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. 2012 Jul;3(7):686-99.
doi: 10.18632/oncotarget.546.

Receptor-targeted therapy of human experimental urinary bladder cancers with cytotoxic LH-RH analog AN-152 [AEZS- 108]

Karoly Szepeshazi  1 , Andrew V Schally, Gunhild Keller, Norman L Block, Daniel Benten, Gabor Halmos, Luca Szalontay, Irving Vidaurre, Miklos Jaszberenyi, Ferenc G Rick
Affiliations

Receptor-targeted therapy of human experimental urinary bladder cancers with cytotoxic LH-RH analog AN-152 [AEZS- 108]

Karoly Szepeshazi et al. Oncotarget. 2012 Jul.

Abstract

Many bladder cancers progress to invasion with poor prognosis; new therapeutic methods are needed. We developed a cytotoxic LH-RH analog, AN-152 (AEZS-108) containing doxorubicin (DOX), for targeted therapy of cancers expressing LHRH receptors. We investigated the expression of LH-RH receptors in clinical bladder cancers and in HT-1376, J82, RT-4 and HT-1197 human bladder cancer lines. The effect of analog, AN-152, on growth of these tumor lines xenografted into nude mice was analyzed. Using molecular and functional assays, we also evaluated the differences between the effects of AN-152, and DOX alone. We demonstrated the expression of LH-RH receptors on 18 clinical bladder cancers by immunohistochemistry and on four human urinary bladder cancer lines HT-1376, J82, RT-4 and HT-1197 by Western blotting and binding assays. AN-152 powerfully inhibited growth of these bladder cancers in nude mice. AN-152 exerted greater effects than DOX and was less toxic. DOX activated strong multidrug resistance mechanisms in RT-4 and HT-1197 cancers, while AN-152 had no or less such effect. PCR assays and in vitro studies revealed differences in the action of AN-152 and DOX on the expression of genes involved in apoptosis. These results suggest that targeted cytotoxic LH-RH analog, AN-152 (AEZS- 108), should be examined for treatment of patients with LH-RH receptor positive invasive bladder cancers.

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Conflict of interest statement

Dr. A.V. Schally is listed as co-inventor on the Tulane University patents on AN-152, but this study was experimental. No potential conflict of interest exists for other authors.

