Biochemical Pharmacology 85 (2013) 486–496

b-Phenylethyl isothiocyanate reverses platinum resistance by a GSH-dependentmechanism in cancer cells with epithelial-mesenchymal transition phenotype

Wen-jing Wu a,b,1, Yan Zhang a,b,1, Zhao-lei Zeng b,1, Xiao-bing Li b, Kai-shun Hu d, Hui-yan Luo a,Jing Yang b, Peng Huang b,c,**, Rui-hua Xu a,b,*a Department of Medical Oncology, Sun Yat-sen University Cancer Center, Guangzhou 510060, Chinab State Key Laboratory of Oncology in South China, Sun Yat-sen University Cancer Center, Guangzhou 510060, Chinac Department of Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USAd Department of Research Center for Medicine, The Second Affiliated Hospital of Sun Yat-sen University, Guanzhou 510120, China

A R T I C L E I N F O

Article history:

Received 22 October 2012

Accepted 26 November 2012

Available online 5 December 2012

Keywords:

Chemo-resistance

PEITC

Glutathione

EMT

Chemotherapy

A B S T R A C T

Platinum (Pt)-based chemotherapy is an important regimen in the clinical treatment of cancer, but

development of drug resistance presents a major challenge. One key mechanism involved in resistance to

Pt drugs is the decrease of intracellular Pt due to the drug efflux through the glutathione (GSH)-mediated

export, and this is particularly significant in cancer cells with stem-cell like properties. In the present

study, we showed that two Pt-resistant human cancer cell lines exhibited stem-cell like EMT properties,

had high cellular GSH and accumulated significantly less cellular Pt compared to their parental cells, and

failed to undergo apoptosis when exposed to Pt at the drug concentrations toxic to the parental cells.

Importantly, we found that the natural compound b-phenylethyl isothiocyanate (PEITC) was able to

effectively abolish this drug resistant mechanism by effective depletion of cellular GSH, leading to a

significant increase in cellular Pt as well as DNA-bound Pt. A combination of PEITC and Pt showed a

striking synergistic anticancer activity both in vitro and in vivo, as evidenced by an increase in drug-

induced apoptosis, a loss of colony formation capacity, and significant suppression of tumor growth in

mice. Taken together, our study shows a promising therapeutic strategy to overcome drug resistance to

platinum-based chemotherapy and may potentially have broad implications in clinical treatment of

cancer.

� 2012 Elsevier Inc. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Biochemical Pharmacology

jo u rn al h om epag e: ww w.els evier .c o m/lo cat e/bio c hem p har m

1. Introduction

Platinum (Pt)-based chemotherapy is among the most widelyused therapeutic regimens for cancer treatment in the past severaldecades, and is effective against a variety of human solid tumorssuch as lung, colorectal, ovarian and bladder cancers [1–5].Although surgical resection still remains the method of choice tocure a majority of solid tumors, many patients at diagnosis presentunresectable tumors in advanced disease stages with extensive

* Corresponding author at: Department of Medical Oncology, Sun Yat-sen

University Cancer Center, Guangzhou 510060, China. Tel.: +86 20 8734 3228;

fax: +86 20 8734 3228.

** Corresponding author at: Department of Molecular Pathology, The University of

Texas MD Anderson Cancer Center, Houston, TX 77054, USA. Tel.: +1 713 834 6044;

fax: +1 713 834 6084.

E-mail addresses: wuwj@sysucc.org.cn (W.-j. Wu), zhangy4@sysucc.org.cn

(Y. Zhang), zengzhl@sysucc.org.cn (Z.-l. Zeng), lixb@sysucc.org.cn (X.-b. Li),

huksh@mail2.sysu.edu.cn (K.-s. Hu), luohy@sysucc.org.cn (H.-y. Luo),

yangjing@sysucc.org.cn (J. Yang), phuang@mdanderson.org (P. Huang),

xurh@sysucc.org.cn (R.-h. Xu).1 These authors contributed equally to this work.

0006-2952/$ – see front matter � 2012 Elsevier Inc. All rights reserved.

http://dx.doi.org/10.1016/j.bcp.2012.11.017

local/regional tumor invasion or distal metastasis. In these cases,chemotherapy using proper combination of anticancer agentsplays a major role in clinical treatment of advanced cancers.Platinum-based chemotherapy is effective in suppressing cancergrowth and in some cases even inducing tumor regression.Unfortunately, certain cancers do not respond to the initialtreatment due to intrinsic drug resistance, while other tumorsthat show initial response often develop drug resistance eventual-ly, leading to treatment failure [6–9]. Recent study suggest that asmall subpopulation of cancer cells with stem-cell like properties(also known as tumor-initiating cells) are intrinsically resistant tochemotherapy and radiotherapy due in part to their high DNArepair capacity, high ability to export drug out of the cells, and highexpression of certain cell survival molecules [10–13]. Thepersistence of viable stem-cell like cancer cells after chemotherapyhas been considered as a major reason for development of drugresistance and disease recurrence [10,14]

Several mechanisms are involved in Pt-resistance, includingreduced accumulation of cellular Pt, increased repair of DNA damageand suppression of apoptosis induction [8,9,15,16]. Drug resistantcell lines have been established to investigate the potential

