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Preclinical report
Statil suppresses cancer cell growth and proliferation by the inhibition of tumor marker AKR1B10 Zhe Caoa,b, Boping Zhoua,b, Xinchun Chena,b, Dan Huangc, Xiuli Zhangc, Ziqi Wangc, Hua Huanga,b, Yuhong Wangc and Deliang Caoa,b,c,d Aldo-keto reductase 1B10 (AKR1B10) is an oncogenic carbonyl reductase that eliminates a,b-unsaturated carbonyl compounds/lipid peroxides and mediates retinoic acid signaling. Targeted inhibition of AKR1B10 activity is a newly emerging strategy for cancer therapy. This study evaluated the inhibitory activity of a small chemical statil towards AKR1B10 and tested its antiproliferative activity in breast (BT-20) and lung (NCI-H460) cancer cells that express AKR1B10. Experimental results showed that statil inhibited AKR1B10 enzyme activity efficiently, with an IC50 at 0.21±0.06 lmol/l. Exposing BT-20 and NCI-H460 cells to statil and diclofenac, a selective AKR1B10 inhibitor, led to dose-dependent inhibition of cell growth and proliferation and plating efficiency. At higher doses (50 lmol/l or higher), statil induced cell death with apoptotic characteristics, such as DNA fragmentation and Annexin-V staining. Furthermore, statil enhanced the susceptibility of cells to acrolein, an active substrate of AKR1B10. Taken together, these data suggest that statil possesses potent antiproliferative activity by inhibiting
c 2014 AKR1B10 activity. Anti-Cancer Drugs 25:930–937 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
the activation of the polycyclic aromatic hydrocarbon in smoke and malignant development of interstitial pneumonia in smokers [9,18,19]. In mammary epithelial cells, AKR1B10 is upregulated with their malignant transformation and blocks the ubiquitin-dependent degradation of acetyl-CoA carboxylase-a, a rate-limiting enzyme in de-novo fatty acid synthesis, promoting lipid synthesis [20,21]. In breast tumors, AKR1B10 is upregulated in ductal carcinoma in situ, infiltrating carcinoma and metastatic and recurrent cancers [17]. Furthermore, AKR1B10 expression in breast cancer is correlated positively with the tumor size and lymph node metastasis, but inversely with disease-related survival, being a potential prognostic factor and target for the prevention and the treatment of this disease [17,22]. Very recently, AKR1B10 was found to be secreted through a heat shock protein 90a-mediated lysosomal protein secretory pathway, and increased in the serum of breast cancer patients, being a potential serum biomarker for this disease [23,24].
Aldo-keto reductase 1B10 (AKR1B10), also named aldose reductase-like-1, is a monomeric enzyme that can efficiently detoxify dietary and cellular a,b-unsaturated carbonyl compounds/lipid peroxides, protecting host cells from carbonyl lesions [1–6]. AKR1B10 is also active to alltrans-retinal, a precursor of the signaling molecule retinoic acid that regulates cell proliferation and differentiation, and to polycyclic aromatic hydrocarbon, an environmental procarcinogen [7–12]. Recent studies from our and other laboratories have shown that AKR1B10 can reduce the C13 ketonic group in daunorubicin and idarubicin, leading to chemoresistance of cancer cells to these cytostatic agents [13–15], suggesting that AKR1B10 is an important carbonyl reductase involved in cell growth and proliferation and in drug resistance. AKR1B10 was identified from human hepatocellular carcinomas [1], but was recently found to be overexpressed in several human tumors, such as lung and breast carcinomas [16,17]. In the lung bronchial epithelium, AKR1B10 is induced by cigarette smoke, participating in Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.anti-cancerdrugs.com). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
Anti-Cancer Drugs 2014, 25:930–937 Keywords: aldo-keto reductase 1B10, breast and lung cancer, [3-(4-bromo-2-fluorobenzyl)-4-oxo-3h-phthalazin-1-yl] acetic acid, cancer therapy, diclofenac a Guangdong Key Laboratory for Diagnosis & Treatment of Emerging Infectious Diseases, bShenzhen Key Laboratory of Infection & Immunity, Shenzhen Third People’s Hospital, Guangdong Medical College, Shenzhen, cState Key Laboratory of Chinese Medicine Powder and Medicine Innovation in Hunan (incubation), Division of Stem Cell Regulation and Application, Hunan University of Chinese Medicine, Hunan, China and dDepartment of Medical Microbiology, Immunology, & Cell Biology, Simmons Cooper Cancer Institute, Southern Illinois University School of Medicine, Springfield, Illinois, USA
Correspondence to Deliang Cao, PhD, Department of Medical Microbiology, Immunology, & Cell Biology, Simmons Cooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794, USA Tel: + 1 217 545 9703; fax: + 1 217 545 9718; e-mail: [email protected] or Hua Huang, MD, Guangdong Key Laboratory for Diagnosis & Treatment of Emerging Infectious Diseases, Shenzhen 518033, China Tel/fax: + 86 755 6123 8983; e-mail: [email protected] Received 7 November 2013 Revised form accepted 27 March 2014
Targeted silencing of AKR1B10 gene or inhibition of AKR1B10 protein by small chemical inhibitors is a newly emerging strategy for developing novel cancer therapeutic modalities [25–28]. AKR1B10 has high structural homology and substrate similarity to aldose reductase (AR, also named AKR1B1 in nomenclature system) [1,29]. DOI: 10.1097/CAD.0000000000000121
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Statil inhibits cell proliferation Cao et al. 931
Scheme 1
O O−H Br
N ⎥ N
O
F
The chemical structure of statil (C10H8N2O3.C8H8BrF, MW: 407.24).
