Disorazoles comprise a family of 29 macrocyclic polyketides isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. The major fermentation product, disorazole A1, was previously found to irreversibly bind to tubulin and to have potent cytotoxic activity against tumor cells, possibly due to its highly electrophilic epoxide moiety. To test this hypothesis, we synthesized the epoxide-free disorazole C1 and found it retained potent antiproliferative activity against tumor cells, causing prominent G2/M phase arrest and inhibition of in vitro tubulin polymerization. Furthermore, disorazole C1 produced disorganized microtubules at interphase, misaligned chromosomes during mitosis, apoptosis, and premature senescence in the surviving cell populations. Using a tubulin polymerization assay, we found disorazole C1 inhibited purified bovine tubulin polymerization with an IC50 of 11.8 ± 0.4 μM and inhibited [3H]vinblastine binding uncompetitively with a Ki of 4.5 ± 0.6 μM. We also found uncompetitive inhibition of [3H]dolastatin 10 binding by disorazole C1 with a Ki of 10.6 ± 1.5 μM, indicating that disorazole C1 bound tubulin uniquely among known antimitotic agents. Disorazole C1 could be a valuable chemical probe for studying the process of mitotic spindle disruption and its relationship to premature senescence.
The disorazoles comprise a family of 29 closely related macrocyclic polyketides isolated in 1994 from the fermentation broth of the gliding myxobacterium Sorangium cellulosum. Disorazoles A1, E and C1 have shown exceptional biological activities toward inhibiting the proliferation of human cancer cell lines in picomolar and nanomolar concentrations through the disruption of microtubule polymerization. This review gives a brief introduction describing the biosynthesis and the significance of the disorazoles as a new class of microtubulin disruptors. Another portion of the review focuses on the biology of the disorazoles, specifically disorazole A1 and C1, and their antiproliferative efficacy against animal and human tumor cell lines, as well as the available SAR data. The majority of the discussion addresses synthetic efforts, including partial syntheses of various disorazoles and a summary of the total synthesis of disorazole C1.
Microtubule-poisoning drugs, such as Paclitaxel (or Taxol, PTX), are powerful and commonly used anti-neoplastic agents for the treatment of several malignancies. PTX triggers cell death, mainly through a mitotic arrest following the activation of the spindle assembly checkpoint (SAC). Cells treated with PTX slowly slip from this mitotic block and die by mitotic catastrophe. However, cancer cells can acquire or are intrinsically resistant to this drug, posing one of the main obstacles for PTX clinical effectiveness. In order to override PTX resistance and increase its efficacy, we investigated both the enhancement of mitotic slippage and the block of mitotic exit.
To test these opposing strategies, we used physiological hyperthermia (HT) to force exit from PTX-induced mitotic block and the anaphase-promoting complex/cyclosome (APC/C) inhibitor, proTAME, to block mitotic exit. We observed that application of HT on PTX-treated cells forced mitotic slippage, as shown by the rapid decline of cyclin B levels and by microscopy analysis. Similarly, HT induced mitotic exit in cells blocked in mitosis by other antimitotic drugs, such as Nocodazole and the Aurora A inhibitor MLN8054, indicating a common effect of HT on mitotic cells. On the other hand, proTAME prevented mitotic exit of PTX and MLN8054 arrested cells, prolonged mitosis, and induced apoptosis. In addition, we showed that proTAME prevented HT-mediated mitotic exit, indicating that stress-induced APC/C activation is necessary for HT-induced mitotic slippage.
Finally, HT significantly increased PTX cytotoxicity, regardless of cancer cells’ sensitivity to PTX, and this activity was superior to the combination of PTX with pro-TAME. Our data suggested that forced mitotic exit of cells arrested in mitosis by anti-mitotic drugs, such as PTX, can be a more successful anticancer strategy than blocking mitotic exit by inactivation of the APC/C.
mitotic block; mitotic catastrophe; hyperthermia; APC/C; proTAME; taxanes; aurora A inhibitors; antimitotic drug resistance
The total synthesis of a bis-cyclopropane analog of the antimitotic natural product (-)-disorazole C1 was accomplished in 23 steps and 1.1% overall yield. A vinyl cyclopropane cross-metathesis reaction generated a key (E)-alkene segment of the target molecule. IC50 determinations of (-)-CP2-disorazole C1 in human colon cancer cell lines indicated low nanomolar cytotoxic properties. Accordingly, this synthetic bioisostere represents the first biologically active disorazole analog not containing a conjugated diene or polyene substructure element.
