Yi-Chen Wu

Induction of Centrosome Amplification during Arsenite-Induced Mitotic Arrest in CGL-2 Cells

Arsenite-induced mitotic abnormalities result in mitotic death in several cancer cell lines. However, how arsenite induces these effects is not known. We have previously shown that arsenite induces mitotic arrest, mitotic abnormalities, and mitotic death in CGL-2 cells. To further delineate the mechanism of action of arsenite, we examined its effect on centrosome duplication and the possible link between centrosome dysregulation and arsenite-induced mitotic death. Immunofluorescence staining of gamma-tubulin revealed that centrosome amplification was induced in arsenite-arrested mitotic cells but not in nocodazole-arrested cells. When S phase-enriched cells were treated with arsenite, they progressed into and arrested at mitosis and then formed supernumerary centrosomes. A further increase in arsenite-induced centrosome amplification was seen during the prolonged mitotic arrest. The arsenite-induced supernumerary centrosomes might result from uneven fragmentation of centrosome, overexpression of pericentriolar materials, and inhibition of centrosomal coalescence during mitosis. Furthermore, termination of mitotic arrest by treatment of arsenite-arrested mitotic cells with cyclin-dependent kinase 1 inhibitors or by suppression of spindle checkpoint function by small interfering RNA-mediated silencing of BubR1 or Mad2 markedly reduced the induction of centrosome amplification and mitotic death in arsenite-treated cells. These results indicate that centrosome amplification is induced in arsenite-arrested mitotic CGL-2 cells in a spindle checkpoint-dependent manner and is involved in the induction of arsenite-induced mitotic death.

Heat shock protein inhibitors, 17-DMAG and KNK437, enhance arsenic trioxide-induced mitotic apoptosis

Arsenic trioxide (ATO) has recently emerged as a promising therapeutic agent in leukemia because of its ability to induce apoptosis. However, there is no sufficient evidence to support its therapeutic use for other types of cancers. In this study, we investigated if, and how, 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG), an antagonist of heat shock protein 90 (HSP90), and KNK437, a HSP synthesis inhibitor, potentiated the cytotoxic effect of ATO. Our results showed that cotreatment with ATO and either 17-DMAG or KNK437 significantly increased ATO-induced cell death and apoptosis. siRNA-mediated attenuation of the expression of the inducible isoform of HSP70 (HSP70i) or HSP90alpha/beta also enhanced ATO-induced apoptosis. In addition, cotreatment with ATO and 17-DMAG or KNK437 significantly increased ATO-induced mitotic arrest and ATO-induced BUBR1 phosphorylation and PDS1 accumulation. Cotreatment also significantly increased the percentage of mitotic cells with abnormal mitotic spindles and promoted metaphase arrest as compared to ATO treatment alone. These results indicated that 17-DMAG or KNK437 may enhance ATO cytotoxicity by potentiating mitotic arrest and mitotic apoptosis possibly through increased activation of the spindle checkpoint.

Requirement of a functional spindle checkpoint for arsenite-induced apoptosis.

To understand the potential influence of spindle checkpoint function in response to arsenic trioxide (ATO)‐induced apoptosis observed in cancer cell lines, we examined the correlation between activation of the spindle checkpoint and susceptibility to ATO‐induced apoptosis in 10 cancer cell lines lacking functional p53. The ability to functionally activate the spindle checkpoint in each cancer cell line was assessed by the induction of mitotic arrest after Taxol treatment. Bromodeoxyuridine (BrdU) pulse‐chase analysis of Taxol‐treated cell lines with low mitotic arrest showed that they were not arrested at mitosis but divided abnormally, confirming that spindle checkpoint activation was impaired in these cell lines. Our results demonstrate that apoptosis was significantly induced by ATO in cancer cell lines with functional activation of the spindle checkpoint and substantial induction of mitotic arrest. Cell lines with negligible mitotic arrest exhibited little ATO‐induced apoptosis. However, no such correlation was observed following treatment of cells with camptothecin, a topoisomerase I inhibitor. Furthermore, attenuation of the spindle checkpoint function by small interfering RNA‐mediated silencing of BubR1 and Mad2 in cancer cells that were susceptible to ATO‐induced mitotic arrest and apoptosis greatly reduced the induction of mitotic arrest and apoptosis by ATO and increased the formation of micronuclei or multinuclei in survived cells. The marked correlation between ATO‐induced mitotic arrest and apoptosis indicates that the induction of apoptosis by ATO was highly dependent on the functional activation of the spindle checkpoint in cancer cells lacking normal p53 function. J. Cell. Biochem. 105: 678–687, 2008.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest.

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.

