Modulation of ghrelin axis influences the growth of colonic and prostatic cancer cells in vitro Hanna £awnicka

(1)

Modulation of ghrelin axis influences the growth of colonic and prostatic cancer cells in vitro

Hanna £awnicka¹*, Gabriela Me³eñ-Mucha¹*, Ewelina Motylewska¹, S³awomir Mucha², Henryk Stêpieñ¹

1Department of Immunoendocrinology,²Department of Clinical Endocrinology, Chair of Endocrinology, Medical University of Lodz, Dr. Sterling 3, PL 91-425 £ódŸ, Poland

Correspondence: Gabriela Me³eñ-Mucha, e-mail: gabriela.melen-mucha@umed.lodz.pl

Abstract:

Background: The risk of different cancers seems to be associated with obesity. Moreover, low ghrelin levels observed in obese peo- ple may be implicated in cancer development and progression. The aim of this study was to examine the direct effects of both forms of ghrelin (acylated and unacylated) and ghrelin receptor type 1a antagonist (D-Lys-GHRP-6) on the growth of murine colon cancer MC38 and human prostate cancer DU145 cell lines in vitro.

Methods: The cells were cultured for 72 h in the presence of rat or human acylated ghrelin (rG, hG), human unacylated ghrelin (hUAG), D-Lys-GHRP-6 (GHS-RA) applied either alone or jointly. The cell line growth was assessed by the colorimetric Mosmann method.

Results: hUAG (10⁶, 10⁷and 10¹⁰M) inhibited MC38 cancer cell growth and, at some concentrations (10⁸, 10⁹, 10¹⁰M), enhanced the antineoplastic effect of GHS-RA (10⁴M). In turn, GHS-RA evoked a biphasic effect on MC38 cancer growth: inhibitory at 10⁴M and stimulatory at 10⁵and 10⁶M. Moreover, GHS-RA at the highest examined concentration (10⁴M) enhanced the cytostatic effect of FU. Human acylated and unacylated ghrelin and GHS-RA inhibited DU145 cancer growth with moderate and different potencies. A dose-response effect was observed for the inhibitory action of hG together with the synergistic effect of hUAG and GHS-RA.

Conclusion: The obtained results indicate an involvement of the ghrelin axis in the growth regulation of colon and prostate cancers and may suggest new therapeutic options for these neoplasms.

Key words:

ghrelin, ghrelin receptor antagonist, colon and prostate cancer cell line

Abbreviations: DU146 – androgen-independent prostate cancer cell line, FU – fluorouracil, G – acylated or octanoylated ghrelin, GH – growth hormone, GHRH – growth hormone releasing hormone, GHS-R – growth hormone secretagogue receptor, GHS-RA – ghrelin receptor type 1a antagonist (D-Lys-GHRP-6), hG – human acylated ghrelin, hUAG – human unacylated ghrelin, IGF-I – insulin like growth factor-I, MC38 – murine colon 38 cells, PCa – prostate cancer, rG – rat acylated ghrelin, UAG – unacylated or desoctanoylated or desacyl ghrelin

Introduction

Increased risk of the development of several cancers including colon and prostate cancers, and the risk of cancer-related mortality, seem to be connected with obesity [16]. This unfavorable relationship is observed in patients from the United States [4] and

Pharmacological Reports 2012, 64, 951959 ISSN 1734-1140

* £awnicka H. and Me³eñ-Mucha G. contributed equally to this work

(2)

position towards cancer development and growth.

Moreover, the unsatisfactory effectiveness of currently available standard therapies for colon and androgen- independent prostate cancers results in continuous searching for new and more potent therapeutic options.

The incidence of colorectal cancer has demon- strated an upward trend in many industrialized countries for more than 30 years [43]. Moreover, colon cancer is usually diagnosed at advanced stages, when it is usually fatal. For several years, fluorouracil (FU) remains the drug of choice and the only chemothera- peutic agent available for the treatment of advanced stages of colorectal cancer [35]. During the last dec- ade, several new drugs have been applied for the treatment of metastatic colon cancer, among them two monoclonal antibodies targeting VEGF and EGF receptors approved by FDA in 2004 [19, 36].

