Human renal cell carcinoma expresses distinct binding sites for growth hormone-releasing hormone

Abstract

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the proliferation of various human cancers in vitro and in vivo by mechanisms that include apparent direct effects through specific binding sites expressed on tumors and that differ from pituitary human GHRH (hGHRH) receptors. In this study, GHRH antagonist JV-1-38 (20 microgram/day per animal s.c.) inhibited the growth of orthotopic CAKI-1 human renal cell carcinoma (RCC) by 83% and inhibited the development of metastases to lung and lymph nodes. Using ligand competition assays with (125)I-labeled GHRH antagonist JV-1-42, we demonstrated the presence of specific high-affinity (K(d) = 0.25 +/- 0.03 nM) binding sites for GHRH with a maximal binding capacity (B(max)) of 70.2 +/- 4.1 fmol/mg of membrane protein in CAKI-1 tumors. These receptors bind GHRH antagonists preferentially and display a lower affinity for hGHRH. The binding of (125)I-JV-1-42 is not inhibited by vasoactive intestinal peptide (VIP)-related peptides sharing structural homology with hGHRH. The receptors for GHRH antagonists on CAKI-1 tumors are distinct from binding sites detected with (125)I-VIP (K(d) = 0.89 +/- 0.14 nM; B(max) = 183.5 +/- 2.6 fmol/mg of protein) and also have different characteristics from GHRH receptors on rat pituitary as documented by the insignificant binding of [His(1),(125)I-Tyr(10), Nle(27)]hGHRH(1-32)NH(2). Reverse transcription-PCR revealed the expression of splice variants of hGHRH receptor in CAKI-1 RCC. Biodistribution studies demonstrate an in vivo uptake of (125)I-JV-1-42 by the RCC tumor tissue. The presence of specific receptor proteins that bind GHRH antagonists in CAKI-1 RCC supports the view that distinct binding sites that mediate the inhibitory effect of GHRH antagonists are present on various human cancers.

Figure 1

Representative example of the saturation of ¹²⁵I-JV-1–42 binding to CAKI-1 tumor membrane fraction. The total (▾), nonspecific (■), and specific (●) binding is plotted as a function of radiolabeled peptide concentration. Specific ¹²⁵I-JV-1–42 binding (●) was experimentally determined as the difference between total binding and nonspecific binding in parallel assays in the absence and presence of 1 μM unlabeled JV-1–42. (Inset) Representative Scatchard plot derived from specific ¹²⁵I-JV-1–42 binding to the membrane fraction isolated from CAKI-1 human RCC. Specific binding was determined as described above. Each point represents the mean of four experiments, done in triplicate.

Figure 2

Ligand-binding specificity of GHRH antagonist JV-1–42 binding. Competition for binding of radioligand ¹²⁵I-JV-1–42 to membrane fractions of CAKI-1 human RCC was determined in the presence of increasing concentrations of JV-1–36 (■), JV-1–38 (□), MZ-5–156 (▿), hGHRH(1–44)NH₂ (○), hGHRH(1–29)NH₂ (●), and [His¹,Nle²⁷]hGHRH(1–32)NH₂ (). VIP (♦) as well as glucagon, PACAP, JV-1–53, and PG 97–269 and other unrelated peptides, such as luteinizing hormone-releasing hormone, epidermal growth factor, [Tyr¹¹]somatostatin-14, [Tyr⁴]bombesin, and IGF-I, did not displace the radioligand (data not shown). One hundred percent specific binding is defined as difference between binding in absence and in presence of 10⁻⁵ M JV-1–42. Each data point represents mean of at least two experiments, done in duplicate or triplicate.

Figure 3

Representative Scatchard plot derived from specific ¹²⁵I-VIP binding to the membrane fraction isolated from CAKI-1 human RCC. Specific binding was determined as described in the legend of Fig. 1. Each point represents the mean of three experiments, done in triplicate.

Figure 4

Ligand-binding specificity of VIP binding. Competition for binding of radioligand ¹²⁵I-VIP to membrane fractions of CAKI-1 human RCC was determined in the presence of increasing concentrations of VIP (○), JV-1–53 (▾), PG 97–269 (▿), JV-1–36 (■), JV-1–38 (♦), JV-1–42 (⋄), and MZ-5–156 (□). hGHRH(1–44)NH₂ (▴), hGHRH(1–29)NH₂, [His¹,Nle²⁷]hGHRH(1–32)NH₂, and glucagon as well as unrelated peptides, such as luteinizing hormone-releasing hormone, epidermal growth factor, [Tyr¹¹]somatostatin-14, [Tyr⁴]bombesin, and IGF-I, did not displace the radioligand (data not shown). One hundred percent specific binding is defined as the difference between binding in absence and in presence of 10⁻⁵ M VIP. Each data point represents mean of at least two experiments, done in duplicate or triplicate.

Figure 5

RT-PCR analysis of mRNA of GHRH receptor and its splice variants in human pituitary adenoma and CAKI-1 human RCC. Poly(A)⁺ RNA was reverse transcribed and amplified in PCR with primers for exon 3–4 (group 1), exon 7–8 (group 2), and intron 3–exon 8 (group 3) of human GHRH receptor gene. The secondary PCR products were separated electrophoretically on 1.5% agarose gels and stained with ethidium bromide. The PCR products were of the expected size of 144 bp (exon 3–4) (group 1); 147 bp (exon 7–8) (group 2); as well as 720 bp, 566 bp, and 335 bp (intron 3–exon 8) (group 3). Lanes: M, 100-bp DNA molecular weight marker; P, human pituitary adenoma; C, CAKI-1 RCC; −, RT-negative control from the mixture of poly(A)⁺ RNA from human pituitary adenoma and CAKI-1 RCC.

Figure 6

The effect of GHRH antagonist JV-1–38 on growth of primary tumors (A) and development of metastases to the lymph nodes and lung (B) in nude mice implanted orthotopically with CAKI-1 RCC. Treatment with JV-1–38 (20 μg/day s.c.) was started 1 week after implantation and lasted for 30 days. Bars, SE; *, P < 0.01 vs. control.