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DR HANNA KOMAROWSKA (Orcid ID : 0000-0001-9085-4330) DR MAREK RUCHALA (Orcid ID : 0000-0002-6296-7220)
Article type : 2 Original Article - Europe, excluding UK
Poznan, Poland Tel +48 618691333 Accepted Article Fax +48 618691682
Ghrelin as a potential molecular marker of adrenal carcinogenesis: in vivo and in vitro evidence.
Short title: Ghrelin expression in adrenal tumors.
Hanna Komarowska1, Marcin Ruciński2, Marianna Tyczewska2, Nadia Sawicka -Gutaj1, Marta Szyszka2, Aleksandra Hernik1, Anna Klimont1, Paulina Milecka2, Laura Migasiuk1, Mateusz Biczysko3, Ilona Idasiak-Piechocka4, Marek Karczewski5, Marek Ruchała1
1 Department of Endocrinology, Metabolism and Internal Medicine, Poznan University of Medical Science, Poznan, Poland;
2 Department of Histology and Embryology, Poznan University of Medical Science, Poznan, Poland;
3 Department of General, Endocrinological and Gastroenterological Surgery, Poznan University of Medical Science, Poznan, Poland;
4 Department of Nephrology, Transplantology and Internal Medicine, Poznan University of Medical Science, Poznan, Poland;
5 Department of General and Transplantation Surgery, Poznan University of Medical Science,
Correspondence: Hanna Komarowska, MD, PhD Department of Endocrinology, Metabolism and Internal Medicine, Poznan University of Medical Science, 49 Przybyszewskiego St., 60-355 Poznań, Poland. E-mail: hkomar@ump.edu.pl
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cen.13725
Conflict of interest: The author reports no conflicts of interest in this work.
This work was supported by the National Science Centre (Poland) allocated on the basis of the decision number DEC-2013/11/B/NZ4/04746.
Summary
Context: Adrenal tumors belong to one of the most prevalent neoplasms. It is a heterogeneous group with different etiology, clinical manifestation, and prognosis. Its histopathologic diagnosis is difficult and identification of differentiation markers for tumorigenesis is extremely valuable for diagnosis.
Design: To assess ghrelin expression and the relationship among ghrelin, IGF2, and the clinicopathological characteristics of adrenal tumors. To investigate the influence of ghrelin on ACC cell line proliferation.
Materials and methods: Expression of ghrelin and IGF2 in a total of 84 adrenal tissue samples (30 adenoma, 12 hyperplasia, 8 myelolipoma, 20 pheochromocytoma, 7 carcinoma, 7 unchanged adrenal glands) were estimated. Every operated patient from whom samples were obtained underwent clinicopathological analysis. All the parameters were compared among the groups examined and correlations between these were estimated.
H295R cell line was incubated with ghrelin to assess its effect on proliferation and migration rate.
Results: The highest ghrelin expression was observed in carcinoma samples and the lowest in the control group. Ghrelin expression was 21 times higher in carcinoma (p =. 017) and 2.4 times higher in adenoma (p =. 029) compared with controls. There were no statistically significant differences between myelolipoma (p =. 093) and pheochromocytoma (p =. 204) relative to the control. Ghrelin level was significantly higher in carcinoma compared to adenoma (p =. 049) samples. A positive correlation between ghrelin and IGF2 expression was observed only in myelolipoma (p =. 001).
Ghrelin at concentrations of 1x10 M and 1x10 8M significantly stimulated proliferation and migration rate in the H295R cell line.
Conclusion: Ghrelin appears to be an essential factor in driving adrenal tumors development.
