CYP2S1 and CYP2W1 Mediate 2-(3,4-Dimethoxyphenyl)-5- Fluorobenzothiazole (GW-610, NSC 721648) Sensitivity in Breast and Colorectal Cancer Cells

Published OnlineFirst August 10, 2011; DOI: 10.1158/1535-7163.MCT-11-0391

Molecular Cancer Preclinical Development Therapeutics

Boon Shing Tan1, Kai Hung Tiong1, Ashwin Muruhadas2, Nirmal Randhawa2, Heng Lungh Choo1, Tracey D. Bradshaw4, Malcolm F.G. Stevens4, and Chee-Onn Leong3

Abstract Both 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F-203; NSC 703786) and 2-(3,4-dimethoxyphenyl)-5-fluorobenzothiazole (GW-610; NSC 721648) are antitumor agents with novel mechanism(s). Previous studies have indicated that cytochrome (CYP) P450 1A1 is crucial for 5F-203 activity. In the present study, we investigated the functional role of 2 newly identified CYP P450 enzymes, CYP2S1 and CYP2W1, in mediating antitumor activity of benzothiazole compounds. We generated isogenic breast cancer (MDA-MB-468, MCF-7) and colorectal cancer (CRC; KM12 and HCC2998) cell lines depleted for CYP1A1, CYP2S1, or CYP2W1. The sensitivity of these cells to 5F-203 and GW-610 was then compared with vector control cells. 5F-203 exhibited potent activity against breast cancer cells, whereas GW-610 was effective against both breast and colorectal cancer cells. CYP1A1 was induced in both breast cancer and CRC cells, while CYP2S1 and CYP2W1 were selectively induced in breast cancer cells only following treatment with 5F-203 or GW-610. Depletion of CYP1A1 abrogated the sensitivity of breast cancer and CRC cells to 5F-203 and GW-610. Although depletion of CYP2S1 sensitized both breast cancer and CRC cells toward 5F-203 and GW-610, CYP2W1 knockdown caused marked resistance to GW-610 in CRC cells. Our results indicate that CYP-P450 isoforms, with the exception of CYP1A1, play an important role in mediating benzothiazole activity. CYP2S1 appears to be involved in deactivation of benzothiazoles, whereas CYP2W1 is important for bioactivation of GW-610 in CRC cells. Because CYP2W1 is highly expressed in colorectal tumors, GW-610 represents a promising agent for CRC therapy. Mol Cancer Ther; 10(10); 1982–92. 2011 AACR.

Introduction CYP1A1-catalyzed bioactivation to electrophilic species in sensitive phenotypes (8). A study comparing Previously, we have described the potent and selec- efficacy of 2-(4-amino-3-methylphenyl)-5-fluoroben- tive antitumor activity of 2-(4-aminophenyl)benzothia- zothiazole (5F-203; Fig. 1A), lysyl amide (Phortress), zoles in vitro (1, 2) and in vivo (3–5). Sensitivity to these and doxorubicin in mammary xenografts revealed agents is clustered within breast [irrespective of estro- Phortress to be equiactive against 6 xenograft models, gen receptor (ER) status] and ovarian human tumor outperforming doxorubicin in 1 model (5). Significant models and correlates with cytochrome P450 1A1 in vivo antitumor activity has also been seen in IGROV- (CYP1A1) inducibility within these tumors (6, 7). As 1 ovarian xenografts. Worldwide, 1.38 million women aryl hydrocarbon receptor (AhR) ligands, aminophe- were diagnosed with breast cancer in 2008. Although nylbenzothiazoles act as both inducer and substrate for the 10-year survival rate is greater than 75%, 458,000 women were killed by this disease in the same year. Ovarian cancer represents 2% of all cancers diagnosed Authors' Affiliations: 1School of Postgraduate Studies and Research, and has a 5-year survival rate of less than 40%. Thus, 2School of Medicine, 3School of Pharmacy and Health Sciences, Interna- discovery and development of novel agents to treat 4 tional Medical University, Bukit Jalil, Kuala Lumpur, Malaysia; and School these malignancies are required. Phortress is currently of Pharmacy, University of Nottingham, Nottingham, United Kingdom undergoing clinical evaluation under the auspices of Note: Supplementary material for this article is available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Cancer Research UK. Subsequent synthesis of a series of dimethoxyphe- B.S. Tan and K.H. Tiong contributed equally to this work. nylbenzothiazoles led to identification of a novel anti- Corresponding Author: Chee-Onn Leong, School of Pharmacy and Health Sciences, International Medical University, 126 Jalan 19/155B, tumor pharmacophore bearing oxygen substituents. Bukit Jalil, 57000 Kuala Lumpur, Malaysia. Phone: 603-2731-7528; Fax: Theleadcompoundofthisseries, 2-(3,4-dimethoxy- 603-8656-7229; E-mail: [email protected] phenyl)-5-fluorobenzothiazole (GW-610; NSC 721648; doi: 10.1158/1535-7163.MCT-11-0391 Fig. 1A), elicits strikingly selective in vitro growth- 2011 American Association for Cancer Research. inhibitory activity after 48-hour exposure to GW-610;

