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Aromatase and comparative response to its inhibitors in two types of endometrial cancer
Journal of Steroid Biochemistry & Molecular Biology 95 (2005) 71–74
Aromatase and comparative response to its inhibitors in two types of endometrial cancer夽 L. Berstein a,∗ , A. Kovalevskij a , T. Zimarina a , S. Maximov a , E. Gershfeld a , D. Vasilyev a , S. Baisheva b , A. Baymakhasheva b , J.H.H. Thijssen c a
Laboratory of Oncoendocrinology, N.N. Petrov Research Institute of Oncology, St. Petersburg 197758, Russia b Research Institute of Oncology and Radiology, Almaaty 480078, Kazahstan c ASL Endocrinology, University Medical Center, Utrecht 3508 AB, The Netherlands
Abstract Aromatase activity (AA) was evaluated totally in 80 tumors collected from primary endometrial cancer (EC) patients. All patients were divided into cases belonging to the types I or II of EC (respectively, 50 and 30 observations). Samples of malignant endometrium from type II demonstrated inclination to the higher AA in comparison with type I samples; the difference reached level of statistical significance in non-smoking patients (p = 0.02). Although no positive correlation was revealed between AA in EC tissue and percentage of cells with DNA damage in normal endometrium from the same patients, the rate of DNA damage (percent of comets, comet’s tail average length, etc.) was higher in intact endometrium collected from patients with type II of the disease. In 19 tumor samples, CYP19 gene expression was evaluated by RT-PCR and level of mRNA signal demonstrated positive correlation with AA (Rs = +0.63, p = 0.05) in the whole this material. Of note, though, CYP19 mRNA expression was not revealed in six cases, and all of them belonged to the type I of disease. Finally, in 23 EC patients (15 with type I and 8 with type II of the disease) effects of 2 weeks treatment with letrozole (10 pts) and exemestane (13 pts) were evaluated in neoadjuvant setting. Although diminishing of endometrial M-echo signal and the increases in FSH and LH concentration after treatment were more pronounced in type I patients, decrease in tumor PR content (p = 0.04) was more revealing in patients with type II of EC; besides, the decreases in AA in tumor tissue by the end of treatment were noted predominantly in patients with lower body weight (BMI <27.5). Thus, although type II of EC is frequently considered as hormone-independent, increased ability of this type of the tumor to estrogen biosynthesis (at CYP19 gene and protein level) may lead to the reconsideration of such conclusion and warrants further investigation. The search of possible ethnic differences in AA and in the biologic response to aromatase inhibitors in EC can be of importance too. © 2005 Elsevier Ltd. All rights reserved. Keywords: Aromatase; CYP19; Activity; Expression; Aromatase inhibitors; Endometrial cancer; Types
1. Introduction Intratumoral estrogen biosynthesis may be considered as a reserve in understanding of the mechanisms of endometrial cancer (EC) development and progression and in elaboration of measures for the increase in efficiency of its treatment. Although no completely deprived of controversy [6], the leading contemporary concept claims that normal human endometrium does not contain aromatase mRNA transcripts, 夽 Presented
at the VIIth International Aromatase Conference: AROMATASE 2004, Edinburgh, Scotland, UK, 6–8 September 2004. ∗ Corresponding author. Tel.: +7 812 596 8654; fax: +7 812 596 8947. E-mail address: [email protected] (L. Berstein). 0960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.04.008
and ability to synthesize estrogens is characteristic only for endometrial cancer itself [7,12]. Meanwhile, aromatase activity (AA) in EC tissue has been evaluated much rarely than in breast cancer, and to our knowledge practically no comparative studies have been performed relating levels of aromatase activity and CYP19 mRNA within malignant endometrium [13]. The important feature of EC is its heterogeneity. Several lines of evidence suggest that two different variants of EC can be distinguished [1,5,8,14]. One of these variants is traditionally called hormone-dependent or estrogen-dependent and is designated as type I. Another variant is usually considered hormone-independent or estrogen-independent and called otherwise as type II. Each of these types is
72
L. Berstein et al. / Journal of Steroid Biochemistry & Molecular Biology 95 (2005) 71–74
characterized by rather specific complex of pathological, genetic and clinical features of the tumor as well as by a set of typical characteristics of the host [5,8,14]. The principal aim of present study was to compare AA and CYP19 gene expression in tumor tissue in cases belonging to the types I or II of endometrial cancer, as well as to evaluate response of such patients and their neoplasms to the short-term pre-operational treatment with aromatase inhibitors.
