Original Article Synergistic Effect of Allyl Isothiocyanate (AITC) on Cisplatin Efficacy in Vitro and in Vivo

Am J Cancer Res 2015;5(8):2516-2530 www.ajcr.us /ISSN:2156-6976/ajcr0010340

Original Article Synergistic effect of allyl isothiocyanate (AITC) on cisplatin efficacy in vitro and in vivo

Xiang Ling1,4,6, David Westover1, Felicia Cao1*, Shousong Cao2, Xiang He1*, Hak-Ryul Kim1, Yuesheng Zhang3, Daniel CF Chan5, Fengzhi Li1

Departments of 1Pharmacology & Therapeutics, 2Medicine, 3Chemoprevention, Roswell Park Cancer Institute, Buffalo, New York 14263, USA; 4Canget BioTekpharma LLC, Buffalo, New York 14203, USA; 5Division of Medical Oncology, University of Colorado Denver, Denver, Colorado 80045 USA; 6Department of Pharmacology, School of Pharmacy, Nantong University, Nantong, Jiangsu Province, China. *FC was a Roswell Summer student then and XH was a PhD student from Sichuan University trained in the FL lab then. Received May 18, 2015; Accepted June 30, 2015; Epub July 15, 2015; Published August 1, 2015

Abstract: Although in vitro studies have shown that isothiocyanates (ITCs) can synergistically sensitize cancer cells to cisplatin treatment, the underlying mechanisms have not been well defined, and there are no in vivo demonstra- tions of this synergy. Here, we report the in vitro and in vivo data for the combination of allyl isothiocyanate (AITC), one of the most common naturally occurring ITCs, with cisplatin. Our study revealed that cisplatin and AITC combination synergistically inhibits cancer cell growth and colony formation, and enhances apoptosis in association with the downregulation of antiapoptotic proteins Bcl-2 and survivin. Importantly, the in vivo combination treatment suppresses human tumor growth in animal models without observable increases in toxicity (body weight loss) in comparison with single agent treatment. Furthermore, our data revealed that addition of AITC to cisplatin treatment changes the profile of G2/M arrest (e.g. increase in M phase cell number) and significantly extends the duration of G2/M arrest in comparison with cisplatin treatment alone. To explore the underlying mechanism, we found that AITC treatment rapidly depletes b-tubulin. Combination of AITC and cisplatin inhibits the expression of G2/M checkpoint- relevant proteins including CDC2, cyclin B1 and CDC25. Together, our findings reveal a novel mechanism for AITC enhancing cisplatin efficacy and provides the first in vivo evidence to support ITCs as potential candidates for devel- oping new regimens to overcome platinum resistance.

Keywords: Cisplatin, AITC, combination index (CI), survivin, Bcl-2, microtubule, cancer cells, human tumor mouse model