1982 Mol Cancer Ther; 10(10) October 2011

Role of CYP2S1 and CYP2W1 in GW-610 Antitumor Activity

A 5F-203 GW-610 CH 3 OMe F N F N NH OMe 2 S S 120 120

Figure 1. Selective growth- 100 100 inhibitory activities of 5F-203 and GW-610 and CYP1A1, CYP2S1, 80 80 and CYP2W1 basal expression in breast cancer and CRC cells. A, 60 60 breast cancer cells (MDA-MB-468 MDA-MB-468 and MCF-7), CRC cells (KM12 and MDA-MB-468 % Viability 40 MCF-7 % Viability 40 HCC2998), and human lung MCF-7 KM12 fibroblast cells (MRC-5) were KM12 20 HCC2998 20 HCC2998 treated with different MRC-5 MRC-5 concentrations of 5F-203 and 0 0 GW-610 for 72 hours and cell 1E-080 0.00001 0.01 10 viability was determined by MTT 1E-080 0.00001 0.01 10 assays. Points represent the Concentration (μmol/L) Concentration (μmol/L) mean SD of 3 independent experiments. B, basal mRNA expression of CYP1A1, CYP2S1, and CYP2W1 in MDA-MB-468, B C MCF-7, KM12, and HCC2998 cells as determined by real-time 10 qPCR. C, CYP1A1, CYP2S1, and CYP1A1 CYP2W1 protein expression in 8 CYP2S1 CYP1A1 MDA-MB-468, MCF-7, KM12, and CYP2W1 HCC2998 cells. 6 CYP2S1

4 mRNA levels 2 CYP2W1

Log 2 α-Actin 0 MDA-MB-468 MCF-7 KM12 HCC2998

exquisite sensitivity was observed in colon as well as hypomethylationinthebodyofCYP2W1 results in certain non–small-cell lung, breast, ovarian, and renal enhanced protein expression. Edler and colleagues cancer cell lines in the NCI human-derived 60 cell line reported that 36% CRCs expressed high CYP2W1 pro- < panel (GI50 values 100 nmol/L; Supplementary tein levels, which correlated with worse clinical out- Fig. S1). Thus, the spectrum of activity of this bis-methyl come (P ¼ 0.007; ref. 13). Thus, expression of CYP2W1 is ether extends to human cell lines derived from intrac- an independent prognostic factor in patients with stage table colorectal carcinomas (CRC). A potent AhR ligand II or stage III CRC. CYP2S1 expression is found in K ¼ ( i 6.8 nmol/L), GW-610 is sequestered exclusively epithelial cells that are targets for carcinogen exposure and rapidly by sensitive breast and CRC cells only including epithelia of the respiratory and gastrointes- (9, 10). tinal tracts (common sites of tumorigenesis; refs. 14–16). CRC is the third most common cancer worldwide and Indeed, strong CYP2S1 expression has been detected in caused more than 600,000 deaths globally in 2008. tumors of epithelial origin (including CRC and lung; Successful surgery underlies the 5-year survival rate refs. 14, 17, 18). statistics which hover around 50% for both sexes. Thus, In this article, we describe experiments undertaken to for inoperable CRC, discovery of novel chemotherapeu- elucidate the roles, if any, of novel AhR-inducible CYP tic agents is imperative. It has recently been reported isoforms 2S1 and 2W1 in biotransformation of GW-610 that novel AhR-inducible cytochrome P450 2W1 and 5F-203, in 4 human-derived carcinoma cell lines–- þ (CYP2W1) is overexpressed in human colon cancer ER MCF-7, ER MDA-MB-468 breast, and HCC2998, tissue (11, 12). CYP2W1 expression is tumor-specific KM12 CRC cells. The role of CYP1A1 in benzothiazole and regulated by DNA methylation status, that is, bioactivation has been compared.