Prostate cancer (PCa) is currently the most com- monly diagnosed malignancy, and the second cause of cancer-related deaths in men from western industrialized countries [30, 42]. While localized prostate cancer is usually curable by either radical prostatectomy or radiotherapy, 20% of PCa patients with organ- confined disease fail local therapy and develop incur- able metastatic disease. Moreover, a further 15% of PCa patients present with metastatic disease at time of diagnosis [7]. Due to the androgen-dependent nature of PCa, androgen deprivation therapy remains the only systemic treatment option in advanced disease.

Although the majority of cases respond to androgen ablation, the progression to androgen independence eventually occurs in almost all PCa patients [30].

Moreover, the cases not cured by surgery become androgen-independent, rendering anti-androgen therapy ineffective and finally becoming untreatable with current therapies [13]. Thus, the experimental and clinical studies on new drugs for androgen-independent prostate cancer are presently performed but have different results [14, 15, 33].

Ghrelin is a novel gut-brain peptide first isolated in 1999 from the stomach as an endogenous ligand for growth hormone secretagogue receptor (GHS-R) [22].

Besides its strong stimulatory effect on growth hormone (GH) release, ghrelin modulates the secretion of other pituitary hormones, influences appetite and weight gain, modulates some functions of the gastrointestinal tract and changes the growth processes of neoplastic tissues (see [24] for review). Ghrelin exists

predominates in circulation and, although it seems to be inactive in the regulation of GH secretion [2], it is active in other processes regulated by ghrelin, such as adipogenesis [40] and neoplastic growth influencing proliferation and apoptosis [1, 5, 21, 44]. Since the first publication reporting the antiproliferative effect of ghrelin, researchers have focused on the role of the ghrelin-GHS-R system in several neoplasms. It was found that some cancers possess binding sites for ra- diolabeled ghrelin mostly other than GHS-R1a [1, 5, 6]. Moreover, several studies have found ghrelins to have both antiproliferative [1, 5, 6] and proliferogenic effects [12, 21, 44] on various cancer cell lines. Re- cently, some interesting associations between ghrelin and neoplastic diseases have been reported, such as the increased ghrelin levels in patients with cancer as compared with gender-matched noncancer controls [17], ghrelin resistance in the treatment of anorexia in tumor-bearing mice [41] and the involvement of ghrelin/GHS-R axis in invasiveness and migration of some cancer cells [11, 12].

It is supposed that the increased risk of gastrointestinal malignancies, especially colorectal cancer, is associated with obesity, and seems to be related to environ- mental, rather than genetic, factors. The mechanism ac- companying obesity includes hyperinsulinemia or insulin resistance along with elevated leptin and decreased ghrelin serum levels [4]. This is especially interesting, since ghrelin is a potent regulator of the GH/IGF-I axis, which is frequently implicated in the development of several neoplasms, including colon cancer [3]. We hypothesized that the changed levels of leptin and ghrelin, acting as two main regulators of energy homeostasis, are also likely to play a role in carcinogenesis. Thus, the aim of our study was to examine the direct effects of both forms of ghrelin (G and UAG) and the ghrelin receptor type 1a antagonist (D-Lys-GHRP-6; GHS-RA), applied alone or jointly, on the growth of colon and prostate cancer cell lines.

Materials and Methods

Murine Colon 38 (MC38, established from transplantable murine Colon 38 tumor, which was chemically induced in C57BL/6 female mice and classified as

(3)

a grade III adenocarcinoma before being adopted to growth in vitro) [8] and human prostate DU145 (es- tablished from a tumor removed from the metastatic brain lesion of a 69-year-old man with androgen- independent prostate carcinoma) [38] cancer cells were used in the study.