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1. Introduction
Adrenal tumors are one of the most prevalent neoplasms comprising a heterogeneous group of malignancies with various clinical presentations and prognoses. The proper diagnosis of adrenal gland tumors is essential for subsequent management. Greater than 70% of tumors are discovered incidentally during radiologic examinations, and usually these lesions are benign, hormonally inactive, less than 3-4 cm in diameter, and do not require treatment. Other adrenal tumors may cause various symptoms depending on hormonal activity and malignancy. Aldosterone-producing tumors (APT), and equally frequent cortisol-producing tumors (CPT) are found in 2%-9% of all adrenal tumors, unlike androgen-producing tumors which are relatively rare. 1-3 Tumors arising from the adrenal medulla are termed pheochromocytomas, and approximately 10%-15% of these tumors are of hereditary origin.4 Other changes observed in the adrenals are myelolipomas that comprise about 8% of all adrenal tumors and are characterized by the predominance of mature adipocytes. Adrenal metastases mostly from kidney, lung, breast, and colorectal cancers are present in up to 20% of patients with known malignant epithelial tumors at autopsy. Finally, adrenal hyperplasia refers to growth of the adrenal glands, which can be classified according to inheritance or morphology.2
Adrenal cortical carcinoma (ACC) is a rare and highly aggressive cancer. The clinical manifestations of this neoplasm depend on the hormonal activity of the tumor. It has been found that 50%-80% of adult patients manifest symptoms of hypercortisolemia, 40%-60% androgenization, and extremely rarely hyperaldosteronism.5-7 In hormonally inactive ACC cases, symptoms are related to the presence of a tumor mass or distant metastases. The histopathological diagnosis of ACC is problematic. Most often, the diagnosis is based on Weiss criteria with Aubert’s modifications.8 However, diagnostic pitfalls are very common, and one out of ten tumors is misclassified.9
There are four clinical stages of ACC. Complete surgical resection is still the most important curative treatment for adrenal cancer and mitotane is the only approved drug for ACC. However, the response to therapy is unpredictable and efficacy of treatment is unsatisfactory. Since the prognosis for these patients is poor, emerging new drugs hold promise for ACC. Insulin-like growth factor 1 (IGF1) receptor inhibitors and multiple tyrosine kinase inhibitors directed at several intracellular and extracellular targets are of particular interest. As with mitotane, the efficacy of this treatment varies from patient to patient for unknown reasons.10
Knowledge of the molecular mechanisms involved in adrenocortical tumorigenesis is still insufficient. The most common molecular alterations in ACC are overexpression of insulin-like growth factor 2 (IGF2) and constitutive activation of the Wnt/b-catenin signaling pathway. Activation of the insulin- like growth factor 1 receptor (IGF1R) by IGF2 leads to cell proliferation by activation of the
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PI3K/AKT/mTOR cascade and the RAS-MAPK pathway.11 Many studies have reported different expression levels of IGF1 and IGF2 in ACC compared to adrenocortical adenomas (ACA).10,12 IGF2 expression is low or absent in the beginning of oncogenesis, which may suggest that other signaling pathways may also play a role in ACC tumorigenesis.13,14
Ghrelin is an endogenous ligand that stimulates the secretion of growth hormone.15 Ghrelin is predominantly produced and secreted by endocrine cells of the gastric mucosa, and it circulates in two forms: acylated and unacylated.16 Initially, unacylated ghrelin had been considered as the inactive form, but currently it is known that both forms exhibit biological activity. Ghrelin expression has also been demonstrated in a number of organs and tumors.17-18 Widely distributed, ghrelin regulates different functions in the body and influences energy homeostasis and GH release.19 Recent data indicate that ghrelin regulates a number of processes related to cancer progression and may be a critical factor in metastasis development.20
The development of ACC is probably caused by autocrine overexpression of different growth factors. It is known that overexpression of a single gene cannot act as the only driver of malignant adrenocortical tumorigenesis, and many accompanying factors may play a role in this process.
The aim of our study was to analyze the expression of ghrelin and IGF2 in a large group of adrenal tumors and healthy adrenals. Moreover, we investigated the relationship between ghrelin and IGF2 expressions and clinicopathological characteristics of adrenal gland tumors including their hormonal activity. To find a causal relationship between ghrelin overexpression and adrenal malignancy, we performed an in vitro study investigating the influence of ghrelin on adrenocortical cancer cell line proliferation (H295 R).