2. Materials and methods 2.1. Patients Tumor samples from 80 patients with EC were evaluated. Age of the patients varied between 38 and 80, and 72 patients were postmenopausal. According to existing surgical classification, stage of the disease varied between FIGO IA-IIIC. Morphologically mostly (approximately in 90% of cases) endometrioid adenocarcinomas were presented in this material. To distinguish between patients with types I and II of EC mainly the following criteria were used: absence or presence of infertility/low pregnancy number, late menopause, obesity, hypertension, hyperlipidemia, uterine myoma, endometriosis, ovarian theca hypertrophy, well-, modestly- or low-differentiated carcinomas, superficial (less than 5 mm) or deep invasion into myometrium, involvement of regional lymph nodes. As proposed, patients who are infertile, obese, hypertensive, hyperlipidemic, have menopause timing ≥53 years, myoma or endometriosis and well-differentiated nodenegative adenocarcinoma with superficial invasion (or are characterized with more than 60% of these features) belong to the type I of disease [5,8]. As a result, it was considered that among studied group of patients 50 women had type I of EC and 30 women type II. In 23 of 80 aforementioned EC patients (15 with type I and 8 with type II of the disease), after the tissue specimens for aromatase activity determination were received during diagnostic biopsy, effects of 2 weeks treatment with aromatase inhibitors letrozole (2.5 mg/day, 10 pts) and exemestane (25 mg/day, 13 pts) were evaluated in neoadjuvant setting.
CYP19 gene expression was studied in 19 samples of malignant endometrium, in which AA has been measured as well. Total RNA was isolated with guanidinium thiocyanate – phenol – chloroform method and checked by spectrophotometry and electrophoresis. Reverse transcription was performed in standard way and its efficiency was verified with PCR with primers to GAPDH-gene. Expression of the coding site of aromatase mRNA was evaluated examining the region across exons II and III with primers described by Koos et al. [9]—sense: 5 -GAATATTGGAAGGATGCACAGACT3 , antisense: GGGTAAAGATCATTTCCAGCATGT-3 . The conditions of reaction were the following: 37 cycles, denaturation—30 s at 95◦ , annealing—30 s at 61◦ , synthesis—1.5 min at 72◦ . The products were separated in the polyacrylamide gel and visualized by ethidium bromide staining. The expected size of CYP19 product was 293 bp and as a positive control RNA from human placenta or breast cancer was used. Results were presented in a semiquantitative manner using arbitrary or conditional units: 1 (“−” negative signal), 2 (“±” weak positive signal) and 3 (“+” strong positive signal). In 12 primary patients (6 with type I of EC and another 6 with type II), samples of normal endometrium were collected simultaneously with cancer specimens with the aim to compare aromatase activity in the tumor with DNA damage rate in macroscopically normal endometrial tissue. DNA damage was evaluated on the basis of COMET assay in the modification suitable for cells isolated from solid tissues [17]. Effect of neoadjuvant treatment of 23 EC patients with aromatase inhibitors (see above, Section 2.1) has been determined. For that, clinical, sonographic, morphologic, cytologic and hormonal-metabolic parameters (blood estradiol, LH, FSH, glucose and cholesterol levels; tumor aromatase activity and steroid hormone receptors (ER and PR) content by methods used in our laboratory [4]) were evaluated before and after the course. Statistical analysis was performed by methods allowing for means, standard errors, χ2 -values and Spearman correlation on the basis of SigmaPlot program. The differences with p ≤ 0.05 were considered as significant.
