Induction of Retinoid X Receptor Activity and Consequent Upregulation of p21WAF1/CIP1 by Indenoisoquinolines in MCF7 Cells

Abstract

Retinoid X receptor (RXR) has been targeted for the chemoprevention and treatment of cancer. To discover potential agents acting through RXRs, we utilized an RXR response element-luciferase reporter gene assay. Following extensive screening, 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride (AM6-36) was found to induce RXRE-luciferase activities. AM6-36 inhibited cyclooxygenase-2 expression and anchorage-independent growth with 12-O-tetradecanoylphorbol 13-acetate-stimulated JB6 Cl41 cells, induced the expression of CD38 in HL-60 cells, and attenuated the growth of N-methyl-N-nitrosourea-induced mammary tumors in rats. Consistent with other reports describing the anti-proliferative effects of RXR agonists in breast cancers, AM6-36 showed growth inhibition with cultured MCF7 breast cancer cells, accompanied by G2/M phase arrest at lower concentrations and enhanced S phase arrest at higher concentrations. Based on DNA microarray analysis, AM6-36 up-regulated the expression of CDKN1A, a target gene of RXR, by 35-fold. In accord with this response, the expression of the corresponding protein, p21WAF1/CIP1, was increased in the presence of AM6-36. Induction of p21 by AM6-36 was abrogated following transient knock-down of RXRα, demonstrating the effect of AM6-36 on the expression of p21 is closely related to modulation of RXRα transcriptional activity. Intestinal permeability was suggested with Caco-2 cells, and limited metabolism resulted when AM6-36 was incubated with human liver microsomes. Oral administration with rats resulted in 0.8 μg/ml, 4.3 μg/g, and 0.3 μg/g in serum, liver, and mammary gland, respectively. In sum, these data suggest AM6-36 is a promising lead for the treatment or prevention of breast cancer and provide a strong rationale for testing in more advanced anti-tumor systems.

Skeletal muscle PGC-1α controls whole-body lactate homeostasis through estrogen-related receptor α-dependent activation of LDH B and repression of LDH A

“…2 E and F). Interestingly, the administration of AM6-36, a drug that binds to RXR responsive elements and mimics the effects of RXRs, has recently been reported to abrogate Myc expression (26,27). Indeed, also in our experimental context, pharmacological inhibition of RXRs by HX-531 was sufficient to restore Myc (Fig.…”

Induction of Apoptosis by 3-Amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline via Modulation of MAPKs (p38 and c-Jun N-terminal Kinase) and c-Myc in HL-60 Human Leukemia Cells

“…13 As currently described, we have examined the effect of AM6-36 on HL-60 cell proliferation as well as cell cycle distribution. Clearly, AM6-36 demonstrated potent inhibition of cell proliferation via accumulation of cells in the subG1 and G2/M phases of the cell cycle.…”

“…Of a total of 5000 substances tested, only one, our indenoisoquinoline 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11 H -indeno[1,2- C ]isoquinoline dihydrochloride (AM6-36; 1 ), was found to induce the transactivation of the RXR response element (RXRE) in a cell-line based dual luciferase assay system. 13 RXRE is one of the cis -acting hormone response elements in the promoter region of target genes, which consists of two repeated consensus hexamers (AGGTCA) separated by one nucleotide, and can be transactivated by RXRs. 14 Both naturally occurring and chemically synthesized rexinoids, including 9- cis -retinoic acid, AGN194204, CD3254, LG100268, LGD1069 (bexarotene), and SR11237 have shown promise in the prevention and treatment of cancer by inducing differentiation and apoptosis.…”

Identification, Synthesis, and Biological Evaluation of the Metabolites of 3-Amino-6-(3′-aminopropyl)-5H-indeno[1,2-c]isoquinoline-5,11-(6H)dione (AM6–36), a Promising Rexinoid Lead Compound for the Development of Cancer Chemotherapeutic and Chemopreventive Agents

“…The assay was performed as described previously by Park et al 23 with a slight modification. Briefly, after the transient transfection of pRXRE (100 ng/well), phRXRα (50 ng/well), and pRL (3 ng/well) by Lipofectamine™ 2000 for 24 h, COS-1 cells were incubated with compounds or vehicle (DMSO) for additional 12 h. Cells were lysed and the RXRE transcriptional activities of compounds were determined by measuring the luciferase activities using Dual-Luciferase ® Reporter Assay System.…”

“…The indenoisoquinoline 3-amino-6-(3'-aminopropyl)-5,6-dihydro-5,11-dioxo-11 H -indeno[1,2- c ]isoquinoline (AM6-36, 23 3 ) was synthesized in our laboratory and found through screening to bind to RXRα and induce apoptosis in MCF-7 breast cancer cells. 23 Since these data suggest that 3 is a promising lead compound for the treatment or prevention of breast cancer, drug development studies were carried out that included evaluation of metabolic stability, intestinal permeability, metabolic transformation by human liver microsomes and human hepatocytes, binding to human plasma proteins, and preliminary disposition studies in rats.…”