The Screening Of The Inhibitor Targeting For NF-kB Pathway Based On Reporter Gene And Anti-tumor Activity Research

Colorectal cancer (CRC) is one of the primary malignant tumor of harm to human health, with colorectal cancer being the second leading cause of cancer death. In recent years, with the change of diet, the incidence rate is gradually increased, while the onset is getting younger. In recent years, great progress has been made in the development of chemotherapy for advanced CRC, but prolonged survival is not obvious, adverse drug reactions and other issues yet to have a breakthrough, therefore, treatment of advanced CRC requires new strategies to replace or complement current therapies. NF-κB involved in the regulation of transcription of a variety of apoptosis-related and proliferation-related genes, which play an important role in the development of colon cancer.NF-κ B was to be a ubiquitous transcription factor present in all cell types. In its resting stage, this factor resides in the cytoplasm as a heterotrimer consisting of p50, p65, and I κ Bα. On activation, the I κ Bα protein, an inhibitor of NF-κB, undergoes phosphorylation, ubiquitination, and degradation. p50and p65are then released to be translocated to the nucleus, bind specific DNA sequences present in the promoters of various genes, and initiate transcription.In this paper, a inhibitors screening model based on reporter gene targeted for NF-κB pathway is successfully established, a variety of compounds are investigated on their effects on LPS-induced NF-κ B activation, and a compounds targeting the NF-κ B pathway is successfully screened:Triptolide derivatives(LLDT-246). With such as Western blot biology detection method evaluate its impact on moleculars associated with the NF-κ B signaling pathways, explore the anti-tumor activity and its induction NF-κ B signaling pathways. In this assay system, a variety of compounds were investigated on their effects on LPS-induced NF-κ B activation. Several compounds were found to have inhibitory effects on LPS-induced NF-κ B activation.1. Screening for NF-κ B pathway targeting inhibitors based on reporter gene assayScreening for NF-κ B pathway targeting inhibitors based on reporter gene assay is successfully established, after a positive drug the NF-κ B specificity inhibitor PDTC to validate the model. In this assay system, a variety of compounds are investigated on their effects on LPS-induced NF-κ B activation. Several compounds are found to have inhibitory effects on LPS-induced NF-κ B activation.2. The effect of triptolide derivatives LLDT-246on NF-κ B signaling pathways Triptolide derivatives LLDT-246can suppresses LPS-induced and constitutive activation of NF-κ B in HCT116cells, suppresses the phosphorylation of I κ B, but has no effect on the expression of I κ B. Inhibition TNFa-induced transport into the nucleus of the p65, LLDT-246is found to have significant inhibitory effects on the expression of NF-κ B target genes (Cyclin D1, Mcl-1, Bcl-2), and further verified that LLDT-246is a inhibition targeted for NF-κ B.3. The anti-tumor activity of triptolide derivatives LLDT-246and its targeting for NF-κ B signaling pathwaysSRB assay results show that, LLDT-246has a strong growth inhibition of HCT116, HT-29the two colon cancer cells, LLDT-246has a strong anti-proliferative activity in vitro, further study found that LLDT-246leads to Caspase-9, Caspase-3and PARP cleaved in HCT116cells. These results suggest that the mitochondrial apoptosis pathway is an important way of HCT116cell cytotoxicity mediated by LLDT-246. LLDT-246and TNF-a have a synergistic effect on three colon cancer cells apoptosis when they co-administered. The mechanism of LLDT-246induced NF-κ B pathway may involve its effects on the PI3K/AKT classic signaling pathway, thereby inhibits the activity of p-GSK3β, p-mTOR; LLDT-246can also inhibits the activity of p-STAT3. But had no effect on the expression of p-JNK and p-ERK in HCT116cells.Taken together, Screening for NF-κ B pathway targeting inhibitors based on reporter gene assay is successfully established. Several compounds are found to have inhibitory effects on LPS-induced NF-κ B activation. LLDT-246targeting inhibit NF-κ B pathway and has strong anti-tumor activity on the three colorectal cancer cells by using cell culture, western blot and other modern pharmacology and molecular biology detection methods. The mechanism of LLDT-246induced NF-κB pathway may be associated with the classic PI3K/AKT signaling pathway, inhibits the activity of p-GSK3β,p-mTOR, p-STAT3, thereby inhibits the activity of NF-κB. The data in the present study not only prompt the development potential and value treatment of inflammation-associated tumors, but also support the pharmacological basis of the use of triptolide derivatives LLDT-246as traditional herbal medicines for the treatment of anti-inflammation and anti-tumor. Provide a new way of thinking for the development of targeted therapy of colorectal cancer and other inflammation-associated tumors.