Human aldo-keto reductase 1C3, type 2 3α-hydroxysteroid dehydrogenase (HSD)/type 5 17β-HSD (AKR1C3) is known to be involved in steroid, prostaglandin and lipid aldehyde metabolism. The role of AKR1C3 in the radiosensitivity to X-rays of human non-small-cell lung cancer (NSCLC) cells was explored. In this study, a specific small interfering RNA (siRNA) to target the AKR1C3 gene was used. A suite of readouts including cell survival were determined using a colony formation assay; apoptosis evaluated by Annexin V expression levels, irradiation-induced cytotoxicity established using a MTT cell viability assay and cell cycle distribution measured by flow cytometry were used in characterizing the role of the AKR1C3 gene. Although AKR1C3 was significantly overexpressed in both our radioresistant subclone cells and NSCLC tissues, a specific AKR1C3 siRNA significantly enhanced cell radiosensitivity and was concomitant with decreased expression of this gene. Furthermore, reduced interleukin-6 (IL-6)-mediated radioresistance was observed when siRNA was used to knock down AKR1C3 activity. This AKR1C3-mediated radioresistance was correlated with an arrest in the G2/M cell cycle and a decreased induction of apoptosis. AKR1C3 may present a potential therapeutic target in addressing radioresistance of NSCLC, and in particular in IL-6-mediated radioresistance.
Radiation therapy (RT) is an integral part of modern cancer management. More than 50% of all newly diagnosed cancer patients worldwide receive RT alone or in combination with chemotherapy or surgery at some point in the course of their treatment.1 Of all cancers affecting the lung, non-small-cell lung cancer (NSCLC) accounts for ∼80% of these cases, and RT is considered the preferred treatment approach. However, intrinsic and/or acquired resistance to RT is increasingly recognized as a significant impediment to effective cancer treatment. The underlying mechanism(s) associated with this intrinsic and/or acquired resistance of the cancerous tissue to radiation treatment remains uncertain.
Human aldo-keto reductase (AKR) 1C3, type 2 3α-hydroxysteroid dehydrogenase (HSC)/type 5 17β-HSD (AKR1C3) is known to be involved in the metabolism of steroids, prostaglandins (PGs) and lipid aldehydes.2, 3 Specifically, AKR1C3 catalyzes the reduction of 4-androstene-3,17-dione to testosterone and estrone to 17β-estradiol in target tissues, which in turn promotes the proliferation of hormone-dependent prostate and breast cancers, respectively. AKR1C3 also catalyzes the reduction of PGH2 to PGF2α and PGD2 to 9α,11β-PGF2, which limits antiproliferative PG formation including 15-deoxy-Δ12,14-PGJ2, and contributes to a proliferative signaling response. AKR1C3 is overexpressed in a wide variety of cancers, including breast and prostate cancer.4, 5, 6
In a previous study,7 two radioresistant NSCLC subclones, A549/R and SPCA1/R, were established after sequential sublethal irradiation of A549 and SPCA1 cells. Unexpectedly, we found that AKR1C3 was significantly upregulated in radioresistant cancer cells, suggesting that AKR1C3 may be a mediator of acquired tumor radioresistance in NSCLC. In this follow-on study, we examined the role of AKR1C3 and its mechanism of action in conferring radioresistance in the NSCLC cell lines.