to statil or diclofenac at indicated concentrations for 72 h. Viable cells were detected using an MTT Cell Proliferation kit (Roche, Indianapolis, Indiana, USA). 3H-thymidine incorporation into DNA was determined by pulsing 1 104 cells/well in 24-well plates with 5.0 mCi/well of 3 H-thymidine (MP Biochemicals, Solon, Ohio, USA) for 2 h [36]. Cells were washed with PBS and lysed in 0.4 ml of 15% trichloroacetic acid on ice. After washing twice with 15% trichloroacetic acid, acidic-insoluble materials were dissolved in 50 ml of 0.1 N NaOH. An aliquot (10 ml) was used to determine protein amounts using protein assay reagents (Bio-Rad, Hercules, California, USA), and the remaining was subjected to radioactivity measurements. 3H-thymidine incorporation into DNA was expressed as cpm/mg protein. Plating efficiency
In past decades, AR has been targeted for the prevention and the treatment of diabetic complications, and numerous aldose reductase inhibitors (ARIs) have been developed and used in diabetic patients [30–32]. Interestingly, some ARIs possess inhibitory activity to AKR1B10 [10,33,34]. In this study, we found that an ARI statil ([3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-yl] acetic acid) (Scheme 1) inhibited AKR1B10 enzyme activity efficiently and suppressed the growth and proliferation of breast (BT-20) and lung (NCI-H460) cancer cells, indicating the potential to develop AKR1B10 inhibitors as cancer therapeutic agents.
Materials and methods Cell culture
BT-20 and NCI-H460 cells were purchased from American Type Culture Collection (ATCC, Manassas, Maryland, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heated fetal bovine serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 371C, 5% CO2. Cells at 85–90% confluence were passaged by trypsinization. AKR1B10 recombinant protein purification and enzyme activity
AKR1B10 recombinant protein was prepared and verified for its enzyme activity as described previously [1]. AKR1B10 inhibition by statil was tested using 0–20 mmol/l DLglyceraldehyde as a substrate. The Lineweaver–Burk plots of 1/velocity versus 1/substrate concentrations were obtained using the GraphPad Prism 4 (Graph Pad Software, La Jolla, California, USA), and IC50, the inhibitor concentration leading to 50% inhibition of AKR1B10 activity, was determined by constructing sigmoidal dose–response curves (GraphPad Prism 4). MTT and 3H-thymidine incorporation assays
MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole] assays were conducted as described previously [35]. In brief, cells (2–3 103/well) were seeded in 96-well plates overnight and then exposed
Cells were suspended at 100 cells/ml and seeded at 150–200 cells per 60-mm culture dish, in triplicate. Cells were incubated in regular medium overnight and then fed with fresh medium containing statil at the concentrations indicated. After incubation for 14 days, colonies were visualized by fixing in 10% formalin for 10 min and staining with 0.01% crystal violet, and were then photographed and counted. Plate efficiency (%) was calculated as (colony number/seeded cell number) 100. Apoptotic body and DNA fragmentation assay
Cells at 70% confluence were exposed to statil for 24 h, and then 1 mg/ml of 40 ,6-diamidino-2-phenylindole (DAPI) was added into the medium for 30 min. Stained nuclei were photographed at 350 nm excitation wavelength and 470 nm emission wavelength. Nuclei in five areas of high power ( 40) were counted and evaluated for apoptosis. For DNA fragmentation, cells were plated at 5.0 105 cells per 60-mm cell culture dish and cultured overnight. After being exposed to statil at indicated concentrations for 24 h, cells were harvested and lysed in 0.5% Triton-X 100, 5 mmol/l Tris-HCl (pH 7.4), 5 mmol/l EDTA for 20 min on ice, followed by centrifugation at 10 000g for 15 min. Supernatants were incubated with 100 mg/ml of proteinase K (A & B Applied Biosystems, California, USA) in 0.5% SDS for 2 h at 561C. DNA was extracted with phenol–chloroform and precipitated with ethanol. DNA pellets were dissolved in 20 mmol/l Tris-HCl (pH 7.4) with 50 mg/ml of RNase A and incubated at 371C for 1 h. DNA products were separated on 2% agarose gel. Flow cytometry
Cells (6 105 per 60-mm cell culture dish) were exposed to statil at concentrations indicated for 24 h. The medium was removed gently, and cells were washed with cold PBS and then trypsinized. Cells in PBS and trypsin digestion were pooled, washed with PBS twice at 1200 rpm for 10 min, and then subjected to immediate propidium iodide (PI) and Annexin-V-FITC staining for 10 min in the dark. A FACScan analysis was performed using a FACScan cytometer (Becton Dickinson, San Jose, California, USA).
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932 Anti-Cancer Drugs 2014, Vol 25 No 8
diclofenac also suppressed cell growth and proliferation (Fig. 2a). The suppression by statil on cell growth and proliferation was further confirmed by DNA synthesis and plating efficiency. Using 3H-thymidine to label the newly synthesized DNA, we found that the DNA synthesis of NCI-H460 cells was significantly reduced by statil at 50 mmol/l or higher concentrations (P < 0.05) (Fig. 2b). Clonogenic growth is a key feature of cancer cells. We tested the plating efficiency of NCI-H460 affected by statil, and found that the plating efficiency was reduced to a larger extent compared with the vector control (P < 0.01; Fig. 2c). Similar results were observed in BT20 cells (Supplementary figure S1, Supplemental digital content 1, http://links.lww.com/ACD/A64). These data suggest that statil can inhibit cancer cell growth and proliferation effectively.