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Materials and Methods Table 1. IC50 values of 5F-203 and GW-610 in breast cancer and CRC cells Cell lines and cell culture

MDA-MB-468 and MCF-7 humanbreast cancercell lines IC50, mmol/L and MRC-5 human embryonic lung fibroblast cells were purchased from the American Type Culture Collection. 5F-203 GW-610 KM12 and HCC2998 CRC cell lines were kind gifts of the National Cancer Institute, Bethesda, MD. All cells were MDA-MB-468 0.052 0.010 0.073 0.010 expanded on receipt and tested for mycoplasma using MCF-7 0.761 0.166 0.363 0.051 MycoAlert Mycoplasma Detection Assay (Lonza) before KM12 >10 0.079 0.015 banking in liquid nitrogen. All cells were maintained in HCC2998 >10 0.079 0.007 RPMI-1640 medium containing 10% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin (Sigma-Aldrich) at 37C, 5% CO . Cells underwent passage in culture for described previously (8, 19, 20). Cells were seeded in 2 3 less than a month and no further authentication was done 96-well plates for 24 hours at a density of 2 10 cells per by the authors. well. Concentrated (100 mmol/L) solutions of 5F-203 and GW-610 were prepared in dimethyl sulfoxide. Cell via- MTT cell proliferation assay bility after 72 hours of drug exposure was determined by

Concentration–response curves and IC50 values were cell-mediated MTT reduction. Cell growth and drug determined using the MTT cell viability assay as activity was determined by measuring absorbance at

A 5F-203 GW-610 16 16 Control Control 14 14 100 nmol/L 100 nmol/L 12 12 1 μmol/L 1 μmol/L 10 10 8 8 6 6 Fold change Fold Fold change Fold CYP1A1 4 4 2 2 0 0 Figure 2. Induction of CYP1A1, MDA-MB- MCF-7 KM12 HCC2998 MDA-MB- MCF-7 KM12 HCC2998 468 468 CYP2S1, and CYP2W1 mRNA in breast cancer and CRC cells B following treatment with 5F-203 and GW-610. MDA-MB-468, 8 Control 8 Control 100 nmol/L 100 nmol/L MCF-7, KM12, and HCC2998 1 μmol/L 1 μmol/L cells were treated with 100 nmol/L 6 6 or 1 mmol/L of 5F-203 or GW-610 for 48 hours followed by 4 4 measurement of mRNA expression by qPCR. A, CYP1A1 CYP2S1 Fold change Fold Fold change Fold 2 2 was induced by 5F-203 and GW- 610 in all cell lines tested, whereas CYP2S1 (B) and CYP2W1 (C) were 0 0 MDA-MB- MCF-7 KM12 HCC2998 MDA-MB- MCF-7 KM12 HCC2998 induced only in breast cancer cell 468 468 lines after 5F-203 and GW-610 treatment. Bars represent the C 7 7 mean SD of 3 independent Control Control experiments. 6 6 100 nmol/L 100 nmol/L 5 1 μmol/L 5 1 μmol/L

4 4

3 3 Fold change Fold Fold change Fold CYP2W1 2 2

1 1

0 0 MDA-MB- MCF-7 KM12 HCC2998 MDA-MB- MCF-7 KM12 HCC2998 468 468

1984 Mol Cancer Ther; 10(10) October 2011 Molecular Cancer Therapeutics

Role of CYP2S1 and CYP2W1 in GW-610 Antitumor Activity

MDA-MB-468 MCF-7

5F-203 GW-610 5F-203 GW-610

Control 100 nmol/L1 μmol/L Control 100 nmol/L1 μmol/L Control 100 nmol/L1 μmol/L Control 100 nmol/L1 μmol/L CYP1A1

Figure 3. Induction of CYP1A1, CYP2S1 CYP2S1, and CYP2W1 protein in breast cancer and CRC cells following treatment with 5F-203 CYP2W1 and GW-610. All cells were treated m with 100 nmol/L or 1 mol/L of α-Actin 5F-203 or GW-610 for 48 hours and their protein isolated as described in Materials and Methods. Immunoblots were KM12 HCC2998 incubated overnight with primary antibodies in 5% bovine serum 5F-203 GW-610 5F-203 GW-610 albumin followed by chemiluminescence detection. Each blot was transferred and mol/L mol/L mol/L mol/L exposed separately. Control 100 nmol/L1 μ Control 100 nmol/L1 μ Control 100 nmol/L1 μ Control 100 nmol/L1 μ CYP1A1