Materials and methods
Materials, cell line and tissue specimen from human patients
The human lung adenocarcinoma cell lines SPCA1 (obtained from China Centre for Type Culture Collection, CCTCC, Wuhan, China) and A549 (from American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco’s modified Eagle’s medium (GIBCO, Carlsbad, CA, USA) supplemented with glutamine (2 mmol l−1), antibiotics (penicillin/streptomycin, 10 units ml−1) and 10% heat-inactivated fetal bovine serum (GIBCO) at 37 °C in 5% CO2. Cells were passaged every 2–3 days to maintain exponential growth. Recombinant human interleukin-6 (IL-6) was purchased from R&D Systems (Minneapolis, MN, USA). Anti-AKR1C3 antibody was purchased from Abcam (Cambridge, MA,USA). Anti-β-actin monoclonal antibody was purchased from Biovision (Milpitas, CA, USA). Secondary anti-mouse or anti-rabbit antibodies coupled to horseradish peroxidase were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Tissue specimens of NSCLC were obtained from Department of Surgery at Shandong Cancer Hospital and Institute under the approval of the Institutional Review Board. The ethics committee of Shandong Cancer Hospital and Institute specifically waived the need for consent. A total of 27 specimens of NSCLC were obtained from patients who underwent surgical resection from 2008 to 2010 in the Department of Surgery at Shandong Cancer Hospital and Institute (Jinan, China). Of these samples, 20 were from male patients with a mean age of 60 years old (45–73 years); 7 from female patients with same mean age (46–74 years). They were pathologically diagnosed as stage I or II NSCLC, with Eastern Cooperative Oncology Group performance (ECOG) status of 0–2. Out of 27 patients, 12 (44.4%) were diagnosed with squamous cell cancer, 8 (29.6%) with adenocarcinoma, 4 (14.8%) with NOS (not otherwise specified) NSCLC and 3 (11.1%) with large-cell neuroendocrinal cancer. Before surgical therapy, none of the patients had received neoadjuvant chemotherapy, RT or immunotherapy. Tissue specimens were appraised immediately after surgery, and part of the specimen was used for pathological examination, whereas the remaining part was snap-frozen with liquid nitrogen and stored at −80 °C for gene expression analysis.
An aliquot of total RNA was purified to isolate mRNA using the Illumina (EI Segundo, CA, USA) Total Prep RNA Amplification kit by Ambion (Austin, TX, USA). Purified mRNA was converted to biotinylated double-stranded complementary DNA and hybridized to Illumina Human-6 v2 Expression BeadChip following the manufacturer’s protocol. Analysis of the genetic expression was performed using GeneSpring GX software, by performing nonparametric t-test (Mann–Whitney rank test) without any error correction on the samples. Genes were considered statistically significant at P<0.05.
RNA extraction and real-time reverse transcriptase-PCR (RT-PCR) analysis
Total RNA was isolated from culture cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance the with manufacture’s procedures. Reverse transcription was performed at 37 °C for 15 min in a volume of 10 μl containing 2 μl of 5 × PrimeScript buffer, 0.5 μl of PrimeScript RT Enzyme Mix I, 0.5 μl of random 6-mer primers and 0.5 μl of Oligo dT primer (TaKaRa, Dalian, China). Reverse transcription enzyme was inactivated at 85 °C for 5 s. The complementary DNA products were used for real-time RT-PCR.
Specific primers for real-time PCR (Table 1) were designed using Primer Express software (Applied Biosystems, Carlsbad, CA, USA) for each mRNA that preferentially spanning intron–exon boundaries to avoid amplification of genomic DNA. Reactions were performed in 25 μl volumes, containing 12.5 μl of 2 × SYBR Premix Ex Tag, 5 μM of specific primers and 200 ng of complementary DNA template. Amplifications were carried out on an ABI Prism 7000 Sequence Analyzer (Grand Island, NY, USA) with the following cycle program: 1 cycle: 95 °C/10 s; 40 cycles: 95 °C/5 s and 60 °C/1 min. The relative gene expression level was calculated by comparing threshold cycle (Ct) values of samples to that of the reference. All data were normalized to β-actin. Accordingly, ΔCt=(mean Ct value of gene)–(mean Ct value of β-actin), and ΔΔCt=ΔCt (selected cells)–ΔCt (parental cells). The relative gene expression in a particular sample was then given by the following: 2-ΔΔCt value.
Cells were lysed in ice-cold lysis buffer (0.15 M NaCl, 50 mM Tris-Cl, pH 7.4, 2 mM EDTA, 5 mM dithiothreitol, 0.5% Triton 100, 0.2 mM phenylmethylsulfonyl fluoride, 1 μg ml−1 aprotinin), supernatants recovered and the total cellular protein concentration was determined with the method of Bradford. Equal amounts of protein were loaded, and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis before electrotransfer to a polyvinylidene difluoride membrane (Amersham Biosciences, Buckinghamshire, UK), and then the membrane was probed with the appropriate antibodies.
