Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 17 β-hydroxysteroid dehydrogenase, is responsible for intratumoral androgen biosynthesis, contributing to the development of castration-resistant prostate cancer (CRPC) and eventual chemotherapeutic failure. Significant upregulation of AKR1C3 is observed in CRPC patient samples and derived CRPC cell lines. As AKR1C3 is a downstream steroidogenic enzyme synthesizing intratumoral testosterone (T) and 5α-dihydrotestosterone (DHT), the enzyme represents a promising therapeutic target to manage CRPC and combat the emergence of resistance to clinically employed androgen deprivation therapy. Herein, we demonstrate the antineoplastic activity of a potent, isoform-selective and hydrolytically stable AKR1C3 inhibitor (E)-3-(4-(3-methylbut-2-en-1-yl)-3-(3-phenylpropanamido)phenyl)acrylic acid (KV-37), which reduces prostate cancer cell growth in vitro and in vivo and sensitizes CRPC cell lines (22Rv1 and LNCaP1C3) toward the antitumor effects of enzalutamide. Crucially, KV-37 does not induce toxicity in nonmalignant WPMY-1 prostate cells nor does it induce weight loss in mouse xenografts. Moreover, KV-37 reduces androgen receptor (AR) transactivation and prostate-specific antigen expression levels in CRPC cell lines indicative of a therapeutic effect in prostate cancer. Combination studies of KV-37 with enzalutamide reveal a very high degree of synergistic drug interaction that induces significant reduction in prostate cancer cell viability via apoptosis, resulting in >200-fold potentiation of enzalutamide action in drug-resistant 22Rv1 cells. These results demonstrate a promising therapeutic strategy for the treatment of drug-resistant CRPC that invariably develops in prostate cancer patients following initial treatment with AR antagonists such as enzalutamide. Mol Cancer Ther; 17(9); 1833–45. ©2018 AACR.

Prostate cancer is the third (1) leading cause of mortality among American men and has the highest incidence of all cancers reported in the United States (2). Surgical removal of the prostate by radical prostatectomy and radiotherapy to resect the localized tumor are standard therapeutic interventions (3). The treatment of advanced and metastatic forms of prostate cancer relies heavily on androgen deprivation therapy (ADT) that involves surgical (orchiectomy) and/or chemical castration (4). Chemical agents such as gonadotropin-releasing hormone agonists [Leuprolide (5), goserelin (6), and buserelin (7)] and antiandrogens (bicalutamide; ref. 8) retard prostate cancer cell proliferation and lead to remission. After an initial response to ADT that results in castrate levels of circulating androgens, the tumor adapts, giving rise to a more aggressive and fatal disease known as castration-resistant prostate cancer (CRPC), which is characterized by molecular changes that include an increase in the expression of androgen-synthesizing enzymes and reactivation of androgen signaling (9, 10). Such adaptations can lead to relapse despite the presence of circulating castrate levels of androgens (11, 12).

Abiraterone acetate (AA) was approved by the FDA for CRPC treatment in 2011 and acts by inhibiting P450c17 (13), an upstream enzyme in the steroid biosynthetic pathway, and is effective in CRPC where tumor progression is dependent on intracrine androgen synthesis that consequently activates AR signaling (14, 15). However, to prevent hypertensive crisis due to accumulation of desoxycorticosterone (DOC) in the adrenal gland, coadministration with prednisone is imperative (16). Enzalutamide (ENZ) is another clinically used therapeutic that exerts potent androgen receptor (AR) antagonistic activity by inhibiting AR nuclear translocation, coactivator recruitment, and AR binding to androgen response elements (ARE; ref. 17). The chemotherapeutic is effective up to stage 3 CRPC where proliferation is dependent on AR activation that occurs even in the absence of an androgen ligand (15). After an initial response to AA or ENZ, resistance develops rapidly due to increases in intratumoral androgen biosynthesis (18), AR overexpression, AR mutation that makes the receptor ligand promiscuous, or the appearance of AR splice variants that makes the AR constitutively active in the absence of the ligand-binding domain (15, 19, 20). Due to tumor heterogeneity and the emergence of adaptive pathways of androgen biosynthesis (21), combined with frequent mutations in AR and androgen biosynthetic enzymes (10, 22), the prognosis for patients that have advanced to CRPC remains bleak. Thus, there is an urgent need for novel therapeutics that can delay or reverse the inevitable emergence of resistance.

Type 5 17β-hydroxysteroid dehydrogenase, also known as aldo–keto reductase 1C3 (AKR1C3), acts downstream in the steroidogenesis pathway and plays a pivotal role in the pathogenesis and progression of CRPC by catalyzing the conversion of weak androgen precursors to the potent AR ligands: testosterone (T) and 5α-dihydrotestosterone (5α-DHT; ref. 23). Also known as prostaglandin (PG) F synthase, AKR1C3 catalyzes the conversion of PGD2 to 11β-PGF and PGF prostanoids (24). An increase in PGF leads to activation of the FP receptor and consequently induces proliferation and radiation resistance in prostate cancer cells (25).

Clinically, AKR1C3 has been shown to be the most upregulated steroidogenic enzyme in CRPC patients (26). Apart from inducing a CRPC phenotype, AKR1C3 also mediates resistance to ENZ and AA by providing a source of intratumoral androgens, and this resistance can be surmounted somewhat by indomethacin (INDO), a known AKR1C3 inhibitor that also inhibits COX isozymes (27, 28). As the enzyme acts in the final steps of the androgen synthesis pathway in the prostate (16), AKR1C3 inhibitors would not cause the accumulation of DOC in the adrenal gland and would not have to be coadministered with prednisone. These properties make AKR1C3 an attractive target for managing CRPC disease progression as well as countering therapeutic resistance. The related isoforms AKR1C1 and AKR1C2 share high homology with AKR1C3, function to eliminate DHT, and should not be inhibited (29). Numerous AKR1C3 inhibitors with diverse scaffolds are known; however, none have made their way into the clinic (30–32). Considerable interest has developed in the role of AKR1C3 in a variety of cancers of the breast (33), lung (34), and colon (35).

