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Revista médica de Chile

versión impresa ISSN 0034-9887

Rev. méd. Chile vol.144 no.12 Santiago dic. 2016

http://dx.doi.org/10.4067/S0034-98872016001200009 

ARTÍCULO DE INVESTIGACIÓN

 

GRP78 levels, regional fat distribution and endometrial cáncer

Niveles de GRP78, distribución regional del tejido adiposo y cáncer endometrial

 

Răzvan Ciortea, Andrei M. Malutan, Lenuta M. Angheluta, Carmen E. Bucuri, Maria P. Rada, Dan Mihu

Department of Obstetrics and Gynecology, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania

Correspondencia a:


Background: The association of obesity with endometrial cancer is supported by the presence of endoplasmic reticulum (ER) stress in the adipocyte. Glucose-regulated protein 78 (GRP78) is a marker for ER stress. This protein is a central regulator of ER stress due to its major anti-apoptotic role. It plays an important role in tumor development, progression and chemoresistance. Aim: To look for an association between android and gynoid obesity, plasma GRP78 levels and endometrial cancer. Material and methods: Forty four patients with endometrial cancer aged 72 ± 6 years and 44 healthy women aged 55 ± 9 years were studied. Android and gynoid fat distribution were determined by dual X-ray absorptiometry and plasma GRP78 levels were measured. Results: GRP78 plasma levels were significantly higher in patients with endometrial cancer as compared to the control group. Android fat distribution had a positive correlation with plasma GRP78 levels (p<0.01). Gynoid fat had a negative correlation with plasma GRP78 levels (p<0.01). Conclusions: GRP78 levels are associated with the distribution of adipose tissue and are higher in patients with endometrial cancer.

Key words: Adipose Tissue; Endometrial Neoplasms; Obesity; Endoplasmic Reticulum Stress.


Antecedentes: La asociación de obesidad con cáncer endometrial puede depender de la presencia de estrés del retículo endoplásmico (RE) en el adipocito. La proteína 78 regulada por glucosa (GRP78) es un marcador de estrés del RE. Esta proteína regula el estrés de RE gracias a su rol antiaopoptótico. Ella juega un rol en el desarrollo, progresión y quimio-resistencia de tumores. Objetivo: Buscar una asociación entre obesidad androide o ginoide, niveles plasmáticos de GRP78 y cáncer endometrial. Material y métodos: Se estudiaron 44 mujeres con cáncer endometrial de 72 ± 6 años and 44 mujeres sanas de 55 ± 9 años. La distribución androide o ginoide de la grasa fue determinada por densitometría radiológica de doble fotón (DEXA) y se midieron los niveles plasmáticos de GRP78. Resultados: Los niveles de GRP78 fueron significativamente más altos en mujeres con cáncer endometrial. Se observó una correlación positiva entre la distribución de grasa androide y los niveles de GRP78 (p< 0.01). Se observó una correlación negativa entre distribución de grasa ginoide y niveles de GRP78. Conclusiones: Los niveles de GRP78 se correlacionan con la distribución del tejido adiposo y son mayores en mujeres con cáncer endometrial.


 

Starting from the idea that in modern society the prevalence of obesity is continuously increasin g, that adipose tissue is directly correlated with several disorders, and that there are a series of paraclinical investigations specific to adipose tissue, adipose tissue dysfunction is currently considered an individual pathological condition.

In economically developed countries, endometrial cancer (EC) is associated with obesity in a proportion of 40%1. The hypothesis of the alteration of the estrogen-progesterone balance is used to support the relationship between obesity, endogenous steroid hormones and the risk of EC, but it cannot explain the effects on the entire population, given that not all obese women develop endometrial abnormalities. The identification of biological markers that indicate an increased risk for the development or recurrence of EC in obese women might be useful for decreasing EC mortality and morbidity.

The strong association between obesity and endometrial cancer can be due to the development of stress endoplasmic reticulum (ER) in adipocytes. Stress ER is an adaptive reaction, which normally occurs in any cell that is required to process more molecules than usual per time unit. In this situation, there is a risk that part of the incompletely processed molecules (post-translational molecular folding, twisting, packing) may generate an adaptive reaction in ER that consists in the occurrence of molecular conformational defects, which might prevent traffic through this structure2.

Glucose-regulated protein ​​78 (GRP78) is in the endoplasmic reticulum and it is an important regulator of the unfolded protein response3. GRP78 supports cell survival, having anti-apoptotic properties4. GRP78 levels in visceral adipocytes are correlated with the stage of EC, but also with the survival of these patients, and might be clinically useful as a predictor for endometrial cancer5.

Material and Methods

The study is a case-control analysis that includes 2 groups of patients: group I : 44 patients diagnosed with endometrial cancer. Group II: 44 patients without gynecological pathology or inflammatory disorders.

The diagnosis of endometrial cancer was made after histopathological examination that analyzed the tissue material obtained following endometrial biopsy. All patients enrolled in the study had endometrial adenocarcinoma, endometrioid type, grade I or II.