Transfection of siRNA and irradiation
The primers used in generating the double-stranded small interfering RNA (siRNA) probe for AKR1C3 interference studies (sense, 5′-GGAACUUUCACCAACAGAUtt-3′; antisense, 5′-AUCUGUUGGUGAAAGUUCCtc-3′) as well as the nonspecific control siRNA probe (sense, 5′-GAUCUACAUACGAGCACUAtt-3′; antisense, 5′-UAGUGCUCGUAUGUAGAUCtc-3′) were synthesized by Ambion. Cells (5 × 105) were seeded in 25 cm2 flasks 24 h before transfection in attaining 50–60% confluence, and then cultured in serum-free OptiMEM (Invitrogen) for 4 h. Lipofectamine 2000 complexes were prepared in serum-free medium using 30 nM of AKR1C3-specific siRNA or control siRNA with 30 μl of Lipofectamine 2000 (Invitrogen) per well following the manufacturer’s recommended protocol. After 20 min of incubation, complexes were added dropwise to cells and incubated at 37 °C, 5% CO2, After 6 h, cells were returned to complete growth media (Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal calf serum). At 24 h after transfection, cells were collected to evaluate gene expression or irradiated at room temperature using a linear accelerator (6-MV X-ray, CLINAC 2100C, Varian, Palo Alto, CA, USA) with a dose rate of 3 Gy min−1.
Assay for radiosensitivity
Cell survival after X-ray irradiation was measured by clonogenic assay. Cells plated in 60 mm tissue culture dish were irradiated at concentrations ranging from 0 to 12 Gy. The appropriate plating density was aimed to produce 20–100 surviving colonies per well. These cells were incubated at 37 °C for 10–14 days (three plates/radiation concentration). After fixation with acetic acid–methanol (1:4) and staining with diluted crystal violet (1:30), colonies consisting of 50 cells were counted under a light microscope. Results from the triplicate plates were averaged and divided by initial seeded cells to yield survival rate of clones for each concentration, and the surviving fraction was determined. All survival curves represent a minimum of three independent experiments. The sensitizer enhancement ratio (SER) at 1% survival level in cells was defined as SER=mean inactivation dose (transfected with si-control)/mean inactivation dose (transfected with si-AKR1C3). SER >1 indicates radiosensitization.
Detection of apoptotic cells
Cell apoptosis was evaluated using the Annexin V–FITC Apoptosis Detection Kit (BD Biosciences Pharmingen, San Jose, CA, USA) followed by fluorescence-activated cell sorting (FACS) analysis. Cells were treated with trypsin–EDTA in phosphate-buffered saline at pH 7.5, washed with normal medium and cold phosphate-buffered saline, and then resuspended in 1 × binding buffer. Annexin V (5 μl) and propidium iodide (10 μl) were added to the cells, vortexed and incubated for 15 min in the dark. Finally, cells were diluted with 1 × binding buffer (400 μl), and samples evaluated by flow cytometry (FACS Calibur, BD Biosciences) using Cellquest software.
MTT cell viability assay
The effect of irradiation was also evaluated by conventional MTT cell viability assay, and results are presented as a percentage of the control. Briefly, 1 × 104 cells per well were seeded in 96-well plates and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 8 h. Following radiation treatment (12 Gy), 10 μl of 5 g l−1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution was added and incubated for 4 h at 37 °C. Formazan crystals that form were solubilized with 100 μl of acidified (0.01 M HCl) 10% sodium dodecyl sulfate for 24 h. Absorbance was read on a Bio-Rad 680 microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm and results reported relative to a reference wavelength of 630 nm.