The cells were cultured as monolayers in culture flasks (Nunc Eas Y flask 25 cm², NUNC) in RPMI 1640 medium (Sigma), supplemented with: Hepes buffer (Sigma) – 25 mM for MC38 and 10 mM for DU145; 100 U/ml penicillin and 100 µg/ml strepto- mycin solution (Sigma); sodium bicarbonate (Sigma) – 2 g/l for MC38 and 1.5 g/l for DU 145; fetal calf serum (Biochrom) – 5% for MC38 and 10% for DU145; L-glutamine (Sigma) – 4 mM for MC38 and 6 mM for DU145; and further for DU145 with: 0.1 mM Non-essential amino acid solution (Sigma) and 1 mM sodium pyruvate (Sigma). The cells were maintained in a humidified atmosphere of 95% air and 5% CO₂at 37°C and harvested weekly after 3-min incubation at 37°C in the presence of trypsin-EDTA at concentrations of 0.05% (MC38) and 0.02% (DU145), in Hanks-balanced salt solution (Sigma). Thereafter, the cells were collected, rinsed three times in culture medium, centrifuged and seeded at a density of 2 × 10⁵ (MC38) or 1 × 10⁵ (DU145) cells/5 ml fresh medium. After one of the subsequent trypsinization procedures, MC38 cells were plated at a density of 2 × 10⁴cells/ well and DU 145 at density of 1 × 10⁴ cells/well into 96-well microplates (96 Cell Culture Cluster Dish, Nunclon MicroWell Plates, NUNC) in complete culture medium and preincubated for 24 h (5% CO₂, 37°C, 95% humidity). Following this, the cells were cultured for a further 72 h in the presence of various concentrations of the test substances, applied either alone or jointly as follows:

1) for MC38 cells:

– rat acylated ghrelin (rG, BACHEM) at final concentrations of 10⁵– 10¹⁰M;

– human acylated ghrelin (hG, BACHEM) at final concentrations of 10⁵– 10¹⁰M;

– human unacylated ghrelin (hUAG, BACHEM) at final concentrations of 10⁵– 10¹⁰M;

– ghrelin receptor type 1a antagonist (GHS-RA; D- Lys-GHRP-6; Sigma) at concentrations of 10⁴– 10⁶M;

– fluorouracil (FU, Roche) at a final concentration of 2.5 µg/ml;

2) for DU 145 cells:

– human acylated ghrelin (hG, BACHEM) at final concentrations of 10⁶– 10⁸M;

– human unacylated ghrelin (hUAG, BACHEM) at final concentrations of 10⁵and 10⁷M;

– ghrelin receptor antagonist (GHS-RA; D-Lys- GHRP-6; Sigma) at a final concentration of 10⁴M.

The cell growth was assessed by the colorimetric Mosmann method, using the EZ4Y kit (Easy for You, The 4^thGeneration Non Radioactive Cell Proliferation

& Cytotoxity Assay, Biomedica Gruppe, Austria, Bellco Biomedica, Poland) according to the manufac- turer’s instructions. The absorbance (OD, optical density) of each sample was measured with an ELISA reader at a wavelength of 450 nm.

Statistical analysis

The results are expressed as the average percentage of the optical density of the control groups (not shown) and represent mean values ± SEM of independent experiments with 6 to 10 replicates per each group.

Comparisons of individual groups were evaluated by analysis of variance (one-way ANOVA) following LSD test (least significant differences). Differences were considered significant if p < 0.05.

Results

The effect of ghrelin axis modulators on the growth of murine MC38 colon cancer cells

D-Lys-GHRP-6, used as the ghrelin receptor type 1a antagonist (GHS-RA), evoked biphasic effect on the growth of the MC38 line: inhibitory at 10⁴ M and stimulatory at 10⁵ and 10⁶ M concentrations. At a concentration of 10⁴ M, GHS-RA caused the strongest inhibitory effect of all of the examined substances, apart from fluorouracil (FU). The inhibitory effect of GHS-RA was reproducible (in 5 different experiments) and strong, with the inhibition of growth by 26 to 39% as compared to controls. This concentration of GHS-RA even enhanced the cytotoxic effect of FU by 9%. However, at two other examined concentrations (10⁵and 10⁶M) GHS-RA slightly but significantly increased the rate of MC38 cell growth by 14%

and 10%, respectively, as compared to the controls, but did not change the cytotoxic effect of FU (Fig. 1).

