Abstract

Introduction

Every year more than 225,000 women are diagnosed with ovarian cancer and an estimated 140,000 deaths are caused by ovarian cancer worldwide (¹1. Kuan AS, Teng CJ, Wu HH, Su VY, Chen YT, Chien SH, et al. Risk of ischemic stroke in patients with ovarian cancer: a nationwide population-based study. BMC Med 2014; 12: 53, doi: 10.1186/1741-7015-12-53.
https://doi.org/10.1186/1741-7015-12-53... ). Five-year survival for patients with ovarian cancer is 92% if the tumor is detected at an early, localized stage. However, early diagnosis is rare; 85% of ovarian cancers are detected at a more advanced stage because symptoms are easily missed until the disease has spread to other body regions (²2. Nurkalem C, Celik H, Dagli F, Gurates B, Kavak B, Dogan Z, et al. Ghrelin and obestatin expression in serous ovarian tumours. Gynecol Endocrinol 2012; 28: 941-944, doi: 10.3109/09513590.2011.650753.
https://doi.org/10.3109/09513590.2011.65... ). Thus, the overall five-year survival for all women diagnosed with epithelial ovarian carcinoma is only 44% (²2. Nurkalem C, Celik H, Dagli F, Gurates B, Kavak B, Dogan Z, et al. Ghrelin and obestatin expression in serous ovarian tumours. Gynecol Endocrinol 2012; 28: 941-944, doi: 10.3109/09513590.2011.650753.
https://doi.org/10.3109/09513590.2011.65... ,³3. Xu Y, Pang X, Dong M, Wen F, Zhang Y. Ghrelin inhibits ovarian epithelial carcinoma cell proliferation in vitro. Oncol Rep 2013; 30: 2063-2070.). Because of the overall high mortality of ovarian cancer, women at increased risk may be counseled to undergo prophylactic (and possibly unnecessary) oophorectomy (⁴4. Milewski L, Wojtowicz K, Roszkowski PI, Barcz E, Ziarkiewicz-Wroblewska B, Kaminski P, et al. Expression of ghrelin and its receptors in ovarian endometrioma. Gynecol Endocrinol 2012; 28: 310-313, doi: 10.3109/09513590.2011.631628.
https://doi.org/10.3109/09513590.2011.63... ).

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHSR). It is a 28-amino acid peptide produced from a pre-prohormone with a length of 117 amino acids. The mature form of ghrelin can undergo several post-translational modifications, including addition of a fatty-acid chain (n-octanoic acid) to the serine 3 residue (⁵5. Kojima M, Kangawa K. Ghrelin: from gene to physiological function. Results Probl Cell Differ 2010; 50: 185-205.). Ghrelin has been shown to stimulate secretion of growth hormone, and to have orexigenic and adipogenic effects (⁶6. Kojima M, Kangawa K. Ghrelin: more than endogenous growth hormone secretagogue. Ann N Y Acad Sci 2010; 1200: 140-148, doi: 10.1111/j.1749-6632.2010.05516.x.
https://doi.org/10.1111/j.1749-6632.2010... ). Most ghrelin is produced in the stomach by a distinct group of endocrine cells located within the gastric oxyntic mucosa (⁷7. Kojima M, Kangawa K. Structure and function of ghrelin. Results Probl Cell Differ 2008; 46: 89-115, doi: 10.1007/400_2007_049.
https://doi.org/10.1007/400_2007_049... ,⁸8. Kojima M. The discovery of ghrelin - a personal memory. Regul Pept 2008; 145: 2-6, doi: 10.1016/j.regpep.2007.09.023.
https://doi.org/10.1016/j.regpep.2007.09... ), with smaller amounts produced by other organs. Small amounts of ghrelin have also been observed elsewhere in the gastrointestinal tract and pancreas, and its activity influences the metabolism of glucose and lipids.