Introduction Previous studies showed that cisplatin and a new synthetic isothiocyanate (ITC) derivate, Cisplatin (cis-diamminedichloroplatinum II, ethyl 4-isothiocyanatobutanoate (E-4IB), can CDDP) is one of the most widely used antican- synergistically inhibit cell growth in both ovarian cer drugs [1]; however, its clinical efficacy is cancer cell line A2780 and its cisplatin-resis- often limited by primary or secondary acquired tant variant A2780/CP in vitro [12]. Further resistance. Several mechanisms are involved studies found that this synergistic effect is in cisplatin resistance development, including related to increased intracellular platinum accumulation, glutathione level depletion and reduced drug uptake, increased cellular thiol/ mitochondrial membrane potential dissipation folate levels and increased DNA repair [2-4]. [13]. These events were also accompanied with Current studies show that activation of anti- changes in apoptosis and cell cycle related apoptotic pathways may also contribute to the pathways [13]. Later studies found that not only resistance phenotype [5-11]. A combination of E-4IB but also indol-3-ethyl isothiocyanate cisplatin with other therapeutic agents to (homoITC), benzyl isothiocyanate (BITC) and enhance tumor sensitivity and decrease phenethyl isothiocyanate (PEITC) could syner- unwanted systemic toxicity is an attractive area gistically enhance cell sensitivity to cisplatin of study. treatment in vitro, and confirmed that this syn- Synergistic interaction of AITC with cisplatin ergistic effect is independent of cisplatin sensi- realize a synergistic effect is reachable in vivo tivity [14-16]. Di Pasqua, et al. recently pub- and that AITC is a good candidate for in vivo lished on the sensitization of lung cancer cells combination study. to cisplatin by naturally occurring isothiocyanates, and showed that structural variations In the present study, we sought to determine among the isothiocyanates affected their abili- whether AITC could enhance cancer cell sensi- ty to sensitize cancer cells, and this correlated tivity to cisplatin treatment in vitro and in vivo with their ability to deplete b-tubulin [17]. and further explore its mechanisms. Although accumulated evidence suggests that Material and methods several natural or synthetic ITCs could sensitize cancer cells to cisplatin treatment, the detailed Cell culture and reagents underlying mechanisms remain largely unde- fined. Furthermore, to the best of our knowl- Human ovarian cancer cell line 2008 (a gift edge, there is no evaluation of the effect of ITCs from Dr. Kunle Odunsi, Roswell Park Cancer and cisplatin combination in tumor growth in Institute), human lung cancer cell lines HOP62 vivo. (a gift from Dr. Daniel Chang, University of Colorado) and A549 (ATCC) were maintained in Allyl isothiocyanate (AITC) is one of the most RPMI 1640 medium supplied with 10% fetal common naturally occurring ITCs and is found bovine serum (FBS, Atlanta Biologicals, in many cruciferous vegetables [18]. It has Lawrenceville, GA), penicillin (100 units/ml) been reported that AITC inhibits various types and streptomycin (0.1 µg/ml) (Invitrogen, Grand of cancer cell growth through multiple mecha- Island, NY). Cells were routinely subcultured nisms such as apoptosis induction and cell twice a week and maintained in a humidified cycle arrest [19-22], and has very good oral bio- incubator with 5% CO at 37°C. Cisplatin was availability [23, 24]. Further studies found that 2 purchased from VWR (West Chester, PA). AITC, short-term treatment with AITC inhibits cancer IGEPAL CA-630, phenylmethyl sulfonyl fluoride, cell growth with IC50 values in the low µM monoclonal anti-β-tubulin antibody, polyclonal range, even in drug resistant cancer cells [19, anti-actin antibody and goat peroxidase-conju- 25, 26]. Interestingly, AITC was shown to be gated anti-rabbit IgG antibody were purchased more toxic to cancer cells than to either normal from Sigma (St. Louis, MO). Survivin (FL-142), human epithelial cells or de-transformed cyclin B1 and polyclonal p-histone H3 (Ser10) human cancer cells [21, 27, 28]. The IC50 value antibodies were purchased from Santa Cruz of AITC in normal human bladder epithelial cells (Santa Cruz, CA). Monoclonal Cdc2 and Cdc25C is approximately 10 times higher than that in antibodies were purchased from Epitomics human bladder cancer cells [28]. Studies have (Burlingame, CA). Alexa Fluor® 594 goat anti- also demonstrated that 10 micromoles AITC rabbit IgG and Alexa Fluor® 488 goat anti- given through intraperitoneal injection (three mouse IgG were purchased from Invitrogen times per week for three weeks) could signifi- (Carlsbad, CA). Antibodies against Bcl-2 and cantly inhibit PC-3 human prostate cancer activated caspase 3 (SA320) were purchased xenografts in athymic mice with no apparent from Cell Signaling (Beverly, MA). MTT toxicity [29], suggesting an effective concentra- (3-[4,5-dimethylthiazol-2-yl]-2,5,-diphenyltetra- tion of AITC for tumor inhibition is achievable in zolium bromide) and leupeptin were purchased vivo. An earlier study also showed that the peak from USB (Cleveland, OH). Cell Death Detection plasma concentrations of AITC in the blood of ELISA assay kit was purchased from Roche both mice and rats, following a single oral dose (Indianapolis, IN). of [14C] AITC at 25 and 250 μmol/kg, were approximately 40 µM and 500 µM, respectively MTT cell viability assay [23, 24], well above the range used to achieve synergistic response with cisplatin in our study. The inhibitory effect of cisplatin (CDDP) and Additional studies revealed that ITCs could be AITC, alone and in combination, on cell growth concentrated in cancer cells through GSH con- was determined by MTT cell viability assay. jugation [30, 31], suggesting that intracellular Viable cells (2500 cells per well) were plated in concentration of AITC can be much higher than each well in 96-well plates. After an overnight extracellular or plasma concentration. Taken incubation, cells were treated with and without together, these prior studies suggest that the cisplatin and/or AITC at various concentrations minimum concentration of AITC required to fully and incubated for 96 hours. MTT, a colorimetric

2517 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin substrate, was added to a final concentration bination that also inhibits x% (i.e. isoeffective). of 0.4 mg/ml to each well. Cells in 96-well The (Dx)1 and (Dx)2 can be readily calculated plates were further incubated in a 5% CO2 incu- from the median-effect equation of Chou [34]: 1/m bator at 37°C for 4 hours, and then the medium Dx=Dm[fa/(1-fa)] where Dx is the median-effect was aspirated. The MTT metabolic product dose, fa is the fraction affected, Dm is the medi- formazan was solubilized by adding 200 ul of an-effect dose signifying potency and m is the DMSO to each well. Absorbance in the relevant kinetic order signifying the shape of dose-effect wells was measured at 570 nm using an Ultra curve. A 3-D plot of CI versus concentrations of Microplate Reader (Bio-Tek Instruments). The both Drug #1 and Drug #2 was obtained for experimental results were expressed as the each treated cell line as descried before [35]. mean ± standard deviation (SD). The statistical significance of differences was determined by Propidium iodide (PI) staining and flow cyto- Student’s t-test between two groups. metric analysis