Acrolein cytotoxicity
Cells were plated in 12-well plates at 5 104 cells/well overnight and then fed with fresh medium containing acrolein at 20 or 50 mmol/l, with or without statil (20 mmol/l), for 72 h. Viable cells were trypsinized and counted by trypan blue staining using a Coulter Counter (Beckman Coulter, Brea, California, USA). Statistic analysis
Statistical analysis was performed using Student’s t-test with INSTAT statistical analysis package (Graph Pad Software). Significance was defined as P value less than 0.05.
Results Inhibition of AKR1B10 activity by statil
Using glyceraldehyde as a substrate, we evaluated the inhibitory activity of statil to AKR1B10 and found that statil was a strong inhibitor of AKR1B10 with an IC50 at 0.21±0.06 mmol/l (Fig. 1a). Substrate–velocity plots showed that statil produced a dose-dependent inhibition to AKR1B10 enzyme activity (Fig. 1b), suggesting that statil is an efficient inhibitor.
Induction of apoptotic cell death by statil
In view of the significant inhibition of cell growth and proliferation by statil, we further investigated its effect on cell death/survival. Using PI and Annexin-V staining and FACScan analysis, we estimated the apoptosis induced by statil. As shown in Fig. 3, in the presence of statil at 100 mmol/l, BT-20 cells stained by Annexin-V alone (early apoptotic cells) were significantly increased compared with the vehicle control (P < 0.01), and the cells stained by both Annexin-V and PI (late apoptotic cells) were also higher in statil-treated cells than in the control (P < 0.05), indicating apoptosis induced by statil.
Inhibition of cell growth and proliferation by statil
To test the effect of AKR1B10 inhibition by statil on cell growth and proliferation, we estimated the viability of BT-20 and NCI-H460 cells (expressing AKR1B10 but not AR [20]), using an MTT assay. As shown in Fig. 2a, statil inhibited NCI-H460 cell growth in a dose-dependent manner, and the cell growth rate was significantly decreased when the concentration of statil was at 50 mmol/l or higher (P < 0.05). To confirm the role of AKR1B10 inhibition in cell growth and proliferation, we treated NCI-H460 cells with diclofenac, a selective inhibitor of AKR1B10, and the results showed that
We further assessed apoptotic bodies induced by statil by DAPI staining of nuclei. As shown in Fig. 4a, statil at 100 mmol/l led to significant apoptotic changes of nuclei of NCI-H460 cells, including condensation and fragmentation. A quantification measurement showed that in the presence of 100 mmol/l statil, apoptotic cells were
Fig. 1
(a)
(b)
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The inhibitory activity of statil towards AKR1B10. (a) Inhibition of AKR1B10 activity. In the presence of statil at 0–20 mmol/l, AKR1B10 enzyme activity and IC50 were measured as described in the Materials and methods section. (b) Substrate–velocity plots. DL-glyceraldehyde was used as a substrate at 0–20 mmol/l. Statil was utilized at 0.01–10 mmol/l. AKR1B10 enzyme activity and substrate–velocity plots were analyzed as described in the Materials and methods section. Values represent the average of three independent measurements.
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Statil inhibits cell proliferation Cao et al. 933
Fig. 2
(a)
(b) 120
% of viable cells
100 80
Radioactivity (cpm/μg)
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Statil (100 μmol/l)
0 Statil (μmol/l): 0
50
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Control
Suppression of cell growth and proliferation by statil. NCI-H460 cells that express AKR1B10 were used for growth and proliferation assays as described in the Materials and methods section. (a) Cell growth rate estimated by MTT assays. *P < 0.05 and **P < 0.01 compared with the vehicle control. (b) 3H-thymidine incorporation into DNA. *P < 0.05 compared with the vehicle control. (c) Plating efficiency, indicating clonogenic growth of cells in the presence of statil at the concentrations indicated. Colony formation efficiency (%): (colony number/seeded cell number) 100. **P < 0.01 compared with the vehicle control. All data denote mean±SD.
increased by more than three times compared with the control (Fig. 4b). This phenomenon was confirmed by a DNA fragmentation assay. As shown in Fig. 4c, DNA fragmentation within NCI-H460 cells occurred noticeably in the presence of 100 or 150 mmol/l of statil. Altogether, these data suggest that statil induces apoptosis of breast and lung cancer cells. Statil enhances cell susceptibility to acrolein
We hypothesize that as an AKR1B10 inhibitor, statil may inhibit cell growth and induce apoptosis by inhibiting AKR1B10 enzyme activity and triggering carbonyl stress. Acrolein is a highly reactive a,b-unsaturated aldehyde produced by lipid peroxidation and a strong substrate of AKR1B10 [35,36]. Therefore, we evaluated the cell viability on exposure to acrolein with or without statil. As shown in Fig. 5, addition of statil (20 mmol/l) in the culture enhanced the susceptibility of cells to acrolein significantly, indicating the inhibition of AKR1B10 activity.
