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Hispidulin, an active component from Artemisia vestita, a traditional Tibetan medicinal plant, has been shown to possess anti-inflammatory and anti-oxidative activities. However, the functional role of hispidulin on tumor growth and angiogenesis has not been elucidated. We found that hispidulin significantly inhibited human pancreatic tumor growth in xenograft mice when s.c. treated at a dosage of 20 mg/kg daily, and this effect was accompanied with a potent inhibition on angiogenesis. When examining the cytotoxicity of hispidulin on HUVECs and pancreatic cancer cells in vitro, we found that HUVECs were more susceptible to the treatment, suggesting angiogenesis might be the primary target of hispidulin. Our results further showed that hispidulin inhibited vascular endothelial growth factor (VEGF)-induced cell migration, invasion, and capillary-like structure formation of HUVECs in a dose-dependent manner. In ex vivo and in vivo angiogenesis assays, we showed that hispidulin suppressed VEGF-induced microvessel sprouting of rat aortic rings and corneal neovascularization in C57/BL6 mice. To understand the underlying molecular basis, we next examined the effects of hispidulin on different molecular components in treated HUVECs, and found that hispidulin suppressed the VEGF-triggered activation of VEGF receptor 2, PI3K, Akt, mTOR, and ribosomal protein S6 kinase, but had little effect on focal adhesion kinase or extracellular signal-regulated kinase at an effective concentration. Taken together, our results indicate that hispidulin targets the VEGF receptor 2-mediated PI3K/Akt/mTOR signaling pathway in endothelial cells, leading to the suppression of pancreatic tumor growth and angiogenesis. (Cancer Sci 2011; 102: 219–225)
Cancer of the exocrine pancreas is characterized by extensive local invasion and metastases to the liver, and this aggressive biology translates into a 5-year survival rate of 1–4% for all patients with a diagnosis of pancreatic adenocarcinoma.(1) The current best therapy including surgery, radiation, and chemotherapy has done little to alter the cancer-related deaths of these patients.(2) As one of the hallmarks of cancer, angiogenesis is an essential event involved in tumor progression and metastasis.(3) Antiangiogenic therapy is now considered to be a prospective strategy because endothelial cells are genetically stable, which makes them an ideal therapeutic target compared with cancer cells.(4)
In the process of tumor angiogenesis, vascular endothelial growth factor (VEGF) plays a pivotal role in the regulation of endothelial cell proliferation, migration, and vascular permeability through binding to its receptor tyrosine kinases such as VEGFR1, R2, and R3.(5,6) However, the VEGF signaling involved in angiogenesis is mainly mediated by VEGFR2.(7) Specifically, VEGFR2 activation leads to the activation of diverse intracellular signaling, and the PI3K/Akt signaling pathway is one of them. In angiogenesis, PI3K/Akt kinases are activated by a variety of stimuli in endothelial cells(8) and regulate multiple critical steps by phosphorylating different downstream substrates, such as the mTOR.(9) It has been well documented that mTOR kinase, a central regulator of cell metabolism, growth, proliferation, and survival,(10,11) is activated during various cellular processes such as tumor initiation, progression, and angiogenesis.(12,13) These observations have attracted broad scientific and clinical interest in the mTOR signaling pathway. However, with an increasing understanding of feedback loops exiting in the PI3K/Akt/mTOR pathway, it has been recognized that inhibition on one output is at the expense of activation of the others.(14) For this reason, there is a growing consensus that the inhibition of combined components in the PI3K/Akt/mTOR pathway likely reflects a mechanistic rationale for therapeutic options.(15,16)
Hispidulin, extracted from Artemisia vestita, a traditional Tibetan medicinal plant, has been used traditionally in China for treating various inflammatory diseases.(17) It has been reported that hispidulin is a partial positive allosteric modulator at the benzodiazepine receptor.(18,19) Given orally, it reduces seizures in a gerbil model of epilepsy.(20) Hispidulin also shows antifungal,(21) antiproliferative,(22) antioxidant,(23) and antithrombosis properties.(24) Very recent studies showed that hispidulin could potentiate the apoptosis of human ovarian cancer cells and human glioblastoma multiforme cells.(25,26) However, whether hispidulin has the ability to modulate tumor growth and tumor angiogenesis is still unknown. In this study, we investigated the functional roles of hispidulin in preventing pancreatic cancer growth in xenograft mice, examined the potential molecular targets by inhibiting tumor angiogenesis, and elucidated the signaling pathways mediated by hispidulin in endothelial cells.
