ORIGINAL STUDY
Fimbrial Cells Exposure to Catalytic Iron Mimics Carcinogenic Changes Debora Lattuada, PhD,* Francesca Uberti, PhD,* Barbara Colciaghi, BA,* Vera Morsanuto, MS,* Elena Maldi, MD,Þ Diletta Francesca Squarzanti, MS,þ Claudio Molinari, MD, PhD,þ Renzo Boldorini, MD,Þ Alessandro Bulfoni, MD,* Paola Colombo, MD,* and Giorgio Bolis, MD*
Objective: Recent evidence strongly suggests that the fallopian tube is a site of origin of ovarian cancer. Although histological data show iron deposition in the fallopian tubes, its role remains unclear. To establish whether catalytic iron has a possible role in ovarian carcinogenesis, we isolated human fimbrial secretory epithelial cells (FSECs). Methods: Fimbrial secretory epithelial cells, isolated from women undergoing isteroannessiectomy, were treated with different doses of catalytic iron (0.05Y100 mM) to study cell viability; NO production; p53, Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc protein expressions through Western blot analysis; and immunocytochemistry or immunofluorescence. Results: In FSECs treated with catalytic iron for up to 6 days, we observed an increase in cell viability, NO production, and p53, pan-Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc activations (P G 0.05) in a dose-dependent and time-dependent manner. These same results were also observed in FSECs maintained for respectively 2 and 4 weeks in the absence of catalytic iron after 6 days of stimulation. Conclusions: Our model aimed at studying the main nongenetic risk factor for ovarian cancer, providing an alternative interpretation for the role of menstruation in increasing risk of this pathology. This in vitro model mimics several features of the precursor lesions and opens new scenarios for further investigations regarding the correlation between damages produced by repeated retrograde menstruation carcinogenic stimuli. Key Words: Fimbrial secretory epithelial cells, Catalytic iron, Epithelial ovarian cancer Received October 10, 2014, and in revised form November 24, 2014. Accepted for publication December 08, 2014. (Int J Gynecol Cancer 2015;25: 00Y00)
cancer affects about 204,000 women worldwide O varian every year with an incidence of about 190,000 new
can be defined by biological and genetic analysis; these are as follows: high-grade serous carcinoma, low-grade serous carcinoma, endometrioid carcinoma, mucinous carcinoma, and clear cell carcinoma.1 Three typesVhigh-grade serous carcinoma, endometrioid carcinoma, mucinous carcinomaVare
*Department of Obstetrics and Gynecology, Fondazione IRCCS Ca Granda, Ospedale Maggiore Policlinico, Milan; †Unit of Pathology, Department of Health Sciences, and ‡Physiology Laboratory, Department of Translational Medicine, University of Eastern Piedmont ‘‘Amedeo Avogadro,’’ Novara, Italy.
Address correspondence and reprint requests to Debora Lattuada, PhD, Department of Obstetrics and Gynecology, Fondazione IRCCS Ca` Granda, Ospedale Maggiore Policlinico, Via Manfredo Fanti, 6, 20122 Milan, Italy. E-mail:
[email protected]. Drs Debora Lattuada and Francesca Uberti contributed equally to the study and are cofirst authors. The authors declare no conflicts of interest. Supplemental digital content is available for this article. Direct URL citation appears in the printed text and is provided in the HTML and PDF versions of this article on the journal’s Web site (www.ijgc.net).
