Celia A. Kanashiro

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

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 μg/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.

Synergistic inhibition of growth of lung carcinomas by antagonists of growth hormone-releasing hormone in combination with docetaxel

We investigated the effect of antagonists of growth hormone-releasing hormone (GHRH) MZ-J-7-138 and JV-1-92 on H460 human non-small cell lung carcinoma (NSCLC) xenografted orthotopically into nude mice. Treatment with MZ-J-7-138 or JV-1-92 inhibited orthotopic growth of H460 NSCLC by 52–65% (P < 0.001) and was associated with a significant decrease in protein expression of K-Ras, cyclooxygenase-2 (Cox-2) and phospho-Akt (pAkt). In other experiments, treatment with MZ-J-7-138 or docetaxel reduced tumor volume of s.c. xenografted H460 human NSCLC by 30–36% (P < 0.01). The combination of MZ-J-7-138 and docetaxel resulted in a synergistic growth inhibition of H460 NSCLC xenografts of 63%. MZ-J-7-138 alone or in combination with docetaxel significantly reduced protein levels of K-Ras, Cox-2, and pAkt by 56–63%. Docetaxel given singly diminished the protein levels only of Cox-2 and did not affect K-Ras and pAkt. High-affinity binding sites, mRNA, and protein expression of pituitary GHRH receptors and its splice variant (SV) 1 were found in H460. H460 NSCLC cells contained GHRH peptide, and its growth was significantly inhibited in vitro by 10 μM MZ-J-7-138 (P < 0.001). Serum insulin-like growth factor 1 (IGF1) was not reduced by either GHRH antagonists. These findings suggest that antiproliferative effects of GHRH antagonists in H460 NSCLC are associated with down-regulation of K-Ras, Cox-2, and pAkt. In conclusion, GHRH antagonists in combination with docetaxel synergistically inhibit growth of H460 NSCLC and the expression of K-ras, Cox-2, and pAkt, which might abrogate the signal transduction pathways for cell growth stimulation and therapeutic resistance.

Growth inhibition of non-small-cell lung carcinoma by BN/GRP antagonist is linked with suppression of K-Ras, COX-2, and pAkt

Bombesin (BN) or gastrin-releasing peptide (GRP) can stimulate the growth of neoplasms such as breast cancer and small-cell lung carcinoma (SCLC). Antagonists of BN/GRP have been shown to inhibit these cancers. We evaluated whether antagonists of BN/GRP can suppress the growth of human non-SCLC (NSCLC) xenografted into nude mice. The effect of the administration of BN/GRP antagonist RC-3940-II on the growth of H460 and A549 NSCLC cell lines orthotopically xenografted into the intrapulmonary interstitium was examined. Protein levels of K-Ras, COX-2, Akt/pAkt, WT p53, Erk1/2, and lung resistance-related protein (LRP) in tumors were analyzed by Western blot analaysis, and receptors for BN/GRP were investigated by radioligand-binding studies. The effect of RC-3940-II on the proliferation of H460 and A549 cells in vitro was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. High-affinity receptors for BN/GRP were found on tumors. Treatment with RC-3940-II significantly (P < 0.001) inhibited growth of H460 and A549 NSCLC xenografts by 30–50% and led to an improved performance status, compared with controls. In H460 NSCLC, the antitumor effect was associated with a significant (P < 0.001) reduction in protein levels of K-Ras, COX-2, pAkt, and pERK1/2 and with a major augmentation in the expression of WT p53, compared with controls. In A549 NSCLC, pAkt and LRP were significantly down-regulated. Our findings demonstrate the efficacy of BN/GRP antagonist RC-3940-II for the treatment of NSCLC. The suppression of K-Ras, COX-2, pAkt, and LRP, as well as the up-regulation of WT p53 might contribute to the antitumor action of BN/GRP antagonists.

Suppression of growth of H-69 small cell lung carcinoma by antagonists of growth hormone releasing hormone and bombesin is associated with an inhibition of protein kinase C signaling