From all studied forms of ghrelin (rG, hG and hUAG) only human unacylated ghrelin significantly

Ghrelin/GHS-R axis and colon and prostate cancers

Hanna £awnicka et al.

(4)

line (Figs. 2 and 3). Rat acylated ghrelin did not modulate the effects of FU or GHS-RA (10⁴ M) (Fig. 2). In turn, hUAG at concentrations of 10⁶, 10⁷ and 10¹⁰M inhibited MC38 cell growth and at some concentrations (10⁸– 10¹⁰ M) enhanced the inhibitory effect of ghrelin receptor antagonist (GHS-RA at

used in this study, was found to evoke the strongest oncostatic effect on MC38 colon cancer cells out of the 3 concentrations examined (25, 2.5 and 0.25 µg/ml). FU at a concentration of 2.5 µg/ml inhibited the growth of MC38 in 5 performed cultures by 61–69% as compared to the controls (Figs. 1–4).

Fig. 1. The effect of ghrelin receptor antagonist (GHS-RA) applied alone or jointly with fluorouracil (FU) on the growth of Colon 38 cancer cells, % of Control (the mean ± SEM), *p < 0.05 vs.C, ^ p < 0.05 vs. FU 2.5 µg/ml

Fig. 2. The effect of rat ghrelin (rG) applied alone or jointly with fluorouracil (FU) or ghrelin receptor antagonist (GHS-RA) on the growth of Colon 38 cancer cells, % of Control (the mean ± SEM), * p < 0.05 vs. C

(5)

The effect of ghrelin axis modulators on the growth of human DU145 prostate cancer cells

All the members of the ghrelin axis and its modulator (hG, hUAG, GHS-RA) inhibited DU145 prostate cancer cell growth with moderate and varied potencies.

For the inhibitory action of hG, a dose-response effect was observed (10⁶– 10⁸M), with the strongest inhibition evoked by the lowest examined concentration (14% of growth inhibition). hUAG at concentrations

of 10⁵and 10⁷M, and GHS-RA at a concentration of 10⁴ M significantly decreased DU145 cell growth, determined as 10% growth inhibition for hUAG both concentrations and 14% growth inhibition for GHS- RA. Moreover, hUAG and GHS-RA (10⁴M) applied together acted synergistically and evoked the strongest inhibitory effect, estimated as the inhibition of DU145 cell growth by 17 and 20% by GHS-RA plus 10⁵ M of hUAG and by GHS-RA plus 10⁷ M of hUAG, respectively (Fig. 5).

Fig. 3. The effect of acylated ghrelin (hG) on the growth of Colon 38 cancer cells, % of Control (the mean ± SEM)

Fig. 4. The effect of desoctanoylated ghrelin (hUAG) applied alone or jointly with fluorouracil (FU) or ghrelin receptor antagonist (GHS-RA) on the growth of Colon 38 cancer cells, % of Control (the mean ± SEM), * p < 0.05 vs. C, ^ p < 0.05 vs. GHS-RA

(6)

Since D-Lys-GHRP-6 used as the growth hormone secretagogue receptor type 1a antagonist did not modify the action of acylated ghrelin (rat and human), it should not be treated as an antagonist, at least in this experimental conditions. However unexpectedly, this substance enhanced the inhibitory effect of hUAG in both examined cancer cell lines.

Discussion

The superiority of non-cytotoxic targeting strategies over traditional chemotherapy is widely discussed nowadays and seems to be a promising option for the therapy of advanced stages for several cancers. Al- though colorectal cancer does not belong to hormone- dependent neoplasms, experimental evidence shows that its growth is modified via several hormones or hormone receptor modulators such as gastrin and its analogs, gastrin/cholecystokinin receptor (CCK-A and CCK-B) antagonist [25, 26, 37], somatostatin and its analogs [28, 29], melatonin [29], estrogens and selective estrogen receptor modulators [25, 26, 31, 32]

or GHRH antagonists [39]. Moreover, some of these substances enhanced even the cytotoxic effect of FU on colon cancer growth [25, 26, 28, 31]. Similar findings concerning the effects of non-cytotoxic therapy have been observed for androgen-independent prostate cancer cells [5, 14, 15].

