Comparison of Cancer Cell Survival Triggered by Microtubule Damage after Turning Dyrk1B Kinase On and O?
Liwen Li,Yin Liu,Qiu Zhang,Hongyu Zhou,Yi Zhang,and Bing Yan*
School of Chemistry and Chemical Engineering,Shandong University,Jinan,China250100
*Supporting Information
rapidly repair microtubules,relieve cell G2/M arrest for42%
viability by10-fold.That is,the dual inhibitor is10times
ords a novel drug discovery strategy by targeting both the
single therapeutic agent.
machinery that involves multiple signaling molecules and intertwined signaling pathways.The molecular interactions that are involved may be partially deciphered using techniques such as gene knockout or speci?c inhibitors.Blocking survival signaling pathways or allowing cells to transmit survival signals may have dramatically di?erent e?ects on cell function.Cell survival that is triggered by microtubule damage is one such example.Microtubules form the cytoskeleton together with micro?laments and intermediate?laments,which are important in several cellular processes.Microtubules participate in cytoskeleton formation,intracellular transport,and cell division. Because of their crucial role in cell division,microtubules are a clear target for anticancer chemotherapeutic drugs.Micro-tubule-damaging agents,such as vinca alkaloids and taxanes, disrupt microtubule dynamics,leading to cell cycle arrest at the G2/M phase and apoptosis.1,2Cell survival that is triggered by microtubule damage is likely regulated by a balancing act between prosurvival and proapoptotic signals.Kinases are known to play a role in the transmission of cellular signals.For example,the activation of extracellular signal-regulated kinases (ERKs)3?5and protein kinase B(PKB,also known as Akt)6,7is related to cell survival,whereas Raf serine/threonine kinases (Raf-1)8?10and c-Jun N-terminal kinases(JNKs)11?15are associated with cytotoxicity after microtubule damage.How-ever,the involvement of other kinases in the cell death and survival that are triggered by microtubule damage remains to be elucidated.
Cellular communication is achieved through a network of signaling proteins and pathways.Due to the complexity of the cellular signaling network,a molecular probe typically targets one or more cellular proteins.16?18A comparison of cellular responses triggered by these speci?cally targeted molecules may reveal valuable information regarding cell function and the signaling machinery.The most valuable evaluation of a cellular process is the ability to measure di?erential e?ects when one probe triggers a signaling process but a second probe inhibits it, as reported in this investigation.
To investigate the association between microtubule damage and cell survival,we utilized a pair of small molecular probes (probes1and2)(Figure1).Both molecules are tubulin polymerization inhibitors.Probe1also inhibits Dual speci?city tyrosine-phosphorylation-regulated kinase1B(Dyrk1B,also known as Mirk)to generate a di?erent cellular phenotype. Dyrk1B is a serine/threonine kinase that is widely expressed in various cells.Dyrk1B mediates cell survival in many solid tumors,19?25such as rhabdomyosarcomas,21pancreatic ductal adenocarcinoma,20and colon carcinomas.25Friedman and colleagues reported that Dyrk1B knockdown enhanced cell killing by the microtubule inhibitor nocodazole in pancreatic ductal adenocarcinoma,demonstrating that Dyrk1B may mediate cell survival in response to microtubule damage.20 However,the mechanism responsible for Dyrk1B-induced cell survival after microtubule damage remains unclear.
On the basis of the di?erent cellular responses to probes1 and2,we reveal the molecular details of the cell survival signaling that is coordinated by Dyrk1B in response to microtubule damage.We additionally quanti?ed the di?erential cellular responses of Dyrk1B-mediated cell survival in terms of
Received:July24,2013
Accepted:December30,2013
Published:December30,2013
recovered cell viability,microtubule integrity,cell cycle,and suppressed apoptosis.
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RESULTS AND DISCUSSION
Molecular Probes 1and 2Equally Inhibit Tubulin Polymerization but Demonstrate Dramatically Di ?erent Inhibition of Cell Viability.To identify speci ?c targeting molecular probes,we investigated a series of compounds containing a thiazolidinone core structure.26We previously showed that compounds from this class possess anticancer activity by targeting tubulin polymerization.27,28Using a spectroscopic assay,probes 1and 2(Figure 1)were identi ?ed as potent tubulin polymerization inhibitors from several thiazolidinone compounds with only small variations in their side chain groups (Supplementary Figure 1).In the in vitro tubulin polymerization assay,time-dependent tubulin polymer-ization,which was indicated by ?uorescence enhancement due to the incorporation of a ?uorescent reporter into microtubules,was monitored in real time (Figure 1a and b and Supplementary Figure 1b).The half maximal inhibitory concentration (IC 50)values for tubulin polymerization of probes 1and 2(Figure 1c)were determined to be 4.0±0.6and 5.6±0.3μM,respectively,and did not demonstrate a signi ?cant di ?erence as analyzed by Student ’s t test,which
indicated that probes 1and 2inhibit tubulin polymerization to a similar extent.
Microtubules are an anticancer target,and chemotherapeutic agents can kill cancer cells through the inhibition of tubulin polymerization.1,2Although probes 1and 2demonstrated similar abilities for the inhibition of tubulin polymerization,whether they also demonstrate comparable e ?ects with respect to cancer cell killing is unknown.To evaluate the anticancer activities of the two probes,the IC 50values of probes 1and 2were determined in the human embryonal rhabdomyosarcoma RD,colorectal carcinoma HCT116,and breast carcinoma MDA-MB-231cell lines.In all three cell lines,probe 1demonstrated substantially greater potency than probe 2(Supplementary Table 1).For example,the IC 50values for probes 1and 2were determined to be 0.65±0.13and 6.22±0.92μM,respectively,in the RD cells,indicating that probe 1was 10-fold more potent than probe 2.
