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Conversion of estrone to 17-beta-estradiol in human non-small-cell lung cancer cells in vitro
Biomedicine & Pharmacotherapy 66 (2012) 530–534
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Original article
Conversion of estrone to 17-beta-estradiol in human non-small-cell lung cancer cells in vitro Hanna Drzewiecka, Paweł P. Jagodzinski * Poznan´ University of Medical Sciences, Department of Biochemistry and Molecular Biology, 6 S´wie˛cickiego St., 60-781 Poznan´, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 January 2012 Accepted 29 February 2012
It has recently been suggested that, in addition to genetic and environmental factors and tobacco exposure, estrogens also may be an independent risk factor in the development of lung cancer. Therefore, we evaluated the transcript and protein levels of 17-beta-hydroxysteroid-dehydrogenase type 1 (HSD17B1), and the conversion of estrone (E1) to 17-beta-estradiol (E2) in human non-small cell lung cancer (NSCLC) Calu-6, Calu-1 and A549 cells. In our work, we established the presence of HSD17B1 transcripts and proteins in all examined NSCLC cells. Moreover, we demonstrated that human NSCLC Calu-6, Calu-1 and A549 cells are able to convert weak estrogen E1 to highly potent E2 in vitro. Our results indicate that NSCLC cells are able to produce E2 from E1. ß 2012 Elsevier Masson SAS. All rights reserved.
Keywords: HSD17B1 Lung cancer 17-beta-estradiol
1. Introduction Despite the development of new treatment methods, lung cancer continues to be the leading cause of death worldwide, with more than one million cases reported among men and women annually [1]. The overall prognosis for this cancer is poor because of late diagnosis, which disqualifies patients for surgical resection [2,3]. Primary lung cancer is derived from epithelial cells and can be distinguished into two major types: non-small cell lung cancer (NSCLC), and small cell lung cancer (SCLC). NSCLC accounts for approximately 80% of all lung cancers and includes the following histological types: squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and other rare types [4]. Squamous cell carcinoma tends to occur more frequently in men and is connected to a history of smoking, whereas adenocarcinoma is the most common form in women [5,6]. Currently, an upward trend in NSCLC incidence is observed among women [7,8]. Lung cancer in young women exhibits rapid growth and a more aggressive biology than in postmenopausal women [9,10]. Furthermore, the longterm use of hormone replacement therapy (HRT) seems to be correlated with a poor survival rate of patients with lung cancer [11,12]. Therefore, gender may be an additional independent risk factor, apart from tobacco exposure and genetic and environmental factors, in carcinogenesis of the lung. This observation may place the estrogens to the fore of lung cancer risk factors, but the role of estrogens in the incidence of this disease remains elusive. * Corresponding author. E-mail address: [email protected] (P.P. Jagodzinski). 0753-3322/$ – see front matter ß 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2012.02.006
Estrogen affects its target tissues by the classical, liganddependend activation of signaling pathways or via non-genomic actions using membrane-associated estrogen receptors ERs and/or a member of the G protein-coupled receptor superfamily, GPCR30 [13,14]. The classical pathway is mediated by estrogen receptors, ERa and ERb, which belong to the nuclear steroid hormone receptor superfamily. ERb is the predominant subtype present in normal lung tissue, and a great majority of NSCLC tumors are positive for ERb, regardless of gender [7,15–22]. Moreover, the non-genomic signalling pathway using the GPR30 receptor has also been identified in lung cancer cells [23]. Although 17-betaestradiol (E2) is mainly produced in the ovaries [24], this steroid hormone can be also locally synthesized from its precursors in various tissues in both women and men [25]. In peripheral tissue, estrone (E1) can be formed by the aromatase or steroid sulfatase (STS) pathways [26,27] and subsequently reduced by 17-betahydroxysteroid-dehydrogenase type 1 (HSD17B1) to E2 [28]. It has recently been demonstrated that HSD17B1 may exhibit a crucial role in the development of estrogen-dependent diseases [29–33]. To date, the metabolism of androgens and estrogens in the lungs has been focused on the aromatase and STS pathways and their pivotal role in intratumoral estrogen synthesis in NSCLC cells [18,34–37]. However, little is known about HSD17B1 expression in lung cancer cells and their ability to convert E1 to E2. Therefore, we evaluated the levels of HSD17B1 transcript and protein and the production of E2 from E1 in human NSCLC Calu-6, Calu-1 and A549 cells. In addition to being a precursor of E2, E1 can also exert an impact on NSCLC cells by acting through ERs [38], so we also explored the influence of both E1 and E2 on the proliferation of Calu-6, Calu-1 and A549 cells.