This is the first study demonstrating the antineoplastic effect of unacylated ghrelin and the biphasic activity of D-Lys-GHRP-6 (strong inhibition and weak stimulation) on the growth of MC38 colon can- cer cells in vitro. D-Lys-GHRP-6 was found to have a strong inhibitory effect at a concentration of 10⁴M, with the effect on the cell line growth estimated as 61–74% of the controls, and confirmed in 5 separate experiments. Taking into account that D-Lys-GHRP-6 is only a biomodulator but not a cytotoxic agent, its inhibitory effect seems to be very potent, especially so when compared to the most effective cytostatic concentration of FU, which inhibited MC38 colon cancer cell growth to 21–29% of the controls. Moreover, it is worth mentioning that D-Lys-GHRP-6 administered at a concentration of 10⁴ M together with FU even enhanced the cytostatic effect of the latter, and applied together with UAG at concentrations of 10^8,9,10 M, caused the inhibition of cancer cell growth by up to 49% of the controls’ growth (the strongest inhibitory effect evoked by the members or modulators of ghrelin axis). The inhibitory activity of UAG noticed in our study was rather weak (it was found to inhibit of MC38 cells growth by 8 or 10% as compared to the controls) but reproducible and statis- tically significant. The effectiveness of the selected hUAG concentrations (10^6,7,10 M and inactivity of 10^8,9 M) towards MC38 cancer cells may be ex- plained by the existence of two different binding sites mediating its effects. The lack of any growth inhibi-

tate cancer cells, % of Control (the mean ± SEM), * p < 0.05 vs. C,

#p < 0.05 vs. hG 10⁶M,^##p < 0.05 vs.

hG 10⁶& hG 10⁷M,^Vp < 0.05 vs.

GHS-RA, ^ p < 0.05 vs. hUAG 10⁵M,

^^ p < 0.05 vs. hUAG 10⁷M

(7)

tory effect associated with human and rat acylated ghrelin on the MC38 line observed in our study is in- consistent with the recently published results of He et al., who found that a ghrelin dose dependently inhibited FU-induced apoptosis in HT-29 cells [18]. How- ever, in this study, the effect of UAG neither D-Lys- GHRP-6 on HT-29 line was not examined.

Moreover, the probable role of ghrelin axis distur- bances in colon carcinogenesis was suggested much earlier in an elegant study performed on gastrin knock- out mice. In this experimental model, increased vis- ceral adiposity observed at an early age resulted in abdominal obesity accompanied by increased leptin and decreased ghrelin plasma levels in mutant mice at 7- months old (as in obese people). These hormonal changes were suggested by the authors to contribute to increased colon carcinogenesis in response to carcinogen, azoxymethane [9]. This data are in accordance with our working hypothesis and partially with our results demonstrating the inhibitory effect of UAG on colon cancer cell growth in an in vitro model. In addi- tion, it agrees with the findings of our earlier studies which have shown that leptin promotes the growth of MC38 cancer cells and, surprisingly, enhances the inhibitory effect of FU applied at lower doses [27].

The potential role of ghrelin in prostate cancer pathogenesis is discussed in the literature [24]. The results of our study concerning the effect of ghrelin peptides on DU145 prostate cancer cells growth are in agreement with or complete other authors’ observa- tions. Cassoni et al. [5] and Jeffery et al. [20] demon- strated the expression of GHS-R 1a and 1b mRNAs on DU145 cells. Moreover, immunohistochemical staining for the GHS-R 1a and ghrelin was positive in the four prostate cancer cell lines, including DU145 [20]. It is worth mentioning that the normal prostate cDNA library expressed GHS-R1a, but not the 1b isoform or ghrelin [20]. Our results are concordant with those of Casoni et al. [5], who observed that acylated ghrelin and desacyl (unacylated) ghrelin inhibited DU145 cell proliferation.

However, studies performed on other prostate cancer lines showed contradictory results. Jeffery et al.