CYP2S1

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α-Actin

570 nm using a TECAN Infinite F200 plate reader protease inhibitors in PBS). Total protein (50 mg) (Mannedorf).€ was subjected to SDS-PAGE followed by immunoblotting. Primary polyclonal antibodies against Quantitative real-time PCR analysis CYP1A1 (ab3568; 1:1,000), CYP2S1 (ab69650; 1:1,000), Total RNA from cells was extracted using the Qiagen and CYP2W1 (ab76666; 1:100) were obtained from RNA Isolation Kit (Qiagen) and first-strand cDNA was Abcam. Mouse monoclonal antibody against a-actin synthesized using High Capacity RNA-to-cDNA Master (clone C-2; 1:250) was obtained from Santa Cruz Mix (Applied Biosystems) according to the man- Biotechnology. ufacturer’s instructions. Gene expression levels were mea- sured by real-time quantitative PCR (qPCR) using the Lentiviral production and transduction FastStart Universal SYBR Green Master Reagent (Roche) Lentiviral short-hairpin RNA (shRNA) constructs and a Biorad iQ5 real-time PCR detector system (Bio-Rad). targeting CYP1A1, CYP2S1, and CYP2W1 were pur- Data analysis was carried out using Biorad iQ5 Optical chased from Sigma-Aldrich. High-titer lentiviral System Software V1.0. The specific forward and reverse stocks were generated by cotransfection with pack- primer sequences are shown in Supplementary Table S1. aging plasmids psPAX2 (Addgene plasmid 12260) The conditions for all qPCR reactions were as follows: 3 and envelope plasmids pMD2.G (Addgene plasmid minutes at 94C followed by 40 seconds at 94C, 40 12259) into HEK-293T cells as reported previously seconds at 60C, and 25 seconds at 72C for 40 cycles. (19–23). The shRNA target sequences for CYP1A1, The expression data was normalized against GAPDH as CYP2S1, and CYP2W1 are shown in Supplementary housekeeping gene. Table S2. Stable pools were generated by transduction of 2 independent lentiviral shRNA constructs target- Protein isolation and Western blot analysis ing each CYP isoform in the breast cancer and Protein lysates from cells were extracted in ice- CRC cells, followed by brief drug selection with cold lysis buffer (0.75% NP-40, 1 mmol/L DTT, and puromycin.

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A MDA-MB-468 MCF-7 KM12 HCC2998

Figure 4. Efficient knockdown of CYP1A1 CYP1A1, CYP2S1, and CYP2W1 by lentiviral shRNA. Stable pool of α -Actin isogenic cell lines depleted for CYP1A1 (A), CYP2S1 (B), or CYP2W1 (C) were generated by lentiviral shRNA transduction B targeting the individual cytochrome P450 isoform followed by brief drug selection. CYP2S1 The resulting cells that stably express the cytochrome-P450 α -Actin shRNA isoforms were subjected for immunoblotting to confirm the degree of knockdown. Efficient and stable knockdown of endogenous CYP1A1, CYP2S1, C and CYP2W1 in MDA-MB-468, MCF-7, KM12, and HCC2998 cells was noted. CYP2W1