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2. Materials and methods 2.1. Antibodies and reagents Rabbit polyclonal anti-HSD17B1 antibody (Ab) (H-158), rabbit polyclonal antiglyceraldehyde-3-phosphate (GAPDH) Ab (FL-335) and goat antirabbit horseradish peroxidase (HRP)-conjugated Ab were provided by Santa Cruz Biotechnology (Santa Cruz, CA). E1, E2 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. (St.-Louis, MO). 2.2. Cell culture T47D breast cancer cells were used as a positive control for HSD17B1 transcript and protein identification [39]. The human NSCLC A549 and Calu-1 cells, as well as the T47D breast cancer cells, were purchased from the American Type Culture Collection (Rockville, MD). The NSCLC Calu-6 cell line was kindly provided by Dr. Marek Rusin from the Centre of Oncology Maria Sklodowska-Curie Memorial Institute (Gliwice, Poland). A549, Calu-1 and T47D cells were routinely maintained in DMEM medium, Sigma-Aldrich Co. (St.-Louis, MO), while Calu-6 cells were grown in RPMI-1640 medium, Sigma-Aldrich Co. (St.-Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine and gentamicin sulfate salt (50 mg/ml) Sigma-Aldrich Co. (St.-Louis, MO) at 37 8C in humidified air with 5% CO2. Prior to all experiments, cells were maintained for 48 hours in an appropriate phenol red-free medium Sigma-Aldrich Co. (St.-Louis, MO) supplemented with 10% charcoal-dextran-stripped FBS Sigma-Aldrich Co. (St.-Louis, MO). 2.3. Conversion of E1 to E2 in Calu-6, Calu-1 and A549 cells To investigate the ability of NSCLC cells to convert E1 to E2, the Calu-6, Calu-1 and A549 cells were seeded in six-well plates (3.6 105 cells/well). After 24 hours, media were replaced and cells were cultured either in the absence or in the presence of E1 at a concentration of 5 mM for 2, 4, 6, 8, 10, and 12 hours. At the end of incubation periods, 1.0 ml of the medium was collected and the concentration of E2 was evaluated by electrochemiluminescence method using Cobas 6000 Roche Diagnostics GmbH (Mannheim, Germany). The results were presented as the concentration of E2 in culture medium in nM or pM per 1 mg of total cell protein. All experiments were performed in triplicate and the results represent mean SE. 2.4. Reverse transcription (RT) and real-time quantitative PCR (RQPCR) analysis To determine the presence of the HSD17B1 transcript and its levels in Calu-6, Calu-1 and A549 cells we applied RT and RQ-PCR analysis. RNA was isolated by TRI Reagent1 Sigma-Aldrich Co. (St.Louis, MO) according to the method of Chomczynski and Sacchi [40]. RNA quality was determined by denaturing agarose gel electrophoresis, and the concentration was quantified by measuring the optical density (OD) at 260 nm. RNA samples were treated with DNase I and reverse-transcribed into cDNA using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase from Invitrogen (Carlsbad, CA). RQ-PCR was conducted in a Light Cycler1480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany), using SYBR Green I as detection dye. Target cDNA was quantified by the relative quantification method. The quantity of HSD17B1 transcript in each sample was standardized by the porphobilinogen deaminase (PBGD) transcript level. The PCR amplification efficiency for target and reference genes was determined by different standard curves created by consecutive
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dilutions of the cDNA template mixture, as provided in Relative Quantification Manual Roche Diagnostics GmbH (Mannheim, Germany). The HSD17B1 cDNA 75 bp amplicon was amplified employing the primer pair: (50 TGA GGA GGT GGC GGA GGT CTT C 30 ) (forward, nt 702-723) and (50 CGC TCG GTG GTG AAG TAG 30 ) (reverse, nt 759-776). The PBGD cDNA 160 bp amplicon was amplified using the primer pair: (50 GCC AAG GAC CAG GAC ATC 30 ) (forward, nt 676-693) and (50 TCA GGT ACA GTT GCC CAT C 30 ) (reverse, nt 817-835). The HSD17B1 and PBGD cDNA primers were designed based on sequences ENST00000225929 and ENST00000278715, located in Ensembl Genome Browser (www.ensembl.org). For amplification, 1 ml of total (20 ml) cDNA solution was added to 9 ml of Light Cycler1480 SYBR Green I Master mix (1 concentrated) Roche Diagnostics GmbH (Mannheim, Germany), containing 2.5 mM MgCl2 and 0.5 mM primers. A sample of RNA that had not been reverse-transcribed and a notemplate control were included in each batch of samples to provide a negative control in subsequent PCR. Melting-curve analysis and sequencing were applied to confirm the specificity of the amplified products. All experiments were performed in triplicate and RQ-PCR results are expressed as averages of relative expression to control transcript levels in T47D cells. 2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and western blotting Cell lysates were prepared using RIPA buffer Sigma-Aldrich Co. (St.-Louis, MO) containing 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1.0% Igepal, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and protease inhibitor cocktail. Samples were incubated for 1 hour on ice and then centrifuged at 10,000 g for 10 min at 4 8C to remove cell debris. Supernatants were stored at 80 8C before use. The total protein concentration in cell extracts was determined by the Bradford assay. Subsequently, 30 mg of protein was resuspended in the loading buffer and boiled at 99 8C for 5 min. Next, all samples were loaded into each lane and separated on 10% Tris-glycine gel using SDS-PAGE. Gel proteins were transferred to polyvinylidene fluoride (PVDF) membrane, Roche Diagnostics GmbH (Mannheim, Germany), which was blocked with 5% non-fat dry milk in Tris buffered saline/Tween 20. Immunodetection was performed with rabbit polyclonal antiHSD17B1 Ab (H-158) at a dilution of 1:500, followed by incubation with goat anti-rabbit HRP-conjugated Ab (1:5000). Bands were revealed using Pierce ECL Plus Western Blotting Substrate, Thermo Fisher Scientific (Rockford, IL). The membranes were stripped and reblotted with rabbit polyclonal anti-GAPDH Ab (FL-335) at a dilution of 1:2500, followed by goat anti-rabbit HRP-conjugated Ab (1:10,000), to ensure equal protein loading of the lanes. 2.6. MTT cell proliferation assay To determine the effect of E1 and E2 on the proliferation of Calu6, Calu-1 and A549 cells we used MTT cell proliferation assay. The cells were either cultured in phenol red-free RPMI-1640 (Calu-6 cells) or phenol red-free DMEM (Calu-1 and A549 cells) supplemented with 10% charcoal-dextran-stripped FBS for 48 hours, then seeded into 24-well culture plates (6 104 cells/ well). Subsequently, cells were incubated either in the absence or in the presence of E1 (5 mM) and E2 (10 nM) for 24, 48 and 72 hours. After the incubation period, all culture media were removed and 50 ml of the MTT stock solution in phosphate buffered saline (PBS) (5 mg/ml), mixed with 450 ml of the appropriate phenol red-free medium, was added to each well. After 4 hours of incubation, the culture medium was discarded and the blue formazan crystals were dissolved by adding MTT solvent
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was significance (P < 0.05) between the groups. The statistical analysis was performed with STATISTICA 6.0 software. 3. Results and discussion
Fig. 1. Identification of HSD17B1 transcript (A) and protein (B) in human NSCLC Calu-6, Calu-1 and A549 cells. The cells were used for total RNA isolation and reverse transcription. The HSD17B1 transcript levels were determined by RQ-PCR analysis. RQ-PCR results were standardized by PBGD cDNA levels. HSD17B1 transcripts levels in the investigated NSCLC cells are expressed as a multiplicity of the HSD17B1 cDNA levels in positive controls, T47D breast cancer cells. Each sample was determined in triplicate. Cell proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane that was then immunoblotted with rabbit polyclonal anti-HSD17B1 Ab and incubated with goat antirabbit HRP-conjugated Ab. To ensure equal protein loading of the lanes, the membrane was then reblotted with anti-GAPDH Ab, followed by goat antirabbit HRP-conjugated Ab.