2. Material and Methods
A. Studied Tissues
From 77 patients qualified for adrenalectomy due to suspicion of ACC or excessive hormones secretion, adrenal gland tumor samples were obtained. All patients underwent physical examination, laboratory testing, and computer tomography scan before the operation. After surgical removal, sections of the pathologically changed adrenals (~0.5 cm3) were immediately immersed in RNALater followed by Tissue Storage Reagent (Sigma) and stored at -70℃ until RNA isolation was carried out. The results of histopathological examination and other diagnostic criteria were used for the subdivision of collected adrenal gland samples into the following groups: adenoma (N = 30), hyperplasia (N = 12), myelolipoma (N = 8), pheochromocytoma (N = 20), and carcinoma (N = 7). Unchanged adrenal gland samples obtained from kidney donors were used as a control group (N = 7). Clinicopathological characteristics of the study group are presented in Table 1. Table 2 provides characteristics of ACC patients.
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The Bioethical Commission approved the study (decision nr 255/15). Informed written consent was obtained from all adrenalectomized patients, as well as from kidney donors.
B. Laboratory Testing
Fasting serum levels of total ghrelin, cortisol, DHEA-s, ACTH, aldosterone, ARO, total cholesterol, triglycerides (TG), and glucose were evaluated. Aprotinin was added to the samples collected for ghrelin assessment. Serum from blood was obtained by centrifugation and was kept frozen at -70℃ until hormonal level determinations were carried out. Ghrelin was measured by radioimmunoassay using a kit from Phoenix Pharmaceuticals (Cat. No. RK-031-30). The kit contained 1251-labeled bioactive ghrelin as a tracer and rabbit polyclonal antibodies directed against the C-terminus of human ghrelin, which recognized both acylated and nonacylated forms of the peptide. Other hormonal assays were performed by electrochemiluminescence (ECL); glucose, total cholesterol, and triglycerides were measured via spectrophotometric methods with the use of Hitachi Cobas e601analyzer (Roche Diagnostics, Indianapolis, IN).
C. RNA Isolation
The applied methods were described earlier.21,22 Total RNA was extracted using TRI Reagent (Sigma) then purified on columns (RNeasy Mini Kit, Qiagen). The amount of total mRNA was determined by optical density at 260 nm and its purity was estimated by 260/280 nm absorption ratio (higher than 1.8) (NanoDrop spectrophotometer, Thermo Scientific).
D. Reverse Transcription
Reverse transcription was performed using Transcriptor High Fidelity Reverse Transcriptase enzyme blend for high fidelity two-step RT-PCR of RNA (Roche) with Oligo(dT) as primers at a temperature of 45℃ for 40 minutes (Thermocycler UNO II, Biometra, Göttingen, Germany). For a single reaction, 1 ug of total RNA was used. The RT was carried out in standard final volumes (20 uL). After RT, each cDNA-containing sample was diluted with 100 uL of RNase-free water.
E. qPCR
Quantitative PCR was performed by means of the Lightcycler 2.0 instrument (Roche) with the 4.1 software. Using the abovementioned primers, SYBR green detection system was applied as described earlier (33,34). Each 20 uL reaction mixture contained a 2 uL template of cDNA, 0.5 UM of specific primers and a previously determined optimum MgCl2 concentration (3.5 uM per reaction). The LightCycler FastStart DNA Master SYBR Green I mix (Roche) was used. The real-time PCR program included a 10-minute denaturation step to activate the Taq DNA Polymerase, followed by a three-step amplification program: denaturation at 95℃ for 10 s, annealing at 56°℃ for 5 s, and extension at 72℃ for 10 s. The specificity of the reaction products was checked by determination of
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melting points (0.1°C/s transition rate). All samples were amplified in triplicate, and the beta-2- microglobulin (B2M) gene was used as a reference to normalize data.
Primers were designed using the Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA). Primers were purchased from the Laboratory of DNA Sequencing and Oligonucleotide Synthesis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw. (Oligonucleotide sequences for sense (S) and antisense (A) primers for GHRL and IGF2, and sequence of the beta-2-microglobulin (B2M) gene, which served as reference are presented in appendices.)
