Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer
© McGoldrick et al.; licensee BioMed Central Ltd. 2014
Received: 19 September 2013
Accepted: 5 February 2014
Published: 10 February 2014
Esterases are often overexpressed in cancer cells and can have chiral specificities different from that of the corresponding normal tissues. For this reason, ester prodrugs could be a promising approach in chemotherapy. In this study, we focused on the identification and characterization of differentially expressed esterases between non-tumorigenic and tumorigenic prostate epithelial cells.
Cellular lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines, tumorigenic RWPE-2 prostate epithelial cells, and non-tumorigenic RWPE-1 prostate epithelial cells were separated by native polyacrylamide gel electrophoresis (n-PAGE) and the esterase activity bands visualized using α-naphthyl acetate or α-naphthyl-N-acetylalaninate (ANAA) chiral esters and Fast Blue RR salt. The esterases were identified using nanospray LC/MS-MS tandem mass spectrometry and confirmed by Western blotting, native electroblotting, inhibition assays, and activity towards a known specific substrate. The serine protease/esterase oxidized protein hydrolase (OPH) was overexpressed in COS-7 cells to verify our results.
The major esterase observed with the ANAA substrates within the n-PAGE activity bands was identified as OPH. OPH (EC 18.104.22.168) is a serine protease/esterase and a member of the prolyl oligopeptidase family. We found that LNCaP lysates contained approximately 40% more OPH compared to RWPE-1 lysates. RWPE-2, DU145 and PC3 cell lysates had similar levels of OPH activity. OPH within all of the cell lysates tested had a chiral preference for the S-isomer of ANAA. LNCaP cells were stained more intensely with ANAA substrates than RWPE-1 cells and COS-7 cells overexpressing OPH were found to have a higher activity towards the ANAA and AcApNA than parent COS-7 cells.
These data suggest that prodrug derivatives of ANAA and AcApNA could have potential as chemotherapeutic agents for the treatment of prostate cancer tumors that overexpress OPH.
Prostate cancer is the second most frequently diagnosed cancer in men and the second-leading cause of cancer related death in American men . There is an estimated 238,590 new cases of prostate cancer predicted in the US this year and an estimated 29,720 deaths due to prostate cancer . Despite advances in radiation and chemotherapy, prostate cancer is a leading cause of cancer death. Radiation and chemotherapy treatment remain central to prostate cancer treatment. These treatments can, however, produce a number of side effects such as neutropenia [2, 3], urinary and bowel symptoms , hair loss , and fatigue . There is, therefore, a critical need to develop tumor specific therapies for prostate cancer.
We identified oxidized protein hydrolase (OPH), also called N-acylaminoacyl-peptide hydrolase (APEH), as a key esterase that is overexpressed in the tumorigenic LNCaP cell line. OPH is a serine esterase/protease that has a well characterized esterase activity towards α-naphthyl butyrate  and an exopeptidase activity for removing the N-terminally acetylated amino acid residues from peptides/proteins [15–17]. Immunohistochemistry of primary prostate tumor sections indicate that OPH is highly expressed in some prostate tumors (http://www.proteinatlas.org/), suggesting that OPH could have potential as a drug target in prostate cancer. The overexpression of OPH in some prostate cancers suggests that chemotherapeutic prodrugs esters modeled after known ester substrates of OPH (i.e., α-naphthyl N-acetyl-alaninate) have potential in treating some prostate cancers.
