1521-0111/87/1/18–30$25.00 http://dx.doi.org/10.1124/mol.114.093245MOLECULAR PHARMACOLOGY Mol Pharmacol 87:18–30, January 2015Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics

In Vitro Antitumor Mechanism of (E)-N-(2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)pyridin-3-yl)methanesulfonamide s

Tiangong Lu, Charles A. Laughton, Shudong Wang, and Tracey D. BradshawSchool of Pharmacy and Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham, UnitedKingdom (T.L., C.A.L., T.D.B.); and Centre for Drug Discovery and Development, Sansom Institute for Health Research, andSchool of Pharmacy and Medical Sciences, University of South Australia, Adelaide, Australia (S.W.)

Received April 16, 2014; accepted October 14, 2014

ABSTRACTON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate; Rigosertib, Estybon], a styryl ben-zylsulfone, is a phase III stage anticancer agent. This non-ATPcompetitive kinase inhibitor has multitargeted activity, promot-ing mitotic arrest and apoptosis. Extensive phase I/II studieswith ON01910.Na, conducted in patients with solid tumorsand hematologic cancers, demonstrate excellent efficacy.However, issues remain affecting its development. These in-clude incomplete understanding of antitumor mechanisms,low oral bioavailability, and unpredictable pharmacokinetics.We have identified a novel (E)-styrylsulfonyl methylpyridine[(E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide (TL-77)] which has shown im-proved oral bioavailability compared with ON01910.Na. Here,we present detailed cellular mechanisms of TL-77 in comparisonwith ON01910.Na. TL-77 displays potent growth inhibitory activityin vitro (GI50 , 1mM against HCT-116 cells), demonstrating 3- to

10-fold greater potency against tumor cell lines when comparedwith normal cells. Cell-cycle analyses reveal that TL-77 causessignificant G2/M arrest in cancer cells, followed by the onset ofapoptosis. In cell-free conditions, TL-77 potently inhibits tubulinpolymerization. Mitotically arrested cells display multipolarspindles and misalignment of chromosomes, indicating thatTL-77 interfereswithmitotic spindle assembly in cancer cells. Theseeffects are accompanied by induction of DNA damage, inhibitionof Cdc25C phosphorylation [indicative of Plk1 inhibition], anddownstream inhibition of cyclin B1. However, kinase assaysfailed to confirm inhibition of Plk1. Nonsignificant effects onphosphoinositide 3-kinase/Akt signal transduction were ob-served after TL-77 treatment. Analysis of apoptotic signalingpathways reveals that TL-77 downregulates expression of B-celllymphoma 2 family proteins (Bid, Bcl-xl, and Mcl-1) and stim-ulates caspase activation. Taken together, TL-77 represents apromising anticancer agent worthy of further evaluation.

IntroductionAmong the cell-cycle phases, S phase and mitosis (M)

determine how cells divide and transmit genetic informationfrom one cell generation to the next (Schmit and Ahmad, 2007).Despite being the shortest phase of the cell cycle, M orches-trates major changes in multiple cellular components (Chanet al., 2012). Mitotic kinases possess pivotal roles throughoutcell division through regulating a diverse range of signalingpathways and controlling mitotic checkpoints, spindle func-tion, and chromosome segregation. Mitotic kinases havealso been implicated in tumorigenesis; aberrant cellular re-sponses triggered by protein kinase–mediated events arethought to lead to uncontrolled proliferation, aneuploidy,and genetic instability culminating in cancer development(Schmit and Ahmad, 2007). Therefore, mitotic kinases haveattracted significant attention as important targets for cancer

drug discovery. The demonstration of clinical activity of anumber of small-molecule inhibitors for the treatment of cancerhas generated considerable interest in the search for mitotickinase inhibitors (Smits et al., 2006; Chan et al., 2012). Non-ATPcompetitive inhibitors for cancer therapy are of particularinterest to overcome the problems of poor selectivity and drugresistance associated with ATP analogs (Kirkland and McInnes,2009).ON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxy-

styrylsulfonyl)methyl)phenylamino)acetate; also referredto as Rigosertib and Estybon] (Fig. 1), a styryl benzylsulfone,is a non-ATP competitive anticancer agent currently in phaseIII clinical trials. It demonstrates multitargeted activity,and induces mitotic arrest and apoptosis in cancer cells(http://www.onconova.com/product-pipeline/rigosertib.php).Initially it was thought that the antimitotic activity was dueto inhibition of polo-like kinase 1 (Plk1) (Gumireddy et al.,2005). However, subsequent studies did not support a directeffect on Plk1 (Steegmaier et al., 2007). Recent studies reportedthat ON01910.Na uses a dual molecular mechanism of action,

dx.doi.org/10.1124/mol.114.093245.s This article has supplemental material available at molpharm.

aspetjournals.org.

ABBREVIATIONS: Ab, antibody; Akt, protein kinase B; Bcl, B-cell CLL/Lymphoma; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; FITC,fluorescein isothiocyanate; GI50, concentration required to achieve 50% growth inhibition; HUVEC, human umbilical cord vein endothelial cell;mTOR, mammalian target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; ON01910.Na, sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate; PBS, phosphate-buffered saline; PI, propidium iodide; PTEN, phosphataseand tensin homolog; TL-77, (E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide.

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inducing cell-cycle arrest correlated with RanGAP1 hyper-phosphorylation and modulating the phosphoinositide 3-kinase (PI3K)/AKT pathway to promote apoptosis in cancercells (Prasad et al., 2009; Oussenko et al., 2011). Extensivephase I/II studies with ON01910.Na have been conducted inpatients with solid tumors and hematologic cancers, revealingobjective durable efficacy of the drug with good tolerability(Jimeno et al., 2008; Ohnuma et al., 2013). However, a number ofkey issues remain that are affecting its development; theseinclude an incomplete understanding of mechanisms of actionand relatively poor drug oral bioavailability.In a program to identify a novel chemical series of molecules