(0.1 N HCl in isopropanol, 150 ml/well). Next, 100 ml of the solution was transferred into 96-well culture plates and the absorbance was measured at a wavelength of 570 nm with a background reading at 640 nm. All experiments were performed in triplicate and the results are presented as means SE. 2.7. Statistical analysis The difference between control and treated cells was statistically analyzed by unpaired, two-tailed t-test to evaluate if there
Despite the fact that cigarette smoking is still a main cause of lung cancer, estrogens appear to be significant factors in this carcinoma. Clinical data demonstrates that individuals who have never smoked and are diagnosed with this disease are mostly females. Women also seem to be more susceptible to developing lung cancer among smokers, because women have been found to be more vulnerable to the carcinogens found in cigarette smoke [41,42]. The uptake of polycyclic aromatic hydrocarbons, formed as by-products during tobacco consumption, stimulates the activity of cytochrome P450 1B1 (CYP1B1). CYP1B1 is commonly expressed in the human lung and metabolizes E2 to 2- and 4-catechol estrogens, which generate DNA adducts when converted into semiquinones and quinines [43,44]. The formation of DNA adducts contributes to the inactivation of tumour suppressor genes and mutation in proto-oncogenes, which are often detected in lung cancer patients [45]. In our study, we found the presence of HSD17B1 transcript and protein in Calu-6, Calu-1 and A549 cells (Fig. 1). Moreover, we demonstrated that all examined NSCLC cells are able to convert the weak estrogen E1 to the highly potent E2 (Fig. 2). To date, increased HSD17B1 mRNA levels associated with an elevated E2/E1 ratio have been observed in breast cancer [29,30], ovarian tumor [31], endometriosis [32], endometrial hyperplasia [33], and uterine leiomyoma [46]. Recently, the clinical case study by Niikawa et al. reported that patients with NSCLC also have a higher E2 concentration in cancerous tissues as compared to the corresponding non-neoplastic lung tissues from the same patients [4]. Furthermore, high endogenous or exogenous estrogen levels were connected with a poorer clinical outcome for the patients. In addition to their influence on the reproductive system, sexsteroid hormones exert multidirectional action on other tissues. Estrogens promote the development and progression of estrogendepended cancers, as well as markedly affecting the immune system and contributing to some autoimmune diseases [47,48]. Results of experimental studies have indicated that estrogen treatment activates both genomic and non-genomic signaling in lung cancer cells [49,50]. Although previous studies with murine
Fig. 3. Effect of E1 and E2 on the proliferation of human NSCLC Calu-6 (A), Calu-1 (B) and A549 (C) cells. To determine the effect of E1 and E2 on the proliferation of Calu-6, Calu-1 and A549 cells we used the MTT cell proliferation assay. Cells were cultured in an appropriate phenol red-free medium for 48 hours, followed by seeding into 24-well culture plates. Subsequently, cells were incubated either in the absence or in the presence of and E1 (5 mM) and E2 (10 nM) for 24, 48 and 72 hours, respectively. After the incubation period, all culture media were removed and the MTT assay was carried out. All experiments were performed in triplicate and results are presented as the mean SE from three independent experiments.
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Fig. 2. Conversion of E1 to E2 in human NSCLC Calu-6 (A), Calu-1 (B) and A549 (C) cells. Cells were cultured in phenol red-free RPMI-1640 (Calu-6 cells) or phenol red-free DMEM (Calu-1 and A549 cells) for 48 hours, followed by incubation either in the absence or in the presence of E1 at a concentration of 5 mM. After 2, 4, 6, 8, 10 and 12 hours, 1.0 ml of medium was collected and the concentration of E2 was determined by electrochemiluminescence method. The results were expressed as E2 concentration in culture medium in nM or pM per 1 mg of total cell protein. Each sample was determined in triplicate and results are presented as the mean SE from three independent experiments.
knockout models showed the indispensability of ERb for normal lung structure and function [51,52], a higher expression of ERb was detected in lung adenocarcinoma [7,15] and provided a new line of evidence for estrogen involvement in lung cancer pathogenesis. Additionally, cross-signalling between ERs and an epidermal growth factor receptor in lung cancer has been established in recent years [16,18,53–55], highlighting the necessity of targeting both of these pathways. Several previous studies have confirmed the presence of estrogen-stimulated NSCLC cell growth and proliferation in vitro and in vivo and indicated that anti-estrogens were able to inhibit those events [16,19,54,56–58]. A549 is a lung adenocarcinoma cell line and Calu-1 is a squamous lung cancer cell line, both derived from males, whereas Calu-6 is an anaplastic lung carcinoma cell line derived from a female [59–61]. We observed that neither E2 at a concentration of 10 nM nor E1 at a concentration of 5 mM had an effect on cellular proliferation of Calu-6, Calu-1 and A549 cells (Fig. 3). Our results are consistent with Miki et al., who did not observe E2 mediated cell proliferation in A549 cells [62]. In contrast, Niikawa et al. used A549 cells transfected with either ERa or ERb expression vectors to demonstrate the positive effect of E2 on the proliferation of these cells [4]. Moreover, our observation that Calu-1 did not respond proliferatively to E2 and E1 may be secondary to findings that E2 has no impact on proliferation of lung adenocarcinoma cells derived from males [19,63]. However, we were not able to confirm growth stimulation of Calu-6 cells after estrogen exposure, and this observation is contrary to earlier studies [22]. This inconsistent result may be due to differences in the sensitivity of the tests used in our study compared to the tests used in earlier studies. We used the MTT proliferation assay, whereas the 5-bromo-2-deoxyuridine assay, which was applied in the above-mentioned study, is over 10 times more sensitive [64]. The results of our study demonstrated the presence of HSD17B1 and the conversion of E1 to E2 in human NSCLC Calu-6, Calu-1 and A549 cells. However, further studies are required to clarify the changes in expression of HSD17B1 during lung carcinogenesis and its role in converting E1 to E2.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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Original article
Conversion of estrone to 17-beta-estradiol in human non-small-cell lung cancer cells in vitro Hanna Drzewiecka, Paweł P. Jagodzinski * Poznan´ University of Medical Sciences, Department of Biochemistry and Molecular Biology, 6 S´wie˛cickiego St., 60-781 Poznan´, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 January 2012 Accepted 29 February 2012
It has recently been suggested that, in addition to genetic and environmental factors and tobacco exposure, estrogens also may be an independent risk factor in the development of lung cancer. Therefore, we evaluated the transcript and protein levels of 17-beta-hydroxysteroid-dehydrogenase type 1 (HSD17B1), and the conversion of estrone (E1) to 17-beta-estradiol (E2) in human non-small cell lung cancer (NSCLC) Calu-6, Calu-1 and A549 cells. In our work, we established the presence of HSD17B1 transcripts and proteins in all examined NSCLC cells. Moreover, we demonstrated that human NSCLC Calu-6, Calu-1 and A549 cells are able to convert weak estrogen E1 to highly potent E2 in vitro. Our results indicate that NSCLC cells are able to produce E2 from E1. ß 2012 Elsevier Masson SAS. All rights reserved.
