Elsevier

Cellular Signalling

Volume 66, February 2020, 109492
Cellular Signalling

Ghrelin induces autophagy and CXCR4 expression via the SIRT1/AMPK axis in lymphoblastic leukemia cell lines

https://doi.org/10.1016/j.cellsig.2019.109492Get rights and content

Highlights

  • Ghrelin upregulates SIRT1 expression during the induction of autophagy and CXCR4 in Jurkat and Molt-4 Cells.

  • SIRT1 is essential for ghrelin-induced autophagy in Jurkat and Molt-4 cells.

  • Ghrelin-induced autophagy is related to SIRT1/AMPK axis in Jurkat and Molt-4 cells.

  • Ghrelin receptor (GHS-R1a) expression and CXCR4 expression are related to SIRT1/AMPK axis in Jurkat and Molt-4 cells.

  • Ghrelin has the opposite effect on proliferation and apoptosis of Jurkat and Molt-4 cells.

Abstract

T cell acute lymphoblastic leukemia (T-ALL) is one of the most frequent malignancies in children, and the CXCR4 receptor plays an important role in the metastasis of this malignancy. Ghrelin is a hormone with various functions including stimulation of the release of growth hormone and autophagy in cancer cells. Moreover, SIRT1 and AMPK (AMP-activated protein kinase) stimulate expression of proteins involved in autophagy. On the other hand, autophagic cell death can be an alternative target for cancer therapy, in the absence of apoptosis. The relationship between ghrelin and the SIRT1/AMPK axis and the resulting effects on autophagy, apoptosis, proliferation, and expression of CXCR4 and the ghrelin receptor (GHS-R1a), in Jurkat and Molt-4 human lymphoblastic cell lines was not previously clear. Here we demonstrate that SIRT1 expression is upregulated during the induction of autophagy by ghrelin, an effect that is inhibited by inactivation of SIRT1/AMPK axis. In addition, ghrelin can affect CXCR4 and GHS-R1a expression. In conclusion, this work reveals that ghrelin induces autophagy, invasion, and downregulation of ghrelin receptor expression via the SIRT1/AMPK axis in lymphoblastic cell lines. However, in these cell lines ghrelin-induced autophagy does not lead to cell death due to weak induction of apoptosis.

Introduction

Derived from “ghre”, the proto-Indo-European root of the word “grow”, the peptide hormone ghrelin was named due to its involvement in the control of the hunger response [1]. The human preproghrelin protein consist of 117 amino acids shares 82% homology with the rat's protein. Ghrelin is a 28-amino acid peptide acylated hormone esterified with octanoic acid on Ser 3, and is primarily produced by X/A-like cells in the stomach and is also a potent trigger for growth hormone secretion. Another type of ghrelin is a non-octanoylated form (desacylated ghrelin) that it does not activate the GHS-R receptor but does have other effects such as cardiac [2], anti-ghrelin and inhibition of hepatic glucose output [3,4]. The ghrelin receptor is a G-protein-coupled receptor, known as the growth hormone secretagogue receptor [5]. This peptide and its receptor exhibit pleiotropic roles and thus, are expressed in many cells such as, spleen, thymus, liver, lymph nodes and immune cells [5,6]. Ghrelin decreases apoptosis, causes rises and falls in proliferation, as well as invasion and autophagy of cancer cells. Also, studies on hepatocytes and adipocytes cells have shown that ghrelin operates as an anti-inflammatory factor to reduce autophagy in acute hepatitis, non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH). Nonetheless, the precise mechanism by which ghrelin induces autophagy in T-acute lymphoblastic leukemia cells remains unclear [1,[7], [8], [9], [10], [11], [12]].

Similar to other types of malignancies, one feature of T-ALL is the penetration of cancer cells into peripheral blood and into other organs of the body (metastasis), such as lymph nodes, liver, spleen, lung, skin, intestine as well as the creation of a repertoire of malignant lymphoblasts [13,14]. In hematological cancers, such as in ALL malignant lymphoblasts, the CXCR4 receptor and its ligand, CXCL12, also known as Stromal-Derived Factor (SDF), play an important role in metastasis [13,[15], [16], [17]].