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cases diagnosed annually worldwide.4,5 Mortality rates is high, especially in those country where effective methods for early detection are not available. Five main ovarian cancer subtypes
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characterized by morphological resemblance to 3 healthy mucosal tissues, namely, fallopian tube epithelium, endometrial glands, and endocervical epithelium, all of which show mu¨llerian differentiation.4,6,7 Recent studies have hypothesized fallopian tubes, in particular, secretory epithelial cells present in the distal site of tubes, to be the site where most serous ovarian neoplasms develop.6,8Y11 This hypothesis is supported by many evidences, including studies on the link between follicular fluid and fallopian tube epithelial cells12 and on the prevalence of occult fallopian tubes cancer in women with germ line BRCA gene mutation.13,14 The fallopian tube epithelium is composed of 2 specialized cell types, secretory and ciliated cells. In the presence of serous ovarian cancer, Piek et al15 observed a shift toward the secretory phenotype, with complete loss of ciliated cells and the acquisition of proliferative capacity. Successive studies have also demonstrated a high incidence of serous tubal intraepithelial carcinomas in the fallopian tubes, but none in the ovary, indicating that the fimbria is the preferred site of serous carcinogenesis in BRCA-positive women.16Y19 The fimbria is located next to ovarian surface epithelium and thus is exposed to the same inflammatory agents that affect salpinx during the retrograde menstruation. In the ‘‘incessant menstruation’’ hypothesis, Vercellini et al8 suggest that the fimbriae, being chronically wetted by peritoneal fluid and by refluxed blood products contained in the pouch of Douglas, are exposed to the action of free radicals and other metabolites that could promote carcinogenesis. This condition would generate chronic inflammation, rapid cell division, and consequent increase in the potential DNA errors in loci encoding tumor suppressor gene in the fallopian tubes. Blood from ovulation and retrograde menstruation could represent the possible pathogenetic pathway leading to the development of different cancer histotypes in fertile women’s fallopian tubes. These findings could explain why fimbria is a site of most serous malignancies.8,20Y22 Cellular iron homeostasis is regulated by cytosolic iron regulatory proteins that bound structural elements named iron-response elements present in the messenger RNA of the major proteins involved, such as transferrin receptor and ferritin.23 In a chronic condition, high concentrations of heme and free iron (catalytic iron) derived from lysis of red blood cells by macrophages are able to exceed the capacity of ferritin to sequester iron leading to oxidative injury. This mechanism generates oxygen-free radicals (ROS) in the cells. This condition results in numerous carcinogenic DNA mutations or loss, genetic instability, overexpression of specific oncogenes, and down-regulation of tumor suppressor genes.8,24Y27 A recent study confirming these findings has shown that ovarian endometriotic cysts are rich in catalytic iron, leading to increased oxidative DNA damage of its epithelia.28 Carcinogenicity of iron is demonstrated in many experimental studies; for example, in mice, the exposition to iron oxide dust caused pulmonary tumors; the injection of iron dextran induced soft tissue sarcoma; and the intraperitoneal injection of iron chelates produced renal cell carcinoma.21,28,29 Finally, in a model of intestinal epithelia using Caco-2 cells, iron accumulation was demonstrated to correlate with oxidative damage to proteins and DNA, resulting in loss of cell viability. These findings confirm the importance of iron in the development of colon cancer.23 To evaluate the possible role of catalytic iron in the pathogenesis of ovarian
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carcinoma, in vitro primary fimbrial secretory epithelial cells (FSECs) were analyzed after exposition to Fe3+.
MATERIALS AND METHODS Samples Fresh fallopian tube fimbriae were obtained from women who attended the endoscopic surgical service of the II Department of Obstetrics and Gynecology of the Fondazione IRCCS Ca` Granda, Ospedale Maggiore Policlinico (Milan, Italy) to undergo isteroannessiectomy for ovarian cancer and benign pathology without comorbidity. All subjects were in premenopause and had not received any type of hormonal or drug therapy for at least 3 months. Samples were obtained under written consent from 33 women after informing them that donated fimbriae would be used for research purposes, and they gave a written consent. Approval for this study was granted by the local human institutional investigation committee.