Herein, we report on the actions of a novel, potent, isoform-selective and metabolically stable AKR1C3 inhibitor, KV-37 (Fig. 1A; refs. 36–38), in androgen-sensitive prostate cancer cell lines and a xenograft model. We have previously reported AKR1C3 inhibitors that act synergistically with etoposide and daunorubicin as chemotherapeutic agents in a panel of leukemia cell lines (38). The AKR1C3 inhibitor KV-37 exerts high synergistic drug interaction in combination with ENZ, which is superior to that seen with INDO. Mechanistic studies reveal that KV-37 causes apoptotic cell death in ENZ-resistant prostate cancer cell lines with a consequent reduction in PSA levels, and in vivo studies demonstrate >50% reduction in tumor growth without observable toxicity.

Figure 1.

KV-37 induces potent antitumor activity in prostate cancer cell lines. A, Structure of KV-37. Percentage cell viability of 22Rv1 cells cultured in (B) normal media, (C) CSS media, and (D) normal media supplemented with 10 nmol/L Δ4-androstenedione, and (E) CSS media supplemented with 10 nmol/L Δ4-androstenedione. Percentage cell viability of LNCaP cells cultured in (F) normal media and (G) CSS media. H, IC50 values of KV-37 determined in 22Rv1 and LNCaP cells. I, Comparative expression of AKR1C3 in prostate cancer cell lines and normal prostate cells (left) and overexpression of AKR1C3 in 22Rv1 cells when cultured in CSS media (right). Data shown as mean ± SD of at least three independent experiments, n = 6. NM, normal media.

Figure 1.

KV-37 induces potent antitumor activity in prostate cancer cell lines. A, Structure of KV-37. Percentage cell viability of 22Rv1 cells cultured in (B) normal media, (C) CSS media, and (D) normal media supplemented with 10 nmol/L Δ4-androstenedione, and (E) CSS media supplemented with 10 nmol/L Δ4-androstenedione. Percentage cell viability of LNCaP cells cultured in (F) normal media and (G) CSS media. H, IC50 values of KV-37 determined in 22Rv1 and LNCaP cells. I, Comparative expression of AKR1C3 in prostate cancer cell lines and normal prostate cells (left) and overexpression of AKR1C3 in 22Rv1 cells when cultured in CSS media (right). Data shown as mean ± SD of at least three independent experiments, n = 6. NM, normal media.

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Cell culture and reagents

22Rv1 and LNCaP cells were purchased from the American Type Culture Collection (ATCC in 2016) and cultured in RPMI 1640 supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (39). LNCaP1C3 cells overexpressing AKR1C3 were generated by stable transfection of AKR1C3 plasmid as previously described (12). WPMY-1 cells were purchased from the ATCC (2017) and maintained in DMEM media supplemented with 5% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. All cell lines were authenticated via short tandem repeat analysis and tested for mycoplasma using the MycoAlert mycoplasma detection kit as per the manufacturer's instructions (Texas cancer cell repository) in May 2017, showing no contamination. Cell line use was limited to passage nine or lower. Where indicated, cells were also cultured in charcoal-stripped (CSS) media prepared by supplementing RPMI 1640 without phenol red with charcoal-stripped FBS. All cells were maintained at 37°C in a humidified incubator with 5% carbon dioxide. ENZ or MDV3100 (catalog no. 50-101-3979) and INDO (catalog no. AAA1991006) were purchased from Fisher Scientific. Stock solutions (200 mmol/L) of ENZ, INDO, baccharin, KV-32, and KV-37 were prepared in DMSO and were serially diluted for cell culture treatments to a final concentration range of 1 to 200 μmol/L, maintaining the final DMSO concentration at less than 1%.

Inhibition of testosterone production by AKR1C3 inhibitors

Systems (200 μL) containing 100 mmol/L potassium phosphate (pH 7.0), 0.9 mmol/L NADPH, 1.2 to 40 μmol/L 4-androstene-3,17-dione (Δ4-AD; containing 2.6 nmol/L [3H]-4-androstene-3,17-dione), KV-37 (0–10 μmol/L), and 0.3 to 10 μmol/L inhibitor plus 1.65 μmol/L AKR1C3 were incubated at 37°C, and samples were prepared as previously reported (40). Quantification of the individual steroid analytes was achieved by integrating the radioactivity corresponding to each peak in the radiochromatogram and representing the radioactivity as a percentage of the total corrected cpm for the organic fraction from which it was derived.

Stability testing of AKR1C3 inhibitors

Baccharin, KV-37, and a related ester analogue KV-32 (2 μmol/L solution in DMSO) were incubated with male CD-1 murine liver S9 fractions containing a NADPH-generating system for 0 to 240 minutes at 37°C. Reactions were quenched with 0.5 mL (1:1) of acetonitrile (ACN) containing 0.2% formic acid and 100 ng/mL internal standard (IS; final conc. = 50 ng/mL). Samples were vortexed for 15 seconds, incubated at room temperature for 10 minutes, and spun for 5 minutes at 2,400 rpm. The supernatant (0.9 mL) was then transferred to an Eppendorf tube, clarified by centrifugation for 5 minutes at 13,200 rpm at 4°C, and transferred to an HPLC vial for analysis by Qtrap 4000 mass spectrometry using the following parameters: Ion Source/Gas Parameters: curtain gas (CUR) = 35, collisionally activated dissociation (CAD) = medium, ionspray voltage (IS) = 4,500, temperature (TEM) = 600, nebulizing gas GS1 = 70, drying gas GS2 = 70. Buffer A: dH2O + 0.1% formic acid; Buffer B: ACN + 0.1% formic acid; flow rate 1.5 mL/min; column Agilent C18 XDB column, 5 μm packing 50 × 4.6 mm size; 0–1 min 97% A, 1–2.5 min gradient to 99% B, 2.5–3.5 min 99% B, 4–4.1 min gradient to 97% A, 4.1–4.5 min 97% A; IS: Warfarin (in MeOH, transition 307.3 to 160.9). Ion transitions followed were 363.1 [M-H] to 186.9 for baccharin and KV-32, and 362.1 [M-H] to 318.1 for KV-37.