Autophagy is a process of self-digestion during which cytoplasmic components, such as protein aggregates, damaged organelles and lipid vesicles are sequestered into structures called autophagosomes. Autophagosomes fuse with lysosomes leading to degradation of their contents [[18], [19], [20], [21]]. The microtubule-associated protein 1 light chain 3 (LC3) is the main autophagy regulator that regulates main stages in the autophagic system such as autophagic membrane development, autophagic cargo recognition, and autophagic lysosome integration [[22], [23], [24], [25]]. The conversion of LC3 I to LC3 II through proteolytic cleavage and lipidation is a mark of mammalian autophagy [20,26]. Another indication of autophagy is expression of Sequestosome 1 (p62/SQSTM1), which is degraded by autophagy [27,28]. Other products of genes related to autophagy (ATGs), such as ATG3 and Beclin1 (also known as ATG6/Vps30), are also essential to the process [29]. Mutations in the vital molecules of the apoptosis process often show that these cancer cells resistance to drugs. Indeed, autophagic cell death can be an alternative target for cancer treatment, in the absence of apoptosis [30,31].

SIRT-1 is an NAD+-dependent class III histone deacetylase that plays an important role in controlling gene expression, repairing DNA damage and inducing apoptosis, metastasis, metabolism, growth of tumors, aging and autophagy [[32], [33], [34], [35]]. SIRT1 promotes autophagy through signaling pathway of adenosine monophosphate-activated protein kinase (AMPK) or through deacetylation of ATG5, ATG7, ATG8 (LC3) or forkhead box O1 (FOXO1) [36].

AMP-activated protein kinase (AMPK) is a key metabolic energy sensor maintained in eukaryotes along the evolutionary scale that measures alterations in the intracellular ration of AMP to ATP to regulate cell metabolism and maintain homeostasis of energy [37,38]. AMPK enhances SIRT1 activity by increasing the level of cellular NAD+ resulting in deacetylation and modulation of downstream SIRT1 activity. The AMPK-induced SIRT1-mediated deacetylation of autophagy-related proteins describes many of the convergent biological effects of AMPK and SIRT1 on autophagy [39].

Considering the connections reviewed between ghrelin and SIRT1 and AMPK proteins in signaling pathways of cellular homeostasis and inflammation in cancerous and healthy cells [40]. In this study, we investigated the metastasis and autophagy inducing effects of ghrelin in Jurkat and Molt-4 cells and explored the mechanisms by which ghrelin induces these effects through the SIRT1/AMPK axis.

Section snippets

Agents and antibodies

Ghrelin hormone (HOR-297) was purchased from Prospection and dissolved in distilled water (diH2O) before addition to the cell cultures. Dorsomorphin (Compound C) and EX-527 were purchased from Cayman Chemical. MTS was purchased from Promega. FITC Annexin V Apoptosis detection kit (556547) was purchased from BD Biosciences. Mouse monoclonal antibodies against GHS-R1 (FITC conjugated), LC3 was purchased from Santa Cruz Biotechnology. Rabbit monoclonal or polyclonal antibodies against SIRT1 and

Ghrelin induces proliferation of Molt-4 Cells

The previous study demonstrated that 10 nmol/L ghrelin induces autophagy in Liver cells [21]. We treated cells with 10 nmol/L and 5 μmol/L ghrelin and have shown by Western blotting that 10 nmol/L ghrelin induces autophagy in both Jurkat and Molt-4 cells. To examine the effects of ghrelin on cell proliferation, we treated Jurkat and Molt-4 cells with 10 nmol/L ghrelin for 6 and 24 h and measured cell viability using the MTS assay. As shown in Fig. 1, Fig. 2, cell proliferation increased

Discussion

Despite advances in leukemia treatment, intensified treatments undertaken to increase survival rates have led to an increased incidence of adverse events, and as a result 30–50% of children experience treatment failure [47,48]. Studies have shown that chemokines and their receptors, such as CXCR4, are involved in the formation of metastases as well as the formation of the malignant lymphoblast repertoire [[15], [16], [17]]. On the other hand, an important goal of anticancer therapies is to

Conclusion

The results of the present study show that ghrelin induces autophagy, invasion (CXCR4 expression), and downregulation of ghrelin receptor (GHS-R1a) expression in a time-dependent through activation of the SIRT1/AMPK axis in Jurkat and Molt-4 cells. However, due to the weak apoptosis, proliferation, and invasion observed at 6 and 24 h, ghrelin-induced autophagy is not autophagic cell death. Ghrelin also significantly increased proliferation in Molt-4 cells. Our findings indicate that the

Funding

This work was supported by the Medical University of Shahrekord with grant-funded number 2707.

Author contributions statement

MGS, MSJ and HS conceived the study; MH designed research; MH, MJS, MNS and AS performed research; MH, MGS, MSJ analyzed data; and MH wrote the manuscript with a minor contribution from other authors. All authors read and approved the final manuscript.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgment

The authors are grateful to the staff of Students Research Committee, Shahrekord University of Medical Sciences, Shahrekord, Iran.