α -Actin

Results DNA damage (6, 8, 24). However, the expression of the newly identified extrahepatic CYP2S1 and CYP2W1 in Selective sensitivity of cancer cells to 5F-203 and breast cancer and CRC cell lines was not investigated. GW-610 Using highly sensitive real-time qPCR and chemilumi- To show the selective antitumor activity of 5F-203 and nescence immunoblotting, we showed that CYP1A1, GW-610, we compared their antiproliferative activities in CYP2S1, and CYP2W1 mRNA and protein are expressed breast cancer cells (MDA-MB-468 and MCF-7), CRC cells in breast cancer (MDA-MB-468 and MCF-7) and CRC (KM12 and HCC2998), and normal lung fibroblasts (KM12 and HCC2998) cells but not in MRC-5 lung fibro- (MRC-5). As shown in Fig. 1A, 5F-203 elicits activity blast (data not shown). The highest expression of against MDA-MB-468 and MCF-7 breast cancer cell lines CYP1A1 and CYP2W1 was observed in MDA-MB-468 < m (IC50 1 mol/L) whereas CRC cells, KM12 and and MCF-7 cells, respectively (Fig. 1B and C). Interest- > HCC2998, were relatively resistant to 5F-203 (IC50 10 ingly, high expression of CYP2S1 was observed mainly mmol/L; Table 1). Similarly, GW-610 also shows potent in the CRC cells, whereas only very low CYP2S1 protein antitumor activity against the 2 breast cancer cell lines. levels are present in breast cancer cells (Fig. 1B and C). However, unlike 5F-203, GW-610 also potently inhibits When basal expression of these CYP isoforms was the growth of KM12 and HCC2998 CRC cells (IC50 values compared with 5F-203 and GW-610 cellular sensitivity, < 0.1 mmol/L). These results are consistent with the NCI- no direct correlation was observed. These results sug- 60 cell lines screen, which showed selectivity of 5F-203 gest that, although the CYP isoforms might play an and GW-610 against different tumor types (Supplemen- important role in the bioactivation of the compounds, tary Figs. S1 and 2). Notably, MRC-5 embryonic lung their endogenous levels might not be a good predictive fibroblasts are inherently resistant to 5F-203 and GW-610, marker for treatment response. demonstrating the selective growth-inhibitory activity of CYP1A1, CYP2S1, and CYP2W1 are induced benzothiazole agents. Estimated IC50 values of 5F-203 and GW-610 in various cell lines are shown in Table 1. by 5F-203 and GW-610 Potent AhR ligands, 5F-203 and GW-610 have been Expression of CYP1A1, CYP2S1, and CYP2W1 in shown to activate AhR and induce CYP1A1 expression in breast and CRC cell lines MCF-7 cells (25, 26). Recently, human CYP2S1 and Previous studies have shown that CYP1A1 is expressed CYP2W1 isoforms have also been shown to be transcrip- in breast cancer cell lines and is essential for bioactivation tionally regulated by AhR (14, 27, 28). As such, we of 5F-203 to active metabolites that are capable of causing investigated whether 5F-203 and GW-610 are able to

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Role of CYP2S1 and CYP2W1 in GW-610 Antitumor Activity

5F-203 GW-610 120 120 Vector 100 100 1A1si-1 1A1si-2 80 80

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Figure 5. Knockdown of CYP1A1 60 60 reduced 5F-203 and GW-610 sensitivity in breast cancer MCF-7 40 40 and CRC cells. Cells depleted 20 20 for CYP1A1 were treated with 1 nmol/L to 10 mmol/L of 5F-203 0 0 or GW-610 for 72 hours and their 120 120 cell viability was determined by 100 100

MTT assay. Bars represent the % Viability mean SD of 3 independent 80 80 experiments. Note that the effects of knockdown were consistently 60 60 reproduced using 2 independent shRNA constructs that target KM-12 40 40 different regions of the same gene. 20 20

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Concentration (nmol/L) induce CYP2S1 and CYP2W1 expression. To test this expression decreased following treatment of HCC2998 hypothesis, MDA-MB-468, MCF-7, KM12, and cells with 5F-203 (Fig. 2B and C). These results show that HCC2998 cells were treated with 100 nmol/L and 1 5F-203 and GW-610 are capable of inducing CYP1A1 in mmol/L 5F-203 or GW-610 for 48 hours and their mRNA both breast cancer and CRC cells, whereas CYP2S1 and and protein isolated for qPCR and immunoblotting anal- CYP2W1 were induced only in breast cancer cells. Thus, yses, respectively. the differential regulation of CYP1A1, CYP2S1, and InductionofCYP1A1mRNAandproteinwasob- CYP2W1 in breast cancer and CRC cells might play served in all cell lines on treatment of cells with 5F- an important role in the regulation of 5F-203 and 203 and GW-610, including KM12 and HCC2998 CRC GW-610 antitumor activity. cells which are inherently resistant to 5F-203 (Fig. 2A). CYP2S1 and CYP2W1 mRNA and protein expression CYP1A1 is required for the antitumor activity were also induced in MDA-MB-468 and MCF-7 breast of 5F-203 and GW-610 cancer cells following 5F-203 and GW-610 treatment To further investigate the role of CYP1A1, CYP2S1, (Figs. 2B and C and 3). In stark contrast, no induction and CYP2W1 in the regulation of 5F-203 and GW-610 of CYP2S1 or CYP2W1 was observed in KM12 and antitumor activities, we generated stable cell lines HCC2998 CRC cells. In fact, CYP2S1 and CYP2W1 after depletion of individual CYP isoforms. Cellular