Cell viability assays

Cells were seeded at a density of 10,000 cells/well in 96-well plates and were incubated in either normal media (24 hours) or CSS media (48 hours). Treatments with ENZ, INDO, KV-37, or combinations of ENZ and KV-37 were made with or without 10 nmol/L Δ4-AD (AKR1C3 substrate) and incubated at the indicated time points (24, 48, 72, and 96 hours). For pretreatment experiments, cells were treated with KV-37 for 24 hours followed by the addition of ENZ and incubated for a further 72 hours. Cell viability was determined by the MTS tetrazolium dye assay as described previously (38). No intrinsic absorbance was noted with either KV-37 or ENZ in the MTS assay.

Inhibition of testosterone production in LNCaP1C3 cells

LNCaP1C3 cells (1.5 × 106 cells per well) were placed in 2 mL of phenol red–free RPMI, 5% CD-FBS, 2 mmol/L l-glu, and 1% P/S. Cells were incubated with DMSO, or 10 or 30 μmol/L KV-37 for 30 minutes at 37°C. After the preincubation, 100 nmol/L 3H Δ4-AD final concentration was added to the wells and cells were incubated for 24 hours at 37°C. Cell media were transferred into labeled borosilicate glass tubes after 48 hours and extracted with 2 mL ethyl acetate. Samples were vortexed for 30 seconds and placed in a −20°C freezer for 1 hour to separate the phases. The frozen samples were allowed to thaw at room temperature for 10 minutes and centrifuged for 20 minutes before the organic phase was extracted and the process repeated. The 4 mL organic layer extract was then dried under vacuum for radiochromatography. The aqueous fractions were dried under vacuum for 30 minutes to evaporate any leftover organic solvent residue. Note that 1% glacial acetic acid aqueous solution (25 μL) was added to the samples to adjust the pH to 6.5, followed by addition of 18 μL of 25 U/μL Escherichia coli β-glucuronidase. Samples were placed in a water bath at 37°C for 24 hours. Samples were then extracted as before. All samples were resuspended in 100 μL of ethyl acetate and vortexed for 30 seconds to mix. Using 10 μL capillary tubes, each sample was spotted onto a multi-channel UNIPLATE 20 × 20 cm TLC plate and analyzed as described before (32). The peak of testosterone observed in LNCaP cells represented the basal formation of testosterone that was independent of AKR1C3, because LNCaP cells are AKR1C3 null. In the presence of 10 mmol/L AKR1C3 inhibitor, the amount of testosterone formed in the LNCaP1C3 cells was identical to the basal level in LNCaP cells showing complete inhibition of AKR1C3-mediated testosterone production.

Apoptosis assay

Annexin V/FITC apoptosis assay was performed using a kit and according to the manufacturer's instructions (BD Biosciences, catalog no. 556547). Cells were seeded at a density of 0.2 × 106 cells/well in 24-well plates and incubated in CSS media for 48 hours followed by treatment with KV-37 for 48 and 72 hours. For pretreatment experiments, cells were treated with KV-37 for 24 hours followed by the addition of ENZ and incubated for a further 72 hours. After appropriate treatments, the samples were analyzed by flow cytometery (Accuri C6).

Western blotting

Whole cell lysates were prepared using 4% (w/v) CHAPS in urea-tris buffer. Protein was quantified and electrophoresed as described previously (41). The membranes were probed with primary antibodies overnight against AR (Cell Signaling Technology; #5153P, rabbit mAb), PSA (Cell Signaling Technology; #5877S, rabbit mAb), AKR1C3 (Sigma; #A6229, mouse mAb), C-PARP (Cell Signaling Technology; #5625S, Rabbit mAb), C-caspase3 (Cell Signaling Technology; #9661S, rabbit mAb), and actin (Sigma; #A5441, mouse mb) followed by incubation with anti-mouse (Sigma; #SAB4600224) or anti-rabbit (Perkin Elmer; #NEF812001EA) secondary antibody (1:2,000) horseradish peroxidase conjugate for 2 hours and detected by chemiluminescence (42). All the antibodies from Cell Signaling Technology were diluted 1:1,000, AKR1C3 (1:500), and actin (1:2,000). Bands were quantified by densitometry using ImageJ software.

Tumor xenograft and pharmacokinetic study

All animal experiments were carried out according to approved Institutional Animal Care and Use Committee protocols at the University of Texas Southwestern Medical Center (Dallas, TX). Eighteen 6- to 8-week-old female NOD-SCID mice (low circulating testosterone) were implanted with 4 × 106 22Rv1 cells in a volume of 0.1 mL in the left flank. When tumor volume reached approximately 100 mm3 (day 13), mice were randomly divided into two groups. One group was administered 20 mg/kg KV-37 QD intraperitoneally, and the other group was given vehicle control (10% DMSO, 20% PEG400, 0.5% tween 80, 69.5% carbonate buffer, pH = 9.2). Tumor volumes were measured twice a week with Vernier calipers, and tumor volume was calculated as (L × W2) × 3.14)/6 (41). On day 34, mice were humanely euthanized, and tumors were collected, weighed, and frozen in liquid nitrogen after taking pictures. Tumors were lysed for Western blot analysis. Whole blood was collected in an Eppendorf tube with ACD anti-coagulant for plasma separation for pharmacokinetic evaluation of KV-37.

Pharmacokinetic evaluation of KV-37

For Standards, 98 μL of blank plasma was added to an Eppendorf and spiked with 2 μL of IS (Warfarin). For QCs, 98.8 μL blank plasma was added to an Eppendorf and spiked with 1.2 μL of IS. Note that 100 μL of plasma was mixed with 200 μL of methanol containing 0.15% formic acid and 37.5 ng/mL IS (IS final conc. = 25 ng/mL). The samples were vortexed for 15 seconds, incubated at room temperature for 10 minutes, and spun at 13,200 rpm in a standard microcentrifuge. The supernatant was then analyzed by LC-MS/MS as described above to test the stability of the AKR1C3 inhibitors.

Statistical analyses and quantification of degree of synergism

Experiments were repeated at least thrice, and the statistical significance was calculated using the Student t test. A P value of <0.05 was considered statistically significant. IC50 values were calculated by GraphPad prism software. Combination treatments of KV-37 with ENZ were analyzed by CompuSyn software (Biosoft) based on the Chou–Talalay method. Combination index (CI) and dose reduction index (DRI) values were generated to evaluate the degree of synergistic drug interaction.