2.2. Experimental procedures
3. Results
Aromatase activity in tumor tissue was estimated by measuring of 3 H2 O release from 3 H-1--androstenedione (NEN, Boston, MA; specific activity, 25.4 Ci/mM) as described [15]. Briefly, the reaction mixture (which contained tumor homogenate, NADPH regeneration system and labeled androgenic precursor) was incubated for 2 h at 37 ◦ C. Then reaction was stopped by adding 5 vol of cold chloroform and 5% suspension of activated charcoal (Norit A) was added to the water phase. The fraction containing 3 H2 O was separated by centrifugation, and counting was performed with dioxane scintillator. Results were presented in fM/mg protein/h.
AA varied in the specimens of malignant endometrium between 0 and 28.4 fM/mg protein/h; in 43.1% of samples it was 5 fM/mg protein/h and less and in 16.3% higher than 15 fM/mg protein/h. Average value of AA in tumor specimens from patients belonging to the type II of EC was higher in comparison with type I cases; this difference reached the level of statistical significance in non-smoking patients (Table 1). Interestingly, prevalence of the specimens with AA value ≤5 fM/mg protein/h has been in patients with type II of EC significantly lower (27 ± 8%) than in type I cohort (54 ± 7%, p = 0.02).
L. Berstein et al. / Journal of Steroid Biochemistry & Molecular Biology 95 (2005) 71–74 Table 1 Aromatase activity in endometrial cancer tissue in patients belonging to types I and II of the disease Group
All patients Type I Type II p (I vs. II)
Aromatase activity (fM/mg protein/h) All samples
Samples from non-smoking patients
8.29 ± 0.77 (n = 80) 7.39 ± 0.95 (n = 50) 10.28 ± 1.19 (n = 30) 0.062
7.98 ± 0.84 (n = 70) 6.52 ± 1.03 (n = 44) 10.63 ± 1.30 (n = 26) 0.02
73
increase in FSH (+32.7% versus +12.2%) as well as LH concentration by the end of neoadjuvant treatment with aromatase inhibitors were more pronounced in type I patients, the decrease in tumor PR content (−65.5% versus −28.9%, p = 0.04) was more revealing in patients with type II of EC. Besides, a decrease in tumor AA in the course of treatment was noted predominantly in patients with lower body weight (BMI < 27.5), e.g. with a host feature more peculiar for patients with type II of the disease [5,8].
4. Discussion
Fig. 1. Distribution of the data on CYP19 expression in endometrial cancer tissue. Vertical line—number of samples. Horizontal line—groups of patients (all, n = 19; type I, n = 13, type II, n = 6). Results of the evaluation are included into two groups: negative expression (−), positive expression (weak + strong, or ± and +). Difference in the distribution of these results between types I and II specimens is significant, χ2 = 4.05 (p = 0.044).
In the whole group of studied samples, AA and CYP19 mRNA signal correlated with each other (Rs = +0.63, p < 0.05); this correlation was more pronounced in type I samples (Rs = +0.67) than in type II material (Rs = +0.33). At the same time, expression of CYP19 mRNA was not revealed in six cases of EC, and all of them belonged to the type I of disease (Fig. 1). No positive correlation was revealed between AA in EC tissue and percentage of cells with DNA damage in normal endometrium from the same patients. Nevertheless, the rate of DNA damage (percent of comets, comet’s tail average length, etc.) demonstrated inclination for the higher levels in intact endometrium collected from patients with type II of the disease (Table 2). Finally, while diminishing in the average value of endometrial M-echo signal (−30.2% versus −11.6%) and the