Experimental Protocol in Fe3+-Treated FSECs Experiments were performed using physiological concentrations of catalytic iron on FSEC ranging from low doses as previously used in another study on colon carcinoma cells study to high doses mirroring the usual contents of endometriotic cysts.23,30 Fimbrial secretory epithelial cells were incubated for 2 hours in Dulbecco’s Modified Eagle’s Medium without red phenol supplemented with 1% penicillin/streptomycin, 2 mM L-glutamine, and 0.5% fetal bovine serum before and during the treatment with different physiological concentrations of Fe3+ (iron III nitrate nonahydrate; Sigma, Milan, Italy) (low doses: 0.05, 0.075, and 0.1 mM; high doses: 50, 75, and 100 mM, respectively). In the first set of experiments, the stimulations were maintained for up to 144 hours and were stopped and analyzed every 24 hours.23,31 In the second set of experiments, cells were stimulated uninterruptedly for 144 hours, and then maintained in complete medium for 2 and 4 weeks. We performed many tests on cell preparation, cell viability, nitric oxide production, Western blotting, immunocytochemistry, immunohystochemistry, and immunofluorescence (see Data, Supplemental Digital Content 1, http://links.lww.com/IGC/A264). In addition, statistical analysis was reported in Supplemental Digital Content 1, http://links.lww.com/IGC/A264.
RESULTS Detection of Morphology, Cell Viability, and Oxidative Stress in FSEC Treated With Different Iron Concentrations Because, as reported in literature, the fimbriae of the fallopian tubes are reported to be the primary source of pelvic (ovarian, tubal, or peritoneal) serous carcinomas and intracellular reactive iron is a possible cause of different carcinomas (such as colon), we tried to determine whether FSECs are susceptible to carcinogenic changes after treatment with catalytic iron. First, the effects of different physiological concentrations of catalytic iron (low doses: 0.05, 0.075, and 0.1 mM; high doses: 50, 75, and 100 mM) on FSECs were examined every * 2015 IGCS and ESGO
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FIGURE 1. Catalytic iron effects on cell viability and oxidative stress in FSEC. A, Cell vitality (MTT test) and (B) NO production (Griess assay) in FSEC treated with both low and high doses of Fe3+ during 144 hours checking every 24 hours are reported. C, Cell viability (MTT test) and (D) NO production (Griess assay) in FSEC treated with both low and high doses of Fe3+ for 6 days plus 2 free weeks. Data are representative of 8 technical replicates performed on 8 FSECs and expressed as mean (SD) (%) in respect to control cells. *P G 0.05 versus control sample. * 2015 IGCS and ESGO
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FIGURE 2. Densitometric analysis of p53, pan-Ras, Ki67, c-Myc, and PAX8 in FSEC. A, Cells maintained in the presence of both low and high doses of catalytic iron for 144 hours and checked every 24 hours are reported. B, FSEC treated for 6 days in the presence of both low and high doses of iron plus 2 free weeks are shown. C, Cells stimulated with high doses of catalytic iron for 6 days plus 4 free weeks are reported. Protein extracts were analyzed by immunoblotting with specific antibodies against the indicated proteins. Densitometry of proteins was obtained, normalizing the expression of antibodies to A-actin detection indicated as ratio. Data were expressed as mean (SD) (indicated by error bars) of 6 technical replicates performed on 6 FSECs and normalized to control. *P G 0.05 versus control. 24 hours for a total period of 144 hours. Morphological cell integrity of the samples is reported in Figures, Supplemental Digital Content 2, http://links.lww.com/IGC/A265; typical epithelial features were maintained. The cells acquired an elongated phenotype after 48 hours treatment with low doses and immediately in the presence of high doses. In addition, evident damages were observed in the presence of high doses of catalytic iron between 120 and 144 hours, in particular, cell alteration, with evident loss of cell border, which is a clear indication of plasma membrane oxidative damage (see Figures, Supplemental Digital Content 2, http://links.lww.com/IGC/A265, which demonstrates cell morphology). Cell viability was assessed by MTT test in a subset of FSEC (Fig. 1A). In the presence of both low and high doses of catalytic iron, the percentage
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of proliferating cells was significantly reduced from 48 to 72 hours. After 96 hours, we observed significant increase in viability (P G 0.05) that increased even further, amplified during the last 2 days in comparison to control cells. These effects resulted more evident, in a dose-dependent manner, in the presence of high doses of catalytic iron (P G 0.05). In addition, in these samples, the reduction on cell viability was statistically significant after 24 hours (Fig. 1A). Finally, because catalytic iron is a well-known initiator of oxidative stress, the relative extent of oxidative stress in the iron-loaded FSEC was determined through the analysis of NO released in culture supernatants. As shown in Figure 1B, in the presence of all doses of catalytic iron, the NO production was statistically increased (P G 0.05) compared with control during * 2015 IGCS and ESGO
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all 6 days of treatment and particularly in the presence of high doses of catalytic iron after 72 hours.