W.- Wu et al. / Biochemical Pharmacology 85 (2013) 486–496 487

mechanisms. Cisplatin-resistant A549/CDDP cells established fromhuman non-small cell lung cancer (NSCLC) cell line A549 were foundto over express Bcl-2 and copper-transporting P-type adenosinetriphosphatase (ATP7A), leading to less Pt accumulation in the cells[17]. Human colon cancer cell line THC8307/L-OHP, which isresistant to oxalipatin, was found to have lower expression of thepro-apoptotic genes such as STK17A and BNIP3, and higherexpression of anti-apoptotic molecules such as PSAP and GDIA1[18]. The ATP-binding cassette (ABC) efflux transporters play a majorrole in drug resistance, and the ABCB1 (p-glycoprotein/MDR1) andABCC2 (MRP2) subfamily is involved in the development of cisplatinresistance [19–21]. Previous studies demonstrated that ABCC-mediated efflux seems to require glutathione (GSH), which serves asa substrate for conjugation with cisplatin to form a Pt(GS)2 conjugatefor ABCC-mediated efflux, leading to an reduction in cellularplatinum [22,23]. Consistently, previous studies showed that anincrease in cellular GSH was associated with cisplatin resistance[8,24–27]. Before binding to DNA, most of the platinum binds tonucleophilic molecules such as GSH to form a Pt(GS)2 conjugate,which is then exported out of the cells. Although this conjugationmay cause a depletion of the antioxidant reserve and leads tooxidative stress that favored DNA damage by cisplatin, GSHeffectively reduces the amount of reactive cisplatin and thus limitsits anticancer activity [8,24–27]. As such, an increase of GSHsynthesis in cancer cells, especially in stem-cell like cancer cells [28],would be expected to cause resistance to Pt drugs.

b-Phenylethyl isothiocyanate (PEITC), a natural compoundfound in consumable cruciferous vegetables, has been shown tohave chemoprevention activity in several experimental models[29–31]. Recent studies suggest that this compound is able topreferentially kill cancer cells and exhibits promising therapeuticactivity in vitro and in vivo [32–37]. A key mechanism of PEITCaction is to induce severe ROS accumulation in cancer cells bycausing rapid depletion of cellular GSH [32]. The therapeuticselectivity of PEITC is due in part to the different redox statebetween cancer cells (high ROS generation) and normal cells (lowbasal ROS output) and thus a depletion of GSH would cause moresevere ROS accumulation and damage to cancer cells [32,38].

Considering the important role of GSH in mediating cisplatin-resistance and the ability of PEITC to effectively deplete GSH, wehypothesized that this compound might be useful as a novel agentto overcome Pt-resistance. Using two pairs of Pt-resistant humancancer cell lines, we showed that the drug resistant cells exhibitcertain stem-cell like properties with a significant increase incellular GSH and a decrease in intracellular accumulation of Pt andits binding to DNA. We further demonstrated that PEITC wasindeed able to abolish such drug resistance by depleting GSH, andsignificantly enhanced the therapeutic activity in vitro and in vivo.

2. Materials and methods

2.1. Chemicals and reagents

b-Phenylethyl isothiocyanate (PEITC), metaphosphoric acid, 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium (MTT), 2-vinylpyridine, triethanolamine, ethanol, nitric acid and CremophorEL were purchased from Sigma–Aldrich (St. Louis, MO). PEITC wasdissolved in dimethyl-sulfoxide (DMSO) and freshly diluted inculture media before use. The final DMSO concentration in mediawas less than 0.1% (v/v). CM-H2DCF-DA was purchased fromInvitrogen (Carlsbad, CA).

2.2. Cells and cell culture

Non-small cell lung cancer cell line A549 and cisplatin resistantA549/CDDP cells derived from A549 cell line were cultured in RPMI

1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetalbovine serum (Invitrogen, Carlsbad, CA) as described previously[17]. Self-established colon carcinoma cell line THC8307 andoxaliplatin resistant THC8307/L-OHP cells derived from THC8307cell line were gifts from Institute of Hematology (CAMS & PUMC,Tianjin, China), and were cultured in RPMI 1640 with 10% fetalbovine serum. These two Pt-resistant cells were established bystepwise drug selection from the parental cells as describedpreviously [17,18]. Human colon cancer cell line HCT116(purchased from ATCC) and human ovarian cancer cell lineA2780 (obtained from Sun Yat-sen University Cancer Center)were maintained in RPMI 1640 supplemented with 10% fetalbovine serum.

2.3. Cytotoxicity and flow cytometry analysis

Cell growth inhibition was determined using MTT reagent in 96-well plates. After 24–72 h of drug incubation, 20 mL MTT reagentwas added to each well and incubated for an additional 4 h and thenthe supernatant was removed. The cell pellets were dissolved in200 mL DMSO. Absorbance was determined using a MultiSkan platereader (Thermo, Helsinki, USA) at a wavelength of 570 nm. Apoptoticor necrotic cell death was determined by flow cytometric analysis ofcells double stained with Annexin V-FITC and propidium iodide (PI)using an apoptosis kit (KeyGEN, Nanjing, China).

2.4. Proliferation assay

Cell growth rate was detected by MTT proliferation assay. Cellswere seeded in 96-well plate at a density of 1 � 103 cells per well.After 24–96 h’s incubation, cell growth rate was detected by MTTassay. Each experiment was performed in triplicate.

2.5. Colony formation assay

Cells were plated in 6-well plates (4 � 102 cells per well),cultured with single or combined drug treatment for 10 days. Thecolonies were stained with 1% crystal violet for 30 min afterfixation with 10% formaldehyde for 15 min.

2.6. Detection of cellular ROS and glutathione

Basal or drug treated cellular ROS contents were measured byincubating cells (2 � 105 cells) with 1 mM CM-H2DCF-DA for60 min and analyzed by flow cytometry as described in ref. [32]. Aglutathione assay kit (Cayman Chemical, Ann Arbor, MI) was usedto measure cellular glutathione. After preparing cell extracts bysonication and deproteination, GSH was determined as describedin ref. [32].

2.7. Wound healing and in vitro invasion assay

Cells were trypsinized and seeded equally into 6-well plates togrow to reach almost total confluence in 24 h followed by non-serum starvation for another 24 h. The cell monolayer were then bescratched with a sterile 100 mL pipette tip. After scratching, thecells were washed with PBS and then cultured with serum-freemedium. Cell migration images were captured at time points of 0 h,18 h and 36 h by inverted microscope (100�). Cell invasion wasdetected by transwell assay according to the vendor’s instructions(BD Biosciences, San Jose, CA).