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required for benzothiazole bioactivation (Fig. 5 and Table 2. IC50 values of 5F-203 and GW-610 in CYP1A1-, CYP2S1-, or CYP2W1-depleted Table 2). breast cancer and CRC cells CYP2S1 deactivates 5F-203 and GW-610 in breast

a and CRC cells IC50, mmol/L In contrast to CYP1A1 knockdown, depletion of CYP2S1 5F-203 GW-610 increased the sensitivity of MDA-MB-468, KM12, and HCC2998 to 5F-203 and GW-610 (Fig. 6 and Table 2). This MDA-MB-468 sensitization, however, was not observed in MCF-7 cells, Vector 0.058 0.017 0.059 0.009 which expressed only low CYP2S1 levels (Fig. 1C). Nota- CYP1A1si-1 0.877 0.071 0.799 0.275 bly, depletion of CYP2S1 in KM12 and HCC2998 cells, CYP1A1si-2 0.682 0.032 1.635 0.419 which are inherently resistant to 5F-203, caused a marked CYP2S1si-1 0.004 0.001 0.006 0.001 increase in sensitivity to 5F-203, suggesting that CYP2S1 CYP2S1si-2 0.006 0.001 0.009 0.002 might be involved in deactivation of benzothiazoles. CYP2W1si-1 0.092 0.003 0.092 0.020 CYP2W1si-2 0.084 0.005 0.097 0.022 CYP2W1 mediates GW-610 sensitivity in colorectal MCF-7 cancer cells Vector 0.811 0.198 0.467 0.141 Although depletion of CYP1A1 or CYP2S1 affected the CYP1A1si-1 >10 7.756 1.333 sensitivity of both breast and CRC cells to 5F-203 and CYP1A1si-2 >10 8.296 1.449 GW-610, CYP2W1 depletion impacted only GW-610 sen- CYP2S1si-1 0.889 0.141 0.433 0.079 sitivity in CRC cells. As shown in Fig. 7 and Table 2, CYP2S1si-2 0.716 0.200 0.490 0.278 knockdown of CYP2W1 reduced sensitivity to GW-610 in CYP2W1si-1 3.810 1.960 0.772 0.144 HCC2998 and KM12 more than 10- to 100-fold, respec- CYP2W1si-2 4.930 2.217 0.840 0.101 tively. However, no such effect was observed in KM12 CYP2W1-depleted breast cancer cells following treat- Vector >10 0.080 0.007 ment with GW-610. Moreover, there was no change in CYP1A1si-1 >10 0.761 0.127 sensitivity of breast cancer or CRC cells to 5F-203 follow- CYP1A1si-2 >10 0.897 0.024 ing CYP2W1 depletion (Fig. 7). These results suggest that CYP2S1si-1 4.118 1.208 0.009 0.001 CYP2W1 specifically mediates GW-610 sensitivity in CYP2S1si-2 6.218 0.855 0.008 0.001 CRC cells and might explain the distinctive selectivity CYP2W1si-1 >10 7.512 0.881 of GW-610 toward CRC cells. CYP2W1si-2 >10 7.989 0.918 HCC2998 Discussion Vector >10 0.060 0.010 CYP1A1si-1 >10 >10 Both 5F-203 and GW-610 are representative molecules CYP1A1si-2 >10 >10 of 2 series of antitumor agents distinct from all mecha- CYP2S1si-1 4.984 0.859 0.022 0.009 nistic classes of chemotherapeutic agents in current clin- CYP2S1si-2 8.873 0.047 0.034 0.022 ical use (8, 24, 29, 30). Exquisitely potent and highly CYP2W1si-1 >10 0.443 0.009 selective 5F-203 antitumor activity has been observed CYP2W1si-2 >10 0.554 0.010 in certain human tumor-derived models in vitro (including breast, renal, and ovarian cell lines) and in vivo against a Cells were treated with 5F-203 or GW-610 for 72 hours. breast and ovarian xenograft models (1, 5, 31). GW-610, Cell viability was determined by MTT assay. Values repre- on the other hand, exhibits a broader antitumor spectrum sent mean SD of at least 3 independent experiments. to include CRC and non–small-cell lung cancer, in addi- tion to breast, renal, and ovarian cancer cell lines (25). Because 5F-203 and GW-610 are potent ligands for sensitivity to 5F-203 and GW-610 was compared with AhR and have been postulated to be metabolically corresponding isogenic vector control cells. The mRNA activated by CYP1A1 leading to antitumor activities, and protein levels of CYP1A1, CYP2S1, and CYP2W1 we investigated whether other AhR-inducible cyto- in MDA-MB-468, MCF-7, KM12, and HCC2998 follow- chrome (CYP) P450 isoforms might be mediating the ing gene knockdown were confirmed by qPCR and sensitivity of cancer cells to benzothiazole compounds. immunoblotting, respectively (Fig. 4; Supplementary Using sensitive qPCR and immunoblotting, we showed Fig. S3). that the 5F-203 and GW-610 sensitivity in breast cancer Next, we tested whether knockdown of CYP1A1, and CRC cells does not correlate with CYP1A1 and CYP2S1, or CYP2W1 affected cellular sensitivity to CYP2W1 basal expression (Fig. 1A; Table 1). Interest- 5F-203 and GW-610. As expected, depletion of ingly, the 2 CRC cell lines, KM12 and HCC2998, which CYP1A1 markedly reduced the sensitivity of cancer were resistant to 5F-203 (but sensitive to GW-610) cells to 5F-203 and GW-610, suggesting that CYP1A1 is expressed high basal level of CYP2S1, suggesting that