Arsenic Trioxide Induces Abnormal Mitotic Spindles Through a PIP4KIIγ/Rho Pathway

Arsenite-induced spindle abnormalities result in mitotic cell apoptosis in several cancer cell lines, but how arsenite induces these effects is not known. Evidence to date has revealed that arsenite activates Rho guanosine triphosphatases (GTPases). Because Rho GTPases regulate spindle orientation, chromosome congression, and cytokinesis, we therefore examined the involvement of Rho GTPases and their modulators in arsenite-induced mitotic abnormalities. We demonstrated that arsenic trioxide (ATO) disrupted the positioning of bipolar mitotic spindles and induced centrosome and spindle abnormalities. ATO increased the level of the active guanosine triphosphate-bound form of Rho. Inhibition of Rho-associated protein kinases (ROCKs) by Y-27632 ameliorated ATO-induced spindle defects, mitotic arrest, and cell death. These results indicate that ATO may induce spindle abnormalities and mitotic cell death through a Rho/ROCK pathway. In addition, screening of a human kinase and phosphatase shRNA library to select genes that mediate ATO induction of spindle abnormalities resulted in the identification of phosphatidylinositol-5-phosphate 4-kinase type-2 gamma (PIP4KIIγ), a phosphatidylinositol 4,5-biphosphate (PIP2) synthesis enzyme that belongs to the phosphatidylinositol phosphate kinase (PIPK) family. Sequestration of PIP2 by ectopic overexpression of the pleckstrin homology domain of phospholipase C-δ1 protected cells from ATO-induced cell death. Furthermore, depletion of PIP4KIIγ, but not other isoforms of the PIPK family, not only reduced Rho GTPase activation in ATO-treated cells but also alleviated ATO-induced spindle defects, mitotic arrest, and mitotic cell apoptosis. Thus, our results imply that ATO induces abnormalities in mitotic spindles through a PIP4KIIγ/Rho pathway, leading to apoptosis of mitotic cells.

Glyfoline induces mitotic catastrophe and apoptosis in cancer cells.

Glyfoline exhibits cytotoxic activity in vitro and antitumor activity in mice bearing murine or human solid tumors, but the underlying mechanisms are unknown. In our study, we found that glyfoline inhibited cell growth and induced accumulation of mitotic cells in human cancer cell lines. Glyfoline induced the appearance of spindle abnormalities, chromosome mis‐segregation, multipolar cell division and multiple nuclei, all of which are indicative of mitotic catastrophe. However, glyfoline did not bind to DNA and did not inhibit or stabilize tubulin polymerization, but slightly increased the resistance of mitotic spindles to nocodazole‐induced disassembly. In addition, microtubule aster formation was significantly enhanced in the extract prepared from glyfoline‐arrested mitotic cells compared to that from synchronized mitotic cells. When Eg5, a mitotic kinesin that plays an essential role in establishing mitotic spindle bipolarity, was inhibited using S‐trityl‐cysteine in glyfoline‐treated cells, formation of spindle multipolarity, multipolar cell division, and multinuclei was significantly reduced. After glyfoline‐mediated arrest of cells at mitosis, considerable poly(ADP‐ribose) polymerase degradation was induced and the number of annexin V‐positive cells significantly increased, indicating that glyfoline ultimately induces apoptosis. Small interfering RNA‐mediated silencing of the spindle checkpoint proteins BUBR1 and MAD2 markedly reduced induction of mitotic cell accumulation, but did not affect glyfoline‐induced mitotic catastrophe and apoptosis. Thus, glyfoline induces mitotic catastrophe probably by enhancing microtubule aster formation and subsequent apoptosis in cancer cells independently of spindle checkpoint function.

Inhibition of the Heat Shock Response by PI103 Enhances the Cytotoxicity of Arsenic Trioxide

Heat shock factor 1 (HSF1) is a key regulator of the cytoprotective and anti-apoptotic heat shock response and can be activated by arsenite. Inhibition of HSF1 activation may therefore enhance the cytotoxicity of arsenic trioxide (ATO). We show that ATO induced HSF1 phosphorylation at serine 326 (S326) and induced HSF1-dependent expression of heat shock proteins (HSPs) 27 and 70 in cultured cells. HSF1 significantly reduced cell sensitivity to ATO by reducing apoptosis. Disruption of HSF1 function not only reduced ATO induction of HSP27 and 70 but also enhanced ATO cytotoxicity by elevating apoptosis. These results reveal that HSF1 activation and the resulting induction of HSPs may protect cells from ATO cytotoxicity. The diminished expression of HSPs and hypersensitivity to ATO in cells stably depleted of HSF1 was rescued by ectopic expression of wild-type HSF1 but not an S326A substitution mutant, indicating that phosphorylation at S326 was critical for the protective effect of HSF1. Simultaneous treatment of cells with ATO and PI103, an inhibitor of members of the phosphatidylinositol 3-kinase (PI3K) family, suppressed not only ATO-induced expression of an HSP70 promoter-reporter construct and endogenous HSP70 but also phosphorylation of HSF1 S326. PI103 considerably reduced HSF1 transactivation in ATO-treated cells but had only a limited effect on HSF1 nuclear translocation and DNA binding. Furthermore, PI103 enhanced ATO cytotoxicity in an HSF1-dependent manner. Thus, inhibition of S326 phosphorylation by PI103 blocks the transactivation of HSF1 and may consequently suppress ATO induction of the heat shock response and sensitize cells to ATO.

