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, 2012, 518437

Regulator of G-Protein Signaling 5 Reduces HeyA8 Ovarian Cancer Cell Proliferation and Extends Survival in a Murine Tumor Model

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Regulator of G-Protein Signaling 5 Reduces HeyA8 Ovarian Cancer Cell Proliferation and Extends Survival in a Murine Tumor Model

Molly K Altman et al. Biochem Res Int.

Abstract

The regulator of G-protein signaling 5 (RGS5) belongs to a family of GTPase activators that terminate signaling cascades initiated by extracellular mediators and G-protein-coupled receptors. RGS5 has an interesting dual biological role. One functional RGS5 role is as a pericyte biomarker influencing the switch to angiogenesis during malignant progression. Its other functional role is to promote apoptosis in hypoxic environments. We set out to clarify the extent to which RGS5 expression regulates tumor progression-whether it plays a pathogenic or protective role in ovarian tumor biology. We thus constructed an inducible gene expression system to achieve RGS5 expression in HeyA8-MDR ovarian cancer cells. Through this we observed that inducible RGS5 expression significantly reduces in vitro BrdU-positive HeyA8-MDR cells, although this did not correlate with a reduction in tumor volume observed using an in vivo mouse model of ovarian cancer. Interestingly, mice bearing RGS5-expressing tumors demonstrated an increase in survival compared with controls, which might be attributed to the vast regions of necrosis observed by pathological examination. Additionally, mice bearing RGS5-expressing tumors were less likely to have ulcerated tumors. Taken together, this data supports the idea that temporal expression and stabilization of RGS5 could be a valuable tactic within the context of a multicomponent approach for modulating tumor progression.

Figures

Figure 1
Figure 1
RGS5 inducible expression in HeyA8-MDR cells reduces proliferation. (a) HeyA8-MDR pTet dual RGS5 inducible cells were seeded in a multiple 6-well and cultured in medium with tetracycline-free fetal bovine serum without doxycycline for 24, 48, and 72 hours. Cells were harvested; the RNA was isolated and processed for qRT-PCR using primers to detect RGS5 expression resulting from the Tet-Off Advanced inducible system. Without the presence of doxycycline or other members of the tetracycline antibiotics, RGS5 expression was observed. (b) HeyA8-MDR cells were plated in a 96-well plate at a density of 3,000 cells per well. Half of the plate was grown in medium containing regular FBS with doxycycline and the other half containing doxycycline-free media. Cells were then incubated at 37°C for 72 hours to allow for RGS5-inducible gene expression. Prior to fixation, HeyA8 MDR cells were pulse-treated for 1 hour with BrdU and then treated for 4 hours with the indicated cell cycle arrest compounds. Cells were then fixed and stained for proliferation and nuclear morphology. Representative images taken using the Cellomics ArrayScan VTI HCS Reader and are shown here. (c) High-content scanning analysis software was used to determine the average number of cells per field.
Figure 2
Figure 2
Expression of RGS5 in ovarian tumors did not significantly reduce the rate of growth, but did increase the overall survival time of mice bearing such tumors. (a) Female athymic nude mice were given intraperitoneal injections of Extracel containing approximately 5 million cells of either HeyA8 MDR pTet Advanced Vector (N = 10) or HeyA8-MDR pTet Dual RGS5 expressing cells (N = 10) per 0.2 μL injection. All mice were monitored for tumor formation and measurements of weight and stomach circumference were routinely taken. The graph plots tumor volume (mm3). (b) Mice bearing HeyA8-MDR pTet Dual RGS5-expressing tumors had a significant increase in survival time (in days, *P = 0.0143, compared to their pTet Advanced Vector alone counterparts). (c) Blood was collected from mice at necropsy. The serum-containing fraction was isolated and measured for vascular endothelial growth factor. Results are nonsignificant (ns) between the groups.
Figure 3
Figure 3
Mice bearing RGS5-expressing ovarian tumors displayed less frequent tumor ulceration. The number of mice with ulcerated tumors observed at necropsy was recorded (N = 10 per group). Tumor sizes were relatively similar between the two groups.
Figure 4
Figure 4
RGS5-expressing ovarian tumors increase the number of CD31-positive vessel-like structures. Tumor specimens were sectioned and prepared on slides for immunofluorescence. (a) Tumor sections were imaged for CD31 expression (red, 20x magnification). (b) CD31-positive vasculature was quantified from compiled whole tumor images (N = 4 vector, N = 4 RGS5) using CellSens and GraphPad Prism software. **P < 0.01, comparing vector versus RGS5.

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References

    1. Zhou J, Moroi K, Nishiyama M, et al. Characterization of RGS5 in regulation of G protein-coupled receptor signaling. Life Sciences. 2001;68(13):1457–1469. - PubMed
    1. Chen C, Zheng B, Han J, Lin SC. Characterization of a novel mammalian RGS protein that binds to Gα proteins and inhibits pheromone signaling in yeast. The Journal of Biological Chemistry. 1997;272(13):8679–8685. - PubMed
    1. Seki N, Sugano S, Suzuki Y, et al. Isolation, tissue expression, and chromosomal assignment of human RGS5, a novel G-protein signaling regulator gene. Journal of Human Genetics. 1998;43(3):202–205. - PubMed
    1. Xiao B, Zhang Y, Niu WQ, Gao PJ, Zhu DL. Haplotype-based association of regulator of G-protein signaling 5 gene polymorphisms with essential hypertension and metabolic parameters in Chinese. Clinical Chemistry and Laboratory Medicine. 2009;47(12):1483–1488. - PubMed
    1. Chang YPC, Liu X, Kim JDO, et al. Multiple genes for essential-hypertension susceptibility on chromosome 1q. American Journal of Human Genetics. 2007;80(2):253–264. - PMC - PubMed

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