F. Cell Proliferation Assay
The study used an electrical impedance based technique-the Real Time Cell Analyzer (RTCA, Roche Applied Science, GmbH, Penzberg, Germany). The system consists of an RTCA Analyzer, RTCA SP Station, and RTCA Software. The system measures real-time changes in electrical impedance across incorporated sensor electrode arrays placed on the bottom of 16-well chamber slide plates (E-plate 16). As described by the manufacturer, the main read-out of the RTCA is a dimensionless parameter named as “Cell Index.” The impedance measurements provides quantitative information about the biological status of the cells, including cell number, viability, and morphology. Studies were performed on the H295R human adrenocortical cell line originating from ATCC. Cells were cultured in H295R complete media containing DMEM/F12, supplemented with 2% Ultroser G and ITS (insulin transferrin selenium). The cells were seeded on a 16-well standard electronic plate (E-Plate® 16). At 24 h of culture, active (acylated) ghrelin was added (Sigma-Aldrich, St. Louis, Missouri ) in concentrations of 1x10 6 M, 1x10 8 M, and 1x10-10 M, and cells were incubated for at least 98 h. Cell index was recorded every 15 min and the exemplary chart plot is shown in Fig.3A. Subsequently, this cell index was normalized (normalized cell index) to the time of peptide addition. Each experiment was repeated at least three times.
G. Cell Migration Assay
Cell migration assay was also carried out for H295R cells using RTCA equipment; however, the electronic cell invasion and migration plates (CIM-Plate 16) were used. The CIM-Plates 16 system was composed of an upper and a lower chamber. The upper chamber had 16 wells, which are sealed with membrane containing gold electrode arrays on the bottom side of the membrane. The lower chamber serves as reservoir for media containing ghrelin at concentrations 1x10 6 M, 1x10 8 M, and 1x10-10 M (chemoattractant) as well as clear medium (control). The H295R cell line (10,000/well) was seeded in the upper chambers. The cells, passing through the pores of the membrane toward the chemoattractant (ghrelin), caused a change in the impedance that was recorded every 15 min for the subsequent 48 h (Fig.3B). Obtained cell index was normalized (normalized cell index) at the beginning of the experiment. Each experiment was repeated at least three times.
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H. Statistical Analysis
All statistical analyses were performed using R programming language and software environment complemented by ggplot2 library for graph drawing. In order to make total data distribution more symmetrical, normalized expression values were log transformed and subsequently divided in to control (healthy) and different types of adrenal tumor groups. Each of the groups was present on a boxplot, where median value, interquartile range, minimum and maximum values, and outliers are shown. Individual log-transformed relative expression values were superimposed on the appropriate boxplot and presented as dots. Kruskal-Wallis statistical test was used to determine whether the difference in ghrelin and IGF2 gene expression of the studied groups was statistically significant. If the Kruskal-Wallis test showed significance, a post-hoc nonparametric pairwise Wilcoxon rank sum statistical test was performed. The resulting p-values were adjusted for multiple testing using Benjamini-Hochberg correction. Chi-squared test was used to compare descriptive characteristics between groups. Corrected p-values equal or lower than .05 were considered as statistical significant.
Association between expression of ghrelin and IGF2 was tested for each of the individual experimental groups using Pearson’s product-moment correlation coefficient (PPMCC) test. Paired- log transformed IGF2 and ghrelin expression values were presented as scatter plots with a line of best fit. The 95% confidence interval level for predictions based on linear models was also shown.
3. Results
A. Laboratory testing
There was a statistically significant difference in age, sex, tumor diameter, ACTH, DHEAs, glucose, and total cholesterol among the studied groups. Results are presented in Table 1. There is no evidence that ghrelin concentration or expression depends on age or sex.23 So, it is unlikely this difference is relevant to our study.
We have observed a positive correlation between ghrelin expression and ghrelin serum level in the whole group (R = 0.45, p = . 03). Ghrelin expression was also associated with total cholesterol serum level in patients with adenomas (R = 0.73, p = . 04). We did not find any other association in the whole group or subgroups.
B. Expression of ghrelin mRNA
Expression of ghrelin mRNA, measured by qPCR, was detectable in all of the analyzed samples (Fig.1). The result of Kruskal-Wallis statistical test was statistically significant for both ghrelin (p = .015) and IGF2 (p = 1.3x106) mRNA levels between study groups and controls and that allows post- hoc pairwise Wilcoxon rank sum statistical test to be performed between each of analyzed groups.