Discussion AKR1B10 is an important reductase that has recently been recognized to play an oncogenic role in cancer development and progression [17]. Development and characterization of AKR1B10 inhibitors emerges as a novel approach for cancer
treatment [27]. Recently, AKR1B10-specific small molecules have been developed and show the capability to increase the sensitivity of lung cancer cells A549 to doxorubicin [28]. However, ARIs with inhibitory activity to AKR1B10 should still hold potential value. AR is a ratelimiting enzyme of the polyol pathway, reducing glucose to sorbitol [37]. In diabetic hyperglycemia, the glucose flux through this pathway renders AR a pathogenic factor by inducing sorbitol accumulation and osmolytic stress, fructose-mediated glycosylation, and redox changes, and thus a treatment target for diabetic complications [38,39]. Up to date, a large number of ARIs with diverse structures have been developed and tested in diabetic clinics, building up important clinical databases [30–32] that considerably facilitate fluid translation to cancer clinics once the anticancer activity of ARIs is proven. This study investigated and demonstrated the inhibitory activity of statil towards AKR1B10 and its capability of inhibiting cell growth and proliferation, being a potential anticancer agent. Statil showed efficient suppression of cell growth and proliferation in both breast cancer cells BT-20 and lung cancer cells NCI-H460 at micromolar levels. Furthermore, the antiproliferative activity of statil by inhibiting AKR1B10 was confirmed by diclofenac, a selective AKR1B10 inhibitor
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Anti-Cancer Drugs 2014, Vol 25 No 8
Fig. 3
(a) 105
Propidium iodide (PI)
Statil (100 μmol/l)
Control 3.9 %
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Staining percentage (%)
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Statil (100 μmol/l)
Apoptosis induced by statil. BT-20 cells were exposed to statil at the concentrations indicated for 24 h. A FACScan analysis was conducted as described in the Materials and methods section. (a) Cell distribution images; (b) Annexin-V staining percentage. Values represent mean±SD from three independent measurements. PI, propidium iodide. *P < 0.05 and **P < 0.01 compared with the vehicle control.
with an IC50 at 1.9 mmol/l for AKR1B10 and greater than 100 mmol/l for AKR1B1 [40]. Statil demonstrated particular activity in suppressing clonogenic growth, which is a critical feature of transformed cells, and reflects the growth properties of individual cells, such as viability and proliferation [41]. Therefore, clonogenic growth is a growth advantage of a transformed cell developed during carcinogenesis and a prerequisite of tumor formation. The anticlonogenic growth activity of statil may render it a potent effect in preventing cancer cell invasion and metastasis. At concentrations of 100 mmol/l or higher, statil induced cellular nuclear condensation and fragmentation, a feature of apoptotic cell death, and this phenomenon was confirmed by Annexin-V staining and FACScan analysis, which showed a significant increase of early apoptotic cells when the cells were exposed to statil at 100 mmol/l for 24 h. These data suggest that statil may be a potent antiproliferative agent.
It is noted that statil inhibits AKR1B10 activity at submicromolar concentrations with an IC50 at 0.21 mmol/l, but significant cell proliferative inhibition and apoptosis induced by statil were observed at 50 mmol/l or higher concentrations. These discrepancies may be due to the differences in experimental systems and in measurement parameters. First, the inhibitory activity of statil was tested in vitro using purified AKR1B10 protein under optimal conditions for the inhibitory estimates, whereas the assessment of cell proliferation and apoptosis by statil was conducted in cell cultures, in which multiple factors, such as membrane transportation and intracellular metabolism, may affect the action of statil towards cellular AKR1B10. Second, the assessed parameters of cell proliferation and apoptosis are effects secondary to the inhibition of AKR1B10 enzyme activity by statil. This may also be affected by multiple factors, such as the cellular apoptotic response to AKR1B10 inhibition.
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Statil inhibits cell proliferation Cao et al. 935
Fig. 4
(a)
Statil (100 μmol/l)
Control (b)
(c) 30
Statil (μmol/l): 0
100
150
∗∗
25 % of apoptotic cells
50
20 15 10 5 0 Control
Statil (100 μmol/l)
Apoptotic bodies and DNA fragmentation induced by statil. NCI-H460 cells were exposed to statil at the concentrations indicated and subjected to apoptotic analysis as described in the Materials and methods section. (a) DAPI staining (1.0 mg/ml), indicating condensed/fragmented nuclei (arrows; 40 magnification). (b) Percentage of apoptotic cells. Cells in five fields of high power ( > 200 nuclei) were estimated, and cells with deformed or fragmented nuclei were counted as apoptotic cells. **P < 0.01 compared with the vehicle control. (c) DNA ladder, showing DNA fragmentation.
Acrolein (CH2 = CH–CHO, 2-propenal) is a highly active a,b-unsaturated carbonyl compound. Acrolein can form alkylated adducts through interaction with nucleophiles at the C-3 double bond (Michael addition), the C-1 carbonyl group (Schiff ’s base formation), or both C-3 and C-1 with 1,4 addition [42]. Therefore, acrolein can interact with cysteine, histidine, and lysine residues of proteins and with nucleophilic sites in DNA, resulting in protein dysfunction, proteasome inhibition, and DNA mutations and breaks [43,44]. These reactions and resulting consequences constitute the basis of cytotoxicity and genotoxic events (mutagenesis and carcinogenesis) in cells exposed to acrolein. As a substrate of AKR1B10, acrolein was used to test the response of cells to statil. Our results showed that statil enhanced the susceptibility of cells to acrolein significantly by the
inhibition of AKR1B10. Reactive carbonyl compounds, including acrolein, 4-hydroxynoneal, and methylglyoxal, are constantly produced inside the cell during amino acid and carbohydrate metabolism, as well as by lipid oxidation in oxidative stress. The acrolein sensitivity enhanced by statil indicates that statil may suppress cell growth and proliferation by triggering a carbonyl stress in response to the inhibition of AKR1B10.
Conclusion
This study found that the ARI statil can efficiently inhibit AKR1B10 enzyme activity, affect cellular response to electrophilic carbonyls, and suppress cancer cell growth and proliferation, being a potential therapeutic agent of cancer.
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Fig. 5
120 Acrolein Acrolein + statil
% of cell viability
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Effect of statil on the cell susceptibility to acrolein. NCI-H460 cells (5 104 cells/well of 12-well plates) were exposed to acrolein at the concentrations indicated, with or without statil (50 mmol/l) for 24 h. Viable cells were trypsinized and counted by trypan blue staining. *P < 0.05 compared with statil-free cells exposed to acrolein at the same concentrations.