possessing similar modes of action, but improved pharmaceu-tical properties compared with ON01910.Na, we have designedand synthesized a class of (E)-styrylsulfonyl methylpyridines.One of the lead compounds with improved oral bioavailability(F 5 56% oral dosing at 10 mg/kg in mice; SupplementalTable 1) (Lu et al., 2014), designated as TL-77 [(E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide] (Fig. 1), has been investigated for itscellular mechanism of action. Utilizing biochemical and cell-based assays, we show that TL-77 arrests cells at the G2/Mphase in cancer cells, followed by the onset of apoptosis.Treatment of HCT-116 cells with TL-77 inhibits mitoticspindle assembly, evoking formation of multipolar spindles andmisalignment of chromosomes, which is similar to the pheno-type observed with Plk1-depleted cells. In cell-free conditions,TL-77 potently inhibits tubulin polymerization. Mitotic markers,Cdc25C and cyclin B1, were investigated to explore the mo-lecular consequences of TL-77 treatment. Phosphorylation ofCdc25C (Ser198) has been suggested to serve as a biomarkerto track inhibition of Plk1. TL-77 inhibits phosphorylation ofCdc25C and downregulates cyclin B1 in cancer cells. Theseobservations reveal that TL-77 induces spindle abnormalitiesand may suppress the activity of Plk1 in cancer cells. TL-77causes downregulation of antiapoptotic proteins, and inducescaspase-dependent apoptosis, which are consistent with theeffects of ON01910.Na. Unlike ON01910.Na, however, TL-77causes preferential toxicity in cancer cells when compared withnormal cells. PI3K/Akt/mammalian target of rapamycin (mTOR)signal transduction is minimally disturbed in tumor cells afterTL-77 treatment.

Materials and MethodsInhibitor Compounds. TL-77 was synthesized in our laboratory

as reported previously (Lu et al., 2014), and the purity and structureconfirmation is reported in SupplementalMethod 1. ON01910.Nawaspurchased from Allichem LLC (Baltimore, MD).

Cell Culture. All cell lines were obtained from the American TypeTissue Culture Collection, expanded, and stored in the liquid nitrogencell bank at the Centre for Biomolecular Sciences, University ofNottingham (Nottingham, UK). All cancer cell lines were maintainedin RPMI 1640 medium with 10% fetal bovine serum (FBS). Humandermal microvascular endothelial cells (HMEC-1) were maintained inEndothelial Cell Growth Medium 2 with 10% FBS. Human umbilicalcord vein endothelial cells (HUVECs) were maintained in EndothelialCell Growth Medium 2 with 10% FBS. All cell lines were incubated at37°C in a humidified, 5% CO2 atmosphere.

Selection of Cell Lines. HCT-116 colorectal and A2780 ovariancell lines were chosen to represent two common and intractable cancers,among the most common malignancies worldwide. They are commonlyused as model systems for studying cancer pathways and for developingnew therapeutic approaches. HCT-116 cells are well characterized by anintact DNA damage checkpoint and normal p53 responses (Vogel et al.,2004). Upregulated expression of seven genes responsible for DNAdamage signaling was observed in HCT-116 cells, including Cdc25C(Duldulao et al., 2012). In addition, HCT-116 cells are reported to exhibithigh Plk1 expression and activity (Herz et al., 2012). Both HCT-116 andA2780 cell lines have been validated in the scientific literature to bemitotic spindle checkpoint proficient (Birk et al., 2012). A2780 cellspossess confirmed biallelic inactivation of phosphatase and tensinhomolog (PTEN; 9 base pair deletion in exon 5 and 37 base pair deletionin exon 8) (Wu et al., 2011), and therefore, deregulated PI3K signaltransduction (Burger et al., 2011). In view of intracellular effects andpotential targets of ON01910.Na reported to date, these two cell lineswere used in this study for investigation of the mechanism(s) of action ofTL-77.

MTT Cytotoxicity Assays. Standard MTT (thiazolyl blue; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays wereperformed as reported previously (Wang et al., 2004). In brief, cellswere seeded (1500–3000 cells/well) in 96-well plates and allowed toattach overnight. Cells were incubated for 96 hours with inhibitorsusing a range of concentrations to generate dose-response curves. Theconcentration required to cause 50% growth inhibition (GI50) wasdetermined using nonlinear regression analysis.

Cell Cycle Analysis and Detection of Apoptosis. Cell-cycleeffects and induction of apoptosis were examined in cancer cells aswell asnontransformed cells. Sample preparation, staining, and analyses wereperformed following the protocol provided by BD (BD Biosciences, SanJose, CA). HCT-116 and A2780 cells were seeded at a density of 6 � 105.Following overnight incubation then treatment with inhibitors at ap-propriate concentrations and for specific time points, cells were collected,pelleted, and washed twice with cold (4°C) phosphate-buffered saline(PBS). For cell-cycle analyses, cells were centrifuged at 1200 rpm for5 minutes at 4°C and resuspended in 0.4 ml of fluorochrome solutioncontaining 50 mg/ml propidium iodide (PI) in 0.1% sodium citrate plus0.1% Triton X-100, protected from light and stored at 4°C overnight.

For determination of apoptotic populations, annexinV/PI stainingwas used. Firstly, 1 � 105 cells were centrifuged at 1200 rpm at 4°C.Supernatants were discarded again and the cell pellet was resus-pended in 100 ml of binding buffer and 5 ml of annexin V–fluoresceinisothiocyanate (FITC). Samples were incubated at room temperaturefor 15 minutes in the dark, and 400 ml of binding buffer was added,together with 10 ml of PI (50 ml/ml in PBS). Samples were kept on ice,protected from light for a further 10 minutes, and analyzed usingfluorescence-activated cell sorting after vortex within 1 hour.

Cell-cycle analyses and quantification of apoptosis were conductedon a Beckman Coulter EPICS-XL MCL flow cytometer (BeckmanCoulter Inc, Indianapolis, IN) and data analyzed using EXPO32software (Beckman Coulter Inc).

Mitotic Index Assay. Mitotic index was determined using theCellomics Mitotic Index Kit (Thermo Fisher Scientific, Waltham, MA)following the manufacturer’s instructions. Fluorescence was measuredat 460 nm at room temperature using a Leica DMIRE2 fluorescentmicroscope (Leica Microsystems, Wetzlar, Germany) to calculate themitotic index, which is the percentage of cell nuclei stained with the

Fig. 1. Chemical structures of ON01910.Na [sodium (E)-2-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)phenylamino)acetate] and TL-77[(E)-N-(2-methoxy-5-((2,4,6-trimethoxystyrylsulfonyl)methyl)pyridin-3-yl)methanesulfonamide].

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mitosis-specific antibody (anti-phospho-histone H3) versus total cellnuclei stainedwithHoechst. Vinblastine (0.5mM)was used as a positivecontrol, and dimethylsulfoxide (DMSO; 0.2%) was used as a negativecontrol. Assays were carried out using duplicate plates.

