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Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction.

Cheng KC, Huang HC, Chen JH, Hsu JW, Cheng HC, Ou CH, Yang WB, Chen ST, Wong CH, Juan HF - BMC Genomics (2007)

Bottom Line: In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha.The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment.Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Taiwan University, Taipei 106, Taiwan. jerekcheng@gmail.com

ABSTRACT

Background: Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells.

Results: Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach.

Conclusion: Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

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The expression of TNF-α in F3- or LPS-induced THP-1 cells. 105 cells/mL concentrations of THP-1 cells were seeded in 96-well microplates and incubated overnight. Then the cells (1.25 × 104) were treated with F3 at dosages indicated as 1 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and with LPS at the dose of 1 μg/mL, respectively. The same volume of medium was used as control After 24 hours, the supernatants were collected and in vitro TNF-α activity was determined using Human TNF-α Immunoassay Kit (Quantikine®, RD systems). TNF-α expression induced by F3 (100 μg/mL and 200 μg/mL) is similar to that of LPS (1 μg/mL). From this TNF-α expression data, we calculated the EC50 (50% effect concentration) of F3-induced to be around 10 μg/mL. The error bars indicate SD from triplicate independent experiments.
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Figure 1: The expression of TNF-α in F3- or LPS-induced THP-1 cells. 105 cells/mL concentrations of THP-1 cells were seeded in 96-well microplates and incubated overnight. Then the cells (1.25 × 104) were treated with F3 at dosages indicated as 1 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and with LPS at the dose of 1 μg/mL, respectively. The same volume of medium was used as control After 24 hours, the supernatants were collected and in vitro TNF-α activity was determined using Human TNF-α Immunoassay Kit (Quantikine®, RD systems). TNF-α expression induced by F3 (100 μg/mL and 200 μg/mL) is similar to that of LPS (1 μg/mL). From this TNF-α expression data, we calculated the EC50 (50% effect concentration) of F3-induced to be around 10 μg/mL. The error bars indicate SD from triplicate independent experiments.

Mentions: Upon the binding of TNF-α to TNFR1, monocytic cells are triggered to undergo apoptosis. This critical regulatory process is accomplished by activating the caspase cascade that results in the degradation of various important cellular proteins. Previous reports showed that lipopolysaccharide (LPS) could markedly stimulate the cytokine expression, especially TNF-α [37]. Compared with the TNF-α expression in LPS-induced THP-1 cells, we could estimate the optimal effect concentration of F3. We treated THP-1 cells with different F3 concentrations (1, 10, 50, 100, 200 μg/mL) and LPS (1 μg/mL) for 24 hours, and measured their TNF-α expressions. Figure 1 demonstrates that F3 was dose dependent in the activation of TNF-α expression. TNF-α expression stimulated by F3 at 100 μg/mL and 200 μg/mL was similar to that of LPS at 1 μg/mL. From these TNF-α expression data, we calculated the EC50 (50% effect concentration) of F3-induced effectiveness to be around 10 μg/mL. If 70% of TNF-α is expressed in F3-induced cells compared to LPS-induced cells, then 30 μg/mL of F3 is required to achieve the same effectiveness. For further experiments, we used 30 μg/mL of F3 to treat THP-1 cells.


Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction.

Cheng KC, Huang HC, Chen JH, Hsu JW, Cheng HC, Ou CH, Yang WB, Chen ST, Wong CH, Juan HF - BMC Genomics (2007)

The expression of TNF-α in F3- or LPS-induced THP-1 cells. 105 cells/mL concentrations of THP-1 cells were seeded in 96-well microplates and incubated overnight. Then the cells (1.25 × 104) were treated with F3 at dosages indicated as 1 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and with LPS at the dose of 1 μg/mL, respectively. The same volume of medium was used as control After 24 hours, the supernatants were collected and in vitro TNF-α activity was determined using Human TNF-α Immunoassay Kit (Quantikine®, RD systems). TNF-α expression induced by F3 (100 μg/mL and 200 μg/mL) is similar to that of LPS (1 μg/mL). From this TNF-α expression data, we calculated the EC50 (50% effect concentration) of F3-induced to be around 10 μg/mL. The error bars indicate SD from triplicate independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211495&req=5

Figure 1: The expression of TNF-α in F3- or LPS-induced THP-1 cells. 105 cells/mL concentrations of THP-1 cells were seeded in 96-well microplates and incubated overnight. Then the cells (1.25 × 104) were treated with F3 at dosages indicated as 1 μg/mL, 10 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, and with LPS at the dose of 1 μg/mL, respectively. The same volume of medium was used as control After 24 hours, the supernatants were collected and in vitro TNF-α activity was determined using Human TNF-α Immunoassay Kit (Quantikine®, RD systems). TNF-α expression induced by F3 (100 μg/mL and 200 μg/mL) is similar to that of LPS (1 μg/mL). From this TNF-α expression data, we calculated the EC50 (50% effect concentration) of F3-induced to be around 10 μg/mL. The error bars indicate SD from triplicate independent experiments.
Mentions: Upon the binding of TNF-α to TNFR1, monocytic cells are triggered to undergo apoptosis. This critical regulatory process is accomplished by activating the caspase cascade that results in the degradation of various important cellular proteins. Previous reports showed that lipopolysaccharide (LPS) could markedly stimulate the cytokine expression, especially TNF-α [37]. Compared with the TNF-α expression in LPS-induced THP-1 cells, we could estimate the optimal effect concentration of F3. We treated THP-1 cells with different F3 concentrations (1, 10, 50, 100, 200 μg/mL) and LPS (1 μg/mL) for 24 hours, and measured their TNF-α expressions. Figure 1 demonstrates that F3 was dose dependent in the activation of TNF-α expression. TNF-α expression stimulated by F3 at 100 μg/mL and 200 μg/mL was similar to that of LPS at 1 μg/mL. From these TNF-α expression data, we calculated the EC50 (50% effect concentration) of F3-induced effectiveness to be around 10 μg/mL. If 70% of TNF-α is expressed in F3-induced cells compared to LPS-induced cells, then 30 μg/mL of F3 is required to achieve the same effectiveness. For further experiments, we used 30 μg/mL of F3 to treat THP-1 cells.

Bottom Line: In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha.The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment.Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Science, National Taiwan University, Taipei 106, Taiwan. jerekcheng@gmail.com

ABSTRACT

Background: Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells.

Results: Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach.

Conclusion: Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

Show MeSH
Related in: MedlinePlus