Although idea on the heterogeneity of EC and existence of its two variants (one of which is traditionally considered as hormone/estrogen dependent and another one as hormone/estrogen independent) became rather popular during last 40–50 years [1,5,8,14], nobody studied by present the aromatase activity and aromatase gene signal separately in tumor samples belonging to types I or II of the disease. Meanwhile, this approach might help to solve, at least partly, the problem whether hormonal imbalance associated with the risk of endometrial cancer and excessive estrogenic stimulation is typical only for so called type I of EC, or such notion is not completely correct. The principal data received in present study demonstrate that tumor specimens collected from endometrial cancer patients with type II of the disease are characterized with higher AA and CYP19 gene signaling (Table 1, Fig. 1). The rate of DNA damage in macroscopically normal endometrium of these patients shows tendency to the increase (Table 2). Besides, the same patients are ready to respond in a specific manner, i.e. by the decrease in tumor PR content, to the shortterm treatment with aromatase inhibitors. Additionally, in one of the previous reports the tendency to increased AA in lowdifferentiated tumors (which are more prevalent among type II patients) was revealed [18]. Of note also, that according to our recent observations the ratio of incidence of CYP19 A6A6 genotype (presenting a combination of more long and probably more biologically active allelic polymorphisms) to the frequency of A1A6 and A3A6 genotypes was higher in type II patients (1.0) than in females with type I of the disease (0.3) [3]. Thus, it may be suggested that more aggressive clinically and frequently receptor-negative type II of endometrial cancer [4,5,8,14,16] is associated with the signs of intratumoral
Table 2 Results of COMET assay with macroscopically normal endometrium in patients belonging to types I and II of endometrial cancer Group
Type I Type II p
Parameter Cells with comets (%)
Average length of tail in cells with comets, cond. units
Average length of tail in 100 counted cells, cond. units
16.7 ± 5.8 26.8 ± 15.1
14.50 ± 3.05 40.25 ± 14.68 0.09
5.74 ± 1.52 13.82 ± 7.15
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hyperproduction of estrogens both at the level of aromatase protein and gene expression. These data additionally support earlier made assumptions that: (1) EC of type II actually may be also estrogen dependent—with the dependence of neoplastic transformation of endometrium and tumor growth progression primarily of locally produced estrogens [4] and (2) two aforementioned variants of EC have to be considered as a clinical equivalents of the two types of hormonal carcinogenesis, e.g. promotional and genotoxic [2,10,11]. An association of intratumoral aromatase activity with menopausal status, clinical stage of disease, tumor differentiation and rate of invasion among cases belonging to the types I or II of EC deserves additional study. The search of possible ethnic differences in intratumoral aromatase activity and in the biologic response to aromatase inhibitors in EC can be of importance too. Acknowledgement The study was supported by grants from INTAS (01-434) and RFBR.
References [1] L.M. Berstein, Excretion of classical estrogens and total phenolsteroids in endometrial cancer patients, Cand. Med. Sci. Thesis. St. Petersburg (1967) 1–250. [2] L.M. Berstein, Hormonal Carcinogenesis, Nauka Publishers, St. Peterburg, 2000, 199 pp. [3] L.M. Berstein, E.N. Imyanitov, T. Zimarina, A. Kovalevskij, S. Maximov, P. Pujol, J.H.H. Thijssen, Allelic polymorphism of steroidogenic enzymes in two types of endometrial cancer, in: International Conference on, Sex steroids: an update on estrogen and progesterone actions. Role in cancer, ageing and in reproductive medicine (from research to clinical practice), Montpellier, France, April, 2004, p. 32. [4] L. Berstein, S. Maximov, E. Gershfeld, I. Meshkova, V. Gamajunova, E. Tsyrlina, A. Larionov, A. Kovalevskij, D. Vasilyev, Neoadjuvant therapy of endometrial cancer with the aromatase inhibitor letrozole: endocrine and clinical effects, Eur. J. Obstetr. Gynecol. Reprod. Biol. 105 (2002) 161–165.