Caspase-3/7 Assay. Caspase activity was determined using theApo-ONE Homogeneous Caspase-3/7 Assay (Promega, Madison, WI),following the manufacturer’s instructions. Fluorescence was mea-sured at 460 nm at room temperature using an EnVision multilabelplate reader (PerkinElmer, Waltham, MA).

Western Blot Analysis. Western blot analyses were performed ina standard manner (Mahmood and Yang, 2012). The separated proteinswere electroblotted to membranes (Bio-Rad Laboratories, Hercules, CA),and thesewere probedwith specific primary antibodies (1° Abs). Anti-Akt,anti–phosphor-Akt (Ser473), anti-Bid, anti-Bax, anti–Bcl-xl, anti-Cdc25C,anti–phosphor-Cdc25C (Ser198), anticyclin B1, anticyclin D1, anti-Mcl-1,anti-mTOR, anti–phosphor-mTOR (Ser2448), anti–[poly (ADP-ribose)polymerase] (anti-PARP), anti-phosphor–phosphoinositide-dependentprotein kinase 1 (PDK1) (Ser241), anti–phosphor-PRAS40 (proline-richAkt substrate of 40 kDa) (Thr246), and anti-PTEN Abs were obtainedfromCell Signaling Technology, Inc (Danvers,MA). Anti–g-H2AX (H2Ahistone family, member X) Ab was from Millipore, Inc (Darmstadt,Germany). Both antimouse and antirabbit IgG horseradish peroxidase–conjugated 2° Abs were obtained from Dako (Dako Denmark A/S,Cambridge, UK).

Tubulin Polymerization Assay. The Tubulin PolymerizationHTS Assay Kit (97% pure tubulin, porcine; Cytoskeleton, Denver, CO)was used according to the manufacturer’s protocol. The kit-based assayis an adaptation of a method (Rodriguez-Jimenez et al., 2010) wherepolymerization is monitored by optical density enhancement as itoccurs. In brief, test compounds and their corresponding vehicles werepipetted into a 96-well microtiter plate, then tubulin reaction mixturewas quickly added to the wells, and tubulin polymerization wasinitiated and monitored at 37°C for 1 hour by recording fluorescenceabsorbance at 340 nm. The tubulin reaction mixture was composed of80 mM PIPES (pH 6.9), 1 mM MgCl2, 1 mM EGTA, 1.0 mM GTP, and2 mg/ml of highly purified porcine brain tubulin heterodimer.

ImmunofluorescenceMicroscopy. HCT-116 cells (5� 104 cells/well)plated on 12-well chamber slides were treated without or with com-pounds for 24 hours. After treatment, cells were fixed in 3.7% form-aldehyde in PBS for 15 minutes, and permeabilized with 0.3% TritonX-100 in PBS for 1 hour with blocking of nonspecific binding sites using1% bovine serum albumin. Fixed cells were then incubated with an-timousea-tubulin Ab (1:50 dilution) for 2 hours at room temperature andthen exposed to the 2° Ab (FITC-conjugated goat antimouse IgG at 1:100dilution), followed by DNA staining with Hoechst. Photomicrographswere obtained using a Leica TCS SP2 Confocal Spectral Microscope.

Statistical Analysis. All experiments were performed in tripli-cate and repeated at least twice. Student’s t tests for paired sampleswere used to determine statistical significance. P # 0.05 wasconsidered to be statistically significant.

ResultsTL-77 Is a Potent Antiproliferative Agent. MTT as-

says were used to assess the antiproliferative activity ofTL-77 against a panel of 10 human tumor cell lines and threeuntransformed cell lines after 96-hour exposure (Table 1).Similar sensitivity was observed for cells with different p53and retinoblastoma protein status for both compounds. TL-77displayed potent antiproliferative activity with GI50 (theconcentration of TL-77 required to inhibit growth by 50%determined by MTT assay) values ranging between 317 and875 nM in tumor cell lines irrespective of tissue origin. Thetwo compounds were also tested against nontransformedcells, HMEC-1, HUVEC, and MRC-5. TL-77 exhibited modestselectivity: as illustrated in Table 1, TL-77 demonstrated.2-fold greater potency in cancer cell lines over normal cells.Optimally, 10-fold greater selectivity was observed (A2780GI50 5 317 nM; MRC-5 GI50 5 3144 nM). Overall, ON01910.Na was a more potent suppressor of tumor cell proliferationagainst the same carcinoma cell lines (GI50 5 7–70 nM); how-ever, little or no selectivity between the cancer and non-transformed human cells was detected (GI50 5 30–51 nM innontransformed cells). Time-dependent antiproliferative ef-fects were further observed for both compounds as exemplifiedin HCT-116 cells (Fig. 2). Following 24-hour exposure to TL-77or ON01910.Na, GI50 values of 4 and 0.06 mM, respectively, inHCT-116 cells and 2 and 0.25 mM, respectively, in A2780 cellswere observed and adopted as agent concentrations used insubsequent assays.TL-77 Induces Cancer Cell Cycle Arrest at G2/M. By

inhibiting Plk1 activity in cancer cells, ON01910.Na inducesmitotic arrest characterized by spindle abnormalities, leadingto apoptotic death (Gumireddy et al., 2005). Therefore, theeffects of the two compounds on tumor cell-cycle progressionwere assessed by flow cytometry in two well characterizedcancer cell lines, HCT-116 and A2780 (Fig. 3, A and B). Theseresults showed stark and time-dependent G2/M cell-cycle

TABLE 1Antiproliferative activity of TL-77 against a panel of cell linesData expressed as the mean 6 standard deviation of three independent experiments.

Human Cell Lines 96 h–MTT, GI50 6 S.D.