p53, pan-Ras, ERK/MAPK, PI3K/Akt, Ki67, and c-Myc Analysis in Cells and Tissues Expressions of p53, pan-Ras, Ki67, and c-Myc were investigated in FSECs treated with low and high doses catalytic
iron by Western blotting and immunofluorescence or immunocytochemistry for 6 consecutive days (Figs. 2A, 3Y4). As reported in Figures 2A and 3, in the presence of low doses of catalytic iron, p53, pan-Ras, Ki67, and c-Myc activations were observed in a dose-dependent and time-dependent manner, and these effects were more evident in the presence of high doses of catalytic iron (P G 0.05). The highest expression
FIGURE 3. p53, pan-Ras, Ki67, and c-Myc Western blot analysis in FSEC. Cells were treated for up to 144 hours with different concentrations of catalytic iron (low doses: 0.05, 0.075, and 0.1 mM; high doses: 50, 75, and 100 mM) checking every 24 hours. Protein extracts were analyzed by immunoblotting with specific antibodies against the indicated proteins. Data represent an example of 6 technical replicates performed on 6 FSECs. * 2015 IGCS and ESGO
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FIGURE 4. PAX8, pan-Ras, Ki67, and c-Myc staining after treatment with catalytic iron. FSECs that grew on chamber slide were stimulated with both low and high doses of catalytic iron for 144 hours. On the left, the score of positive cells for each protein selected is reported. The ratio reported a mean (SD) (%) of positive cells counted in 12 different areas. Data reported are mean of 6 technical replicates performed on 6 FSECs. *P G 0.05 versus control cells. On the right, representative pictures, obtained through microscopy at original magnification of 40, are shown. The scale reported in the first column is to be considered valid for the same antibody.
of p53, pan-Ras, Ki67, and c-Myc was shown at 144 hours (PG 0.05), both for 0.1 mM and 100 mM (65%Y190%, 19%Y76%, 56%Y990%, 20%Y29%, respectively), compared with control values. In addition, we observed ERK1/2 and Akt phosphorylations in a dose-dependent and time-dependent manner, and these effects were more evident in the presence of high doses of catalytic iron, confirming pan-Ras activation (data not shown). Similar data were also observed during immunofluorescence (p53) and immunocytochemistry experiments (pan-Ras, Ki67, c-Myc) performed in FSECs (Fig. 4). The results revealed the presence of these proteins activated in a different dose-dependent and time-dependent manner; in particular, these effects were more evident in samples treated with high doses of catalytic iron. After 6 days, the cells mimicked carcinogenic changes, without loss of the specific marker for secretory epithelial cells PAX8 (Figs. 2A, 3Y4), independently from the iron concentrations used. Furthermore, the presence of the proteins analyzed before was also investigated in healthy fimbrial and serous ovarian carcinomas G3 tissues by
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immunohistochemistry (see Figures, Supplemental Digital Content 3, http://links.lww.com/IGC/A266, which demonstrates the different expression between healthy fimbrial and serous ovarian carcinomas G3 tissues). Healthy fimbrial tissues showed a negative staining for p53, pan-Ras, and c-Myc and expressed cytoplasmic positivity in epithelial surface for PAX8, as already stated in the literature, and focal positivity for Ki67 (see Figures, Supplemental Digital Content 3A, http://links.lww.com/IGC/A266). On the contrary, in tumoral tissues, a focal positivity in neoplastic cells for p53 and pan-Ras, an intense and diffuse nuclear staining in neoplastic cells for Ki67, and a cytoplasmic positivity for c-Myc were observed (see Figures, Supplemental Digital Content 3B, http://links.lww.com/IGC/A266).