Porcine liver esterase (PLE), digitonin, α-naphthyl acetate, fast blue RR salt, goat anti-rabbit HRP conjugate polyclonal antibody, and diisopropyl fluorophosphate (DFP) were purchased from Sigma Chemical Company (St. Louis, MO). Novex Tris-glycine native sample buffer, NuPAGE LDS sample buffer, Novex Tris-glycine gels, NativeMark unstained protein standards, Protein A agarose beads, penicillin/streptomycin solution, and geneticin (G418) were purchased from Invitrogen (Grand Island, NY). Precision plus protein standards were purchased from Bio-Rad (Hercules,CA); the BCA kit and the In-gel tryptic digestion kit were purchased from Pierce (Rockford, IL); ZipTipU-C18 tips were purchased from Millipore (Billerica, MA); 3,3′,5,5′-tetramethylbenzidine (TMB) was purchased from Promega (Madison, WI); rabbit polyclonal anti-AARE antibody was purchased from Abcam (Cambridge, MA); superose 12 column (10/300 GL) was purchased from GE Healthcare (Pittsburgh, PA); (TransIT-LT1 transfection reagent was purchased from Mirus Bio (Madison, WI). pCDNA3.1(+) vector encoding OPH-Flag was a kind gift from Dr. M. Hayakawa (Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan).
R- and S- isomers of ANAA (Figure 2) were synthesized and purified as previously described  and stored at -20°C. Stock solutions of 100 mM α-naphthyl acetate were prepared in DMSO and stored at -20°C. The synthesis of N-acetyl-alanyl-p-nitroanilide (AcApNA) was guided by a previously published procedure . AcApNA was synthesized by adding 20 ml of dichloromethane to a solution of anhydrous dimethylformamide (0.51 ml) and 0.865 g of N-acetyl-L-alanine. The mixture was cooled to -20°C with an acetone-dry ice bath. Thionyl chloride (0.485 ml) was added dropwise to the cooled mixture. After 20 min, a cold solution (-20°C) of 0.828 g 4-nitroanaline and 1.82 ml of triethylamine in 10 ml of dichloromethane was added dropwise to the N-acetyl-L-alanine solution. The resulting mixture was maintained at 0°C for 2 h. After concentration, the mixture was extracted with ethyl acetate (2 × 30 ml). The organic layer was washed with 4 N HCl (2 × 40 ml) and NH4Cl aqueous solution (40 ml), and dried over MgSO4. After filtration and concentration of the organic layer, the residue was purified using column chromatography with hexane, then 30-50% acetone in hexane, affording 0.248 g of the final product (16%). M.P. was found to be 194-196°C. The M.P. has been previously reported as 192-197°C ).
Cell culture and lysates
RWPE-1 (CRL-11609), RWPE-2 (CRL-11610), LNCaP (CRL-1704), DU-145 (HTB-81), PC-3 (CRL-1435) and COS-7 (CRL-1651) cell lines were purchased from American Type Culture Collection (Manassas, VA), cultured according to ATCC’s instructions and supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were detached from the 75 cm3 cell culture flasks after reaching 80% confluence by washing the cells with PBS followed by the addition of 0.25% trypsin. The detached cells were centrifuged at 500 × g for 5 mins and washed with PBS to remove trypsin. Cells were centrifuged a second time and pellets stored at -80°C. Cell pellets of each cell line were lysed using 2% (wt/vol) digitonin in PBS on ice with vortexing every two minutes. After 10 min of incubation on ice, the lysates were centrifuged at 18,000 × g for 5 min at 4°C and the supernatant collected. Protein concentrations were determined with the BCA kit using the manufacturer’s instructions.
n-PAGE esterase activity profiles
Cell lysates containing 120 μg of protein were mixed with an equal volume of 2X Novex Tris-glycine native sample buffer and applied to a Novex 10-20% or 6% Tris-glycine gel. NativeMark unstained protein standards were used as migration markers. Gels were electrophoresed under native conditions at 4°C using 20 mA/gel for 270 min for the 10-20% gel or 180 min for the 6% gels. For inhibition assays, the gel lanes were separated and immersed in either 0.1 M sodium phosphate buffer, pH 6.5 or sodium phosphate buffer containing 50 μM DFP for 10 min. The gels were then stained for esterase activity by immersing them in 30 ml of 0.1 M sodium phosphate buffer, pH 6.5 containing 10 mg Fast Blue RR Salt and 800 μM α-naphthyl acetate or 800 μM ANAA isomer. Bands were developed at room temperature for 30 min followed by 3 washes with distilled water. The migration markers were stained with Coomassie blue and destained overnight in 10% acetic acid. Gels were scanned with an Epson Perfection V750 PRO scanner.