Inhibitors of AKR1C3 from various structural classes [flavones (30), jasmonates (31), and NSAIDS (23, 33)] have previously been described. We adopted a cinnamic acid derivative “baccharin” and conducted structure–activity relationship (SAR) studies to identify lead AKR1C3 inhibitors (37, 38). The cinnamic acid derivative KV-37 (Fig. 1A) was chosen for further studies as it demonstrated potent AKR1C3 inhibition (IC50 = 66 nmol/L) and selectivity (109-fold over AKR1C2; ref. 38). In order to delineate the preliminary mechanism of action, we tested its mode of AKR1C3 inhibition. We find that KV-37 displayed competitive enzyme inhibition versus AKR1C3 when Δ4-AD was used as substrate, yielding a Ki value of 3.0 μmol/L for the E.NADPH.KV-37 complex. The difference between this Ki value and the IC50 value seen in the preliminary inhibitor screen is consistent with the lower Ki value for the E.NADP+.inhibitor complex which is seen with other AKR1C3 inhibitors when they are screened in the oxidation direction (Supplementary Fig. S1; ref. 40). We also examined the mode of action of KV-37 in androgen-dependent 22Rv1 cells (high AKR1C3 expression) and LNCaP cells (low AKR1C3 expression). The 22Rv1 cell line shows increased expression of AKR1C3 when grown in CSS media and possesses intrinsic resistance to ENZ and AA (27). LNCaP cells stably overexpressing AKR1C3 (LNCaP1C3) were used to further validate the effect of KV-37 on the target (12). Further, WPMY-1 cells, a nonmalignant prostate stromal cell line, devoid of AKR1C3 expression (Fig. 1I), were employed to evaluate the selective antineoplastic effects of KV-37.

KV-37 inhibits AKR1C3 activity in prostate cancer cell lines by inhibiting the conversion of Δ4-AD to testosterone

KV-37 was used to inhibit the production of testosterone in LNCaP1C3 cells following β-glucuronidase treatment of the aqueous phase. At a concentration of 10 μmol/L, >80% inhibition of testosterone production is blocked and the residual testosterone produced is that seen in untransfected LNCaP cells (Supplementary Fig. S2).

KV-37 exhibits excellent plasma stability above that of the parent and other derivatives

Baccharin undergoes rapid hydrolysis to yield the inactive phenol drupanin (AKR1C3 IC50 = 15 μmol/L; ref. 37). In order to compare the hydrolytic stability of lead compounds, baccharin and its derivatives were subjected to incubation with mouse S9 fractions in the presence of phase I cofactors from 0 to 240 minutes. Aliquots were withdrawn and analyzed by LC-MS at various time points. As expected, baccharin and a related ester analogue (KV-32) were readily hydrolyzed (Supplementary Fig. S3). The amide-based inhibitor KV-37 displayed remarkable stability (t1/2 = >240 minutes).

AKR1C3 inhibitor KV-37 exhibits greater reduction of prostate cancer cell viability compared with baccharin

Baccharin and its analogue KV-37 were screened for antineoplastic effects in a panel of human prostate cancer cell lines (LNCaP, 22Rv1, and LNCaP1C3). Baccharin consistently displayed no toxicity among all the cell lines tested, up to concentrations as high as 100 μmol/L (Supplementary Fig. S4). This is in agreement with previous studies (38). However, treatment with KV-37 demonstrated a greater dose and time-dependent reduction in cell viability in androgen-dependent 22Rv1 and LNCaP cell lines. The antineoplastic effect was greater in 22Rv1 cells due to the higher expression of AKR1C3 as compared with the LNCaP cell line (Fig. 1B–H). For 22Rv1 cells, a significant decline in IC50 values (P = 0.0015, <0.0001, <0.0001 at 24, 48, and 72 hours, respectively) was observed after KV-37 treatment when the cells were cultured in CSS media as compared with normal media (Fig. 1B, C, H). This is attributable to the overexpression of AKR1C3 when cells are grown in media devoid of androgens (Fig. 1I). This drives the cellular phenotype to more closely resemble clinical castrate conditions, where the cancer cells are dependent on AKR1C3 for survival and proliferation. Culture in CSS media supplemented with an AKR1C3 substrate, 10 nmol/L Δ4-AD, led to a slight reduction in the antitumor effect and a significant increase in IC50 values (P = <0.0001, <0.0009, <0.0001 at 24, 48, and 72 hours, respectively) as compared with those observed in nonsupplemented CSS media (Fig. 1C, E, H). Addition of 10 nmol/L Δ4-AD to CSS culture mimics the serum level of this androgen in CRPC patients (12, 43, 44). As Δ4-AD is a substrate for AKR1C3, this observation suggests that the reduced antineoplastic activity of KV-37 is due to the enzymatic formation of T and DHT and corroborates AKR1C3 as the inhibitor target. The antitumor activity of KV-37 was also evaluated in normal media supplemented with 10 nmol/L Δ4-AD as a control experiment to directly measure the difference in dose responses between normal and CSS media (Fig. 1D and H).

AKR1C3 inhibitor KV-37 sensitizes ENZ-resistant prostate cancer cells to ENZ by exerting a synergistic drug effect

The 22Rv1 cell line is intrinsically resistant to ENZ (27). The dose–response curve of ENZ in 22Rv1 cells displays increasing resistance upon culture in CSS media (Supplementary Fig. S5), which are accompanied by an increase in AKR1C3 expression (Fig. 1I). LNCaP cells that are sensitive to ENZ-induced cytotoxicity were used as controls to compare the effect of KV-37 in combination with ENZ. To test whether the sensitivity of 22Rv1 cells to ENZ could be restored with KV-37, 22Rv1 and LNCaP cells were pretreated with KV-37 for 24 hours followed by exposure to ENZ. The combination demonstrated a remarkable synergistic effect in 22Rv1 cells at both 48 and 72 hours after ENZ exposure in normal as well as CSS media (CI = 0.19 and 0.07, respectively; Fig. 2A–C, I). Concentrations as low as 1 μmol/L of KV-37 were capable of sensitizing 22Rv1 cells to only 1 μmol/L of ENZ and reduced viability by more than 50%. Either agent employed alone did not exert any antitumor effect. Among cultures supplemented with 10 nmol/L Δ4-AD in normal and CSS media, the synergistic drug interaction was maintained (CI = 0.33 and 0.10, respectively; Fig. 2D–F, I). Quantification of degree of synergism was made using the Chou–Talalay method (45). Up to a 200-fold reduction in ENZ dosing was achieved in 22Rv1 cells (Fig. 2I). LNCaP cells however, due to their low expression of AKR1C3, displayed a lack of synergistic drug effect at the concentrations tested, in both normal and CSS media, and the interaction was merely additive (CI = 1.12 and 1.14, respectively; Fig. 2G–I). On the contrary, cotreatment experiments did not show any adjuvant effect in either cell line cultured in normal or CSS media, with or without Δ4-AD supplementation (Supplementary Fig. S6). This observation suggests that the activity of AKR1C3 needs to be inhibited prior to ENZ treatment in 22Rv1 cells for therapeutic effect.