We investigated the effects of antagonists of growth hormone‐releasing hormone (GHRH) alone and in combination with bombesin/gastrin‐releasing peptide (BN/GRP) antagonist RC‐3940‐II on the growth of H‐69 human small cell lung carcinoma (SCLC) xenografted into nude mice. Since the activation of the signaling pathways involving protein kinase C (PKC) and the subsequent steps involving mitogen‐activated protein kinase (MAPK) and c‐fos and c‐jun oncogenes are known to be important mechanisms implicated in cellular growth, we investigated how the blockade of tumoral GHRH receptor splice variants and BN/GRP receptors by these antagonists could interfere with these intracellular signaling pathways. Treatment with GHRH antagonists JV‐1‐65 or MZ‐J‐7‐110 for 4 weeks significantly (p<0.05) decreased the tumor volume by 22.7±3.0% and 36.7 ± 3.6%, respectively, as compared to controls. A larger decrease in tumor volume of 73.0 ± 9.5% (p<0.01) was produced by BN/GRP antagonist RC‐3940‐II and its combination with JV‐I‐65 caused the greatest tumor reduction of 91.0 ± 9.8% (p<0.01) vs. controls. H‐69 SCLC tumors expressed α‐, βII‐, δ‐ and η‐PKC isoforms. Antagonists of GHRH and BN/GRP decreased significantly (p<0.05) the expression of βII‐ and δ‐, but not of α‐ and η‐PKC isoforms. They also inhibited MAPK levels, the effects being significant (p<0.05) in the groups that received BN/GRP antagonist. In addition, expression of c‐fos and c‐jun mRNA was reduced after combined treatment with JV‐1‐65 and RC‐3940‐II. The proliferation of H‐69 SCLC cells “in vitro” was also significantly inhibited after incubation of cells with GHRH antagonist, PKC inhibitors or MAPK inhibitor. These findings suggest that the anti‐proliferative effects of antagonists of GHRH and BN/GRP on H69‐SCLC involve an inhibition of the signaling pathways of specific PKC isoforms, MAPK and c‐fos and c‐jun oncogenes.

Targeted chemotherapy with cytotoxic bombesin analogue AN-215 inhibits growth of experimental human prostate cancers

We developed a powerful cytotoxic analogue of bombesin AN‐215, in which the bombesin (BN)‐like carrier peptide is conjugated to 2‐pyrrolino doxorubicin (AN‐201). Human prostate cancers express high levels of receptors for BN/gastrin releasing peptide (GRP) that can be used for targeted chemotherapy. The effects of targeted chemotherapy with cytotoxic BN analogue AN‐215 were evaluated in nude mice bearing subcutaneous xenografts of DU‐145, LuCaP‐35, MDA‐PCa‐2b and intraosseous implants of C4‐2 human prostate cancers. Intraosseous growth of C4‐2 tumors was monitored by serum PSA. BN/GRP receptors were evaluated by 125I‐[Tyr4]BN binding assays and RT‐PCR. The effects of AN‐215 on apoptosis and cell proliferation were followed by histology, and the expression of Bcl‐2 and Bax protein was determined by Western blot analysis. Targeted analog AN‐215 significantly inhibited growth of subcutaneously implanted DU‐145, LuCaP‐35 and MDA‐PCa‐2b prostate cancers by 81% to 91% compared to controls, while cytotoxic radical AN‐201 was less effective and more toxic. Serum PSA levels of mice bearing intraosseous C4‐2 prostate tumors were significantly reduced. In LuCaP‐35 tumors administration of BN antagonist RC‐3095 prior to AN‐215 blocked the receptors for BN/GRP and inhibited the effects of AN‐215. High affinity receptors for BN/GRP and their m‐RNA were detected on membranes of all 4 tumor models. Therapy with AN‐215, but not with AN‐201, decreased the ratio of Bcl‐2/Bax in DU‐145 and the expression of antiapoptotic Bcl‐2 in LuCaP‐35 tumors. The presence of BN/GRP receptors on primary and metastatic prostate cancers makes possible targeted chemotherapy with AN‐215 for the treatment of this malignancy.

Antagonists of bombesin/gastrin-releasing peptide decrease the expression of angiogenic and anti-apoptotic factors in human glioblastoma

We have investigated the antitumor effects and the mechanism of action of antagonists of bombesin/gastrin-releasing peptide (GRP), RC-3940-II and RC-3940-Et, on the growth of U-118MG human malignant glioma xenografted into nude mice. Tumors volume was measured weekly, and after 6 weeks of treatment with GRP antagonists the tumors were analyzed by Western blot assays for the expression of vascular endothelial growth factor (VEGF), protein kinase C (PKC)-&agr;, the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax. A radioreceptor assay was used to characterize the receptors for bombesin/GRP. Specific high-affinity receptors for bombesin were found in U-118MG tumors, and their growth was reduced by 52.5% by RC-3940-II and 72.6% by RC-3940-Et (both p<0.01). The tumor doubling time was prolonged by 4.6 and 12 days after treatment with RC-3940-II and RC-3940-Et, respectively, compared to controls (p<0.05). Both antagonists caused a significant (p<0.05) decrease of about 28% in the levels of VEGF protein and a reduction of approximately 35% in the expression of PKC&agr;. The relative ratio of Bcl-2:Bax was also diminished by around 70% by both analogs, indicating a net apoptotic gain and the efficacy of treatment. Our results suggest that bombesin/GRP antagonists, RC-3940-II and RC-3940-Et, could be of value for the treatment of human glioblastomas.