Semi-purified OPH from rat liver
OPH was semi-purified from 100 g of rat liver using the method described by Tsunasawa  with the following modifications. After elution from the hydroxyapatite column, the OPH fractions were combined and subjected to gel filtration on a Superose12 column (10/300 GL) using a Biologic Duo Flow protein purification system (Bio-Rad, Hercules,CA). Fractions were eluted with 50 mM sodium phosphate buffer, pH 7 containing 1 mM EDTA and 0.2 M NaCl at a rate of 0.5 ml/min in 0.5 ml fractions. Fractions that contained OPH activity were combined and stored at -20°C. The pooled semi-purified OPH was analyzed by mass spectroscopy to verify that no other esterases or proteases were present.
Overexpression of OPH in COS-7 cells
COS-7 cells were transfected using TransIT-LT1 transfection reagent and the vector pCDNA3.1(+) encoding OPH with a Flag tag using the transfection reagent’s manufacturer’s instructions. COS-7 cells overexpressing OPH were selected using 1 mg/ml G418 over a three week period. Cells surviving selection were termed COS-7-OPH for further experiments and were maintained with 1 mg/ml G418.
LC/MS-MS mass spectroscopy
Protein bands were individually excised from the n-PAGE gel and cut into small pieces using a scalpel. The gel pieces were destained, disulfide bonds reduced, unmodified thiol groups alkylated, and the proteins digested with trypsin overnight using the In-Gel Tryptic Digestion Kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. After digestion, the liquid containing the peptides from each band was transferred to a 1.5 ml tube. The peptides were further extracted from each gel piece by covering gel piece with extraction buffer consisting of formic acid/acetonitrile/water (5:50:45, v/v/v) for 10 min then collecting the liquid and adding it to the appropriate 1.5 ml tube. The peptides in the vial inserts were completely dried using a DNA Speed Vac Concentrator (Thermo Fisher Scientific, Asheville, N.C.). Peptides were rehydrated with 0.1% formic acid and further purified using ZipTipU-C18 tips according to manufacturer’s instructions. Peptides eluted from zip tips were transferred to vial inserts. The peptides in the vial inserts were completely dried using the Speed Vac Concentrator and then rehydrated in a volume of 4 μl of formic acid/acetonitrile/water (0.1:20:79.9, v/v/v). A volume of 2 μl of each sample was trapped by a picofrit column packed with C18 (New Objective, Inc., Woburn, MA) and equilibrated in 0.1% formic acid in water/acetonitrile (98:2, v/v). Peptides were then eluted with a gradient of 2 to 40% of solvent B containing 0.1% formic acid in acetonitrile over 60 min at a flow rate of 200 nL/min. Eluted peptides were analyzed by electrospray ionization using a LTQ-XL ion trap mass spectrometer (Thermo Fisher). Mass spectrometry (MS) data were acquired using data-dependent acquisition with a series of one full scan followed by a zoom scan and then a MS/MS scan of the ions. The dynamic exclusion duration was 30 ms. Proteins were identified from each MS raw data file using the SEQUEST search algorithm (Thermo Fisher Scientific) and the SwissProt/UNIPROT database through the Bioworks browser, version 3.3.
Cell lysates containing 90 μg of protein were mixed with NuPAGE LDS Sample Buffer, heated to 90°C, and applied to a Novex 10-20% Tris-Glycine gel. Precision Plus Protein Standards were used for molecular weight markers. Gels were electrophoresed for 90 min at 125 V in 1X Novex Tris-Glycine SDS running buffer. Gels were then electrophoretically transferred to a nitrocellulose membrane for 90 min at 25 V. The membrane was probed with 1:1000 rabbit polyclonal anti-AARE (OPH) antibody (ab84694, Abcam) or anti-GAPDH (ab9485, Abcam) overnight at 4°C, 1:2000 anti-rabbit IgG conjugated to HRP (A0545-1ML, Sigma) was used as the secondary antibody and incubated for 1 hour. Membranes were washed with PBS containing 0.05% Tween 20. Peroxidase was detected using 3,3′,5,5′-tetramethylbenzidine according to manufacturer’s instructions.