Origin Destination ON01910.Na TL-77

mM

Colon carcinoma HCT-116 (p53+, Rb+) 0.070 6 0.003 0.570 6 0.023HCT-116 (p532, Rb+) 0.017 6 0.002 0.860 6 0.086

Breast carcinoma MCF-7 (p53+,ER+) 0.050 6 0.010 0.780 6 0.025MDA-MB 231(p532,ER2) 0.055 6 0.015 0.671 6 0.056

Melanoma MDA-MB 468 (p532,Rb–) 0.039 6 0.015 0.509 6 0.130MDA-MB 435 (p532,Rb+) 0.012 6 0.002 0.592 6 0.128

Ovarian carcinoma A2780 0.062 6 0.004 0.317 6 0.179Cervical carcinoma Hela 0.012 6 0.001 0.682 6 0.112Pancreatic carcinoma Mia-paca II 0.007 6 0.002 0.463 6 0.208

PANC-1 0.039 6 0.006 0.875 6 0.274Microvascular endothelial HMEC-1 0.051 6 0.002 1.681 6 0.270Umbilical vein endothelial HUVEC 0.030 6 0.001 1.110 6 0.270Lung Fibroblast MRC-5 0.049 6 0.025 3.144 6 0.832

ER, estrogen receptor; Rb, retinoblastoma protein.

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block (enhanced numbers of cells with 4 N DNA content) aftertreatment of both HCT-116 and A2780 cells with bothcompounds at GI50 values. Particularly in HCT-116 cells,TL-77 caused significant G2/M accumulation (.50%) as early

as 6 hours, whereas ON01910.Na caused the same effect(∼50%G2/M arrest) after a longer exposure period ($12 hour).Cell-cycle arrest was induced more rapidly when concen-trations increased to 2 � GI50 for both compounds (effects in

Fig. 2. TL-77 and ON01910.Na display time-dependentantiproliferative effects in human cancer cells. MTTgrowth inhibition assay time-course experiments werecarried out against human HCT-116 colon cancer cells.The data given are mean values derived from at leastthree replicates (n = 3 per experiment) 6 S.D.

Fig. 3. TL-77 and ON01910.Na induce G2/Marrest in HCT-116 and A2780 cells. HCT-116(A) and A2780 (B) cells were treated with GI50concentrations of TL-77 or ON01910.Na. Cellswere fixed, stained with propidium iodide, andDNA content was analyzed by flow cytometry.

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HCT-116 cells are shown in Supplemental Fig. 1). Treatmentof cells from both cell lines .18 hours resulted in accumula-tion of events containing sub-G1 DNA content, which is anindication of cell death, suggesting that the induction ofapoptosis might be a consequence of cell-cycle arrest.To further characterize the cell cycle effect after TL-77

exposure, a mitotic index assay which indicates the ratiobetween the number of cells inmitosis and the total number ofcells was performed (Fig. 4). Both HCT-116 and A2780 cellswere treated with TL-77 or ON01910.Na for a period of 6 or12 hours. As shown in Fig. 4, A and C, TL-77 increased mitoticindex in a time- and dose-dependent manner in both cell lines.Following 6-hour exposure to TL-77 (0.5–2 � GI50), 15–20%of cells resided in mitosis compared with ,5% in controlsamples. An increased mitotic index (25%) was further ob-served in HCT-116 cells exposed to 4 � GI50 TL-77 (Fig.4A). After 12-hour treatment with TL-77, a dramatic dose-dependent increase in mitotic index was observed in HCT-116cells (.35%; 1–4 � GI50 TL-77), whereas a gradual climb ofmitotic population was observed in A2780 cells (∼35%; 4 �GI50 TL-77). Meanwhile, ON01910.Na caused less dramaticmitotic arrest in both cell lines as shown in Fig. 4, B and D. Atconcentrations .0.06 mM, a gentle dose-dependent increasein mitotic index was observed at both 6 and 12 hours. In HCT-116 cells, only at 4 � GI50 value (0.24 mM ON01910.Na) wasa time-dependent divergence observed (mitotic index 17% and27% after 6 and 12 hours, respectively).Plots of mitotic indices (detailing percentage of mitotic cells

in a given population) compared with data from cell cycleanalyses (detailing G2/M events) are provided in Fig. 5. Bysubtracting mitotic indices from G2/M values, the populationof cells in the G2 phase could be obtained. Whereas TL-77evoked a dramatic mitotic block in both cell lines after 6-to12-hour exposure (Fig. 5, A and C), the G2 block induced byTL-77 was less significant. In HCT-116 cells, numbers of TL-77–treated (4–8 mM) cells in the G2 phase remained stable at 6hours; ∼20% increased G2 events were observed when TL-77exposure extended to 12 hours at 2 � GI50 (8 mM) (Fig. 5A). Incontrast, ON01910.Na-treated cells revealed less significantmitotic blocks but a notable G2 arrest (Fig. 5, B and D). At 6hours, ON01910.Na-treated HCT-116 cells started to arrest in

the G2 phase (∼10%) at 2 � GI50, whereas a steady raise wasobserved at both GI50 (∼20%) and 2 � GI50 (∼29%) in A2780cells. Furthermore, after 12-hour treatment, ON01910.Nasharply increased the numbers of cells in the G2 phase (∼20–30%) at both concentrations in both cell lines. These resultsare consistent with cell-cycle observations (Fig. 3A) wheremore rapid cell cycle arrest was observed in cells exposed toTL-77.TL-77 Inhibits Tubulin Polymerization. A biochemical

assay was adopted to then examine whether either compoundaffected tubulin polymerization (Fig. 6). Two known com-pounds, paclitaxel and nocodazole, were used as positive andnegative controls, respectively. Paclitaxel binds to tubulin tosuppress dissociation of microtubules (Díaz et al., 1998), andpromotes polymerization of purified tubulin. In contrast,nocodazole inhibits polymerization by binding to b-tubulinand preventing formation of one of the two interchaindisulfide linkages between tubulins (Vasquez et al., 1997;Chu and Ng, 2004). We compared the two test compounds’effects on microtubule polymerization at GI50 and 1.5 � GI50for TL-77, and GI50 and 2 � GI50 for ON01910.Na. As shownin representative traces in Fig. 6A, TL-77 acted (in a similarmanner to nocodazole) as a microtubule suppressor at 4 and6 mM. Similar results were obtained with ON01910.Na at 0.06and 0.12 mM (Fig. 6B). These data indicate both compoundspotently suppress tubulin polymerization in a manner similarto nocodazole at tested concentrations.TL-77 Perturbs Spindle Assembly in Cancer Cells. To

examine whether TL-77 could induce abnormal mitoticspindles, we examined the spindle architecture using confocalmicroscopy. Immunofluorescence staining using anti–a-tubulinantibody was performed in HCT-116 and A2780 cells. After12-hour DMSO treatment, interphase cells were observed withbipolar mitotic spindles. As shown in Fig. 7, both 4 and 8 mMTL-77 caused remarkable inhibition of microtubule assembly intreated cells. The phenotypes caused by TL-77 includedabnormal mitotic spindle formation with bi-, tri-, or multipolarspindles; chromosome misalignment; and complete loss ofcoordination in mitotic spindle assembly. Meanwhile, ab-normal mitotic spindle formation was also observed in cellsafter ON01910.Na treatment. However, instead of generating