Cell colony formation assay Cancer cells were treated with or without AITC and cisplatin (CDDP) alone or in combination To determine the long-term effect of AITC and for 12, 16, 24, 48 and 72 hours, then harvest- cisplatin (CDDP) combination, colony formation ed by trypsinization and washed with PBS. Cells assays were carried out. 2008 cells and HOP62 (~1 × 106) were resuspended in 5 ml 70% etha- cells were trypsinized and seeded into 6-well nol. After the initial fixation, cells were suspend- plates (200 cells per well). After being attached ed in 0.5 ml PBS containing 25 μg/ml PI, 0.2% to the plate, the cells were treated with and Triton X-100 and 40 μg/ml RNase A, incubated without cisplatin and AITC, alone or in combina- for at least 30 minutes at 4°C with PI and then tion for 96 hours. Culture medium containing analyzed by flow cytometry as previously drugs in each well was replaced with fresh described [36]. Data from flow cytometry were medium 96 hours after treatment. Cells were analyzed using WinList software (Verity allowed to grow for 3 weeks. Colonies were Software House Inc., Topsham, ME) and pre- then fixed with ice-cold methanol for 10 min sented as a percentage of total cells in G2/M and stained with 0.5% crystal violet solution in phase. Triplicate assays were performed. The 25% methanol for 10 min. The stained 6-well experimental results were expressed as the plates were carefully rinsed with ddH2O several mean ± standard deviation (SD). The statistical times and photographed. The colony numbers significance of differences was determined by were counted on a GS-800 Calibrated Student’s t-test between two groups. Densitometer using the Quantity One software (Bio-Rad, Hercules, CA) and reported as the Cell death detection ELISA assay average of triplicate wells. Apoptosis was measured by quantification of Determination of combination index (CI) for the histone-complex DNA fragments (mono- AITC and cisplatin (CDDP) treatment combina- and oligonucleosomes) using the Cell Death tion Detection ELISA assay kit (Roche, Mannheim, Germany) according to the manufacturer’s Effects of cisplatin and AITC combination on instructions. 2008 cells were seeded in tripli- growth inhibition were analyzed by the cates in 96-well plates and treated with and Combination Index (CI) equation developed by without AITC and cisplatin (alone or in combina- Chou-Talalay [32, 33] using the CalcuSyn pro- tion) for 48 hours. The levels of mono- and oli- gram (Biosoft, Cambridge, UK). The general gonucleosomes released into the cytoplasm equation for the classic isobologram is given were measured at 405 nm, using an ABTS solu- by: CI=(D)1/(Dx)1 + (D)2/(Dx)2 where CI<1 indi- tion as a blank control. The Enrichment factor cates synergism; CI=1 indicates additive effect, was calculated using the following formula: and CI>1 indicates antagonism; (Dx)1 and (Dx)2 Enrichment factor=A405 drug treated samples in the denominators are the doses (or concen- (apoptotic cells)/A405 no treatment. The exper- trations) of D1 (drug #1, for example, cisplatin) imental results were expressed as the mean ± and D2 (drug #2, for example, AITC) alone that standard deviation (SD). The statistical signifi- gives x% inhibition, whereas (D)1 and (D)2 in the cance of differences was determined by numerators are the doses of D1 and D2 in com- Student’s t-test between.

2518 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin

Figure 1. Combination of cisplatin (CDDP) with AITC inhibits cancer cell growth. A and B. 2008 ovarian cancer cells and Hop62 lung cancer cells were treated with different concentrations of AITC and cisplatin alone or in combination. After 96-hour treatment, MTT assay was used to determine cell growth status. Each bar is the mean ± SD from at least 3 independent parallel experiments. C and D. Colony formation assays. Two hundred 2008 cells or Hop62 cells were seeded per well in six-well plates and treated with indicated doses of cisplatin and/or AITC for 96 hours and then cultured in medium without drugs. Photographs were taken three weeks after treatment. E and F. Colonies were stained and counted three weeks after treatment. Each bar is the mean ± SD from 3 independent triplicates. *Significant difference compared to single drug treatment alone (P<0.01); otherP values are marked on the relevant images. Cisplatin for 2.5 µg/ml, 5 µg/ml and 10 µg/ml is equivalent to cisplatin for 8.3 µM, 16.7 µM and 33.3 µM.

Triple staining of DNA, p-histone H3 and PBS, cells were incubated in PBS containing β-tubulin, and fluorescence microscopy obser- Alexa Fluor® 594 goat anti-rabbit IgG (1:2000) vation and Alexa Fluor® 488 goat anti-mouse IgG antibody (1:2000) for 45 min at room tempera- 2008 cells and A549 cancer cells were seeded ture, followed by staining with 4,6-diamidino- on round glass cover slips which were coated 2-phenylindole (DAPI) at a final concentration of with 2% gelatin in 12-well plates. Sixteen hours 0.5 µg/ml in PBS for 15 min. Cover slips were after AITC and/or cisplatin treatment, cells mounted onto glass slides with Gel/MountTM were fixed with 4% paraformaldehyde in PBS for solution (Biomedia, Foster City, CA). Cell mor- triple staining of DNA, β-tubulin and p-histone phology was visualized and digitally captured H3 (Ser10), a mitotic marker. After one hour fixa- using a Zeiss Axiovert 100 M Fluorescence tion, cells were blocked/permeabilized with Microscopy System. PBS containing 2% BSA and 0.2% Triton X-100 for 30 minutes, and then incubated in PBS con- Double staining of p-histone H3 and DNA taining 1% BSA, an anti-β-tubulin antibody (1:2000) and an anti-p-histone H3 antibody Sixteen hours after drug treatment (see above), (1:2000) for 60 min at 37°C. After washing with cells were double-stained for DNA and p-his-