Keywords: HSD17B1 Lung cancer 17-beta-estradiol
1. Introduction Despite the development of new treatment methods, lung cancer continues to be the leading cause of death worldwide, with more than one million cases reported among men and women annually [1]. The overall prognosis for this cancer is poor because of late diagnosis, which disqualifies patients for surgical resection [2,3]. Primary lung cancer is derived from epithelial cells and can be distinguished into two major types: non-small cell lung cancer (NSCLC), and small cell lung cancer (SCLC). NSCLC accounts for approximately 80% of all lung cancers and includes the following histological types: squamous cell carcinoma, adenocarcinoma, large cell carcinoma, and other rare types [4]. Squamous cell carcinoma tends to occur more frequently in men and is connected to a history of smoking, whereas adenocarcinoma is the most common form in women [5,6]. Currently, an upward trend in NSCLC incidence is observed among women [7,8]. Lung cancer in young women exhibits rapid growth and a more aggressive biology than in postmenopausal women [9,10]. Furthermore, the longterm use of hormone replacement therapy (HRT) seems to be correlated with a poor survival rate of patients with lung cancer [11,12]. Therefore, gender may be an additional independent risk factor, apart from tobacco exposure and genetic and environmental factors, in carcinogenesis of the lung. This observation may place the estrogens to the fore of lung cancer risk factors, but the role of estrogens in the incidence of this disease remains elusive. * Corresponding author. E-mail address: [email protected] (P.P. Jagodzinski). 0753-3322/$ – see front matter ß 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2012.02.006
Estrogen affects its target tissues by the classical, liganddependend activation of signaling pathways or via non-genomic actions using membrane-associated estrogen receptors ERs and/or a member of the G protein-coupled receptor superfamily, GPCR30 [13,14]. The classical pathway is mediated by estrogen receptors, ERa and ERb, which belong to the nuclear steroid hormone receptor superfamily. ERb is the predominant subtype present in normal lung tissue, and a great majority of NSCLC tumors are positive for ERb, regardless of gender [7,15–22]. Moreover, the non-genomic signalling pathway using the GPR30 receptor has also been identified in lung cancer cells [23]. Although 17-betaestradiol (E2) is mainly produced in the ovaries [24], this steroid hormone can be also locally synthesized from its precursors in various tissues in both women and men [25]. In peripheral tissue, estrone (E1) can be formed by the aromatase or steroid sulfatase (STS) pathways [26,27] and subsequently reduced by 17-betahydroxysteroid-dehydrogenase type 1 (HSD17B1) to E2 [28]. It has recently been demonstrated that HSD17B1 may exhibit a crucial role in the development of estrogen-dependent diseases [29–33]. To date, the metabolism of androgens and estrogens in the lungs has been focused on the aromatase and STS pathways and their pivotal role in intratumoral estrogen synthesis in NSCLC cells [18,34–37]. However, little is known about HSD17B1 expression in lung cancer cells and their ability to convert E1 to E2. Therefore, we evaluated the levels of HSD17B1 transcript and protein and the production of E2 from E1 in human NSCLC Calu-6, Calu-1 and A549 cells. In addition to being a precursor of E2, E1 can also exert an impact on NSCLC cells by acting through ERs [38], so we also explored the influence of both E1 and E2 on the proliferation of Calu-6, Calu-1 and A549 cells.