ETOC: Despite the use of antimitotic drugs, our understanding of the stress response, especially during mitotic arrest, is lacking. We report a molecular mechanism resulting in DNA damage during mitotic arrest that occurs via the apoptotic machinery but in the absence of cell death; this mechanism triggers p53 induction after cells slip from mitotic arrest.
Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1. We investigated the cause of this induction. We detected DNA damage late in mitotic arrest and also after slippage. This damage was inhibited by treatment with caspase inhibitors and by stable expression of mutant, noncleavable inhibitor of caspase-activated DNase, which prevents activation of the apoptosis-associated nuclease caspase-activated DNase (CAD). These treatments also inhibited induction of p53 after slippage from prolonged arrest. DNA damage was not due to full apoptosis, since most cytochrome C was still sequestered in mitochondria when damage occurred. We conclude that prolonged mitotic arrest partially activates the apoptotic pathway. This partly activates CAD, causing limited DNA damage and p53 induction after slippage. Increased DNA damage via caspases and CAD may be an important aspect of antimitotic drug action. More speculatively, partial activation of CAD may explain the DNA-damaging effects of diverse cellular stresses that do not immediately trigger apoptosis.
Microtubule drugs are effective anti-cancer agents, primarily due to their ability to induce mitotic arrest and subsequent cell death. However, some cancer cells are intrinsically resistant or acquire a resistance. Lack of apoptosis following mitotic arrest is thought to contribute to drug resistance that limits the efficacy of the microtubule-targeting anti-cancer drugs. Genetic or pharmacological agents that selectively facilitate the apoptosis of mitotic arrested cells present opportunities to strengthen the therapeutic efficacy.
Methodology and Principal Findings
We report a natural product Celastrol targets tubulin and facilitates mitotic cell death caused by microtubule drugs. First, in a small molecule screening effort, we identify Celastrol as an inhibitor of neutrophil chemotaxis. Subsequent time-lapse imaging analyses reveal that inhibition of microtubule-mediated cellular processes, including cell migration and mitotic chromosome alignment, is the earliest events affected by Celastrol. Disorganization, not depolymerization, of mitotic spindles appears responsible for mitotic defects. Celastrol directly affects the biochemical properties of tubulin heterodimer in vitro and reduces its protein level in vivo. At the cellular level, Celastrol induces a synergistic apoptosis when combined with conventional microtubule-targeting drugs and manifests an efficacy toward Taxol-resistant cancer cells. Finally, by time-lapse imaging and tracking of microtubule drug-treated cells, we show that Celastrol preferentially induces apoptosis of mitotic arrested cells in a caspase-dependent manner. This selective effect is not due to inhibition of general cell survival pathways or mitotic kinases that have been shown to enhance microtubule drug-induced cell death.
Conclusions and Significance
We provide evidence for new cellular pathways that, when perturbed, selectively induce the apoptosis of mitotic arrested cancer cells, identifying a potential new strategy to enhance the therapeutic efficacy of conventional microtubule-targeting anti-cancer drugs.
The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.
Microtubule-targeting drugs induce mitotic delay at pro-metaphase by preventing the spindle assembly checkpoint to be satisfied. However, especially after prolonged treatments, cells can escape this arrest in a process called mitotic slippage. The mechanisms underlying the spindle assembly checkpoint and slippage are not fully understood. It has been generally accepted that during mitosis there is a temporary shutdown of high-energy-consuming processes, such as transcription and translation. However, the synthesis of specific proteins is maintained or up-regulated since protein synthesis is necessary for entry into and progression through mitosis.
