Inhibition of mutant p53 expression and growth of DMS-153 small cell lung carcinoma by antagonists of growth hormone-releasing hormone and bombesin

Abstract

We investigated the effects of growth hormone-releasing hormone (GHRH) antagonists, JV-1-65 and JV-1-63, and bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC-3940-II on DMS-153 human small cell lung carcinoma xenografted into nude mice. Treatment with 10 microg/day JV-1-65 or RC-3940-II decreased tumor volume by 28% (P < 0.05) and 77% (P < 0.01), respectively, after 42 days compared with controls. Combination of JV-1-65 and RC-3940-II induced the greatest inhibition of tumor proliferation (95%; P < 0.01), suggesting a synergism. Western blotting showed that the antitumor effects of these antagonists were associated with inhibition of the expression of the mutant tumor suppressor protein p53 (Tp53). Mutation was detected by sequence analysis of the p53 gene at codon 155: ACC [Thr] --> CCC [Pro]. Combination of JV-1-65 and RC-3940-II decreased the levels of mutant p53 protein by 42% (P < 0.01) compared with controls. JV-1-65, JV-1-63, and RC-3940-II, given singly, reduced mutant p53 protein expression by 18-24% (P < 0.05). Serum insulin-like growth factor (IGF)-I levels were diminished in animals receiving GHRH antagonists. mRNA levels for IGF-II, IGF receptor-I, GRP receptor, and EGF receptor in tumors were significantly decreased by combined treatment with JV-1-65 and RC-3940-II. DMS-153 tumors expressed mRNAs for GHRH and GHRH receptor splice variants 1 and 2, suggesting that GHRH could be an autocrine growth factor. Proliferation of DMS-153 cells in vitro was stimulated by GRP and IGF-II and inhibited by JV-1-65. This study indicates that GHRH antagonists and BN/GRP antagonist inhibit the growth of DMS-153 small cell lung carcinoma concomitantly with the expression of mutant Tp53, which might uncouple the signal transduction pathways for cell growth stimulation.

Fig. 1.

Tumor volumes in nude mice bearing DMS-153 human SCLC during treatment with the GHRH antagonists JV-1-65, JV-1-63, and BN/GRP antagonist RC-3940-II alone or in combination with JV-1-65, all at a dose of 10 μg/day per animal. Vertical bars indicate SEM. *, P < 0.05 vs. control; **, P < 0.01 vs. control; †, P < 0.05 vs. RC-3940-II.

Fig. 2.

Expression of mRNA for GHRH (A) and GHRH-SV receptors (B) in DMS-153 SCLC tumors. Poly(A) RNAs from individual tumor samples were subjected to RT-PCR analysis, electrophoresed on agarose gel, and stained with ethidium bromide. (A) RT-PCR analysis shows a product of 322 bp for GHRH. (B) PCR products of 720 bp for SV1 and 566 bp for SV2 were detected. Lane M, 100-bp DNA molecular marker; lane 1, untreated tumor; lane 2, tumor treated with RC-3940-II; lane 3, tumor treated with JV-1-65; lane 4, tumor treated with RC-3940-II and JV-1-65; lane 5, tumor treated with JV-1-63; lane 6, positive control from LNCaP human prostate cancer cells; lane 7, negative control.

Fig. 3.

RT-PCR analysis of IGF-II and IGFR-I in samples of DMS-153 SCLC tumors. PCR products were of the expected sizes of 538 bp for IGF-II, 447 bp for IGFR-I, and 459 bp for β-actin. Lane M, 100-bp DNA molecular weight marker; lanes 1-5, untreated animals; lanes 6-9, animals treated with RC-3949-II; lanes 10-13, animals treated with JV-1-65; lanes 14-16, animals treated with RC-3940-II + JV-1-65; lanes 17-20, animals treated with JV-1-63.

Fig. 4.

Expression of mRNA for GRPR and EGFR in samples of DMS-153 SCLC. PCRs yielded products the size of 159 bp for GRPR, 400 bp for EGFR, and 459 bp for β-actin. Lane M, 100-bp DNA molecular mass marker; lanes 1-5, untreated animals; lanes 6-9, animals treated with RC-3940-II; lanes 10-12, animals treated with RC-3940-II + JV-1-65.

Fig. 5.

Expression of mutant p53 in DMS-153 SCLC tumors. Protein matched samples of tumor tissues from mice untreated or treated with JV-1-65, JV-1-63, and RC-3940-II alone or combined with JV-1-65 were submitted to Western blotting assays. Whole tissue homogenate was examined for p53 by using specific antibody (1:1,000) and immunoblot analysis. (A) Three representative tumors from each group are shown. (B) Percentages of protein measurements of mutant p53 are presented as means ± SEM of six to eight tumor samples from each group. Values were normalized with the expression of β-actin. *, P < 0.05 vs. control; **, P < 0.01 vs. control.

Fig. 6.

Effects of GHRH, GRP, their antagonists, and IGF-I and IGF-II on cell proliferation of DMS-153 human SCLC line in vitro. Cultured cells were treated for 96 h with GHRH, GHRH antagonist JV-1-65, GRP, BN/GRP antagonist RC-3940-II, IGF-I, and IGF-II at different concentrations. Cell growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. Data are expressed as percentage T/C values, where T represents absorbance of treated cells and C represents the absorbance of control cells. The measured absorbance is proportional to the number of living cells.