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2. Materials and methods 2.1. Antibodies and reagents Rabbit polyclonal anti-HSD17B1 antibody (Ab) (H-158), rabbit polyclonal antiglyceraldehyde-3-phosphate (GAPDH) Ab (FL-335) and goat antirabbit horseradish peroxidase (HRP)-conjugated Ab were provided by Santa Cruz Biotechnology (Santa Cruz, CA). E1, E2 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. (St.-Louis, MO). 2.2. Cell culture T47D breast cancer cells were used as a positive control for HSD17B1 transcript and protein identification [39]. The human NSCLC A549 and Calu-1 cells, as well as the T47D breast cancer cells, were purchased from the American Type Culture Collection (Rockville, MD). The NSCLC Calu-6 cell line was kindly provided by Dr. Marek Rusin from the Centre of Oncology Maria Sklodowska-Curie Memorial Institute (Gliwice, Poland). A549, Calu-1 and T47D cells were routinely maintained in DMEM medium, Sigma-Aldrich Co. (St.-Louis, MO), while Calu-6 cells were grown in RPMI-1640 medium, Sigma-Aldrich Co. (St.-Louis, MO) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine and gentamicin sulfate salt (50 mg/ml) Sigma-Aldrich Co. (St.-Louis, MO) at 37 8C in humidified air with 5% CO2. Prior to all experiments, cells were maintained for 48 hours in an appropriate phenol red-free medium Sigma-Aldrich Co. (St.-Louis, MO) supplemented with 10% charcoal-dextran-stripped FBS Sigma-Aldrich Co. (St.-Louis, MO). 2.3. Conversion of E1 to E2 in Calu-6, Calu-1 and A549 cells To investigate the ability of NSCLC cells to convert E1 to E2, the Calu-6, Calu-1 and A549 cells were seeded in six-well plates (3.6 105 cells/well). After 24 hours, media were replaced and cells were cultured either in the absence or in the presence of E1 at a concentration of 5 mM for 2, 4, 6, 8, 10, and 12 hours. At the end of incubation periods, 1.0 ml of the medium was collected and the concentration of E2 was evaluated by electrochemiluminescence method using Cobas 6000 Roche Diagnostics GmbH (Mannheim, Germany). The results were presented as the concentration of E2 in culture medium in nM or pM per 1 mg of total cell protein. All experiments were performed in triplicate and the results represent mean SE. 2.4. Reverse transcription (RT) and real-time quantitative PCR (RQPCR) analysis To determine the presence of the HSD17B1 transcript and its levels in Calu-6, Calu-1 and A549 cells we applied RT and RQ-PCR analysis. RNA was isolated by TRI Reagent1 Sigma-Aldrich Co. (St.Louis, MO) according to the method of Chomczynski and Sacchi [40]. RNA quality was determined by denaturing agarose gel electrophoresis, and the concentration was quantified by measuring the optical density (OD) at 260 nm. RNA samples were treated with DNase I and reverse-transcribed into cDNA using Moloney Murine Leukemia Virus (M-MLV) Reverse Transcriptase from Invitrogen (Carlsbad, CA). RQ-PCR was conducted in a Light Cycler1480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany), using SYBR Green I as detection dye. Target cDNA was quantified by the relative quantification method. The quantity of HSD17B1 transcript in each sample was standardized by the porphobilinogen deaminase (PBGD) transcript level. The PCR amplification efficiency for target and reference genes was determined by different standard curves created by consecutive
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dilutions of the cDNA template mixture, as provided in Relative Quantification Manual Roche Diagnostics GmbH (Mannheim, Germany). The HSD17B1 cDNA 75 bp amplicon was amplified employing the primer pair: (50 TGA GGA GGT GGC GGA GGT CTT C 30 ) (forward, nt 702-723) and (50 CGC TCG GTG GTG AAG TAG 30 ) (reverse, nt 759-776). The PBGD cDNA 160 bp amplicon was amplified using the primer pair: (50 GCC AAG GAC CAG GAC ATC 30 ) (forward, nt 676-693) and (50 TCA GGT ACA GTT GCC CAT C 30 ) (reverse, nt 817-835). The HSD17B1 and PBGD cDNA primers were designed based on sequences ENST00000225929 and ENST00000278715, located in Ensembl Genome Browser (www.ensembl.org). For