The anthropometric measurements performed were: body mass index (BMI), calculated by the formula BMI = weight (kg)/[height (m)]², and abdominal circumference (AC), measured in orthostatism, at umbilical level. The height of the subjects was accurately measured (error less than 1 mm), by the stretching procedure for height measurement of the Society for the Development of Kinanthropometry, using an anthropometer fixed to the wall (222 model; Seca GMBH, Hamburg, Germany). Body mass was determined with an accepted error of 20 g using a calibrated electronic scale (FW-150K model; A&D Mercury, Thebarton, Australia).

These patients underwent dual-energy X-ray absorptiometry (DXA) examination with a GE Prodigy Lunar device, which measured the android and gynoid adipose tissue content. The “android” and “gynoid” regions were defined using the software provided by the producer. The android region has a lower limit at the upper limit of the pelvis and an upper limit at the junction of the lower 1/5 with the upper 4/5 of the pelvis-to-chin distance. Lateral limits are represented by the internal limits of the hands. The gynoid region has an upper limit at the upper part of the greater trochanters and a lower limit defined at the distance equal to twice the height of the android region. Lateral limits are represented by the external limits of the legs.

From each subject included in the study, 6 ml fasting blood were taken by venous puncture and collected in test tubes without anticoagulant for the determination of plasma GRP78 levels. The serum obtained by centrifugation was divided and stored in 600 µl freezing tubes at a temperature of –600 C until the tests were performed, in order to avoid repeated freezing-thawing cycles. The serum GRP78 concentration was determined by the sandwich ELISA technique using the Human GRP78 Immunoassay MBS031039 kit, R&D Systems, USA. The detection rate of this kit is 0,1µg/ml. No significant cross reactivity or interference between human GRP78 and analogues were found.

Case-control studies imply an appropriately pairing of subjects. Age was an independent factor associated with GRP78 levels, so it did not influenced pairing of subjects included in this study.

The informed consent of all patients was obtained. The study was conducted under the tenets of Helsinki declaration. The Ethical Committee of the University of Medicine and Pharmacy Iuliu Hatieganu, Cluj-Napoca, approved the study.

All parameters were included in the study database. Normal distribution was tested with the Kolmogorov-Smirnov test. Normally distributed variables are presented as mean ± standard deviation; non-normally distributed as median (interquartile range). For comparison of two means the t-test or Mann-Whitney test was used. For relationship analysis between two variables, Pearson’s or Spearman correlation coefficient (r) was used. To control for age partial correlation and multivariate linear regression the stepwise method was used. Statistical analysis was performed using SPSS 15.0.

Results

The characteristics of the patients included in the study are described in Table 1.

Table 1. Characteristics of the patients included in the study

 

Plasmatic level of GRP78 was significant higher in patients with endometrial cancer compared to the control group.

Android fat was in a statistically significant, positive linear correlation with the plasma GRP78 level (Figure 1). The correlation coefficient between android fat and the plasma GRP78 level was R = 0.4431, p < 0.0001. This coefficient suggests an influence of 44% in plasma GRP78 level variance due to android fat.

 

Figure 1. Correlation between GRP78 and android fat.

Increased gynoid fat was correlated also with low plasma GRP78 levels (Figure 2) with a correlation coefficient of 0.4792, p < 0.0001.

 

Figure 2. Correlation between GRP78 and gynoid fat.

In order to establish the influence of the other variables on GRP78, the following variables were entered in regression: age, menarche, menopause, android fat, age, menopause and android fat proved to be independent factors associated with GRP78 level (Table 2).

Table 2. Multivariate linear regression analysis

Discussion and conclusions

Depending on the predominance of adipose tissue and on its distribution, obesity is android and gynoid. Android obesity is characterized by the predominance of adipose tissue in the central region of the body, in the abdominal wall and mesentery. Gynoid obesity is characterized by the predominance of adipose tissue in the lower part of the body, in the buttocks and thigh region. Android obesity is associated with hyperinsulinemia, decreased glucose tolerance, diabetes mellitus, increased secretion of androgens, testosterone, as well as decreased sex hormone binding globulin (SHBG) levels6. All these pathological conditions associated with android obesity are risk factors for endometrial cancer. This study supports the idea that android obesity is a risk factor for endometrial cancer, as there is a statistically significant difference in abdominal fat between the two groups.

Women with android obesity have an increased adrenal gland activity, with the secondary increase of ACTH and cortisol secretion, associated with hyperinsulinemia, decreased glucose tolerance, diabetes mellitus, an increase of androgen and testosterone secretion, as well as a reduction of SHBG7.

The association of android obesity with hyperinsulinemia can be explained by three possible mechanisms8:

  • Android obesity is more sensitive to catecho­lamines and less sensitive to insulin, which induces an increase of free fatty acid concentrations and consequently, hyperglycemia.

  • Androgens inhibit insulin action at hepatic and peripheral level.

  • Hepatic insulin extraction is inhibited by androgens and free fatty acids.