2.8. Western blotting

Western blotting was carried out as described in ref. [39].Briefly, cells were lysed in MCLB (50 mM Tris–HCl, pH 8.0, 2 mM

Fig. 1. Effect of Pt-based drug on Pt-sensitive and resistant cell growth and viability. (A and B) Sensitivities of A549/CDDP cells and THC8307/L-OHP cells to cisplatin or

oxaliplatin compared with their parental Pt-sensitive A549 cells and THC8307 cells after 24–72 h of treatment using an MTT assay. Points, mean (n = 3); bars, SEM. (C and D)

Flow cytometric analysis of cell death in A549/CDDP and THC8307/L-OHP cells compared with their parental cells after treated with cisplatin or oxaliplatin using Annexin V

and PI double stained. Bar graphs representing % of apoptosis cells are shown. Columns, mean (n = 3); bars, SEM; **P < 0.01.

W.- Wu et al. / Biochemical Pharmacology 85 (2013) 486–496488

Fig. 2. Pt-resistant cells present stem-cell like EMT properties. (A and B) 24 h of transwell-matrigel penetration assay done in Pt sensitive and resistant cells, and the

representative micrographs (up panel) and quantification (down panel) from at least three independent experiments were shown. **P < 0.01. (C and D) Wound-healing assay

done in the same cells, and the representative micrographs at different time points and the quantification of wound healing rates were shown on the bar graphs, comparing

the wound width of each sample to that of the 0-h control. (E) Expression of EMT makers E-cadherin, N-cadherin and vimentin in the Pt-resistant cells and their parental cells

was detected by western blotting (n = 3) and the quantitative data was shown. The expression of b-catenin, which is involved in stem cell self-renewal and differentiation,

was also measured in the same cells for comparison (n = 3) and the quantitative data was shown. (F) Expression of stem cell biomarkers OCT-4 and Sox2 in the drug-resistant

cells and their parental cells was detected by western blotting (n = 3) and the quantitative data was shown.

W.- Wu et al. / Biochemical Pharmacology 85 (2013) 486–496 489

dithiothreitol, 5 mM EDTA, 0.5% Nonidet P-40, 100 mM NaCl,1 mM microcystin, 1 mM sodium orthovanadate, 2 mM phenyl-methylsulfonyl fluoride, protease (Sigma, St. Louis, MO) sup-plemented with a phosphatase inhibitor mixture (Invitrogen,Carlsbad, CA) and clear lysates (40 mg) were resolved by SDS-PAGE and transferred to PVDF membranes for western blottingusing either ECL detection reagents (KeyGEN, Nanjing, PRC).Rabbit monoclonal anti-E-cadherin (1:1000), rabbit polyclonalanti-Oct-4 (1:1000), rabbit polyclonal anti-Sox2 (1:1000),mouse anti-vimentin, mouse monoclonal anti-gH2AX (1:1000)antibodies were purchased from Cell Signaling Technology

(Danvers, MA). Mouse monoclonal anti-N-cadherin antibody(1:2000) was purchased from BD Biosciences (San Jose, CA).Mouse monoclonal anti-GAPDH antibody (1:10,000) was pur-chased from Kang Cheng (Shanghai, PRC). Rabbit monoclonalanti-a-tubulin antibody (1:20,000) was purchased from Abcam(MA, USA).

2.9. Assay of total cellular cisplatin content and DNA-bond Pt

The measurement of total intracellular content of Pt wasdone as described previously [40]. Briefly, after treatment with

Fig. 3. Cell growth curves of Pt-sensitive cells and Pt-resistant cells. (A) Cell proliferation rate of A549/CDDP cells and A549 cells by MTT assay. Points, mean (n = 3); bars, SEM.

(B) Cell proliferation rate of THC8307/L-OHP cells and THC8307 cells by MTT assay. Points, mean (n = 3); bars, SEM.

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PEITC and/or Pt, cells were harvested, washed with PBS and cellnumbers were counted. The cell pellets were then digested innitric acid and sent for Pt analysis using Agilent 7500ceinductively coupled plasma mass spectrometry (ICP-MS) (Agi-lent). For DNA-binding Pt content, cells with different treat-ments were harvested and washed with cold PBS twice bycentrifugations. Then the cell pellets were re-suspended in200 ml cold water to make single cell suspension. Then an equalvolume of 2� lysing buffer (2% SDS, 20 mM EDTA, 200 mM NaCl,20 mM Tris, pH 8.0) was added to the cell suspension, followedby addition of 20 mL of protease K (stock solution 2 mg/mL) tothe 400 mL cell lysate. Samples were mixed well and incubatedat 45 8C for overnight. Then 1.2 mL of 100% cold ethanol wasadded to the 0.4 mL sample to precipitate DNA. Samples werekept in freezer (-20 8C) overnight and then spun to recover theDNA in the pellet, and washed twice with 75% ethanol bycentrifugations. The DNA in the pellets was sent for Ptanalysis using Agilent 7500ce ICP-MS (Agilent).