In this work we investigated whether the mitotic arrest caused by the mitotic checkpoint is independent of transcription and translation. By using immunofluorescent microscopy and western blotting, we demonstrate that inhibition of either of these processes induces a shortening of the mitotic arrest caused by the nocodazole treatment, and ultimately leads to mitotic slippage. Our western blotting and RTQ-PCR results show that inhibition of transcription during mitotic arrest does not affect the expression of the spindle checkpoint proteins, whereas it induces a significant decrease in the mRNA and protein levels of Cyclin B1. The exogenous expression of Cyclin B1 substantially rescued the mitotic phenotype in nocodazole cells treated with the inhibitors of transcription and translation.
This work emphasizes the importance of transcription and translation for the maintenance of the spindle assembly checkpoint, suggesting the existence of a mechanism dependent on cyclin B1 gene regulation during mitosis. We propose that continuous transcription of mitotic regulators is required to sustain the activation of the spindle assembly checkpoint.
Andrographolide (Andro) suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi) has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose) polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC), alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC.
Anti-mitotic chemotherapeutic agents such as taxanes activate the spindle assembly checkpoint (SAC) to arrest anaphase onset, but taxane-exposed cells eventually undergo slippage to exit mitosis. The therapeutic efficacy of taxanes depends on whether slippage after SAC arrest culminates in continued cell survival, or in death by apoptosis. However, the mechanisms that determine these outcomes remain unclear. Here, we identify a novel role for cyclin G1 (CCNG1), an atypical cyclin. Increased CCNG1 expression accompanies paclitaxel-induced, SAC-mediated mitotic arrest, independent of p53 integrity or signaling through the SAC component, BUBR1. CCNG1 overexpression promotes cell survival after paclitaxel exposure. Conversely, CCNG1 depletion by RNA interference delays slippage and enhances paclitaxel-induced apoptosis. Consistent with these observations, CCNG1 amplification is associated with significantly shorter post-surgical survival in patients with ovarian cancer who have received adjuvant chemotherapy with taxanes and platinum compounds. Collectively, our findings implicate CCNG1 in regulating slippage and the outcome of taxane-induced mitotic arrest, with potential implications for cancer therapy.
mitotic checkpoint; cyclin G1; anti-mitotic drugs; cancer therapy
Cancer relies upon frequent or abnormal cell division but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the anti-mitotic drug paclitaxel was the same in tumors as in cell culture; observed the impact of paclitaxel (Ptx) on the tumor response as a whole; and evaluated the single-cell pharmacodynamics of paclitaxel (by in vivo pharmacodynamic microscopy [IPDM]). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Paclitaxel treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation and apoptosis. Compared to cell culture, the peak mitotic index in tumors exposed to paclitaxel was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less pro-apoptotic than cell culture. The morphologies associated with mitotic arrest were dose- and time-dependent, thereby providing a semi-quantitative, single-cell measure of pharmacodynamics. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings demonstrate that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single cell level.
pharmacodynamics; in vivo microscopy; therapeutics; mitosis
The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.
Here we describe mitotic slippage in yeast bub2Δ mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.
The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.
Anaphase promoting complex/cyclosome (APC/C); Bub2; Cdh1; mitotic exit network (MEN); mitotic slippage; Saccharomyces cerevisiae; securin
Kinesin-5 inhibitors (K5Is) are promising anti-mitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using time-lapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and non-cancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G1, with little cell death. Several cancer cell lines either died during mitotic arrest, or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility, and long-term responses after transient drug exposure in MCF7 breast cancer cells. While many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared to washout after spontaneous slippage, likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype.
experimental therapeutics; mitotic drugs; Kinesin-5; live-cell imaging
Reovirus type 3 Dearing strain (ReoT3D) has an inherent propensity to preferentially infect and destroy cancer cells. The oncolytic activity of ReoT3D as a single agent has been demonstrated in vitro and in vivo against various cancers, including colon, pancreatic, ovarian and breast cancers. Its human safety and potential efficacy are currently being investigated in early clinical trials. In this study, we investigated the in vitro combination effects of ReoT3D and chemotherapeutic agents against human non-small cell lung cancer (NSCLC).