Acknowledgements This work was supported by the National Natural Science Foundation of China (81272918 for D.C.).
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Conflicts of interest
There are no conflicts of interest.
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Preclinical report
Statil suppresses cancer cell growth and proliferation by the inhibition of tumor marker AKR1B10 Zhe Caoa,b, Boping Zhoua,b, Xinchun Chena,b, Dan Huangc, Xiuli Zhangc, Ziqi Wangc, Hua Huanga,b, Yuhong Wangc and Deliang Caoa,b,c,d Aldo-keto reductase 1B10 (AKR1B10) is an oncogenic carbonyl reductase that eliminates a,b-unsaturated carbonyl compounds/lipid peroxides and mediates retinoic acid signaling. Targeted inhibition of AKR1B10 activity is a newly emerging strategy for cancer therapy. This study evaluated the inhibitory activity of a small chemical statil towards AKR1B10 and tested its antiproliferative activity in breast (BT-20) and lung (NCI-H460) cancer cells that express AKR1B10. Experimental results showed that statil inhibited AKR1B10 enzyme activity efficiently, with an IC50 at 0.21±0.06 lmol/l. Exposing BT-20 and NCI-H460 cells to statil and diclofenac, a selective AKR1B10 inhibitor, led to dose-dependent inhibition of cell growth and proliferation and plating efficiency. At higher doses (50 lmol/l or higher), statil induced cell death with apoptotic characteristics, such as DNA fragmentation and Annexin-V staining. Furthermore, statil enhanced the susceptibility of cells to acrolein, an active substrate of AKR1B10. Taken together, these data suggest that statil possesses potent antiproliferative activity by inhibiting
c 2014 AKR1B10 activity. Anti-Cancer Drugs 25:930–937 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Introduction
the activation of the polycyclic aromatic hydrocarbon in smoke and malignant development of interstitial pneumonia in smokers [9,18,19]. In mammary epithelial cells, AKR1B10 is upregulated with their malignant transformation and blocks the ubiquitin-dependent degradation of acetyl-CoA carboxylase-a, a rate-limiting enzyme in de-novo fatty acid synthesis, promoting lipid synthesis [20,21]. In breast tumors, AKR1B10 is upregulated in ductal carcinoma in situ, infiltrating carcinoma and metastatic and recurrent cancers [17]. Furthermore, AKR1B10 expression in breast cancer is correlated positively with the tumor size and lymph node metastasis, but inversely with disease-related survival, being a potential prognostic factor and target for the prevention and the treatment of this disease [17,22]. Very recently, AKR1B10 was found to be secreted through a heat shock protein 90a-mediated lysosomal protein secretory pathway, and increased in the serum of breast cancer patients, being a potential serum biomarker for this disease [23,24].
Aldo-keto reductase 1B10 (AKR1B10), also named aldose reductase-like-1, is a monomeric enzyme that can efficiently detoxify dietary and cellular a,b-unsaturated carbonyl compounds/lipid peroxides, protecting host cells from carbonyl lesions [1–6]. AKR1B10 is also active to alltrans-retinal, a precursor of the signaling molecule retinoic acid that regulates cell proliferation and differentiation, and to polycyclic aromatic hydrocarbon, an environmental procarcinogen [7–12]. Recent studies from our and other laboratories have shown that AKR1B10 can reduce the C13 ketonic group in daunorubicin and idarubicin, leading to chemoresistance of cancer cells to these cytostatic agents [13–15], suggesting that AKR1B10 is an important carbonyl reductase involved in cell growth and proliferation and in drug resistance. AKR1B10 was identified from human hepatocellular carcinomas [1], but was recently found to be overexpressed in several human tumors, such as lung and breast carcinomas [16,17]. In the lung bronchial epithelium, AKR1B10 is induced by cigarette smoke, participating in Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.anti-cancerdrugs.com). c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0959-4973
Anti-Cancer Drugs 2014, 25:930–937 Keywords: aldo-keto reductase 1B10, breast and lung cancer, [3-(4-bromo-2-fluorobenzyl)-4-oxo-3h-phthalazin-1-yl] acetic acid, cancer therapy, diclofenac a Guangdong Key Laboratory for Diagnosis & Treatment of Emerging Infectious Diseases, bShenzhen Key Laboratory of Infection & Immunity, Shenzhen Third People’s Hospital, Guangdong Medical College, Shenzhen, cState Key Laboratory of Chinese Medicine Powder and Medicine Innovation in Hunan (incubation), Division of Stem Cell Regulation and Application, Hunan University of Chinese Medicine, Hunan, China and dDepartment of Medical Microbiology, Immunology, & Cell Biology, Simmons Cooper Cancer Institute, Southern Illinois University School of Medicine, Springfield, Illinois, USA
Correspondence to Deliang Cao, PhD, Department of Medical Microbiology, Immunology, & Cell Biology, Simmons Cooper Cancer Institute, Southern Illinois University School of Medicine, 913 N. Rutledge Street, Springfield, IL 62794, USA Tel: + 1 217 545 9703; fax: + 1 217 545 9718; e-mail: [email protected] or Hua Huang, MD, Guangdong Key Laboratory for Diagnosis & Treatment of Emerging Infectious Diseases, Shenzhen 518033, China Tel/fax: + 86 755 6123 8983; e-mail: [email protected] Received 7 November 2013 Revised form accepted 27 March 2014
Targeted silencing of AKR1B10 gene or inhibition of AKR1B10 protein by small chemical inhibitors is a newly emerging strategy for developing novel cancer therapeutic modalities [25–28]. AKR1B10 has high structural homology and substrate similarity to aldose reductase (AR, also named AKR1B1 in nomenclature system) [1,29]. DOI: 10.1097/CAD.0000000000000121
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Statil inhibits cell proliferation Cao et al. 931
Scheme 1
O O−H Br
N ⎥ N
O
F
The chemical structure of statil (C10H8N2O3.C8H8BrF, MW: 407.24).