2.10. In vivo therapeutic study

All animal experiments were conducted according to theguidelines of the Institutional Animal Care and Use Committeeunder approved protocol. A549/CDDP cells (1 � 107) wereinjected subcutaneously into flanks of BALB/c nude mice (4–5weeks old). When palpable tumors reached about 35 mm3 after 2weeks, mice were randomized to treatment and control groups.Mice of single drug therapy groups were injected i.p. with cisplatin(5 mg/kg per week) and PEITC (25 mg/kg, bid, on the same day ofCDDP treatment, also once per week). For drug combinationchemotherapy, mice were intraperitoneal injected with PEITC(25 mg/kg) 5 h before and after the injection of cisplatin (5 mg/kg), once per week. Control group received 200 mL of solvent(ethanol:Cremophor EL:PBS at the ratio of 1:1:8 by volume). Afterthe first course of treatment (week 3), the mice were allowed aweek of recovering time (without drug treatment), and the secondand third courses of treatment started on week 5 and week 6,respectively. Tumor volumes were measured twice a weekusing a caliper, and were calculated using the formulaV = (width2 � length)/2. Body weights were also recorded. Whenthe tumor volumes of the control group reached 1200 mm3, micewere sacrificed according to the animal study protocol. The tumorxenografts were isolated and their weights were measured andrecorded.

2.11. Statistics

Statistical analysis was performed using the Student’s t test, andthe results were expressed as mean � SD or mean � SEM. Signifi-cance levels were set at P < 0.05.

3. Results

3.1. Platinum-resistant cells exhibited stem-cell like EMT property

Two human platinum-resistant cancer cell lines, A549/CDDPcells derived from non-small cell lung cancer cell line A549 andTHC8307/L-OHP derived from human colon cancer cell lineTHC8307 [17,18], were used in this study. To confirm theirplatinum-resistant phenotype, we first used MTT assay to comparethe sensitivities of these two cell lines with their parental cells tocisplatin (CDDP) or oxaliplatin (L-OHP). The results showed thatCDDP and L-OHP inhibited cell proliferation in a time- andconcentration-dependent manner in the four cell lines with differentsensitivity (Fig. 1A and B). The IC50 values of CDDP (72-h incubation)were 3.2 mM and 21.3 mM for A549 cells and A549/CDDP cells,respectively, indicating that A549/CDDP cells were about 6-foldmore resistant to CDDP compared to the parental cells (Fig. 1A).Similarly, THC8307/L-OHP cells (IC50 = 9.4 mM) were about 18-foldmore resistant to L-OHP than THC8307 cells (IC50 = 0.5 mM). Thedrug resistant phenotype was further confirmed by flow cytometryanalysis, using annexin-V and PI double staining to assess cellularapoptotic response under high dose of CDDP or L-OHP. As shown inFig. 1C, incubation of the parental A549 cells with 40 mM CDDP for24 h resulted in 25% cell death, whereas the same drug treatment inA549/CDDP cells only caused 1% cell death. Consistently, incubationof THC8307 cells with 40 mM L-OHP for 24 h caused a massive celldeath (50.4%), while this drug treatment induced only 2.8% cell deathin THC8307/L-OHP cells (Fig. 1D).

Interestingly, compared to their respective parental cell lines, thetwo Pt-resistant cell lines displayed certain features of epithelial–mesenchymal transition (EMT). For instance, both A549/CDDP andTHC8307/L-OHP cells displayed a mesenchymal-like morphologyand appeared more motile (Fig. 2A and B). Functional analysisshowed a significant increase in cell mobility, as evidenced by anelevated ability to invade through matrix gel in a Transwell-Matrigelassay (Fig. 2A and B, lower panels, P < 0.01). In vitro wound healingassay also showed an increased rate of cell migration in the two Pt-resistant cell lines (Fig. 2C and D). The Pt-resistant cells showedsimilar growth rates compared to their parental cell lines (Fig. 3),suggesting that the increase in cell invasion and migration observedin the Transwell-Matrigel assay and wound-healing assay was notdue to increase in cell proliferation. Western blotting analysisshowed a substantial down-regulation of E-cadherin (epithelialmarker) and an increased expression of vimentin and N-cadherin(mesenchymal marker) in the Pt-resistant cells (Fig. 2E). Bothparental THC8307 cells and the drug-resistant THC8307/L-OHP cellsexhibited no expression of E-cadherin (Fig. 2E). Interestingly, theexpression of stem cell markers OCT-4 and SOX2 also increased inboth drug-resistant cell lines compared to their respective parentalcells (Fig. 2F).

Fig. 4. Different redox state in Pt-sensitive and resistant cells, and depletion of glutathione by PEITC. (A and B) Basal cellular GSH in Pt-resistant cells (A549/CDDP cells (A);

THC8307/L-OHP cells (B)) compared with Pt sensitive cells. Columns, mean (n = 3); bars, SD; **P < 0.01. (C and D) Cellular GSH of the same cells after incubation with cisplatin

or/and PEITC. Columns, mean (n = 3); bars, SD; *P < 0.05; **P < 0.01, compared with control. (E and F) Increased cellular ROS in A549/CDDP and THC8307/L-OHP cells treated

with Pt drugs or/and PEITC by flow cytometry analysis using 1 mM DCF-DA. Representative histograms were shown.

W.- Wu et al. / Biochemical Pharmacology 85 (2013) 486–496 491

3.2. Increased cellular glutathione in the Pt-resistant cancer cells and

its depletion by PEITC

Based on the important role of glutathione (GSH) in drugresistance, we postulated that the Pt-resistant cells with EMTphenotype might have an elevated level of GSH and thus affecttheir cellular ROS and drug sensitivity. To test this possibility, we

measured the GSH levels in the drug resistant A549/CDDP andTHC8307/L-OHP cells in comparison with their respective parentalcells. The results showed that the drug-resistant cells had higherglutathione levels than their parental cells (Fig. 4A and B, P < 0.01).

The increase of GSH in A549/CDDP and THC8307/L-OHP cellssuggests that this biochemical alteration might play an importantrole in drug resistance due to the significant role of GSH in cellular

Fig. 5. Efficient killing of platinum resistant cells by PEITC combined with cisplatin or oxaliplatin. (A and B) Flow cytometric analysis of cell death in Pt resistant cells (A549/

CDDP cells (A); THC8307/L-OHP cells (B)) treated with Pt drug or/and PEITC using Annexin-V and PI double stained. Columns, mean (n = 3); bars, SD; **P < 0.01. (C and D) The

histogram shows the colony formation efficiency of the same cells treated with Pt drug or/and PEITC. Columns, mean (n = 3); bars, SD; **P < 0.01.