This peptide also has less well-understood roles in other tissues and organs. Ghrelin expression has been reported at low levels in the brain, pituitary gland, kidneys, and thyroid gland, as well as in several areas of the reproductive system, including the placenta, testes, and ovaries (⁹9. Anderson LL, Jeftinija S, Scanes CG, Stromer MH, Lee JS, Jeftinija K, et al. Physiology of ghrelin and related peptides. Domest Anim Endocrinol 2005; 29: 111-144, doi: 10.1016/j.domaniend.2005.02.033.
https://doi.org/10.1016/j.domaniend.2005... ). A recent report describes ghrelin expression at mRNA and peptide levels in the ovaries of adult rats. mRNA levels of ghrelin in rat ovaries were monitored throughout the estrous cycle to better understand physiologic regulation of expression of the ghrelin gene in ovaries (¹⁰10. Camina JP. Cell biology of the ghrelin receptor. J Neuroendocrinol 2006; 18: 65-76, doi: 10.1111/j.1365-2826.2005.01379.x.
https://doi.org/10.1111/j.1365-2826.2005... ). Despite persistent expression of the signal throughout all stages of the estrous cycle, mRNA levels of ghrelin change in a cyclic fashion, with lowest expression occurring in the proestrus phase, and maximum values in the diestrous phase (¹¹11. Yeh AH, Jeffery PL, Duncan RP, Herington AC, Chopin LK. Ghrelin and a novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogen-activated protein kinase in prostate cancer. Clin Cancer Res 2005; 11: 8295-8303, doi: 10.1158/1078-0432.CCR-05-0443.
https://doi.org/10.1158/1078-0432.CCR-05... ).

In many (though not all) normal tissues, ghrelin stimulates cell proliferation and protects against apoptosis (⁷7. Kojima M, Kangawa K. Structure and function of ghrelin. Results Probl Cell Differ 2008; 46: 89-115, doi: 10.1007/400_2007_049.
https://doi.org/10.1007/400_2007_049... ). In contrast, there are numerous reports of ghrelin-mediated inhibition of the growth of cancer cells (⁹9. Anderson LL, Jeftinija S, Scanes CG, Stromer MH, Lee JS, Jeftinija K, et al. Physiology of ghrelin and related peptides. Domest Anim Endocrinol 2005; 29: 111-144, doi: 10.1016/j.domaniend.2005.02.033.
https://doi.org/10.1016/j.domaniend.2005... ), including the ovarian cancer cells known as HO-8910 (²2. Nurkalem C, Celik H, Dagli F, Gurates B, Kavak B, Dogan Z, et al. Ghrelin and obestatin expression in serous ovarian tumours. Gynecol Endocrinol 2012; 28: 941-944, doi: 10.3109/09513590.2011.650753.
https://doi.org/10.3109/09513590.2011.65... ). There are conflicting reports describing positive and negative effects of ghrelin and other growth hormone-stimulating molecules on the growth of tumor cells in vitro (¹²12. Northrup R, Kuroda K, Duus EM, Barnes SR, Cheatham L, Wiley T, et al. Effect of ghrelin and anamorelin (ONO-7643), a selective ghrelin receptor agonist, on tumor growth in a lung cancer mouse xenograft model. Support Care Cancer 2013; 21: 2409-2415, doi: 10.1007/s00520-013-1800-0.
https://doi.org/10.1007/s00520-013-1800-... ,¹³13. Lundholm K, Gunnebo L, Korner U, Iresjo BM, Engstrom C, Hyltander A, et al. Effects by daily long term provision of ghrelin to unselected weight-losing cancer patients: a randomized double-blind study. Cancer 2010; 116: 2044-2052, doi: 10.1002/cncr.24917.
https://doi.org/10.1002/cncr.24917... ). The activity of several signaling pathways, including mitogen-activated protein kinase (MAPK) pathways, have been implicated in these processes. We investigated if ghrelin exerts its inhibitory effects on HO-8910 cells through GHSR activation and the downstream activity of MAPKs.