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Ghrelin expression level was normalized to that of reference beta-2-microglobulin (B2M) gene. The lowest ghrelin expression level was observed in healthy adrenal gland samples. Normalized median expression level was equal to 7.17x10-5. Ghrelin expression was higher in tumor samples as compared to the control group. In adenoma samples, ghrelin expression was 2.4 times higher (median = 1.72x10-4) than in healthy adrenals, and the difference reached statistical significance (p = .029). The most significant ghrelin overexpression was observed in carcinoma (p = . 017) tissue samples, where ghrelin expression was 21 times higher than in the control group. Hyperplasia samples exhibited a sevenfold increase in ghrelin expression in comparison to healthy adrenals, but the difference was not statistically significant (p = . 139). We have not found any statistically significant differences in ghrelin expression between myelolipoma (p = . 093) and pheochromocytoma (p = . 204) samples relative to the control; however, statistically significant increase of ghrelin mRNA was found in carcinoma samples when compared to adenoma (p = . 049) samples.
In Fig. 2, the expression of IGF2 mRNA is shown. IGF2 expression was also detectable in all samples and, similar to ghrelin, the lowest expression of IGF2 mRNA was noted in the control group. IGF2 expression in other samples (adenoma, pheochromocytoma, carcinoma) was significantly higher. In adenoma samples, the expression level was 11 times higher as compared to the control adrenals (p = . 005). In myelolipoma, IGF2 expression level was 4.66 times higher than controls but without statistical significance (p = . 063). However, the highest level of IGF2 mRNA was noted in carcinoma (208 times higher) and pheochromocytoma (133 times higher) samples relative to the control group (p = . 004 and p = . 004, respectively). Moreover, expression of IGF2 mRNA in pheochromocytoma was also higher than that observed in adenoma, hyperplasia, and myelolipoma (Fig. 2) samples and was statistically significant. IGF2 expression in hyperplasia group was similar to the control group (p = .163).
Figure 3 presents the scatter plots for the results of each experimental group obtained using the PPMCC test. A strong positive correlation between IGF2 and ghrelin expressions was observed in the myelolipoma group (R = 0.92, p = . 001), whereas this was not found in control (R = 0.43, p = . 33), adenoma (R = 0.17, p = . 357), carcinoma (R=0.02, p = . 972), hyperplasia (R=0.36, p = . 254), and pheochromocytoma (R = 0.04, p = . 872) samples.
C. Proliferation and Migration Assay
In this study, we were interested in using the RTCA platform to monitor the effects of acylated ghrelin on proliferation and migration of the H295R human adrenocortical cell line. As documented by Fig. 4, ghrelin concentrations of 1x10 6 M and 1x10 8 M stimulated proliferation and migration in the H295R cell line, while lower ghrelin concentration (1x10-10 M) was ineffective.
4. Discussion
To date, we analyzed ghrelin mRNA expression level in the largest ever evaluated group of adrenal tumors. Overexpression of ghrelin was found in adrenocortical tumors, as shown by the significantly high levels in ACC. Previously, only two papers have investigated ghrelin expression in adrenal tumors, and their results were contradictory. Barzon et al. demonstrated higher ghrelin expression level in all types of benign adrenal tumors (n = 34) and unchanged or slightly decreased expression in adrenal cancers (n = 6) compared to the healthy glands (n = 14). A wide variability of ghrelin expression among all samples was emphasized by the authors. Interestingly, the expression of ghrelin in two androgen-secreting adenomas was different: very high level of ghrelin mRNA was detected in a sporadic case, while normal level was found in a sample arising from congenital adrenal hyperplasia due to 21-hydroxylase deficiency.24 Different results were published by Ueberberg et al., who examined 13 pheochromocytomas and 43 benign adrenal tumors.25 Similar ghrelin mRNA expressions were found in normal adrenal glands, CPT, and nonfunctional adenomas. Ghrelin expression levels decreased in APT and in pheochromocytomas.
Methods of obtaining control adrenals might partially explain differences in achieved results. We obtained control adrenal glands from kidney donors, similar to Barzon et al., whereas Ueberberg et al. extracted RNA from adrenals obtained during autopsy. It is well known that the transcriptome profile is quite unstable and might be dramatically changed during postmortem examination.26,27 The method of sample storage might be even more important. Our samples were immediately immersed in RNALater Tissue Storage Reagent while Barzon and Ueberberg froze samples in liquid nitrogen. The unexpected lowering of ghrelin mRNA expression reported by Barzon et al. might be related not only to a rapid ghrelin degradation, but also to clinical variability, potential hormonal activity of ACC, and treatments given before operation, which were not presented in discussed studies.