Fig. 4. Cell-cycle status detected by mi-totic index assay in cancer cells. HCT-116and A2780 cells were treated with concen-trations of TL-77 (A and C, respectively) orON01910.Na (B and D, respectively) andincubated for 6 and 12 hours. Afterfixation in 4% paraformaldehyde, cellswere treated with phospho-histone H3antibody and Hoechst. The cells were ana-lyzed by confocal laser microscopy. Verti-cal bars represent the means 6 S.D. of atleast two independent experiments (n = 3per experiment). *Values significantly(P # 0.05) different from DMSO vehiclecontrol.

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multipolar spindles, cells exposed to ON01910.Na exhibitedfragmented microtubules, resulting in prevention of celldivision. Paclitaxel and nocodazole were used in this study aspositive and negative controls (Supplemental Fig. 2). Cellstreated with these two compounds displayed stabilized ordepolymerized microtubules, respectively.

TL-77 Downregulates Phosphorylation of Cdc25C. Ineukaryotic cells, the initiation of mitosis is triggered byactivation of the Cdc2/cyclin B1 complex, the “mitosis-promoting factor” (Yang et al., 1998). Cdc25C, a substrate ofPlk1, regulates Cdc2/cyclin B1 activity and controls entryinto mitosis (Toyoshima-Morimoto et al., 2002; Schmit and

Fig. 5. Cell-cycle status of TL-77 andON01910.Na plotted based on mitoticindex and cell cycle. Plots of mitotic indexcompared with the data from cell-cycleanalysis of TL-77 [HCT-116 cells (A) andA2780 cells (C)] or ON01910.Na [HCT-116cells (B) and A2780 cells (D)]. The pop-ulation mean of cells in G2 phase wasobtained by subtracting the mean ofmitotic indices from the mean of G2/Mvalues at corresponding concentrations,mG2 = mG2/M 2 mMitosis. The popula-tion of cells in G2/M phase and mitosiswere considered as two random varia-bles. The standard deviation is adopted

as SD12 ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffis21 ∕ n1 þ s2

2 ∕ n2

q(Shahbaba,

2012), where s21 and s2

2 are the populationvariances for the population of cells inG2/M and mitotic, respectively.

Fig. 6. Tubulin polymerization assay. Paclitaxel (PAC)and nocodazole (NOC) were used as additional controlsknown to enhance tubulin polymerization and depolymer-ization, respectively. TL-77 (A) and ON01910.Na (B) arecompared with control. OD, optical density.

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Ahmad, 2007). Previous work reported that ON01910.Nainhibits activation of the phosphatase Cdc25C as one of itsprimary intracellular effects (Gumireddy et al., 2005; Jimenoet al., 2009). With emphasis on events upstream of Cdc25C,recent work identified the effect of ON01910.Na on DNAdamage response signal pathways throughout the cell cycle(Oussenko et al., 2011).Therefore, we next examined the effects of TL-77 on Cdc25C

and cyclin B1 in HCT-116 and A2780 using Western blotanalysis. Treatment with TL-77 for 8 hours dramaticallyreduced phosphorylation of Cdc25C at Ser198 in bothHCT-116 and A2780 cells (Fig. 8). Similarly, ON01910.Natreatment also caused reduced levels of phosphor-Cdc25C.Although we found that both compounds minimally affectedexpression of cyclin B1 after 8-hour treatment in both celllines, suppression of cyclin B1 was detected in TL-77–treatedcells (16 mM) as well as ON01910.Na-treated cells (0.24 mM)after 24-hour treatment.Inhibition of PI3K/Akt/mTOR Signal Transduction

Caused by TL-77. ON01910.Na has been reported to inhibitPI3K/Akt/mTOR signal transduction as the major target withdownregulation of cyclin D1 translation and activation ofapoptosis (Prasad et al., 2009). To determine whether TL-77modulates PI3K/Akt/mTOR signaling, we used Western blotto examine expression of key effectors of the Akt/mTORpathway. HCT-116 and A2780 cells were treated with TL-77in parallel with ON01910.Na. We first examined the effect ofTL-77 on expression of the major cell-cycle regulator cyclinD1. As shown in Fig. 9, after 8-hour treatment, expression ofcyclin D1 was significantly inhibited in ON01910.Na-treatedcells at 0.24 mM (4 � GI50) and TL-77–treated cells at 8 mM(2 � GI50) in HCT-116 cells, whereas only minor inhibition of

cyclinD1 expressionwas observed for both compounds at 4�GI50in A2780 cells.Akt, a family of serine/threonine kinases, is the primary

receptor of PI3K-initiated signaling and controls essentialcellular activities through phosphorylation of a number ofdownstream effectors (Chang et al., 2003; Shaw and Cantley,2006). Through PDKs, PI3K activates Akt, which in turnphosphorylates (thereby activating) mTOR protein at Ser2448.Western blot analysis detecting phosphorylated Akt Ser473revealed that TL-77 ($4 mM) significantly inhibited Aktphosphorylation in HCT-116 cells, suggesting that TL-77negatively regulates Akt in the same manner as ON01910.Na(Fig. 9). To determine whether TL-77 inhibits effectorsthat function upstream of Akt, we examined phosphor-ylation of PDK1, which is known to have a central functionin Akt activation (Osaki et al., 2004). Western blot analy-sis revealed that phosphorylation of PDK1 was down-regulated in a dose-dependent manner by both compoundsin HCT-116 cells. Complete inhibition of PDK1 phosphor-ylation was observed after 8-hour exposure of cells to 4 � GI50TL-77 (16 mM) and ON01910.Na (0.24 mM) (Fig. 9). However,we found the treatment with both compounds for 8 hoursminimally affected the phosphorylated Akt and PDK1 inA2780 cells.The impact of TL-77 on downstream effectors of Akt was

further investigated. One known Akt substrate is a 40-kDa,proline-rich protein (PRAS40) that binds mTOR to transduceAkt signals to the mTOR complex. Phosphorylation of PRAS40byAkt at Thr246 helps to reduce PRAS40 inhibition ofmTORC1(Sancak et al., 2007). Although we found that ON01910.Nareduced phosphor-PRAS40 at 0.24 mM, which is first reportedhere, only minor inhibition of PRAS40 phosphorylation was

Fig. 7. Induction of spindle abnormalities by TL-77 andON01910.Na in cancer cells. HCT-116 and A2780 cells aretreated with DMSO, TL-77, or ON01910.Na for 12 hours,fixed with 4% paraformaldehyde, and treated with anti–a-tubulin antibody (green) and Hoechst (blue). The cellswere analyzed by confocal laser microscopy.