Native electroblot activity staining and western blotting
Native electroblot activity staining was carried out by electrophoretically transferring proteins from n-PAGE gels to a nitrocellulose membrane at 4°C, followed by the esterase activity staining procedure (see above). Western blots of the n-PAGE gel were carried out by probing a native electroblot as described in the Western blotting methods.
An aliquot of 0.5 ml Protein A agarose beads was coupled with 5 μg of anti-OPH antibody on ice for 30 min. LNCaP cell lysates containing 120 μg of protein was combined with either Protein A agarose beads or anti-OPH conjugated Protein A agarose beads and incubated on ice for 1 hour with gentle mixing every 15 min. The samples were centrifuged for 5 min at 1000 × g and the supernatants were then separated using 6% n-PAGE followed by the esterase activity staining procedure.
OPH activity assay
Aliquots of 20 μL of cell lysates containing either 4.5 μg/μl of protein, or 0.5 unit PLE, or 12.5 ng/μl semi-purified rat liver OPH, or PBS were added in triplicate to a 96 well microplate. One unit of PLE is defined as the amount of PLE that will hydrolyze 1.0 μmole of ethyl butyrate to butyric acid and ethanol per min at pH 8.0 at 25°C. An assay cocktail of 220 μL 0.1 M sodium phosphate buffer, pH 6.5 and 10 μl of 100 mM AcApNA was added to each well giving a final AcApNA concentration of 4 mM. The release of p-nitroaniline was monitored with a microplate reader at a wavelength of 405 nm for 10 min at room temperature. The concentration of p-nitroaniline was calculated using a molecular extinction coefficient of 7530 M-1 cm-1.
Cell culture esterase staining
LNCaP, RWPE-1, COS-7, and COS-7-OPH were seeded in triplicate in 24 well cell culture plates at 1x105 cells/well. The plate was incubated at 37°C in a CO2 incubator overnight. Staining solutions of 0.1 M sodium phosphate buffer, pH 6.5 containing 10 mg Fast Blue RR Salt and 800 μM α-naphthyl acetate or 800 μM α-naphthyl N-acetylalaninate isomer were prepared immediately prior to cell staining. The cell media was removed from each well and 500 μl of staining solution was added to each well. The cells were incubated at 37°C in 5% CO2 for 20 minutes. The staining solution was removed and replaced with 500 μl of PBS. The cells were observed at 100x magnification and digitally photographed using a MOTIC inverted phase contrast microscope equipped with a Nikon Coolpix E4300 4-megapixel camera (Martin Microscope, Easley, SC). The percent area threshold of staining was measured using ImageJ, v1.44o (NIH, Bethesda, MD).
Data were analyzed by analysis of variance (ANOVA) followed with the Scheffe test for significance with P < 0.05 using SPSS 19.0 for Windows (Chicago, Illinois). Results were expressed as the mean ± SD of at least three experiments. In all figures, letters that are not the same are significantly different with P < 0.05.
The research conducted in this study adhered to US NIH ethical guidelines. All the human cell lines studied were purchased from the American Type Culture Collection and such studies are not considered human subjects research because the cell lines are publicly available and all of the information known about the cell lines is also publicly available. No experimental animals were used in the studies reported here.
Differential esterase activity between non-tumorigenic RWPE-1 and tumorigenic LNCaP cells
Our first objective was to determine if non-tumorigenic prostate cells have a different n-PAGE esterase activity profile compared to tumorigenic prostate cells and to characterize any chiral ester substrate preferences.