Cell cycle analysis
Cells (1 × 106) were fixed in 70% ethanol, at −20 °C, overnight. Cells were then centrifuged at 1000 r.p.m. for 5 min and washed once in phosphate-buffered saline. Cells were resuspended in 1 ml of propidium iodide/Triton X-100 staining solution with RNase A (0.1% Triton X-100, 200 μg ml−1 DNase-free RNase A, 20 μg ml−1 propidium iodide) and stained for 30 min at room temperature. Analysis was Flow cytometry analysis was carried out on a FACS Calibur instrument (Becton Dickinson, Bedford, MA, USA). The distribution of cell cycle phases was determined using ModiFit software (Topsham, ME, USA). Two independent experiments were carried out.
Statistical analysis was performed using the t-test. The results with P-values of <0.05 were considered to be statistically significant.
Increased levels of AKR1C3 are present in radioresistant cell lines
In previous studies, following eight rounds of sublethal irradiation, two radioresistant NSCLC cell subclones were established (A549/R and SPCA1/R).7 Comparison of A549/R and SPCA1/R model cell lines to the parent cell line identified the AKR1C3 gene as one of the most responsive and highly expressed genes in the microarray panel (Figure 1a). Quantitative RT-PCR and western blot analysis were used to validate the changes in AKR1C3 gene expression observed by microarray. AKR1C3 mRNA (Figure 1b) and protein (Figure 1c) levels were significantly upregulated in A549/R and SPCA1/R cells. AKR1C3 protein level in A549/R and SPCA1/R were 6.2-fold and 3.5-fold greater than that observed in the parental cells, respectively.
Human NSCLC tissue overexpress AKR1C3
Although AKR1C3 has been reported to be overexpressed in a wide variety of cancers, including breast and prostate cancer,4, 5, 6 to date there has been no reports of altered expression of this protein in lung cancers. Therefore, to determine if AKR1C3 was also involved in human NSCLC, 27 human NSCLC cancerous tissue samples along with paired paracancerous tissues were analyzed by western blot analysis. In 19 of the 27 cases analyzed, the AKR1C3 protein level was higher than levels found in the paired paracancerous tissues (Figure 2). On an average, the AKR1C3 proteins were approximately threefold higher in cancerous tissues compared with those detected in the paracancerous samples.
AKR1C3 knockdown increases radiosensitivity in NSCLC cells
To further confirm the role of AKR1C3 in regulating NSCLC cell radiosensitivity, both the parental A549 and A549/R cell lines were transiently transfected with a specific AKR1C3 siRNA (si-AKR1C3) to knock down its expression. At 24 h after transfection, AKR1C3 expression was markedly inhibited (>90% at mRNA level), as shown in Figures 3a and b. Furthermore, a clonogenic survival assay showed that knockdown of AKR1C3 in these cell lines resulted in fewer surviving colonies than controls with increasing doses of radiation (Figure 3c). A significant increase of radiosensitivity was observed in both A549 and A549/R cells transfected with si-AKR1C3 relative to si-control transfected cells at 12 Gy (P<0.05). Si-AKR1C3 caused a radiosensitization with a SER of 1.13 in A549 cells and a SER of 1.21 in A549/R cells. Results from this analysis suggest that irradiation-mediated upregulation of the AKR1C3 gene is linked to radioresistance and that by blocking this gene, tumors can effectively be interrupted.
AKR1C3 knockdown increases radiation-induced apoptosis
An apoptosis assay was also performed to establish the impact of AKR1C3 on mediating the radiosensitivity of NSCLC cell lines. Apoptosis in cells exposure to X-ray irradiation (10 Gy dose) at indicated time (0, 48, 96 or 120 h) was determined using a standard Annexin V assay. A significant increase in irradiation-induced apoptosis was observed for both A549 and A549/R cells transfected with si-AKR1C3, compared with the cells transfected with the si-control (Figure 4). This response was selective for the AKR1C3 gene as transfection of siRNA itself did not induce apoptosis.
