Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

Abstract

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.

Keywords: peptide analogs; targeted therapy.

Fig. 1.

(A) The effect of treatment with the GHRH agonist JI-34 (50 µg/kg/d or 1 µg/mouse/d) (J) and doxorubicin (DOX; D) (13 µmol/kg/wk) alone and in combination on the growth of U-87 MG, human GBM tumors xenotransplanted to nude mice. Numbers at labels represent the number of successfully implanted tumors. (B) Final weights of necropsied tumor samples compared with the control. (C) Numbers at the end of each curve show the tumor doubling times. *P < 0.05 vs. control.

Fig. 2.

The effect of single exposure (A) or repeated exposure (B) to GHRH agonist JI-34 (1 µM) (J) doxorubicin (100 nM DOX; D), and their combination on the proliferation of U-87 MG cells in vitro. *P < 0.05 vs. control, ⁺P < 0.05 vs. DOX. For conditions see Materials and Methods and Results. (C) Phase-contrast images of the U-87 MG cell cultures. Images are shown at 400× magnification with 20-megapixel resolution. Representative sections of the visual field were cropped and fitted. (Scale bars, 100 µm.)

Fig. 3.

The effect of the combination treatment with the GHRH agonist JI-34 + DOX on viability and apoptosis (A), calcein retention (B), and the expression of bFGF, TGFβ, and the tumor suppressor p53 (C), in vitro. For conditions see Materials and Methods and Results. *P < 0.05 vs. control. DOX, doxorubicin; FGFb, fibroblast growth factor basic; TGFβ, transforming growth factor β. *P < 0.05 vs. control, ⁺P < 0.05 vs. DOX.

Fig. 4.

Western blot images (A) and integrated density values (IDVs) (B) for the expression of GHRH receptors and differentiation antigens in necropsied in vivo samples. GHRH-R, pituitary type growth hormone releasing hormone receptor; SV1, splice variant-1 of GHRH receptor; DOX or D, doxorubicin; J, JI-34; GFAP, glial fibrillary acidic protein. *P < 0.05 vs. control.

Fig. 5.

GHRH agonist induces the differentiation of U-87 MG cells in reduced serum-containing medium in vitro. Cells were treated with GHRH agonist JI-34 (1 µM) or DMSO in 0.1% FBS-containing growth medium. Phase-contrast images of live cells were taken after 2 d and then cells were fixed and stained for the differentiation markers, GFAP and nestin. The GHRH agonist-treated cells possess a higher tendency toward outgrowth of projections; moreover, high levels of GFAP were detected in these cells, indicative of glial differentiation. The protein level of nestin did not change notably following the treatment with GHRH agonist. Nuclei were stained with DAPI. (Scale bars, 100 µm.)