Cantharidin-induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2Aα

Abstract

Cantharidin, a natural vesicant, inhibits the activity of several PPP family phosphatases, displays antitumor activity, and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, doseresponse studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that before apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G 1 -S or S-G 2 phase progression. In contrast, studies with double thymidinesynchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G 2 or early M phase. Immunostaining indicates that cell cycle arrest occurs before the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live cell imaging with time-lapse microscopy shows that cantharidin disrupts the metaphase alignment of chromosomes and produces a prolonged mitotic arrest, with the onset of apoptosis occurring before the onset of anaphase. To explore the contribution of individual phosphatases, antisense oligonucleotides and small interfering RNA were developed to suppress the expression of cantharidinsensitive phosphatases. The suppression of PP2AA, but not PP2AB, is sufficient to induce metaphase arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10-to 15-hour delay, the cells enter anaphase, suggesting that an additional cantharidin-sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis.

Inhibition of serine/threonine phosphatase PP2A enhances cancer chemotherapy by blocking DNA damage induced defense mechanisms

“…Small molecules derived from cantharidin (a vesicant originally extracted from beetles) or its demethylated homolog (norcantharidin) mimic the effects of okadaic acid (16,17). Modest clinical benefit of cantharidin is constrained by urologic toxicity, and norcantharidin, although less toxic, has limited effectiveness (17).…”

“…Small molecules derived from cantharidin (a vesicant originally extracted from beetles) or its demethylated homolog (norcantharidin) mimic the effects of okadaic acid (16,17). Modest clinical benefit of cantharidin is constrained by urologic toxicity, and norcantharidin, although less toxic, has limited effectiveness (17). We synthesized a series of norcantharidin derivatives and characterized their antiphosphatase and anticancer activity in vitro (18) and selected LB1.2 for more detailed study in vivo alone and in combination with TMZ, the standard drug for the palliative treatment of GBM.…”

LB100, a small molecule inhibitor of PP2A with potent chemo- and radio-sensitizing potential

“…[59][60][61] Inhibition of PP2A with either of these compounds results in abnormal mitotic figures characteristic of mitotic catastrophe, a form of cell death distinct from apoptosis. [62][63][64] To overcome the toxicities of okadaic acid and cantharidins, a competitive small molecule inhibitor of PP2A called LB100 (Lixte Biotechnology, East Setauket, NY) was recently developed and was studied in a variety of pre-clinical cancer models. These pre-clinical studies demonstrated that LB100 exhibits potent chemo-and radio-sensitizing properties, which has prompted interest in evaluation of LB100 in human clinical trials.…”

Non-ionic surfactant vesicles simultaneously enhance antitumor activity and reduce the toxicity of cantharidin

“…7 Moreover, CTD has been shown to increase the level of Bax, inhibit the expression of B-cell lymphoma 2 and survivin, 8 and regulate the production of reactive oxygen species, subsequently inducing apoptotic cell death. 9 However, the toxicity of CTD, including the damage to mammals' urinary system, hematemesis, dysuria, liver congestion, and the risk of aborticide, remains a great challenge for its further development in clinical applications. 1 Therefore, scientists have persisted in the search to maximize its potential, and many CTD derivatives and several novel drug-delivery systems have been developed.…”