to statil or diclofenac at indicated concentrations for 72 h. Viable cells were detected using an MTT Cell Proliferation kit (Roche, Indianapolis, Indiana, USA). 3H-thymidine incorporation into DNA was determined by pulsing 1 104 cells/well in 24-well plates with 5.0 mCi/well of 3 H-thymidine (MP Biochemicals, Solon, Ohio, USA) for 2 h [36]. Cells were washed with PBS and lysed in 0.4 ml of 15% trichloroacetic acid on ice. After washing twice with 15% trichloroacetic acid, acidic-insoluble materials were dissolved in 50 ml of 0.1 N NaOH. An aliquot (10 ml) was used to determine protein amounts using protein assay reagents (Bio-Rad, Hercules, California, USA), and the remaining was subjected to radioactivity measurements. 3H-thymidine incorporation into DNA was expressed as cpm/mg protein. Plating efficiency
In past decades, AR has been targeted for the prevention and the treatment of diabetic complications, and numerous aldose reductase inhibitors (ARIs) have been developed and used in diabetic patients [30–32]. Interestingly, some ARIs possess inhibitory activity to AKR1B10 [10,33,34]. In this study, we found that an ARI statil ([3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-yl] acetic acid) (Scheme 1) inhibited AKR1B10 enzyme activity efficiently and suppressed the growth and proliferation of breast (BT-20) and lung (NCI-H460) cancer cells, indicating the potential to develop AKR1B10 inhibitors as cancer therapeutic agents.
Materials and methods Cell culture
BT-20 and NCI-H460 cells were purchased from American Type Culture Collection (ATCC, Manassas, Maryland, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% heated fetal bovine serum, 2 mmol/l glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 371C, 5% CO2. Cells at 85–90% confluence were passaged by trypsinization. AKR1B10 recombinant protein purification and enzyme activity
AKR1B10 recombinant protein was prepared and verified for its enzyme activity as described previously [1]. AKR1B10 inhibition by statil was tested using 0–20 mmol/l DLglyceraldehyde as a substrate. The Lineweaver–Burk plots of 1/velocity versus 1/substrate concentrations were obtained using the GraphPad Prism 4 (Graph Pad Software, La Jolla, California, USA), and IC50, the inhibitor concentration leading to 50% inhibition of AKR1B10 activity, was determined by constructing sigmoidal dose–response curves (GraphPad Prism 4). MTT and 3H-thymidine incorporation assays
MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole] assays were conducted as described previously [35]. In brief, cells (2–3 103/well) were seeded in 96-well plates overnight and then exposed
Cells were suspended at 100 cells/ml and seeded at 150–200 cells per 60-mm culture dish, in triplicate. Cells were incubated in regular medium overnight and then fed with fresh medium containing statil at the concentrations indicated. After incubation for 14 days, colonies were visualized by fixing in 10% formalin for 10 min and staining with 0.01% crystal violet, and were then photographed and counted. Plate efficiency (%) was calculated as (colony number/seeded cell number) 100. Apoptotic body and DNA fragmentation assay
Cells at 70% confluence were exposed to statil for 24 h, and then 1 mg/ml of 40 ,6-diamidino-2-phenylindole (DAPI) was added into the medium for 30 min. Stained nuclei were photographed at 350 nm excitation wavelength and 470 nm emission wavelength. Nuclei in five areas of high power ( 40) were counted and evaluated for apoptosis. For DNA fragmentation, cells were plated at 5.0 105 cells per 60-mm cell culture dish and cultured overnight. After being exposed to statil at indicated concentrations for 24 h, cells were harvested and lysed in 0.5% Triton-X 100, 5 mmol/l Tris-HCl (pH 7.4), 5 mmol/l EDTA for 20 min on ice, followed by centrifugation at 10 000g for 15 min. Supernatants were incubated with 100 mg/ml of proteinase K (A & B Applied Biosystems, California, USA) in 0.5% SDS for 2 h at 561C. DNA was extracted with phenol–chloroform and precipitated with ethanol. DNA pellets were dissolved in 20 mmol/l Tris-HCl (pH 7.4) with 50 mg/ml of RNase A and incubated at 371C for 1 h. DNA products were separated on 2% agarose gel. Flow cytometry
Cells (6 105 per 60-mm cell culture dish) were exposed to statil at concentrations indicated for 24 h. The medium was removed gently, and cells were washed with cold PBS and then trypsinized. Cells in PBS and trypsin digestion were pooled, washed with PBS twice at 1200 rpm for 10 min, and then subjected to immediate propidium iodide (PI) and Annexin-V-FITC staining for 10 min in the dark. A FACScan analysis was performed using a FACScan cytometer (Becton Dickinson, San Jose, California, USA).
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932 Anti-Cancer Drugs 2014, Vol 25 No 8
diclofenac also suppressed cell growth and proliferation (Fig. 2a). The suppression by statil on cell growth and proliferation was further confirmed by DNA synthesis and plating efficiency. Using 3H-thymidine to label the newly synthesized DNA, we found that the DNA synthesis of NCI-H460 cells was significantly reduced by statil at 50 mmol/l or higher concentrations (P < 0.05) (Fig. 2b). Clonogenic growth is a key feature of cancer cells. We tested the plating efficiency of NCI-H460 affected by statil, and found that the plating efficiency was reduced to a larger extent compared with the vector control (P < 0.01; Fig. 2c). Similar results were observed in BT20 cells (Supplementary figure S1, Supplemental digital content 1, http://links.lww.com/ACD/A64). These data suggest that statil can inhibit cancer cell growth and proliferation effectively.