Figure 2.

Pretreatment with KV-37 sensitizes prostate cancer cell lines to the antitumor effects of ENZ. Percentage cell viability of prostate cancer cells when pretreated with KV-37 for 24 hours followed by ENZ treatment at indicated concentrations and time points in (A) 22Rv1 cells cultured in normal media and (B and C) 22Rv1 cells cultured in CSS media, (D) 22Rv1 cells cultured in normal media supplemented with 10 nmol/L Δ4-androstenedione and (E and F) 22Rv1 cells cultured in CSS media supplemented with 10 nmol/L Δ4-androstenedione, and (G) LNCaP cells cultured in normal media and (H) LNCaP cells cultured in CSS media. I, Quantification of the degree of synergism. CI and DRI values for KV-37 and ENZ combination treatments in prostate cancer cells. CI and DRI indices generated using CompuSyn software. Data shown as mean ± SD of at least three independent experiments, n = 6.

Figure 2.

Pretreatment with KV-37 sensitizes prostate cancer cell lines to the antitumor effects of ENZ. Percentage cell viability of prostate cancer cells when pretreated with KV-37 for 24 hours followed by ENZ treatment at indicated concentrations and time points in (A) 22Rv1 cells cultured in normal media and (B and C) 22Rv1 cells cultured in CSS media, (D) 22Rv1 cells cultured in normal media supplemented with 10 nmol/L Δ4-androstenedione and (E and F) 22Rv1 cells cultured in CSS media supplemented with 10 nmol/L Δ4-androstenedione, and (G) LNCaP cells cultured in normal media and (H) LNCaP cells cultured in CSS media. I, Quantification of the degree of synergism. CI and DRI values for KV-37 and ENZ combination treatments in prostate cancer cells. CI and DRI indices generated using CompuSyn software. Data shown as mean ± SD of at least three independent experiments, n = 6.

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AKR1C3 overexpression in LNCaP cells confers resistance to the antitumor effects of ENZ that is reversed upon KV-37 treatment

The LNCaP1C3 cell line, stably overexpressing AKR1C3, was used to evaluate the activity of KV-37 alone and in combination with ENZ to further validate AKR1C3 as the target. KV-37 induced a dose- and time-dependent reduction in cell viability of LNCaP1C3 cells (Fig. 3A and table insert). The antitumor drug effect increased further as compared with 22Rv1 and low AKR1C3 expressing LNCaP cells at all time points tested. These observations demonstrate that the bioactivity of KV-37 is AKR1C3-dependent and further corroborates AKR1C3 as the compound's target. Next, a dose–response curve of ENZ was evaluated in LNCaP1C3 cells that revealed the generation of a resistant phenotype as observed in previous findings (27). The 72-hour IC50 of ENZ increased to 150 μmol/L in LNCaP1C3 cells as compared with 50 μmol/L in sensitive LNCaP cells (Fig. 3B; Supplementary Fig. S5). To overcome this drug resistance, cells were treated with a combination of KV-37 and ENZ as cotreatments for 72 hours or as 24-hour pretreatments with KV-37 followed by ENZ exposure for a further 72 hours. Cotreatment experiments showed a moderate degree of drug synergism (CI = 0.54; Fig. 3C). Among the pretreatment experiments, a very high degree of synergistic dug interaction was observed (CI = 0.14), and concentrations as low as 1 μmol/L of KV-37 were able to sensitize the cells to ENZ-induced cytotoxicity. As a result, the effective ENZ concentration to reduce prostate cancer cell viability was reduced by 25-fold and yielded an IC50 of 8.19 μmol/L for the drug combination (Fig. 3D). These findings strongly suggest that KV-37, by virtue of its AKR1C3-inhibiting properties, is able to resensitize ENZ-resistant prostate cancer cells that are high expressers of AKR1C3 toward the antineoplastic effects of ENZ.

Figure 3.

AKR1C3 overexpression in LNCaP cells (LNCaP1C3) increases the antineoplastic activity of KV-37 and induces resistance to ENZ that is overcome by pretreatment with KV-37. Percentage cell viability of LNCaP1C3 cultured in CSS media after (A) KV-37 treatment at indicated time points and concentrations IC50 values, (B) ENZ treatment at indicated time points and concentrations, (C) cotreatment with KV-37 and ENZ for 72 hours, and (D) pretreatment with KV-37 followed by ENZ exposure for 72 hours. Insert depicts the parameters for quantifying the degree of synergism. Data shown as mean ± SD of at least three independent experiments, n = 6.

Figure 3.

AKR1C3 overexpression in LNCaP cells (LNCaP1C3) increases the antineoplastic activity of KV-37 and induces resistance to ENZ that is overcome by pretreatment with KV-37. Percentage cell viability of LNCaP1C3 cultured in CSS media after (A) KV-37 treatment at indicated time points and concentrations IC50 values, (B) ENZ treatment at indicated time points and concentrations, (C) cotreatment with KV-37 and ENZ for 72 hours, and (D) pretreatment with KV-37 followed by ENZ exposure for 72 hours. Insert depicts the parameters for quantifying the degree of synergism. Data shown as mean ± SD of at least three independent experiments, n = 6.