[5] J.V. Bokhman, Two pathogenetic types of endometrial carcinoma, Gynecol. Oncol. 15 (1983) 10–17. [6] J. Brosens, H. Verhoeven, R. Campo, L. Gianaroli, S. Gordts, J. Hazekamp, L. Hagglund, T. Mardesic, E. Varila, J. Zech, I. Brosens, High endometrial aromatase P450 mRNA expression is associated with poor IVF outcome, Hum. Reprod. 19 (2004) 352–356. [7] S.E. Bulun, K. Economos, D. Miller, E.R. Simpson, CYP19 (aromatase cytochrome P450) gene expression in human malignant endometrial tumors, J. Clin. Endocrinol. Metab. 79 (1994) 1831– 1834. [8] G. Emons, G. Fleckenstein, B. Hinney, A. Huschmand, W. Heyl, Hormonal interactions in endometrial cancer, Endocr. Relat. Cancer 7 (2000) 227–242. [9] R.D. Koos, P.K. Banks, S.E. Inkster, W. Yue, A.M. Brodie, Detection of aromatase and keratinocyte growth factor expression in breast tumors using reverse transcription-polymerase chain reaction, J. Steroid Biochem. Mol. Biol. 45 (1993) 217–225. [10] J.G. Liehr, Dual role of oestrogens as hormones and pro-carcinogens: tumour initiation by metabolic activation of oestrogens, Eur. J. Cancer Prev. 6 (1997) 3–10. [11] R.J. Santen, Endocrine-responsive cancer, in: P.R. Larsen, et al. (Eds.), Williams’ Textbook of Endocrinology, W.B. Saunders Comp., Philadelphia, 2003, pp. 1797–1833. [12] H. Sasano, N. Harada, Intratumoral aromatase in human breast, endometrial, and ovarian malignancies, Endocr. Rev. 19 (1998) 593–607. [13] H. Sasano, K. Kaga, S. Sato, A. Yajima, H. Nagura, N. Harada, Aromatase cytochrome P450 gene expression in endometrial carcinoma, Br. J. Cancer 74 (1996) 1541–1544. [14] M.E. Sherman, Theories of endometrial carcinogenesis: a multidisciplinary approach, Mod. Pathol. 13 (2000) 295–308. [15] N. Tilson-Mallett, S.J. Santner, P.D. Feil, R.J. Santen, Biological significance of aromatase activity in human breast tumors, J. Clin. Endocrinol. Metabol. 57 (1983) 1125–1128. [16] D. Tritz, M. Pieretti, S. Turner, D. Powell, Loss of heterozygosity in usual and special variant carcinomas of the endometrium, Hum. Pathol. 28 (1997) 607–612. [17] S. Tsuda, Y. Kosaka, M. Murakami, H. Matsuo, N. Matsusaka, K. Taniguchi, Y.F. Sasaki, Detection of nivalenol genotoxicity in cultured cells and multiple mouse organs by the alkaline single-cell gel electrophoresis assay, Mutat. Res. 415 (1998) 191– 200. [18] K. Watanabe, H. Sasano, N. Harada, M. Ozaki, H. Niikura, S. Sato, A. Yajima, Aromatase in human endometrial carcinoma and hyperplasia, immunohistochemical, in situ hybridization, and biochemical studies, Am. J. Pathol. 146 (1995) 491–500.
Aromatase and comparative response to its inhibitors in two types of endometrial cancer夽 L. Berstein a,∗ , A. Kovalevskij a , T. Zimarina a , S. Maximov a , E. Gershfeld a , D. Vasilyev a , S. Baisheva b , A. Baymakhasheva b , J.H.H. Thijssen c a
Laboratory of Oncoendocrinology, N.N. Petrov Research Institute of Oncology, St. Petersburg 197758, Russia b Research Institute of Oncology and Radiology, Almaaty 480078, Kazahstan c ASL Endocrinology, University Medical Center, Utrecht 3508 AB, The Netherlands
Abstract Aromatase activity (AA) was evaluated totally in 80 tumors collected from primary endometrial cancer (EC) patients. All patients were divided into cases belonging to the types I or II of EC (respectively, 50 and 30 observations). Samples of malignant endometrium from type II demonstrated inclination to the higher AA in comparison with type I samples; the difference reached level of statistical significance in non-smoking patients (p = 0.02). Although no positive correlation was revealed between AA in EC tissue and percentage of cells with DNA damage in normal endometrium from the same patients, the rate of DNA damage (percent of comets, comet’s tail average length, etc.) was higher in intact endometrium collected from patients with type II of the disease. In 19 tumor samples, CYP19 gene expression was evaluated by RT-PCR and level of mRNA signal demonstrated positive correlation with AA (Rs = +0.63, p = 0.05) in the whole this material. Of note, though, CYP19 mRNA expression was not revealed in six cases, and all of them belonged to the type I of disease. Finally, in 23 EC patients (15 with type I and 8 with type II of the disease) effects of 2 weeks treatment with letrozole (10 pts) and exemestane (13 pts) were evaluated in neoadjuvant setting. Although diminishing of endometrial M-echo signal and the increases in FSH and LH concentration after treatment