Studies On The Anti-tumor Effects And Mechanism Of Andrographolide Derivatives

Andrographolide (AD) is one of the major active constituents from Andrographis [Andrographis paniculata (Burm. f) Nees], a traditional Chinese herbal medicine. In recent years, AD, as a lead compound for the synthesis of anti-tumor and anti-viral drugs, has become one of the hot spots in the new drug development area. To improve the cytotoxic activity on tumor cells, a lot of derivatives of AD modified in the sites of 3,19-hydroxies and/or 8,17 double bonds have been synthesized by many researchers. However, the in vivo anti-tumor activities of the known derivatives have not been enhanced efficiently. In our previous studies, it was found that two C-15-substituted AD derivatives, AD-3 and AD-2, could significantly inhibit the adhesion between tumor cells and vascular endothelial cells actived by LPS, the migration of tumor cells and the increasing vascular permeability, compared with AD.In this study, the in vivo anti-angiogenesis effects of AD-2 and AD-3 were investigated by the chick chorioallantoic membrane model (CAM assay) and the H22 tumor-bearing mice model; their in vitro anti-angiogenesis effects were studied by the two-dimensional and three-dimensional migration models. The effects on inhibiting the growth of tumor tissue were also evaluated with both H22 and S180 tumor-bearing mice models. The expression of some cytokines in the tumor tissues and serum of the tumor-bearing mice were evaluated with the immunohistochemistry method and ELISA, respectively, to clarify the complex mechanism of AD-2 and AD-3 on regulating the tumor microenvironment, anti-angiogenesis and enhancing immunity of the mice. These efforts will establish favourable groundwork for the synthesis of novel andrographolide derivatives, even for the development of anti-cancer drugs. The main results are as follows:1. The in vivo anti-tumor effect of AD-2 and AD-3 were significantly stronger than that of the parent compound AD (P<0.05). AD-3 significantly decreased the weight of tumor tissues by 53.38±6.83% in the dosage of 0.85 mmol/kg (ig). It also significantly inhibited the spread of solid tumor under the dosages of 0.85 mmol/kg (ig) and 1.10 mmol/kg (ig), compared either with the model group or the AD group (P <0.01). These results illustrated that the anti-tumor activity in vivo could be significantly improved by modifying AD in the C-15 site, witch will contribute to the synthesis of AD derivatives with stronger anti-cancer activities.2. AD-2 and AD-3 showed good anti-angiogenic effects in vitro and in vivo. The vascular branch number in the CAM was significantly reduced about 75% and 56% by AD-2 and AD-3, respectively, compared with that of the model group (P<0.05). In the H22 tumor bearing mice treated with AD-2 (1.35 mmol/kg, ig) and AD-3 (1.00 mmol/kg, ig), the vascular index of the tumor tissue were 3.14±1.69 and 3.30±1.43, separately, which were significantly decreased (P<0.05) when compared with that of the mice in model group (4.82±2.43) and AD group (5.10±2.13).The results about mechanism studies demonstrated that AD-2 and AD-3 could deter the formation of microvessels in tumor tissues by significantly inhibiting the expression of vascular endothelial growth factor (VEGF), increasing the expression of matrix metalloproteinase inhibitor-1 (TIMP-1), and then inhibiting the migration of vascular endothelial cell ECV304.3. AD-2 and AD-3 significantly inhibited the NF-κB activation by down-regulating the expression of IL-1β, PGE2 and iNOS in the serum of the S180 tumor bearing mice, then inhibited the proliferation and angiogenesis of the malignant tumor tissues. Meanwhile, the secretion of tumor necrosis factor (TNF-a) also could be significantly enhanced by them to directly inhibit the tumor growth.4. Unlike cyclophosphamide, the weight increase of mice was not influenced by AD-2 or AD-3, and their immunity index of thymus and spleen had been strengthened in a certain level. The results indicated that AD-2 and AD-3 were able to enhance the immunity of organisms, which was possibly related with their comprehensive effects on anti-tumor.In conclusion, andrographolide derivatives, AD-2 and AD-3, could inhibit the formation of microvessels, the growth and diffusion of the solid tumor tissues through up-regulating the expression of TNF-a and TIMP-1, down-regulating the expression of VEGF, IL-1(3, PGE2 and iNOS, inhibiting the NF-κB pathway which could promote the tumor genesis and development in the tumor microenvironment. Therefore, AD-2 and AD-3, especially the AD-3, are potentially to be developed as the new anti-tumor drugs, which can regulate the tumor microenvironment and inhibit the tumor development with great efficiency and low toxicity.