Cell Viability and Oxidative Stress Up to 6 Days of Treatment Plus 2 Free Weeks A second set of experiments was performed to verify whether all these effects were stabilized in absence of * 2015 IGCS and ESGO
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catalytic iron. For this purpose, cells were maintained only in complete medium for 2 weeks after treatment with Fe3+. In the presence of low doses of catalytic iron, cell viability measured by MTT test showed an increase (Fig. 1C); this effect was immediately observed in the samples treated for 24 hours and was amplified in a time-dependent manner during all the period of treatment. In addition, cell viability was inversely proportional to doses of catalytic iron used. In the presence of high doses of catalytic iron, cell viability was statistically increased; these effects were observed after 48 hours after treatments with 50 to 75 mM of catalytic iron and 72 hours after treatment with 100 mM. High doses effects after 72 hours were also amplified in a time-dependent manner (P G 0.05) and were
inversely proportional to the concentration used. Furthermore, the nitric oxide production observed in the samples treated with low doses of catalytic iron (Fig. 1D) revealed a minimal variation during all the stimulation period. On the contrary, high doses effects showed a significant decrease in nitric oxide production in a dose-dependent and time-dependent manner.
p53, pan-Ras, Ki67, and c-Myc Analysis in FSEC After 6 Days of Treatment Plus 2 and 4 Free Weeks At the end of the 6 days of treatment, the cultures were maintained for 2 and 4 weeks without catalytic iron to analyze
FIGURE 5. p53, pan-Ras, Ki67, and c-Myc Western blot analysis in FSEC treated with catalytic iron. A, FSEC treated for 6 days in the presence of both low and high doses of iron plus 2 free weeks are shown. B, Cells stimulated with high doses of catalytic iron for 6 days plus 4 free weeks are reported. Protein extracts were analyzed by immunoblotting with specific antibodies against the indicated proteins. Data represent an example of 6 technical replicates performed on 6 FSECs. * 2015 IGCS and ESGO
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whether protein expression persisted. As reported previously, p53, pan-Ras, Ki67, and c-Myc showed a significant increase both after 2 (Figs. 2B, 5A) and 4 free weeks (Figs. 2C, 5B) (P G 0.05). In particular, after 2 free weeks, the effects were dose-dependent and more evident in the presence of high doses of catalytic iron (Figs. 2B, 5A). The highest expression of p53, pan-Ras, Ki67, and c-Myc was shown at 100 mM (P G 0.05), both for 2 and 4 free weeks from catalytic iron (186%Y213%, 122%Y140%, 17%Y20%, 80%Y97%, respectively), compared with control values. These data were also confirmed by immunofluorescence (p53) and immunocytochemistry experiments (pan-Ras, Ki67, c-Myc) (Fig. 6). Finally,
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we verified PAX8 positivity indicating that primary culture did not phenotypically change over time.
DISCUSSION The pathogenesis of epithelial ovarian cancer has remained unclear for many years, largely because of the uncertainty regarding the cell of origin of this disease.32,33 Recent data show the fallopian tube secretory epithelial cells to be a site of origin of most serous ovarian cancer.20 Many evidences support this hypothesis; these are as follows; ~80%
FIGURE 6. PAX8, pan-Ras, Ki67, and c-Myc staining in FSEC treated for 6 days in the presence of iron treatment plus 2 and 4 free weeks. A, The panel represents the effects of 6 days in the presence of high doses of catalytic iron plus 2 free weeks and (B) plus 4 free weeks. On the left, the score of positive cells for each protein selected is reported. The ratio reports a mean (SD) (%) of positive cells counted in 12 different areas. Data reported are means of 4 technical replicates performed on 4 FSECs. *P G 0.05 versus control cells. On the right, representative pictures, obtained through microscopy at original magnification of 40, are shown. The scale reported in the first column is to be considered valid for the same antibody.