were more pronounced in type I patients, decrease in tumor PR content (p = 0.04) was more revealing in patients with type II of EC; besides, the decreases in AA in tumor tissue by the end of treatment were noted predominantly in patients with lower body weight (BMI <27.5). Thus, although type II of EC is frequently considered as hormone-independent, increased ability of this type of the tumor to estrogen biosynthesis (at CYP19 gene and protein level) may lead to the reconsideration of such conclusion and warrants further investigation. The search of possible ethnic differences in AA and in the biologic response to aromatase inhibitors in EC can be of importance too. © 2005 Elsevier Ltd. All rights reserved. Keywords: Aromatase; CYP19; Activity; Expression; Aromatase inhibitors; Endometrial cancer; Types
1. Introduction Intratumoral estrogen biosynthesis may be considered as a reserve in understanding of the mechanisms of endometrial cancer (EC) development and progression and in elaboration of measures for the increase in efficiency of its treatment. Although no completely deprived of controversy [6], the leading contemporary concept claims that normal human endometrium does not contain aromatase mRNA transcripts, 夽 Presented
at the VIIth International Aromatase Conference: AROMATASE 2004, Edinburgh, Scotland, UK, 6–8 September 2004. ∗ Corresponding author. Tel.: +7 812 596 8654; fax: +7 812 596 8947. E-mail address: [email protected] (L. Berstein). 0960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.04.008
and ability to synthesize estrogens is characteristic only for endometrial cancer itself [7,12]. Meanwhile, aromatase activity (AA) in EC tissue has been evaluated much rarely than in breast cancer, and to our knowledge practically no comparative studies have been performed relating levels of aromatase activity and CYP19 mRNA within malignant endometrium [13]. The important feature of EC is its heterogeneity. Several lines of evidence suggest that two different variants of EC can be distinguished [1,5,8,14]. One of these variants is traditionally called hormone-dependent or estrogen-dependent and is designated as type I. Another variant is usually considered hormone-independent or estrogen-independent and called otherwise as type II. Each of these types is
72
L. Berstein et al. / Journal of Steroid Biochemistry & Molecular Biology 95 (2005) 71–74
characterized by rather specific complex of pathological, genetic and clinical features of the tumor as well as by a set of typical characteristics of the host [5,8,14]. The principal aim of present study was to compare AA and CYP19 gene expression in tumor tissue in cases belonging to the types I or II of endometrial cancer, as well as to evaluate response of such patients and their neoplasms to the short-term pre-operational treatment with aromatase inhibitors.
2. Materials and methods 2.1. Patients Tumor samples from 80 patients with EC were evaluated. Age of the patients varied between 38 and 80, and 72 patients were postmenopausal. According to existing surgical classification, stage of the disease varied between FIGO IA-IIIC. Morphologically mostly (approximately in 90% of cases) endometrioid adenocarcinomas were presented in this material. To distinguish between patients with types I and II of EC mainly the following criteria were used: absence or presence of infertility/low pregnancy number, late menopause, obesity, hypertension, hyperlipidemia, uterine myoma, endometriosis, ovarian theca hypertrophy, well-, modestly- or low-differentiated carcinomas, superficial (less than 5 mm) or deep invasion into myometrium, involvement of regional lymph nodes. As proposed, patients who are infertile, obese, hypertensive, hyperlipidemic, have menopause timing ≥53 years, myoma or endometriosis and well-differentiated nodenegative adenocarcinoma with superficial invasion (or are characterized with more than 60% of these features) belong to the type I of disease [5,8]. As a result, it was considered that among studied group of patients 50 women had type I of EC and 30 women type II. In 23 of 80 aforementioned EC patients (15 with type I and 8 with type II of the disease), after the tissue specimens for aromatase activity determination were received during diagnostic biopsy, effects of 2 weeks treatment with aromatase inhibitors letrozole (2.5 mg/day, 10 pts) and exemestane (25 mg/day, 13 pts) were evaluated in neoadjuvant setting.
