase (PP) inhibitors, cantharidin (CN), endothall (EN) and okadaic acid (OA), and blast pathogen ( Magnaporthe grisea) attack, but surprisingly not by jasmonic acid (JA). Interestingly, the OsAPX2 inductions were more potent and rapid than OsAPX1, whose expression increased gradually with time. These inductions were influenced by light signal(s), and showed requirement for certain de novo synthesized protein factor(s). Intriguingly, OsAPX1/2 mRNAs expression manifested a clear rhythmicity under light/dark cycle. Differential regulation of the OsAPX1/2 expressions by diverse environmental cues, including pathogen, and their developmental regulation in both young and reproductive stages strongly suggests their role in stress-signaling pathways and development in rice.

Cloning and characterization of a jasmonate inducible rice ( Oryza sativa L.) peroxidase gene, OsPOX, against global signaling molecules and certain inhibitors of kinase-signaling cascade(s)

We report the characterization of a rice peroxidase ( OsPOX) gene, identified from screening a jasmonic acid (JA)-treated rice seedling leaf cDNA library, whose nucleotide sequence was essentially similar to the previously cloned pathogen inducible OSPER/POX22.3. However, it remains totally uncharacterized against global signaling molecules mediating defense/stress response(s), with little information on its regulation. OsPOX showed a weak constitutive expression in rice seedling leaf, and was highly responsive to cut. The cut inducible OsPOX transcript was further enhanced by the global signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (using the ethylene generator ethephon, ET). Strong induction of OsPOX expression by protein phosphatase 2A inhibitors, cantharidin (CN) and endothall (EN), and use of staurosporine alone or with JA, CN and EN suggested a role for the kinase-signaling cascades in its regulation. These inductions were light/dark-, and time-dependent, and required a certain de novo synthesized protein factor(s). Surprisingly, among other phytohormonal components of the signaling cascades, abscisic acid (ABA) down regulated the induced OsPOX expression, whereas a cytokinin, kinetin (KN) strongly induced its expression. As the JA induced OsPOX expression was almost abolished by SA and ABA, but slightly enhanced with KN, an interaction among these signaling molecules in modulating OsPOX expression is suggested. Present study clearly demonstrates that signaling molecules known to act as or generate cellular signals mediating the plant's response to stress/pathogen infection modulate the OsPOX expression.

Light-dependent induction of OsPR10 in rice ( Oryza sativa L.) seedlings by the global stress signaling molecule jasmonic acid and protein phosphatase 2A inhibitors

The OsPR10 gene was first shown to be weakly induced only 3 days after treatment with a herbicide, probenazole (N. Midoh, M. Iwata, Cloning and characterization of a probenazole-inducible gene for an intracellular pathogenesis-related protein in rice, Plant Cell Physiol. 37 (1996) 9–18), and remains uncharacterized till date. We provide first evidence for the induction of OsPR10 by the global signaling molecule, jasmonic acid (JA), and two protein phosphatase (PP) 2A inhibitors, cantharidin (CN) and endothall (EN). These inductions were light- and time-dependent, and also specific to the dose of applied JA, CN and EN. OsPR10 was not responsive to cut, and was expressed mainly in leaves and leaf sheaths, but not in roots. Furthermore, OsPR10 induction was blocked completely by cycloheximide, indicating requirement of de novo protein synthesis in its induction. Two signal transducers, salicylic acid (SA) and abscisic acid (ABA) induced OsPR10, and a cytokinin, kinetin (KN) also strongly up-regulated its expression, under light. Simultaneously applied staurosporine, a serine/threonine protein kinase inhibitor, strongly enhanced the JA inducible OsPR10 expression. The inducible nature of OsPR10 was affected significantly by a combination of applied JA, with varying concentrations of SA, ABA and KN. Induction of OsPR10 was followed by the appearance of necrotic lesions/coalescing necrotic regions clearly visible at 48 h under light, with CN and EN, but not with JA at a similar time period. Finally, using two-dimensional polyacrylamide gel electrophoresis, we identified a new member of the OsPR10 protein family, the OsPR10-3 protein. This represents the first systematic report in rice demonstrating that the inducible OsPR10 expression is light dependent, and may be regulated via a kinase-signaling cascade.

