D261Inhibits The Growth Of NSCLC Cells And Its Mechanism

The morbidity and mortality of lung cancer today is one of the highest growing of various cancers which seriously harm to human’s health and life. In China, the mortality of lung cancer in the city has been ranked first in the tumor death. Non-small cell lung cancer (NSCLC) is the most common type, observed in approximately85%of patients, making it a major public health concern.With the increase of lung cancer, the effect of traditional treatment does not improve significantly, as well as one of the main reasons is usually diagnosed at an advanced stage. In the chemical class of anticancer drugs, the vast majority of active ingredients are natural anti-tumor lead compounds, further modified, in order to achieve medicinal purposes. Therefore more options to treat lung cancer should be needed, small molecules extracted from natural products is the main source of new drugs.In this study, we attempt to analyze the D261, a novel nature compound, which is able to target non-small cell lung cancer. Our study showed that D261could effectively suppress the growth of NCI-H460and A549. This assay initially discussed the researches and developments of D261and simply explained its principles and functions of anticancer. 1. The effect of D261on non-small cell lung cancer cellsIn this study, we used CCK-8assay to detect cell viability of a variety of tumor cells, A549, NCI-H460, MCF7, HepG2and KGN which treated on D261(0.3125-20uM); we used CFSE staining assay to detect cell proliferation; we also used cell clone assay to explore D261impact on the role of cell clones. Experimental results showed that, D261can inhibit non-small cell lung cancer cell A549, NCI-H460cell viability in a dose-dependent manner. D261can effectively inhibit the two types of non-small cell lung cancer cell proliferation. Cell cloning experiments showed that2uM and8uM of D261effectively inhibited the clone growth in the long,-term. In summary, D261can effectively inhibit the non-small cell lung cancer cell growth.2. Mechanism of D261on cell growth inhibitionInhibition of cell growth mainly from two aspects, one is the effect of promoting cell apoptosis and the other is inhibiting the cell cycle. We studied on these two aspects to explore how D261works. Disorders of cell cycle play an important role in the process of tumor development. Therefore, the tumor is a class of cellular cyclical disease. Main molecules involved in cell cycle regulation are cyclin, cyclin-dependent kinase (CDK) and cyclin dependent kinase inhibitors (CKIs). CDK plays a positive role of regulation, while CKIs act as a negative control. Studies have shown that excessive activation of the PI3K/Akt signaling pathway in NSCLC is very common, which regulate cell cycle and cell proliferation. Phosphorated Akt regulate cyclin D1kinase by glycogen synthase-3β (glucose syntase kinase-3β, GSK3β) to prevent cyclin Dl down. In addition, phosphorated Akt can inhibit the expression of CKIs p27kip1and p21CIP1/WAF1. Akt phosphorylation express abnormal in about50%-70%of NSCLC. Now there are a series of specific drugs that target this pathway.First, we detect the effect of apoptosis of D261. After incubation with D261for24h and48h, apoptosis was detected by Annexin V and propidium Iodide staining. The results showed that the treatment of the D261did not significantly alter apoptosis on A549and NCI-H460. However, we found cell cycle arrest in G1phase. cyclin D1, cyclin El, CDK2and CDK4protein are important in cell cycle G1phase to S phase node. We detected these proteins by western blot. The results showed that the expression of cyclin D1and CDK2/4were decreased with increasing concentration of D261, and the cell cycle arrest in the G1phase. We also detected pAkt/Akt expression by western blot analysis. It turned out that the decline in the expression of phosphorylated AKT, and increased expression of p21.3. D261inhibits tumor growth in vivoIn order to evaluate the effect of D261on tumor suppression in vivo, we established a nude mouse model. NCI-H460cells were diluted to4×106cells/100μl using growth medium. The100μl of cell suspension was injected subcutaneously into right flank area of each mouse. After3days, mice were divided into two groups:the experimental group (5mg/kg D261, i.p.) and control group (DMSO in saline, i.p.). The body weight and tumor volumes of the mice were monitored every3days. Results showed that the5mg/kg of D261did not affect body weight of mice, but significantly decrease tumor volume and weight.