Acrolein cytotoxicity
Cells were plated in 12-well plates at 5 104 cells/well overnight and then fed with fresh medium containing acrolein at 20 or 50 mmol/l, with or without statil (20 mmol/l), for 72 h. Viable cells were trypsinized and counted by trypan blue staining using a Coulter Counter (Beckman Coulter, Brea, California, USA). Statistic analysis
Statistical analysis was performed using Student’s t-test with INSTAT statistical analysis package (Graph Pad Software). Significance was defined as P value less than 0.05.
Results Inhibition of AKR1B10 activity by statil
Using glyceraldehyde as a substrate, we evaluated the inhibitory activity of statil to AKR1B10 and found that statil was a strong inhibitor of AKR1B10 with an IC50 at 0.21±0.06 mmol/l (Fig. 1a). Substrate–velocity plots showed that statil produced a dose-dependent inhibition to AKR1B10 enzyme activity (Fig. 1b), suggesting that statil is an efficient inhibitor.
Induction of apoptotic cell death by statil
In view of the significant inhibition of cell growth and proliferation by statil, we further investigated its effect on cell death/survival. Using PI and Annexin-V staining and FACScan analysis, we estimated the apoptosis induced by statil. As shown in Fig. 3, in the presence of statil at 100 mmol/l, BT-20 cells stained by Annexin-V alone (early apoptotic cells) were significantly increased compared with the vehicle control (P < 0.01), and the cells stained by both Annexin-V and PI (late apoptotic cells) were also higher in statil-treated cells than in the control (P < 0.05), indicating apoptosis induced by statil.
Inhibition of cell growth and proliferation by statil
To test the effect of AKR1B10 inhibition by statil on cell growth and proliferation, we estimated the viability of BT-20 and NCI-H460 cells (expressing AKR1B10 but not AR [20]), using an MTT assay. As shown in Fig. 2a, statil inhibited NCI-H460 cell growth in a dose-dependent manner, and the cell growth rate was significantly decreased when the concentration of statil was at 50 mmol/l or higher (P < 0.05). To confirm the role of AKR1B10 inhibition in cell growth and proliferation, we treated NCI-H460 cells with diclofenac, a selective inhibitor of AKR1B10, and the results showed that
We further assessed apoptotic bodies induced by statil by DAPI staining of nuclei. As shown in Fig. 4a, statil at 100 mmol/l led to significant apoptotic changes of nuclei of NCI-H460 cells, including condensation and fragmentation. A quantification measurement showed that in the presence of 100 mmol/l statil, apoptotic cells were
Fig. 1
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The inhibitory activity of statil towards AKR1B10. (a) Inhibition of AKR1B10 activity. In the presence of statil at 0–20 mmol/l, AKR1B10 enzyme activity and IC50 were measured as described in the Materials and methods section. (b) Substrate–velocity plots. DL-glyceraldehyde was used as a substrate at 0–20 mmol/l. Statil was utilized at 0.01–10 mmol/l. AKR1B10 enzyme activity and substrate–velocity plots were analyzed as described in the Materials and methods section. Values represent the average of three independent measurements.
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Statil inhibits cell proliferation Cao et al. 933
Fig. 2
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Suppression of cell growth and proliferation by statil. NCI-H460 cells that express AKR1B10 were used for growth and proliferation assays as described in the Materials and methods section. (a) Cell growth rate estimated by MTT assays. *P < 0.05 and **P < 0.01 compared with the vehicle control. (b) 3H-thymidine incorporation into DNA. *P < 0.05 compared with the vehicle control. (c) Plating efficiency, indicating clonogenic growth of cells in the presence of statil at the concentrations indicated. Colony formation efficiency (%): (colony number/seeded cell number) 100. **P < 0.01 compared with the vehicle control. All data denote mean±SD.
increased by more than three times compared with the control (Fig. 4b). This phenomenon was confirmed by a DNA fragmentation assay. As shown in Fig. 4c, DNA fragmentation within NCI-H460 cells occurred noticeably in the presence of 100 or 150 mmol/l of statil. Altogether, these data suggest that statil induces apoptosis of breast and lung cancer cells. Statil enhances cell susceptibility to acrolein
We hypothesize that as an AKR1B10 inhibitor, statil may inhibit cell growth and induce apoptosis by inhibiting AKR1B10 enzyme activity and triggering carbonyl stress. Acrolein is a highly reactive a,b-unsaturated aldehyde produced by lipid peroxidation and a strong substrate of AKR1B10 [35,36]. Therefore, we evaluated the cell viability on exposure to acrolein with or without statil. As shown in Fig. 5, addition of statil (20 mmol/l) in the culture enhanced the susceptibility of cells to acrolein significantly, indicating the inhibition of AKR1B10 activity.