Inhibition of AKT enhances mitotic cell apoptosis induced by arsenic trioxide

Accumulated evidence has revealed a tight link between arsenic trioxide (ATO)-induced apoptosis and mitotic arrest in cancer cells. AKT, a serine/threonine kinase frequently over-activated in diverse tumors, plays critical roles in stimulating cell cycle progression, abrogating cell cycle checkpoints, suppressing apoptosis, and regulating mitotic spindle assembly. Inhibition of AKT may therefore enhance ATO cytotoxicity and thus its clinical utility. We show that AKT was activated by ATO in HeLa-S3 cells. Inhibition of AKT by inhibitors of the phosphatidyl inositol 3-kinase/AKT pathway significantly enhanced cell sensitivity to ATO by elevating mitotic cell apoptosis. Ectopic expression of the constitutively active AKT1 had no effect on ATO-induced spindle abnormalities but reduced kinetochore localization of BUBR1 and MAD2 and accelerated mitosis exit, prevented mitotic cell apoptosis, and enhanced the formation of micro- or multi-nuclei in ATO-treated cells. These results indicate that AKT1 activation may prevent apoptosis of ATO-arrested mitotic cells by attenuating the function of the spindle checkpoint and therefore allowing the formation of micro- or multi-nuclei in surviving daughter cells. In addition, AKT1 activation upregulated the expression of aurora kinase B (AURKB) and survivin, and depletion of AURKB or survivin reversed the resistance of AKT1-activated cells to ATO-induced apoptosis. Thus, AKT1 activation suppresses ATO-induced mitotic cell apoptosis, despite the presence of numerous spindle abnormalities, probably by upregulating AURKB and survivin and attenuating spindle checkpoint function. Inhibition of AKT therefore effectively sensitizes cancer cells to ATO by enhancing mitotic cell apoptosis.

Novel 2-substituted quinolin-4-yl-benzenesulfonate derivatives: synthesis, antiproliferative activity, and inhibition of cellular tubulin polymerization.

A series of 2‐substituted quinolin‐4‐yl‐benzenesulfonate derivatives were synthesized for the purpose of evaluating antiproliferative activity. Structure–activity relationships of the newly synthesized compounds against human lymphoblastic leukemia and various solid tumor cell growths in culture are discussed. Of these derivatives, 2‐phenyl‐6‐pyrrolidinyl‐4‐quinoline sulfonate analogues 10 f, 10 g, and 10 k, and 4′‐nitrophenyl sulfonate 10 m exhibit superior cytotoxicity over other sulfonates. The antiproliferative activities of these compounds correlate well with their abilities to induce mitotic arrest and apoptosis. Mechanistic studies indicate that they target the vinblastine binding site of tubulin and inhibit cellular tubulin polymerization. Hence, these compounds induce the formation of aberrant mitotic spindles and mitotic arrest, resulting in intensive apoptosis. The tested compounds were shown to be poor substrates for membrane multidrug resistance transporters. The present studies suggest that these newly synthesized compounds are promising tubulin polymerization inhibitors and are worthy of further investigation as antitumor agents.

Derivatives of 6-cinnamamido-quinoline-4-carboxamide impair lysosome function and induce apoptosis

Autophagy is a lysosomal degradative process that protects cancer cells from multiple types of stress. In this study, we synthesized a series of derivatives of 6-cinnamamido-quinoline-4-carboxamide (CiQ), and investigated their effects on the proliferation and autophagy of cancer cells in vitro. These derivatives effectively inhibited the proliferation of a broad spectrum of cancer cell lines. Further study revealed that CiQ derivatives may induce autophagy and result in disruption of autophagy propagation. Consequently, these derivatives triggered massive apoptosis, as evidenced by caspase-9 activation and PARP cleavage. Blockage of autophagy by depletion of autophagy related gene ATG5 or BECN1 considerably alleviated CiQ-induced cell death, indicating that autophagy may mediate CiQ-induced cell death. Furthermore, treatment with CiQ derivatives increased lysosome membrane permeability (LMP) and enhanced accumulation of ubiquitinated proteins, which collectively indicate impaired lysosome function. In addition, treatment of cells with CiQ derivatives activated extracellular signal-regulated kinase (ERK); abrogation of ERK activation, either by treating cells with U0126, an inhibitor of mitogen-activated protein/ERK kinase 1 (MEK1), or by ectopically overexpressing a dominant-negative MEK1, significantly reduced CiQ derivative-induced LMP, LC3 and p62 accumulation, and cytotoxicity. These results indicate that CiQ derivatives activate ERK and disrupt lysosome function, thereby altering autophagic flux and resulting in apoptotic cell death.