Tubulin Polymerization/Dyrk1B Dual Inhibitor Is 10-Fold More Potent in the Inhibition of Cell Viability than a Tubulin Polymerization Inhibitor Alone.With an approximately similar inhibition of tubulin polymerization,probes 1and 2demonstrated a dramatically di ?erent inhibition on cancer cell proliferation.This ?nding raised the possibility that other biological targets may be involved in the regulation of the cell death or survival triggered by microtubule damage.Human kinases play a pivotal role in cellular signaling,including apoptosis and cell survival.Thus,we ?rst quantitatively measured the inhibition of a panel of 96human kinases by probes 1and 2.
Kinase inhibition by probes 1and 2was assessed using a competition assay that quantitatively measured the ability of a compound to compete with an immobilized active site-directed ligand in binding to the kinase active site using the KINOMEscan platform.29,30At 5μM,probe 1strongly inhibited Dyrk1B,but probe 2did not demonstrate a noticeable inhibition of the entire panel of kinases (Supplementary Tables 2and 3).To corroborate this ?nding,we further determined the binding constants (K d )of probes 1and 2for Dyrk1B binding (Figure 2a).The K d of probe 1was determined to be 42±2nM,whereas probe 2did not bind Dyrk1B at a concentration of up to 30μM.In addition to Dyrk1B binding,we determined the inhibition of Dyrk1B kinase activity by these two https://www.doczj.com/doc/025614812.html,ing the HotSpot platform,31Dyrk1B activity was determined by measuring the radioactivity of the phosphory-lated substrate after the incubation of Dyrk1B with 33P-γ-ATP,the Dyrk1B substrate,and either probe 1or 2.Probe 1strongly inhibited Dyrk1B kinase activity with an IC 50of 36.7±4.4nM.In contrast,probe 2did not inhibit Dyrk1B activity up to a concentration of 10μM (Figure 2b).These results were consistent with the kinase binding results.Collectively,these results demonstrated that a tubulin polymerization/Dyrk1B dual inhibitor (probe 1)demonstrated a 10-fold higher e ?cacy in the inhibition of cell viability than a tubulin polymerization-only inhibitor (probe 2).
We next asked whether probe 1also inhibits Dyrk1B activity in living cells.To address this question,Dyrk1B activity was investigated using an immune complex kinase assay.20,21In this assay,each reaction contained three major components:Dyrk1B kinase,the Dyrk1B substrate (the recombinant p27kip1,referred to as p27hereafter),and 32P-ATP.Because activated Dyrk1B phosphorylates p27at Ser-10,recombinant p27was used as a selective Dyrk1B substrate in this assay.32The extent of p27phosphorylation directly re ?ects
Dyrk1B
Figure 1.Inhibition of tubulin polymerization by probes 1and 2.Time-dependent tubulin polymerization assay in response to 10μM nocodazole and various concentrations of probes 1(a)and 2(b).The ?uorescence enhancement represents tubulin polymerization after a ?uorescent reporter was incorporated into microtubules during polymerization.The maximum polymerization reaction velocity (V max )of the growth phase was determined according to the maximum slope of the ?uorescence enhancement between 10and 40min.(c)The inhibition of tubulin polymerization by probes 1and 2at various concentrations was determined from the V max as follows:inhibition =1?V max (treatment)/V max (DMSO).The IC 50values represent the mean ±SEM from three independent experiments.
kinase activity.Because probe 1inhibited cell viability more potently in the RD cell line than in the other cell lines,we performed the immune complex kinase assay in RD cells.RD cells were ?rst treated with probe 1or 2.Subsequently,Dyrk1B kinase was immunoprecipitated from the RD cell lysate.The immunoprecipitated Dyrk1B (IP-Dyrk1B)was then incubated with p27and 32P-ATP.The enzymatic activity of Dyrk1B was determined from the radioactivity of phosphorylated p27(32P-p27).In Figure 3a,the band intensity of 32P-p27represents the enzymatic activity of Dyrk1B (row 1),and the IP-Dyrk1B band represents the total amount of the immunoprecipitated Dyrk1B (row 2).The bottom row (total p27)indicates that comparable amounts of p27were added in each reaction.To correct for small variations in the amount of Dyrk1B assayed in each reaction,the ratio of the band intensities of 32P-p27(the total IP-Dyrk1B activity)and IP-Dyrk1B (the amount of IP-Dyrk1B)were quantitatively determined using ImageJ software.This ratio represented the normalized Dyrk1B kinase activity (Figure 3b).Both probes 1and 2increased Dyrk1B activity at 24h,indicating that microtubule damage initially triggered a signaling pathway that led to the activation of Dyrk1B kinase,not the Dyrk1B overexpression (row 2in Figure 3a).However,the increase in Dyrk1B activity in RD cells treated with probe 1was much lower than that after being treated with probe 2.This result was consistent with the ?nding that probe 1also bound to the active site of Dyrk1B and inhibited its enzymatic activity (Figure 2).The dual targeting property of probe 1has numerous precedents.Many drugs or clinical compounds have been found to bind to multiple protein targets with divergent primary sequences.For example,a novel Alzheimer ’s
compound is reported to inhibit both acetylcholinesterase and monoamine oxidase.33Moreover,the antiretroviral drug cosalane targets integrase,protease,gp120,and reverse transcriptase.34Furthermore,an antitumor compound targets both HSP90and tubulin in the treatment of non-small-cell lung cancer.28Therefore,the tertiary structure of a protein,not its primary sequence,may play an important role in determining protein function and its interactions with other molecules.The tertiary structures of the two proteins may both contain binding sites for probe 1or for di ?erent faces of the probe 1molecule.Early studies have indicated that Dyrk1B may be a survival factor in solid tumor cell lines,including RD cells.19?25Our results demonstrated that the microtubule damage induced by probes 1and 2triggered cell survival through Dyrk1B activation,whereas probe 1simultaneously inhibited Dyrk1B (Figure 3c).This ?nding also explained our observation that probe 1was more potent than probe 2in the suppression of cancer cell viability.