2519 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin

2520 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin

Figure 2. Combinational regimen of AITC and cisplatin (CDDP) changes the time-dependent G2/M arrest profile compared to single drug treatment. A. 2008 cells were treated with or without AITC and/or cisplatin for the duration as shown. Cells (~1 × 106) were fixed and prepared for analysis with flow cytometry (20,000 events per sample) as noted in Materials and Methods. Raw data were then analyzed using WinList software. B and D. 2008 cells or A549 cells were seeded on round glass cover slips coated with 2% gelatin in 12-well plates and treated with or without AITC and cisplatin for 24 hours. Cells were then incubated first with a p-histone H3 antibody and then with a fluorescent secondary antibody (pink), and finally stained with DAPI (blue) and visualized under fluorescent microscope. C. Expression of p-H3 in 2008 cells was determined by Western blotting. Actin is presented to verify protein loading. E. Cell cycle distribution in A549 cells was determined by flow cytometry at 16 hours. Cisplatin for 5 µg/ml and 10 µg/ ml is equivalent to cisplatin for 16.7 µM and 33.3 µM. tone H3. For double staining of DNA and p-his- Human tumor xenograft mouse model and tone H3, cells were fixed/blocked as described treatment above, and then incubated in PBS containing 1% BSA and an anti-p-histone H3 antibody All experiments were performed following the (1:2000) for 60 min at room temperature. After IACUC-approved mouse protocol. washing with PBS, cells were incubated in PBS Animal work followed our previously published containing Alexa Fluor® 594 goat anti-rabbit protocols [37]. Briefly, A549 lung cancer cells IgG (1:2000) for 45 min at room temperature, were first used to establish tumor mass. followed by staining with DAPI at a final concen- Specifically, tumor cells grown in culture medi- tration of 0.5 μg/ml in PBS for 10 min. um were harvested by trypsinization, washed Coverslips were mounted on glass slides with twice in ice-cold PBS, and adjusted to 2 × 107 Gel/Mount™ solution (Biomedia, Foster City, viable cells/ml. A 0.1-0.2 ml cell suspension CA). Cell morphology was visualized and digi- was injected subcutaneously into the right flank tally captured as described above. of female athymic nude mice to establish suffi- Western blot analysis cient tumor size in about 3 weeks. After tumor tissues were isolated from established tumors, Cells treated with and without cisplatin (CDDP) a piece of non-necrotic tumor tissues (30-40 and/or AITC were lysed in RIPA buffer contain- mg) were subcutaneously transplanted into the ing 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% flank area of 20 female athymic nude mice. sodium deoxycholate, 0.1% SDS, and 50 mM Mice were then randomly divided into 4 groups Tris, pH 8.0. Fifty µg total protein from each (5 mice per group): a control group, an AITC sample were heated at 95°C for 5 minutes group, a cisplatin group and a combination after mixing with equal volume of 2X SDS load- group. Based on the previous studies of AITC ing buffer. Samples were separated on 12-15% [29] and in consideration of the clinical rele- SDS-polyacrylamide gel electrophoresis (SDS- vance, we chose per oral (p.o.) instead of using PAGE) gels and electrotransferred to Pure the intraperitoneal (i.p.) route for AITC adminis- Nitrocellulose Membranes (Bio-Rad, Hercules, tration at a dose of 50 mg/kg formulated in CA). The membrane was then blocked in 5% vegetable oil, once every other day for two skim milk in TBS-T buffer (20 mM Tris/HCl pH weeks starting from the day on which 30-40 7.5, 0.137 M NaCl, and 0.1% Tween 20) at room mg tumor masses were subcutaneously temperature for 2-3 hours. Next, the membrane implanted at the flank area on individual mice. was incubated with different primary antibod- Seven days after tumor implantation (defined as day 0) on which xenograft tumors reached ies in TBS-T containing 5% BSA overnight at 200-250 mm3, a one-time sub-maximal toler- 4°C in the range of dilutions from 1:500 to ated dose (MTD, 6 mg/kg) of cisplatin in a clini- 1:2000. After washing with TBS-T, the mem- cally used formulation was injected via the brane was incubated in TBS-T buffer containing mouse tail vein. Tumor length (L) and width (W) 5% skim milk and corresponding secondary were measured using a vernier caliper every antibody (1:5000) for 45-60 minutes at room other day on Days 0, 2, 4, 6, 8, 10, 12, 14, 16, temperature with shaking. Protein of interest 18, 20, 22, 24, 26 and 28. The tumor volume was detected using Western Lightning Plus-ECL (v) was calculated using the formula: v=0.5 (L × (Perkin Elmer, Waltham, MA) and visualized by W2). The mean tumor volume at each time point various times (30-120 seconds) of exposure. was derived from 5 mice in each group. Actin was detected as the internal control to normalize total protein loading for each Antitumor efficacy was assessed by 1) maxi- sample. mum tumor growth inhibition (MTGI), which is

2521 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin

Figure 3. G2/M arrest resulted from AITC and cisplatin (CDDP) combination treatment is associated with G2/M profile changes and microtubule depletion. (A and E) 2008 ovarian cancer cells and A549 lung cancer cells were treated with AITC for 0, 8, 16, 24, 48, and 72 hours and then lysed. Immunoblotting was carried out with antibodies for β-tubulin and p-Histone H3 in 2008 cells (A) and A549 (E). (B) 2008 cells were also treated with AITC at 1, 10, and 100 µM for 24 hours and then lysed. Immunoblotting was carried out with antibodies for β-tubulin and p-Histone H3 to determine the response to varying doses of AITC. (C and F) investigation of morphological changes related to AITC and cisplatin treatment. 2008 cells and A549 cells were treated with or without AITC and/or cisplatin for 18 hours. Cells were then incubated first with antibodies to p-histone H3 and β-tubulin and then with secondary fluorescent antibodies (pink=p-histone H3, green=β-tubulin) and finally stained with DAPI (blue) and visualized under a fluorescent microscope. (D) 2008 ovarian cancer cells were treated with different concentrations of AITC and cisplatin alone or in combination for 48 hours and then lysed. Immunoblotting was performed for G2/M transition pathway-relevant proteins β-tubulin, CDC2, CDC 25, and cyclin B1. Actin protein expression shown in (A-E) is the internal controls for verifying protein loading. the mean tumor weight difference for treated ment (Day 0); 3) partial tumor response (PR), group (MTWTG) versus mean tumor weight for which was expressed as when tumor weight untreated control group (MTWCG) at the same was reduced at least 50% initial tumor size on time [the calculated formula is MTGI=(MTWTG– Day 0; and 4) complete tumor response (CR), MTWCG)÷MTWCG × 100%]; 2) the tumor dou- which was defined as inability to detect tumor bling time (TDT), which was defined as the via palpitation at the initial tumor-transplanted mean time for the tumor reaching twice its ini- site. All images were made using Sigma Plot tial weight from the time beginning the treat- software. The differences among different