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detoxification and in export of platinum. Thus, we postulated that adepletion of GSH in these cells might overcome such drugresistance. To explore this possibility, we used PEITC, a naturalcompound capable of rapidly depleting cellular GSH throughconjugation with GSH (27), to test its effect on cellular GSH in thedrug resistant cells. Our results showed that 5 mM PEITC caused adepletion of GSH in A549/CDDP and THC8307/L-OHP cells within6–12 h in the presence or absence of Pt drugs (40 mM CDDP forA549/CDDP cells; 40 mM L-OHP for THC8307/L-OHP cells) (Fig. 4Cand D, P < 0.05), whereas Pt drugs alone did not cause anysignificant change in cellular GSH contents (Fig. 4C and D).

Depletion of GSH in both A549/CDDP and THC8307/L-OHP celllines by PEITC led to substantial increase in cellular ROS, detectedby flow cytometry analysis using DCF-DA as a redox-sensitive dye(Fig. 4E and F).

3.3. PEITC sensitized the Pt-resistant cells by restoring cellular drug

accumulation

Based on the increased GSH in Pt resistant cells which could bedepleted by PEITC efficiently, we suggested a combination effect ofPEITC and Pt drugs. To test this possibility, we firstly used PEITC

Fig. 6. Efficient killing of cancer cells by PEITC combined with cisplatin or oxaliplatin. (A) Flow cytometric analysis of cell death in HCT116 cells treated with oxaliplatin or/and

PEITC using Annexin-V and PI double stained. Columns, mean (n = 3); bars, SD; **P < 0.01. (B) Flow cytometric analysis of cell death in A2780 cells treated with cisplatin or/and

PEITC using Annexin-V and PI double stained. Columns, mean (n = 3); bars, SD; **P < 0.01.

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together with cisplatin or oxaliplatin and tested their cytotoxiceffect on A549/CDDP cells and THC8307/L-OHP. As shown inFig. 5A, a sub-toxic concentration of CDDP (40 mM) or PEITC(5 mM) alone did not cause significant cell death in A549/CDDPcells at either 24 h or 48 h, combination of both compounds led to asignificant increase of cell death, with 22% and 47% dead cell at 24 hand 48 h, respectively. The observed % cell death was significantlyhigher than the calculated additive effect (Fig. 5A, right panels,P < 0.01). Similarly, PEITC was able to significantly sensitizeTHC8307/L-OHP cells to oxaliplatin treatment (Fig. 5B, P < 0.01).The synergistic effect of PEITC in combination with cisplatin oroxaliplatin was then observed in colony formation assays in bothdrug-resistant cell lines (Fig. 5C and D, P < 0.01). Moreover, in twoother Pt sensitive cancer cell lines, which were HCT116 coloncancer cells and A2780 ovarian cancer cells, the synergistic effectwas also observed (Fig. 6).

We further tested if the synergetic cytotoxic effect was due tohigh Pt accumulation as a consequence of GSH depletion leading toless export of Pt from the cells. Quantitative analysis of Pt showedthat after 12 h or 24 h incubation with 40 mM CDDP, the totalintracellular Pt (Fig. 7A) and the DNA-bound Pt (Fig. 7B) were threefold higher in cisplatin-sensitive A549 cells than in A549/CDDPcells, confirming that reduced cellular platinum content was a keymechanism of cisplatin resistance. Importantly, addition of 5 mMPEITC significantly increased the Pt in A549/CDDP cells. Both thetotal intracellular Pt and DNA-bound Pt increased significantlycompared with the cells treated with CDDP alone (Fig. 7A and B,P < 0.01). Similar results were also observed in THC8307/L-OHPcells (Fig. 7C and D, P < 0.01). Western blotting analysis revealedDNA damage at 12 h and was more severe at 24 h when cells weretreated with PEITC and Pt combined using g-H2AX expression as anindicator (Fig. 7E and F). g-H2AX expression after treatment with

PEITC was likely due to elevated cellular ROS induced by PEITC,leading to ROS-mediated DNA damage (Fig. 4E and F).

3.4. Synergistic therapeutic effect of PEITC and CDDP in vivo

The synergistic effect of CDDP and PEITC observed in vitro

prompted us to further test the in vivo therapeutic effect of thisdrug combination. BALB/c nude mice were inoculated with thedrug-resistant cell line A549/CDDP subcutaneously (1 � 107 cellsper injection site). Drug treatment started when the tumorxenografts reached an average volume of 35 mm3. The controlgroup received 200 mL of solvent, while the treatment groupsreceived CDDP (5 mg/kg, i.p., once per week), PEITC (25 mg/kg, bid,on the same day as CDDP, once per week), or their combination. Asshown in Fig. 8A, treatment of the mice with CDDP as a single agentexhibited only a moderate suppression of tumor growth(P = 0.036); PEITC alone at the dosage of 25 mg/kg slightlydecreased the tumor weight but this effect was not statisticallysignificant (P = 0.127). Strikingly, combination of both compoundsshowed a dramatic synergistic anticancer effect that reduced theaveraged tumor size by more than 80% (Fig. 8A). The potent in vivo

therapeutic effect of CDDP and PEITC combination was furtherevident by the flat tumor growth curve (Fig. 8B). This drugcombination caused a slight decrease in body weight, which wasreversible (Fig. 8C). Overall, combination of PEITC and CDDPexhibited highly synergistic therapeutic activity against the CDDP-resistant tumor.

