Spliceostatin A blocks angiogenesis by inhibiting global gene expression including VEGF

Abstract

Spliceostatin A (SSA) is a methylated derivative of an antitumor natural product FR901464, which specifically binds and inhibits the SF3b spliceosome sub-complex. To investigate the selective antitumor activity of SSA, we focused on the regulation of vascular endothelial growth factor (VEGF) mRNA, since VEGF is a key regulatory component in tumor angiogenesis and known for the intricate regulation of mRNA processing, such as alternative splicing. We found that in HeLa cells SSA reduced the amount of both mRNA and protein of VEGF. Spliceostatin A not only inhibited the splicing reaction of VEGF pre-mRNA but also reduced the total amount of VEGF's transcripts, while SSA affected GAPDH mRNA to a lesser extent. Given a significant reduction in VEGF gene expression, SSA was expected to possess anti-angiogenic activity in vivo. Indeed, SSA inhibited cancer cell-derived angiogenesis in vivo in a chicken chorioallantoic membrane (CAM) assay. The inhibition of angiogenesis with SSA was abolished by addition of exogenous VEGF. We also performed global gene expression analyses of HeLa cells and found that the expression levels of 38% of total genes including VEGF decreased to <50% of the basal levels following 16 h of SSA treatment. These results suggest that the global interference of gene expression including VEGF in tumor cells is at least one of the mechanisms by which SSA (or FR901464) exhibits its strong antitumor activity.

Figure 1

Chemical structure of FR901464, spliceostatin A (SSA) and its inactive derivative, acetyl‐spliceostatin A (Ac‐SSA).

Figure 2

Spliceostatin A (SSA) suppressed vascular endothelial growth factor (VEGF) production. (a) HeLa cells were treated with 200 nM SSA or inactive acetylated‐SSA (Ac‐SSA) for the indicated times, and RT‐PCR analysis was performed using primers that detect both pro‐angiogenic VEGF₁₆₅ (∼200‐bp) and anti‐angiogenic VEGF₁₆₅b, (∼150‐bp). (b) HeLa cells were treated with 200 nM SSA or FR901464 (FR) for 3 or 12 h, and then western blot analysis using anti‐VEGF antibody was performed. The GAPDH was a loading control. (c) HeLa cells were treated with vehicle (0.1% methanol; MeOH), 200 nM FR901464, 200 nM SSA or Ac‐SSA for the indicated times. The levels of secreted VEGF protein in the medium were determined by ELISA and were normalized by cell numbers in the same dishes. The results are expressed as percentages of the control cells treated with vehicle as the means ± SD of three independent experiments, each performed in duplicate. ***P < 0.001; *P < 0.05.

Figure 3

Spliceostatin A (SSA) inhibited production of vascular endothelial growth factor (VEGF) mRNA in a dose‐dependent manner. HeLa cells treated with various concentrations of SSA or inactive acetylated‐SSA (Ac‐SSA) for 6 h. RT‐qPCR analyses were performed for mature mRNA (a), pre‐mRNA (b) and the sum of both (c) of VEGF, as well as for mature mRNA of GAPDH (d). The results are the means ± SD of three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 4

Comparison among spliceostatin A (SSA), FR901464 and ACR in their ability to suppress in vivo blood vessel formation induced by HeLa cells in chicken chorioallantoic membrane (CAM) tissues (gelatin sponge‐CAM assay). (a) HeLa cells were delivered at 3 × 10⁵ cells per embryo onto the top of the CAM at day 8 using a gelatin sponge implant, supplemented with or without SSA (2 nM), FR901464 (0.2, 2 and 20 nM) and acyclic retinoid (ACR) (0.05, 0.5 and 5 μM) as a positive control. At day 12, newly formed microvessels sprouting towards the implant were observed under the microscope. Representative results are shown. g. s., represents the area of gelatin sponge; c. a., represents the count area meaning the area in which we counted microvessels. Black scale bars represent 500 μm. (b) The numbers of the microvessels inside the same area (surrounded by squares) were counted. Each datum represents the average ±SD (n = 6–7). **P < 0.01; *P < 0.05. NS, not significant.

Figure 5

Suppression of in vivo blood vessel formation induced by HeLa cells in chicken chorioallantoic membrane (CAM) tissues by spliceostatin A (SSA). (a) HeLa cells were delivered at 3 × 10⁵ cells per embryo onto the top of the CAM at day 8 using a gelatin sponge implant, supplemented with or without 2 nM SSA, acetylated‐SSA (Ac‐SSA) or 100 ng/mL vascular endothelial growth factor (VEGF). At day 12, newly formed microvessels sprouting towards the implant were observed under the microscope. Representative results are shown. g. s., represents the area of gelatin sponge; c. a., represents the count area meaning the area in which we counted microvessels. (b) The numbers of the microvessels inside the same area were counted. Each datum represents the average ±SD (n = 6–7). ***P < 0.001; *P < 0.01.

Figure 6

(a) HeLa cells were treated with 200 nM spliceostatin A (SSA) for the indicated times, and chromatin immunoprecipitation (ChIP) analysis with anti‐RNA polymerase II (Pol II) antibody phosphorylated at Ser‐2 or Ser‐5 in C‐terminal domain (CTD) (red or blue lines, respectively) was performed. Schematic representation of the designed primer pairs targeting seven different sites of the vascular endothelial growth factor (VEGF) gene region used in ChIP are shown above as small squares. The intensity of signals of each sample was calculated from the ratio to those of the input sample. The experiments were performed independently at least three times, and a representative result is shown. (b) Summary of the results of the microarray analyses in HeLa cells treated with 200 nM SSA for 3 or 16 h. Total numbers of genes found on Human Genome U133 Plus 2.0 GeneChip expression arrays (Affymetrix, Santa Clara, CA, USA) are presented as either no change (gray), decreased (black) or increased (white).