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KV-37 induces cell death in prostate cancer cells via apoptosis

To elucidate the primary mechanism of cell death, 22Rv1 and LNCaP1C3 cells cultured in CSS media were treated with increasing doses of KV-37. After 48- and 72-hour time periods after treatment, the cells were harvested and stained with Annexin V/PI to measure apoptotic cell percentage. The percentage of the apoptotic cell population increased with KV-37 concentration in a dose- and time-dependent manner (Fig. 4A; Supplementary Fig. S7). Consistent with the results from the cytotoxicity screening, the effect was significantly greater in the LNCaP1C3 cell line as compared with the 22Rv1 cells at both time points (P = <0.05 at 25 μmol/L KV-37 and <0.003 at 50 μmol/L KV-37), underscoring the significance of AKR1C3 overexpression in prostate cancer. In order to further corroborate these findings, Western blot analysis was conducted after inhibitor treatment following an identical dosing schedule. The results, in agreement with Annexin V/PI assay, consistently show a dose-dependent increase in the levels of C-caspase3 and C-PARP with higher expression at 72 hours as compared with 48-hour incubation time, among both 22Rv1 and LNCaP1C3 cell lines (Fig. 4C). Taken together, these data strongly suggest apoptosis as the primary mechanism of antitumor action for KV-37.

Figure 4.

KV-37 induces apoptosis in prostate cancer cells alone and potentiates the degree of apoptosis when combined with ENZ. A, Dose- and time-dependent increase in apoptotic cell population after treatment with KV-37 in indicated cell lines as analyzed by Annexin V/PI costaining. B, Increase in apoptotic cell population after treatment with indicated concentrations of KV-37, ENZ, and a 24-hour pretreatment combination of KV-37 and ENZ in indicated cell lines analyzed 72 hours after ENZ exposure by Annexin V/PI costaining. C, Western blot showing an increase in C-caspase3 and C-PARP levels after treatment with KV-37 at indicated concentration and time points in prostate cancer cells. D, Western blot showing a greater increase in C-caspase3 and C-PARP levels with a 24-hour pretreatment combination of KV-37 and ENZ in prostate cancer cells analyzed 72 hours after ENZ exposure. Data shown as mean ± SD of at least three independent experiments, n = 3. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; and ns, nonsignificant.

Figure 4.

KV-37 induces apoptosis in prostate cancer cells alone and potentiates the degree of apoptosis when combined with ENZ. A, Dose- and time-dependent increase in apoptotic cell population after treatment with KV-37 in indicated cell lines as analyzed by Annexin V/PI costaining. B, Increase in apoptotic cell population after treatment with indicated concentrations of KV-37, ENZ, and a 24-hour pretreatment combination of KV-37 and ENZ in indicated cell lines analyzed 72 hours after ENZ exposure by Annexin V/PI costaining. C, Western blot showing an increase in C-caspase3 and C-PARP levels after treatment with KV-37 at indicated concentration and time points in prostate cancer cells. D, Western blot showing a greater increase in C-caspase3 and C-PARP levels with a 24-hour pretreatment combination of KV-37 and ENZ in prostate cancer cells analyzed 72 hours after ENZ exposure. Data shown as mean ± SD of at least three independent experiments, n = 3. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; and ns, nonsignificant.

Close modal

Combination of KV-37 with ENZ potentiates the degree of apoptosis in prostate cancer cells

To determine whether the effect of KV-37 to induce apoptosis in 22Rv1 cells persists in the presence of ENZ, cells were pretreated with KV-37 followed by ENZ incubation for 72 hours. The cells were subjected to apoptosis analysis by Annexin V/PI costaining. In 22Rv1 cells, a combination of 10 μmol/L KV-37 with 25 μmol/L ENZ displayed a significant increase (P = <0.05) in apoptotic cell percentage as compared with 25 μmol/L ENZ treatment alone, whereas a combination of 25 μmol/L KV-37 with 25 μmol/L ENZ displayed an even more significant (P = <0.0001) increase in apoptotic cell percentage as compared with either treatment alone (Fig. 4B, left plot; Supplementary Fig. S7). Percent apoptosis was also increased in LNCaP1C3 cells wherein both 10 and 25 μmol/L KV-37 plus 25 μmol/L ENZ demonstrated a significant (P = <0.05 and <0.01, respectively) increase in percent apoptosis levels than with either treatment alone (Fig. 4B, right plot; Supplementary Fig. S7). These findings were further confirmed by Western blot analysis showing a greater increase in C-caspase3 and C-PARP levels in drug combination experiments performed with 10 μmol/L KV-37 and 25 μmol/L ENZ in 22Rv1 (Fig. 4D, left plot) and LNCaP1C3 cells (Fig. 4D, right plot). These observations complement the cytotoxicity studies of the drug combinations and further strengthen the hypothesis of apoptotic cell death induced by KV-37 in prostate cancer cells.

Molecular changes following KV-37 treatment in prostate cancer cells

Western blot analysis was conducted to measure the expression levels of AKR1C3, AR, and PSA. A slight decrease in the expression level of AKR1C3 was noted in 22Rv1 cells at 48 hours, whereas after 72-hour AKR1C3 expression increased, both changes were observed at 25 and 50 μmol/L KV-37 concentrations (Fig. 5A). In LNCaP1C3 cells, a decrease in AKR1C3 expression was noted at only 50 μmol/L concentration at 48 hours. On the contrary, no change in AKR1C3 expression was observed in the LNCaP1C3 cell line at all concentrations tested at 72 hours after KV-37 exposure (Fig. 5B). Because AKR1C3 serves as a coactivator for AR (46), it was prudent to measure AR levels after treatment. We observed that expression of AR complements AKR1C3 levels and was found to be decreased after 48 hours of treatment, whereas at 72 hours, no change in AR levels was observed in both 22Rv1 and LNCaP1C3 cell lines. By contrast, a concentration-dependent decline in PSA levels was observed with KV-37 in both cell lines at 48- and 72-hour treatments. Such findings signify a beneficial therapeutic outcome in prostate cancer after treatment with KV-37 (Fig. 5A and B).

Figure 5.

Molecular changes in AR signaling following KV-37 treatment. Treatment with KV-37 alone at indicated concentration and time points in (A) 22Rv1 and (B) LNCaP1C3 cells cultured in CSS media. Treatment with indicated concentrations of KV-37, ENZ, and a 24-hour pretreatment combination of KV-37 and ENZ in (C) 22Rv1 cells and (D) LNCaP1C3 cells cultured in CSS media analyzed 72 hours after ENZ exposure.

Figure 5.