4. Discussion

Platinum-based chemotherapy is an important treatmentregimen used in clinical management of various cancers of

Fig. 7. PEITC caused an increase in Pt accumulation in Pt-resistant cancer cells. (A and B) Total intracellular Pt content in Pt resistant cells (A549/CDDP cells (A); THC8307/L-

OHP cells (B)) after 12 h and 24 h incubation with Pt drug or/and PEITC. Columns, mean (n = 3); bars, SD; **P < 0.01; the column labeled with an ‘‘�’’ indicate no Pt was

presented. (C and D) DNA-binding Pt content in Pt resistant cells (A549/CDDP cells (C); THC8307/L-OHP cells (D)) after 12 h and 24 h incubation with cisplatin or/and PEITC.

Columns, mean (n = 3); bars, SD; **P < 0.01. (E and F) Western blotting analysis of g-H2AX expression induced by platinum or/and PEITC in the same cells after 12–24 h drug

exposure (n = 3) and the quantitative data was shown.

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different tissue types, and development of drug resistance toplatinum treatment presents a major challenge [1–9]. Even withthe newer generation of platinum drugs such as oxaliplatin, cross-resistance has still been observed [41,42]. Thus, understanding theunderlying mechanisms responsible for platinum resistance anddeveloping new strategies to overcome such drug resistance arehighly important. Cancer cells may become resistant to Pt drugsthrough multiple mechanisms such as acquisition of highercapacity to repair DNA damage caused by platinum, neutralizationof Pt toxicity, and an increase in drug export. Glutathione (GSH) isthe most abundant cellular antioxidant and plays a major role inpromoting cell survival and causing resistance to Pt drugs, owing toits ability to form conjugates with Pt compounds and thus

neutralize the drug toxicity and promote their export [26]. Theimportant role of GSH in drug resistance is further underscored bythe recent finding that cancer stem cells (CSC), which are known tobe resistant to chemotherapeutic agents and radiation [43,44],seem to have higher capacity to synthesize GSH. Thus, abrogationof the GSH-mediated drug resistant mechanism may constitutean effective strategy to improve the activity of Pt-basedchemotherapy.

Our study revealed that two Pt-resistant cell lines, A549/CDDPand THC8307/L-OHP, had significantly higher cellular GSH levelscompared to their parental cells. Interestingly, these drug-resistantcells also exhibited certain features of stem-cell like EMTproperties, exhibiting mesenchymal cell morphology, increase in

Fig. 8. PEITC combined with cisplatin suppressed tumor growth in vivo. (A) On day 60 after chemotherapy, xenografts were excised from mice and weighed, as shown in the

right panel. Each dot represented a tumor weight, the mean tumor weights of each group were indicated by solid lines (left panel; n = 8), and P values were obtained using the

Student’s t-test. (B) The volume of tumors were measured and recorded twice a week, and tumor growth curve was created for each group (n = 8). Dots, mean; bars, SD;

***P < 0.001. (C) The weight of mice were measured and recorded twice a week, and the weight curve was created for each group (n = 8). Dots, mean; bars, SD.

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cellular motility, decrease in E-cadherin expression, and increasedexpression of vimentin and N-cadherin. These data support thenotion that Pt-resistance is associated with epithelial to mesen-chymal transitions and that the stem-cell like cancer cellsintrinsically have a higher level of GSH. Although the role ofGSH in drug resistance has been known for some time [26], thefindings that GSH was higher in stem-cell like cancer cells withdrug-resistant phenotype and that depletion of GSH significantlyincreased the therapeutic activity against these cells add a newdimension in our understanding of drug resistance mechanismsand new intervention strategies. Considering the important role ofcancer stem cells in drug resistance and disease recurrence,development of new strategies to effectively kill this subpopula-tion of cancer cells is of particular importance for improving theoutcomes of cancer treatment.

In vitro studies had suggested that certain isothiocyanates(ITCs) seem able to enhance the cytotoxic effect of cisplatininvolving various mechanisms such as activation of ERK, inhibitionof NF-kB, and induction of b-tubulin degradation [40,45,46].However, the in vivo synergetic effect remains unknown and thereare no reports on sensitization of oxaliplatin by ITCs. In the currentstudy, we firstly employed the cisplatin and oxaliplatin resistantcell lines to investigate the role of PEITC, one of the ITCs, inovercoming Pt resistance both in vitro and in vivo. Our studydemonstrated that PEITC efficiently reversed Pt-resistance thoughdepletion of cellular GSH. This conclusion was supported bymultiple lines of evidence, which include: (1) a sub-toxicconcentration of PEITC (5 mM) was able to cause a depletion of50% cellular GSH in 6 h; (2) this depletion of GSH led to a significantincrease of cellular Pt accumulation as well as higher Pt bound toDNA by approximately 100%; (3) combination of a sub-toxicconcentration of PEITC with cisplatin or oxaliplatin significantly

enhanced their cytotoxic effect against the Pt-resistant cells both in

vitro and in vivo.It should be noted that PEITC, through its ability to accumulate

in the cells and conjugate with GSH to promote its export [33], isable to deplete cellular GSH within several hours (Fig. 4C–Fig. 3D).This is quite different from the kinetics of GSH depletion inducedby buthionine sulfoximine (BSO), which inhibits GSH synthesis andthus causes slow GSH depletion due to the ability of cells to re-usethe existing glutathione by recycling the oxidized GSSG to GSH(reduced form) by the glutathione reductase system. A slowdepletion of GSH may allow sufficient time for the cancer cells toadapt to such redox changes and up-regulate their survivalmechanisms, whereas a rapid depletion of GSH is likely to be moreeffective in disabling this drug resistant mechanism. This mayexplain why BSO is an effective inhibitor but yet shows limitedanticancer activity in clinical trials [47]. As such, it is possible thatPEITC may show superior anticancer activity compared to BSO andlikely to be effective in overcoming Pt-resistance.