To further con ?rm that the inhibition of both tubulin polymerization and Dyrk1B facilitate the inhibition of cell viability compared with the inhibition of tubulin polymerization alone,we subsequently investigated the potency of the inhibition of cell viability using a combination of a tubulin polymerization-only inhibitor (probe 2)and a Dyrk1B-only inhibitor (probe 3).Probe 3bound to Dyrk1B with a K d of 255±35nM (Supplementary Figure 2b)and inhibited Dyrk1B activity with an IC 50of 31.4±6.6nM (Supplementary Figure 2c)but did not a ?ect tubulin polymerization (IC 50>50μM,Supplementary Figure 1).The combined e ?ect of probes 2and 3was evaluated using an F a -CI plot.The fraction a ?ected (F a )represents the inhibition of RD cell viability in this assay.This combination index o ?ers a quantitative evaluation of the synergistic (CI <1),antagonistic (CI >1)or addictive (CI =1)e ?ect of the two inhibitors.35The results indicated that the CI value of the combination was 0.66at 50%of the e ?ective dose (F a =0.5)(Supplementary Figure 3).This result provided strong evidence for the synergism in cell inhibition through the combination of probes 2and 3and further demonstrated that the inhibition of both tubulin polymerization and Dyrk1B activity can simultaneously synergistically enhance the potency of the inhibition of cell viability.
Because cells possess sophisticated survival machinery,a single molecular probe may induce multiple signaling responses and complicate the explanation of cellular responses.In this investigation,a pair of molecular probes that selectively activate or inhibit a speci ?c signaling pathway was utilized.This approach simpli ?es the explanation of cellular responses,reveals the molecular mechanism,and quanti ?es the magnitude of a speci ?c signaling event.
Ideally,the entire proteome should be screened to assess the speci ?c targets of a molecular probe.In our investigation,we identi ?ed the targets of probes 1and 2based on our previous knowledge of this class of compounds (tubulin-targeting)and the screening of a large panel of kinase proteins.Probe 2was used as a tubulin polymerization inhibitor,and probe 1was used as a tubulin polymerization/Dyrk1B dual inhibitor to investigate the association between kinase signaling and microtubule damage.The inhibition of tubulin polymerization induces microtubule damage and a series of dysfunctions in the cells.However,microtubule dysfunction triggers a Dyrk1B-coordinated signaling event that mediates cell survival,as observed in the cells that were treated with probe 2.Because probe 1inhibits Dyrk1B in addition to the inhibition of
tubulin
Figure 2.Inhibition of Dyrk1B activity by probe 1but not by probe 2.(a)Using the KINOMEscan platform,the K d was determined using a competition assay that quantitatively measured the abilities of probes 1and 2to compete with an immobilized,Dyrk1B active-site directed ligand.As the kinases were tagged by DNA,the concentration at which the kinase bound to the immobilized ligand was quanti ?ed using qPCR.(b)Concentration-dependent inhibition of Dyrk1B kinase activity by probe 1or 2.In the assay,the Dyrk1B kinase reaction was performed using a Dyrk1B substrate and 33P-γ-ATP.Radioisotope-labeled substrates were quantitatively measured to determine the kinase activity.The results represent the mean ±SEM from two independent experiments.
polymerization,this Dyrk1B-coordinated signaling pathway is blocked after microtubule damage.The comparison of the di ?erential cellular responses from cells treated with these two probes qualitatively and quantitatively aids in the character-ization of cell survival that is triggered by microtubule damage.Dyrk1B-Coordinated Cell Survival Involves Micro-tubule Repair and Recovery from Cell Cycle Arrest.The cell survival pathway is complicated and may be multipronged.Taking advantage of dual and single targeting probes (1and 2,respectively)enabled us to examine Dyrk1B-coordinated cell survival after microtubule damage.