CYP19 gene expression was studied in 19 samples of malignant endometrium, in which AA has been measured as well. Total RNA was isolated with guanidinium thiocyanate – phenol – chloroform method and checked by spectrophotometry and electrophoresis. Reverse transcription was performed in standard way and its efficiency was verified with PCR with primers to GAPDH-gene. Expression of the coding site of aromatase mRNA was evaluated examining the region across exons II and III with primers described by Koos et al. [9]—sense: 5 -GAATATTGGAAGGATGCACAGACT3 , antisense: GGGTAAAGATCATTTCCAGCATGT-3 . The conditions of reaction were the following: 37 cycles, denaturation—30 s at 95◦ , annealing—30 s at 61◦ , synthesis—1.5 min at 72◦ . The products were separated in the polyacrylamide gel and visualized by ethidium bromide staining. The expected size of CYP19 product was 293 bp and as a positive control RNA from human placenta or breast cancer was used. Results were presented in a semiquantitative manner using arbitrary or conditional units: 1 (“−” negative signal), 2 (“±” weak positive signal) and 3 (“+” strong positive signal). In 12 primary patients (6 with type I of EC and another 6 with type II), samples of normal endometrium were collected simultaneously with cancer specimens with the aim to compare aromatase activity in the tumor with DNA damage rate in macroscopically normal endometrial tissue. DNA damage was evaluated on the basis of COMET assay in the modification suitable for cells isolated from solid tissues [17]. Effect of neoadjuvant treatment of 23 EC patients with aromatase inhibitors (see above, Section 2.1) has been determined. For that, clinical, sonographic, morphologic, cytologic and hormonal-metabolic parameters (blood estradiol, LH, FSH, glucose and cholesterol levels; tumor aromatase activity and steroid hormone receptors (ER and PR) content by methods used in our laboratory [4]) were evaluated before and after the course. Statistical analysis was performed by methods allowing for means, standard errors, χ2 -values and Spearman correlation on the basis of SigmaPlot program. The differences with p ≤ 0.05 were considered as significant.
2.2. Experimental procedures
3. Results
Aromatase activity in tumor tissue was estimated by measuring of 3 H2 O release from 3 H-1--androstenedione (NEN, Boston, MA; specific activity, 25.4 Ci/mM) as described [15]. Briefly, the reaction mixture (which contained tumor homogenate, NADPH regeneration system and labeled androgenic precursor) was incubated for 2 h at 37 ◦ C. Then reaction was stopped by adding 5 vol of cold chloroform and 5% suspension of activated charcoal (Norit A) was added to the water phase. The fraction containing 3 H2 O was separated by centrifugation, and counting was performed with dioxane scintillator. Results were presented in fM/mg protein/h.
AA varied in the specimens of malignant endometrium between 0 and 28.4 fM/mg protein/h; in 43.1% of samples it was 5 fM/mg protein/h and less and in 16.3% higher than 15 fM/mg protein/h. Average value of AA in tumor specimens from patients belonging to the type II of EC was higher in comparison with type I cases; this difference reached the level of statistical significance in non-smoking patients (Table 1). Interestingly, prevalence of the specimens with AA value ≤5 fM/mg protein/h has been in patients with type II of EC significantly lower (27 ± 8%) than in type I cohort (54 ± 7%, p = 0.02).
L. Berstein et al. / Journal of Steroid Biochemistry & Molecular Biology 95 (2005) 71–74 Table 1 Aromatase activity in endometrial cancer tissue in patients belonging to types I and II of the disease Group
All patients Type I Type II p (I vs. II)
Aromatase activity (fM/mg protein/h) All samples
Samples from non-smoking patients
8.29 ± 0.77 (n = 80) 7.39 ± 0.95 (n = 50) 10.28 ± 1.19 (n = 30) 0.062
7.98 ± 0.84 (n = 70) 6.52 ± 1.03 (n = 44) 10.63 ± 1.30 (n = 26) 0.02
73
increase in FSH (+32.7% versus +12.2%) as well as LH concentration by the end of neoadjuvant treatment with aromatase inhibitors were more pronounced in type I patients, the decrease in tumor PR content (−65.5% versus −28.9%, p = 0.04) was more revealing in patients with type II of EC. Besides, a decrease in tumor AA in the course of treatment was noted predominantly in patients with lower body weight (BMI < 27.5), e.g. with a host feature more peculiar for patients with type II of the disease [5,8].
