Effects Of Compound BENC-511 On Suppressing Non-small Cell Lung Cancer Invasion And Metastasis

Non-small cell lung cancer (NSCLC) is the foremost cause of cancer-related mortality, with high incidence rates, robust metastatic propensity, and acquired resistance to therapy. Most patients present with advanced or metastatic disease and approximately 30% of patients with completely resected node-negative NSCLC develop tumor recurrence even die, so that development of efficient anti-metastasis agents is an important research challenge. More recent trials suggested that more than 60% NSCLC patients overexpress epidermal growth factor receptor (EGFR) and initial therapy with a tyrosine kinase inhibitor (TKI) instead of chemotherapy may be the best choice of treatment, while no drugs have shown increased overall survival. The most challenge is that cancers harboring active or overexpressed receptor tyrosine kinases (RTKs) such as EGFR or HER2 can display resistance to TKIs through PI3K signaling. Mounting researches demonstrated that PI3K/Akt signal pathway played crucial role in NSCLC metastasis. In the front of metastasis site of most NSCLC patients, there has find an aberrant amplification of PIK3CA gene, overexpressed PI3K protein and abnormal activation of downstream signal pathway, suggesting that anti-cancer drugs targeting some key factors in this pathway is popular in researches on cancer prevention and cancer targeted therapy. Now there has some PI3K pathway inhibitors apply to part of clinical patients, while the development of small molecular inhibitors of this signal pathway still hot in drug development field.Small molecular compound, S14161 (8-Ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene), is 3-nitro chromene racemic compound, has potent PI3K inhibition effect, but because of its chiral structures and poor solubility limited its further application. In order to develop better compound, the laboratory of Prof. ZP Liu, in ShandongUniversity, simplified the structure of S14161, removed the 4-fluorophenyl, and acquired BENC-511 (6-bromo-8-ethoxy-3-nitro-2H-chromene) with better activation. In previous study, it has documented that both of them has strong inhibition effect on PI3K/Akt signal pathway. BENC-511 can significantly inhibit the phosphorylation of Akt and downstream factors. It is effective on inhibiting leukemia and multiple myeloma tumor growth and inducing cancer cells apoptosis with low toxicity. However, the effects of BENC-511 on lung cancer especially highly metastatic non-small lung cancer invasion and metastasis are not clear.The aim of this research is to explore the activities of BENC-511 on invasion and metastasis of NSCLC in vitro and in vivo. Furthermore, demonstrate its mechanism by detecting the expression of PI3K signal related proteins and interaction of proteins. Our study complements the profile of anti-cancer activity of BENC-511 and proposes novel possible targets of BENC-511 that enlighten new idea about development of anti-cancer metastasis drugs.Methods and resultsIn A549 and HUVEC cell lines, we used wound healing assay to test the inhibitory effects of BENC-511(5,10 μmol/L) and S14161 (10 μmol/L) on cells migration. Then we used the Transwell system to verify the inhibitory effects of BENC-511 (1.25,2.5, 5,10 μmol/L) on A549 cells invasion. To further understand the effect of BENC-511 on tumor cell migration in vivo, we established hematogenic lung metastasis mice model and A549-luc-GFP experimental lung metastasis mice model through injecting B16BL6 cells or A549-luc-GFP respectively into the tail veins of syngeneic C57BL/6J mice or nude mice. Mice were then daily treated with 20 and 40 mg/kg BENC-511 by gavage administration for two weeks. In addition, we detected the effects of BENC-511 on inhibiting tumor growth in A549-luc-GFP xenografts model.We further estimated the activity of MMP-2 and MMP-9 in A549 cells treated with BENC-511 (1.25-40 μmol/L) by gelatin zymography. Western blotting assay were used to detect the expression of MMP-9, MMP-2, EGFR, PI3K, Akt, mTOR and their active form, and GSK-3β, p-GSK-3β, β-catenin and ZEB1 in nucleus.Furthermore, we found that H460, A549, H1299 are three NSCLC cell lines with rise of migration propensity and express different level of ZEB1, β-catenin, EGFR. We used MTT assay to estimate the different sensitivity of BENC-511 on these cell lines and calculated the IC50. The result of Western blotting showed the expression of ZEB1 in three cell lines treated with BENC-511 was inhibited to different degrees. To further understand the link between ZEB1 and the effect of compound, we used shRNA to knockdown ZEB1 in A549 cells and detected the expression of MMP-9, MMP-2 in both protein and mRNA level. Cell viability and invasion of A549 cells that were interfered with shRNA were detected by MTT assay and Transwell assay. We also tested the effects of BENC-511 on A549 cells after knockdown of ZEB1.ConclusionsOur results show that BENC-511 has inhibitory effects on A549 cells migration and invasion by decreasing the expression and activity of MMP-9, and this effect is more potent than S14161. It decreases the expression of main components in PI3K signaling and inhibits EGFR phosphorylation. The inhibition of BENC-511 on NSCLC cells relates to the invasive propensity and expression of ZEBl, β-catenin and EGFR. Knockdown of ZEB 1 can promote the effects of BENC-511.