AKR1C3 may function as an effector in IL-6-mediated radioresistance
AKR1C3 is generally related to hormone-dependent cancer such as prostate and breast but not NSCLC. In efforts to begin to unravel the mechanism(s) of action of AKR1C3 in the context of NSCLC radiosensitivity, we addressed the putative role of IL-6 in mediating this response. Precedence for examining the role of this cytokine is based on previous studies that reported IL-6-associated increases of AKR1C3 promoter activity with concomitant increases in gene expression.8 In addition, irradiation corresponded with increased levels of IL-6 that were linked with increased cell proliferation and associated radioresistance.9 The addition of IL-6 to A549 cells resulted in increased AKR1C3 protein expression (Figure 5a) and the presence of this cytokine conferred radioresistance to the cells (Figure 5b). This IL-6-induced radioresistance was significantly interrupted (P<0.05) in irradiated A549 cells (10 Gy) when the AKR1C3 expression was selectively blocked by siRNA transfection strategies. Figure 5c illustrates a significant reduction in cell viability in si-AKR1C3-transfected A549 cells (59.7 to 39.9% viability at day 6, P<0.01). This reduced cell viability correlated with increased apoptosis in si-AKR1C3-transfected A549 cells (at day 2 12.27% apoptosis relative to 7.89% in control cells, P<0.05). These results suggest a role for AKR1C3 as a radiosensitivity mediator that may be a critical effector in IL-6-mediated radioresistance in human NSCLCs.
Radiosensitization induced by AKR1C3 knockdown is associated with G2/M arrest
IL-6 has a direct stimulatory effect on the growth of many tumor cells via the activation of several signaling pathways as well as promoting tumor cell entry into the cell cycle.10 AKR1C3 catalyzes the reduction of PGH2 to PGF2α and PGD2 to 9α,11β-PGF2, which limit the formation of anti-proliferative PGs, and contribute to proliferative signaling. Here the cell cycle distribution in si-AKR1C3-transfected A549 cells was observed and compared with the controls. The transfection of si-AKR1C3 accelerated cells into S and G2/M phase obviously (Figure 6). At 24 h after transfection, percentage of G2/M phase in A549 cells transfected with si-AKR1C3 were 26.20, and 21.69% in controls (data not shown). After irradiation with 10 Gy X-ray, more and more cells entered G2/M phase and this difference remained for at least 24 h (77.13% vs 68.93% at 24 h). These findings accounted for that AKR1C3 knockdown in A549 cells increase sensitivity to irradiation.
The aldo-keto reductases (AKRs) comprise a functionally diverse family of proteins encoded by 15 distinct genes.11 Members of the AKR superfamily are generally located in the cytosol, function as monomeric proteins, share a common (α/β)8-barrel structural motif and exhibit NAD(P)(H)-dependent oxidoreductases activity. Four human AKR1C isoforms have been cloned and characterized, including AKR1C1 (20α (3α)-hydroxysteroid dehydrogenase (HSD)),12 AKR1C2 (type 3 3α-HSD),13, 14 AKR1C3 (type 2 3α/type 5 17β-HSD)15, 16 and AKR1C4 (type 1 3α-HSD).14 Natural substrates for these enzymes include steroids, PGs and lipid aldehydes.17 Based on enzyme kinetics, AKR1C3 possesses 3α-HSD, 3β-HSD, 17β-HSD and 11-ketoprostaglandin reductase activities, and catalyzes estrogen, progesterone, androgen and PG metabolism.2, 16, 18 As a result, AKR1C3 is capable of indirectly regulating ligand access to various nuclear receptors, including estrogen receptor, progesterone receptor, androgen receptor and peroxisome proliferator-activated receptor, and regulating trans-activation activities of these nuclear receptors through intracrine actions.4 The presence of AKR1C3 has been demonstrated in steroid hormone-dependent cells including breast cells,3 endometrial cells,19 prostate cells and Leydig cells.20 De-regulated expression of AKR1C3 has been demonstrated in multiple types of hormone-related cancers, including breast cancer,5 endometrial cancer19 and prostate cancer.6, 21 A variant allele of AKR1C3 decreases the risk of lung and prostate cancers.22 AKR1C3 was recently reported to mediate the metabolic activation of a clinical trial antitumor agent PR104.23
Of the many genes whose expression was altered in our microarray analysis of radioresistant NSCLC cell lines, AKR1C3 emerged as one of the most dramatically upregulated of the genes identified (Figure 1). We have shown that AKR1C3 is also overexpressed in human NSCLC tissue samples (Figure 2), which implicates the role of this gene product in enhancing the malignant potential of lung cancers. Specifically in this study, we investigated the role that AKR1C3 may play in mediating radiation resistance commonly exhibited by tumor cells, which remains a significant challenge in treating this pleiotropic disease. Transient attenuation of AKR1C3 expression by siRNA resulted in an increased rate of radiation-induced apoptosis (Figure 3) and may provide a new therapeutic approach in overcoming radiosensitivity associated with lung cancer treatments.