ReoT3D alone exerted significant cytolytic activity in 7 of 9 NSCLC cell lines examined, with the 50% effective dose, defined as the initial virus dose to achieve 50% cell killing after 48 hours of infection, ranging from 1.46 ± 0.12 ~2.68 ± 0.25 (mean ± SD) log10 pfu/cell. Chou-Talalay analysis of the combination of ReoT3D with cisplatin, gemcitabine, or vinblastine demonstrated strong synergistic effects on cell killing, but only in cell lines that were sensitive to these compounds. In contrast, the combination of ReoT3D and paclitaxel was invariably synergistic in all cell lines tested, regardless of their levels of sensitivity to either agent. Treatment of NSCLC cell lines with the ReoT3D-paclitaxel combination resulted in increased poly (ADP-ribose) polymerase cleavage and caspase activity compared to single therapy, indicating enhanced apoptosis induction in dually treated NSCLC cells. NSCLC cells treated with the ReoT3D-paclitaxel combination showed increased proportions of mitotic and apoptotic cells, and a more pronounced level of caspase-3 activation was demonstrated in mitotically arrested cells.
These data suggest that the oncolytic activity of ReoT3D can be potentiated by taxanes and other chemotherapeutic agents, and that the ReoT3D-taxane combination most effectively achieves synergy through accelerated apoptosis triggered by prolonged mitotic arrest.
BACKGROUND: At therapeutic concentrations, the antineoplastic agent taxol selectively perturbs mitotic spindle microtubules. Taxol has recently been shown to induce apoptosis, similar to the mechanism of cell death induced by other antineoplastic agents. However, taxol has shown efficacy against drug-refractory cancers, raising the possibility that this pharmacological agent may trigger an alternative apoptotic pathway. MATERIALS AND METHODS: The kinetics and IC50 of mitotic (M) block, aberrant mitosis, and cytotoxicity following taxol treatment were analyzed in human cell lines as well as normal mouse embryo fibroblasts (MEFs) and MEFs derived from p53-null mice. Apoptosis was followed by DNA gel electrophoresis and by in situ DNA end-labeling (TUNEL). RESULTS: Taxol induced two forms of cell cycle arrest: either directly in early M at prophase or, for those cells progressing through aberrant mitosis, arrest in G1 as multimininucleated cells. TUNEL labeling revealed that DNA nicking occurred within 30 min of the arrest in prophase. In contrast, G1-arrested, multimininucleated cells became TUNEL positive only after several days. In the subset of cells that became blocked directly in prophase, both wt p53-expressing and p53-null MEFs responded similarly to taxol, showing rapid onset of DNA nicking and apoptosis. However, p53-null MEFs progressing through aberrant mitosis failed to arrest in the subsequent G1 phase or to become TUNEL positive, and remained viable. CONCLUSIONS: Taxol induces two forms of cell cycle arrest, which in turn induce two independent apoptotic pathways. Arrest in prophase induces rapid onset of a p53-independent pathway, whereas G1-block and the resulting slow (3-5 days) apoptotic pathway are p53 dependent.
Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.
Variability in cell-to-cell behavior within clonal populations can be attributed to the inherent stochasticity of biochemical reactions. Most single-cell studies have examined variation in behavior due to randomness in gene transcription. Here we investigate the mechanism of cell fate choice and the origin of cell-to-cell variation during mitotic arrest, when transcription is silenced. Prolonged mitotic arrest is commonly observed in cells treated with anti-mitotic drugs. Cell fate during mitotic arrest is determined by two alternative pathways, one promoting cell death, the other promoting cyclin B1 degradation, which leads to mitotic slippage and survival. It has been unclear whether these pathways are mechanistically coupled or independent. In this study we experimentally uncoupled these two pathways using zVAD-fmk to block cell death or Cdc20 knockdown to block slippage. We then used time-lapse imaging to score the kinetics of single cells adopting the remaining fate. We also used kinetic simulation to test whether the behaviors of death versus slippage in cell populations where both pathways are active can be quantitatively recapitulated by a model that assumes stochastic competition between the pathways. Our data are well fit by a model where the two pathways are mechanistically independent, and cell fate is determined by a stochastic kinetic competition between them that results in cell-to-cell variation.
The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.
When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37°C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.
