Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple myeloma cells
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 Ganoderma lucidum causes apoptosis in leukemia, lymphoma and multiple myeloma cells
Abstract Over many centuries, herbal remedies have treated a variety of ailments. This empiric observational approach has produced a number of leads for formulated medicines. Ganoderma lucidum extract was screened for its anti-proliferative activity using a panel of 26 human cancer cell lines. The six most sensitive hematologic cell lines were: HL-60 (ED50 26 μg/ml), U937 (63 μg/ml), K562 (50 μg/ml), Blin-1 (38 μg/ml), Nalm-6 (30 μg/ml) and RPMI8226 (40 μg/ml). Cell cycle analyses revealed a G2/M arrest, most prominently in HL-60 cells. Four hematopoietic cell lines (HL-60, Blin-1, U937, RPMI8226) were examined for apoptosis, which ranged between 21 and 92%. After exposure to G. lucidum extract, HL-60 cells became multinucleated with an increased DNA content. These results indicate that G. lucidum extract has a profound activity against leukemia, lymphoma and multiple myeloma cells and may be a novel adjunctive therapy for the treatment of hematologic malignancies. Keywords
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Fig. 1. (a) HPLC chromatogram of G. lucidum extract showing ganoderic acid C2 standard eluting at 32.9 min. (b) HPLC chromatogram of ganoderic acid C2 standard eluting at 32.9 min. (c) HPLC chromatogram of G. lucidum extract spiked with ganoderic acid C2 standard confirming its presence at 32.9 min. Figure options
 
Fig. 2. Growth arrest of hematologic cell lines induced by G. lucidum extract. Cells included: panel a, acute myeloid leukemia (HL-60, U937); panel b, erythroid chronic myeloid leukemia (K562); panel c, acute lymphoblastic leukemia (Blin-1, Nalm-6); panel d, multiple myeloma (RPMI8226). Cells of each line were treated with G. lucidum (10, 20, 40, 60, 80 and 100 μg/mg) for 96 h. MTT assay was performed. Viable cells were expressed as a percentage of untreated control cultures for each line. Results represent the mean ± S.D. of three different experiments performed in triplicates. Figure options
 
Fig. 3. Induction of G2/M arrest in HL-60 cells by G. lucidum extract. Panel a shows cell cycle analysis by flow cytometry after PI staining for untreated control cells. Panel b depicts cell cycle analysis for cells treated with G. lucidum, 100 μg/ml for 72 h. Increasing number of HL-60 cells in the G2/M phase of the cell cycle were detected. Horizontal and vertical axis represent DNA content and cell number, respectively. Percentages of the different cell cycle phases are calculated for the population of cells within the dashed lines. Representative of one of two experiments with similar results. Figure options
 
Fig. 4. Induction of apoptosis in HL-60 and U937 cells by G. lucidum extract. HL-60 (panel a) and U937 (panel b) cells were cultured with G. lucidum (50, 100, 150 and 200 μg/mg) for 72 h, stained with FITC-conjugated Annexin V and propidium iodide (PI). Percentage of apoptotic cells was measured by flow cytometry in comparison to diluent-treated control cells. Bar graphs reflect the cells exclusively stained with FITC. Results represent the mean ± S.D. of three different experiments. Figure options
 
Fig. 5. Induction of mitochondrial membrane collapse in U937 cells cultured with G. lucidum extract. HL-60 (panel a) and U937 (panel b) cells were incubated with G. lucidum for 48 h. Mitochondrial membrane potential of cells treated with G. lucidum and diluent treated control cells was assessed by flow cytometry after staining with JC-1. JC-1 dimers fluoresce red in stable mitochondria and form green fluorescent monomers when the mitochondrial membrane is decreasing in potential. The decrease of the red/green fluorescence reflects increasing apoptotic cells. Results represent ratios of the mean of three different experiments. Figure options
 
Fig. 6. Upregulation of p21WAF1 and p27KIP1 by G. lucidum extract in U937 cells. U937 cells were treated with increasing doses of G. lucidum extract (50, 100, 150 and 200 μg/ml) for 48 and/or 72 h. Protein lysates were analyzed by Western blot with p21WAF1 (panel a) or p27KIP1 (panel b) specific antibodies. The blots were stripped and rehybridized with a GAPDH antibody as control for equal loading. Figure options
 
Fig. 7. Multinucleation of HL-60 cells caused by G. lucidum extract. Untreated HL-60 cells (panel a) and HL-60 cells treated with G. lucidum (100 μg/mg, 96 h) (panel b). Arrowheads point to several of the multinucleated cells present in the photograph. Cells were cytocentrifuged, fixed, stained as described in Section 2 (magnification 400×). Figure options
Table 1. Human cancer cell lines examined for anti-proliferative effects of G. lucidum extract  
The six most sensitive cell lines are in bold. View Within Article Table 2. Inhibition of the proliferation of hematopoietic cell lines by G. lucidum extract
Cells were cultured for 96 h in the presence of 10, 20, 40, 60, 80 or 100 μg/ml of G. lucidum, and cell growth was analyzed by MTT assay. Percent growth in experimental wells compared to untreated control wells was graphed, and the effective dose which inhibited 50% growth (ED50), was calculated for each cell line. View Within Article  
Corresponding author at: Division of Hematology/Oncology, Davis Building 5065, Cedars-Sinai Medical Center, David Geffen School of Medicine at UCLA, 8700 Beverly Boulevard, Los Angeles, CA 90048, United States. Tel.: +1 310 423 7759; fax: +1 310 423 0225. Yüklə 36,65 Kb. Dostları ilə paylaş:
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