Molecular changes in AR signaling following KV-37 treatment. Treatment with KV-37 alone at indicated concentration and time points in (A) 22Rv1 and (B) LNCaP1C3 cells cultured in CSS media. Treatment with indicated concentrations of KV-37, ENZ, and a 24-hour pretreatment combination of KV-37 and ENZ in (C) 22Rv1 cells and (D) LNCaP1C3 cells cultured in CSS media analyzed 72 hours after ENZ exposure.

Close modal

Combination of KV-37 with ENZ downregulates AR expression that increases after ENZ treatment

To determine the mechanism of synergistic interaction between KV-37 and ENZ, Western blot analysis was performed on AR, AKR1C3, and PSA. In contrast to previous reports that indicate AR degradation in 22Rv1 cells (47), treatment of 25 μmol/L ENZ alone increased AR expression in both 22Rv1 and LNCaP1C3 cells signifying drug resistance. By contrast, a combination treatment of 25 μmol/L ENZ with 10 μmol/L KV-37 reduced the expression to less than control levels (Fig. 5C and D). Consistent with inhibitor treatment alone, the combination produced no change in AKR1C3 expression levels. However, the combination of 10 μmol/L KV-37 with 25 μmol/L ENZ completely abrogates PSA expression in 22Rv1 cells and reduced PSA to control levels in LNCaP1C3 cells. These data suggest apoptotic cell death as a primary mechanism of synergistic antitumor effect between the two compounds leads to a decline in AR and PSA expression.

Antitumor effect and initial pharmacokinetics of KV-37 in a 22Rv1 xenograft model

Treatment with KV-37 at 20 mg/kg per day in female mice harboring 22Rv1 xenografts significantly (P = 0.0003) inhibited tumor volume by greater than 50% and tumor weight by 35% (P = 0.0179; Fig. 6A–C) as compared with vehicle-treated controls. No significant change in body weight was observed (Fig. 6D), indicative of a nontoxic effect. On day 34, mice were euthanized and blood collected at 10, 120, and 360 minutes after KV-37 administration at 20 mg/kg and pharmacokinetic parameters analyzed. The peak plasma concentration (Cmax) was determined to be 86.5 μg/mL, and Tmax, AUC, Vz/F, and CL/F were calculated (Fig. 6E). Further, Western blot analyses of the tumor samples were conducted that showed no significant upregulation of AKR1C3 expression in KV-37–treated tumors as compared with nontreated controls (Fig. 6F).

Figure 6.

KV-37 induces tumor growth inhibition in prostate cancer xenografts. Reduction in (A) prostate tumor volume and (B) prostate tumor weight in 22Rv1 murine xenografts after treatment with 20 mg/kg KV-37. C, Representative images showing a reduction in tumor size after treatment with 20 mg/kg KV-37. D, Mice body weight after KV-37 or vehicle treatment. E, Pharmacokinetic parameters determined in plasma after treatment with 20 mg/kg KV-37. F, Expression of AKR1C3 in tumor lysates (left plot), and each blot indicates tumor lysate from individual mouse. Bar graph representation of quantified blots (right plot).

Figure 6.

KV-37 induces tumor growth inhibition in prostate cancer xenografts. Reduction in (A) prostate tumor volume and (B) prostate tumor weight in 22Rv1 murine xenografts after treatment with 20 mg/kg KV-37. C, Representative images showing a reduction in tumor size after treatment with 20 mg/kg KV-37. D, Mice body weight after KV-37 or vehicle treatment. E, Pharmacokinetic parameters determined in plasma after treatment with 20 mg/kg KV-37. F, Expression of AKR1C3 in tumor lysates (left plot), and each blot indicates tumor lysate from individual mouse. Bar graph representation of quantified blots (right plot).

Close modal

INDO displays diminished cell viability reduction and a weak synergistic drug action compared with the more potent inhibitor KV-37

INDO (AKR1C3 Ki = 8 μmol/L for the E.NADPH.Indomethacin complex) has been shown to reverse ENZ resistance in prostate cancer models (27). The dose–response curve of INDO showed a weak inhibition of cell viability at 96-hour incubation, IC50 = 112.8 μmol/L (Supplementary Fig. S8A). Further, we also administered INDO as a 24-hour pretreatment to LNCaP1C3 cells followed by ENZ exposure for 72 hours. A moderate synergistic drug action was observed (CI = 0.32; DRI = 6.4; Supplementary Fig. S8B) as compared with the very high degree of synergism with KV-37 under identical conditions (CI = 0.14; DRI = 24.8). These findings further bolster the concept that AKR1C3 inhibition by KV-37 sensitizes ENZ-resistant prostate cancer cells to the chemotherapeutic, and is superior to INDO.

KV-37 exhibits no toxicity alone, or in combination with ENZ, in WPMY-1 prostate cells

The nonmalignant prostate stromal cell line WPMY-1 was chosen to evaluate the toxicity of KV-37. Consistent with the observation that WPMY-1 cells are low expressers of AKR1C3, the reduction of cell viability induced by KV-37 was minimal with 96-hour IC50 = 80 μmol/L. At concentrations of 1, 10, and 25 μmol/L of KV-37, those used in our study of prostate cancer cells, no reduction in WPMY-1 cell viability was observed, an observation consistent with no loss of body weight in mice xenografts upon 20 mg/kg daily dosing. Further, pretreatment with KV-37 for 24 hours followed by ENZ exposure for 72 hours resulted in no loss of cell viability (Supplementary Fig. S8C and S8D).

Prostate tumors resistant to AA and ENZ are characterized by overexpression of steroidogenic enzymes where AKR1C3 is among the most overexpressed (26, 48, 49). Thus, in situ or intratumoral androgen biosynthesis is a predominant factor of therapeutic failure with second-line ADT (13, 48). Overexpression of AKR1C3 by stable transfection in prostate cancer cells imparted ENZ resistance which was overcome by AKR1C3 knockdown by shRNA. INDO, a weak AKR1C3 inhibitor, was also able to rescue xenografts from ENZ and AA resistance (27, 28). Continuing our efforts to identify potent and selective AKR1C3 inhibitors, we synthesized and characterized KV-37, a nanomolar inhibitor of AKR1C3 with >100-fold isoform selectivity and metabolic stability (38). We demonstrate that the synergistic effects of KV-37 with ENZ in reducing prostate cancer cell viability are much greater than that seen with INDO (Supplementary Fig. S9) and that the effects of KV-37 are mediated by AKR1C3 inhibition.