4. Discussion
Fig. 1. Distribution of the data on CYP19 expression in endometrial cancer tissue. Vertical line—number of samples. Horizontal line—groups of patients (all, n = 19; type I, n = 13, type II, n = 6). Results of the evaluation are included into two groups: negative expression (−), positive expression (weak + strong, or ± and +). Difference in the distribution of these results between types I and II specimens is significant, χ2 = 4.05 (p = 0.044).
In the whole group of studied samples, AA and CYP19 mRNA signal correlated with each other (Rs = +0.63, p < 0.05); this correlation was more pronounced in type I samples (Rs = +0.67) than in type II material (Rs = +0.33). At the same time, expression of CYP19 mRNA was not revealed in six cases of EC, and all of them belonged to the type I of disease (Fig. 1). No positive correlation was revealed between AA in EC tissue and percentage of cells with DNA damage in normal endometrium from the same patients. Nevertheless, the rate of DNA damage (percent of comets, comet’s tail average length, etc.) demonstrated inclination for the higher levels in intact endometrium collected from patients with type II of the disease (Table 2). Finally, while diminishing in the average value of endometrial M-echo signal (−30.2% versus −11.6%) and the
Although idea on the heterogeneity of EC and existence of its two variants (one of which is traditionally considered as hormone/estrogen dependent and another one as hormone/estrogen independent) became rather popular during last 40–50 years [1,5,8,14], nobody studied by present the aromatase activity and aromatase gene signal separately in tumor samples belonging to types I or II of the disease. Meanwhile, this approach might help to solve, at least partly, the problem whether hormonal imbalance associated with the risk of endometrial cancer and excessive estrogenic stimulation is typical only for so called type I of EC, or such notion is not completely correct. The principal data received in present study demonstrate that tumor specimens collected from endometrial cancer patients with type II of the disease are characterized with higher AA and CYP19 gene signaling (Table 1, Fig. 1). The rate of DNA damage in macroscopically normal endometrium of these patients shows tendency to the increase (Table 2). Besides, the same patients are ready to respond in a specific manner, i.e. by the decrease in tumor PR content, to the shortterm treatment with aromatase inhibitors. Additionally, in one of the previous reports the tendency to increased AA in lowdifferentiated tumors (which are more prevalent among type II patients) was revealed [18]. Of note also, that according to our recent observations the ratio of incidence of CYP19 A6A6 genotype (presenting a combination of more long and probably more biologically active allelic polymorphisms) to the frequency of A1A6 and A3A6 genotypes was higher in type II patients (1.0) than in females with type I of the disease (0.3) [3]. Thus, it may be suggested that more aggressive clinically and frequently receptor-negative type II of endometrial cancer [4,5,8,14,16] is associated with the signs of intratumoral
Table 2 Results of COMET assay with macroscopically normal endometrium in patients belonging to types I and II of endometrial cancer Group
Type I Type II p
Parameter Cells with comets (%)
Average length of tail in cells with comets, cond. units
Average length of tail in 100 counted cells, cond. units
16.7 ± 5.8 26.8 ± 15.1
14.50 ± 3.05 40.25 ± 14.68 0.09
5.74 ± 1.52 13.82 ± 7.15
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hyperproduction of estrogens both at the level of aromatase protein and gene expression. These data additionally support earlier made assumptions that: (1) EC of type II actually may be also estrogen dependent—with the dependence of neoplastic transformation of endometrium and tumor growth progression primarily of locally produced estrogens [4] and (2) two aforementioned variants of EC have to be considered as a clinical equivalents of the two types of hormonal carcinogenesis, e.g. promotional and genotoxic [2,10,11]. An association of intratumoral aromatase activity with menopausal status, clinical stage of disease, tumor differentiation and rate of invasion among cases belonging to the types I or II of EC deserves additional study. The search of possible ethnic differences in intratumoral aromatase activity and in the biologic response to aromatase inhibitors in EC can be of importance too. Acknowledgement The study was supported by grants from INTAS (01-434) and RFBR.
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