However, cautions should be exercised in considering such drugcombination for clinical treatment of cancer patients due topotential toxic side effects. Our in vivo study revealed thattreatment of mice with CDDP and PEITC combined caused smallbut observable loss of mouse body weight which was reversible,whereas no weight loss was seen in mice treated with eithercompound alone. Further investigation is required to test theoptimal drug combination conditions (drug dosage and injectionschedule) that produce effective anticancer activity with minimumtoxic side effect.

In summary, our study revealed that Pt-resistant cancer cellswith EMT-like properties have a significant increase in theircellular GSH, which appeared to play a key role in drug resistance.We further showed that rapid depletion of GSH by PEITC was able

W.- Wu et al. / Biochemical Pharmacology 85 (2013) 486–496496

to increase cellular accumulation of Pt and effectively overcomethe drug resistant mechanism. Combination of PEITC and Ptexhibited significant synergetic anticancer activity against the Pt-resistant cells both in vitro and in vivo. Our study identified apromising therapeutic strategy in overcoming drug resistance ofplatinum-based chemotherapy. Further research work in this areais needed to evaluate the feasibility of using such drug combina-tions in clinical treatment of cancers that are resistant toconventional platinum therapy.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was funded by National Nature Science Foundation ofChina (No. 30672408 to Xu RH). We thank Prof. Li X of Tianjin LifeScience Research Center for providing us with THC8307 cells andTHC8307/L-OHP cells.

References

[1] Rosenberg B, VanCamp L, Trosko JE, Mansour VH. Platinum compounds: a newclass of potent antitumour agents. Nature 1969;222:385–6.

[2] Spigel DR, Greco FA. Chemotherapy in metastatic and locally advanced non-small cell lung cancer. Semin Surg Oncol 2003;21:98–110.

[3] Vasey PA, Paul J, Birt A, Junor EJ, Reed NS, Symonds RP, et al. Docetaxel andcisplatin in combination as first-line chemotherapy for advanced epithelialovarian cancer. Scottish Gynaecological Cancer Trials Group. J Clin Oncol1999;17:2069–80.

[4] Kim SW, Suh C, Lee SD, Kim WS, Kim DS, Kim WD, et al. Weekly low dosepaclitaxel and cisplatin as first-line chemotherapy for advanced non-small celllung cancer. Lung Cancer 2003;41:221–6.

[5] Giacchetti S, Perpoint B, Zidani R, Le Bail N, Faggiuolo R, Focan C, et al. Phase IIImulticenter randomized trial of oxaliplatin added to chronomodulated fluo-rouracil-leucovorin as first-line treatment of metastatic colorectal cancer. JClin Oncol 2000;18:136–47.

[6] Korita PV, Wakai T, Shirai Y, Matsuda Y, Sakata J, Takamura M, et al. Multidrugresistance-associated protein 2 determines the efficacy of cisplatin in patientswith hepatocellular carcinoma. Oncol Rep 2010;23:965–72.

[7] Wang J, Zhou JY, Zhang L, Wu GS. Involvement of MKP-1 and Bcl-2 in acquiredcisplatin resistance in ovarian cancer cells. Cell Cycle 2009;8:3191–8.

[8] Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, et al. Molecularmechanisms of cisplatin resistance. Oncogene 2011.

[9] Koike K, Kawabe T, Tanaka T, Toh S, Uchiumi T, Wada M, et al. A canalicularmultispecific organic anion transporter (cMOAT) antisense cDNA enhancesdrug sensitivity in human hepatic cancer cells. Cancer Res 1997;57:5475–9.

[10] Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer2005;5:275–84.

[11] Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stemcells promote radioresistance by preferential activation of the DNA damageresponse. Nature 2006;444:756–60.

[12] Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY. CD133+ HCC cancer stem cellsconfer chemoresistance by preferential expression of the Akt/PKB survivalpathway. Oncogene 2008;27:1749–58.

[13] Wulf GG, Wang RY, Kuehnle I, Weidner D, Marini F, Brenner MK, et al. Aleukemic stem cell with intrinsic drug efflux capacity in acute myeloidleukemia. Blood 2001;98:1166–73.

[14] He K, Xu T, Goldkorn A. Cancer cells cyclically lose and regain drug-resistanthighly tumorigenic features characteristic of a cancer stem-like phenotype.Mol Cancer Ther 2011;10:938–48.

[15] Ishida S, McCormick F, Smith-McCune K, Hanahan D. Enhancing tumor-specific uptake of the anticancer drug cisplatin with a copper chelator. CancerCell 2010;17:574–83.

[16] Yashiro M, Inoue T, Nishioka N, Matsuoka T, Boland CR, Hirakawa K. Allelicimbalance at p53 and microsatellite instability are predictive markers forresistance to chemotherapy in gastric carcinoma. Ann Surg Oncol2009;16:2926–35.

[17] Li ZH, Qiu MZ, Zeng ZL, Luo HY, Wu WJ, Wang F, et al. Copper-transporting P-type adenosine triphosphatase (ATP7A) is associated with platinum-resis-tance in non-small cell lung cancer (NSCLC). J Transl Med 2012;10:21.

[18] Tang H, Liu YJ, Liu M, Establishment Li X. gene analysis of an oxaliplatin-resistant colon cancer cell line THC8307/L-OHP. Anticancer Drugs2007;18:633–9.

[19] Yamasaki M, Makino T, Masuzawa T, Kurokawa Y, Miyata H, Takiguchi S, et al.Role of multidrug resistance protein 2 (MRP2) in chemoresistance and clinicaloutcome in oesophageal squamous cell carcinoma. Br J Cancer 2011;104:707–13.

[20] Tiwari AK, Sodani K, Dai CL, Ashby Jr CR, Chen ZS. Revisiting the ABCs ofmultidrug resistance in cancer chemotherapy. Curr Pharm Biotechnol2011;12:570–94.