[20] and Yeh et al. [44] noted that PC3 (androgen- independent line) and LNCaP (androgen-dependent) cell proliferation was stimulated by ghrelin. The effect of D-Lys-GHRP-6 on prostate cancer growth was not examined in these studies. Hence, our results showing the biphasic effect of D-Lys-GHRP-6 on DU145 cancer cells growth, complements the previ-

ous data concerning the role of the ghrelin axis in prostate cancer proliferation.

Since UAG does not bind to GHS-R1a, it might be speculated that the anti-growth effect of this form of ghrelin seems to be mediated via another receptor sub- type than GHS-R1a. In addition, it cannot be excluded that the biphasic effect of D-Lys-GHRP-6 is mediated through GHS-R1a and another type of receptor.

Since in our study the growth of both cancer cell lines was determined by the modified Mosmann’s method, reflecting the number of viable and metaboli- cally active cells, the cellular growth inhibition re- vealed by this method should be interpreted as the inhibition of cell proliferation, or enhancement of cell apoptosis, or changes in both of these processes.

Based on the results of ours and other studies, FU in- deed inhibited the growth of the colon cancer line through both mechanisms, induction of apoptosis and inhibition of proliferation [10, 28].

Summing up, the different members of the ghrelin axis, and D-Lys-GHRP-6 used as the ghrelin receptor type 1a antagonist, affect the growth of MC38 colon cancer and DU145 PCa cell lines with diverse potencies. Their effect depends on the type of the cancer cell line, the applied substance and the concentration used. The inhibitory effect of human UAG on the growth of both cancer lines seems to be mediated via another type of GHS-R than type 1a, because this form of ghrelin does not bind to this receptor subtype.

D-Lys-GHRP-6 used in this study as a growth hormone secretagogue receptor type 1a antagonist caused rather unexpected effects and did not modify the action of acylated ghrelin of rat or human in an antago- nistic way. Thus, under these experimental conditions, D-Lys-GHRP-6 should not be treated as an antagonist. Surprisingly, D-Lys-GHRP-6-enhanced the inhibitory effect of hUAG in both examined cancer cell lines. However, the interpretation of our and other data concerning ghrelins effects on the growth of can- cer cell lines in vitro is rather difficult due to the fact that ghrelin and its receptor are present in some cancer lines including DU145 [20].

Taken together, these results suggest that ghrelin system plays a role in the control of the growth of colon and prostate androgen-independent cancers, and hint the possibility of targeting its activity in the fu- ture therapy of these neoplasms.

Acknowledgment:

The study was supported by grant No.

502-03/1-153-03/502-14-002 from the Medical University of Lodz.

(8)

Palù G.: Loss of growth hormone secretagogue receptor 1a and overexpression of type 1b receptor transcripts in human adrenocortical tumors. Oncology, 2005, 68, 414–421.

2.Broglio F, Gottero C, Benso A, Prodam F, Destefanis S, Gauna C, Maccario M et al.: Non-acylated ghrelin does not possess the pituitaric and pancreatic endocrine activity of acylated ghrelin in humans. J Endocrinol Invest, 2003, 26, 192–196.

3.Bustin SA, Jenkins PJ: The growth hormone-insulin-like growth factor-I axis and colorectal cancer. Trends Mol Med, 2001, 7, 447–454.

4.Calle EE, Rodriguez C, Walker-Thurmond K, Thun MJ:

Overweight, obesity, and mortality from cancer in a pro- spectively studied cohort of U.S. adults. N Engl J Med, 2003, 348, 1625–1638.

5.Cassoni P, Ghé C, Marrocco T, Tarabra E, Allia E, Catapano F, Deghenghi R et al.: Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines. Eur J Endocrinol, 2004, 150, 173–184.

6.Cassoni P, Papotti M, Ghè C, Catapano F, Sapino A, Graziani A, Deghenghi R et al.: Identification, characterization, and biological activity of specific receptors for natural (ghrelin) and synthetic growth hormone secreta- gogues and analogs in human breast carcinomas and cell lines. J Clin Endocrinol Metab, 2001, 86, 1738–1745.

