Antagonists of growth hormone-releasing hormone receptor induce apoptosis specifically in retinoblastoma cells

Abstract

Retinoblastoma (RB) is the most common intraocular cancer in children worldwide. Current treatments mainly involve combinations of chemotherapies, cryotherapies, and laser-based therapies. Severe or late-stage disease may require enucleation or lead to fatality. Recently, RB has been shown to arise from cone precursor cells, which have high MDM2 levels to suppress p53-mediated apoptosis. This finding leads to the hypothesis that restoring apoptosis mechanisms in RBs could specifically kill the cancer cells without affecting other retinal cells. We have previously reported involvement of an extrapituitary signaling pathway of the growth hormone-releasing hormone (GHRH) in the retina. Here we show that the GHRH receptor (GHRH-R) is highly expressed in RB cells but not in other retinal cells. We induced specific apoptosis with two different GHRH-R antagonists, MIA-602 and MIA-690. Importantly, these GHRH-R antagonists do not trigger apoptosis in other retinal cells such as retinal pigmented epithelial cells. We delineated the gene expression profiles regulated by GHRH-R antagonists and found that cell proliferation genes and apoptotic genes are down-and up-regulated, respectively. Our results reveal the involvement of GHRH-R in survival and proliferation of RB and demonstrate that GHRH-R antagonists can specifically kill the RB cells.GHRH pathway | GHRH-R antagonist | retinoblastoma | growth hormonereleasing hormone | apoptosis R etinoblastoma (RB) is the most common childhood intraocular malignancy and accounts for approximately 3% of all childhood cancers (1). It has been reported to arise from the cone precursor cells of the retina that transduce light into electrical signals (2). RB is thought to result largely from the loss of function of both copies of RB1 gene located on human chromosome 13q14 (3, 4). Functional disruption of RB1 can be generated by the somatic inactivation of both RB1 alleles, or with a germline RB1 mutation in one allele and a somatic inactivation of the second RB1 allele (5). The RB1 protein acts as a signal transducer connecting cell cycle progression with the transcription machinery (6). There are four steps in the mitotic cycle of a cell: G1, S, G2, and cell division. In the G1 phase, cyclin D is highly expressed, which leads to activation of cyclin-dependent kinases (CDKs) 4 and 6. CDK4 and CDK6 then phosphorylate RB1, inhibiting RB1 binding to the transcription factor E2F (7, 8). As a result, the RB1-free E2F binds to promotors of several genes and turns on their expressions to induce cell cycle progression into S phase, the DNA synthesis phase. Similarly, cells carrying RB1 mutations would also progress into S phase. Normally, this premature progression into S phase would trigger apoptosis to prevent uncontrolled cell proliferation (9). However, it has been reported that the cone precursor cells express high levels of MDM2, a protein that suppresses apoptosis mediated by p53 (2). Therefore, cone precursor cells in patients carrying RB1 mutations pass through the cell cycle faster an...

Expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in human primary conjunctival and pterygium cell lines and in mouse cornea

“…Human ocular cell lines, including retinal pigment epithelial (RPE) cells ARPE-19 (catalogue number: CRL-2302, American Type Culture Collection (ATCC), Manassas, VA) [11,12], lens epithelial cells B3 (catalogue number: CRL-11421, ATCC) [13], primary trabecular meshwork (TM) cells [14], and retinoblastoma cells Y79 (catalogue number: HTB-18, ATCC) [15], have been previously established. We also collected human mesenchymal stem cells (MSCs) from different sources, including bone marrow-derived MSCs (BM-MSCs; catalogue number: 7500; ScienceCell Research Laboratories, Carlsbad, CA) [16], adipose-derived stem cells (ASCs) [17], and periodontal ligament-derived stem cells (PDLSCs) [18,19].…”

Induction of Apoptosis in Pterygium Cells by Antagonists of Growth Hormone–Releasing Hormone Receptors

“…To investigate the molecular pathways regulated by the GHRH-R antagonist in apoptosis, mRNA was extracted from MIA-602-treated cells and subjected to reverse transcription coupled with quantitative real-time PCR (qRT-PCR). In our previous study, 5 we have found that GHRH-R antagonist suppresses ERK1 expression and induces Casp3 expression in retinoblastoma cells. In pterygium cells, we observed that MIA-602 could also significantly suppress ERK1 expression and induce Casp3 expression in a dose-dependent manner (Fig.…”

“…The relative ERK1 and Casp 3 mRNA expression in each sample was calculated as described previously. 5,8…”

“…7 These antagonists decrease cell proliferation and survival in cancer cell lines through pleiotropic antitumor mechanisms such as suppression of AKT/ERK signaling cascades and downregulation of PAK1-STAT3/NF-jB signaling pathway. 10,11 Our previous study 5 also has shown that MIA-602 can induce the expression of Casp3, which induces apoptosis in retinoblastoma cells.…”

“…Similarly, in another ocular disease, namely, retinoblastoma, the transcriptional activity of p53 is also inhibited by MDM2. 4 Despite p53 inactivity, our previous study 5 has shown that antagonists of growth hormonereleasing hormone receptor (GHRH-R) can induce the expression of Caspase 3 (Casp3), which induces apoptosis in retinoblastoma cells. Therefore, we hypothesized that GHRH-R inhibitors could also induce apoptosis in PECs.…”