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Reproductive Toxicology
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Reproductive Toxicology Developmental Basis of Health and Disease
Inhibitory effects of mitotane on viability and secretory activity in mouse gonadotroph cell lines
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Erica Gentilin a,b, Daniela Molea, Teresa Gaglianoª, Mariella Minoia a, Maria Rosaria Ambrosioª, Ettore C. degli Ubertia,b, Maria Chiara Zatelli a, b,*
a Section of Endocrinology, Department of Medical Sciences, University of Ferrara, Italy
b Laboratorio in Rete del Tecnopolo Tecnologie delle Terapie Avanzate (LTTA), University of Ferrara, Italy
ARTICLE INFO
Article history: Received 27 August 2013 Received in revised form 9 January 2014 Accepted 20 January 2014 Available online 31 January 2014
Keywords: Hypogonadism Mitotane LH
FSH
Pituitary function Adrenocortical carcinoma
ABSTRACT
Mitotane represents the mainstay medical treatment for metastatic, inoperable or recurrent adreno- cortical carcinoma. Besides the well-known adverse events, mitotane therapy is associated also with endocrinological effects, including sexual and reproductive dysfunction. The majority of male patients undergoing adjuvant mitotane therapy show a picture of hypogonadism, characterized by low free testos- terone and high sex hormone binding globulin levels and unmodified LH concentrations. Since mitotane has been shown to have direct pituitary effects, we investigated whether mitotane may influence both cell viability and function of gonadotroph cells in the settings of two pituitary cell lines. We found that mitotane reduces cell viability, induces apoptosis, modifies cell cycle phase distribution and secretion of gonadotroph cells. The present data strengthen previous evidence showing a direct mitotane effect at pituitary level and represent a possible explanation of the lack of LH increase following decrease in free testosterone in patients undergoing adjuvant mitotane therapy.
@ 2014 Elsevier Inc. All rights reserved.
1. Introduction
Mitotane is an adrenolytic agent that represents the mainstay medical treatment for metastatic, inoperable or recurrent adreno- cortical carcinoma (ACC) [1,2]. ACC is characterized by a poor prognosis with an overall 5 year survival of less than 35% [3,4]. Since the response rates to systemic treatments are very poor, the majority of patients do not achieve complete remission. Although complete surgical resection offers the best chance of long-term sur- vival, up to 91% of ACC patients relapse despite radical surgery [2], indicating the need for medical therapy. In these settings, mitotane treatment may be chosen as first or second line therapy as well as adjuvant treatment. Serum mitotane levels of 14-20 mg/l have been shown to correlate with therapeutic response [5], but are associated with a variety of adverse events. The latter include biochemical and clinical endocrine and metabolic effects, such as hypercholesterolemia and hypertriglyceridemia, increase in hor- mone binding globulin levels (cortisol-, sex hormone-, thyroid-, and
vitamin D-binding globulins), gynecomastia, sexual dysfunction, slight hyperprolactinemia and central hypothyroidism [6,7].
The mechanism of action of mitotane is not well known and recent evidences focus on apoptotic effects at adrenocortical level. In both H295R and SW13 ACC cell lines, mitotane significantly reduces the activity of the respiratory chain complex IV and induces mitochondrial fragmentation leading to programmed cell death [8]. More recently, Poli et al. confirmed that mitotane at 30-50 p.M exerts its toxic effects on human ACC cell lines inducing the apop- totic process by mitochondria disruption [28]. In addition, mitotane treatment differentially modulates the expression of 117 genes in ACC cells as compared to controls, including genes involved in steroid hormone biosynthesis. However, the function of the major- ity of the dysregulated genes has not been explored in the settings of ACC [9]. Therefore, the mechanism of action of this drug at adreno- cortical level is still a challenging issue, and even more so in other tissues.
Male patients treated with adjuvant mitotane therapy show low free testosterone levels, and need replacement therapy [6]. Mitotane has been demonstrated to increase sex hormone binding globulin (SHBG) levels, but this phenomenon does not explain the clinical picture of hypogonadism. In addition, mitotane concentra- tion has been shown to inversely correlate with free testosterone levels; however, luteinizing hormone (LH) levels did not change accordingly [6], suggesting a defective LH response. The latter may possibly be due to a pituitary damage, in keeping with the
Abbreviation: ACC, adrenocortical carcinoma.
