Detection Of Telomerase Activity In Psoriasis, And A Study On Antipsoriatic Effects Of Camptothecin And Isocamptothecin

Objective: Telomerase is a ribonucleoprotein complex that synthesizes telomeric DNA onto chromosome ends using its RNA component as template, enabling extension of cell lifespan and even immortalization. Telomerase is expressed in most malignant tumors and some proliferative diseases including psoriasis. Psoriasis is a T-cell mediated disorder, with abnormality in keratinocytes proliferation and apoptosis. Telomerase activity is closely related to T-cell activation and keratinocytes proliferation and apoptosis in psoriasis. In our study, we investigated the effects of camptothecin and a novel camptothecin analogue, isocamptothecin on proliferation, apoptosis and telomerase activity in keratinocyte HaCaT; examined their effects on telomerase activity of human umbilical vein endothelial cell ECV304, to explore the mechanism of their antipsoriatic effects; and evaluated the telomerase activity in peripheral blood mononuclear cells (PBMC) from psoriatic patients and its correlation with disease severity, to understand its action in psoriasis. Methods: Cultured HaCaT and ECV304 cells were exposed to camptothecin and isocamptothecin 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. Results: (1) Both camptothecin and isocamptothecin inhibited HaCaT cell proliferation in a time and concentration-dependent manner. The IC50 of camptothecin and isocamptothecin for 24h, 48h and 72h were 1.893μg/ml, 1.029μg/ml, 0.332μg/ml and 81.69μg/ml, 74.63μg/ml, 66.56μg/ml respectively. Observation of the treated cells under inverted microscope and transmission electron microscope revealed characteristic apoptotic morphological changes. Flow cytometry analysis demonstrated that both drugs could arrest HaCaT cells in S phase and induce apoptosis in a concentration-dependent manner. Telomerase activity of HaCaT cells was down-regulated at concentrations both sufficient (1 and 2μg/ml; 70 and 85μg/ml) and insufficient (0.5μg/ml; 55μg/m) to induce apoptosis, and the inhibitory effect correlated with drug concentration. (2) Camptothecin could inhibit telomerase activity of ECV304 cells not only at concentrations sufficient to induce apoptosis (0.125 and 0.5μg/ml) but also at concentrations insufficient to induce apoptosis (0.063μg/m), the inhibitory effect correlated with drug concentration. Isocamptothecin could also inhibit telomerase activity of ECV304. (3) Telomerase activity was significantly increased in PBMC from psoriatic patients (0.94±0.18) compared with that from healthy control (0.49±0.11) (t=8.096, P<0.001). Patients of progressive phase displayed higher levels of telomerase activity( 0.97±0.19 ) than static phase (0.90±0.17), but without statistical significance. A positive correlation was found between telomerase activity of PBMC and PASI score (r=0.889, P<0.001), but no correlation between telomerase activity and duration was found. Conclusions: Camptothecin and isocamptothecin could inhibit proliferation and induce apoptosis in keratinocytes in a concentration-dependent manner. Both drugs could inhibit telomerase activity of vascular endothelial cells and keratinocytes, at concentrations lower than that of apoptosis induction, suggesting that cell apoptosis may be preceded by down-regulation of telomerase activity. The mechanism of antipsoriatic effects of camptothecin may be associated with on the one hand antiproliferation and apoptosis induction in keratinocytes through down-regulation of telomerase activity, and on the other hand antiangiogenesis in the same way. Isocamptothecin may have similar antipsoriatic effects with camptothecin. Telome...