Overespression Of Spermidine Synthase Inhibits Growth And Migration Of Human Caski Cervical Tumor Cells

Objective: To study the effect of SPDS overexpression on polyamine content,proliferation and migration of human Caski cervical cancer cells.Methods The SPDS gene was cloned out from the cDNAs derived from the humanCaski cervical cacer cell by Nest-PCR and cloned into the eukaryotic expression vectorpcDNA3.1(-). Resulted plasmid pcDNA3.1(-)/SPDS and empty plasmid pcDNA3.1(-)were transfected respectively into Caski cells by Lipofectamine-2000reagent to obtainCaski cell line with SPDS overexpression (Caski/SPDS) and control cell line (Caski/3.1).Western blot and RT-PCR were used to determine the expression of SPDS in the proteinand mRNA levels in above cell lines. MTT and FCM were performed to observe the cellproliferation and cell cycle.The polyamine levels were detected by RP-HPLC. The mRNAlevels of polyamine biosynthetic enzymes (ODC, OAZI, AMDC, SMS) and catabolicenzymes (SSAT, SMO, APAO) were measured by QT-RT-PCR. Traswell technique wasused to assay cell migration of the Caski/3.1and Caski/SPDS cells. Western blot assay wasused to determine Annexin A2expression in protein level.Results1) A Caski cell clone with SPDS overexpression (Caski/SPDS) was obtainedsuccessfully. The expression of SPDS in mRNA and protein levels was obviously higher inthe Caski/SPDS cells than that in the Caski/3.1cells.2) The elevated mRNA levels ofODC AMDC SMS SMO SSAT and PAO were observed in the Caski/SPDS cells.3) TheSPDS overexpression could inhibit cell proliferation and effect cell cycle that resulted in anincreased cell ratio in G0/G1phase and decreased cell ratio in S phase.4) The results ofthe RP-HPLC shown that, compared with Caski/3.1cells，putrescine content in theCaski/SPDS cells was dramatically decreased but the contents of spermidine and sperminewere both increased in a small range, and the total content of polyamine was reduced.5)SPDS overexpression dramatically suppressed the Caski/SPDS cells migration and inhibitthe expression of Annexin A2which is a protein factor related to cell migration.6) SPDSoverexpression decreased the sensitivity of the caski cells to the anti-tumor poilyamineanalog BENS.Conclusion：SPDS overexpression reduces the total polyamine content and significanly inhibitscell proliferation and migration in the human caski cervical cacer cell line，which suggests apotential value of SPDS as a molecular target in anti-tumor therapy.