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 μg Cx46 siRNA (n = 6), 30 μg 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.

Figure 1

Y79 cells express Cx46 and thrive under hypoxic conditions. (A) Y79 whole cell lysates probed with rabbit anti-Cx46 antibody and mouse anti-tubulin antibody, demonstrating Cx46 expression in human Y79 retinoblastoma cells. (B) Viability of Y79 cells under normoxia (37°C humidified ambient air, 5% CO₂) and hypoxia (37°C humidified 1% oxygen displaced by N₂, 5% CO₂) was monitored for 7 days and recorded. (C) Total Y79 cell numbers were counted for 7 days under normoxic and hypoxic growth conditions. Data trends from several experiments indicate that Y79 cells express Cx46 and can survive and thrive in hypoxic conditions (error not calculated). Representative experimental data shown, additional data in supplement.

Figure 2

Western blot of siRNA knockdown in Y79 cells in vitro. Y79 cells treated with 250 nM and 15 uL HiPerfect for each siRNA show were grown in normoxic conditions, lysed at the timepoints shown and western blotted for Cx46 expression. Optimal knockdown of Cx46 was shown at 24 hours.

Figure 3

Viability assay of siRNA knockdown in Y79 cells in vitro. Y79 cells treated with 250 nM and 15 uL HiPerfect for each siRNA were subjected to normoxic and hypoxic growth conditions as shown. Viability (A, B) was measured and cell counts (C, D) taken to assess the knockdown effect of Cx46 on Y79 cell growth characteristics. Each data point shown with standard error (n = 4).

Figure 4

Results of anti-Cx46 siRNA dosing and proof-of-concept in vivo study. 4 groups of mice (n = 2) were used in the study to assess the most effective dosing regimen for the primary study. Tumors receiving anti-Cx46 siRNA were allowed to grow until they became a health concern for the mice harboring the tumors, then the mice were euthanized to prevent any further suffering. A dosage of 30 ug/Injection every other day was used for the primary study. Tabulated data for each mouse can be found Supplemental Data Table 1.

Figure 5

Intratumor Cx46 siRNA suppresses the growth of Y79 tumors in vivo. Macroscopic appearance of Y79 tumors in nude mice 14 days post-transplantation of human Y79 retinoblastoma cells in (A) untreated control tumors, or following 5 siRNA treatments, with (B) 30 µg non-silencing siRNA or (C) 30 µg Cx46 siRNA. Arrows outline the representative tumors in the left dorsal region of the mice. Tabulated data for each mouse can be found Supplemental Data Table 2.

Figure 6

Tumor volume in Cx46 siRNA, non-silencing siRNA, and no siRNA treatment groups of mice transplanted with Y79 cells. Tumor volume of the transplanted mice was measured for a maximum of 21 days, with the administration of 30 µg Cx46 siRNA (n = 6), 30 µg non-silencing siRNA (n = 6), or no treatment (n = 6). (A) The calculated tumor volumes were averaged by treatment group and day over the course of the study (mean ± SE). The tumor volumes of Cx46 siRNA treated mice (n = 6) were statistically significant (* = P ≤ 0.05), when compared to non-silencing siRNA treatment group (n = 6) beginning on 8 days after treatment was initiated and continued to be significant throughout the study. The Cx46 siRNA treatment group was statistically different (P ≤ 0.05) from the untreated, no siRNA group (n = 6) beginning on day 6 and was also significant on days 8, 14, and 16. (B) Final measurements were taken on the excised tumors and the volumes were averaged by group. There was a significant reduction in final tumor volume after treatment with Cx46 siRNA (287 mm³ ± 77 mm³, n = 5) when compared to the non-silencing siRNA (894 mm³ ± 218 mm³, n = 5) or untreated (1068 mm³ ± 192 mm³, n = 6) groups (P ≤ 0.03 and P ≤ 0.002, respectively). (C) Cx46 siRNA treated mice had a decreased tumor weight. Final tumor weights of groups treated with Cx46 siRNA, non-silencing siRNA (negative control siRNA) ,or no siRNA, shown as mean tumor weight (mg) ± SE (n = 6 for each group). The weight of tumors treated with Cx46 siRNA (226 mg ± 75 mg) were significantly less than the tumors of mice treated with non-silencing siRNA (700 mg ± 218 mg, * = P ≤ 0.05) or no siRNA (864 mg ± 136 mg, P ≤ 0.01).

Figure 7

Decreased expression of Cx46 with a reciprocal increase in Cx43 protein expression in Y79 tumors of mice treated with Cx46 siRNA. Conversely, tumors treated with a non-silencing siRNA or were untreated showed Cx46 upregulation and Cx43 downregulation. Equal amounts of Y79 tumor homogenate were analyzed for (A) Cx46 and (B) Cx43 where β-actin was used as the loading control. Lane 1: Tumor #14, sham treated tumor; Lane 2: Tumor #5, anti-Cx46 siRNA treated tumor; Lane 3: Tumor #11, AllStars negative control siRNA treated tumor; Lane 4: Y79 cell lysate positive control for Cx46 expression. Representative tumor western blots shown.

Figure 8

Decreased Y79 tumor cell burden in mice treated with Cx46 siRNA. Excised tissues were fixed, embedded, sectioned and then stained with hematoxylin (purple, nuclei) and eosin (pink, cytoplasm and connective tissue). (A) Untreated sham Y79 tumor (cage 5, mouse 2) (B) Cx46 siRNA treated Y79 tumor (cage 2, mouse 2) (C) Non-silencing siRNA treated Y79 tumor (cage 4, mouse 2). The representative images were taken by a board-certified veterinary pathologist and show the microscopic appearance of the Y79 tumors isolated from the xenograft mice, treated with Cx46 siRNA, a non-silencing siRNA, or sham treated for a maximum of 21 days.