* Corresponding author at: Section of Endocrinology, Department of Medical Sci- ences, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy.
Tel .: +39 0532 239618; fax: +39 0532 236514.
E-mail addresses: ztlmch@unife.it, mczat@hotmail.com (M.C. Zatelli).
previously demonstrated toxic effect of mitotane on thyrotroph and corticotroph pituitary cells [7,10]. The lack of LH increase follow- ing decrease in free testosterone suggests that mitotane may affect both the testes and the pituitary, possibly through its estrogen like activity. The latter has been called upon to explain the different bio- chemical picture observed in female patients undergoing adjuvant mitotane therapy, who display unchanged LH, FSH and testosterone levels [6] and are capable of conceiving [11]. The available litera- ture does not report estrogen levels in female patients undergoing mitotane therapy, therefore it is unclear how mitotane affects the pituitary-gonadal axis in females in vivo.
Therefore, the aim of our study is to investigate whether mitotane directly effects gonadotroph cells in terms of apoptosis induction and hormone secretion, evaluating cell viability, cas- pase3/7 activity, cell cycle phase distribution, LH and FSH secretion in two in vitro models.
2. Materials and methods
2.1. Substances
Mitotane (o,p’-DDD) powder (Supelco, Bellefonte, PA) was resuspended in absolute ethanol. Mitotane was used in the in vitro experiments at concentrations of 10, 40, 60 and 80 pM that corre- spond to 3.2, 12.8, 19.2 and 26.6 mg/l, respectively. Serum mitotane levels of 14-20 mg/l that correlate with therapeutic response in ACC patients [5] correspond to 44-62 p.M. All reagents were purchased from Sigma (Milano, Italy) if not otherwise indicated.
2.2. Cell culture
The immortalized mouse gonadotroph cell lines &T3-1 and LBT2 cells were a generous gift of Prof. Pamela Mellon (Dept of Reproduc- tive Medicine and Neurosciences and the Center for Reproductive Science and Medicine, University of California, San Diego, CA). The &T3-1 cell line was immortalized at a relatively early stage of devel- opment and does not produce the gonadotrophin ß-subunits. On the contrary, LBT2 cells express the gonadotrophin @-subunit and the LH and the FSH B-subunits. The cells were grown in DMEM (Invitrogen, Life technologies, Carlsbad, CA) containing 10% char- coal treated fetal bovine serum (EuroClone, Siziano, Italy) and Antibiotic Antimycotic (EuroClone). The cells were maintained at 37 °℃ in 5% CO2 and 95% air.
2.3. Cell viability
Cell viability was assessed by the ATPlite assay (Perkin-Elmer, Waltham, Massachusetts, USA), as previously described [12,13]. Briefly, the cells were seeded at 9 x 103 cells/well in 96-well white plates and then exposed to test substances. After incubation time, substrate solution was added at room temperature directly to the cell culture plates. The plates were shaken at 700 rpm for 2 min, and then measured for luminescent output (Relative Light Units) by Vic- tor3 1420 Multilabel Counter (Perkin-Elmer). Results are expressed as mean value ± standard error percent cell viability vs. control cells in five independent experiments in six replicates.
2.4. Caspase 3/7 assay
Caspase activity was measured using Caspase-Glo 3/7 assay (Promega, Milano, Italy) as described previously [14,15]. Results are expressed as mean value ± standard error percent caspase 3/7 activity vs. control cells in five independent experiments in six replicates.
2.5. Cell cycle analysis
Cell cycle phase distribution analysis was performed by flow cytometry after DNA staining as previously reported [16]. Briefly, 3 x 106 cells were harvested and resuspended in GM solution. Cells were mixed with 70% ice-cold ethanol, added dropwise, and fixed at room temperature overnight. PBS was used for washing the cells that were then resuspended and incubated at room temperature in extraction buffer (CyStain PI Absolute T; Partec Italy Srl, Carate Bri- anza, Italy) for 15 min. Propidium iodide (PI) stain and ribonuclease were added to cell extracts, followed by incubation at room tem- perature overnight. DNA PI-associated fluorescence in all cells was measured by a CyFlow Space cytometer (Partec Italy Srl), capturing a total of 2 x 104 events for each treatment, followed by analysis with FlowMax software (Partec Italy Srl).
