Effect of Sulindac Sulfide on Metallohydrolases in the Human Colon Cancer Cell Line HT-29

Matrix metalloproteinase 7 (MMP7), a metallohydrolase involved in the development of several cancers, is downregulated in the ApcMin/+ colon cancer mouse model following sulindac treatment. To determine whether this effect is relevant to the human condition, HT-29 human colon cancer cells were treated with sulindac and its metabolites, and compared to results obtained from in vivo mouse studies. The expression of MMP7 was monitored. The results demonstrated that sulindac sulfide effectively downregulated both MMP7 expression and activity. Furthermore, activity-based proteomics demonstrated that sulindac sulfide dramatically decreased the activity of leukotriene A4 hydrolase in HT-29 cells as reflected by a decrease in the level of its product, leukotriene B4. This study demonstrates that the effect of sulindac treatment in a mouse model of colon cancer may be relevant to the human counterpart and highlights the effect of sulindac treatment on metallohydrolases.


Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs), such as sulindac, are chemo-preventive reagents towards colorectal cancers [1,2,3]. This effect is consistent with observations in vitro in human colon cancer cells [4], as well as in vivo in the Apc Min/+ mouse model, where a large decrease in tumor burden is observed [5]. The gastrointestinal complications that develop in NSAID users are well documented [6], and represent an obstacle in the use of these drugs as chemo-preventive agents. The pleiotropic effects of sulindac on colon cancer prevention are still unclear and hinder the development of more specific treatments with diminished side effects. Regarding this issue, it has been shown that sulindac treatment causes changes in extracellular matrix remodeling events, including downregulation of matrix metalloproteinase 7 (MMP7) in intestinal adenomas of Apc Min/+ mice [7].
MMP7 belongs to a group of metal ion-dependent proteases. According to the MEROPS database (www.merops.sanger.uk), matrix metalloproteases (MMPs), consisting of 24 members, comprise the M10 subfamily of the MA family of the metallohydrolases clan M. MMPs have been strongly implicated in the development of many cancers. Deletion of MMP2 and MMP9 in mice have been shown to reduce pancreatic tumorigenesis by reducing angiogenesis [8]. Pertinent to this study, MMP7 deletion in Apc Min/+ mice has been shown to strongly reduce intestinal tumor burden [9]. These observations implicate MMP7 as a viable target for the development of novel treatment regimes.
The current study determined the effect of sulindac treatment on MMP7 in the human colon cancer cell line, HT-29, and utilized a global approach to detect altered activities of other metallohydrolases. The results of these investigations are described herein.

Cell culture
HT-29 cells (American Type Culture Collection, Manassas, VA) were maintained in McCoy's 5A medium (Gibco, Grand Island, NY) with 10% fetal bovine serum at 37uC in an atmosphere of 95% air/5% CO 2 . Sulindac, sulindac sulfide, and sulindac sulfone were purchased from Sigma Aldrich (St. Louis, MO). These drugs were diluted in dimethyl sulfoxide (DMSO). DMSO was added to the media at a final concentration of 1% and cells were cultured in 6-well plates. Once cells were 80% confluent, they were treated with various concentrations of sulindac and its metabolites. After 24 hr, the cells were trypsinized and viable cells counted using the trypan blue method.

Gene expression
HT-29 cells, at the time of harvest, were trypsinized, centrifuged, and the cell pellet used to extract RNA following the Qiagen protocol. Concentration and quality of the samples were assessed using the spectral profile obtained from the NanoDrop-1000 (Thermo Scientific, Wilmington, DE). Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed as previously described [7]. Primers and probes designed for human MMP7, MMP25, Trypsin1, and RPL-19 are shown in Table 1.
For RT-PCR studies, total RNA was extracted from HT-29 cells, treated with DMSO or 100 mM sulindac sulfide for 24 hr using the Qiagen protocol. RT-PCR reaction included 2.56 Hotmaster Mix (5 Primer Inc., Gaithersburg, MD), RNase Inhibitor, Multiscribe RT enzyme, passive reference dye ROX, and Sybr Green. Total RNA (25 ng) was revere-transcribed in 30 ml reaction mixture at 48uC for 30 min. The following conditions were used for amplification: pre-denaturation for 5 min at 95uC, 30 cycles of denaturation at 95uC for 20 sec, annealing at 59uC for 20 sec, and extension at 72uC for 25 sec. PCR products were separated by electrophoresis on a 3% agarose gel containing ethidium bromide. A 50 bp DNA ladder (Promega, Madison, WI) was used as the molecular weight marker.