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- Materials and Methods
- Disclosure Statement
In this study, we showed for the first time that hispidulin, an active flavonoid from Artemisia vestita, has a potent ability to suppress human pancreatic tumor growth, an effect achieved to a great extent by angiogenesis inhibition.
In xenograft pancreatic tumor mice, 20 mg/kg/day hispidulin significantly suppressed tumor volume and tumor weight (Fig. 2A,B), without toxic effects on mouse body weight (Fig. 2C). Immunohistochemistry results further revealed that the generation of new blood vessels and the expression of VEGF in the treated group were dramatically reduced when compared with the control groups (Fig. 2D,E). However, whether this phenomenon occurs through effects on the biological function of endothelial cells or cancer cells, or both, was not ascertained. Therefore, we tested the effect of hispidulin on cell viability. Interestingly, we found that the IC50 value of hispidulin in HUVECs was 20 μmol/L, whereas at least 200 μmol/L hispidulin was required to effectively inhibit cell viability in pancreatic cancer cells (PANC-1, PANC-28, and BxPC-3) (Fig. 3B–D), suggesting that endothelial cells were probably the primary target of hispidulin in tumor inhibition. In addition, hispidulin potently inhibited VEGF-induced microvessel sprouting ex vivo and corneal neovascularization in vivo. All of these results indicated that hispidulin is an angiogenesis inhibitor, and blocks multiple steps of angiogenesis.
Accumulating evidence shows that VEGFR2 is the crucial and main receptor mediating angiogenic and vascular permeability activity, whereas VEGFR3 is mainly involved in lymphangiogenic activity.(7) In this study, we found that hispidulin is a VEGFR2 inhibitor. The inhibitory effect of hispidulin on the activation of VEGFR2 was consistent with that on PI3K/Akt/mTOR signaling components. This indicates that VEGFR2 might be the important target of hispidulin in endothelial cells (Fig. 7D). Conversely, VEGFR1 contains a classical tyrosine kinase domain, however, the primary function of VEGFR1 may be as a negative regulator in vascular development.(31) Our results showed that hispidulin significantly suppressed the VEGFR2-mediated PI3K/Akt/mTOR signaling pathway in endothelial cells at 10 μmol/L, which was in accord with the in vitro results that indicated hispidulin could block VEGF-induced endothelial cell migration, invasion, and tubular formation. mTOR kinase, a central regulator of cell metabolism, growth, proliferation, and survival,(10,11) has been implicated in inflammation by regulating signal-dependent translation in platelets, monocytes, and neutrophils.(32,33) A recent study also showed that PI3K/mTOR was an important target for leptin-induced formation of cytoplasmic lipid bodies, enhancing inflammatory mediator production in macrophages.(34) Our results indicated that hispidulin significantly inhibited mTOR kinase and its downstream target, S6 kinase (Fig. 7B), which might provide the reason that hispidulin exerted anti-inflammatory properties in previous reports.(17) However, how hispidulin affects the complex of mTOR kinase is still an open question.
It is documented that PI3K/Akt favors survival through the direct regulation of apoptotic proteins, such as Bcl-2 and Bcl-xL.(35,36) Also, Bcl-2, Bcl-xL, and survivin are nuclear factor-кB regulated gene products and PI3K/Akt can also activate nuclear factor-кB.(37) In this study, we found that hispidulin could inhibit the expression of these cell growth-related proteins in a concentration-dependent manner in both endothelial cells and pancreatic cancer cells (Fig. 3E,F). Recently, endothelial p70S6K has been shown to play an important role in regulating tumor microenvironment and angiogenesis,(38) and our results showed that hispidulin could significantly decrease the activation of S6K; the effective concentration of hispidulin is approximately 10 μmol/L. Consistent with the inhibition of these kinases at the molecular level, angiogenesis was dose-dependently rectified by hispidulin.
In summary, we show for the first time that hispidulin inhibited human pancreatic tumor angiogenesis and tumor growth by targeting the VEGFR2-mediated PI3K/Akt/mTOR signaling pathway. We have reason to believe that hispidulin could be a potential drug candidate for cancer prevention and cancer therapy.