We have also noticed that among the cancer groups, the highest ghrelin expression was detected in case of large ACCs in the fourth clinical stage, which were associated with metastasis and very poor prognosis-with rapid disease progression, without disease- free survival, and with shortest overall survival. On the other hand, ghrelin expression is very low in patient at the same advanced stage (with metastasis to the second adrenal gland), with disease free survival of 60 months, and who are alive to this day. It is possible that ghrelin influences on course of ACC and is one of the factors that promotes cancer metastasis in ACC. Similar association of ghrelin expression and advanced stage or worsening of prognosis have been reported in several cancers in in vitro and in vivo studies, i.e. renal cancer and pancreatic adenocarcinoma.28,29 In contrast, in patients with breast cancer, ghrelin has been suggested to be a good prognostic factor. 30,31
Interestingly, we found an association between the expression of ghrelin in the altered adrenal gland and its serum concentration. It is rather unlikely that circulating ghrelin is secreted by pathologically changed adrenals because our unpublished data show that increased ghrelin serum level in patients with ACC is maintained even after adrenalectomy. We would rather hypothesize,
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that elevated ghrelin blood levels contribute to the changes in the adrenal gland. We also observed correlation between ghrelin expression and cholesterol level in patients with ACA. Previously, we have reported the relationship between ghrelin and lipids in patients with acromegaly. The potential causal relationship between ghrelin overexpression and hypercholesterolemia needs further research.
We also found IGF2 mRNA overexpression in all adrenal tumor types compared to control glands, with its highest level in carcinoma samples. Our observations are consistent with previous studies, which investigated IGF2 expression in ACC. Since IGF2 is a peptide downstream of the ghrelin/GHS-R axis, we speculated that ghrelin stimulated the expression or function of IGF2. Therefore, we analyzed the relationship between ghrelin and IGF2 expressions. However, the association was found only in myelolipoma indicating that ghrelin and IGF2 expression are not coregulated and belong to different adrenal signaling pathways.
Ghrelin and IGF2 mRNA expression variability among adrenal samples was also observed. Interestingly, we found relatively low ghrelin and IGF2 mRNA expressions in hereditary cases: two pheochromocytomas associated with MEN2A presented the lowest ghrelin mRNA in the entire investigated group. Similarly, low levels of ghrelin and IGF2 mRNA expressions were observed in hyperplasia and myelolipoma associated with congenital adrenal hyperplasia. These observations speak for potential downregulation of ghrelin and IGF2 expression if other proliferative factors are involved in the development of adrenal tumors.
Subsequently, we have observed that acylated ghrelin stimulated the proliferation and migration of the adrenocortical tumor cell line H295R at concentrations of 1x10 6 M and 1x10 8 M while 1x10-10 M was ineffective. The influence of ghrelin on the proliferation of adrenal cancer has been investigated by Barzon et al. and Delhanty et al.24,32 In contrast to our results, in the former study, the antiproliferative effect of ghrelin was proven for NCI-H295 at 10-10 to 10 6 M and for SW- 13 at 10 8 to 10 6 M concentration. In addition, the effect was more evident after 72-96 h of exposure to the hormone. In line with our results, Delhanty et al. showed that both acylated or unacylated ghrelin suppressed apoptotic rate in adrenocortical carcinoma cells. 