7.Cooperberg MR, Broering JM, Litwin MS: The contem- porary management of prostate cancer in the United States: lessons from the cancer of the prostate strategic urologic research endeavor (CapSURE), a national disease registry. J Urol, 2004, 171, 1393–1401.

8.Corbett TH, Griswold DP Jr, Roberts BJ, Peckham JC, Schabel FM Jr: Tumor induction relationships in development of transplantable cancers of the colon in mice for chemotherapy assays, with a note on carcinogen struc- ture. Cancer Res, 1975, 35, 2434–2439.

9.Cowey SL, Quast M, Belalcazar LM, Wei J, Deng X, Given R, Singh P: Abdominal obesity, insulin resistance, and colon carcinogenesis are increased in mutant mice lacking gastrin gene expression. Cancer, 2005, 103, 2643–2653.

10.De Angelis PM, Svendsrud DH, Kravik KL, Stokke T:

Cellular response to 5-fluorouracil (5-FU) in 5-FU- resistant colon cancer cell lines during treatment and re- covery. Mol Cancer, 2006, 5, 20–21.

11.Dixit VD, Weeraratna AT, Yang H, Bertak D, Cooper- Jenkins A, Riggins GJ, Eberhart CG, Taub DD: Ghrelin and the growth hormone secretagogue receptor constitute a novel autocrine pathway in astrocytoma motility. J Biol Chem, 2006, 281, 16681–16690.

12.Duxbury MS, Waseem T, Ito H, Robinson MK, Zinner MJ, Ashley SW, Whang EE: Ghrelin promotes pancreatic adenocarcinoma cellular proliferation and invasiveness. Biochem Biophys Res Commun, 2003, 309, 464–468.

Steinberg SM, Jones E et al.: A randomized phase II trial of thalidomide, an angiogenesis inhibitor, in patients with androgen-independent prostate cancer. Clin Cancer Res, 2001, 7, 1888–1893.

15.Figg WD, Liu Y, Arlen P, Gulley J, Steinberg SM, Liewehr DJ, Cox MC et al.: A randomized, phase II trial of ketoconazole plus alendronate versus ketoconazole alone in patients with androgen independent prostate cancer and bone metastases. J Urol, 2005, 173, 790–796.

16.Freeman HJ: Risk of gastrointestinal malignancies and mechanisms of cancer development with obesity and its treatment. Best Pract Res Clin Gastroenterol, 2004, 18, 1167–1175.

17.Garcia JM, Li H, Mann D, Epner D, Hayes TG, Marcelli M, Cunningham GR: Hypogonadism in male patients with cancer. Cancer, 2006, 106, 2583–2591.

18.He XT, Fan XM, Zha XL: Ghrelin inhibits 5- fluorouracil-induced apoptosis in colonic cancer cells.

J Gastroenterol Hepatol, 2011, 26, 1169–1173.

19.Hurwitz H, Fehrenbacher L, Novotny W, Cartwright T, Hainsworth J, Heim W, Berlin J et al.: Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med, 2004, 350, 2335–2342.

20.Jeffery PL, Herington AC, Chopin LK: Expression and action of the growth hormone releasing peptide ghrelin and its receptor in prostate cancer cell lines. J Endocri- nol, 2002, 172, R7–11.

21.Kim SW, Her SJ, Park SJ, Kim D, Park KS, Lee HK, Han BH et al.: Ghrelin stimulates proliferation and dif- ferentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells. Bone, 2005, 37, 359–369.

22.Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a growth hormone releasing acylated peptide from stomach. Nature, 1999, 402, 656–660.

23.Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB: Ghrelin – a hormone with multiple functions. Front Neuroendocrinol, 2004, 25, 27–68.

24.Lanfranco F, Baldi M, Cassoni P, Bosco M, Ghé C, Muc- cioli G: Ghrelin and prostate cancer. Vitam Horm, 2008, 77, 301–324.

25.Me³eñ-Mucha G: Effects of short term treatment with pentagastrin, proglumide, tamoxifen given separately or together with 5-fluorouracil on the growth in the murine transplantable Colon 38 cancer. Neoplasma, 2001, 48, 134–139.

