Celastrol suppresses angiogenesis-mediated tumor growth through inhibition of AKT/mammalian target of rapamycin pathway

Abstract

Understanding the molecular basis and target of traditional medicine is critical for drug development. Celastrol, derived from Trypterygium wilfordii Hook F. ("Thunder of God Vine"), a traditional Chinese medicine plant, has been assigned anticancer activities, but its mechanism is not well understood. Here, we investigated whether Celastrol could inhibit angiogenesis-mediated tumor growth and, if so, through what mechanism. When given s.c. to mice bearing human prostate cancer (PC-3 cell) xenografts, Celastrol (2 mg/kg/d) significantly reduced the volume and the weight of solid tumors and decreased tumor angiogenesis. We found that this agent inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, invasion, and capillary-like structure formation by primary cultured human umbilical vascular endothelial cells (HUVEC) in a dose-dependent manner. Furthermore, Celastrol abrogated VEGF-induced sprouting of the vessels from aortic rings and inhibited vascular formation in the Matrigel plug assay in vivo. To understand the molecular mechanism of these activities, we next examined the signaling pathways in treated HUVECs and PC-3 tumor cells. Celastrol suppressed the VEGF-induced activation of AKT, mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase (P70S6K). Additionally, we found that Celastrol inhibited the proliferation of prostate cancer cells and induced apoptosis, and these effects correlated with the extent of inhibition of AKT/mTOR/P70S6K signaling. Taken together, our results suggest that Celastrol targets the AKT/mTOR/P70S6K pathway, which leads to suppression of tumor growth and angiogenesis.

Figure 1. Celastrol inhibits tumor growth and tumor angiogenesis in xenograft mice

PC-3 cells were injected into 6-week-old BALB/cA nude mice (5×10⁶ cells per mouse). After solid tumors grew to about 130 mm³, the mice were subcutaneously administrated with or without Celastrol (2 mg/kg/d). A, Celastrol inhibited tumor growth as measured by tumor volume. B, as shown by the body weight change in mice, Celastrol had little toxicity in the amount tested. C, solid tumors in the Celastrol-treated mice were significantly smaller than those untreated mice. D, blood vessel staining revealed that Celastrol inhibited tumor angiogenesis. The arrows indicate blood vessels. Columns, mean; bars, standard deviation; **, P < 0.01 vs. control.

Figure 2. Celastrol inhibits VEGF–induced chemotactic motility, capillary-structure formation, and cell viability of endothelial cells

A, Celastrol inhibited HUVEC migration. HUVECs were scratched by pipette and treated with or without 10 ng/mL VEGF and Celastrol. The migrated cells were quantified by manual counting. B, Celastrol inhibited HUVEC invasion. Migrated cells through the membrane were quantified in the Transwell assays. C, Celastrol inhibited the VEGF-induced tube formation of endothelial cells in matrigel. After incubation, endothelial cells were fixed, and tubular structures were photographed (Magnification, ×100). D, Celastrol inhibited the VEGF-induced cell survival of HUVECs. MTS was used to quantify endothelial cell viability. Columns, mean from three different experiments; bars, standard deviation; *, P < 0.05; **, P < 0.01 vs. VEGF alone.

Figure 3. Celastrol inhibits VEGF–induced microvessel sprouting ex vivo

Aortic segments isolated from Sprague-Dawley rats were placed in the Matrigel-covered wells and treated with VEGF in the presence or absence of Celastrol. A, representative photographs of sprouts from the margins of aortic rings. B, sprouts were scored from 0 (least positive) to 5 (most positive) in a double-blinded manner. Columns, mean; bars, standard deviation; **, P < 0.01 vs. VEGF alone.

Figure 4. Celastrol inhibits VEGF–induced angiogenesis in vivo

Six–week–old C57/BL/6 mice were injected with 0.5 mL of Matrigel containing 10 μg Celastrol, 100 ng of VEGF, and 20 units of heparin into the ventral area (n=5 per group). After 6 d, the skin of mice was pulled back to expose the intact Matrigel plugs. A, representative Matrigel plugs were photographed. B, Celastrol inhibited blood vessel formation. The Matrigel plugs were fixed, sectioned, and stained with hematoxylin and eosin (Magnification, ×200). C, infiltrating microvessels with intact red blood cells were quantified by manual counting. Columns, mean; bars, standard deviation; **, P < 0.01 vs. VEGF alone.

Figure 5. Celastrol inhibits the VEGF-triggered activation of VEGFR2 and mTOR pathway in endothelial cells

A, Celastrol suppressed the activation of VEGFR2 triggered by VEGF in HUVECs. The activation of VEGFR2 from different treatments was tested by Western blotting and probed with anti-phospho-VEGFR2 antibody. The relative density of immunoreactivity was measured. Columns, mean from two independent experiments; bars, standard deviation, **, P < 0.01 vs. VEGF alone. B, Celastrol inhibited the activation of AKT/mTOR/S6K pathway in endothelial cells. Proteins from different treatments were probed with specific antibodies. Similar experiments were performed at least three times.

Figure 6. Celastrol induces cell apoptosis and inhibits AKT/mTOR/P70S6K pathway in PC-3 cancer cells

A. Celastrol inhibited cell viability of prostrate PC-3 cancer cells. Cell viability was quantified by MTS assay. Columns, mean from three different experiments; bars, standard deviation, **, P < 0.01 vs. VEGF alone. B. Celastrol induced PC-3 cancer cell apoptosis in concentration-dependent and time-dependent manners by the cleaved-PARP analysis. C, Celastrol suppressed the phosphorylation of mTOR signaling pathway kinases in PC-3 prostate cancer cell. To examine mTOR pathway in prostate tumor cells, normal cultured PC-3 cells (non-starving) were directly treated with indicated dilutions of Celastrol for 4 h. Proteins from different treatments were applied to Western blotting and probed with specific antibodies. Similar data were obtained from three independent experiments.