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Treatment with connexin 46 siRNA suppresses the growth of human Y79 retinoblastoma cell xenografts in vivo.

ABSTRACT: Tumors with a hypoxic component, including human Y79 retinoblastoma cells, express a specific gap junction protein, Connexin 46 (Cx46), which is usually only found in naturally hypoxic tissues such as the differentiated lens. The aim of this study was to investigate if Cx46 downregulation would suppress Y79 tumor formation in vivo. Five-week old nude mice were subcutaneously implanted with human Y79 retinoblastoma cells and treated with intratumor siRNA injections of 30 ?g Cx46 siRNA (n = 6), 30 ?g non-silencing siRNA (n = 6), or no siRNA treatment (n = 6) every 2 days for a maximum of 10 treatments. Tumor volume (TV) was calculated from the recorded caliper measurements of length and width. Excised tumors were measured and weighed. Western blot analyses were performed to evaluate Cx46 and Cx43 expression in tumors which received Cx46 siRNA, non-silencing siRNA, or no siRNA treatment. Tumor histopathology was used to assess tumor features. Cx46 siRNA treated Y79 tumors had a reduced TV (287 mm(3) ± 77 mm(3)) when compared to the tumors of mice receiving the negative control siRNA (894 mm(3) ± 218 mm(3); P ? 0.03) or no siRNA (1068 mm(3) ± 192 mm(3); P ? 0.002). A 6-fold knockdown of Cx46 and a 3-fold rise in Cx43 protein expression was observed from western blots of tumors treated with Cx46 siRNA compared to mice treated with non-silencing siRNA. Knockdown of Cx46 with siRNA had an antitumor effect on human Y79 retinoblastoma tumors in the nude mouse model. The results suggest that anti-Cx46 therapy may be a potential target in the future treatment of retinoblastoma.

SUBMITTER: Burr DB

PROVIDER: S-EPMC3060947 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Treatment with connexin 46 siRNA suppresses the growth of human Y79 retinoblastoma cell xenografts in vivo.

Burr Diana B DB Molina Samuel A SA Banerjee Debarshi D Low Derek M DM Takemoto Dolores J DJ

Experimental eye research 20110212 4

Tumors with a hypoxic component, including human Y79 retinoblastoma cells, express a specific gap junction protein, Connexin 46 (Cx46), which is usually only found in naturally hypoxic tissues such as the differentiated lens. The aim of this study was to investigate if Cx46 downregulation would suppress Y79 tumor formation in vivo. Five-week old nude mice were subcutaneously implanted with human Y79 retinoblastoma cells and treated with intratumor siRNA injections of 30 μg Cx46 siRNA (n = 6), 30 ...[more]

PMID: 21320488

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Project description:Retinoblastoma (Rb), the most common childhood intraocular malignant tumor, is reported to have cancer stem cells (CSCs) similar to other tumors. Our previous investigation in primary tumors identified the small sized cells with low CD133 (Prominin-1) and high CD44 (Hyaluronic acid receptor) expression to be putative Rb CSCs using flow cytometry (FSClo/SSClo/CD133lo/CD44hi). With this preliminary data, we have now utilized a comprehensive approach of in vitro characterization of Y79 Rb cell line following CSC enrichment using CD133 surface marker and subsequent validation to confirm the functional properties of CSCs.The cultured Rb Y79 cells were evaluated for surface markers by flow cytometry and CD133 sorted cells (CD133lo/CD133hi) were compared for CSC characteristics by size/percentage, cell cycle assay, colony formation assay, differentiation, Matrigel transwell invasion assay, cytotoxicity assay, gene expression using microarray and validation by semi-quantitative PCR.Rb Y79 cell line shared the profile (CD133, CD90, CXCR4 and ABCB1) of primary tumors except for CD44 expression. The CD133lo cells (16.1 ± 0.2%) were FSClo/SSClo, predominantly within the G0/G1 phase, formed larger and higher number of colonies with ability to differentiate to CD133hi cells, exhibited increased invasive potential in a matrigel transwell assay (p < 0.05) and were resistant to Carboplatin treatment (p < 0.001) as compared to CD133hi cells. The CD133lo cells showed higher expression of several embryonic stem cell genes (HOXB2, HOXA9, SALL1, NANOG, OCT4, LEFTY), stem cells/progenitor genes (MSI2, BMI1, PROX1, ABCB1, ABCB5, ABCG2), and metastasis related gene- MACC1, when compared to the CD133hi cells.This study validates the observation from our earlier primary tumor study that CSC properties in Rb Y79 cell line are endowed within the CD133lo population, evident by their characteristics- i.e. small sized, dormant in nature, increased colony forming ability, differentiation to CD133hi cells, higher invasiveness potential, drug resistance and primitive gene expression pattern. These findings provide a proof of concept for methodological characterization of the retinoblastoma CSCs with future implications for improved diagnostic and treatment strategies.

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Treatment with connexin 46 siRNA suppresses the growth of human Y79 retinoblastoma cell xenografts in vivo.

Publications

Treatment with connexin 46 siRNA suppresses the growth of human Y79 retinoblastoma cell xenografts in vivo.

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