32 The proliferation was dose-dependent and the highest spinning concentration was 108 M for acylated ghrelin and 10-9 M for unacylated ghrelin. They also suggested the potential influence of type of cell line on the response to ghrelin stimulation. NCI-H295R cells express higher levels of IGF2 than SW-13 cells, resulting in the lowering of ghrelin’s potency.32 Secondly, SW-13 cell lines are considered heterogenic; so NCI-H295R cell line, which we used in our experiment appears to be more desirable for study. We should also emphasize that the effect of ghrelin on cancer cell proliferation is dose- dependent; at concentrations lower than 10 6-10 8 M it stimulates proliferation, while at higher concentrations it displays an inhibitory effect.29,33 Similar discrepancy was reported in breast cancer studies. Cassoni et al.34 reported that ghrelin significantly inhibited cell proliferation, while Jeffrey et al.35 demonstrated the opposite effect. In addition, the short half-life of acylated ghrelin was highlighted in the study. Since acylated ghrelin is rapidly deacylated, an inhibitor of this reaction
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must be used to prevent the rapid deacylation. It is a very important point, which may cause discrepancies among studies. GHS-R1a is known to be activated only by acylated ghrelin. The stimulating effect on cancer cell proliferation was proven for acylated ghrelin.33 GHS-R1a expression was not shown in any of the studies where antiproliferative effect of ghrelin on cancer cells was proven.34 This also suggests that antiproliferative effect is caused by unacylated ghrelin. Further differences in results can explained by the differences in concentrations of ghrelin used for the experiment, the duration of incubation, the possible involvement of ghrelin in different signaling pathways, the type and degree of differentiation of the tumor, the effect of ghrelin acylation, and GHS-R expression.21,
5. Conclusion
In conclusion, we have shown ghrelin and IGF2 overexpressions in adrenal tumors. Increased expression was particularly evident in adrenal carcinoma. Ghrelin and IGF2 are probably proliferative factors for some, but not all types of adrenal tumors. In addition, we have found that acylated ghrelin strongly stimulated proliferation of adrenal cancer cells. Based on our observations and previous reports, we can conclude that acylated ghrelin has an influence on the development of adrenal tumors and in particular of adrenal cancer. The role of unacylated ghrelin is yet to be explained and further studies are needed to elucidate this. The next most important step is examining the influence of ghrelin on proliferation and migration of ACC cells. Since understanding events of ACC development is crucial for designing effective therapeutic agents, further studies investigating molecular alterations in adrenal tumorigenesis are warranted.
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Accepted Article
| igle | Adenoma | Hyperplasia | Myelolipoma | Pheochromocytoma | Carcinoma | p (Kruskal- Wallis or chi squere) |
|---|---|---|---|---|---|---|
| Age [years] | 54 (60.25- 42.5) a | 58 (65-54.75) a | 36 (42.75- 30.75) b | 48 (55-42.75) a | 56 (62.5- 48.5) a | 0.031 |
| Sex [F female; M male] | 28/5 a | 9/4 | 2/6 c | 12/12 | 2/5 | 0.0019 |
| b | d | c | ||||
| Tumor diameter [mm] | 38 (47.75- 21.75) a | 25 (43.25-12.25) | 67.5 (102.2-45.75) | 42 (65-30) a | 105 (145- 81.75) b | 1.486e- 05 |