2.6. LH secretion
LH secretion was evaluated by the LH ELISA kit (Uscn Life Sci- ences, Wuhan, China) in the conditioned culture medium from LBT2 cells. Hormone assays were performed in duplicate after appropriate sample dilutions of medium. The minimum detectable concentration of mouse LH is <0.27 pIU/ml. The absorbance was then recorded using the Wallac 1420 Multilabel Counter (Perkin Elmer, Turku, Finland), as previously described [17]. The assay results were normalized by viable cell number, as deter- mined by the ATPlite assay. Results are expressed as the mean value ± standard error LH concentration in conditioned medium of six experiments in duplicate.
2.7. FSH secretion
FSH secretion was evaluated by the FSH ELISA kit (Uscn Life Sciences) in the conditioned culture medium from LBT2 cells. Hor- mone assays were performed in duplicate after appropriate sample dilutions of medium. The minimum detectable dose of mouse FSH is <0.45 ng/mL. The absorbance was then recorded using the Wallac 1420 Multilabel Counter (Perkin Elmer, Turku, Finland), as previously described [18]. The assay results were normalized by viable cell number, as determined by the ATPlite assay. Results are expressed as the mean value ± standard error FSH concentration in conditioned medium of six experiments in duplicate.
2.8. Statistical analysis
Statistical analysis was performed as previously described [19,20]. Results of experiments are expressed as the mean of at least three independent experiments. Student’s paired or unpaired t-test was used to evaluate individual differences between means. P<0.05 was considered significant in all tests.
3. Results
3.1. Mitotane reduces the viability of gonadotroph cell lines
In order to determine the effects of mitotane on gonadotroph cell viability, &T3-1 and LBT2 cell viability was assessed after 6, 24 and 48 h of treatment in the presence of increasing mitotane concentrations. As shown in Fig. 1a, mitotane in the concentrations range from 40 to 80 p.M significantly reduced &T3-1 cell viability at all incubation intervals tested: after 6 h the reduction in cell viabil- ity was from 11 to 19%, after 24 h from 8 to 20%, and after 48 h from 7 to 31% as compared to control untreated cells. Mitotane treatment also significantly reduced LBT2 cell viability (Fig. 1b). After 6 h and 24 h incubation, mitotane at 60 and 80 p.M significantly reduced cell viability (-14%; p<0.05 and -30%; p<0.01, respectively), while a
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longer incubation time (48 h) was necessary to observe significantly reduced cell viability in the presence of lower drug concentrations (40 µM).
3.2. Mitotane increases caspase 3/7activity in gonadotroph cell lines
To investigate whether mitotane reduces cell viability also by activating apoptosis, caspase 3/7 activity was measured in &T3-1 and LBT2 cells incubated for 6, 24 and 48h in the pres- ence of increasing mitotane concentrations. As shown in Fig. 2a, mitotane in the concentrations range from 40 to 80 p.M signif- icantly increased &T3-1 caspase 3/7 activity by 13-20% at all incubation times. In LBT2 cells, caspase 3/7 activation pattern was different: as shown in Fig. 2b, mitotane potently and significantly induced caspase 3/7 activity at 40-80 p.M after 6 (from 43 to 397%; p<0.01), 24 (from 84 to 553%; p <0.01), and 48 h (from 82 to 303%; p <0.01) as compared to control cells.
3.3. Mitotane modifies cell cycle phase distribution of gonadotroph cell lines
We explored cell cycle progression in viable &T3-1 and LBT2 cells after treatment with increasing mitotane concentrations for 48 h. As shown in Fig. 3a, &T3-1 cells treated with 40 and 60 p.M mitotane displayed an increase in G1 phase (+15 and +12% vs. con- trol cells, respectively; p <0.05) and a decrease in S phase (75 and 60% vs. control cells, respectively; p<0.05) and G2/M phase (-49 and -39% vs. control cells, respectively; p < 0.05). The differences in cell cycle phase distribution observed in cells treated with mitotane
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o.T3-1 caspase 3/7 activity (% vs. Ct)
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80 µM (+8% in G1; - 21% in S; - 31% in G2/M phase) did not reach statistical significance.
