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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.
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