A Study On Antitumor And Psoriatic Effects And Mechanism Of Isocamptothecin

Objective: Camptothecins (CPTs) are DNA topoisomerase I inhibitors which have been extensively used clinically. In the recent years, attentions have been focused on developing effective CPT analogues with lower toxicity. Studies have showed that CPTs could inhibit proliferation, induce apoptosis and promote differentiation in tumor cells, venular endothelial cells and keratinocytes. Iso-camptothecin (Iso-CPT) is a novel CPT analogue. In our study, we investigated the effects of a novel CPT analogue, Iso-CPT and CPT on proliferation, apoptosis and telomerase activity in human squamous carcinoma cell Tca8113, human umbilical venular endothelial cell ECV304 and human keratinocyte HaCaT; to explore the effect and mechanism of their antitumor and antipsoriatic effects; in relation to potential use in treating tumors and psoriasis .As well evaluated the telomerase activity in peripheral blood mononuclear cells (PBMC) from psoriatic patients and its correlation with disease severity, to understand its action in the development of psoriasis. In this study, antisense recombinent plasmid of hTERT gene was constructed and then transfected into human tongue cancer Tca8113 cells in order to study its effect on the telomerase activity and cell growth.Methods: Cultured Tca8113,Hele, HaCaT and ECV304 cells were exposed to Iso-CPT and CPT at different concentrations. Inhibition on cell proliferation was measured by MTT assay. Morphological changes were observed under inverted microscope and transmission electron microscope. Cell cycle and apoptosis were analyzed by flow cytometry. Telomerase activity was detected by TRAP-ELISA method. PBMC form 19 psoriatic patients and 13 normal controls were obtained by density gradient centrifugation. Telomerase activity of PBMC was measured, and its correlation with severity and duration of psoriasis was analyzed. hTERT gene was cloned from Hela cells by RT-PCR. First ,RNA was distilled and reverse transcripted to get cDNA, The recombinant plasmid was identified by PCR, endonuclease cutting and sequence assay. The recombinant plasmid was transfected into Tca8113 cells using liposome. Cell genome DNA was extracted to do PCR and Southern blot in order to assay whether recombinant plasmid was integrated to cell genome. On the base of conventional telomeric repeat amplification protocol(TRAP), a DNA seqence analysis technique was used to assay telomerase activity. Then cell growth curve was drawn to observe effects of hTERT antisense gene on the growth of Tca8113 cells.