Also, there are statistically significant corre­lations between abdominal circumference and insulinemia, which evidences the pathogenic effect of abdominal obesity in insulin resistance and compensatory hyperinsulinemia. Thus, larger abdominal circumference, is related with higher the insulin levels. If there is a significant correlation between abdominal circumference and insulinemia, the authors cannot describe the same correlation between BMI and insulinemia, which shows that abdominal obesity has a high insulin resistance potential, unlike gluteal-femoral obesity9.

Chronic hyperinsulinemia as a risk factor in endometrial cancer is explained at two levels10:

  • At endometrial level, insulin stimulates tumor development by a decrease of IGFBP1 and an increase of IGF1. Insulin may directly trigger tumor transformation through endometrial receptors.

  • At ovarian level, it induces hyperandrogenia, which determines anovulation, resulting in a progesterone deficit.

Insulin and insulin-like growth factor-1 (IGF-1) stimulate protein synthesis, anti-apoptotic proliferation and signaling through the induction of mitogen activated protein kinase (MAPK). GRP78 expression is a target downstream of insulin, recent evidence suggesting that GRP78 and the global protein balance of the endoplasmic reticulum can regulate the insulin sensitivity of the body and can protect cells during acute stress. Obesity and type 2 diabetes mellitus are metabolic disorders characterized by insulin resistance and hepatic steatosis. The presence of ER stress in the context of metabolic syndrome has been documented11,12, and the presence of chaperones might play a key role in the regulation of insulin sensitivity and glucose homeostasis. The administration of chemical chaperones to obese mice reduced stress ER markers, restored glucose levels and insulin homeostasis, while weight gain and the increase of hepatic insulin sensitivity were low13. GRP78 was diminished in the adipose tissue of patients with gastric bypass after weight loss, which means that the relationship between obesity-related stress ER and metabolic dysfunction is present in humans14.

Stress ER has been extensively studied because it can contribute to the development of severe diseases such as neurodegenerative diseases, type 2 diabetes mellitus or different forms of cancer15. Recent studies have demonstrated that endoplasmic reticulum stress leads to the alteration of lipid metabolism and hepatic steatosis, because certain components of the signaling system through the unfolded protein response also play a role in the regulation of lipid metabolism by increasing the synthesis of certain enzymes involved in lipogenesis16.

If adipose cells have a low lipid peroxidation level, cellular division and growth is stimulated. During oxidative reactions, superoxide radical is generated in the body, which reacts with hydrogen peroxide and generates extremely reactive hydroxyl radicals that destroy cellular components. This situation can lead to the development of ER stress and the release of GRP78 from adipocytes17.

The majority of recent studies maintain that chronic inflammation is a risk factor for endometrial cancer. Macrophages in the structure of adipose tissue are morphologically altered (giant cells), presenting an overactivation that is reflected by an increased secretion of cytokines (TNF-α and IL-6). These cytokines are involved in the genesis of endometrial cancer due to their effect of activating the nuclear factor kB (NF-kB), but also due to the development of ER stress with the secondary release of GRP7818.

GRP78 is a central regulator of ER stress due to its ability to control the activation of ER transmembrane stress sensors (IRE1, Perk, and ATF6)19,20. Recent studies have demonstrated that GRP78 plays an important role in tumor development, progression and chemoresistance21.

The interest in GRP78 is based on its different functions, both under normal and pathological conditions. GRP78 regulates intracellular calcium, models ER stress proteins and supports cell survival by an immediate response to insults, having anti-apoptotic properties15. The reduction of apoptosis allows the affected cells to survive and accumulate additional mutations.

The rapid proliferation of cancer cells requires an increase of endoplasmic reticulum activity to facilitate the folding, assembling and transmembrane transport of proteins, thus subjecting the endoplasmic reticulum to stress. The stress to which ER is subjected is the insufficient vascularization and the rapid growth of tumor cells that induce hypoxia, on one hand, and nutrient deprivation that affects protein glycosylation and ATP production, on the other hand22,23.

Cancer cells are characterized by an altered glucose metabolism and the tumor microenvironment is marked by blood flow insufficiency and hypoxia, all these phenomena triggering endoplasmic reticulum stress. Under these conditions, tumor cells overexpress GRP78 and the pro-survival characteristics of GRP78 positively influence tumor progression and chemoresistance24.

In our study, the plasmatic level of GRP78 had a linear positive correlation with the andoid fat and had been increased in EC. Detailed knowledge of the steps of these complex pathways, as well as cross connections between them, is necessary in order to understand the mechanisms of action of existing targeted therapy drugs and to discover new ones25. Determination of android fat in association with the GRP78 level could narrow down the group of patients with EC risk.

Acknowledgments: This paper was published under the frame of The European Social Fund, Human Resources Development Operational Programme 2007-2013, project no. 7135/5/30.04.2014 POSDRU

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Recibido el 21 de mayo de 2016, aceptado el 10 octubre de 2016.

Financial support for this work: This paper was published under the frame of The European Social Fund, Human Resources Development Operational Programme 2007-2013, project no. 7135/5/30.04.2014 POSDRU.

The authors declare no conflict of interest.

Correspondencia a: Ciortea Razvan
Bd 21st Decembrie no. 55-57. Cluj-Napoca, Romania. Tel. 0040748030897
r_ciortea@yahoo.com

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