Material and Methods

Unless specified otherwise, all chemicals and reagents were purchased from Sigma-Aldrich (USA). Antibodies against IgG, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ERK1/2, JNK, p90rsk, phospho-ERK1/2, phospho-JNK and phospho-p90rsk1 (Ser380) were purchased from Millipore (USA). Unless specified otherwise, culture of the ovarian line HO-8910 (Chinese Academy of Sciences, China) took place at 38.5°C with 5% CO₂ under humidified air. The HO-8910 cell line is derived from a 51-year-old Chinese patient with ovarian cancer and ascites in 1994.

RNA extraction and reverse-transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from HO-8910 cells using an RNeasy kit (Qiagen, Germany). RNA samples were treated with RNase-free DNase I to remove contamination of genomic DNA. RNA content of samples was too low to be quantified accurately by spectrometry. Thus, 6.5-µL RNA aliquots were converted to cDNA by reverse transcription, then amplified (Takara Bio, Japan). PCR primers for the ghrelin receptor were: sense, 5′-TCTTCCTTCCTGTCTTCTGTC-3′; antisense, 5′-AGTCTGAACACTGCCACC-3′ (¹⁴14. Caminos JE, Gualillo O, Lago F, Otero M, Blanco M, Gallego R, et al. The endogenous growth hormone secretagogue (ghrelin) is synthesized and secreted by chondrocytes. Endocrinology 2005; 146: 1285-1292, doi: http://dx.doi.org/10.1210/en.2004-1379.
https://doi.org/10.1210/en.2004-1379... ).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay

Initially, cells were grown in 96-well plates (1×10³ cells/well) with ghrelin and D-Lys3-GHRP-6. Control cells were switched from RPMI1640 to Dulbecco’s modified Eagle’s medium (DMEM) containing 0.1% dimethyl sulfoxide (DMSO). At 12, 24, 36, 48, 60 and 72 h after treatment with ghrelin and D-Lys3-GHRP-6, 20 µL of MTT was added to each well to a final concentration of 0.5%. After 4 h incubation at 37°C in the dark, 150 µL DMSO was added to each well for 10 min to dissolve formazan crystals. Absorbance was measured using a microplate reader (ELx800; BioTek, USA) at 490 nm. Experiments were repeated three times. Viability of ghrelin- and D-Lys3-GHRP-6-treated cells was expressed as the percentage of population growth plus standard error of the mean relative to that of untreated control cells. Cell death caused by ghrelin and D-Lys3-GHRP-6 was calculated as a percentage of inhibition using the following formula: Percent inhibition = (1 - mean experimental absorbance/mean control absorbance) ×100.

Assay to determine effective concentrations of ghrelin and D-Lys3-GHRP-6 (ghrelin receptor inhibitor)

Ghrelin was added to HO-8910 growth media to final concentrations of 121, 152, 182, 212, and 242 nM, cells were cultured for 12, 24, 36, 48, 60 and 72 h, and then the growth of HO-8910 cells was analyzed. Once the optimum ghrelin concentration and treatment duration to achieve inhibition were determined, this treatment was repeated with addition of D-Lys3-GHRP-6 to final concentrations of 10^-8, 10^-9, 10^-10, and 10^-11 mg/mL. HO-8910 cells were then cultured for 12, 24, 36, 48, 60 and 72 h, and their growth analyzed.

Western blotting

HO-8910 cells were homogenized and proteins separated by electrophoresis on 8-12% sodium dodecyl sulfate/polyacrylamide gels, and then transferred to immunoblot nitrocellulose membranes. Membranes were blocked for 30 min at room temperature with phosphate-buffered saline (PBS) containing 5% fat-free milk and 0.1% Tween 20. Then, membranes were incubated with primary anti-Rac1 antibody for ≥1 h at room temperature, or overnight at 4°C. Then, membranes were washed thrice with PBS containing 0.1% Tween 20, incubated with peroxidase-conjugated secondary antibodies, and developed using ECL reagent (Pierce, USA).

siRNA design

RNA interference was used to silence expression of ERK1/2 in HO-8910 cells. ERK1/2-siRNA (TGAATTGTATCATCAACAT) was synthesized by Gene Pharma Biotechnology (China).