2522 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin

Figure 4. AITC enhances pro-apoptotic effects induced by cisplatin (CDDP) treatment. 2008 cells were treated with or without AITC and/or cisplatin for 48 hours and then subjected to the following analysis. A. Cells were subjected to flow cytometry to detect sub-G1 cells. B. Cells were lysed and analyzed by an ELI- SA as per manufacturer’s instructions (Cell Death Detection ELISA). Individual bars shown in A and B are the mean ± SD from 3-5 independent parallel assays. C. Cells were lysed and analyzed by immunoblotting for Bcl-2, survivin, and active caspase 3 levels. Cleaved/ activated caspase 3 is present at the 17 kD band. Actin is the internal control to verify protein-loading profiles. Cisplatin for 5 µg/ml and 10 µg/ml is equivalent to cisplatin for 16.7 µM and 33.3 µM. treatment groups were analyzed for signifi- Results cance using one-way analysis of variances (ANOVA) with Tukey-Kramer multiple compari- AITC significantly enhances cancer cell growth son tests. A P-value of <0.05 was considered to inhibition induced by cisplatin (CDDP) in MTT and colony formation assays. be statistically significant. To evaluate the effects of AITC and cisplatin Statistical analysis combination in cell growth in vitro, an MTT via- The experimental results were expressed as bility assay was carried out 72 hours after drug the mean ± standard deviation (SD). The statis- treatment. We found that combination of cispl- tical significance of differences was determined atin with AITC significantly inhibits cell growth in by Student’s t-test in two groups and one-way comparison to cisplatin or AITC treatment alone analysis of variance among multiple groups, in both 2008 ovarian cancer cells and HOP62 with a P-value of 0.05 or less considered lung cancer cells (P<0.05) (Figure 1A and 1B). significant. We next studied the ability of cancer cells to

2523 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin

ther explore if G2/M arrest is involved in combination- enhanced cell growth inhibition, we evaluated the changes in DNA distribution after drug treatment using flow cytometry. Our data showed that in AITC single drug treatment group, significant G2/M arrest starts at 8 hours and reaches the peak at 12-16 hours after treatment (P<0.05) (Figure 2A). The number of cells in G2/M phase begins to significantly decrease at 24 hours post- treatment. In the 10 µg/ml (33.3 µM) cisplatin group, the number of cells in G2/M begins increasing 16 hours after treatment, reaches its peak at 48 hours and begins to decrease 72 hours after cisplatin treatment. In the 5 µg/ml (16.7 µM) cisplatin group, increasing in G2/M phase cells begins at 16 Figure 5. The combination index (CI) profile of cisplatin (CDDP) and AITC treat- hours and decreases 48 ment on growth inhibition. A549 (A) and Hop62 (B) lung cancer cells were treated with cisplatin and AITC at the doses as shown for 72 hours and subjected hours after treatment. How- to MTT assays, and the results were analyzed by for CI using the CalcuSyn soft- ever, G2/M arrest in the ware (Biosoft). Each bar represents the corresponding CI from a specific AITC combination group appears and cisplatin combination treatment. Of note, CI>1 means antagonist effects at 8 hours and reaches its from the combination treatment; CI=1 means additive effects from the com- peak 12 hours after combi- bination treatment; and CI<1 means synergistic effects from the combination treatment; and CI≤0.5 means highly synergistic effects from the combination nation treatment, similar to treatment. the pattern seen in AITC treatment alone. In the late stage combination treat- form colonies on 6-well plates in presence or ment, G2/M arrest levels stay high, much like absence of cisplatin and AITC, alone and in the pattern observed in cisplatin treatment, combination. Consistent with the MTT assay indicating that the duration of G2/M arrest in results, combination of cisplatin with AITC AITC and cisplatin combination group has been resulted in much less colony growth than in extended in comparison with single drug treat- AITC and cisplatin treatment alone (Figure 1C ment alone, which is likely important for the and 1D). Quantitative analysis of colony forma- synergistic effect of combination treatment. tion data showed that inhibition of cancer cell colony formation by the combination of AITC To better understand the extended G2/M and cisplatin could be a synergistic effect arrest induced by combination of AITC and cis- (Figure 1E and 1F). platin (CDDP), we stained cells with DAPI and phospho-histone H3 (p-H3, a marker showing Combination of AITC and cisplatin (CDDP) cells in the M phase), and explored the possibil- modulates cancer cell G2/M transition and ity that AITC and cisplatin combinations G2/M arrest profiles increase cell numbers in the M phase using fluorescent microscope. We observed round, The G2/M checkpoint plays a key role in the p-H3-positive cells in the AITC treatment groups response to cisplatin treatment [38-42]. To fur- (Figure 2B). However, in the cisplatin treatment

