Studis On The Mechanism Of Anti-tumor Metastasis Of Aspirin By Targeting Heparanase

IntroductionCancer metastasis remains the major challenge for cancer therapy, and accounts for most cancer related death. Due to the complexity of the pathological process of metastasis, there is still lack of effective therapeutic solutions. Aspirin, a classical nonsteroidal anti-inflammatory drug, has been in medical use for over a hundred years in broad conditions including fever, pain, rheumatic fever, and inflammatory diseases. Recently, increasing epidemiological investigations have suggested that aspirin may possess anti-metastasis value, which inspired our interests to understand the molecular basis behind. A pilot proteomic study in our laboratory to identify aspirin associated proteins has suggested that heparanase, an enzyme degrading polymeric heparan sulfate at the cell-surface and within the extracellular matrix, may be one of the target proteins of aspirin. This study aimed to confirm this observation and more importantly to investigate whether heparanase inhibition contributes to the anti-metastasis of aspirin.Methods and Results:Section 1. Aspirin inhibits the enzymatic activity of heparanaseA homogeneous time resolved fluorescence (HTRF) assay was used to examine the impact of aspirin on heparanase and demonstrated that aspirin can inhibit the enzymatic activity of heparanase. Streptavidin pulldow to enrich proteins binding to biotin conjugated-aspirin identified heparanase as aspirin binding protein, suggesting aspirin can directly target heparanase. Further, surface plasmon resonance (SPR) assay showed that aspirin binds to a non-heparin binding site of heparanase. Molecular docking of aspirin to heparanase together with mutagenesis approach, discovered that aspirin may bound to Glu225, a residue critical for the enzymatic activity of heparanase.Section 2. Aspirin inhibits heparanase-related effects in vitro.1. The inhibitory effect of aspirin on the migration and invasion.HTRF assay was used to examine the effect of aspirin on heparanase in cancer cells and the results showed that aspirin inhibited the enzyme activity of endogenous heparanase in cancer cells. In parallel, aspirin treatment inhibited migration and invasion of multiple cancer cell lines in a dose-dependent manner. Further, over-expression, depletion or exogenous addition of heparanase was able to rescue the effects of aspirin in terms of cell migration and invasion suggesting a heparanase dependent effect.2. Aspirin abolished heparanase-driven cell adhesion.Aspirin inhibited the internalization process of syndecan-1 stimulated by exogenous addition heparanase, as detected by immunofluorescence approach, which suggested aspirin may affects cell adhesion. Heparanase overexpression contributes to F-actin rearrangement and the formation of pseudopods through activing Rac-1 and Rho-A, and ultimately the overexprssion promotes tumor cells adhesion, which was inhibited by aspirin as well.3. Aspirin suppressed angiogenesis.Aspirin dose-dependently inhibited the release of VEGF by tumor cells, which was reversed by over-expression of heparanase. Knock-down of heparanase abolished the effects of aspirin on VEGF release. Consistently, antagonizing potency against angiogenesis on multiple models. It extensively inhibited HUVEC cells tube formation at non-cytotoxic concentrations with the inhibition rate 60.8% at 4 mM. Meanwhile aspirin can significantly inhibit microvessel budding outgrowth from rat aortic rings and angiogenesis in chicken chorioallantoic membrane model.4. Aspirin inhibited signaling which activated by heparanase.Over-expression of heparanase activated signaling including STAT3, STAT5, Src, Erk, which was inhibited by aspirin in a time and dose-dependent manner. Section 3. Aspirin inhibits tumor metastasis, angiogenesis and growth in vivo.We further evaluated the anti-metastasis and anti-angiogenesis effects of aspirin in vivo. Oral administration of aspirin at 62.5,125,250 mg/kg significantly inhibited B16F10 lung metastasis, with an inhibition rate of 19.9%,49.6% and 65.5%, respectively. In MDA-MB-435 breast cancer xenografts models aspirin inhibited tumor growth with a T/C of 61.69% and 47.79% respectively at the doses of 125mg/kg and 250mg/kg. Further immunohistochemistry (IHC) staining of tissue sections for CD31, a specific marker for endothelial cells, revealed that aspirin treatment significantly diminished the number of microvessels in tumor tissues.Moreover, western blotting analysis proved that aspirin inhibited the introtumoral signaling of STAT3, STAT3, STAT5, Src, Erk. Aaspirin therapy also down-regulated heparanase protein level in tumor tissues in a dose-dependent manner. However, RQ-PCR showed that mRNA of heparanase was up-regulated. These results suggested that aspirin down-regulated heparanase level by promoting the degradation of heparanase, which was supported by the protease inhibitors rotary experiments.Conclusions:In summary, the present study has, for the first time, illustrated heparanse as a new target of aspirin. Aspirin may directly binds to Glu225 region to inhibit its enzymatic activity, thereby impeding tumor metastasis and angiogenesis. Aspirin also inhibits the non-enzymatic activity of heparanase and promotes its degradation. This provides new insights for a better understanding of the mechanisms of aspirin in anti-tumor metastasis effects, and offers new direction for the development of small-molecule inhibitors of heparanase.