26.Me³eñ-Mucha G: The combined effects of tamoxifen or proglumide with 5-fluorouracil on the growth of murine transplantable Colon 38 cancer. GI Cancer, 2001, 3, 383–394.

27.Me³eñ-Mucha G, £awnicka H: Leptin promotes the growth of Colon 38 cancer cells and interferes with the cytotoxic effect of fluorouracil in vitro. Endokrynol Pol, 2007, 58, 2–6.

28.Me³eñ-Mucha G, Winczyk K, Pawlikowski M: Effects of somatostatin analogs octreotide and lanreotide on the proliferation and apoptosis in Colon 38 tumor: interac-

(9)

tion with 5-fluorouracil. Neuroendocrinol Lett, 2000, 21, 137–142.

29.Me³eñ-Mucha G, Winczyk K, Pawlikowski M: Somato- statin analogue octreotide and melatonin inhibit bro- modeoxyuridine incorporation into cell nuclei and en- hance apoptosis in the transplantable murine Colon 38 cancer. Anticancer Res, 1998, 18, 3615–3620.

30.Miyake H, Chi KN, Gleave ME: Antisense TRPM-2 oli- godeoxynucleotides chemosensitize human androgen- independent PC-3 prostate cancer cells both in vitro and in vivo. Clin Cancer Res, 2000, 6, 1655–1663.

31.Motylewska E, Me³eñ-Mucha G: Estrone and progester- one inhibit the growth of murine MC38 colon cancer line. J Steroid Biochem Mol Biol, 2009, 113, 75–79.

32.Motylewska E, Stasikowska O, Me³eñ-Mucha G: The inhibitory effect of diarylpropionitrile, a selective agonist of estrogen receptor beta, on the growth of MC38 colon cancer line. Cancer Lett, 2009, 276, 68–73.

33.Pluta K, Jeleñ M, Morak-M³odawska B, Zimecki M, Artym J, Kociêba M: Anticancer activity of newly syn- thesized azaphenothiazines from NCI’s anticancer screening bank. Pharmacol Rep, 2010, 62, 319–332.

34.Renehan AG, Soerjomataram I, Leitzmann MF: Inter- preting the epidemiological evidence linking obesity and cancer: A framework for population-attributable risk es- timations in Europe. Eur J Cancer, 2010, 46, 2581–2592.

35.Saltz L: Drug treatment of colorectal cancer. Drugs, 1991, 42, 616–627.

36.Schrag D: The price tag on progress – chemotherapy for colorectal cancer. N Engl J Med, 2004, 351, 317–319.

37.Smith JP, Stock EA, Wotring MG, McLaughlin PJ, Zagon IS: Characterization of the CCK-B/gastrin-like receptor in human colon cancer. Am J Physiol, 1996, 271, R797–805.

38.Stone KR, Mickey DD, Wunderli H, Mickey GH, Paul- son DF: Isolation of a human prostate carcinoma cell line (DU 145). Int J Cancer, 1978, 21, 274–281.

39.Szepeshazi K, Schally AV, Groot K, Armatis P, Halmos G, Herbert F, Szende B et al.: Antagonists of growth hormone-releasing hormone (GH-RH) inhibit IGF-II production and growth of HT-29 human colon cancers.

Br J Cancer, 2000, 82, 1724–1731.

40.Thompson NM, Gill DA, Davies R, Loveridge N, Hous- ton PA, Robinson IC, Wells T: Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology, 2004, 145, 234–242.

41.Wang W, Andersson M, Iresjö BM, Lönnroth C, Lund- holm K: Effects of ghrelin on anorexia in tumor-bearing mice with eicosanoid-related cachexia. Int J Oncol, 2006, 28, 1393–1400.

42.Williams H, Powell IJ: Epidemiology, pathology, and ge- netics of prostate cancer among African Americans compared with other ethnicities. Methods Mol Biol, 2009, 472, 439–453.

43.Wingo PA, Tong T, Bolden S: Cancer statistics. CA Can- cer J Clin, 1995, 45, 8–30.

44.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.

Received: November 17, 2011; accepted: March 8, 2012.