Construction And Optimization Of NF-κB Transcription Factor Reporter Gene System And Its Application In Screening TRAIL-promoting Anti-tumor Drug

TRAIL,tumor necrosis factor related apoptosis inducing ligand,is a kind of protein human can secrete.Due to its ability to kill tumor cells while having small cytotoxicity to normal cells,it was regarded as a potential anti-tumor protein drug with valid practical values.However,after the clinical application of TRAIL,some kind of tumors are found to be resistant to TRAIL,and some acquired the resistance after the TRAIL induction,which were originally sensitive to TRAIL induced apoptosis.Thus,these reasons restricted the usage of TRAIL in clinical therapy.At present,the reported underlying mechanisms of TRAIL resistance mainly include the following three aspects,1)the abnormal expression and distribution of TRAIL receptors,2)the abnormality of apoptosis related factors in tumor cells,3)TRAIL activated some non-apoptotic signal pathways,such as NF-κB、MAPK、PI3K/AKT、STAT pathways,while initiating the apoptotic pathway.[Objective]Based on the present mechanism research of TRAIL resistance,we tried to enhance the anti-tumor capability of TRAIL through inhibiting the NF-κB pathway which was considered to hinder the apoptosis inducing ability of TRAIL.The main four parts of the project maybe concluded as:1)the construction and modification of NF-κB reporter gene system;2)screening of traditional Chinese medicine ingrediants can inhibit or decrease NF-κB;3)combination therapy between TRAIL and the ingrediants selected from part 2);4)preliminary mechanism exploration behind the promotion of the effective ingredients from part 3)on TRAIL induced cytotoxicity.[Methods and Results]1.Construction and verification of the NF-κB regulated NLuc luciferase-expressing cell line.Through deleting the original CMV promoter of pQCXIP vector,and then inserting the NF-κB enhancer sequence and NanoLuc luciferase reporter gene sequentially,we constructed the pQCXIP-NF-κB-NLuc expression vector.The constructed pQCXIP-NF-κB-NLuc plasmid with p-VSVG packing plasmid was transfected into human renal epithelial packing cell GP2-293 to prepare the retrovirus.Human cervical cancer cell line HeLa was infected with the prepared retrovirus,and then puromycin positive cells were selected.Furthermore,the cell clone has the strongest response to the stimulation of TNFα,which is the activator of NF-κB pathway,was selected finally.Further results showed it responses to the stimulation of TNFa in a time-and concentration-dependent way,and it indicated that the NF-κB regulated NLuc luciferase-expressing cell line was successfully constructed,and it can be used to the quantification of NF-κB transcriptional activity and in the NF-κB regulation-related drug screening.2.Modification of the assay method of NLuc luciferase.During the application of the constructed cell line,we found the pH of the culture medium and the serum in the culture medium would affect the detection of NLuc luciferase,thus having bad effects of the next drug screening.We verified that the pH and the serum were part of the reason,and then we selected the PBS buffer containing 0.03%(V/V)Triton X-100 as the detection buffer,in which the intensity and stability of NLuc luciferase would be better.3.Screening of the ingredients inhibiting NF-κB.With the constructed cell line and modified assay method,36 crude extractions or monomers of traditional Chinese medicine were assayed,and 12 kinds of which have the potential ability to inhibit the activity or expression of NF-κB.Two monomers,QD6-4 and SKN,were finally chosen to combine with TRAIL in the research of anti-tumor ability,after the exclusion of the ingredients which have strong cytotoxicity or may contribute to the proliferation of the tumor or have been reported in combination with TRAIL4.Combination therapy between TRAIL and selected ingredients in anti-tumor experiments.The cell viability results showed that SKN can obviously enhance the anti-tumor ability of TRAIL in A549 cell line,and synergistic effect was found when SKN was treated at the concentration of 2-8μM.However,it has little effects on COLO205 cell line,and QD6-4,another selected monomer,almost have no effects on both A549 and COLO205 cell lines.In addition,SKN can also promote the killing ability of TRAIL in human colorectal cancer cell line HCT116,human cervical cancer cell line HeLa and human pancreatic cancer cell line PANC-1,and it showed a synergistic effect when treated at the concentration of 2-8μM.5.Exploration of the mechanisms underlying the synergistic effect between SKN and TRAIL on apoptosis inducing.Besides,we preliminarily explored the mechanisms behind the synergistic effect between SKN and TRAIL on tumor killing.Annexin V-PI flow cytometry results indicated that A549 and PANC-1 cells both appeared apparent apoptosis after the combination therapy of SKN and TRAIL,while that treated with SKN or TRAIL alone didn’t show or showed just a slight apoptosis.Caspase 3 and Caspase 8 were both evidently activated after the combination treatment of SKN and TRAIL when compared with the control group,while Caspase 9 almost the same with the control one,which indicated that SKN enhance the tumor killing ability of TRAIL by the extrinsic apoptosis pathway.Furthermore,A549 treated with SKN alone or SKN combined with TRAIL were both showed reduction of p65,which was the core element of NF-κB.SKN and TRAIL combination treatment still inhibit the anti-apoptosis protein Mcl-1、Bcl-2、Bcl-xL,while have no effect on pro-apoptosis protein Bax.In conclusion,SKN may inhibit the NF-κB partly by reducing the amount of p65,and the synergistic effect between SKN and TRAIL on apoptosis maybe related to the anti-apoptosis protein inhibition.[Conclusions]1.We successfully constructed the NF-κB regulated NLuc luciferase-expressing cell line and modified the detection way of NLuc,and it can be used to the quantification of NF-κB transcriptional activity.2.SKN can enhance the anti-tumor ability of TRAIL in A549,HCT116,PANC-1 and HeLa cell lines,and SKN can augument the apoptosis induced by TRAIL in A549 and PANC-1 cells.3.Combinational treatment with SKN and TRAIL in A549 can activate Caspase 3and Caspase 8,while having little effects on Caspase 9.4.Combinational treatment with SKN and TRAIL in A549 can inhibit p65,anti-apoptotic protein Mcl-1、Bcl-2、Bcl-xL,while have no effect on pro-apoptotic protein Bax.