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observed in TL-77–treated HCT-116 cells. This observation wasconfirmed in A2780 cells. Subsequently, we found thatON01910.Na treatment significantly reduced phosphorylationof mTOR in both cell lines. Although treatment with TL-77 for8 hours did not induce changes in the phosphorylation status ofmTOR in HCT-116, a dramatic inhibition was shown at 8 mM inA2780 cells. In addition, we observed that neither compoundaffected expression of PTEN in HCT-116 cells, the 39 phospha-tase major tumor suppressor protein and inhibitory regulator ofPI3K/Akt signaling.TL-77 Induces Cancer Cell Apoptosis. Induction of

apoptosis was initially analyzed by annexin V/PI doublestaining in both HCT-116 and A2780 cancer cells followingtreatment with TL-77 for 24 and 48 hours (Fig. 10, A and B).Both TL-77 and ON01910.Na induced apoptosis in dose- andtime-dependent manners in both cell lines. TL-77 inducedHCT-116 apoptosis at GI50 and 2 � GI50 values (4 and 8 mM,

respectively) $24-hour exposure with maximal effect after48-hour treatment, whereas ON01910.Na triggered apoptosismore slowly than TL-77 (minor effect at 24 hours, clear effect at48 hours). A2780 cells appeared more sensitive to ON01910.Naand TL-77; both TL-77 and ON01910.Na at GI50 effectivelyinduced apoptosis with .30% (24 hours) and .60% (48 hours)of cells going through early apoptosis. These time-dependentobservations are consistent with cell-cycle effects.Molecular mechanisms of TL-77–mediated apoptosis were

further explored in both cell lines. Western blot assays inHCT-116 cells (Fig. 11A) revealed that TL-77 (24-hourtreatment) induced dose-dependent PARP cleavage $4 mM.However, consistent with insensitivity to apoptosis observedin the annexin V/PI dual staining assay, PARP cleavage wasnot observed in cells exposed toON01910.Na,0.24mM(4�GI50).Consistent with annexin V–positive A2780 populations de-tected by flow cytometry (Fig. 10B), ON01910.Na-treated

Fig. 8. Effect of TL-77 and ON01910.Na on cell-cycle regulators. HCT-116 and A2780 cells were incubated with indicated concentrations of TL-77 andON01910.Na for 8 or 24 hours. The cells were lysed and subjected to Western blot analysis using indicated antibodies. b-Actin was used as an internalcontrol for protein loading. The Western blot results were quantified and statistical analysis was performed (*P , 0.05). All of the aforementionedexperiments were repeated three times, and a representative result is shown. P-Cdc25C, phosphor-Cdc25C.

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cells (0.25–1.0mM) showed significant PARP cleavage, whereastreatment with TL-77 indicated only minimal cleavage ofPARP ,8 mM (4 � GI50) (Fig. 11A).PARP cleavage is the result of activation of caspase 3;

therefore, a caspase 3/7 activation assay was performed inHCT-116 cells and HMEC-1 untransformed cells after 24-hourtreatment with either TL-77 or ON01910.Na (Fig. 10C). Thesedata demonstrate that concentrations$2 mM (0.5�GI50) TL-77dose dependently enhanced caspase 3/7 activity compared withDMSO control, confirming an active apoptotic process duringthis period, but no such activity was detected in HMEC-1 cells#20 mM (4 � GI50). In contrast, ON01910.Na significantlyactivated caspase 3/7 at .0.06 mM (GI50) in normal cell linesas well as in normal cells at 0.12 mM (2 � GI50).Caspases and Bcl family proteins comprise the executors of

apoptosis. As apoptosis related to Bcl family members isaffected by the balance between proapoptotic (Bax, Bid) andantiapoptotic (Bcl-xl, Mcl-1) molecules (Adams and Cory,

2007), we tested expression levels of Bcl-2 family members inHCT-116 cells after 24-hour treatment. As shown in Fig. 11B,expression levels of Bid were significantly reduced in TL-77–treated cells compared with controls, whereas ON01910.Natreatment resulted in only a minor effect. Total protein levelsof Bax in HCT-116 cells following treatment with thesecompounds were then examined; we observed that theexpression of Bax was essentially unchanged. Expression ofBcl-xl and Mcl-1, however, was significantly reduced inTL-77–treated cells compared with controls. Treatmentwith TL-77 at 16 mM for 24 hours resulted in the completeinhibition of Bcl-xl protein expression.TL-77 Induces Histone H2AX Phosphorylation. Cell-

cycle checkpoints protect normal cells from tumorigenesis bydelaying progression of the cell cycle until DNA is repaired.H2AX is a variant of histone H2A required to maintaingenomic stability (Celeste et al., 2003). Phosphorylation ofSer139 of the histone human variant H2AX, commonly

Fig. 9. Effect of TL-77 and ON01910.Na on the Akt/mTOR signal transduction pathway. HCT-116 and A2780 cells were incubated with indicatedconcentrations of TL-77 and ON01910.Na for 8 hours. The cells were lysed and subjected toWestern blot analysis using anti–p-AKT antibody, anti–p-mTORantibody, and indicated antibodies. Equal loading was confirmed in each lane by stripping and reprobing the blots with either anti-AKT antibody oranti-mTOR antibody. b-Actin was used as an internal control for protein loading. The Western blot results were quantified and statistical analysiswas performed (*P, 0.05). All of the aforementioned experiments were repeated three times, and a representative result is shown. p-AKT, phosphor-AKT;p-PDK1, phosphor-PDK1; p-PRAS40, phosphor-PRAS40; p-mTOR, phosphor-mTOR.