The phosphatidylinositol 3-kinase (PI3K) pathway plays an important role in many biological processes, including cell cycle progression, cell growth, survival, actin rearrangement and migration, and intracellular vesicular transport. However, the involvement of the PI3K pathway in the regulation of mitotic cell death remains unclear. In this study, we treated HeLa cells with the PI3K inhibitors, 3-methyladenine (3-MA, as well as a widely used autophagy inhibitor) and wortmannin to examine their effects on cell fates using live cell imaging. Treatment with 3-MA decreased cell viability in a time- and dose-dependent manner and was associated with caspase-3 activation. Interestingly, 3-MA-induced cell death was not affected by RNA interference-mediated knockdown (KD) of beclin1 (an essential protein for autophagy) in HeLa cells, or by deletion of atg5 (an essential autophagy gene) in mouse embryonic fibroblasts (MEFs). These data indicate that cell death induced by 3-MA occurs independently of its ability to inhibit autophagy. The results from live cell imaging studies showed that the inhibition of PI3Ks increased the occurrence of lagging chromosomes and cell cycle arrest and cell death in prometaphase. Furthermore, PI3K inhibitors promoted nocodazole-induced mitotic cell death and reduced mitotic slippage. Overexpression of Akt (the downstream target of PI3K) antagonized PI3K inhibitor-induced mitotic cell death and promoted nocodazole-induced mitotic slippage. These results suggest a novel role for the PI3K pathway in regulating mitotic progression and preventing mitotic cell death and provide justification for the use of PI3K inhibitors in combination with anti-mitotic drugs to combat cancer.
Paclitaxel plays a major role in the treatment of ovarian cancer; however, resistance to paclitaxel is frequently observed. Thus, new therapy that can overcome paclitaxel resistance will be of significant clinical importance. We evaluated antiproliferative effects of an antimitotic and antivascular agent BPR0L075 in paclitaxel-resistant ovarian cancer cells. BPR0L075 displays potent and broad-spectrum cytotoxicity at low nanomolar concentrations (IC50 = 2–7 nM) against both parental ovarian cancer cells (OVCAR-3, SKOV-3, and A2780-1A9) and paclitaxel-resistant sublines (OVCAR-3-TR, SKOV-3-TR, 1A9-PTX10), regardless of the expression levels of the multidrug resistance transporter P-gp and class III β-tubulin or mutation of β-tubulin. BPR0L075 blocks cell cycle at the G2/M phase in paclitaxel-resistant cells while equal concentration of paclitaxel treatment was ineffective. BPR0L075 induces cell death by a dual mechanism in parental and paclitaxel-resistant ovarian cancer cells. In the parental cells (OVCAR-3 and SKOV-3), BPR0L075 induced apoptosis, evidenced by poly(ADP-ribose) polymerase (PARP) cleavage and DNA ladder formation. BPR0L075 induced cell death in paclitaxel-resistant ovarian cancer cells (OVCAR-3-TR and SKOV-3-TR) is primarily due to mitotic catastrophe, evidenced by formation of giant, multinucleated cells and absence of PARP cleavage. Immunoblotting analysis shows that BPR0L075 treatment induced up-regulation of cyclin B1, BubR1, MPM-2, and survivin protein levels and Bcl-XL phosphorylation in parental cells; however, in resistant cells, the endogenous expressions of BubR1 and survivin were depleted, BPR0L075 treatment failed to induce MPM-2 expression and phosphorylation of Bcl-XL. BPR0L075 induced cell death in both parental and paclitaxel-resistant ovarian cancer cells proceed through caspase-3 independent mechanisms. In conclusion, BPR0L075 displays potent cytotoxic effects in ovarian cancer cells with a potential to overcome paclitaxel resistance by bypassing efflux transporters and inducing mitotic catastrophe. BPR0L075 represents a novel microtubule therapeutic to overcome multidrug resistance and trigger alternative cell death by mitotic catastrophe in ovarian cancer cells that are apoptosis-resistant.
Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic anti-proliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.