[21] Shi Z, Tiwari AK, Shukla S, Robey RW, Singh S, Kim IW, et al. Sildenafil reversesABCB1- and ABCG2-mediated chemotherapeutic drug resistance. Cancer Res2011;71:3029–41.

[22] Chen HH, Kuo MT. Role of glutathione in the regulation of cisplatin resistancein cancer chemotherapy. Met Based Drugs 2010;2010.

[23] Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC)transporter superfamily. Genome Res 2001;11:1156–66.

[24] Yao KS, Godwin AK, Johnson SW, Ozols RF, O‘Dwyer PJ, Hamilton TC. Evidencefor altered regulation of gamma-glutamylcysteine synthetase gene expressionamong cisplatin-sensitive and cisplatin-resistant human ovarian cancer celllines. Cancer Res 1995;55:4367–74.

[25] Dedoussis GV, Andrikopoulos NK. Glutathione depletion restores the suscep-tibility of cisplatin-resistant chronic myelogenous leukemia cell lines toNatural Killer cell-mediated cell death via necrosis rather than apoptosis.Eur J Cell Biol 2001;80:608–14.

[26] Ishikawa T, Ali-Osman F. Glutathione-associated cis-diamminedichloroplati-num(II) metabolism and ATP-dependent efflux from leukemia cells. Molecularcharacterization of glutathione-platinum complex and its biological signifi-cance. J Biol Chem 1993;268:20116–25.

[27] Chen HH, Song IS, Hossain A, Choi MK, Yamane Y, Liang ZD, et al. Elevatedglutathione levels confer cellular sensitization to cisplatin toxicity by up-regulation of copper transporter hCtr1. Mol Pharmacol 2008;74:697–704.

[28] Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, et al. Association ofreactive oxygen species levels and radioresistance in cancer stem cells. Nature2009;458:780–3.

[29] Cheung KL, Kong AN. Molecular targets of dietary phenethyl isothiocyanateand sulforaphane for cancer chemoprevention. AAPS J 2010;12:87–97.

[30] Hecht SS. Chemoprevention by isothiocyanates. J Cell Biochem Suppl1995;22:195–209.

[31] Szumilo J. Natural compounds in chemoprevention of esophageal squamouscell tumors—experimental studies. Pol Merkur Lekarski 2009;26:156–61.

[32] Trachootham D, Zhang H, Zhang W, Feng L, Du M, Zhou Y, et al. Effectiveelimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism. Blood 2008;112:1912–22.

[33] Trachootham D, Zhou Y, Zhang H, Demizu Y, Chen Z, Pelicano H, et al. Selectivekilling of oncogenically transformed cells through a ROS-mediated mecha-nism by beta-phenylethyl isothiocyanate. Cancer Cell 2006;10:241–52.

[34] Hu Y, Lu W, Chen G, Zhang H, Jia Y, Wei Y, et al. Overcoming resistance tohistone deacetylase inhibitors in human leukemia with the redox modulatingcompound beta-phenylethyl isothiocyanate. Blood 2010;116:2732–41.

[35] Batra S, Sahu RP, Kandala PK, Srivastava SK. Benzyl isothiocyanate-mediatedinhibition of histone deacetylase leads to NF-kappaB turnoff in human pan-creatic carcinoma cells. Mol Cancer Ther 2010;9:1596–608.

[36] Gupta P, Srivastava SK. Antitumor activity of phenethyl isothiocyanate inHER2-positive breast cancer models. BMC Med 2012;10:80.

[37] Xiao D, Singh SV. Phenethyl isothiocyanate sensitizes androgen-independenthuman prostate cancer cells to docetaxel-induced apoptosis in vitro and invivo. Pharm Res 2010;27:722–31.

[38] Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediatedmechanisms: a radical therapeutic approach. Nat Rev Drug Discov2009;8:579–91.

[39] Lv XB, Xie F, Hu K, Wu Y, Cao LL, Han X, et al. Damaged DNA-binding protein 1(DDB1) interacts with Cdh1 and modulates the function of APC/CCdh1. J BiolChem 2010;285:18234–40.

[40] Di Pasqua AJ, Hong C, Wu MY, McCracken E, Wang X, Mi L, et al. Sensitization ofnon-small cell lung cancer cells to cisplatin by naturally occurring isothio-cyanates. Chem Res Toxicol 2010;23:1307–9.

[41] Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat RevDrug Discov 2005;4:307–20.

[42] Stordal B, Pavlakis N, Davey R. Oxaliplatin for the treatment of cisplatin-resistant cancer: a systematic review. Cancer Treat Rev 2007;33:347–57.

[43] Ishikawa F, Yoshida S, Saito Y, Hijikata A, Kitamura H, Tanaka S, et al.Chemotherapy-resistant human AML stem cells home to and engraft withinthe bone-marrow endosteal region. Nat Biotechnol 2007;25:1315–21.

[44] Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: accumulatingevidence and unresolved questions. Nat Rev Cancer 2008;8:755–68.

[45] Wang X, Govind S, Sajankila SP, Mi L, Roy R, Chung FL. Phenethyl isothiocya-nate sensitizes human cervical cancer cells to apoptosis induced by cisplatin.Mol Nutr Food Res 2011;55:1572–81.

[46] d’Avenia M, Rosati A, Belisario MA, Torino M, Torino G, Turco MC, et al. Theexpression of the pro-apoptotic gene Air is inducible in human pancreaticadenocarcinoma cells. J Cell Physiol 2011;226:2207–12.

[47] Bailey HH, Mulcahy RT, Tutsch KD, Arzoomanian RZ, Alberti D, Tombes MB, et al.Phase I clinical trial of intravenous L-buthionine sulfoximine and melphalan: anattempt at modulation of glutathione. J Clin Oncol 1994;12:194–205.