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referred to as g-H2AX, is an early cellular response to theinduction of DNA double-strand breaks. Detection of thisphosphorylation event has emerged as a highly specific andsensitive biomarker for monitoring DNA damage (Mah et al.,2010). Cellular expression of g-H2AX was detected followingtreatment of HCT-116 cells with TL-77 or ON01910.Na for 8and 24 hours, indicative of DNA damage. Western blotanalysis of HCT-116 cells after treatment with TL-77 andON01910.Na for 8 hours showed significant dose-dependentelevation of g-H2AX (Fig. 11C). Following treatment of cellswith either compound for 24 hours, intense bands revealedhigh levels of g-H2AX. These data reveal that early andprolonged phosphorylation of histone H2AX has occurredafter treatment with TL-77 or ON01910.Na.

DiscussionThe majority of anticancer chemotherapy drugs are de-

livered by intravenous or oral routes which impact directly onbioavailability. Improvements in bioavailability have beenfound to enhance efficacy and tolerability and decreaseadverse effects (Dmochowski and Staskin, 2002). By modifyingthe structure of ON01910.Na, we designed and synthesizedTL-77, a novel (E)-styrylsulfonyl methylpyridine. Pharma-cokinetic studies in mice revealed that oral bioavailability ofTL-77 was improved to 56%, compared with 9% for ON01910.Na (Lu et al., 2014). Based on these findings, we hypothesizethat the structural modification may influence positivelyon efficacy and tolerability. Therefore, in this study, we have

Fig. 10. Induction of apoptosis by TL-77and ON01910.Na. HCT-116 (A) and A2780(B) cells were exposed to different con-centrations of TL-77 or ON01910.Na for24 or 48 hours and stained using FITC-conjugated annexin V and propidium iodide,and subjected to flow cytometric analysis asdescribed in the experimental procedures.A1, annexin-negative and PI-positive deadcells; A2, double-positive late apoptotic cells;A3, double negative live cells; A4, annexinV–positive, PI-negative early apoptotic cells.All of the aforementioned experiments wererepeated three times, and a representativeresult is shown. Induction of caspase 3/7activity in cancer cell line HCT-116 andHMEC-1 after treatment with indicatedconcentrations of TL-77and ON01910.Nafor 24 hours. Caspase 3/7 activity was mea-sured according to the assay conditionsdescribed in the experimental procedures.Vertical bars represent the mean 6 S.D.of three replicates. *Values significantly(P , 0.05) different from DMSO vehiclecontrol (two-tailed t test). FL, focusing lens;OD, optical density.

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investigated in detail the cellular mechanisms of action ofTL-77 in comparison with ON01910.Na. TL-77 displays potentgrowth inhibitory activity in vitro (GI50 ,1 mM against colon,breast, ovarian, cervical, and pancreatic carcinoma as wellas melanoma cell lines). Importantly, TL-77 demonstratessignificant selectivity in tumor cell lines over nontransformedHMEC-1, HUVEC, and MRC-5 cells.Cell-cycle and mitotic index analyses reveal that TL-77

evokes rapid mitotic arrest (#6 hours) followed by delayed G2block (12 hours, 8 mM) in HCT-116 cells. In contrast, cellsexposed to ON01910.Na accumulate in the G2 cell-cyclephase, failing to traverse mitosis. TL-77, however, mediatesa rapid mitotic inhibitory effect as an initial event.Mitotic spindle assembly is critical for segregation of chromo-

somes to two daughter cells during mitosis. Several mitosis-specific protein kinases are required to organize the bipolarspindle assembly and chromosome biorientation, including

Cdc2, aurora B, and Plk1 (Peters et al., 2006). Plk1 has beenidentified as a key player for G2/M transition and mitoticprogression in both normal and tumor cells (Schmidt et al.,2006). In late G2 phase and mitosis, Plk1 is essential forcentrosome maturation, separation, and mitotic spindle as-sembly (Eckerdt et al., 2005). Downregulation of Plk1 activ-ity in tumor cells results in mitotic arrest characterized byspindle abnormalities and apoptosis (Gumireddy et al., 2005).Mitotic entry is triggered by a steep increase in cyclin B/Cdc2activity (Lindqvist et al., 2009). Plk1 promotes the activation ofcyclin B/Cdc2 by activating the phosphatase Cdc25C on Ser198(Roshak et al., 2000). Cdc25C has been suggested to serve asa reliable marker to monitor Plk1 activity in a cellular context(Schmidt et al., 2006).ON01910.Na has been reported to inhibit Plk1 and de-

crease expression of Cdc25C (Gumireddy et al., 2005; Jimenoet al., 2009). However, inconsistent effects of ON01910.Na on

Fig. 11. TL-77 inhibits Bcl-2 family of proteins and induces DNA damage. (A) Western blot analyses in HCT-116 and A2780 cells. Cells were treatedwith the indicated concentrations of TL-77 or ON01910.Na for 24 hours. Cell lysates were subjected toWestern blot analyses using anti-PARP antibodiesto assess the cleavage of PARP. (B) Effects on antiapoptotic proteins by Western blot analysis in HCT-116 cells. Cells were treated with indicatedconcentrations of TL-77 or ON01910.Na for 24 hours. Cell lysates were prepared and subjected to Western blot analyses using anti-BID, anti-Bax, anti-Bcl-xl, and Mcl-1 antibodies. b-Actin was used as an internal control for protein loading. (C) Induction of g-H2AX by TL-77 and ON01910.Na. Westernblot analysis was performed in HCT-116 cells. Cells were treated with indicated concentrations of TL-77 or ON01910.Na for 8 or 24 hours. b-Actin wasused as an internal control for protein loading. The Western blot results were quantified and statistical analysis was performed (*P , 0.05). All of theaforementioned experiments were repeated three times, and a representative result is shown.