The emergence of tumors after radiotherapy has been attributed to repopulation of tumors from cells surviving irradiation. Radiation is known to induce multiple biological responses at the cellular and tissue levels mediated often by early activation of cytokine cascades that promote the growth and survival of tumor tissue.24 Proinflammatory cytokines such as IL-6 have been shown to be overexpressed in NSCLC patients undergoing chemoradiation therapy.25 IL-6 has been implicated in the modulation of growth and differentiation in many cancers and is associated with poor prognosis in renal cell carcinoma, ovarian cancer, lymphoma, melanoma and prostate cancer.26 Overexpression of IL-6 protects LNCaP cells from undergoing apoptosis induced by androgen deprivation therapy.27 IL-6 increases the levels of AKR1C3 mRNA and protein expression in both LNCaP and CWR22rv1 cells, and upregulates AKR1C3 promoter activity.8 Results from the current study showed that IL-6 can increase radioresistance and AKR1C3 gene expression in A549 cells. The radioresistance caused by IL-6 was significantly inhibited in cells transfected with AKR1C3 siRNA (Figure 5), suggesting that AKR1C3 is involved in this process and may be a critical modulator in radiosensitivity.
Response to radiation is thought to have multifactorial etiologies. The possible underlying mechanisms by which AKR1C3 may enhance cell survival upon radiation exposure remain to be elucidated. The conventional notion is that AKR1C3 enhances survival of tumor cells primarily through suppression of apoptosis-related cell death. However, there also seemed to be additional mechanisms. By activating Ras/Raf/MEK/Erk1/2, IL-6 stimulates tumor cell proliferation.10, 28 IL-6 which activates STAT-3 (signal transducer and activator of transcription 3) to upregulate the expression of cyclins D1, D2 and B1, and MYC, and downregulate the expression of cdk inhibitor p21Cip1, serves in promoting entry into the cell cycle.10, 29 It is well established that tumor cells are sensitive to radiation-induced cell death when synchronized in the G2/M phase of the cell cycle.30 Apoptosis after G2/M cell cycle arrest could be a key mechanism of the cell-killing effects of irradiation.31, 32, 33 We observed that AKR1C3 siRNA treatment altered the cell cycle distribution, resulting in an increased G2/M fraction (Figure 6). Above finding suggests that blocking ASR1C3 expression in tumor cells may provide shift the cells into a more radiosensitive stage of the cell cycle and thus more responsive to radiation treatment.
The results indicated that AKR1C3 indeed plays an important role in radiosensitivity of NSCLC cancer cells, especially in the IL-6-mediated signaling pathway. AKR1C3-mediated radioresistance may be due to the reduction of G2/M phase arrest associated with radiation treatment and a decrease in radiation-induced apoptosis. This study contributes basic information as to the role of AKR1C3 in radioresistance, which may eventually lead to the validation of AKR1C3 as a predictive biomarker for lung cancer. This study provides valuable initial insight of a protein target that shows potential for developing a new radiosensitive therapeutic strategy.
We thank Maureen Dolan, Guolei Zhou and Nadia Awar for revising the manuscript. This work was supported by grants from the National Science Foundation of China (30801066) and the Natural Science Foundation of Shandong Province (ZR2010HZ002 and ZR2010HM031).