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of BRCA-positive women undergoing prophylactic salpingooophorectomy had an early cancer lesion in the fimbria portion of the fallopian tubes, termed as serous tubal intraepithelial carcinoma; 50% of women with stage III/IV serous cancer had a neoplastic lesion in the fallopian tubes; the early presence of TP53 mutations in neoplasia of the fallopian tubes determined the development of serous ovarian cancer11,34; and precursor lesion (termed as p53 signature) was present in benign fallopian tube secretory epithelial cells leading to DNA damage and p53 mutations.6,34,35 In addition, a model to study follicular fluid effects on fimbria was reported by Bahar-Shany et al,12 proving the molecular basis and the relative site of ovarian cancer pathogenesis.36,37 These observations suggest that the carcinogenic pathway originated in the secretory epithelial cells of fimbria, but the molecular mechanism involved is still to be found. Recent studies hypothesize a common oncogenic pathway and similar pathogenetic mechanisms, such as iron-induced oxidative stress derived from retrograde menstruation7 in epithelial ovarian cancer. Fimbria in the pouch of Douglas of peritoneal cavity is exposed to catalytic iron generated from hemolysis of erythrocytes by pelvic macrophage during retrograde menstruation, a common physiologic event in all menstruating women (defined as incessant menstruation).8 In addition, Seidman37 reports the significant presence of mucosal iron in fallopian tubes in advanced-grade pelvic serous carcinoma, supporting the incessant menstruation hypothesis.38 Oral contraceptive and tubal ligation are well known to reduce the risk of more frequent histotype of ovarian cancer; the reason might be the reduction or absence of retrograde menstruation.8,39 Up on this theoretical scheme, we developed an experimental model in FSEC to explain the involvement of catalytic iron in carcinogenic changes. For this purpose, FSECs were treated with catalytic iron at the doses (both low and high) reported in literature.23,30 In the presence of catalytic iron, FSEC showed an increased cell viability in a dose-dependent and time-dependent manner. Our results were also similar to what Nu´n˜ez et al23 observed in Caco-2 cells treated with low doses of catalytic iron.18 In addition, we showed an oxidative damage in fimbrial cells, as indicated by the increased nitric oxide production in culture supernatants. Finally, the cells showed an elongated phenotype in the presence of catalytic iron in a dose-dependent manner, maintaining the specific marker for secretory epithelial cells PAX8.22,35,40 All these findings show that iron is able to induce oxidative damage without cytotoxic effects in all fimbrial cells tested. To clarify the mechanism activated by catalytic iron, we analyzed whether the principal biomarkers, such as p53, c-Myc, Ras, and Ki67,4,22,35,41 were expressed and activated after exposure to catalytic iron. The results indicated that the expression of these proteins after 6 days in all treatments correlated to an enhanced growth, suggesting a possible epithelial cell transformation. These data were also confirmed by ERK/MAPK and PI3K/Akt analysis, in which increased phosphorylations were observed. In addition, these results were also comparable to what we obtained in paraffinembedded serous ovarian carcinoma G3 tissues. Thus our findings indicate that p53, pan-Ras, Ki67, and c-Myc were able to mimic in FSEC the carcinogenic changes typical of serous ovarian cancer. After both 2 and 4 free weeks, these
activations were not reverted, indicating a stabilization of the signals. Summing up, we proposed an in vitro model of linkage between catalytic iron and FSEC; our study suggests an alternative interpretation for the role of menstruation in increasing the risk of epithelial ovarian cancer. In the future, this model could be used to perform further investigation about the relationship between repeated exposure to catalytic iron and accumulation of preneoplastic changes, and also to discover and develop early biomarkers, and to develop chemoprevention therapies or novel drugs to limit or repair oxidative damages. This innovative experimental model to study the tumorigenic process may greatly impact further understanding of the mechanisms at the basis of epithelial ovarian cancer.
ACKNOWLEDGMENTS The authors thank Ms Mariangela Fortunato for her precious help with the preparation of the article.
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