Prostate esterases identified as OPH have a preference for S-ANAA
Parallel gels stained with S-ANAA or R-ANAA show two prominent and sharp esterase bands at 216 kDa and 198 kDa native protein marker points. Densitometry analysis of the 216 kDa band showed significantly higher esterase activity in the LNCaP cell lysate stained with R-ANAA and S-ANAA compared to all other cell lysates (Figure 4B). Moreover, the 216 kDa band for LNCaP cells showed higher activity with the S-ANAA substrate compared to the R-ANAA substrate. The degree of chiral substrate selectivity was more apparent in the esterase activity of the 198 kDa band (Figure 4C). Densitometry of the 198 kDa band showed a significant preference for S-ANAA substrate in all of the cell lines except DU 145. The LNCaP lysates contained 40-50% higher esterase activity in both bands with S-ANAA substrate than with the R-isomer and a 40-60% higher activity compared to RWPE-1 lysate. RWPE-2 and PC3 lysates had similar staining profiles to RWPE-1, while DU 145 showed less activity compared to RWPE-1.
LC/MS-MS analysis of 198 kDa n-PAGE bands
N-acylaminoacyl-peptide hydrolase (OPH)
OPH Peptides Indentified
K.STHALSE VE VE SDSFMNAVLVVLR.T
LNCaP lysates showed significantly higher levels of esterase activity with α-naphthyl acetate and ANAA substrates compared to non-tumorigenic RWPE-1 and tumorigenic RWPE-2, DU145 and PC3 cell lysates. It appears clear that not all tumorigenic prostate cells contain high levels of OPH activity; however, the human protein atlas (http://www.proteinatlas.org/) indicates that some human tumors overexpress OPH compared to normal prostate tissue. Since the overexpression of OPH in tumors compared to normal prostate tissue is the most ideal situation for OPH targeted prodrugs, we have limited the remainder of this study to the non-tumorigenic RWPE-1 and tumorigenic LNCaP cell lines.
Esterase activity profiles with n-PAGE electroblotting
Electroblot esterase activity bands contain OPH
B and 2
N-acylaminoa cly-peptide hydrolase
B and 3
B and 4
OPH accounts for the all the esterase staining observed with the S-ANAA substrate
OPH present in prostate cell lysates appears as a single MW weight species in SDS-PAGE Westerns
Mammalian OPH has been primarily reported as a homotetramer [16, 24, 25] with each OPH subunit being active within the tetramer. It has been shown that citraconylation of the amino groups of purified OPH tetramer reversibly dissociates the quaternary structure of OPH. When acylated with citraconic anhydride, OPH separated by n-PAGE forms multiple OPH bands . Interestingly, our prostate cell lysates produced four uniformly distributed activity bands when separated by n-PAGE. Some explanations for the multiple OPH activity bands are OPH multi-mers, isoforms, degradation products, protein interactions, and post-translational modifications. OPH isoforms and degradation products appear to be unlikely causes for the multiple bands. Isoforms and degredation products typically result in multiple bands when separated by SDS-PAGE; however, western blots of the prostate lysates reveal a single 80kD OPH band. The interaction of native OPH with other proteins is plausible.
There is evidence that under conditions of oxidative stress OPH translocates to the cell membrane of erythrocytes and degrades oxidized proteins . Similarly, OPH was found to translocate to the aggresome when the proteasome was inhibited in COS-7 cells . High levels of oxidative stress are known to oxidize proteins resulting in protein aggregations that can inhibit the proteasome . LNCaP, DU 145, and PC3 cell lines are reported to have significantly higher free radical production compared to RWPE-1 , which might induce OPH to interact with aggresomal or membrane proteins. Our mass spectrometry analysis of the OPH bands revealed several proteins known to be associated with aggresomes and were consistent with previously published data . We are actively pursuing an explanation for the multiple OPH bands.
