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Molecules 2011, 16 1398 3.8. DNA flow cytometry analysis for cell cycle and apoptosis The harvested Bel-7402 cells were washed twice in phosphate-buffered saline, fixed with 70% ice- cold ethanol for 30 min, washed twice with phosphate-buffered saline, and stained with PI (50 μg/mL) containing RNAase (25 μg/mL) for 30 min. Cell cycle and apoptosis were assayed by DNA content on a flow cytometer. 3.9. Immunoblot assay All immunoblots were performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bis-Tris gel electrophoresis as outlined by the supplier. For total cellular protein, Bel- 7402 cells were lysed in buffer containing 25 mM Hepes, pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 20 mM β-glycerophosphate, 0.5 mM DTT, 1 mM sodium orthovanadate, 0.1 μM okadaic acid, and 1 mM phenylmethylsulfonyl fluoride. Protein concentrations of the cell lysates were determined by lorry method with bovine serum albumin as standard, and the supernatants were boiled in SDS sample buffer for 5 min. Equal amounts of lysate protein were run on 12% SDS– PAGE and electrophoretically transferred to PVDF membrane. After blocking, the blots were incubated with specific primary antibodies (anti-Bcl-2 and anti-Fas antibodies) overnight at 4 °C and further incubated for 1 h with horseradish peroxidase -conjugated respective secondary antibody. Bound antibodies were detected by enhanced chemiluminescence kit with a Lumino Image Analyzer (Founder, Beijing, China). 3.10. Statistical analysis Values are means ±S.D. of three independent experiments. Statistical significance was determined using Student’s unpaired two-tailed t-test (SPSS 10.0 software). P value less than 0.01 was statistically significant. 4. Conclusions In the present study, we found that an intense absorption in UV-vis absorption spectrum of TαPcZn was in the red visible region at 650–680 nm, that green TαPcZn localized to both plasma membrane and nuclear membrane of Bel-7402 cells, and that TαPcZn-PDT significantly resulted in the proliferation inhibition, apoptosis induction, S cell cycle arrest, and down-regulation of Bcl-2 and Fas. Taken together, we conclude that TαPcZn-PDT inhibits the proliferation of Bel-7402 cells by triggering apoptosis and arresting cell cycle. Acknowledgements This project was financial support by the Natural Science Foundation of Heilongjiang Province (No. D200822), the Scientific Research Foundation from the Health Department of Heilongjiang Province(No. 2005-333), the Scientific Research Foundation from the Education Department of Heilongjiang Province(No. 11511441), and the Scientific Research Foundation from Qiqihar (No.SFG06-02 and No. GY05-19).PDF Image | Zinc-Mediated Photodynamic Therapy Inhibits the Proliferation
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