amplification, 1 ml of total (20 ml) cDNA solution was added to 9 ml of Light Cycler1480 SYBR Green I Master mix (1 concentrated) Roche Diagnostics GmbH (Mannheim, Germany), containing 2.5 mM MgCl2 and 0.5 mM primers. A sample of RNA that had not been reverse-transcribed and a notemplate control were included in each batch of samples to provide a negative control in subsequent PCR. Melting-curve analysis and sequencing were applied to confirm the specificity of the amplified products. All experiments were performed in triplicate and RQ-PCR results are expressed as averages of relative expression to control transcript levels in T47D cells. 2.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and western blotting Cell lysates were prepared using RIPA buffer Sigma-Aldrich Co. (St.-Louis, MO) containing 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1.0% Igepal, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and protease inhibitor cocktail. Samples were incubated for 1 hour on ice and then centrifuged at 10,000 g for 10 min at 4 8C to remove cell debris. Supernatants were stored at 80 8C before use. The total protein concentration in cell extracts was determined by the Bradford assay. Subsequently, 30 mg of protein was resuspended in the loading buffer and boiled at 99 8C for 5 min. Next, all samples were loaded into each lane and separated on 10% Tris-glycine gel using SDS-PAGE. Gel proteins were transferred to polyvinylidene fluoride (PVDF) membrane, Roche Diagnostics GmbH (Mannheim, Germany), which was blocked with 5% non-fat dry milk in Tris buffered saline/Tween 20. Immunodetection was performed with rabbit polyclonal antiHSD17B1 Ab (H-158) at a dilution of 1:500, followed by incubation with goat anti-rabbit HRP-conjugated Ab (1:5000). Bands were revealed using Pierce ECL Plus Western Blotting Substrate, Thermo Fisher Scientific (Rockford, IL). The membranes were stripped and reblotted with rabbit polyclonal anti-GAPDH Ab (FL-335) at a dilution of 1:2500, followed by goat anti-rabbit HRP-conjugated Ab (1:10,000), to ensure equal protein loading of the lanes. 2.6. MTT cell proliferation assay To determine the effect of E1 and E2 on the proliferation of Calu6, Calu-1 and A549 cells we used MTT cell proliferation assay. The cells were either cultured in phenol red-free RPMI-1640 (Calu-6 cells) or phenol red-free DMEM (Calu-1 and A549 cells) supplemented with 10% charcoal-dextran-stripped FBS for 48 hours, then seeded into 24-well culture plates (6 104 cells/ well). Subsequently, cells were incubated either in the absence or in the presence of E1 (5 mM) and E2 (10 nM) for 24, 48 and 72 hours. After the incubation period, all culture media were removed and 50 ml of the MTT stock solution in phosphate buffered saline (PBS) (5 mg/ml), mixed with 450 ml of the appropriate phenol red-free medium, was added to each well. After 4 hours of incubation, the culture medium was discarded and the blue formazan crystals were dissolved by adding MTT solvent
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was significance (P < 0.05) between the groups. The statistical analysis was performed with STATISTICA 6.0 software. 3. Results and discussion
Fig. 1. Identification of HSD17B1 transcript (A) and protein (B) in human NSCLC Calu-6, Calu-1 and A549 cells. The cells were used for total RNA isolation and reverse transcription. The HSD17B1 transcript levels were determined by RQ-PCR analysis. RQ-PCR results were standardized by PBGD cDNA levels. HSD17B1 transcripts levels in the investigated NSCLC cells are expressed as a multiplicity of the HSD17B1 cDNA levels in positive controls, T47D breast cancer cells. Each sample was determined in triplicate. Cell proteins were separated by 10% SDS-PAGE and transferred to a PVDF membrane that was then immunoblotted with rabbit polyclonal anti-HSD17B1 Ab and incubated with goat antirabbit HRP-conjugated Ab. To ensure equal protein loading of the lanes, the membrane was then reblotted with anti-GAPDH Ab, followed by goat antirabbit HRP-conjugated Ab.