1988 Mol Cancer Ther; 10(10) October 2011 Molecular Cancer Therapeutics

Role of CYP2S1 and CYP2W1 in GW-610 Antitumor Activity

5F-203 GW-610 120 120 Vector 100 100 2S1si-1 2S1si-2 80 80

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Figure 6. Knockdown of CYP2S1 40 40 MCF-7 increased 5F-203 and GW-610 20 20 sensitivity in MDA-MB-468, KM12, and HCC2998 cells but 0 0 not MCF-7 cells. Cells depleted 120 120 for CYP2S1 were treated with 1 nmol/L to 10 mmol/L of 5F-203 100 100 or GW-610 for 72 hours and their % Viability 80 80 cell viability was determined by MTT assay. Bars represent the 60 60 mean SD of 3 independent 40 40 experiments. KM-12

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CYP2S1 might play a role in 5F-203 or GW-610 benzothiazole to hydroxylated biotransformation pro- resistance (Fig. 1B and C). ducts devoid of activity (6). CYP2S1 (highly expressed To further understand the mechanism of action of 5F- in CRC cells)-catalyzed biotransformation represents a 203 and GW-610, we investigated the inducibility of plausible mechanism of metabolic deactivation (as dis- CYP1A1, CYP2S1, and CYP2W1 following 48-hour expo- cussed below). Interestingly, both CYP2S1 and CYP2W1 sure of cells to benzothiazole analogues. As expected, were also induced by 5F-203 and GW-610 (Figs. 2B and CYP1A1 was strongly induced in mammary carcinoma C and 3). Although AhR-inducible, unlike CYP1A1, cells following treatment with 5F-203 or GW-610 (Figs. 2A CYP2S1, and CYP2W1 further induction was observed and 3). Surprisingly, we also observed strong CYP1A1 only in breast, and not CRC, cancer cells. induction in KM12 and HCC2998 CRC cells which are To investigate the functional roles of CYP1A1, CYP2S1, inherently resistant to 5F-203. These results suggest that and CYP2W1 in mediating sensitivity of cancer cells to 5F-203 might be activated in CRC cells but its activity is 5F-203 and GW-610, we conducted gene knockdown by suppressed. Tumor-specific CYP1B1, for example, has lentiviral shRNA targeting each specific CYP P450 iso- been shown to metabolize 2-(4-amino-3-methylphenyl) forms. As expected, CYP1A1, which was postulated to

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5F-203 GW-610 120 120 Vector 100 100 2W1si-1 2W1si-2 80 80

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20 20 Figure 7. Knockdown of CYP2W1 reduced GW-610 sensitivity in 0 0 CRC cells only. Cells depleted for 120 120 CYP2W1 were treated with 1 nmol/L to 10 mmol/L of 5F-203 or 100 100

% Viability GW-610 for 72 hours and their cell 80 80 viability was determined by MTT assay. Bars represent the 60 60 mean SD of 3 independent experiments. KM-12 40 40

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0 0 01100 101 102 03 104 01100 101 102 03 104