The inhibition of tubulin polymerization seriously disrupts microtubule formation and causes cell cycle arrest in living cells.To evaluate the e ?ects of the probes on microtubules,we labeled α-tubulin proteins with ?uorescent tags and observed the microtubule integrity in RD cells at various time points after the treatment with probe 1or 2(Figure 4and Supplementary Figure 4).As shown in Figure 4a ?d,the microtubules in these cells were straight and long.Microtubule damage,as revealed by fragmented microtubules,was detected 6h after treatment with probe 1,and this damage continued for at least 48h (Figure 4e ?h).However,after the treatment with probe 2,microtubule damage was only detected at 6and 12h (Figure 4i and j).At 24h,the morphology of the microtubules returned to normal (Figure 4k and l).The polymer and monomer fractions of tubulin were isolated for the quantitative analysis of microtubule damage.The ratio of polymeric to monomeric tubulins was reduced after the treatment with probe 1or 2for 12h,indicating that tubulin polymerization was inhibited in the treated cells.However,the ratio returned to a level similar to
that in normal cells (at 0h)at 24h after the treatment with probe 2but not with probe 1(Figure 4m).These results were consistent with the microtubule damage that was observed using immuno ?uorescence microscopy.Probes 1and 2exhibited a similar level of tubulin polymerization inhibitory activity outside the cells but did not induce similar microtubule damage within the cells after 24h.The time point of microtubule repair (24h)after the treatment with probe 2correlated with the time of Dyrk1B activation.Thus,Dyrk1B activation likely induced microtubule repair,which is also consistent with previous reports that Dyrk1B mediated cancer cell survival.19?25
To further clarify whether Dyrk1B or activated Dyrk1B molecules are responsible for microtubule repair,we inves-tigated the e ?ects of probe 3(which is a Dyrk1B-only inhibitor)on tubulin polymerization in RD cells.After treating RD cells with probe 3for various times,no change in the microtubules was detected using immuno ?uorescence micros-copy (Supplementary Figure 5a ?d).These results indicated that without microtubule damage,Dyrk1B kinase did not a ?ect the normal microtubule assembly/disassembly equilibrium.Most likely,the microtubule damage-activated Dyrk1B mediated the microtubule recovery.
Perturbation of the dynamic microtubule assembly/dis-assembly process directly a ?ects cell division and induces G2/M arrest.As shown in Figure 5,the cells exhibited a signi ?cant cell cycle arrest at the G2/M phase 12h after the treatment with probe 1or 2.However,after 24h,a greater number of cells remained arrested at the G2/M phase (65%to 78%)after the treatment with probe 1,whereas the cell
cycle
Figure 3.Cellular Dyrk1B kinase activity induced by probe 1or 2.(a)Dyrk1B was immunoprecipitated (IP)from RD cells using a Dyrk1B monoclonal antibody after treatment with DMSO or 5μM concentration of either 1or 2for various times.The control immunoprecipitation was performed using rabbit IgG.The cellular Dyrk1B kinase activity was determined by measuring the incorporation of 32P into the recombinant Dyrk1B substrate p27kip1(32P-p27,the ?rst row)using SDS-PAGE analysis and autoradiography.The amount of Dyrk1B in each reaction was quanti ?ed using Western blot analysis (IP-Dyrk1B,the second row).The amount of p27kip1in each reaction was analyzed using SDS-PAGE analysis and visualized with Coomassie blue staining (total p27,the third row).The band density of 32P-p27and IP-Dyrk1B was quanti ?ed using ImageJ software and normalized to the band density of the 0-h time point.(b)Dyrk1B activity was quanti ?ed from the ratio of the band densities from the ?rst row (32P-p27)and the second row (IP-Dyrk1B)in panel a using ImageJ software.The results represent the mean ±SEM from two independent experiments.*The Dyrk1B activity of the group treated with probe 2was signi ?cantly di ?erent (P <0.05)from that of the group treated with probe 1at 24h.(c)A schematic diagram illustrating the relationship between microtubule damage and Dyrk1B activation.Probe 1,but not probe 2,inhibited Dyrk1B kinase activity in addition to tubulin polymerization.
arrest at G2/M was relieved (56%to 36%)after the treatment with probe 2.This G2/M arrest and recovery correlated well with the aforementioned microtubule damage and recovery.
Moreover,the time course of microtubule repair and recovery of cell cycle arrest after the treatment with probe 2(24h)correlated with the time point of Dyrk1B activation,
which
Figure 4.Microtubule damage and recovery in RD cells.Immuno ?uorescence microscopy images of RD cells after incubation with DMSO (a ?d)or 5μM concentration of either probe 1(e ?h)or 2(i ?l)at various time points.Detailed views of boxed regions in each image are shown in the following row.Microtubules (green)were labeled with an anti-α-tubulin antibody.Nuclei (blue)were stained using DAPI.The scale for each row is indicated in the ?rst panel.(m)Polymerized and monomeric tubulin fractions were isolated as described in the Methods,and equal amounts of each sample were analyzed using Western blot analysis.The ratio of polymeric to monomeric tubulin fractions was quanti ?ed using densitometric analysis using ImageJ software after normalization to the value at the 0-h time point.
indicated that the Dyrk1B-coordinated cell survival process included microtubule repair and recovery from cell cycle arrest.Microtubule-associated proteins (MAPs)are critical regu-lators of microtubule assembly/disassembly.36?39Because probes 1and 2damage microtubules through the inhibition of tubulin polymerization,we focused on assembly-promoting MAP proteins in our investigation.Several MAPs are classi ?ed as assembly-promoting MAPs,including tau,MAP2,and MAP4.Tau and MAP2are primarily expressed in neurons,40?42whereas MAP4is ubiquitously expressed in non-neuronal cells.43,44Bulinski and colleagues reported that microtubules in mouse L tk ?cells with MAP4overexpression enhanced tolerance to the microtubule depolymerizing agent nocodazole.38Micro-tubules in HeLa cells with MAP4knockdown recover more slowly following treatment with nocodazole.39
Examining the expression level of MAP4,probe 1caused very minor changes in MAP4expression.In contrast to probe 1,the MAP4expression level was increased after the treatment with probe 2,suggesting that the cell survival process required a higher level of MAP4(Figure 6a).The time course of MAP4
expression induced by probe 2correlated well with the time course of Dyrk1B activation.These results indicated that the Dyrk1B-regulated cell survival process involved microtubule repair and recovery from cycle arrest,potentially as a result of the upregulation of MAP4expression (Figure 6b).