Figures

Figure 1
Figure 1. Expression of LH-RH receptors in human bladder carcinomas
The tissue was stained by immunohistochemistry with primary LH-RH receptor antibody. A-C: urothelial bladder carcinoma; D: positive control, anterior pituitary.
Figure 2
Figure 2. Effect of treatment with cytotoxic LH-RH analog, AN-152 (AEZS-108), and doxorubicin (DOX), on growth of human bladder cancers in nude mice
The vertical bars represent SE. Solid arrows show treatments with both cytotoxic compounds, dashed arrow shows treatment only with AN-152 (2d).
Figure 3
Figure 3. Detection LH-RH receptor protein (38 KD) by Western blotting
All four human urinary bladder cancer lines grown in nude mice expressed LH-RH receptors. Representative blots of three independent experiments are shown.
Figure 4
Figure 4. Human urinary bladder cancers grown in nude mice were analyzed with the Human Cancer Drug Resistance & Metabolism RT Profiler PCR Array The vertical bars on the left shows 10-fold change compared to control
*= P<0.05 vs. control. (A)Changes in genes involved in drug resistance. ABCB1: ATP-binding cassette, subfamily B, member 1; ABCC1-6: ATP-binding cassette, subfamily C, members 1-6; ABCG2: ATP-binding cassette, subfamily G, member 2; BAX: BCL2-assiciated X protein; BCL2: B-cell CCL/lymphoma 2; BCL2L1: BCL2-like 1; MVP: Major vault protein; RB1: Retinoblastoma 1; TOP1: Topoisomerase (DNA) I; TOP2A: Topoisomerase (DNA) II alpha; TOP2B: Topoisomerase (DNA) II beta; TP53: Tumor protein p53. (B)Changes in genes involved in drug metabolism. ARNT: Aryl hydrocarbon receptor nuclear translocator; BLMH: Bleomycin hydrolase; CLPTM1L: Cleft lip and palate transmembrane protein 1-like protein (cisplatin resistance-related protein); CYP1A1, CYP1A2, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5: Cytochrome P450, family 1-3, subfamily A-D, polypeptide 1-19; DHFR: Dihydrofolate reductase; EPHX1: Epoxide hydrolase, microsomal (xenobiotic); GSK3A: Glycogen synthase kinase 3 alpha; GSTP1: Glutathione S-transferase pi 1; NAT2: N-acetyltransferase 2; SOD1: Superoxide dismutase 1; SULT1E1: Sulfotransferase family 1E, estrogen-preferring, member 1; TPMT: Thiopurine S-methyltrasferase. (C)Changes in genes involved in DNA repair. 1: APC: Adenomatous polyposis coli; ATM: Ataxia teleangiectasia mutated; BRCA1, BRCA2: Breast cancer 1, 2; ERCC3: Excision repair cross-complementing rodent repair deficiency, complementation group 3 (xeroderma pigmentosum group B complementing); MSH2:MutS homolog 2, colon cancer, nonpopyposis type 1; XPA, XPC: Xeroderma pigmentosum, complementation group A, C. (D)Genes involved in cell cycle. CCND1, CCNE1: Cyclin D1, E1; CDK2, CDK4: Cyclin dependent kinase 2, 4; CDKN1A, CDKN1B, CDKN2A: CDKN2D: Cyclin-dependent kinase inhibitor 1A, 1B, 2A, 2D. (E)Changes in growth factor genes. EGFR: Epidermal growth factor receptor; ERBB2, ERBB3, ERBB4: V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, 3, 4:FGF2: Fibroblast growth factor 2; IGF1R, IGF2R: Insulin-like growth factor 1, 2 receptor; MET: Met proto-oncogene (hepatocyte growth factor receptor). (F)Hormone receptor genes. AR: Androgen receptor;ESR1, ESR2: Estrogen receptor 1, 2; PPARA, PPARD, PPARG: Peroxisome proliferator-activated receptor alpha, beta, gamma; RARA, RARB, RARG: Retioic acid receptor alpha, beta, gamma; RXRA, RXRB: Retinoid X receptor alpha, beta. (G)Genes related to transcription factors. AHR: Aryl hydrocarbon receptor; AP1S1: Adaptor-related protein complex1, sigma 1 subunit; ELK1: ELK1, member of ETS oncogene family; FOS: FBJ murine osteosarcoma viral oncogene homolog; HIF1A: Hypoxia inducible factor 1, alpha subunit; MYC: V-myc myelocytomatosis viral oncogene homolog; NFKB1, NFKB2, NFKBIB, NFKBIE: Nuclear factor of kappa light polypeptide gene enhancer in B cells1, 2, inhibitor beta, epsilon; RELB: V-rel reticuloendotheliosis viral oncogene homolog B; TNFRSF11A: Tumor necrosis factor receptor superfamily, member 11a.
Figure 4
Figure 4. Human urinary bladder cancers grown in nude mice were analyzed with the Human Cancer Drug Resistance & Metabolism RT Profiler PCR Array The vertical bars on the left shows 10-fold change compared to control
*= P<0.05 vs. control. (A)Changes in genes involved in drug resistance. ABCB1: ATP-binding cassette, subfamily B, member 1; ABCC1-6: ATP-binding cassette, subfamily C, members 1-6; ABCG2: ATP-binding cassette, subfamily G, member 2; BAX: BCL2-assiciated X protein; BCL2: B-cell CCL/lymphoma 2; BCL2L1: BCL2-like 1; MVP: Major vault protein; RB1: Retinoblastoma 1; TOP1: Topoisomerase (DNA) I; TOP2A: Topoisomerase (DNA) II alpha; TOP2B: Topoisomerase (DNA) II beta; TP53: Tumor protein p53. (B)Changes in genes involved in drug metabolism. ARNT: Aryl hydrocarbon receptor nuclear translocator; BLMH: Bleomycin hydrolase; CLPTM1L: Cleft lip and palate transmembrane protein 1-like protein (cisplatin resistance-related protein); CYP1A1, CYP1A2, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A4, CYP3A5: Cytochrome P450, family 1-3, subfamily A-D, polypeptide 1-19; DHFR: Dihydrofolate reductase; EPHX1: Epoxide hydrolase, microsomal (xenobiotic); GSK3A: Glycogen synthase kinase 3 alpha; GSTP1: Glutathione S-transferase pi 1; NAT2: N-acetyltransferase 2; SOD1: Superoxide dismutase 1; SULT1E1: Sulfotransferase family 1E, estrogen-preferring, member 1; TPMT: Thiopurine S-methyltrasferase. (C)Changes in genes involved in DNA repair. 1: APC: Adenomatous polyposis coli; ATM: Ataxia teleangiectasia mutated; BRCA1, BRCA2: Breast cancer 1, 2; ERCC3: Excision repair cross-complementing rodent repair deficiency, complementation group 3 (xeroderma pigmentosum group B complementing); MSH2:MutS homolog 2, colon cancer, nonpopyposis type 1; XPA, XPC: Xeroderma pigmentosum, complementation group A, C. (D)Genes involved in cell cycle. CCND1, CCNE1: Cyclin D1, E1; CDK2, CDK4: Cyclin dependent kinase 2, 4; CDKN1A, CDKN1B, CDKN2A: CDKN2D: Cyclin-dependent kinase inhibitor 1A, 1B, 2A, 2D. (E)Changes in growth factor genes. EGFR: Epidermal growth factor receptor; ERBB2, ERBB3, ERBB4: V-erb-b2 erythroblastic leukemia viral oncogene homolog 2, 3, 4:FGF2: Fibroblast growth factor 2; IGF1R, IGF2R: Insulin-like growth factor 1, 2 receptor; MET: Met proto-oncogene (hepatocyte growth factor receptor). (F)Hormone receptor genes. AR: Androgen receptor;ESR1, ESR2: Estrogen receptor 1, 2; PPARA, PPARD, PPARG: Peroxisome proliferator-activated receptor alpha, beta, gamma; RARA, RARB, RARG: Retioic acid receptor alpha, beta, gamma; RXRA, RXRB: Retinoid X receptor alpha, beta. (G)Genes related to transcription factors. AHR: Aryl hydrocarbon receptor; AP1S1: Adaptor-related protein complex1, sigma 1 subunit; ELK1: ELK1, member of ETS oncogene family; FOS: FBJ murine osteosarcoma viral oncogene homolog; HIF1A: Hypoxia inducible factor 1, alpha subunit; MYC: V-myc myelocytomatosis viral oncogene homolog; NFKB1, NFKB2, NFKBIB, NFKBIE: Nuclear factor of kappa light polypeptide gene enhancer in B cells1, 2, inhibitor beta, epsilon; RELB: V-rel reticuloendotheliosis viral oncogene homolog B; TNFRSF11A: Tumor necrosis factor receptor superfamily, member 11a.
Figure 5
Figure 5. Effects of AN-152, and DOX on HT-1376, J82 and RT-4 human urinary bladder cancer cells in vitro
5a: calcein retention in the cells analyzed with MDR Assay Kit and 5b: apoptosis investigated with the Multi-Parameter Apoptosis Assay.
See this image and copyright information in PMC

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