| Sorafenib (Nexavar), is a first multi-kinase inhibitor and one of the most widely used small-molecule oral-targeted drugs. It has been widely used for the treatment of patients with advanced renal cell carcinoma (RCC) and unresectable hepatocellular carcinoma (HCC). It also has shown clinical advantage in many cancers, such as HCC, breast cancer, thyroid cancer, RCC and lung cancers. At present, sorafenib is recommended as the standard first line treatment for advanced HCC, the second-line treatment for advanced RCC, and, more recently, the treatment of late-stage (metastatic) differentiated thyroid cancer. Sorafenib shows potent inhibition on tumor growth by inhibiting serine/threonine kinases (such as Raf-1, B-Raf V600E, wild-type B-Raf and C-Raf), as well as several receptor tyrosine kinases (RTK), including platelet-derived growth factor receptor β (PDGFR-β), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR-1, VEGFR-2 and VEGFR-3), Flt-3, and c-Kit. However, the treatment of sorafenib was often interrupted and/or dose reduced due to some adverse events. The most common adverse effects of sorafenib may impact quality of life, including neutropenia, hypertension, liver dysfunction, kidney injury and so on. In addition, sorafenib is a patent-protected drug with high selling price in China, which increases the cost of Chinse patients to accept treatment. Thus, new derivatives of sorafenib with lower toxicity and higher activity than sorafenib are urgent needed and under research.Due to their good advantages and adverse effect, a series of diarylurea compounds based on the structure of sorafenib were synthesised in Prof. Li Wenbao laboratory. We evaluated the inhibitory effect of 17 compounds against 5 kinds of cancer cells using MTT assay in our primary screening assays. Using inhibition rate (%) and IC50 values (μM) as reference, we screened out 6 compounds including 1118-20、1124-21、1124-15、1125-67、1125-4、1118-41, which exhibited stronger activity against human cancer lines than that of sorafenib. Especially,1118-20 significantly inhibited the growth of 5 kinds of cancer cell lines among them. Thus, we chose 1118-20 as the potential compound to study the underlying mechanisms against HepG2 cells.MTT and clone formation assays showed that 1118-20 exerted stronger inhibitory effect on the proliferation of HepG2 than sorafenib. However,1118-20 weakly suppressed the growth of human normal hepatocyte cell HL-7702 without significant difference; and the cytotoxic effect of 1118-20 was higher on HepG2 than HL-7702. The analysis of cell cycle distribution suggested that 1118-20 could induce HepG2 cells arrested in S cell cycle. Hoechst 33258 staining showed that 1118-20 induced HepG2 cells apoptosis with chromatin condensation, split and marginalization. Annexin-V/PI double staining displayed that 1118-20 could increase ratio of apoptotic cell; western blotting indicated that the expressions of cleaved caspase-9, cleaved caspase-3 and its substrate cleaved PARP were upregulated after 1118-20 treatment. JC-1 and western blotting analysis indicated the decrease of mitochondria membrane potential (△ψm), down-regulation of Mcl-1, increase of ratio of Bax/Bcl-2, which suggested the activation of mitochondria mediated apoptosis. Further study analysis showed that 1118-20 also involved in regulation Wnt/β-catenin pathway, EGFR/PI3K/Akt pathway and Ras/ERK pathway. These results showed that 1118-20 blocked the three signal pathway to inhibit the cancer cells growth.Tumor angiogenesis, emerging as a hallmark of cancer, is essential for the growth and metastatic of cancer cells. Without blood vessels, cancer cells would not acquire adequate nutrition and oxygen and efficiently evacuate the undesired waste products, restricting cancer progression towards malignancy and metastasis. MTT assay indicated that 1118-20 significantly inhibited the proliferation of human umbilical vein endothelial cells HUVEC, with stronger inhibition effect than sorafenib. Scratch assay showed that 1118-20 blunted the migratory property of HUVEC. The levels of active MMP-2 was obviously suppressed by 1118-20, as well as the decrease of active MMP-9. Further study indicated that 1118-20 inhibited the capillary structure formation of HUVEC on 3-D Matrigel. Meanwhile,1118-20 obviously decreased the expression of angiogenic molecules, including FGF-2, VEGF, VEGFR-2, EGFR, and blocked the phosphorylation of VEGFR-2, EGFR, which destroyed the molecular basis of angiogenesis.In conclusion,1118-20 was a promising compound with stronger anticancer activity than sorafenib and with low toxicity. In HepG2 cells,1118-20 could not only induce apoptosis via Bcl-2 family mediated mitochondria apoptosis pathway, but also regulate Wnt/β-catenin pathway, EGFR/PI3K/Akt pathway and Ras/ERK pathway to inhibit the growth of cancer cells.1118-20 also suppressed capillary structure formation to restrict cancer progression. These results support the potential of 1118-20 to be developed as a promising agent for treatment of cancers. |
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