Dyrk1B-Coordinated Cell Survival Triggers Mitochon-drial Translocation of p21for Antiapoptosis.In addition to the inhibition of cell division,microtubule damage induces apoptosis.Therefore,antiapoptosis is a critical event in cell survival.We next asked whether Dyrk1B-coordinated cell survival also mediated antiapoptosis.As shown in Figure 7a,apoptotic cells increased over time after the treatment with probe 1,and the total number of apoptotic cells reached 38%at 48h.However,apoptosis induced by probe 2represented only 11%of the total population.Quantitative analysis of caspase 3activity further con ?rmed this ?nding.Cellular caspase 3activity that was induced by probe 1was nearly 3-fold higher than that induced by probe 2(Figure 7b).The lower number of apoptotic cells re ?ected microtubule damage-induced cell survival after the treatment with probe 2.In contrast,probe 1also inhibited Dyrk1B and Dyrk1B-mediated cell survival.Therefore,probe 1prevented antiapoptosis.
The protein p21inhibits apoptosis only when it is translocated from the nucleus to the mitochondria to block proapoptotic e ?ectors such as procaspase 3.45?48Dyrk1B kinase protected myoblast cells from apoptosis through the phosphorylation of p21at Ser-153,which induced the translocation of p21to the mitochondria and induced the inhibition of caspase 3.49Thus,we hypothesized that the reduced apoptosis of RD cells by probe 2may be associated with the mitochondrial translocation of p21after Dyrk1B activation and that the association of p21translocation with Dyrk1B activation can be veri ?ed by comparing the e ?ects of treatment using probe 1with that of probe 2.To evaluate this hypothesis,we measured the mitochondrial translocation of p21after the treatment with probe 1or 2.RD cells were not perturbed after DMSO treatment,and p21proteins were mainly localized in the nucleus (Figure 7c).However,p21proteins were translocated to the mitochondria 24h after the cells were incubated with probe 2(Figure 7e).Almost no p21translocation was observed after the treatment with probe 1,suggesting that this branch of the survival pathway was
also
Figure 5.RD cells exhibit recovery from G2/M arrest after the treatment with probe 2but not with probe 1.The cell cycle of RD cells that were treated with DMSO (a,d)or 5μM concentration of either probe 1(b,e)or 2(c,f)for 12and 24h was analyzed using ?ow cytometry.The cells were stained with the Guava cell cycle reagent.The percentages of the cells in the G2/M phases represent the mean ±SEM from three independent
experiments.
Figure 6.Induction of MAP4protein expression by probe 2.(a)Western blot analysis of MAP4in RD cell extracts after the treatment with probe 1or 2(5μM)for various times.(b)A schematic model illustrating that the Dyrk1B survival pathway may trigger the upregulation of MAP4expression,which promotes microtubule stabilization.
blocked by probe 1upon Dyrk1B inhibition (Figure 7d).The activation or inhibition of Dyrk1B by probe 2or 1correlated well with p21mitochondrial translocation or the lack thereof,respectively.Quantitative analysis of the p21expression level in
cytoplasmic and nuclear extracts indicated that after the treatment with probe 2but not with probe 1,the level of p21protein in the cytoplasm was higher than that in the nucleus (Figure 7f),which further con ?rmed that p21
proteins
Figure 7.Dyrk1B-induced p21mitochondrial translocation to inhibit apoptosis after the treatment with probe 2but not with probe 1.(a)The percentage of apoptotic cells after the treatment with DMSO or 5μM concentration of either probe 1or 2for various times using ?ow cytometry analysis.(b)The activity of caspase 3in RD cells treated with DMSO or 5μM concentration of either probe 1or 2for 12and 24h was determined.Mitochondria and p21double immuno ?uorescence images of RD cells incubated with DMSO (c),5μM probe 1(d),or 5μM probe 2(e)for 24h are shown.Nuclei (blue)were stained using DAPI.Mitochondria (red)and p21(green)were labeled using anti-Core II and anti-p21antibodies,respectively.The scale bar in all panels represents 50μm.(f)Western blot analysis of p21in the cytoplasmic or nuclear extracts of RD cells after the treatment with DMSO or probe 1or 2(5μM)for 24h.β-actin and lamin A/C were used as loading controls in the cytoplasmic and nuclear extracts,respectively.The ratio of p21to β-actin in the cytoplasmic extracts or p21to lamin A/C in the nuclear extracts was quanti ?ed by densitometric analysis using ImageJ software and normalized to the value of the DMSO group.The ratio of cytoplasmic p21to nuclear p21was plotted.(g)The RD cells were treated with DMSO or probes 1or 2(5μM)for 24h,and p21was immunoprecipitated using an anti-p21antibody.The level of p21phosphorylation was detected with an anti-phosphorylated serine antibody using Western blot analysis.The band intensities of p-Ser and p21were quanti ?ed using ImageJ software.The ratio of these two bands was determined and plotted by normalization to the DMSO group.The results represent the mean ±SEM from three independent measurements.
