Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro

Abstract

Antagonistic analogs of growth hormone-releasing hormone (GHRH) suppress growth of various tumors in vivo. This effect is exerted in part through inhibition of the GHRH-GH-insulin-like growth factor (IGF)-I axis. Nevertheless, because autocrine/paracrine control of proliferation by IGF-II also is a major factor in many tumors, the interference with this growth-stimulating pathway would offer another approach to tumor control. We thus investigated whether GHRH antagonists MZ-4-71 and MZ-5-156 also act on the tumor cells directly by blocking the production of IGF-II. An increase in the IGF-II concentration in the media during culture was found in 13 of 26 human cancer cell lines tested. Reverse transcription-PCR studies on 8 of these cell lines showed that they also expressed IGF-II mRNA. Antagonists of GHRH significantly inhibited the rate of proliferation of mammary (MDA-MB-468 and ZR-75-1), prostatic (PC-3 and DU-145), and pancreatic (MiaPaCa-2, SW-1990, and Capan-2) cancer cell lines as shown by colorimetric and [3H]thymidine incorporation tests and reduced the expression of IGF-II mRNA in the cells and the concentration of IGF-II secreted into the culture medium. Growth and IGF-II production of lung (H-23 and H-69) and ovarian (OV-1063) cancer cells that express mRNA for IGF-II and excrete large quantities of IGF-II also was marginally suppressed by the antagonists. These findings suggest that antagonistic analogs of GHRH can inhibit growth of certain tumors not only by inhibiting the GHRH-GH-IGF-I axis, but also by reducing the IGF-II production and by interfering with the autocrine regulatory pathway.

Figure 1

The effect of antagonistic analogs of GHRH (MZ-4-71 and MZ-5-156) on IGF-II production of human cancer cell lines in culture. Each group consisted of 4–8 wells with 3,000–5,000 cells per well. The cells were exposed to the analogs at 300 nM or 3 μM concentration for 42–97 hours depending on the rate of proliferation of the cell line. Control cultures received medium alone. Changes of IGF-II concentration in the tissue culture media as compared with control groups are plotted as mean ± SEM of 4–6 experiments. Significant differences from the control groups are indicated: ∗, 0.05 > P > 0.01; ∗∗, P < 0.01, two-tailed Student’s t test.

Figure 2

The effects of antagonistic analogs of GHRH on growth of human cancer cells as determined by colorimetric tests (MTT for H-69 and crystal violet for other cells). The experimental conditions were similar to those in Fig. 1. ∗, 0.05 > P > 0.01; ∗∗, P < 0.01, two-tailed Student’s t test.

Figure 3

The effects of antagonistic analogs on [³H]thymidine incorporation into DNA of cultured human cancer cell lines. The cells were exposed to MZ-4-71 or MZ-5-156 for 24 hours, [³H]thymidine was added, and the incubation was continued for 4 hours. Relative [³H] activities of the washed cells (mean ± SEM) are plotted relative to those of control groups. Significant differences from the control groups are indicated. ∗, 0.05 > P > 0.01; ∗∗, P < 0.01, two-tailed Student’s t test.

Figure 4

IGF-II mRNA expression in cultured human cancer cell lines. mRNA from each cell line was amplified by using RT-PCR. After electrophoresis in 2% agarose gel, the product was stained with ethidium bromide. The PCR product was of the expected size (538 bp) for IGF-II. M, pUC18/MspI-digested cDNA marker.

Figure 5

The effect of antagonistic analogs of GHRH on IGF-II mRNA expression in cultured human cancer cell lines. The cells were exposed to the analogs (MZ4 = MZ-4-71, MZ5 = MZ-5-156) at 3 μM concentration for 4 hours. mRNA was extracted from the cells and amplified by using RT-PCR. After electrophoresis in 2% agarose gel, the product was stained with ethidium bromide. For internal control, GAPDH-mRNA was obtained and visualized in the same way from the RNA extracts. The PCR products were of the expected size (538 bp for IGF-II and 207 bp for GAPDH). C, control cells; M, pUC18/MspI-digested cDNA marker.