Western blotting
Proteomes were extracted from HT-29 cells and then treated with sulindac sulfide (100 mM) for 24 hr at 80% confluency. Cells were trypsinized, centrifuged, and then washed in PBS. Cell pellets were then Dounce homogenized and sonicated. The proteome was centrifuged at 100,0006 g to separate the cytosolic and membrane fractions. The secreted fraction was obtained by centrifugation of the cell media at 100,0006 g. The proteome was precipitated by the addition of ammonium sulfate. Samples were passed over a PD-10 desalting column (GE Healthcare, Piscataway, NJ) and protein concentrations were determined using the NanoDrop-1000 (Thermo Scientific, Wilmington, DE). Samples were load onto a 10% polyacrylamide gel, electrophoresed, and then transferred to a PVDF membrane. The membranes were incubated overnight at 4uC with polyclonal rabbit-anti-human MMP7 (Abcam, Cambridge MA), and polyclonal rabbit-antihuman active MMP7 (Abcam) at 1 mg/ml in blocking buffer (PBS, 3% nonfat dry milk, 0.1% Tween 20). For MMP25 and a-Tubulin, rabbit anti-human MMP25 (Abcam) and mouse anti-human a-Tubulin (Santa Cruz Technology), respectively, was used. HRP-conjugated anti-rabbit IgG antibody or HRP-conjugated-anti-mouse IgG (Cell Signaling Technology, Boston MA) in the blocking buffer was used as the secondary antibody. Blots were developed using the LumiGLO reagents (Protein Research Products, Gaithersburg, MA) or the Western Lightning ECL Substrate (Perkin Elmer Inc., Waltham, MA).

Activity-based proteomics for the identification of activated metallohydrolases
Proteomes were adjusted to a concentration of 1 mg/ml in PBS. A cocktail of alkyne probes targeting the active site of metallohydrolases was a generous gift of Benjamin F Cravatt (The Scripps Research Institute, La Jolla, CA). Labeling was performed as previously described [10]. Briefly, probes were added to a final concentration of 1 mM and incubated under UV light for 1 hr. Rhodamine azide was then bound to the probes using click chemistry [11]. Samples were electrophoresed on a 10% polyacrylamide gel and scanned using either a Hitachi FMBio IIe flatbed scanner (MiraBio, Alameda, CA) or the KODAK In-Vivo Multispectral System FX (Carestream Health, Rochester, NY).

Identification of activity-based labeled proteins
Real-size gel scan prints were used as a template to extract the desired bands and scanned afterwards to assure correct slicing of the gel. Each gel plug was reduced with 10 mM dithiothreitol in ammonium bicarbonate and alkylated with 55 mM iodoacetamide. Samples were digested with 0.1 mg trypsin (Promega, Madison, WI) in 100 ml of 10 mM ammonium bicarbonate and incubated overnight at 37uC. Tryptic peptides were extracted from gel plugs with 50% acetonitrile/49.9% H 2 O/0.1% trifluoroacetic acid followed by 100% acetonitrile and finally with 0.1% trifluoroacetic acid.
Tryptic peptides were chromatographed on a 100 mm610 cm C18 column (particle size: 5 mm) and eluted using a linear gradient of 3 to 45% acetonitrile in H 2 O over 70 min at room temperature, at a flow rate of 500 nl/min. The eluent was electro-sprayed into the linear quadrupole ion trap (LTQ) mass spectrometer (Thermo-Fisher Scientific, Waltham, MA). Sequest and X!Tandem algorithms were used to search the database utilizing the international protein index (IPI) mouse database.

Leukotriene B4 enzyme immunoassay
At 80% confluence, HT-29 cells were incubated with either sulindac sulfide or DMSO for 24 hr. The secreted proteome was obtained and concentrated by vacuum centrifugation. Proteomes were adjusted to 5 mg/ml and LTB4 quantified with an enzymatic immunoassay kit (Cayman Chemical, Ann Arbor, MI).