2524 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin group, p-H3 positive cells which reflect the cancer cells, AITC alone could reduce β-tubulin mitotic (M) phase cells clearly decreased expression in a dose- and time-dependent pat- (Figure 2B). In the combination group, there is tern. The level of β-tubulin decreased after 8 no observable decrease in p-H3 positive cells hours of AITC treatment, and recovered at 48 compared to control, and there is an increase hours (Figure 3A and 3E). The β-tubulin in M phase cells in comparison with cisplatin- decrease is associated with p-H3 increase treated cells alone (Figure 2B). These findings (Figure 3A, 3E) and G2/M arrest (Figure 2E). were further confirmed by western blots of This suggests that AITC might induce microtu- p-H3. The results suggest that in the combina- bule depletion and/or dysfunction. To further tion group, p-H3 is unchanged at 5 µg/ml (16.7 demonstrate that microtubule dysfunction and/ µM) cisplatin and slightly decreased in 10 µg/ or depletion is involved in G2/M phase arrest, ml (33.3 µM) cisplatin as compared with no we triple stained 2008 ovarian cancer cells treatment. However, there is an observable with DAPI, p-H3 and β-tubulin after 16 hour increase in comparison to cisplatin treatment treatment (Figure 3C). Regardless of the num- alone (Figure 2C). Intriguingly, flow cytometry- ber of mitotic cells in different groups, during fractionated cells for cell cycle distribution, mitosis in both control and cisplatin groups we both cisplatin (CDDP) and AITC showed a cell see a certain amount of polar microtubules in arrest in the G2/M phase in comparison with the spindles of cells. However, in AITC- control (Figure 2E). This suggests that there is containing groups, the amount of β-tubulin is a largely G2 arrest after cisplatin treatment decreased and these cells failed to establish alone. We know that there are only few M phase microtubule polarity necessary for mitotic divi- cells after cisplatin (CDDP) treatment (Figure sion (Figure 3C). This finding has been further 2B and 2D). Generally speaking, when the cells confirmed by western blot assay Figure( 3D, top were arrested only in G2 phase, there will be an panel). In AITC alone and the AITC-cisplatin M phase decrease just like that shown in cispl- combination group, β-tubulin expression was atin treatment. However, in combination group, decreased, while expression of p-H3 increased M phase cells have not been significantly in comparison with cisplatin treatment alone. decreased (Figure 2B), suggesting combination We further investigated whether AITC and cis- of AITC and cisplatin could arrest the cells in platin combination could induce changes in both G2 and M phase. Similar results were molecular signaling associated with the G2/M observed in A549 cells (Figure 2D and 2E). checkpoint. Combination treatment with AITC and cisplatin decreased CDC2, Cyclin B1 and G2/M arrest induced by AITC and cisplatin CDC25C expression, more than single drug (CDDP) combination treatment is associated treatment alone (Figure 3D), suggesting that with signaling pathway change, microtubule AITC-induced microtubule depletion and dys- depletion and dysfunction function is associated G2/M phase arrest. Next, we searched for an appropriate time point β-tubulin depletion and microtubule dysfunc- Figure after treatment to investigate the mechanism tion were also observed in A549 cells ( 3E 3F that is responsible for G2/M arrest in AITC and and ). cisplatin combination. As shown above, AITC, Inhibition of cell growth via combination of cis- cisplatin, and their combination, induce G2/M platin (CDDP) with AITC is accompanied with arrest in different time-dependent patterns. cell death, caspase 3 activation and decreases However, all regimens have significant effect on in survivin and Bcl-2 expression. G2/M arrest 16 hours after treatment (P<0.05). We therefore concluded that the 16-hour time To study the biological mechanisms by which point offers a good window to study the mecha- AITC and cisplatin combination decreases cell nism of drug combination. It has been reported viability, we first investigated whether combina- that some ITCs analogues could induce micro- tion treatment was inducing cell death. After 72 tubule depletion [43]. We sought to determine hours of combination treatment, cell death was whether cellular skeletal system dysfunction is identified in 2008 ovarian cancer cells using involved in M phase arrest after AITC-containing flow cytometry (sub-G1 DNA content) Figure ( regimen treatment. We first tested if AITC alone 4A) and a cell death ELISA assay that quanti- could deplete the microtubule in cancer cells. fied cytoplasmic DNA-histone complexes As shown in Figure 3A and 3B, in 2008 ovarian (Figure 4B). We found that combination of cis-

2525 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin

the combination treatment of cisplatin with AITC. We therefore used the Combination Index (CI) equation method developed by Chou- Talalay [32, 33] to study the combinational synergistic effects. Our studies revealed that in all the dose ranges used in the combination of the two agents (AITC and cisplatin) for treatment of either A549 and Hop62 lung cancer cells, we obtained the CI≤1 (Figure 5). While only in a few combinational cases the CI is approaching 1, most combinational cases showed the CI is significantly <1. Furthermore, we obtained the CI much smaller than 0.5 for the combinational treatment of AITC at 10-20 µM and cisplatin at 8-16 µM (Figure 5), suggesting a high synergistic effect can be reached.

Combination of cisplatin (CDDP) with AITC enhances the sensitivity of tumor cells to cisplatin treatment in lung cancer xenograft tumor models.