Concentration (nmol/L)

metabolically activate benzothiazoles, is required for 5F- (10-fold) in MDA-MB-468 breast, KM12, and HCC2998 203 and GW-610 activity. Depletion of CYP1A1 from CRC cells. MCF-7 cells, which express very low CYP2S1 breast and CRC cells abrogated the antitumor activity levels, defied this trend (Fig. 6). Interestingly, the 2 CRC of 5F-203 and GW-610 (Figs. 4 and 5). This result is cell lines which expressed high endogenous level of consistent with a previous study which showed that CYP2S1 and are inherently resistant to 5F-203 became cotreatment with a CYP1A1 inhibitor, resveratrol, and sensitive to this agent after depletion of CYP2S1. These 5F-203 dramatically decreased 5F-203–induced CYP1A1 results suggest that CYP2S1 metabolically inactivates expression, correspondingly diminishing 5F-203 antitu- 5F-203 and GW-610 to an as yet unidentified secondary mor activity. Together, these results provide direct evi- metabolite. Reports show that human CYP2S1 plays dence that CYP1A1 induction is crucial for antitumor an important role in detoxification of many toxic activity of benzothiazoles. substrates to less toxic metabolites including ben- Similarly, gene knockdown was also carried out to zo[a]pyrene, 7,12-dimethylbenz[a]anthracene, aflatoxin investigate the role of CYP2S1 and CYP2W1 in 5F-203 B1, naphthalene, and styrene (32). Whether CYP2S1 is and GW-610 activity. Surprisingly, depletion of CYP2S1 targeting inactivation of parental 5F-203 and GW-610 or dramatically increased sensitivity to 5F-203 and GW-610 their CYP1A1-bioactivated electrophilic species remains

1990 Mol Cancer Ther; 10(10) October 2011 Molecular Cancer Therapeutics

Role of CYP2S1 and CYP2W1 in GW-610 Antitumor Activity

to be determined. However, on the basis of the observed offers tremendous potential for translation into a thera- induction of CYP1A1 in 5F-203–resistant CRC cells and peutic agent for CRC. sensitization of these cells following CYP2S1 depletion, it is likely that CYP2S1 metabolically inactivates the elec- Disclosure of Potential Conflicts of Interest trophilic species generated from benzothiazoles. M.F.G. Stevens and T.D. Bradshaw are listed as inventors on patents Knockdown of CYP2W1, on the other hand, signifi- covering the intellectual property of 5F-203 and GW-610. M.F.G. Stevens cantly reduced the sensitivity of CRC cells, but not breast and T.D. Bradshaw are consultants and shareholders of Pharminox Ltd., cancer cells, to GW-610. Interestingly, depletion of which has a commercial interest in antitumor benzothiazoles. CYP2W1 did not affect the sensitivity of breast and Acknowledgments CRC cells to 5F-203, suggesting that CYP2W1 is important for activation of GW-610 in CRC cells only. Thus, The authors would like to acknowledge Dr Geoff Wells (School of these results provide an alternative mechanism for Pharmacy, University of London, UK) for his original synthesis of GW- GW-610 activation distinct from 5F-203. 610. This is part 32 of the series on "Antitumour 2-(4-aminophenyl) benzothiazoles." In conclusion, we have showed that 5F-203 and GW-610 are capable of inducing CYP2S1 and CYP2W1 Grant Support in breast cancer cells. Knockdown of CYP2S1 sensitized both breast cancer and CRC cells to 5F-203 and GW-610, This work was financially supported by research grants from the Interna- suggesting that CYP2S1 is able to catalyze deactivation of tional Medical University (B.S. Tan, K.H. Tiong, H.L. Choo, and C.-O. Leong) and by the International Medical University BMedSci Research Training Program these agents. Knockdown of CYP2W1 markedly reduced (A. Muruhadas and N. Randhawa). GW-610 sensitivity in CRC cells only, implicating an The costs of publication of this article were defrayed in part by the payment important role for CYP2W1 in bioactivation of GW-610 of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. in CRC cells. Because of the exclusive metabolic activation of GW-610 by CYP2W1, which has recently been Received June 3, 2011; revised August 1, 2011; accepted August 3, 2011; shown to be a tumor-specific drug target in CRC, GW-610 published OnlineFirst August 10, 2011.

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1992 Mol Cancer Ther; 10(10) October 2011 Molecular Cancer Therapeutics

CYP2S1 and CYP2W1 Mediate 2-(3,4-Dimethoxyphenyl)-5-Fluorobenzothiazole (GW-610, NSC 721648) Sensitivity in Breast and Colorectal Cancer Cells

Boon Shing Tan, Kai Hung Tiong, Ashwin Muruhadas, et al.

Mol Cancer Ther 2011;10:1982-1992. Published OnlineFirst August 10, 2011.

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