| Objective:To probe into the killing effects and mechanisms of a novel Zinc phthalocyanine photochemotherapeutic agent ZnPcH1 based photodynamic therapy (ZnPcH1-PDT) on acute leukemia cell lines.Methods : Acute lymphoid leukemia cell line Jurkat cells and acute erythroleukemia cell line HEL cells were employed in present study. Leukemia cell lines with 4×106/ml cellular density were re-suspended in RPMI 1640 medium in the presence of different concentrations of ZnPcH1 (0.125, 0.25, 0.5 and 1.0μmol/L), respectively, and incubated for 5h before photo-irradiation. Photo-irradiation (1.9 J/cm2 at 38.3 mW/cm2) was carried out by a LD-670 semiconductor laser (670 nm, Institute of Laser, Tianjin, China). The laser was calibrated by a power meter (TMP 310; Lexel, USA). Meanwhile, three control groups were established, including the RPMI 1640 blank control, the photo-irradiated control and the ZnPcH1 control. The proliferative potency of Jurkat cells and HEL cells were detected by Typan blue stain assay, MTT colorimetric assay and colony formation assay. Several parameters were performed to detect the apoptotic cells, i.e. acridine orange and ethidium bromide stains (AO/EB stains), identification of nucleosomal DNA cleavage by gel electrophoresis and the detection of Annexin-V-FITC/PI by flow cytometry. The levels of Akt, P-Akt, P-GSK3β, P53, Bax, HSP70 and Bcl-2 proteins were determined by Western blot.Results:①The results of trypan blue stain assay, MTT colorimetric assay and colony formation assay showed that PDT presented significant anti-proliferation effects on both Jurkat cells and HEL cells in a dose dependent manner (0.125, 0.25, 0.5 and 1.0μmol/L, p﹤0.01). However, no substantial differences existed among the three control groups.②AO/EB stains showed that at 6h after PDT, the morphology of both Jurkat cells and HEL cells remarkably changed, and Jurkat cells were more sensitive to PDT than HEL cells; post PDT 6h,12h,24h and 36h, the apoptosis proportion of Jurkat cells were 21.4±2.3%, 57.4±1.3%, 7.4±1.1%, 0.8±0.4%, respectively; and that of HEL cells were 12.9±1.9%, 14.5±1.4%, 21.1±1.0%, 23.4±0.6%, respectively; the necrosis percentage of Jurkat cells were 10.3±1.5%, 15.5±2.3%, 71.5±2.4%, 97.3±1.3%, respectively; and that of HEL cells were 2.3±0.6%, 7.1±1.5%, 8.1±1.6%, 20.3±0.4% respectively. DNA extracted from either Jurkat cells or HEL cells presented characteristic"ladders"of DNA fragments that were observed by gel electrophoresis. Annexin-V-FITC/PI double stains showed apoptosis rates increased in a time dependent manner(6h, 12h, 24h and 36h). At the different time point, the apoptosis rates of Jurkat cells were 30.69±4.44%, 36.59±4.09%, 40.63±4.18%, 54.84±4.61%, respectively; and that of HEL cells were 20.76±4.34%, 27.78±4.48%, 39.66±4.28%, 51.75±5.06%, respectively.③Western blot discovered that at different time after PDT treatment the expression of HSP70, Bcl-2, Akt, P-Akt and P-GSK3βproteins(Jurkat) were down-regulated, while that of P53 and Bax proteins were up-regulated, the level of P-GSK3βproteins in HEL cells were not changed after PDT.Conclusion:ZnPcH1-PDT could significantly inhibit the proliferation of both Jurkat cells and HEL cells.and PDT could also induce apoptosis in the two cell lines. Moreover, Jurkat cells were more sensitive to PDT than HEL cells.
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