In order to demonstrate that AITC is a good candidate for in vivo combination with cisplatin (CDDP), we determined whether AITC could sensitize tumor cells to cisplatin treatment using human tumor xenograft mouse models. Figure 6. Inhibition of human tumor xenograft growth As we expected and also consistent with the in animal models by AITC, cisplatin (CDDP) alone and in combination. Experimental procedures are data shown in Figure 5 for the CI study, combi- described in the Methods section. A549 lung can- nation of cisplatin with AITC significantly cer cell line-derived tumor xenograft in nude mouse increases the tumor growth inhibition in com- models was used. (A) effects of AITC, cisplatin alone parison with each compound treatment alone or in combination on the inhibition of human tumor (P<0.05) (Figure 6A), while combination treat- xenograft growth. (B) mouse body weight profiles during treatment in the experiments. The value in each ment did not increase toxicity/body weight loss time point is the mean tumor size (A) or mean body (Figure 6B). Data obtained in our xenograft ani- weight (B) with SD derived from 5 mice. mal model are summarized in Table 1. The maximum tumor growth inhibitions (MTRI) are 46.6 ± 18.3, 30.2 ± 10.1 and 94.3 ± 4.8 for AITC platin and AITC could significantly increase sub- alone, cisplatin alone and their combination in G1 DNA content and cytoplasmic DNA-histone turn, respectively. The tumor doubling times complexes, as compared to those treated with (TDT) are 9.3 ± 1.1, 9.7 ± 1.2 and more than 24 single drug alone (P<0.05) (Figure 4A and 4B). days (Table 1) in AITC, cisplatin and their combi- Apoptosis in cells treated with AITC and cispla- nation in turn, respectively. In the combination tin combination was further investigated by group, 60% of mice had at least 50% reduction in western blot analysis of survivin, Bcl-2, and tumor volume, and 20% of mice had no palpable caspase 3. As shown (Figure 4C), the combina- tumors at the end of the study. Importantly, com- tion treatment increased caspase 3 activation, bination of cisplatin with AITC could significant- and decreased the expression of Bcl-2 and ly enhance the capacity of tumor growth inhibi- survivin. tion without a significant increase in systemic toxicity/body weight loss (P<0.05) (Figure 6; Synergistic inhibitory effects of cisplatin Table 1). (CDDP) and AITC combinational treatment in lung cancer cell growth Discussion

The results shown from Figure 1C and 1D clear- Consistent with the in vitro studies from other ly suggest a possible synergistic effect upon ITCs [12-16], we found that combination of cis-

2526 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin

Table 1. Antitumor activity and toxicity of CDDP ± AITC in nude mice bearing human lung A549 xenografts Antitumor activity Toxicity Treatment MTGI (%) TDT (day) PR (%) CR (%) MWL Lethality A549 lung cancer Control (vehicle) - 5.7 ± 0.2 0 0 2.1 ± 1.8 0 CDDP 6 mg/kg i.v. x 1 30.2 ± 10.1 9.7 ± 1.2 0 0 7.4 ± 3.8 0 AITC 50 mg/kg p.o.every 2 days x 8 46.4 ± 18.3 9.3 ± 1.1 0 0 4.7 ± 2.8 0 CDDP 6 mg/kg + AITC 50 mg/kg 94.3 ± 4.8 >24 60 20 6.1 ± 3.1 0 Note: Antitumor activity and toxicity of AITC ± CDDP (cisplatin) in nude mice bearing human lung A549 xenografts. MTGI: maximum tumor growth inhibition; TDT: tumor doubling time; PR: partial tumor response; CR: complete tumor response; MWL: maximum weight loss of pretreatment body weight. Treatment was initiated 6 days after the tumor transplantation when the tumor weight reaching ~ 200-220 mg (mm3 in volume). platin with AITC significantly sensitizes cancer Substantial evidence shows that G2/M arrest cells to cisplatin-induced cell growth inhibition plays a key role in cell growth inhibition induced in both MTT and colony formation assays by cisplatin treatment [38-42]. In our study we (Figure 1). We further revealed the synergistic found that AITC and cisplatin combination not effect between AITC and cisplatin using combi- only changes the time course of G2/M arrest nation-index (CI) analysis (Figure 5). We defined but also induces both G2 and M phase arrest the dose ranges for realizing synergistic combi- with extending time (Figure 2). Furthermore, nation effects for both cisplatin and AITC which upon G2/M decrease at 72 hours after AITC is reachable in vivo [23, 24], suggesting that and cisplatin combination treatment, there is a this combination could fully realize synergistic significant increase in sub-G1 cells and DNA function in vivo. To make our protocol easier to damage; in contrast, for the cisplatin treatment translate into a clinical model, one time tail vein alone, upon the decrease in G2/M cells, there i.v. injection of cisplatin and daily oral adminis- is much less sub-G1 and DNA damage com- tration of AITC was used for combination treat- pared with AITC and cisplatin combination ment in our studies. We found that treatment in treatment (Figure 2). These observations fur- human tumor animal models with AITC and cis- ther indicate that both cell cycle arrest and platin combination could significantly increase apoptosis induction are involved in cell growth the capacity for tumor growth inhibition in com- inhibition induced by combination of AITC and parison with AITC or cisplatin treatment alone cisplatin. (Figure 6A). Surprisingly, unlike other combination regimens, there is no significant increase Although a full understanding of the mecha- in toxicity (both weight loss) in the combination nism underlying the synergistic interaction group (Figure 6B). between cisplatin and AITC requires more studies, both drugs have been reported to interact In the present study, we also explored whether with tubulin. Cisplatin could change microtu- the addition of AITC could enhance the pro- bule assembly dynamics [49, 50], and AITC and apoptotic effect of cisplatin treatment. Our its analogues could inhibit the expression of data showed that combination of AITC and cis- alpha- and beta-tubulin proteins [51, 52]. We platin could significantly increase sub-G1 dying speculate that the microtubule depletion and cells, DNA damage and caspase activation with dysfunction may be involved in synergistic func- the inhibition of Bcl-2 and survivin expression tion. We found that AITC decreases β-tubulin (Figure 4), suggesting that combination of AITC levels, while cisplatin alone in our experiment and cisplatin could significantly increase pro- has not induced a significant β-tubulin decrease apoptotic response in comparison with single (Figure 3). This is possibly because we are limit- drug treatment alone. This finding is consistent ing the dosing range to clinically relevant con- with our previous studies that revealed that centrations [49]. In the combination of AITC drug resistance or sensitivity is associated with and cisplatin group, β-tubulin was observably anti-apoptotic protein expression [44-48]. decreased, and time course studies for tubulin