Results
The reduced metabolite of sulindac, sulindac sulfide, selectively increases cell death of HT-29 cells HT-29 cells were treated with sulindac, sulindac sulfide, and sulindac sulfone at concentrations ranging from 10 to 1000 mM ( Figure 1). Starting at 250 mM, sulindac sulfide significantly increased cell death, reaching 100% cell death at 500 mM. In contrast, sulindac and sulindac sulfone failed to significantly increase cell death, even when the drugs where added at mM concentrations.
Sulindac sulfide reduces MMP7 at the mRNA, protein, and activity levels It was reported that two days of sulindac treatment is sufficient to downregulate MMP7 in tumors of Apc Min/+ mice [7]. In order to determine which metabolite effectively diminished MMP7, HT-29 cells were treated with sulindac and its metabolites at 100 mM, a concentration not toxic to the cells as demonstrated in figure 1, for 24 hr. The expression profile of MMP7 was compared to the results previously obtained in mouse studies (Figure 2A). Only the active metabolite, sulindac sulfide, was capable of significantly reducing the expression of MMP7. A titration curve using different concentrations of sulindac sulfide revealed that even at 50 mM, MMP7 showed a small (14%), but statistically significant downregulation of expression (p = 0.044) ( Figure 2B). At 100 mM, a decrease of 65% was observed (p = 0.000001). The difference between 100 mM and 200 mM was less dramatic (11%), but was still significantly different between these two concentrations (p = 0.017). Western blotting of MMP7, as well as the active site of MMP7, was performed on cytosolic, membrane, and secreted fractions of HT-29 proteomes after 24 hr of sulindac sulfide treatment at 100 mM ( Figure 2C). In both cases, MMP7 was only detected in the secreted fractions of the proteome. Sulindac sulfide treatment showed a clear reduction of MMP7 in the secreted fraction at a 100 mM concentration. Western blot analysis targeting the active site of MMP7 revealed a complete disappearance of detectable active MMP7 after sulindac sulfide treatment.
In order to determine if other MMPs, other classes of proteases, and the housekeeping gene, RPL-19, were affected by sulindac in HT-29 cells, RT-PCR (whole cell extracts) and Western blot (secreted, cytosolic, and fractions) analyses were performed. mRNA for MMP25, trypsin1, and RPL-19 were not altered after sulindac sulfide treatment ( Figure 3A, B). Western blot analysis of the membrane fraction of these cells confirmed RT-PCR results for MMP25 ( Figure 3C).  Activity-based protein profiling targeting metallohydrolases reveals a decrease in LTA4H activity Proteomes extracted from HT-29 cells after 24 hr of treatment with sulindac sulfide or DMSO were labeled with a probe cocktail targeting the metallohydrolase superfamily and run on a 1D gel. A strong band between 60 and 65 kDa was observed in HT-29 cells treated with DMSO. Sulindac sulfide-treated cells showed a faint band of similar molecular weight. The bands were excised and analyzed by mass spectrometry. The samples were run by linear quadrupole MS/MS (Figure 4) and the results matched with the MEROPS database for metallohydrolases. There were two peptides (LTYTAEVSVPK and LVVDLTDIDPDVAYSSV-PYEK) detected in the ,70 kDa band that matched leukotriene A4 hydrolase (LTA4H). The predicted molecular weight of LTA4H is 69 kDa. LTA4H has previously been shown to be a target for this metalloprotease probe cocktail (10).
In order to validate the reduction in LTA4H activity, an enzymatic immunoassay was used to measure leukotriene B4 (LTB4) levels after sulindac treatment ( Figure 5). Sulindac sulfidetreated cells showed strongly reduced levels of LTB4 when compared to DMSO-treated cells.

Discussion
The present study demonstrated that sulindac sulfide is capable of reducing the RNA and protein levels of MMP7, which is consistent with what was observed in mouse studies [7]. Through activity-based protein profiling, it was demonstrated that sulindac sulfide dramatically reduces the activity of LTA4H. This result was validated by decreased levels of LTB4.
The involvement of metalloproteases in colon cancer has previously been identified but the development of treatment strategies targeting these enzymes has been unsuccessful [12]. One problem is the low specificity of metalloprotease inhibitors that have made it to clinical trials. MMP7, however, has consistently been found to be relevant in colon cancer and its animal models [9,13]. The present study indicated that sulindac sulfide is able to reduce the expression of MMP7 in the human colon cancer cell line HT-29 which was also observed, in vivo, in Apc Min/+ mice [7]. In order to determine if other MMPs or other classes of proteases were also altered, MMP25, a membrane-associated MMP that is highly expressed in cancer cells [14], and trypsin1 were analysed and shown to be unaffected by sulindac sulfide treatment. Additionally, the housekeeping gene RPL-19 was also not affected by this NSAID. Nobiletin, another agent with anticancer properties, has also been shown to downregulate MMP7 [15] in HT-29 cells. Several mechanisms have been proposed by which MMP7 promotes tumor growth. Several of those studies link MMP7 with a disrupted Fas-mediated apoptotic response [16,17,18], and inflammation-related facilitation of tumor growth has been proposed to be caused by MMP7 cleavage of Fas ligand [19]. Zinc metallopeptidases consist of 12 members and belong to the family of metallohydrolases. These proteases have been implicated in several cancers including aminopeptidase N in colon cancer [20], cystinyl aminopeptidase in renal cancer [21], and LTA4H in lung and colon adenocarcinomas [22]. This is the first study linking sulindac to regulating LTA4H. The downregulation of activity of LTA4H, and decrease in tumor burden, after sulindac treatment supports studies in which it has been shown to be upregulated in colon cancer. Furthermore, [6]-gingerol, a natural component with antitumorigenic properties, has been found to suppress cancer growth by LTA4H inhibition [23]. The decrease in LTA4H activity was validated by the observation that lower LTB4 levels were found after sulindac treatment. LTB4 is a well known eicosanoid with chemotactic properties and has been shown to stimulate the proliferation, in vitro, of HT-29 colon cancer cells [24].
There has been no other report of the involvement of sulindac, or any other NSAID, on MMP7 or LTA4H expression. However, in preliminary studies, we have shown that sulindac is not the only NSAID capable of downregulating MMP7, i.e., aspirin (data not shown), which suggests that MMP7 is downregulated by a shared NSAID mechanism [7,25] and not a unique property of sulindac.
In summary, the present study identified two candidates, previously reported to be highly relevant in tumor development, that are altered after sulindac treatment. New treatment modalities selectively targeting MMP7 and LTA4H, individually or in combination, offer new therapeutic approaches that take advantage of the benefits of sulindac treatment, but potentially without the adverse side effects of this drug.