Discussion AKR1B10 is an important reductase that has recently been recognized to play an oncogenic role in cancer development and progression [17]. Development and characterization of AKR1B10 inhibitors emerges as a novel approach for cancer
treatment [27]. Recently, AKR1B10-specific small molecules have been developed and show the capability to increase the sensitivity of lung cancer cells A549 to doxorubicin [28]. However, ARIs with inhibitory activity to AKR1B10 should still hold potential value. AR is a ratelimiting enzyme of the polyol pathway, reducing glucose to sorbitol [37]. In diabetic hyperglycemia, the glucose flux through this pathway renders AR a pathogenic factor by inducing sorbitol accumulation and osmolytic stress, fructose-mediated glycosylation, and redox changes, and thus a treatment target for diabetic complications [38,39]. Up to date, a large number of ARIs with diverse structures have been developed and tested in diabetic clinics, building up important clinical databases [30–32] that considerably facilitate fluid translation to cancer clinics once the anticancer activity of ARIs is proven. This study investigated and demonstrated the inhibitory activity of statil towards AKR1B10 and its capability of inhibiting cell growth and proliferation, being a potential anticancer agent. Statil showed efficient suppression of cell growth and proliferation in both breast cancer cells BT-20 and lung cancer cells NCI-H460 at micromolar levels. Furthermore, the antiproliferative activity of statil by inhibiting AKR1B10 was confirmed by diclofenac, a selective AKR1B10 inhibitor
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Fig. 3
(a) 105
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Apoptosis induced by statil. BT-20 cells were exposed to statil at the concentrations indicated for 24 h. A FACScan analysis was conducted as described in the Materials and methods section. (a) Cell distribution images; (b) Annexin-V staining percentage. Values represent mean±SD from three independent measurements. PI, propidium iodide. *P < 0.05 and **P < 0.01 compared with the vehicle control.
with an IC50 at 1.9 mmol/l for AKR1B10 and greater than 100 mmol/l for AKR1B1 [40]. Statil demonstrated particular activity in suppressing clonogenic growth, which is a critical feature of transformed cells, and reflects the growth properties of individual cells, such as viability and proliferation [41]. Therefore, clonogenic growth is a growth advantage of a transformed cell developed during carcinogenesis and a prerequisite of tumor formation. The anticlonogenic growth activity of statil may render it a potent effect in preventing cancer cell invasion and metastasis. At concentrations of 100 mmol/l or higher, statil induced cellular nuclear condensation and fragmentation, a feature of apoptotic cell death, and this phenomenon was confirmed by Annexin-V staining and FACScan analysis, which showed a significant increase of early apoptotic cells when the cells were exposed to statil at 100 mmol/l for 24 h. These data suggest that statil may be a potent antiproliferative agent.
It is noted that statil inhibits AKR1B10 activity at submicromolar concentrations with an IC50 at 0.21 mmol/l, but significant cell proliferative inhibition and apoptosis induced by statil were observed at 50 mmol/l or higher concentrations. These discrepancies may be due to the differences in experimental systems and in measurement parameters. First, the inhibitory activity of statil was tested in vitro using purified AKR1B10 protein under optimal conditions for the inhibitory estimates, whereas the assessment of cell proliferation and apoptosis by statil was conducted in cell cultures, in which multiple factors, such as membrane transportation and intracellular metabolism, may affect the action of statil towards cellular AKR1B10. Second, the assessed parameters of cell proliferation and apoptosis are effects secondary to the inhibition of AKR1B10 enzyme activity by statil. This may also be affected by multiple factors, such as the cellular apoptotic response to AKR1B10 inhibition.
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Statil inhibits cell proliferation Cao et al. 935
Fig. 4
(a)
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Apoptotic bodies and DNA fragmentation induced by statil. NCI-H460 cells were exposed to statil at the concentrations indicated and subjected to apoptotic analysis as described in the Materials and methods section. (a) DAPI staining (1.0 mg/ml), indicating condensed/fragmented nuclei (arrows; 40 magnification). (b) Percentage of apoptotic cells. Cells in five fields of high power ( > 200 nuclei) were estimated, and cells with deformed or fragmented nuclei were counted as apoptotic cells. **P < 0.01 compared with the vehicle control. (c) DNA ladder, showing DNA fragmentation.
Acrolein (CH2 = CH–CHO, 2-propenal) is a highly active a,b-unsaturated carbonyl compound. Acrolein can form alkylated adducts through interaction with nucleophiles at the C-3 double bond (Michael addition), the C-1 carbonyl group (Schiff ’s base formation), or both C-3 and C-1 with 1,4 addition [42]. Therefore, acrolein can interact with cysteine, histidine, and lysine residues of proteins and with nucleophilic sites in DNA, resulting in protein dysfunction, proteasome inhibition, and DNA mutations and breaks [43,44]. These reactions and resulting consequences constitute the basis of cytotoxicity and genotoxic events (mutagenesis and carcinogenesis) in cells exposed to acrolein. As a substrate of AKR1B10, acrolein was used to test the response of cells to statil. Our results showed that statil enhanced the susceptibility of cells to acrolein significantly by the
inhibition of AKR1B10. Reactive carbonyl compounds, including acrolein, 4-hydroxynoneal, and methylglyoxal, are constantly produced inside the cell during amino acid and carbohydrate metabolism, as well as by lipid oxidation in oxidative stress. The acrolein sensitivity enhanced by statil indicates that statil may suppress cell growth and proliferation by triggering a carbonyl stress in response to the inhibition of AKR1B10.
Conclusion
This study found that the ARI statil can efficiently inhibit AKR1B10 enzyme activity, affect cellular response to electrophilic carbonyls, and suppress cancer cell growth and proliferation, being a potential therapeutic agent of cancer.
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Fig. 5
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Effect of statil on the cell susceptibility to acrolein. NCI-H460 cells (5 104 cells/well of 12-well plates) were exposed to acrolein at the concentrations indicated, with or without statil (50 mmol/l) for 24 h. Viable cells were trypsinized and counted by trypan blue staining. *P < 0.05 compared with statil-free cells exposed to acrolein at the same concentrations.
Acknowledgements This work was supported by the National Natural Science Foundation of China (81272918 for D.C.).
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There are no conflicts of interest.
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