Microtubule inhibitors are widely used in cancer chemotherapy. These drugs characteristically induce mitotic arrest and cell death but the mechanisms linking the two are not firmly established. One of the problems is that cancer cells vary widely in their sensitivity to these agents, and thus comparison of data from different systems is difficult. To alleviate this problem we sought to molecularly induce mitotic death and study its mechanisms, by expressing non-degradable cyclin B (R42A) in HeLa cells. However, this approach failed to induce significant mitotic arrest, Cdk1 activation, or phosphorylation of anti-apoptotic Bcl-2 proteins, all characteristics of cells treated with microtubule inhibitors. Furthermore, cyclin B1-R42A induced rapid cell death, and when expressed in synchronized cells, cell death occurred in G1 phase. Decreasing the plasmid concentration reduced transfection efficiency but restored mitotic arrest and eliminated non-specific death. These results show that inappropriate overexpression of cyclin B1 causes non-specific cell death and suggest caution in its use for the study of mitotic events.
Mcl-1 is one of the major anti-apoptotic members of the Bcl-2 family of apoptotic regulatory proteins. In this study we investigated the role of Mcl-1 in mitotic arrest-induced apoptosis. Vinblastine treatment of KB-3 cells initially resulted in a phosphatase-sensitive mobility shift in Mcl-1 and then subsequent loss of Mcl-1 protein expression which was prevented by MG132, suggesting that phosphorylation triggered proteosome-mediated degradation. Mcl-1 phosphorylation/degradation was a specific response to microtubule inhibition and did not occur in response to lethal concentrations of DNA damaging agents. Vinblastine treatment caused degradation of Mcl-1 in cells in which apoptosis was blocked by Bcl-xL overexpression, indicating that Mcl-1 degradation was not a consequence of apoptosis. A partial reversible phosphorylation of Mcl-1 was observed in synchronized cells traversing mitosis, whereas more extensive phosphorylation and subsequent degradation of Mcl-1 was observed if synchronized cells were treated with vinblastine. Mcl-1 phosphorylation closely paralleled cyclin B expression, and specific cyclin-dependent kinase (Cdk) inhibitors blocked vinblastine-induced Mcl-1 phosphorylation, its subsequent degradation, and improved cell viability after mitotic arrest. Co-immunoprecipitation studies indicated that Mcl-1 was complexed with Bak, but not Bax or Noxa, in untreated cells, and that Bak became activated in concert with loss of Mcl-1 expression. These results suggest that Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis via Mcl-1 phosphorylation, promoting its degradation and subsequently releasing Bak from sequestration.
Mcl-1; Cdk1; microtubule inhibitors; apoptosis; Bcl-2 proteins
Growth suppressive effect of diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, against cultured human cancer cells correlates with checkpoint kinase 1 (Chk1)-mediated mitotic arrest, but the fate of the cells arrested in mitosis remains elusive. Using LNCaP and HCT-116 human cancer cells as a model, we now demonstrate that the Chk1-mediated mitotic arrest resulting from DATS exposure leads to apoptosis. The DATS exposure resulted in G2 phase and mitotic arrest in both LNCaP and HCT-116 cell lines. The G2 arrest was accompanied by down-regulation of cyclin-dependent kinase 1 (Cdk1), cell division cycle (Cdc) 25B, and Cdc25C leading to Tyr15 phosphorylation of Cdk1 (inactivation). The DATS-mediated mitotic arrest correlated with inactivation of anaphase-promoting complex/cyclosome as evidenced by accumulation of its substrates cyclinB1 and securin. The DATS treatment increased activating phosphorylation of Chk1 (Ser317) and transient transfection with Chk1-targeted siRNA conferred significant protection against DATS-induced mitotic arrest in both cell lines. The Chk1 protein knockdown also afforded partial yet statistically significant protection against apoptotic DNA fragmentation and caspase-3 activation resulting from DATS exposure in both LNCaP and HCT-116 cells. Even though DATS treatment resulted in stabilization and Ser15 phosphorylation of p53, the knockdown of p53 protein failed to rescue DATS-induced mitotic arrest. In conclusion, the results of the present study indicate that Chk1-dependence of DATS-induced mitotic arrest in human cancer cells is not influenced by the p53 status and cells arrested in mitosis upon DATS exposure are driven to apoptotic DNA fragmentation.
diallyl Trisulfide; mitotic Arrest; apoptosis