As shown in Fig. 3b, 40 µM mitotane did not significantly influ- ence LBT2 cell cycle phase distribution. On the contrary, cells treated with 60 and 80 p.M mitotane displayed an increase in G1 phase (+21 and +12% vs. control cells, respectively; p <0.05) and a decrease in S phase (-59 and -45% vs. control cells, respectively; p<0.05). G2/M phase fraction was reduced only by mitotane 60 µM (-51% vs. control cells, p < 0.05).
3.4. Mitotane reduces LH and FSH secretion by gonadotroph cell line
To determine the effects of mitotane on hormone secretion, LH and FSH levels were assessed in the conditioned medium from LBT2 cells treated for 6, 24 and 48 h with increasing mitotane concen- trations. As shown in Fig. 4a, after 6 and 24h treatment mitotane significantly reduced LH secretion at 80 p.M (-19% and -20%, respectively; p<0.01). After 48 h, mitotane significantly reduced LH secretion at concentrations ≥10 p.M (from 14 to 58%; p < 0.01).
As shown in Fig. 4b, after 6 and 24 h treatment mitotane signifi- cantly reduced FSH secretion at 80 p.M as compared to control cells (-14% and -19%, respectively; p<0.01). After 48 h, mitotane sig- nificantly reduced FSH secretion at concentrations ≥10 p.M (from 12 to 47%; p<0.05 and p<0.01, respectively).
4. Discussion
Mitotane is frequently used in ACC therapy and it is often reported as a toxic drug with a narrow therapeutic window. The
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identification of the functional aftermath of mitotane treatment in ACC patients is crucial to improve the drug management. Despite most of unwanted effects associated with mitotane therapy are well known, hypogonadism is not fully elucidated. It has been shown that male patients treated with adjuvant mitotane therapy need replacement therapy due to low free testosterone levels that inversely correlate with plasma mitotane concentrations. However, LH levels do not change accordingly [6]. The lack of an increase in LH levels, as expected in the settings of primary hypogonadism due to a direct inhibition of testosterone secretion by the testes, can- not be explained by the induction in SHBG synthesis [21] and by the reduction of 5x-reductase activity [22] observed in patients under- going adjuvant mitotane therapy. On the contrary, the absence of testosterone negative feedback on LH secretion suggests that mitotane may have a central effect. This study provides evidence that mitotane reduces both cell viability and LH/FSH secretion in gonadotroph cells in vitro, strengthening the hypothesis that mitotane may have direct inhibitory effects at pituitary level. These findings may account for the clinical picture suggestive of hypog- onadism observed in male patients exposed to adjuvant mitotane treatment.
Since immortalized human pituitary cell lines are not avail- able, we employed two mouse gonadotroph cell lines: the &T3-1 cells, that do not produce gonadotrophin ß-subunits, and the LBT2 cells, that express the gonadotrophin & subunit and the LH as well
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as the FSH ß subunits. We demonstrated that mitotane rapidly reduces &T3-1 cell viability at the therapeutic concentration of 40 p.M starting from 6 h of exposure. Mitotane also influenced LBT2 cell viability although a 48 h exposure was needed to reduce this parameter at the therapeutic concentration of 40 p.M. Our data indi- cate that mitotane, at therapeutic concentrations, reduces mature gonadotroph cell viability only after a prolonged exposure. Despite LBT2 cells required a longer exposure time to mitotane than &T3- 1 cells to show a reduction in cell viability, the former cell line appeared to be more sensitive to mitotane treatment compared to the latter. The dissimilarity in the response to mitotane may mir- ror the different stage of development of these cell lines. However, the effects of mitotane on cell viability are in agreement with pre- vious reports indicating that mitotane levels equal to, or greater than the therapeutic concentrations reduce cell viability in human functioning [23-25] and nonfunctioning ACC cell lines [26], pitu- itary thyrotroph [7] and adrenocorticotroph cell lines [10]. Our data demonstrate that pituitary cell viability reduction is mirrored by apoptosis activation in keeping with previous reports [7,10,27,28]. We observed that, after 6 and 24h, mitotane 40 p.M significantly increased caspase activity in LBT2 cells, but did not affect cell via- bility, suggesting that short exposure times do not influence the viability of mature gonadotroph cells, but commit them to apopto- sis. The significant reduction in LBT2 cell viability after treatment with 80 p.M compared to 60 p.M mitotane is not mirrored by a fur- ther increase in apoptosis, suggesting that cell death observed with greater mitotane concentrations might not be referred only to an apoptotic mechanism.