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cyclin B1 expression have been described (Jimeno et al., 2009;Prasad et al., 2009; Fantl et al., 2009). From our data, effectiveinhibition of phosor-Cdc25C but minor effects on cyclin B1were observed after 8-hour treatment of both compounds inboth cell lines. This indicates TL-77 suppressed Cdc25Cactivity, and the cyclin B1/Cdc2 complex still remained active.In addition, TL-77 evoked a notable mitotic block from mi-totic index analysis (6- to 12-hour exposure). We thereforeextrapolate that cells eventually entered mitosis during earlystages of treatment (#12 hours). It is more likely that cellsbecome blocked at the mitosis checkpoint with the outcome ofmultipolar spindles and misaligned chromosomes. This led tofurther conjecture that the target of TL-77 might be located atthe kinase-mediating mitotic checkpoint, such as Plk1 (Barret al., 2004), which still requires further clarification. After24-hour treatment, suppressed expression of cyclin B1 wasobserved in ON01910.Na-treated cells (0.24 mM) as well asTL-77–treated cells (16 mM). Moreover, mitotic index analy-ses have shown increasing G2 arrest in cells after treatmentwith both ON01910.Na and TL-77 (2 � GI50 only .12 hours).These data confirm the hypothesis that by inhibiting Cdc25Cactivity, both compounds maintain cyclin B/Cdc2 in an inactivestate after longer exposure to compounds (12–24 hours). Con-sequently, cells fail to traverse mitosis and become blocked inthe G2/M phase of the cell cycle. These observations indicatea multifaceted mechanism culminating in mitotic inhibition incancer cells for both compounds.Immunofluorescence staining of a-tubulin revealed that

TL-77 promoted disassociation of microtubules and inhibitedmitotic spindle assembly, leading to formation of multipolarspindles and misalignment of chromosomes in vitro. This out-come is similar to the phenotype observed in Plk1-depleted cells(Sumara et al., 2004), suggesting TL-77 suppresses activity ofPlk1 in cancer cells. In cell-free conditions, both ON01910.Na(0.06 and 0.12 mM) and TL-77 (4 and 6 mM) inhibitedmicrotubule formation, acting in a similarmanner to nocodazole.During experimental preparation, slight precipitation of TL-77at 8 mM (2� GI50) in H2O solution was observed. Consequently,a concentration of 6 mM (1.5 � GI50) was adopted. Minorinhibition or no effects of ON01910.Na on tubulin polymerizationat 1 and 5 mM, respectively, has previously been reported(Gumireddy et al., 2005; Oussenko et al., 2011). These apparentinconsistencies could be due to experimental differences, such astesting buffer and compound concentrations. But the reproduc-ibility between data sets in this study leads us to conclude thatinhibition of tubulin polymerization is an important molecularmechanism of action of these agents.Although the antimitotic activity of ON01910.Na was

deemed a consequence of Plk1 inhibition, the initial kinase in-hibitory effects of ON01910.Na on Plk1 could not be reproducedby other scientists (Schmidt and Bastians, 2007; Steegmaieret al., 2007) nor by ourselves (Supplemental Table 2). In vitrokinase assays indicated that ON01910.Na failed to signifi-cantly inhibit Plk1 activity ,30 mM (Steegmaier et al., 2007).Furthermore, several reports have confirmed that ON01910.Na inhibits tubulin polymerization; cells with depolymerizedmicrotubules were observed after treatment with higher con-centrations (2.5mM) of ON01910.Na (Peters et al., 2006; Schmidtand Bastians, 2007; Steegmaier et al., 2007). Based on ourobservations, TL-77 as well as ON01910.Na inhibit tubulinpolymerization, inducingmicrotubule abnormalities, andmaysuppress the activity of Plk1.

The PI3K/Akt pathway is a well characterized cell survivalsignaling pathway that blocks apoptosis and promotes sur-vival and growth in cancer cells. From our results, TL-77, aswell as ON01910.Na, downregulates cyclin D1 and inhibitsphosphorylation of Akt and PDK1. Whereas ON01910.Nadownregulates phosphor-mTOR in both cell lines, suppres-sion of phosphor-mTOR is cell line–specific following treat-ment with TL-77 (A2780 only). Also, only ON01910.Nasuppresses PRAS40 significantly in both cell lines. Takentogether, these results indicate that TL-77 inhibits Akt/mTORsignal transduction; however, compared with ON01910.Na,arrest of Akt/mTOR signaling by TL-77 may be less significantand cell line–specific.Dose- and time-dependent apoptosis induced by both com-

pounds, detected by annexin V binding, is closely associatedwith induction of caspase 3/7 activity and PARP cleavage.TL-77 induced rapid apoptosis in HCT-116 cells, whereasON01910.Na only triggered apoptosis after a longer exposureperiod, an observation consistent with cell-cycle effects, in-dicating that cell-cycle arrest may be the apoptotic trigger.Analysis of Bcl-2 family proteins after treatment of cells withTL-77 andON01910.Na revealed that neither compound alteredexpression levels of the proapoptotic molecule Bax. However,expression of Bid was significantly reduced in cells followingTL-77 treatment. This may indicate that the precursor formof Bid translocates to mitochondria, where it induces cyto-chrome c release (Luo et al., 1998), suggesting the involvementof the mitochondrial pathway in TL-77–mediated apoptosis.Among the prosurvival molecules, expression of Bcl-xl andMcl-1 was significantly inhibited by both compounds. Takentogether, these data show that TL-77 and ON01910.Na selec-tively induce G2/M cell-cycle block with ensuing induction ofapoptosis in tumor cells.Preliminary cellular and mechanistic investigations dem-

onstrate that TL-77 evokes excellent antiproliferative activityagainst a wide range of human tumor cell lines. TL-77 causesprofound G2/M cell-cycle arrest with inhibition of Cdc25Cphosphorylation, and induces multipolar spindles and chro-mosome misalignment in cancer cells, which leads to caspase-dependent apoptosis, effects comparable to those elicited byON01910.Na. Compared with ON01910.Na, TL-77 elicitslower toxicity in nontransformed cells, possesses superiororal bioavailability, and mediates rapid mitotic inhibitoryeffects; moreover, TL-77 inhibits the Akt/mTOR survivalsignal transduction pathway as a secondary effect. Theseobservations indicate a multifaceted mechanism culminatingin mitotic inhibition in cancer cells. Therefore, further eval-uation of this promising antitumor agent is justified.

Acknowledgements

The authors thank Dr. Hilary Collins from the Centre for Bio-molecular Sciences, University of Nottingham, UK, who provided theHoechst dye and training for immunofluorescent staining.

Authorship Contributions

Participated in research design: Bradshaw, Lu, Laughton, Wang.Conducted experiments: Lu.Performed data analysis: Lu, Bradshaw, Laughton, Wang.Wrote or contributed to the writing of the manuscript: Lu,

Bradshaw, Laughton, Wang.

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Address correspondence to: Tracey D. Bradshaw, Centre for BiomolecularSciences, University of Nottingham, University Park, Nottingham, NG7 2RD,United Kingdom. E-mail: tracey.bradshaw@nottingham.ac.uk

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