Transfection of siRNA

siRNA transfection was conducted using lipofectamine according to the protocol supplied by Invitrogen (USA). Briefly, 1×10⁵ cells were seeded onto six-well plates containing antibiotic-free medium and incubated overnight. For each well, 5 µL siRNA was mixed with 125 µL Opti-MEM I. The mixture was combined with a solution of 5 µL lipofectamine in 125 µL Opti-MEM I. After 20 min at room temperature, the mixture was applied to cells in an appropriate volume of Opti-MEM I to achieve a final concentration of 100 nM for each siRNA. Negative control group was transfected without siRNA. After incubation for 6 h at 37°C, RPMI1640 supplemented with serum was added to wells. Cells were cultured for an additional 24 h at 37°C before analyses.

Results

GHS-R expression in HO-8910 cells

RT-PCR was used to detect expression of GHSR mRNA in HO-8910 cells. The GHSR (348 bp) was expressed at a high level in HO-8910 cells (Figure 1).

Optimal concentrations and timing of treatment of ghrelin and D-Lys3-GHRP-6 on HO-8910 cells

The MTT assay was used to assess the growth and viability of HO-8910 cells after treatment with varying concentrations of ghrelin, and to determine the appropriate duration of treatment to achieve the desired level of growth inhibition.

Ghrelin was added to HO-8910 media to final concentrations of 121, 152, 182, 212 and 242 nM. Numbers of viable cells were assessed at 12, 24, 36, 48, 60 and 72 h (Figure 2A). The optimal concentration and duration of ghrelin treatment for HO-8910 cells was found to be 182 nM and 24 h, respectively.

Next, we determined the D-Lys3-GHRP-6 concentration needed to prevent the growth inhibition caused by 182 nM ghrelin in HO-8910 cells. HO-8910 cells that had been treated with 182 nM ghrelin were treated with D-Lys3-GHRP-6 (10^-5, 10^-6, 10^-7, 10^-8 ng/mL) and cell numbers evaluated at 24 h (Figure 2B). We found that 10^-6 ng/mL D-Lys3-GHRP-6 could inhibit the growth inhibition caused by 182 nM ghrelin.

Ghrelin inhibited the growth of HO-8910 cells via the ERK1/2 pathway

To ascertain which pathway has a key role in ghrelin-mediated inhibition of the growth of HO-8910 cells, the phosphorylation status of the MAPKs ERK1/2, JNK and p38 was detected at 0, 10, 20, 30 and 60 min (Figure 3) after treatment with 182 nM ghrelin. A decrease in ERK1/2 phosphorylation was greater than that observed for JNK or p38 after 20 min.

When this treatment was repeated with addition of 10^-9 mg/mL D-Lys3-GHRP-6, ERK1/2 phosphorylation was reduced compared with treatment with ghrelin alone. The phosphorylation status of JNK and p38 were not changed significantly (Figure 4).

This result suggested that ERK1/2 has a key role in the ability of ghrelin to block the growth of HO-8910 cells. To verify this finding, siRNA specific for ERK1/2 was used to knock down ERK1/2 expression (Figure 5). Also, the phosphorylation of p^90rsk (p-p^90rsk) was low (Figure 5).

After reduction of ERK1/2 by siRNA knockdown, ghrelin (182 nM) was no longer able to reduce the growth of HO-8910 cells (Figure 6).