were translocated from the nucleus to the cytoplasm in cells treated with probe 2but not with probe 1.Dyrk1B initially induces p21translocation through the phosphorylation of p21at Ser-153.49To verify this e ?ect,we measured the level of phosphorylated p21after the treatment with probes 1and 2.Our results indicated that the phosphorylation of p21was signi ?cantly increased after the treatment with probe 2compared with probe 1(Figure 7g).These results indicated that probe 1was able to inhibit Dyrk1B and the downstream mitochondrial translocation of p21,whereas probe 2induced microtubule damage and then triggered Dyrk1B-coordinated cell survival that involved the mitochondrial translocation of p21,which in turn inhibited caspase 3and reduced apoptosis.Magnitude of Dyrk1B-Coordinated Cell Survival Triggered by Microtubule Damage.The magnitude of cell survival may be partially quanti ?ed using the number of cells that return to a normal cell cycle or that escape apoptosis,which ultimately re ?ects enhanced cell viability.When tubulins are attacked by inhibitors,cells experience a series of insults such as broken microtubules,cell cycle arrest at the G2/M phase,and apoptosis.However,cells swiftly respond to these adverse situations by triggering cell self-repair and survival processes.Dyrk1B is a key regulator in this process.The activation of this signaling pathway results in recovered microtubule function,a return to a normal cell cycle,and the avoidance of apoptosis.With this pair of molecular probes,we
are now able to quantitatively measure the magnitude of survival through the quanti ?cation and comparison of di ?erent cellular outputs when the survival process is fully functional and when it is blocked.
The ?rst indicator for cell survival was the extent of cell cycle arrest at the G2/M phase when the microtubules were damaged.As a result of cell self-repair,37%of the cells were populated in the G2/M phase at 24h after inhibitor treatment,whereas 73%of the cells were populated in the G2/M phase when cell survival was blocked.The net di ?erence of 42%re ?ects self-repair by the cells.Cellular damage and cell cycle arrest subsequently cause apoptosis.With cell survival signaling,probe 2induced only 11%apoptotic cells at 48h.However,the apoptotic cells reached 38%when the cell survival pathway was blocked by probe 1.Cells maintain a delicate balance between damage and repair.The disruption of this balance caused by a tubulin polymerization inhibitor and a tubulin polymerization/Dyrk1B dual inhibitor can also be quantitatively evaluated based upon their inhibition of cell viability.In this regard,probe 1was 10-fold more potent in reducing viable cells (as measured by the IC 50value)than probe 2,which demonstrates the crucial in ?uence of the cell survival process.The magnitude of the di ?erences in cell cycle,apoptosis,and cell viability that were induced by probes 1and 2provides an approximation of the aptitude of cell self-repair and sustainability.These alterations,which were produced after a relatively moderate inhibition
of
Figure 8.Schematic model illustrating the di ?erent molecular interactions between the two probes and the proposed Dyrk1B survival pathway.Microtubule damage triggered by probe 2activates Dyrk1B.The activated Dyrk1B promotes microtubule stabilization,which may be related to the upregulation of MAP4expression.This action recovers cells from G2/M arrest.Dyrk1B activation also phosphorylates p21and promotes its mitochondrial translocation to inhibit apoptosis.Probe 1,however,inhibits both Dyrk1B and tubulin polymerization and prevents the Dyrk1B-mediated cell self-repair and survival process.
cellular Dyrk1B activity,suggest that Dyrk1B-mediated signal transduction impacts multiple molecular and cellular events during this repair process.
Conclusion.We rationalized that through the utilization of a molecular probe to inhibit tubulin polymerization and a second probe to inhibit both tubulin polymerization and microtubule damage-induced cell survival,we may elucidate the mechanism of this cell survival process.By examining the di?erential e?ects of probes1and2,we revealed that Dyrk1B kinase is a key regulator for cell survival after its activation by microtubule damage.Dyrk1B-triggered cell survival involves at least two pathways:(1)microtubule stabilization and cell cycle recovery from G2/M arrest and(2)the mitochondrial translocation of p21to avoid apoptosis through the inhibition of caspase3activity(Figure8).In addition to the elucidation of Dyrk1B-coordinated cell survival signaling pathways,we also report for the?rst time the magnitude of the recovery from the disruption of various cellular functions as a result of cellular self-repair and survival that is triggered by microtubule damage. The cell survival process recovers42%of the cells from G2/M arrest,prevents27%of the cells from undergoing apoptosis, and increases cell viability by10-fold.Without the comparison between the e?ects of a tubulin polymerization inhibitor(probe 2)and a tubulin polymerization/Dyrk1B dual inhibitor(probe 1),the magnitude of the recovered cell function as a result of cellular self-repair and survival is not easily elucidated.In other words,probe1demonstrates10-fold more potent inhibition of cancer cell viability by targeting both tubulin polymerization and the cell survival pathway triggered by microtubule damage. This?nding also a?ords a novel drug discovery strategy to target both the therapeutic target and the cell survival pathway
using a single therapeutic agent.
■METHODS
Molecular Probes and Cell Culture.Probes were synthesized and puri?ed by high-performance liquid chromatography as previous reported26and characterized by1H NMR(400MHz,DMSO-d6)and HR-MS(Support information).
RD cells were cultivated in DMEM medium containing L-glutamine (Gibco)with10%(v/v)fetal bovine serum,100U mL?1penicillin, and100μg mL?1streptomycin.HCT116cells and MDA-MB-231cells were cultivated in RPMI1640medium(Gibco)with10%(v/v)fetal bovine serum,100U mL?1penicillin,and100μg mL?1streptomycin. All of cells were grown in a humidi?ed incubator at37°C(95% humidity,5%CO2).