2527 Am J Cancer Res 2015;5(8):2516-2530 Synergistic interaction of AITC with cisplatin depletion induced by AITC showed that β-tubulin Disclosure of conflict of Interest decrease is negatively associated with p-H3 increase (Figure 3), suggesting that decreased None. β-tubulin levels might be involved in G2/M arrest after AITC and cisplatin combination Abbreviations treatment. In the triple staining β-tubulin, p-H3 AITC, allyl isothiocyanate; CI, combination and DAPI, we found that AITC alone or the AITC index; cisplatin (CDDP), cis-diamminedichloro- and cisplatin combination group, showed that platinum II; DAPI, 4, 6-diamidino-2-phenylin- not only has amount of microtubule protein dole; ITCs, isothiocyanates; MTT, 3-[4, been decreased but also the spindle architec- 5-dimethylthiazol-2-yl]-2, 5,-diphenyltetrazoli- ture has been changed (Figure 3C and 3F), sug- um bromide; PI, propidium iodide; SD, standard gesting that microtubule depletion and dys- deviation; SDS, sodium dodecyl sulfate. function was involved in M phase arrest induced by combination of AITC and cisplatin. Address correspondence to: Drs. Xiang Ling and Our further studies revealed that combination Fengzhi Li, Department of Pharmacology and of AITC and cisplatin could decrease CDC2, Therapeutics, Roswell Park Cancer Institute, Elm CDC25 and cyclin B1 expression levels (Figure and Carlton Streets, Buffalo, NY 14263, USA. Tel: 3D). Taken together, we propose that the com- (716)-845-1565, (716)-881-7470; Fax: (716)-845- bination of AITC and cisplatin induces both G2 8857; E-mail: [email protected] (XL); feng- and M arrest through depletion of microtu- [email protected] (FZL) bules, microtubule dysfunction and G2/M References checkpoint change. [1] Kollmannsberger C, Nichols C and Bokemeyer Our data is in agreement with reports by other C. Recent advances in management of pa- groups. ITCs enhance the sensitivity of a variety tients with platinum-refractory testicular germ of cancer cells to cisplatin treatment [19, 25, cell tumors. Cancer 2006; 106: 1217-1226. [2] Galluzzi L, Vitale I, Michels J, Brenner C, 26]. It is possible to take advantage of the syn- Szabadkai G, Harel-Bellan A, Castedo M and ergistic effects for combinational treatment of Kroemer G. Systems biology of cisplatin resis- both newly diagnosed patients as well as tance: past, present and future. Cell Death Dis patients failing primary chemotherapy. Most 2014; 5: e1257. importantly, our in vivo data showed that AITC [3] Galluzzi L, Senovilla L, Vitale I, Michels J, could significantly enhance the anticancer Martins I, Kepp O, Castedo M and Kroemer G. function of cisplatin without increasing the tox- Molecular mechanisms of cisplatin resistance. icity of the treatment. Therefore, this combina- Oncogene 2012; 31: 1869-1883. tion might provide a novel therapeutic strategy [4] Koberle B, Tomicic MT, Usanova S and Kaina B. in solid tumor treatment in general. Cisplatin resistance: preclinical findings and clinical implications. Biochim Biophys Acta 2010; 1806: 172-182. Acknowledgements [5] Obexer P and Ausserlechner MJ. X-linked inhibitor of apoptosis protein - a critical death This work was sponsored in part by NIH Grants, resistance regulator and therapeutic target for R01CA109481 and R01CA133241 to FL, personalized cancer therapy. Front Oncol R01CA164574 to YZ, P50CA058187 to DC, 2014; 4: 197. and by shared resources supported by NCI [6] Wurstle ML, Zink E, Prehn JH and Rehm M. Cancer Center Core Support Grant to Roswell From computational modelling of the intrinsic Park Cancer Institute (P30CA016056), and by apoptosis pathway to a systems-based analy- a Mesothelioma Foundation (Alexandria, VA) sis of chemotherapy resistance: achieve- grant to FL. We thank other members in the Li ments, perspectives and challenges in sys- Lab for helpful discussion of data during lab tems medicine. Cell Death Dis 2014; 5: e1258. [7] Masood A, Shahshahan MA and Jazirehi AR. meeting. We also thank other faculty and staff Novel approaches to modulate apoptosis resis- scientists including PhD students for their sup- tance: basic and clinical implications in the porting and friendship during carrying out the treatment of chronic lymphocytic leukemia studies of our projects. We also thank Ms. (CLL). Curr Drug Deliv 2012; 9: 30-40. Katherine Plante for editorial revision of this [8] Indran IR, Tufo G, Pervaiz S and Brenner C. manuscript. Recent advances in apoptosis, mitochondria

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