The dual effects of polar methanolic extract of Hypericum perforatum L. in bladder cancer cells

摘要

Introduction and background: We have reported on the polar methanolic fraction (PMF) of Hypericum Perforatum L as a novel photosensitizing agent for photodynamic therapy (PDT) and photodynamic diagnosis (PDD). PMF has been tested in human leukemic cells, HL-60 cells, cord blood hemopoietic progenitor cells, bladder cancers derived from metastatic lymph node (T-24) and primary papillary bladder lesion (RT-4). However, the mechanisms of the effects of PMF on these human cell lines have not been elucidated. We have investigated mechanisms of PMF + light versus PMF-alone (dark experiment) in T-24 human bladder cancer cells. Methods: PMF was prepared from an aerial herb of HPL which was brewed in methanol and extracted with ether and methanol. Stock solutions of PMF were made in DSMO and stored in dark conditions. PMF contains 0.57% hypericin and 2.52% hyperforin. The T24 cell line was obtained from American Type Culture Collection (ATCC). In PDT treatment, PMF (60&mgr;g/ml) was incubated with cells, which were excited with laser light (630nm) 24 hours later. Apoptosis was determined by DNA fragmentation/laddering assay. DNA isolation was performed according to the manufacture's instructions with the Kit (Oncogene Kit#AM41). Isolated DNA samples were separated by electrophoresis in 1.5% in agarose gels and bands were visualized by ethidium bromide labeling. The initial cell cycle analysis and phase distribution was by flow cytometry. DNA synthesis was measured by [3H] thymidine incorporation, and cell cycle regulatory proteins were assayed by Western immunoblot. Results: The results of the flow cytometry showed PMF +light induced significant (40%) apoptosis in T24 cells, whereas Light or PMF alone produced little apoptosis. The percentage of cells in G0/G1 phase was decreased by 25% and in G2/M phase by 38%. The main impact was observed on the S phase which was blocked by 78% from the specific photocytotoxic process. DNA laddering analysis showed that PMF (60&mgr;g/ml) + light at 630nm induced DNA fragmentation in a light dose-dependent manner; in contrast, PMF or light alone did not induce DNA fragmentation. In separate experiments, PMF alone treatment produced a dose-dependent DNA synthesis with a 90% inhibition at a concentration of 25&mgr;g/ml (IC90 = 25&mgr;g/ml). Expression of p53 and p27 cell cycle regulatory proteins was not altered by PMF alone, however, a dose-dependent increase in p21 expression was observed that correlates with PMF concentrations. Cyclin A and cyclin B protein levels showed a clear decrease inverse to the concentration of PMF. In the absence of light treatment, flow cytometry analysis showed that PMF alone results in G0/G1 cell cycle arrest, with a 2-fold increase in G0/G1 cells concomitant with 50% decrease in cells in both S and G2/M phases. However, flow cytometry on PMF alone-treated cells did not show sub G0/G1 peak, further evidence of the lack of apoptosis as a mechanism of effect of PMF in the dark. Conclusions: With respect to light treatment, apoptosis appears to play a vital role in PDT-induced cytotoxicity. The flow cytometry and DNA laddering results revealed that T24 cells demonstrated apoptotic responses in PMF-mediated PDT. Experiments conducted with PMF alone showed a dose-dependent inhibition of DNA synthesis associated with G0/G1 cell cycle arrest and the extract is able to coordinate changes in key cell cycle regulatory proteins in human bladder cancer cells. Both experimental conditions suggest PMF as a potent and effect anti-proliferative agent in cancer chemoprevention and therapy of human urothelial carcinoma cells.