The higher expression of OPH protein in LNCaP cell lysates is reflected by a higher activity towards N-acetyl-alanyl-p-nitroanilide
N-acetyl-alanyl-p-nitroanilide (AcApNA) (Figure 1D) is a specific OPH substrate and is routinely used to measure OPH activity and follow OPH purification from tissue homogenates [16, 24, 25, 28]. The product p-nitroaniline (p-NA) is released upon hydrolysis of AcApNA and its absorbance measured at 405 nm. We found that non-tumorigenic and tumorigenic cell lysates incubated with AcApNA released p-NA at differential rates (Figure 7B). After 10 minutes of incubation with AcApNA, LNCaP lysates released approximately 40% more p-NA than RWPE-1 lysates. PC3 lysates released approximately 15% more p-NA compared to RWPE-1, while the rates for AcApNA hydrolysis were similar for DU 145 and RWPE-1. The activity profile of the prostate cell lysates incubated with the known OPH substrate AcApNA parallel the expression of OPH observed by SDS-PAGE Western blots (Figure 7A) as well as the esterase activity profiles observed for n-PAGE stained with S-ANAA. As expected, porcine liver esterase (PLE) had no activity towards the AcApNA substrate.
The esterase substrates enter prostate cells and have measurable in situesterase activities
Human OHP overexpressed in COS-7 has characteristics similar to that of OPH in the human prostate epithelial cell lines
Discussion and Conclusion
A number of investigators have suggested that chiral ester prodrugs hold the promise of providing more selective anticancer chemotherapy [7–9, 11, 14]. A major requirement for this strategy is the need to identify target esterases that have differential expression or substrate selectivity in cancer cells compared to their normal counterpart. Ideally, esterases targeted for prodrug hydrolysis should be highly expressed in the target tumor cells and/or have a chiral preference different from normal cells. Yamazaki et al. previously found that several cancers displayed hydrolytic preferences for isomers of chiral substrates opposite that of their normal counterparts [13, 14, 32]. However, in the work presented here we found that the esterases of both tumorigenic and non-tumorigenic prostate cells both showed a preference for the S-isomer of α-naphthyl N-acetyl-alaninate (S-ANAA).
Additionally, we have improved upon the work by Yamazaki et al. by identifying a specific esterase that has differential activity towards chiral ANAA substrates. We have used several proteomic techniques to identify OPH in tumorigenic and non-tumorigenic prostate cells. Using an n-PAGE method similar to Yamazaki et al., n-PAGE electroblotting, immunoblotting, inhibition studies and mass spectrometry we have identified OPH in prostate cells and have found that OPH has selective activity towards chiral ANAA substrates.
OPH is a serine protease and a member of the prolyl oligopeptidase (POP) family. Three functions of OPH have been described: (1) an exopeptidase activity that unblocks N-acetyl peptides with a preference for N-acetyl alanyl peptides ; (2) an endopeptidase activity towards oxidized and glycated proteins [28, 33–36] and; (3) an ability to associate with aggresomes when proteasome function is inhibited . Moreover, work by Shimizu et al.  suggests that the proteasome and OPH work coordinately to clear cells of oxidized (carbonylated) proteins. A comprehensive physiological understanding of OPH remains elusive. Nevertheless, the acetylation of the N-terminal α-amine group of proteins is the most common post-translational modification in eukaryotic proteins yet, little is known about the biological role of N-alpha-terminal acetylation, and even less is known about the role of enzymes (like OPH) that catalyze the hydrolysis of an N-terminally acetylated peptide to release an N-acetylamino acid.