(0.1 N HCl in isopropanol, 150 ml/well). Next, 100 ml of the solution was transferred into 96-well culture plates and the absorbance was measured at a wavelength of 570 nm with a background reading at 640 nm. All experiments were performed in triplicate and the results are presented as means SE. 2.7. Statistical analysis The difference between control and treated cells was statistically analyzed by unpaired, two-tailed t-test to evaluate if there
Despite the fact that cigarette smoking is still a main cause of lung cancer, estrogens appear to be significant factors in this carcinoma. Clinical data demonstrates that individuals who have never smoked and are diagnosed with this disease are mostly females. Women also seem to be more susceptible to developing lung cancer among smokers, because women have been found to be more vulnerable to the carcinogens found in cigarette smoke [41,42]. The uptake of polycyclic aromatic hydrocarbons, formed as by-products during tobacco consumption, stimulates the activity of cytochrome P450 1B1 (CYP1B1). CYP1B1 is commonly expressed in the human lung and metabolizes E2 to 2- and 4-catechol estrogens, which generate DNA adducts when converted into semiquinones and quinines [43,44]. The formation of DNA adducts contributes to the inactivation of tumour suppressor genes and mutation in proto-oncogenes, which are often detected in lung cancer patients [45]. In our study, we found the presence of HSD17B1 transcript and protein in Calu-6, Calu-1 and A549 cells (Fig. 1). Moreover, we demonstrated that all examined NSCLC cells are able to convert the weak estrogen E1 to the highly potent E2 (Fig. 2). To date, increased HSD17B1 mRNA levels associated with an elevated E2/E1 ratio have been observed in breast cancer [29,30], ovarian tumor [31], endometriosis [32], endometrial hyperplasia [33], and uterine leiomyoma [46]. Recently, the clinical case study by Niikawa et al. reported that patients with NSCLC also have a higher E2 concentration in cancerous tissues as compared to the corresponding non-neoplastic lung tissues from the same patients [4]. Furthermore, high endogenous or exogenous estrogen levels were connected with a poorer clinical outcome for the patients. In addition to their influence on the reproductive system, sexsteroid hormones exert multidirectional action on other tissues. Estrogens promote the development and progression of estrogendepended cancers, as well as markedly affecting the immune system and contributing to some autoimmune diseases [47,48]. Results of experimental studies have indicated that estrogen treatment activates both genomic and non-genomic signaling in lung cancer cells [49,50]. Although previous studies with murine
Fig. 3. Effect of E1 and E2 on the proliferation of human NSCLC Calu-6 (A), Calu-1 (B) and A549 (C) cells. To determine the effect of E1 and E2 on the proliferation of Calu-6, Calu-1 and A549 cells we used the MTT cell proliferation assay. Cells were cultured in an appropriate phenol red-free medium for 48 hours, followed by seeding into 24-well culture plates. Subsequently, cells were incubated either in the absence or in the presence of and E1 (5 mM) and E2 (10 nM) for 24, 48 and 72 hours, respectively. After the incubation period, all culture media were removed and the MTT assay was carried out. All experiments were performed in triplicate and results are presented as the mean SE from three independent experiments.
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Fig. 2. Conversion of E1 to E2 in human NSCLC Calu-6 (A), Calu-1 (B) and A549 (C) cells. Cells were cultured in phenol red-free RPMI-1640 (Calu-6 cells) or phenol red-free DMEM (Calu-1 and A549 cells) for 48 hours, followed by incubation either in the absence or in the presence of E1 at a concentration of 5 mM. After 2, 4, 6, 8, 10 and 12 hours, 1.0 ml of medium was collected and the concentration of E2 was determined by electrochemiluminescence method. The results were expressed as E2 concentration in culture medium in nM or pM per 1 mg of total cell protein. Each sample was determined in triplicate and results are presented as the mean SE from three independent experiments.
knockout models showed the indispensability of ERb for normal lung structure and function [51,52], a higher expression of ERb was detected in lung adenocarcinoma [7,15] and provided a new line of evidence for estrogen involvement in lung cancer pathogenesis. Additionally, cross-signalling between ERs and an epidermal growth factor receptor in lung cancer has been established in recent years [16,18,53–55], highlighting the necessity of targeting both of these pathways. Several previous studies have confirmed the presence of estrogen-stimulated NSCLC cell growth and proliferation in vitro and in vivo and indicated that anti-estrogens were able to inhibit those events [16,19,54,56–58]. A549 is a lung adenocarcinoma cell line and Calu-1 is a squamous lung cancer cell line, both derived from males, whereas Calu-6 is an anaplastic lung carcinoma cell line derived from a female [59–61]. We observed that neither E2 at a concentration of 10 nM nor E1 at a concentration of 5 mM had an effect on cellular proliferation of Calu-6, Calu-1 and A549 cells (Fig. 3). Our results are consistent with Miki et al., who did not observe E2 mediated cell proliferation in A549 cells [62]. In contrast, Niikawa et al. used A549 cells transfected with either ERa or ERb expression vectors to demonstrate the positive effect of E2 on the proliferation of these cells [4]. Moreover, our observation that Calu-1 did not respond proliferatively to E2 and E1 may be secondary to findings that E2 has no impact on proliferation of lung adenocarcinoma cells derived from males [19,63]. However, we were not able to confirm growth stimulation of Calu-6 cells after estrogen exposure, and this observation is contrary to earlier studies [22]. This inconsistent result may be due to differences in the sensitivity of the tests used in our study compared to the tests used in earlier studies. We used the MTT proliferation assay, whereas the 5-bromo-2-deoxyuridine assay, which was applied in the above-mentioned study, is over 10 times more sensitive [64]. The results of our study demonstrated the presence of HSD17B1 and the conversion of E1 to E2 in human NSCLC Calu-6, Calu-1 and A549 cells. However, further studies are required to clarify the changes in expression of HSD17B1 during lung carcinogenesis and its role in converting E1 to E2.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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