The Effect And Mechanism Of Cryptotanshinone On Angiogenesis

Background and Purpose:In1971, the view of tumor growth and metastasis required angiogenesis and tumor could be prohibited if we inhibited angiogenesis was firstly put forward by Dr. Judah Folkman and his colleagues in Boston Children’s Hospital. Research of anti-angiogenesis has been a hot spot for scholars all over the world for years. Huoxuehuayu is a vital rule of treatment in Chinese medicine. Traditional Chinese medicine (TCM) served as Huoxuehuayu could exert anti-tumor effect by inhibiting angiogenesis. Danshen is the most frequently used Huoxuehuayu TCM in clinical which plays an extremely critical role in anti-tumor therapy. Cryptotanshinone (CT) is one of the major anti-tumor ingredients also been reported as a potential anti-angiogenic agent. However, the anti-angiogenesis mechanism of CT remains to be elucidated. In this study, we used CT as a molecular template to observe its effect on angiogenesis and explore the underlying mechanism in order to provide a brand new perspective for the anti-angiogenesis and anti-tumor study of TCM.Experimental Approach:(1) In this study, we first used Cytoscape3.0.3software to establish the anti-tumor network of Danshen in order to explore the promising anti-tumor angiogenesis target. Then, we examined the effect of compounds in Danshen on the secretion of TNF-a. Furthermore, we used COREMINE Medical to retrospectively analyze latest progress.(2) We employed the transgenic zebrafish angiogenesis assay and Matrigel plug assay in mice to examine the effect of CT on angiogenesis in vivo as well as capillary tube formation assay and vascular sprouting assay to examine the effect of CT on angiogenesis in vitro. We also employed the BrdU immunofluorescence staining to explore the effect of CT on proliferation as well as wound healing assay and Transwell to examine the migration of HUVECs.(3) In order to probe the targets of CT, we used western blot examine the effect of Notch signaling and VEGF related sprouting and TLR4in HUVECs. The change of VEGF secretion was further measured by ELISA. Besides, the mRNA and protein levels of TNF-a were measured by real-time PCR and ELISA respectively. Moreover, TNFRs were further examined by western blot.(4) In terms of mechanism, we first examined the effect of CT on TNF-a mRNA stability by realtime PCR. Then vectors containing full length of TNF-a3’-UTR were constructed and transfected into the HUVEC cells, the effect of CT on TNF-a3’-UTR were examined by luciferase reporter gene. Furthermore, westernblot and immunofluorescence staining were employed to investigate the effect of CT on translocation of HuR. Moreover, TNF-a upstream signaling pathways treated with CT were measured by luciferase assay, western blot and immunofluorescence staining.(5) We used transgenic zebrafish tumor model to investigate the effect of CT on tumor growth and tumor angiogenesis.Key Results:(1) Results from data mining illustrated that TNF-a might be the major anti-tumor target of Danshen. Anti-TNF-a might be the basis of anti-tumor and anti-angiogenesis effect of Danshen. Moreover, Cryptotanshinone (CT) showed to be a promising anti-TNF-a angent. Furthermore, the anti-angiogenesis effect of CT has been barely reported, especially the study of CT on spouting was totally blank.(3) CT could significantly inhibit LPS and VEGF induced angiogenesis in vivo and in vitro.(4) CT could also decreased proliferation and migration of HUVECs in vitro.(5) However, CT had no obvious effects on the expression of sprouting-related Notch signaling and VEGF or the secretion of VEGF, as well as the expression of TLR4, which is the pattern recognition receptor of LPS. CT indeed does-dependently inhibited the TNF-α levels. Moreover, CT further could decrease the expression of TNFR1rather than TNFR2.(6) In order to understand the underlying mechanism of CT downregulating TNF-a, we first found that LPS could extend the half life of TNF-a mRNA, CT, however, remarkably decreased LPS-induced half life extension even below control. We then constructed vector containing TNF-a3’UTR and found that CT could decrease the fluorescence values in a dose dependent manner. Moreover, CT also decreased LPS induced the cytoplasm translocation of HuR. CT exerted the effects through inhibiting the relative promoter activity of NF-κB and phosphorylation of NF-κB and STAT3signaling pathway rather than affecting the relative promoter activity of STAT3or P38%Akt、Erk signaling pathways.(7) CT could further inhibit tumor growth in zebrafish and neo-sprouting.Conclusions and Implications:The outcomes of this study are as following:(1) CT could inhibit neovascular sprouting angiogenesis in zebrafish and Matrigel plug angiogenesis in mice in vivo. CT also could inhibit tube formation and sprouting angiogenesis of HUVECs in vitro. Moreover, CT could prohibit proliferation and migration of HUVECs in vitro.(2) CT could downregulate TNF-a level and inhibit the expression of TNFR1of HUVECs.(3) CT downregulated TNF-a level by inhibiting HuR regulated TNF-a mRNA stability, which is achieved by inhibiting the NF-κB and STAT3signaling pathways.(4) CT also exerted an inhibitory effort on tumor growth and sprouting angiogenesis. This project helped us illustrate the acted mechanisms of CT on angiogenesis and provided a new way and visual angle for us to research the effect of TCM on angiogenesis and tumor.