Identification Of SKP2 Inhibitor Through High Throughput Screening

Background:Aberrant gene expression is one of the most important character of cancer,usually drives proliferation,differentiation and transfer of malignant tumor cells.Imbalance of cell cycle is an unneglectable factor of canceration.Prostate cancer is regarded as one of the most frequent non-cutaneous male malignancies,also the second leading cause of cancer-related death for men.Several studies have pointed out that aberrant expression of SKP2 gene which closely related to cell cycle regulation plays an important role in the development of prostate cancer.SKP2 gene over-expression has been found in a variety of human cancer cells,besides,the ectopic expression of SKP2 gene can also promote the occurrence of cancer.All of those suggest abnormal expression of SKP2 gene has the vital significance in the development of cancers,and inhibition of SKP2 gene over-expression is clearly a promising strategy in therapies of cancer.Transcription therapy for cancer which intends to rectify aberrant of gene expression through direct intervention in the transcription process is a hot spot of gene therapy.As more and more targeted genes to determine,and the continuous improvement of new drug development technology,transcription treatment is now developing rapidly.How to screen out effective anti-cancer drugs from a large number of different kinds compounds,is an important point in drug development.Reporter gene assay has made the high-throughput screening(HTS)simple,economic and efficient for compounds screen.However,randomly-integrated reporters are often epigenetically silenced by flanking condensed chromatin.Considering that problem,Yan developed an Internal Ribosome Entry Site based in-situ report gene assay,by which report gene can be designed and selectively inserted into a gene sequence most close to the location of endogenous gene coding region downstream,and co-expressed with the endogenous gene under the regulation of authentic mechanism.This new type report gene assay can avoid the disadvantages of traditional methods,and its effectiveness remains to be more demonstrated.Objects: Considering the high incidence,mortality,lacking effective therapy of prostate cancer,as well as the importance of Skp2 gene over-expression in prostate cancer,we expect to use Yan's report gene assay technology to screen out compounds that can be used to rectify aberrant of Skp2 gene expression during transcription process.Then verify those compounds' validity and try to explore the mechanism.Through these efforts,we try to provide new strategy of treatment in prostate cancer.Methods: Firstly we constructed Skp2-FLuc DU145 cell line with the help of Yan's new report gene assay technology.Then we screened more than 8000 compounds from NCI libraries,She's lab and Chemibrige commercial chemical library using Skp2-FLuc DU145 cell line.At last we verify those compounds' validity by real-time PCR,western blot assay,MTT assay.Results: With the help of the new report gene assay mentioned above,we successfully screened 10 compounds after luciferase assay and 1 compound(48X from NCI libraries).We affirmed the compound's effect of reduce the over-expression of Skp2 gene in prostate cancer cell line by western blot assay.Besides,the effect of compound is both dose-dependent and time-dependent.MTT assay showed that the compound has influences on the proliferation of prostate cancer cells.Conclusion: The Internal Ribosome Entry Site based in-situ report gene assay developed by Yan is feasible and effective to screen out usefull compounds for cancer treatment.Compound 48X(NCI libraries)can reduce the over-expression of SKP2 gene in prostate cancer,and also restrain the proliferation of prostate cancer cells.