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

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.

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.

Figure 2

Characterization of F3 induced cell death in human THP-1 cells. Phase-contrast microscopy was used to detect the morphology of the control (A) and F3-treated THP-1 cells (B). Cell shrinkage, shape irregularity, and cellular detachment were observed in F3-treated cells, but not in the control. The control (C) and F3-treated THP-1 cells (D) were stained with 4, 6-diamidino- 2-phenylindole (DAPI). (E) The percentage of chromatin condensed cells. There was apparent difference in cell morphology between the un-treated and F3-treated THP-1 cells.

Figure 3

The scatter plot depicting the separation of control (0 and 6 hours) and F3-treated (6 and 24 hours) samples based on the first two principle components derived from the gene expression profiles measured by microarray experiments.

Figure 4

The flow chart for the microarray data analysis. We used Affymetrix HG-U133A chip GeneChip oligonucleotide microarray. Initial data analysis was performed using Affymetrix Microarray Suite v5.0 software, setting the scaling of all probe sets to a constant value of 500 for each GeneChip. Additional data analysis was performed using GeneSpring v 5.1 (Silicon Genetics Inc., Redwood City, California). Genes with a 2-fold change in differential expression between THP-1 control and F3- or LPS-treated THP-1 cells were selected for mapping significantly disturbed biological pathways. The pathway of apoptosis induction through the DR3 and DR4/5 death receptors was shown to be very significant in F3-treated THP-1 cells. F3–6 h and F3–24 h indicate the F3-treated THP1 cells after 6 hours and 24 hours, respectively. LPS-24 h indicates the LPS-treated THP1 cells after 24 hours. C-0 h and C-6 h indicate the control THP1 cells (without any treatment) in 0 hour and 6 hours, respectively.

Figure 5

Scatter plot of the gene expressions in the repeated microarray experiments. The filtered probe intensities of gene expression of one experiment were plotted on the x axis while the intensities of the other experiment were plotted on the y axis. Each gene was represented by a single dot in the scatter plot. The upper three scatter plots showed no difference between control experiments for 0 hour and 6 hours. These results showed the consistency in duplicate microarray experiments.

Figure 6

CASP3 and CASP 7 were cleaved into active forms after F3 treatment in THP-1 cells. After THP-1 cells were treated with F3 for 0, 6, 12, 24 hours, we detected proforms and active forms of CASP3 and CASP7 using western blotting. CASP3 and CASP7 were activated after F3 treatment.

Figure 7

Fold change in time-course gene expression of F3- or LPS-induced THP-1 cells by Q-PCR. First, THP-1 cells were treated with F3 or LPS for different periods of time and were collected at different time points. Total RNA was isolated from cell lines using TRIzol reagent. First-strand cDNA synthesis were performed with 5 μg of total RNA in a volume of 20 μl with 1 μl ThermoScript™ Reverse Transcriptase (Invitrogen) and 1 μl oligo(dT). Extracted first-strand cDNAs were analyzed using BioRad iCycler iQ Real-Time Detection System with SYBR Green dye (Molecular Probes, Eugene, OR). Software designed for the BioRad iCycler will aid in analyzing collected data. mRNA expression of these genes were normalized to RNA content for each sample by using GADPH gene products as internal controls. Relative expression was calculated as the ratio of expression from each F3-treated THP-1 cells in comparison to untreated THP-1 cells (control). The error bar came from n > 3. The p values indicate the statistical significance of different time-course gene expression profiles between F3 and LPS treatment.

Figure 8

Fold change in time-course gene expression of F3- or LPS-induced THP-1 cells by Q-PCR. (continued from Figure 7)

Figure 9

Comparisons between oligonucelotide microarray and Q-PCR results. (A) THP-1 cells were treated with F3 for 6 hours (A) and 24 hours (B) and with LPS for 24 hours (C). The gene expressions showed consistent trends between the microarray and Q-PCR results. The error bar came from n > 3.

Figure 10

Proposed F3-induced cell death pathways in THP-1 cells. Based on our self-developed software for the reconstruction of gene networks in addition to literature research, we proposed plausible cell death pathways induced by F3 in THP-1 cells. F3 may induce death receptor ligands (TNF-α and TRAIL) to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades. Lastly, cell shrinkage and apoptosis occur.