Discussion

Ghrelin is produced predominantly by the stomach, though smaller amounts are produced by the bowel, pancreas, pituitary, kidneys, and placenta. The GHSR is a typical G protein-coupled seven-transmembrane receptor (¹⁵15. Fujitsuka N, Asakawa A, Amitani H, Hattori T, Inui A. Efficacy of ghrelin in cancer cachexia: clinical trials and a novel treatment by rikkunshito. Crit Rev Oncog 2012; 17: 277-284, doi: 10.1615/CritRevOncog.v17.i3.50.
https://doi.org/10.1615/CritRevOncog.v17... ). Several studies have identified the protective effects of ghrelin (⁴4. Milewski L, Wojtowicz K, Roszkowski PI, Barcz E, Ziarkiewicz-Wroblewska B, Kaminski P, et al. Expression of ghrelin and its receptors in ovarian endometrioma. Gynecol Endocrinol 2012; 28: 310-313, doi: 10.3109/09513590.2011.631628.
https://doi.org/10.3109/09513590.2011.63... ,¹⁶16. Bai R, Zhao P, Cao G, Wen S, Li Q, Meng Q. Ghrelin promotion of oocyte maturation via ERK1/2 pathway in ovis aries. Cell Mol Biol 2012; 58 (Suppl): OL1797-OL1802. ¹⁷17. Hosoda H, Kangawa K. [Role of ghrelin in cancer]. Nihon Rinsho 2004; 62 (Suppl 9): 421-423. ¹⁸18. Rak A, Szczepankiewicz D, Gregoraszczuk EL. Expression of ghrelin receptor, GHSR-1a, and its functional role in the porcine ovarian follicles. Growth Horm IGF Res 2009; 19: 68-76, doi: 10.1016/j.ghir.2008.08.006.
https://doi.org/10.1016/j.ghir.2008.08.0... ), but little is known about the effects of ghrelin on ovarian cancer cells or their mechanism of action. We found that the mechanism through which ghrelin inhibits the growth of HO-8910 cells involves the ERK1/2 branch of MAPK pathways. Maximum inhibition of the growth of HO-8910 cells was achieved using 182 nM ghrelin for 24 h. We also demonstrated that this effect of ghrelin was mediated through its receptor, the GHSR (¹⁹19. Kamegai J, Wakabayashi I, Miyamoto K, Unterman TG, Kineman RD, Frohman LA. Growth hormone-dependent regulation of pituitary GH secretagogue receptor (GHS-R) mRNA levels in the spontaneous dwarf Rat. Neuroendocrinology 1998; 68: 312-318, doi: 10.1159/000054379.
https://doi.org/10.1159/000054379... ), which were shown to be expressed at high levels in HO-8910 cells. Experiments in which the GHSR inhibitor D-Lys3-GHRP-6 (10^-6 ng/mL) blocked ghrelin-mediated growth inhibition provided further support for the role of the GHSR.

To gain further insight into the mechanism by which ghrelin inhibits growth of HO-8910 cells, we evaluated the activity of signaling pathways downstream of the GHSR. MAPKs are a family of serine/threonine kinases that includes ERK, JNK and p38. These kinases are involved primarily in activation of the nuclear transcription factors that control the proliferation, differentiation and apoptosis of cells (²⁰20. Delhanty PJ, van der Eerden BC, van der Velde M, Gauna C, Pols HA, Jahr H, et al. Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a. J Endocrinol 2006; 188: 37-47, doi: 10.1677/joe.1.06404.
https://doi.org/10.1677/joe.1.06404... ). Our study suggests that ghrelin inhibits the growth of HO-8910 cells via the ERK signaling pathway, and not through activation of JNK or p38. We found that 20–60 min of ghrelin treatment was required to inhibit ERK phosphorylation, so stimulation was time-dependent. Furthermore, GHSR blockade by chemical inhibition and silencing of ERK by siRNA suppressed ghrelin-mediated inhibition of the growth of HO-8910 cells.

Overall, our study suggests that the ghrelin/GHSR signaling pathway attenuates the growth of HO-8910 cells mainly through an ERK-dependent pathway. Thus, ghrelin could be a target for ovarian cancer therapy. Despite this promising finding, further study is necessary before clinical application is considered.

Acknowledgments

This work was supported by a grant from the Inner Mongolia Natural Science Foundation (#2013MS11115).

References

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