References (77)

  • M. Komatsu et al.

    Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice

    Cell

    (2007)
  • X. Ou et al.

    SIRT1 deficiency compromises mouse embryonic stem cell hematopoietic differentiation, and embryonic and adult hematopoiesis in the mouse

    Blood

    (2011)
  • S. Yanagi et al.

    The homeostatic force of ghrelin

    Cell Metab.

    (2018)
  • D. Fatehi et al.

    SRT1720, a potential sensitizer for radiotherapy and cytotoxicity effects of NVB-BEZ235 in metastatic breast cancer cells

    Pathol. Res. Pract.

    (2018)
  • R. Huang et al.

    Deacetylation of nuclear LC3 drives autophagy initiation under starvation

    Mol. Cell

    (2015)
  • T.C. Abshire et al.

    Weekly polyethylene glycol conjugated L-asparaginase compared with biweekly dosing produces superior induction remission rates in childhood relapsed acute lymphoblastic leukemia: a pediatric oncology group study

    Blood

    (2000)
  • E. Talero et al.

    Expression patterns of sirtuin 1-AMPK-autophagy pathway in chronic colitis and inflammation-associated colon neoplasia in IL-10-deficient mice

    Int. Immunopharmacol.

    (2016)
  • W.Y. Chen et al.

    Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53-dependent DNA-damage responses

    Cell

    (2005)
  • G. Eades et al.

    miR-200a regulates SIRT1 expression and epithelial to mesenchymal transition (EMT)-like transformation in mammary epithelial cells

    J. Biol. Chem.

    (2011)
  • Z. Gao et al.

    Inhibition of transcriptional activity of c-JUN by SIRT1

    Biochem. Biophys. Res. Commun.

    (2008)
  • P. Oberdoerffer et al.

    SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging

    Cell

    (2008)
  • K.A. Anderson et al.

    Hypothalamic CaMKK2 contributes to the regulation of energy balance

    Cell Metab.

    (2008)
  • M. Goebel-Stengel et al.

    The ghrelin activating enzyme ghrelin-O-acyltransferase (GOAT) is present in human plasma and expressed dependent on body mass index

    Peptides

    (2013)
  • X. Hu et al.

    CXCR4-mediated signaling regulates autophagy and influences acute myeloid leukemia cell survival and drug resistance

    Cancer Lett.

    (2018)
  • F. Broglio et al.

    Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans

    J. Clin. Endocrinol. Metab.

    (2004)
  • C. Gauna et al.

    Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes

    J. Clin. Endocrinol. Metab.

    (2005)
  • A. Rodríguez et al.

    The ghrelin O-acyltransferase-ghrelin system reduces TNF-α-induced apoptosis and autophagy in human visceral adipocytes

    Diabetologia.

    (2012)
  • Y. Mao et al.

    Ghrelin reduces liver impairment in a model of concanavalin a-induced acute hepatitis in mice

    Drug Des. Devel. Ther.

    (2015)
  • S. Ezquerro et al.

    Ghrelin Reduces TNF-α-Induced Human Hepatocyte Apoptosis, Autophagy, and Pyroptosis: Role in Obesity-Associated NAFLD

    J. Clin. Endocrinol. Metab.

    (2019)
  • S. Ezquerro et al.

    Acylated and desacyl ghrelin are associated with hepatic lipogenesis, β-oxidation and autophagy: role in NAFLD amelioration after sleeve gastrectomy in obese rats

    Sci. Rep.

    (2016)
  • D. Mukherjee et al.

    The role of chemokine receptor CXCR4 in breast cancer metastasis

    Am. J. Cancer Res.

    (2013)
  • D.A. Sipkins et al.

    In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment

    Nature

    (2005)
  • J.A. Burger et al.

    CXCR4 antagonists: targeting the microenvironment in leukemia and other cancers

    Leukemia.

    (2009)
  • L.A. Pitt et al.

    Abstract A06: the chemokine receptor CXCR4 is essential for the maintenance of T cell acute lymphoblastic leukemia

    Tumor Microenvironment

    (2015)
  • A. Zlotnik

    Involvement of chemokine receptors in organ-specific metastasis

  • D.J. Klionsky

    Autophagy: from phenomenology to molecular understanding in less than a decade

    Nat. Rev. Mol. Cell Biol.

    (2007)
  • N. Mizushima

    Autophagy: process and function

    Genes Dev.

    (2007)
  • Y. Mao et al.

    Ghrelin attenuated lipotoxicity via autophagy induction and nuclear factor-kappaB inhibition

    Cell. Physiol. Biochem.

    (2015)
  • View full text