We demonstrate that KV-37 inhibits the activity of AKR1C3 in prostate cancer cells by inhibiting the conversion of Δ4-AD to testosterone. Crucially, KV-37 exerts a preferential antineoplastic effect in AKR1C3-overexpressing 22Rv1 and LNCaP1C3 cells as compared with the low expressing LNCaP cell line and nonmalignant WPMY-1 cells. Furthermore, culture of 22Rv1 cells in CSS media, which are devoid of androgens, upregulates AKR1C3 expression further and results in greater susceptibility to KV-37--induced antineoplastic effects. Because the physiologic androgen level in clinical castrate conditions is of the order of 1 to 10 nmol/L, CSS was supplemented with 10 nmol/L Δ4-androstenedione to further mimic the CRPC phenotype (44). KV-37 displayed an equally robust inhibition of cell viability in such cultures. The 22Rv1 cell line is inherently resistant to ENZ, whereas LNCaP cells became resistant to ENZ when AKR1C3 was stably expressed in this cell line. Pretreatment with KV-37 restored the sensitivity of 22Rv1 cells to ENZ and provided a strong synergistic effect resulting in >200-fold reduction in chemotherapeutic dose under conditions that mimic the CRPC disease phenotype. The strong synergistic effect was maintained in LNCaP1C3 cells upon pretreatment with KV-37. A moderate synergistic effect was also observed in these cells if KV-37 was coadministered with ENZ. By contrast, as LNCaP cells are low expressers of AKR1C3, only an additive effect was observed, an observation that may be ascribed to the general toxicity of KV-37 toward cancer cells as evidenced previously in leukemic cell lines (38).

Insights into the mechanism of apoptosis mediated by KV-37 were obtained by measuring an increase in the apoptotic markers C-caspase3 and C-PARP, as well as a dose-dependent increase in Annexin V/PI after KV-37 treatment. The observation that percent apoptotic cells were greater in LNCaP1C3 cells at any given concentration, compared with 22Rv1 cells, corroborates AKR1C3 as the target of KV-37. In combination drug treatments, 25 μmol/L ENZ was employed, as this corresponds to the minimum steady-state plasma concentration of 12 μg/mL of the drug, the physiological concentration achieved clinically in prostate cancer patients at 150 mg/day dosing regimen (17). A subtherapeutic concentration of KV-37 (10 μmol/L), which exerts no more than 20% reduction in prostate cancer cell viability, was chosen for the drug combination treatments. In combination drug treatments, Annexin V/PI staining demonstrated a significant increase in the percentage of apoptotic cells compared with either treatment alone, which was confirmed by an increase in C-caspase3 and C-PARP levels. These data indicate that apoptotic cell death is a primary mechanism of the synergistic drug effect. Androgen-dependent gene expression was analyzed after KV-37 treatment, and a dose-dependent reduction in PSA levels was observed. Although the activity of AKR1C3 was inhibited by KV-37, the protein expression levels either remained unchanged or slightly increased in 22Rv1 cells, after treatment, indicating a feedback increase in AKR1C3 expression. Interestingly, we observed an increase in the AR expression levels after treatment with ENZ alone in both 22Rv1 and LNCaP1C3 cell lines, which is one of the signatures of drug resistance. A combination of ENZ with KV-37 reduced AR expression to control levels or less. Further, treatment with KV-37 alone in 22Rv1 murine xenografts reduced tumor volume by more than 50%, whereas no change in mouse body weight was observed. In order to compare the degree of synergistic drug interaction exhibited by the weak AKR1C3 inhibitor INDO (Ki = 8 μmol/L) with KV-37 (Ki = 3 μmol/L), pretreatment experiments with INDO were conducted in LNCaP1C3 cells followed by ENZ treatment that revealed a moderate degree of drug synergism, much lower than that achieved with KV-37. This study serves as proof of concept that AKR1C3 inhibition has the potential to overcome ENZ resistance and corroborates previous findings (27). Further, we also analyzed the activity of KV-37 alone and in combination with ENZ in the nonmalignant human prostate stromal cell line WPMY-1. No reduction in cell viability up to 25 μmol/L was observed, whereas no cell viability reduction in combination experiments was noted, indicating selectivity to cells overexpressing AKR1C3.

In conclusion, we highlight the potential of KV-37 as a combination therapy with ENZ for CRPC because it retards ENZ-resistant prostate cancer cell growth and induces apoptosis. Furthermore, it is conceivable from the results of this study that the structurally novel AKR1C3 inhibitor KV-37 scaffold can be developed as a stand-alone therapeutic for prostate cancer management.

K. Verma has ownership interest (including stock, patents, etc.) in a provisional patent application describing KV-37. T.M. Penning reports receiving commercial research grant from Forendo and other commercial research support from Constellation, and is a consultant/advisory board member for Research Institute for Fragrance Materials, Markey Cancer Center, Mailman School of Public Health, Columbia U, and EOSHI, Rutgers University. P.C. Trippier has ownership interest (including stock, patents, etc.) in a provisional patent application describing KV-37. No potential conflicts of interest were disclosed by the other authors.

Conception and design: K. Verma, N. Gupta, T.M. Penning, P.C. Trippier

Development of methodology: K. Verma, N. Gupta, T. Zang, P. Wangtrakluldee, S.K. Srivastava, T.M. Penning, P.C. Trippier

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Verma, N. Gupta, T. Zang, P. Wangtrakluldee, T.M. Penning

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Verma, N. Gupta, T. Zang, P. Wangtrakluldee, S.K. Srivastava, T.M. Penning, P.C. Trippier

Writing, review, and/or revision of the manuscript: K. Verma, T. Zang, P. Wangtrakluldee, S.K. Srivastava, T.M. Penning, P.C. Trippier

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Zang

Study supervision: K. Verma, S.K. Srivastava, T.M. Penning, P.C. Trippier

We are indebted to Drs. Noelle Williams and Sukesh Voruganti at the University of Texas Southwestern Medical Center Preclinical Pharmacology Core for conducting in vivo pharmacokinetic studies and tumor measurements.

Financial support for this work was provided by Texas Tech University Health Sciences Center (to P.C. Trippier).

The costs of publication of this article were defrayed in part by the payment 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.

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