| Summary: | 博士 === 中國醫藥學院 === 中國藥學研究所 === 90 === We have investigated the effects of two pine needles extracts, RPS (Soxlet extract from Pinus densiflora) and FP (Soxle extract from Pinus morrisonicola), two ethanol extracts of raw propolis (obtained from China and Brazil), and Pycnogenol on human leukemia cells HL-60, U937 and K562. A dose-dependent decrease in cell proliferation was observed in all extracts investigated, but with different degrees of inhibition. The IC50 of RPS, FP, and Pycnogenol were 166.07, 209.01 and 200.02 μg/ml for HL-60; 93.96, 100.18 and 40.26 μg/ml for U937; and 177.29, 199.07 and 100.98 μg/ml for K562 cells, respectively. The IC50 of propolis from China were 30.32, 300.16 and 90.21 μg/ml for HL60, U937 and K562, respectively. The IC50 of propolis from Brazil were 150.48, 350.35 and 180.09 μg/ml for HL60, U937 and K562, respectively.
In order to increase the amounts of flavonoids and to get rid of the wax-like materials and other undesired compounds, which may cause allergic reaction, raw propolis was extracted by various methods. Nine active propolis flavonoids were analyzed to indicate the amounts of active compounds in propolis. Most of the extracts exhibited a positive inhibitory effect. The inhibitory effects on the cell viability of leukemia cancer cells were also investigated. The amount of the total flavonoids in ReE extract of propolis from China were the highest one (92022 μg/g). The IC50 of ReE extract was the lowest one compared to the other propolis extracts. The IC50s were 16.80, 24.30 and 6.20 μg/ml for HL60, U937 and K562, respectively. These results indicated that the quantity of flavoids in ReE extract may be related to its significant growth-inhibiting effects.
Further studies were performed to investigate the mechanisms involved in the growth-inhibiting effects on leukemia cells. The results from DAPI staining, subnuclei analysis, and DNA fragmentation assay indicated the induction of apoptosis in HL-60 cells by RPS and Pycnogenol . Cell cycle analysis revealed the significant increase of cell number in G0/G1 phase by RPS or Pycnogenol. Based on the changes of cell morphology, the increase of NBT reduction, NSE activity, and CD-11b-positive cells, we suggested that that RPS and Pycnogenol at a dose below 100 μg/ml has the ability to induce HL-60 to differentiation into monocytic lineage. The RPS or Pycnogenol induced apoptosis is mediated by activating caspase 3 activity. One hour pretreatment with of z-DEVD-fmk, a caspase 3 inhibitor, not only decreased the activity of caspase 3 but also reduced the percentage of apoptotic cells by RPS and Pycnogenol.
In addition, RPS or Pycnogenol at a concentration up to 250 μg/ml did not showed significant cytotoxicity in normal human cell lines examined and in Con A stimulated PBMC. The dual effects of RPS and Pycnogenol on differentiation and apoptosis suggest that they may be potential agents in the treatment of leukemia.
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