Characterization of a rice (Oryza sativa L.) Bowman–Birk proteinase inhibitor: tightly light regulated induction in response to cut, jasmonic acid, ethylene and protein phosphatase 2A inhibitors

The Bowman–Birk (BB) family of proteinase inhibitors (PI), initially reported from legume seeds, and thereafter also from wounded alfalfa and maize leaves appear to be regulated in similar ways as the extensively characterized PI I and PI II family from dicots. Here, we report a first characterization of the expression profiles of a rice (Oryza sativa L. cv. Nipponbare) BBPI gene, OsBBPI, which is part of a multigene family as demonstrated by genomic Southern hybridization. OsBBPI was found to be rapidly induced in rice seedling leaf in response to cut, exogenous jasmonic acid (JA), and two potent protein phosphatase 2A (PP2A) inhibitors, cantharidin (CN) and endothall (EN), in a light/dark-, time- and dose-dependent manner; this induction was completely inhibited by cycloheximide (CHX), indicating a requirement for de novo protein synthesis in its induction. Surprisingly, dark strongly up regulated cut-, JA-, CN-, and EN-induced OsBBPI expression, with the strongest enhancement observed with JA. A simultaneous application of a serine/threonine protein kinase inhibitor staurosporine (ST) did not affect significantly the JA-, CN-, and EN-induced OsBBPI transcript. Besides JA, it was found that the ethylene generator ethephon (ET) also had an enhancing effect on OsBBPI transcript, suggesting a direct effect of ethylene on OsBBPI expression. However, a simultaneous application of salicylic acid (SA) and abscisic acid (ABA), with JA, respectively, completely blocked OsBBPI gene expression, whereas kinetin (KN) was only partially effective. To the best of our knowledge, complete inhibition of JA-induced OsBBPI expression by SA is the first report in monocots, and with ABA in plants. Taken together, these results suggest that among the phytohormones tested here, JA and ethylene play important role(s) in regulating OsBBPI expression, with an intimate interaction with light signals. Finally, that the induced OsBBPI expression follows a kinase-signaling cascade is implied by the use of PP2A inhibitors.

Jasmonate, salicylate, protein phosphatase 2A inhibitors and kinetin up-regulate OsPR5 expression in cut-responsive rice ( Oryza sativa)

The rice OsPR5 gene, initially identified in response to pathogen attack, remains completely uncharacterized against global signaling molecules involved in defense/stress. Here, we provide evidence that OsPR5 expression, which is weakly constitutive in rice seedling leaves, is responsive to cut, and is up-regulated by jasmonic acid (JA), salicylic acid (SA), and protein phosphatase (PP) 2A inhibitors, cantharidin (CN) and endothall (EN), in a light/dark-, time-, and dose-dependent manner. Furthermore, except for the cut control, which showed considerable increase in the OsPR5 transcript with cycloheximide (CHX), a consistent partial suppression in the JA, SA, CN and EN induced OsPR5 transcript levels by CHX might suggest a requirement for de novo protein synthesis downstream of these chemical agents. Moreover, co-application of SA, ABA, and kinetin (which also up-regulated the OsPR5 transcript) with JA was found to further modulate the induced mRNA level of OsPR5. These results provide insight into the regulation of OsPR5 expression by signaling molecules and their co-application, and additionally implied a role for phosphorylation/dephosphorylation events in its regulation.

Rice (Oryza sativa L.) OsPR1b Gene Is Phytohormonally Regulated in Close Interaction with Light Signals

Strategies evolved by plants to counteract a variety of biotic/abiotic stresses include induction of genes encoding pathogenesis-related (PR) protein, in particular the PR class 1 (PR1) gene family, widely used in stress response studies. In spite of its immense importance as a PR family member, and an accepted gene marker in plant disease/defense in dicots, little is known about rice PR1 genes. Recently, we cloned and characterized the first OsPR1a (rice acidic PR1) gene (Agrawal et al. (2000) Biochem. Biophys. Res. Commun. 274, 157–165). Here, we report characterization of a rice basic PR1 (OsPR1b) gene, identified from screening a cDNA library prepared from jasmonic acid (JA)-treated rice seedling leaf, providing detailed and valuable insights into rice PR1 gene expression. The deduced amino acid sequence of OsPR1b reveals only 63.1% homology with the OsPR1a protein, whereas Southern blot analyses indicate that OsPR1b is a multigene family. The JA-inducible OsPR1b gene was also up-regulated by salicylic acid (SA), abscisic acid (ABA), and kinetin (KN). Furthermore, protein phosphatase inhibitors, cantharidin (CN) and endothall (EN) strongly induced the OsPR1b transcript. However, OsPR1b was not cut-responsive, diagrammatically opposite to cut inducibility of OsPR1a. This induction was light-, time-, and dose-dependent, as demonstrated by using, JA, CN, and EN, and completely inhibited by cycloheximide, but not by tetracycline. The simultaneous application of SA, and ABA, with JA, respectively, showed almost complete inhibition of the JA-induced OsPR1b transcript by 200 μM SA or ABA, but not by 100 μM concentrated solutions, suggesting a potential interaction among JA, SA, and ABA, whereas KN dramatically enhanced JA-induced OsPR1b transcript upon simultaneous application. Moreover, a simultaneous application of staurosporine enhances JA-, CN-, and EN-induced OsPR1b transcript, in particular with CN. Finally, a comparative analysis with the OsPR1a gene gives us insight into the differential regulation of the PR1 gene family, while proposing OsPR1 genes as important gene markers in rice, with potential use(s) in analyzing plant defense responses.