Our finding that OPH in non-tumorigenic and tumorigenic prostate cell lines have a greater hydrolytic preference for the S-ANAA isomer of ANAA is consistent with previous observations that OPH has a preference for small peptides with Ac-L-alanine (Ac-S-alanine) compared to Ac-D-alanine (Ac-R-alanine) . OPH activity bands were not observed with the α-naphthyl acetate substrate while distinct activity bands were visualized using α-naphthyl N-acetyl-alaninate substrates. In addition, OPH has high specificity for AcApNA and Ac-Ala-β-naphthylamide . AcApNA and Ac-Ala-β-naphthylamide are structurally similar to ANAA; the main differences being the 4-nitrobenzene of AcApNA and the peptide bond of Ac-Ala-β-naphthylamide. AcApNA, Ac-Ala-β-naphthylamide, and ANAA contain N-terminal acetylated alanine. Taken together, this family of small N-acetyl-alaninate substrates appears to be good models for future OPH targeted prodrug designs.
We found OPH to be differentially expressed in the LNCaP prostate cancer cell line compared to non-tumorigenic RWPE-1 cell lines and that tumor cells overexpressing OPH might be responsive to prodrug derivatives of α-naphthyl N-acetyl-alaninate. We have found that OPH expression in prostate cancer cells can vary widely. DU-145 and PC3 lysates showed slightly diminished levels of OPH compared to RWPE-1 while LNCaP contained nearly twofold more OPH than RWPE-1. OPH protein expression levels appear to vary in other cancers as well. Serine protease activity profiling performed by Jessani et al. shows that OPH is highly active in several melanoma and breast cancer cell lines ; however, Scaloni previously reported that OPH was deleted or under-expressed in a number of small cell lung carcinomas . We have screened other tumorigenic cell lines for OPH activity and have found that several aggressive colon cancer and melanoma cell lines exhibit significantly higher OPH activity compared to their non-tumorigenic counterparts. Further OPH expression profiling of normal prostate cell lines, prostate cancer cell lines, and primary prostate tissues is needed to determine which prostate cancers might be suitable for OPH-targeted therapies.
OPH has recently been proposed as a potential target for the development of anticancer drugs . Histological data in the Human Protein Atlas shows that OPH can be strongly expressed in some cases of colorectal, breast, prostate, ovarian, endometrial and liver cancers . OPH has a well-documented substrate specificity towards N-acetylated-L-alaninate esters . Our results suggest that α-naphthyl N-acetyl-alaninate substrates could be used to rapidly determine levels of active OPH in non-tumorigenic and tumorigenic cells and tissues. Naphthyl substrates are routinely used to differentiate chronic myelogenous leukemia (CML) from leukemoid reaction and to distinguish other myeloproliferative disorders [42, 43]. Yamazaki et al. demonstrated that a substrate similar to S-ANAA, N-methoxycarbonylalaninate, could be used to visualize esterase activity in cryostat tissue sections . Our cell culture activity staining of RWPE-1, LNCaP, and COS-7-OPH cells with S-ANAA suggest that S-ANAA may be useful to screen cells and tissues for relative OPH activity. Screening normal and tumorigenic cells or tissues with S-ANAA may aid in identifying candidates for OPH directed therapies.
In conclusion, we have found that cell lysates from non-tumorigenic RWPE-1 cells and several tumorigenic prostate cell lines display differential esterase activity profiles when visualized with α-naphthyl acetate or chiral α-naphthyl N-acetyl-alaninate substrates. Our n-PAGE results show that tumorigenic LNCaP, DU 145, and PC3 cell lysates contain higher general esterase activity when visualized with α-naphthyl acetate compared to non-tumorigenic RWPE-1 cell lysates. In addition, we found that the OPH activity of prostate cell lysates could be visualized by staining with chiral α-naphthyl N-acetyl-alaninate substrates, and that OPH has a hydrolytic preference for the S-isomer of ANAA. LNCaP lysates in particular showed the highest esterase activity with all of the ester substrates tested and contained the highest OPH activity measured with AcApNA. The results of this study indicate that ester prodrugs designed after S-ANAA or AcApNA may be a promising therapeutic approach to prostate cancers that overexpress OPH.
We thank Dr. Makio Hayakawa for generously providing the pCDNA3.1(+) vector encoding OPH-Flag. This work was supported by East Tennessee State University’s School of Graduate Studies Research Grant.
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