Colorectal cancer represents the fourth commonest malignancy, and constitutes a major cause of significant morbidity and mortality among other diseases. In this study, the novel compound, HMQ18–22 (a novel taspine analog) significantly reduced angiogenesis of CAM and mouse colon tissue, inhibited cell migration and tube formation and decreased phosphorylation of VEGFR2, VEGFR1, Akt, PKCα and PLCγ-1. Most importantly, HMQ18–22 inhibited tumor cell growth in xenografted model in athymic mice. These results suggest that HMQ18–22 can be a useful therapeutic candidate for colon cancer intervention.
HMQ18–22 was synthesized by our group for cancer drug development. Our data showed that HMQ18–22 significantly blocked angiogenesis using CAM model. Angiogenesis plays a vital role in growth, intravasation and metastatic spread of cancer. Inhibition of angiogenesis provides a good chance of preventing cancer from becoming malignant. Tumor angiogenesis shows a markedly increasing proliferation of endothelial cell and has significant functional and structural differences in the vascular plexus. In solid tumors, angiogenesis is well characterized as a critical step for growth, invasion and metastasis.3, 22, 23
To further validate our results of HMQ18–22, we established a new colon angiogenesis tissue model. In this model, the tissues cultured from colon were grown in a three-dimensional matrix (usually fibrinogen or matrigel). In this model, new capillary vessels erupted from the tissue in matrigel, and new erupted endothelial cells grew from tissues in fibrinogen proliferated and formed tube. These results generated by total nutritive index difference between fibrinogen and matrigel. The different type from the two culture mediums in the colon tissue model could be used to assess angiogenesis and tube formation at tissue and organ Level. HMQ18–22 inhibited the sprouting of new vessel from the tissue and tube formation by the erupted endothelial cells, further supporting the notion that HMQ18–22 suppressed the neovascularization.
The VEGF/VEGFR pathway was an important regulator in tumor angiogenesis. It has led to the development of novel antiangiogenic agents. The various downstream signaling molecular players, such as PI3K/AKT, MAPK and PLCγ
/PKC, have the specific functions including cellular proliferation, migration, angiogenesis and apoptosis.16, 17, 18
1), a very important member of phospholipase C (PLC) families, is upregulated in many cancer tissues and cancer cell lines and has been found to participate in many physical processes such as cell proliferation, motility, differentiation, cytoskeleton rearrangement and so forth.24, 25
There is an intimate relation between PLC-γ
1 and PKCα
. Activated PLC-γ
1 can catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diaeylglyeerol (DAG). DAG and IP3 are second messengers to activate PKC. A decrease in the extent of tyrosine phosphorylation of PLC-γ
1 has also been proved to be positively regulated by PKCα
So, downregulation of surface receptor expression represents a mechanism in which decreased PLC might block PKC activation. Furthermore, in the signaling pathway, PI3K/AKT mediated cell proliferation, migration, invasion, angiogenesis and metastasis.28
Akt is a kinase in the signal transduction, making it a potential target. The Akt/PKB kinase has been implicated in the genesis and/or progression of numerous human tumors, because AKT has a wide range of downstream targets that regulate endothelial cell functions such as migration, growth, proliferation and apoptosis.29
HMQ18–22 acted on VEGFR and inhibited the phosphorylation of VEGFR by p-VEGFR AlphaScreen assay. However, VEGFR kinase activity was not affected by HMQ18–22, indicating that the inhibition of VEGFR phosphorylation is not due to inhibition on kinase of VEGFR2 Kinase. Using a phospho-antibody array to screen the potential target in the VEGF/VEGFR pathway, we found that the phosphorylation of VEGFR2, VEGFR1, Akt, PKCα
-1 were inhibited by HMQ18–22. We further confirmed the array data by western blots using antiphosphorylation antibodies against VEGFR2, VEGFR1, Akt, PKCα
-1 in culture cells and in vivo
human colon cancer mouse models. Knockdown of VEGFR2, VEGFR1, Akt, PKCα
-1 by siRNA significantly attenuated tumor inhibitory effects of HMQ18–22. Taken together, these results suggest that HMQ18–22 inhibits tumor angiogenesis and tumor growth by downregulating the phosphorylation signaling of VEGFR2, VEGFR1, Akt, Raf1, PKCα
-1. A compound exerts its action by entering the cell or acting on the cell membrane receptor with good affinity. In previous reports, taspine could enter the cell or act on the cell membrane receptor determined by HPLC-MS and cell membrane chromatography.30
HMG18–22 is a derivative of taspine, so HMG18–22 should exert its inhibition and downregulate the phosphorylation signaling by entering the lovo cells and being with good affinity with VEGFR-1 and VEGFR-2.
In conclusion, our results demonstrate that HMQ18–22 might be a novel angiogenesis inhibitor that reduces angiogenic responses in vivo and in vitro by blocking VEGFR signaling pathways. The effectiveness of HMQ18–22 in disrupting colorectal tumor growth provides a promising anticancer agent for further clinical trial for HMQ18–22 to colorectal cancer treatment.