| a | b | |||||
| Sporadic (s)/ hereditary (h) | 33/0 | 12/1 | 6/2 | 22/2 | 7/0 | 0.0873 |
| Cortisol [nmol/I] | 478.5 (591- 428.2) | 709 (778.5-672.2) | 534.5 (720.2-437.2) | 462.5 (621.8-438.8) | 391.5 (500.5- 226.5) | 0.077 |
| ACTH [pg/ml] | 5.5 (12.6-2.06) a | 18.4 (30.89-10.26) | 24.13 (383.3-16) | 26.23 (37.13-19.15) | 48.81 (80.32- 26.34) b | 1.119e- 05 |
| b | a,b | b | ||||
| DHEAS [ug/dl] | 35.5 (75.5-21.25) a | 83 (115.2-58) a,b | 84.5 (154.5-19) a,b | 113 (183-84) b | 43 (301- 23.25) a | 0.0225 |
| Glucose [mg/dl] | 95 (102.8- 86.75) a | 110 (153.5-99) a | 96 (98.5-87.5) a | 101 (119-90) b | 86.5 (96.75-85) a | 0.031 |
| Total cholesterol [mg/dl] | 236 (260-215 a | 169 (187.5-163.8) b | 187 (229-161) a,b | 205 (232-183.5) a,b | 239,5 (257,25- 217,5) a,b | 0.027 |
| TAG [mg/dl] | 101.5 | 149 | 121 | 114 | 114 (159- 90) | 0.902 |
| (224.2- 81.25) | (150-99.25) | (130-109) | (168.5-95) |
Total ghrelin [M]
1.2×10-10 (1.62×10-10_ 7.256×10-11)
8.1×10-11 (1.75x10-10_ 8.15x10-11)
2.93×10-10 (3.36×10-10_ 1.72×10-10)
2.52x10-11 (7.68x10-11_ 1.93x10-11)
7.12x10-11 (3.17×10” 10
0.248
-5.61x10 11)
Accepted Article
Accepted Article
| Age [years] | Sex [F female; M male] | Tumor size [mm] | Clinical stage | Distant metastases | Survival (months) | Follow- up period (months) | Hormone production | |
|---|---|---|---|---|---|---|---|---|
| ACC1 | 40 | F | 210 | III | - | Up till now | 17 | DHEAS androstendione |
| ACC2 | 58 | M | 125 | II | - | Up till now | 18 | DHEAS androstendione cortisol |
| ACC3 | 68 | M | 125 | IV | Liver, lungs, lymph nodes | 6 | 6 | - |
| ACC4 | 67 | M | 57 | IV | Adrenal, bones | ? | ? | cortisol |
| ACC5 | 44 | F | 62 | IV | Adrenal | Up till now | 75 | - |
| ACC6 | 51 | M | 200 | IV | Liver, lungs, lymph nodes | 19 | 19 | cortisol |
| ACC7 | 56 | M | 175 | IV | Lungs, lymph nodes, bones | Up till now | 18 | - |
Accepted Article
0 -
Ghrelin
ACC: 3
log(relative expression level)
ACC: 2
-5 -
ACC: 6 00 ACC: 7
ACC: 100 ACC: 4
ACC: 5
-10 -
-15 -
Control -
Adenoma -
Hyperplasia -
Myelolipoma -
Pheochromocytoma -
Carcinoma -
| Adenoma | Carcinoma | Control | Hyperplasia | Myelolipoma | |
|---|---|---|---|---|---|
| Carcinoma | 0.049 | - | - | - | - |
| Control | 0.029 | 0.017 | - | - | - |
| Hyperplasia | 0.358 | 0.356 | 0.139 | - | - |
| Myelolipoma | 0.326 | 0.656 | 0.093 | 0.697 | - |
| Pheochromocytoma | 0.626 | 0.093 | 0.204 | 0.248 | 0.224 |
Accepted Article
log(relative expression level)
IGF2
ACC: 6
0
ACC: 2
ACC: 5
ACC: 1
ACC: 3
-5
ACC: 4 ACC: 70
-10 -
Control -
Adenoma -
Hyperplasia -
Myelolipoma -
Pheochromocytoma -
Carcinoma -
| Adenoma | Carcinoma | Control | Hyperplasia | Myelolipoma | |
|---|---|---|---|---|---|
| Carcinoma | 0.026 | - | - | - | - |
| Control | 0.005 | 0.004 | - | - | - |
| Hyperplasia | 0.421 | 0.021 | 0.163 | - | - |
| Myelolipoma | 0.376 | 0.027 | 0.063 | 1 | - |
| Pheochromocytoma | 2e-04 | 1 | 0.004 | 0.004 | 0.007 |
Accepted Article
Adenoma
Carcinoma
Control
-4 -
-6 -
0 -
-7 -
-6
-5-
-8
-8.
log(IGF2)
R=0.17
R=0.02
R= 0.43
p=0.357
p= 0.972
-9 -
p= 0.33
-12.5
-10.0
-7.5
-5.0
-2.5
-7.5
-5.0
-2.5
0.0
-11.0 -10.5 -10.0 -9.5
-9.0
Hyperplasia
Myelolipoma
Pheochromocytoma
0.0 -
-2.5-
-2-
0 -
-5.0-
-4 -
-7.5-
-6-
-5 -
R= 0.36
R=0.92
R= 0.04
-10.0-
p=0.254
-8 -
p= 0.001
-10 -
p= 0.872
-9
-6
-3
-10
-8
-6
-4
-2
-12
-10
-8
-6
log(Ghrel)
4.9
Normalized Cell Index
A
3.9
2.9
1.9
0.9
-0.1
ghrelin 10-6 M
0
14
28
42
56
70
84
98
Time (in Hour)
ghrelin 10-8 M
1.400
ghrelin 10-10 M
Normalized Cell Index
B
control
1.275
1.150
1.025
0.9
0
8
16
24
32
40
48
Time (in Hour)