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.

Cantharidin effects on protein phosphatases and the phosphorylation state of phosphoproteins in mice.

Our earlier studies indicated that the action of cantharidin (CA) in mice is associated with binding to protein phosphatase 2A in liver cytosol and inhibition of its phosphorylase a phosphatase activity. In this investigation, we find that CA totally inhibits the phosphorylase a phosphatase activity in mouse liver, muscle, and skin cytosol at 5000 nM, with IC50s of 110-250 nM. About 50% of the phosphorylase a phosphatase activity of brain cytosol is sensitive to CA with an IC50 of approximately 80 nM and the remaining half is not inhibited even at 5000 nM. Intraperitoneal treatment of mice with CA leads to a dose-dependent decrease in phosphorylase a phosphatase activity with the aforementioned tissues displaying differential CA sensitivity. At 60 min after a 10 mg/kg CA dose, there is 90-95% inhibition of phosphorylase a phosphatase activity in liver and skin cytosol, 50% in muscle cytosol, and almost no inhibition in brain cytosol. The phosphorylation state of several phosphoproteins examined with tissue cytosol and [gamma-32P]ATP is increased by CA, in a concentration-dependent manner, as follows: endogenous glycogen phosphorylase a in muscle both in vitro and in vivo, and unidentified phosphoproteins in brain (approximately 34 and approximately 75 kDa) and skin (approximately 34 kDa) in vitro. These findings confirm the importance of protein phosphatases as primary targets of CA action in a variety of mouse tissues and, more generally, the possible use of CA and its analogs to investigate and potentially control some processes modulated by the reversible phosphorylation of proteins.

Effects of CGRP in different models of mouse ear inflammation

The anti-inflammatory activity of calcitonin gene-related peptide (CGRP) has been studied in cutaneous inflammation induced by croton oil (CO), arachidonic acid (AA), tetradecanoylphorbol acetate (TPA) or cantharidin (CA). Our results show that mouse ear inflammation induced by CO, AA or TPA is decreased by topical administration of CGRP, whereas that induced by CA is not affected. The dose-response and temporal analysis of CGRP effect show that the maximal activity is present at the dose of 30 pmol/ear and when administered 30 min after the irritating agent. Moreover, pretreatment with capsaicin is able to mimic the anti-inflammatory effect of exogenous CGRP, while simultaneous administration of CGRP and capsaicin produces a reduced response. Our results suggest that CGRP released from sensory

Protein phosphatase 2A and its [ 3H]cantharidin/[ 3H]endothall thioanhydride binding site : Inhibitor specificity of cantharidin and ATP analogues

The target site for cantharidin (CA) and its analogues was isolated recently from mouse liver and identified as protein phosphatase 2A (PP2A) in the heterodimeric form known as PP2A 2. The most toxic CA analogue, endothall thioanhydride (ETA) (mouse i.p. LD 50 0.3 mg/kg), appears to have the same binding site in mouse liver and brain based on studies comparing [ 3H]ETA and [ 3H]CA. ATP and its nonhydrolyzable analogues and pyrophosphate and related compounds including phosphonoformic acid inhibited both [ 3H]CA and [ 3H]ETA binding with IC 50 values ranging from 2 to 81 μM. As with CA itself, the most potent inhibitors have two negatively charged groups in close proximity to each other. Inhibition of [ 3H]CA binding by 5,5'-dithiobis(2-nitrobenzoic acid) and stimulation by N-ethylmaleimide indicated the involvement of a thiol site in the CA-binding domain. CA and three analogues (cantharidic acid, palasonin and endothall) inhibited PP2A and protein phosphatase 1 (PP1) but not PP2B or PP2C. The catalytic subunit of PP2A was 5- to 12-fold more sensitive to these CA analogues than the catalytic subunit of PP1. CA and the herbicide endothall also inhibited spinach leaf PP1 and PP2A and, at 50 μM, decreased the PP2A-mediated light-induced activation of nitrate reductase in intact spinach leaves by 62 and 56%, respectively. This is consistent with PP2A as their site of action in plants, and indicates the potential use of CA analogues as pharmacological probes to investigate cellular processes that are regulated by reversible protein phosphorylation in vivo.

Cantharidin: Novel effects on plants

Cantharidin: Novel effects on plants

Poisoning by Cantharidin

Poisoning by Cantharidin

Poisoning by cantharidin.

Poisoning by cantharidin.

A case of cantharidin poisoning.

A case of cantharidin poisoning.

The constitution of cantharidin

The constitution of cantharidin

Carpenterʼs Oil of Cantharidin.

Carpenterʼs Oil of Cantharidin.