Tubulin Polymerization Assay.Tubulin polymerization assay was performed using a Tubulin Polymerization Assay Kit(Cytoske-leton)following a protocol provided by manufacturer.Small molecule probes and nocodazole were dissolved in DMSO separately.After adding the probes to the assay solution,DMSO’s volume was0.1%(v/ v)in the solution.An equal volume of DMSO was used as a control (in all assays,compounds were dissolved in DMSO and DMSO’s volume was0.1%(v/v)in the solution).The?uorescence was monitored every2min for a total of50min by a SpectraMax M5 microplate reader(Molecular Devices).Microtubule formation after the addition of DMSO(negative control)is represented by three phases:nucleation(0?8min),growth(10?40min),and equilibrium phase(40?50min).V max is identi?ed as the maximum slope of the time??uorescence curve between10and40min.Tubulin polymer-ization inhibition is calculated using the following formula:inhibition= 1?V max(treatment)/V max(DMSO).The IC50values are determined by SigmaPlot10.0program(Systat Software,Inc.).
Cell Viability Assay.Cell viability was determined by the CellTiter-Glo luminescent cell viability assay(Promega).Cells(3×104mL?1)were seeded in96-well?at-bottom plates.After24h,cells were treated with probes or an equal volume of DMSO at various concentrations.The cell viability was determined at48h by quantifying the amount of ATP,which is proportional to metabolic active cells.Cell viability was calculated using the following formula: cell viability=L treatment/L control.The IC50values were determined by the SigmaPlot10.0program(Systat Software,Inc.).
Binding/Inhibition of a Panel of Kinases.Primary kinase screen and K d of probes with Dyrk1B kinase were performed and measured by KINOMEscan platform.Probes1and2in DMSO at various concentrations were prepared and shipped to KINOMEscan Division (DiscoverRx Corporation)for KINOMEscan’s assay.KINOME scan is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized,active-site directed ligand.The assay is performed by combining three components:DNA-tagged kinase,immobilized ligand,and a test compound.Binding reactions were assembled by the three components in1×binding bu?er[20%(v/v)SeaBlock,0.17×PBS, 0.05%(v/v)Tween20and6mM DTT].The reactions were incubated at RT with shaking for1h,and the liganded a?nity beads were washed with wash bu?er[1×PBS,0.05%(v/v)Tween20].The beads were then resuspended in elution bu?er[1×PBS,0.05%(v/v) Tween20,0.5μM ligand]and incubated at RT with shaking for30 min.The kinase concentration in the eluates was measured by qPCR, which indicated that the ability of test compounds to compete with the immobilized,active-site directed ligand.
For primary kinase screen,probes1and2were prepared as40×stocks in100%DMSO and directly diluted into the assay.The?nal concentration of probe1or2was5μM.The results were reported as “%Ctrl”,which was calculated using the following formula:(test compound signal?positive control signal)/(DMSO signal?positive control signal)×100.The lower%Ctrl indicated the stronger binding to kinase active site.Kinases with%Ctrl<1were selected as kinases inhibited strongly by probes.For K d’s of probes with Dyrk1B kinase, probes1and2were prepared in100%DMSO at100×?nal test various concentrations and subsequently diluted to1×in the assay [?nal DMSO concentration=2.5%(v/v)].K d’s were calculated with a dose(x-axis)?qPCR signal(y-axis)curve using the Hill equation(the Hill Slope was set to?1):
=+
?
+
y
K x
background
signal(max)background
1(/)
d
Hill Slope
Dyrk1B Kinase Activity in Vitro.IC50values for inhibition of Dyrk1B kinase activity in vitro for probes1and2were determined by 33P Kinase HotSpot method.Probes1and2in DMSO at various concentrations were prepared and shipped to Reaction Biology Corporation for this assay.A peptide called“Dyrktide”with sequence [RRRFRPASPLRGPPK]was used as Dyrk1B’s substrate.The substrate was prepared in freshly Base Reaction Bu?er[20mM Hepes(pH7.5),10mM MgCl2,1mM EGTA,0.02%(v/v)Brij35, 0.02mg mL?1BSA,0.1mM Na3VO4]and then incubated with Dyrk1B,our probes,and33P-ATP(10μCiμL?1)for2h at RT.The reactions were spotted onto P81ion exchange paper(Whatman no. 3698-915).After the?lters were washed in0.75%(v/v)phosphoric acid,the radioactivity of the phosphorylated substrates in each reaction was detected,which re?ects directly kinase activity.
Cellular Protein Preparation.Cells were incubated with5μM of probes or an equal volume of DMSO for various times.The total cellular protein was extracted by M-PER Mammalian Protein Extraction Reagent(Thermo).The cytoplasmic and nuclear extracts were prepared by NE-PER Nuclear and Cytoplasmic Extraction Reagents(Thermo).
Immune Complex Dyrk1B Kinase Assays.Dyrk1B kinase activity in live cells was determined by immune complex kinase assays of immunoprecipitated Dyrk1B(IP-Dyrk1B).Aliquots of cell lysate were immunoprecipitated with24μL of Dyrk1B antibody(D40D1, Cell Signaling)overnight at4°C.Normal rabbit IgG(sc-2027,Santa Cruz)was used as a negative control.The complexes were collected after incubating for2h with50μL of protein A/protein G agarose(sc-2003,Santa Cruz)at4°C.The sample was washed with ice-cold PBS three times and ice-cold kinase bu?er(9802,Cell Signaling).The
complex pellets were then suspend in25μL of kinase bu?er containing6nmol cold ATP(9804,Cell Signaling),7μCi of32P-α-ATP(BLU503H,PerkinElmer,MA,USA),and2.5μg of recombinant p27(ATGen Ltd.)as substrate for15min at30°C before analyzed by SDS-PAGE and autoradiography.