Our results demonstrate that mitotane induces an accumu- lation in G1 phase, reaching the greatest effect at therapeutic
concentrations in both cell lines. A previous report showed that mitotane treatment reduces cell viability by an alteration of cell cycle in many cell types such as ACC, glioblastoma, medulloblas- toma, ovarian cancer and breast cancer cell lines [24]. Our data indicate that gonadotroph cells that survive mitotane treatment display a derangement in cell cycle regulation that may eventually lead to a functional alteration. Indeed, mitotane reduced LH and FSH secretion by viable LBT2 cells after a 48 h exposure at concentra- tions below the lower limit of the therapeutic window. Our results are in line with previous reports, showing that mitotane reduces the secretory activity of H295R adrenocortical cell line [24,26], T&T1 thyrotroph cell line [7] and AtT20/D16v-F2 adrenocorticotroph cell line [10].
We point out that mitotane inhibits cell survival and function of many pituitary cytotypes underlining a generalized, but specific, pituitary toxic effect [7,10]. Since metabolic activation is needed for mitotane action [29], the sensitivity of pituitary cells to mitotane treatment may suggest an increased ability of these cells to metab- olize and activate the drug.
It is well known that mitotane has the capability to bind pro- teins, phospholipids and DNA in vitro [30], suggesting that mitotane may interact with an essential molecular target for pituitary cell function.
Pinpointing which tissues are the target of mitotane effects will expedite the understanding of its mechanism of action. This study gives evidences for mitotane toxic effects on gonadotroph cells suggesting that this drug acts on the gonadal axis at differ- ent levels. We demonstrated that mitotane reduces cell viability and induces caspase 3/7 activity, modifies the cell cycle phase distribution and LH/FSH secretion of viable cells. These data are in line with mitotane biological effects shown in adreno- cortical cells and in thyrotroph and corticotroph cells, where mitotane molecular targets have not been identified, yet. The latter is a limitation of the study design and will be addressed by further investigation, on the basis of the findings provided also by our data. Our results indeed point to a similar mecha- nism of action of mitotane at both pituitary and adrenocortical level.
However, the molecular mechanism by which mitotane impairs pituitary function needs further investigation.
5. Conclusions
Our results demonstrate that gonadotroph cells are sensitive to mitotane treatment. Not only mitotane reduces cell viabil- ity and induces caspase 3/7 activity, but also modifies the cell cycle phase distribution and LH/FSH secretion of viable cells. Overall, the present data strengthen previous evidence indicat- ing a direct mitotane effect at pituitary level and represent a possible explanation of the absence of testosterone negative feed- back on LH secretion in patients undergoing adjuvant mitotane therapy. However, these results do not exclude other periph- eral effects of the drug. Mitotane affects gonadotroph function at drug concentrations that represent the therapeutic window in clinical settings, highlighting the clinical relevance of these data.
Ethical approval
The authors declare that the experiments comply with the cur- rent laws of the country in which they were performed.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Transparency document
The Transparency document associated with this article can be found in the online version.
Acknowledgments
This work was supported by grants from the Italian Min- istry of Education, Research and University (FIRB RBAP11884M, RBAP1153LS, 2010TYCL9B_002), Fondazione Cassa di Risparmio di Ferrara, and Associazione Italiana per la Ricerca sul Cancro (AIRC) in collaboration with Laboratorio in rete del Tecnopolo “Tecnologie delle terapie avanzate” (LTTA) of the University of Ferrara. All fund- ing sources had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report and in the decision to submit the article for publication.
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