Preparation of Polymer and Monomer Fractions of Tubulin. RD Cells were treated with5μM concentration of probe1or2for various time.The polymer and monomer fractions of tubulin were prepared as described previously.50In brief,the cells were trypsinized and collected before they were washed with microtubule-stabilizing bu?er(MSB;0.085M Pipes,pH6.9,1mM MgSO4,2mM EGTA and 4M glycerol)and then extracted with MSB containing0.5%(v/v) Triton X-100and protease inhibitors.After3min at37°C,the extract (monomeric tubulin fraction)was gently collected.The remaining polymeric tubulin fraction was dissolved in0.5%(w/v)SDS bu?er [0.5%(w/v)SDS,25mM Tris,pH6.8and protease inhibitor]for10 min on ice.Equal protein amounts of each fraction were separated by SDS-PAGE followed by Western blot analysis using an anti-α-tubulin antibody.
Western Blot.Cell lysates or immunoprecipitations were separated by SDS-PAGE using NuPAGE gradient Bis-Tris gels(4?12%) (Invitrogen)and then transferred to a nitrocellulose membrane using iBlot dry blotting system(Invitrogen).After being blocked with block bu?er(LI-COR Biotechnology)for2h,the membrane was incubated with primary antibody againstα-tubulin,MAP4,β-actin, Dyrk1B,p21or lamin A/C(2125,Cell Signaling,A-3,Santa Cruz,A-5316,Sigma-Aldrich,D40D1,Cell Signaling,H-164,Santa Cruz,346, Santa Cruz)overnight at4°C following by washing with PBST[PBS with0.5%(v/v)Tween20].The membrane was then incubated with secondary antibodies goat anti-mouse or goat anti-rabbit(LI-COR Biotechnology or Santa Cruz)for1h at RT,followed by visualization using Odyssey System(LI-COR Biotechnology)or Western chemiluminescence kit(Bio-Rad).
Immuno?uorescence Microscopy.RD cells were seeded in22×22mm2coverslips(Corning Life Sciences)for24h and treated with 5μM concentration of probe1or2in DMSO for various times.The cells were washed with PBS and then?xed with4%(v/v) paraformaldehyde for10min.After washing with PBS(0.1M) three times,cells were blocked with1%(w/v)BSA in PBS for20min at RT.Then,antibodies againstα-tubulin,p21,and Complex III subunit Core2(2125,Cell Signaling,H-164,Santa Crus,MS304-SP, MitoSciences)were incubated with cells in a humid chamber overnight at4°C.After washing with PBS(0.1M)three times,secondary antibodies,goat anti-rabbit IgG FITC and goat anti-mouse IgG TRITC(Jackson ImmunoResearch),were incubated with cells for20 min at37°C.Cells were mounted with mounting medium containing DAPI(Vector Lab)after washing with0.1M PBS three times and then observed with a?uorescence microscope(Olympus DP70, Olympus Corporation).
Flow Cytometry Analysis.Flow cytometry analysis was performed on a Guava EasyCyte Mini?ow cytometry system (Millipore).Cells were incubated with5μM probes or an equal volume of DMSO for various times before they were trypsinized, aspirated,and counted.An equal number of cells was resuspended in Guava cell cycle reagent(Millipore)and incubated for30min at RT in the dark,followed by?ow cytometry analysis.For cell apoptosis analysis,an equal number of cells was incubated with Guava nexin reagent(Millipore)for20min at RT in the dark,followed by?ow cytometry analysis.
Caspase3Activity Assay.Caspase3activity was determined using a Caspase3/CPP32Colorimetric Assay Kit(k106-100, BioVision).RD cell lysates were prepared after treatment of5μM probes or an equal volume of DMSO for12or24h.Cellular protein (200μg)was used for caspase3activity assays.The absorbance of samples was read by SpectraMax M5microplate reader(Molecular Devices)at400nm.The relative caspase3activity was determined by comparing with the control group.
p21Phosphorylation Detection.RD cell lysates were prepared after treatment of5μM probes and DMSO for24h.The p21protein was immunoprecipitated by p21antibody(H-164,Santa Cruz),and normal rabbit IgG was used as control.The level of serine phosphorylation and was detected by Western blot with serine
phosphorylation antibody(16B4,Santa Cruz).
■ASSOCIATED CONTENT
*Supporting Information
Data for tubulin inhibitor screen,data for the combination of probes2and3,additional data for microtubule damage and recovery,kinase binding assay data,and analytical character-izations of all probes.This material is available free of charge via
the Internet at https://www.doczj.com/doc/025614812.html,.
■AUTHOR INFORMATION
Corresponding Author
*E-mail:drbingyan@https://www.doczj.com/doc/025614812.html,.
Notes
The authors declare no competing?nancial interest.■ACKNOWLEDGMENTS
We thank T.Chen,J.Wu,and L.Liu(St.Jude Children’s Research Hospital,TN,USA)for providing cell lines and some technical assistance and Q.Mu,G.Su,and P.Jiao for stimulating discussion.This research was supported by the National Natural Science Foundation of China(90913006,
21077068,and21137002).
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