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Effects of triterpenes from Ganoderma lucidum on protein expression profile of HeLa cells.

ABSTRACT

To elucidate the cytotoxicity mechanism of Garnoderma triterpenes, a chemoproteomic study using five purified ganoderic acids, ganoderic acid F (GAF), ganoderic acid K (GAK), ganoderic B (GAB), ganoderic acid D (GAD) and ganoderic acid AM1 (GAAM1) was conducted. GAF, GAR, GAB, GAD and CAAM1 treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with [IC.sub.50] values of 19.5 [+ or -] 0.6 [micro]M, 15.1 [+ or -] 0.5 [micro]M, 20.3 [+ or -] 0.4 [micro]M, 17.3 [+ or -] 0.3 [micro]M. 19.8 [+ or -] 0.7 [micro]M, respectively. The protein expression profiles of HeLa cells treated with each ganoderic acid at dose of 15 [micro]M for 48 h were checked using two-dimensional electrophoresis (2-DE). The possible target-related proteins of ganoderic acids, i.e. proteins with same change tendency in all five ganoderic acids-treated groups compared with control, were identified using MALDI-TOF MS/MS. Twelve proteins including human interleukin-17E, eukaryotic translation initiation factor 5A (elF5A), peroxiredoxin 2, ubiquilin 2, Cu/Zn-superoxide dismutase, 14-3-3 beta/alpha, TPM4-ALK fusion oncoprotein type 2, PP2A subunit A PR65-alpha isoform, nucleobindin-1, heterogeneous nuclear ribonucleoprotein K, reticulocalbin 1 and chain A of DJ-1 protein were identified. Ganoderic acids might exert their cytotoxicity by altering proteins involved in cell proliferation and/or cell death, carcinogenosis, oxidative stress, calcium signaling and ER stress.

Keywords: Ganoderic acids Cytotoxicity Chemoproteomic 2-DE Mass spectrometry

Introduction

Ganoderma lucidum, also called as 'Lingzhi', is a medicinal mushroom that has been used as a home remedy of traditional Chinese medicine in Asian countries for over 2000 years and is also popularly accepted as a dietary supplement in Western countries (Sliva, 2004). In traditional Chinese medicine, it was believed to have potential in preserving the human vitality and promoting longevity. Though the mechanism of its announced effect on human vitality and longevity is still not clear, modern pharmacological studies did show that Ganoderma lucidum might be useful in the prevention or treatment of a variety of diseases including cancer, heart disease and infection (Wachtel-Galor et al., 2004). The anticancer effects of Ganoderma lucidum include inhibiting tumor growth, anti-angiogenesis, antimetastasis, immuno-enhancement and etc (Yuen and Gohel, 2005). Among these effects, the immuno-regulating effect of Ganoderma polysaccharides and the cytotoxic effect of Ganoderma triterpenes were of particular interest (Yeung et al., 2004). In our previous studies, we checked the immuno-regulating effects of Ganoderma polysaccharides on mononuclear cells (Ma et al., 2008) and thymus atrophy caused by cyclophosphamide (Ma et al., 2009). In the present study, we focused on studying the cytotoxicity mechanism of Ganoderma triterpenes.

The cytotoxicity of Ganoderma triterpenes were reported to include inhibiting growth, inducing apoptosis and causing cell cycle arrest of cancer cells (Lin et al., 2003; Yang, 2005; Kimura et al., 2002; Min et al., 2000). While, the target-related proteins of Ganoderma triterpenes were still not fully clear. For a comprehensive analysis of the molecular targets of Ganoderma triterpenes, we used a small-scaled chemoproteomic approach to identify their possible target-related proteins in cancer cells. One of the categories of chemoproteomic approaches in drug research is focused application that often involves the detailed study of a single class of compounds with known biological activity, aiming to identify a mechanism of action or the use of a compound series in target validation (Hall, 2006). Thus, in the present study, five purified ganoderic acids (Fig. 1), which are the main components of Canoderma triterpenes according to our previous analysis result (Yang et al., 2007), were used to treat human cervical carcinoma HeLa cells. Then, 2-DE system which could run six 2-DE gels at the same time was employed to check the protein expression profiles of HeLa cells underwent treatment of the five ganoderic acids or solvent control. The detected proteins with significant change in expression level under treatment of these ganoderic acids might be considered as the possible target-related proteins of ganoderic acids. The change of protein expression detected in proteomic result was further confirmed using Western blotting analysis.

[FIGURE 1 OMITTED]

Materials and Methods

Materials and reagents

Ganoderic acids including ganoderic acid F (GAF), ganoderic acid K (GAK), ganoderic B (GAB), ganoderic acid D (GAD), and ganoderic acid AM1 (GAAM1) (Fig. 1) were isolated and purified from fruit bodies of Ganoderma lucidum as reported before (Wang et al., 2006; Yang et al., 2007). The purity of the ganoderic acids was more than 98%. All reagents used in 2-DE were bought from Bio-Rad Laboratories (Hercules, CA, USA).

Cell culture and Cytotoxicity assay

The HeLa human cervical carcinoma cell line (CCL-2) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cells were maintained in Minimum essential medium (Life Technologies, Gaithersburg, MD, USA) complemented with 2 mM L-glutamine, 1.5 g/1 sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% fetal bovine serum, 100 [micro]g/ml streptomycin and 100 units/ml penicillin (Invitrogen, Karlsruhe, Germany). The cultures were maintained in a humidified atmosphere of 5% [CO.sub.2] at 37[degrees]C. The cells were passaged at preconfluent densities with a solution containing 0.5 mM EDTA and 0.05% trypsin(Life Technologies).

The cytotoxity of ganoderic aicds was examined using a calorimetric tetrazolium (MTT) assay as reported before (Liu et al., 2005). Briefly, cells were plated in 96-well flat-bottomed plates (Corning, Acton, MA, USA) at a density of 1 x [10.sup.3] cells/well in complete medium (with 10% fetal bovine serum) and attached overnight. Then, the media were changed into fresh complete medium (with 10% fetal bovine serum) containing various amounts of ganoderic acids for 48 h and control wells were treated with 0.1% DMSO solvent control. After the incubation, 20 [micro]l of the dye (3, [4,5-dimethylthiazol-2-yl-] diphenyltetrazolium bromide, 5 mg/ml), MTT, was added to each well and the plates were incubated for 3 h at 37[degrees]C. Then, 100 [micro]l of lysis buffer (20% sodium dodecyl sulfate [SDS] in 50% N,N-dimethylformamide, containing 0.4% [v:v] 1N HCL and 0.5% [v:v] 80% acetic acid) was added to each well and incubated overnight for 16 h. At the end of culture, cell viability was determined by measuring the mitochondrial-dependent conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolically active cells. The optical density (proportional to the number of live cells) was assessed with a Genios Microplate Reader (Tecan, Research Triangle Park, NC, USA) at 570 nm. Each experiment was carried out in triplicate and results of three independent experiments were used for statistical analysis. [IC.sub.50] value (half-maximal inhibitory concentration) was calculated using the Logit method.

2-DE, image analysis and MALDI-TOF MS/MS

2-DE, image analysis and MALDI-TOF MS/MS were carried out similar to our previous reports (Ma et al., 2008; Yue et al., 2008a). For sample preparation, cells were incubated for 48 h with each ganoderic acid at dose of 15 [micro]M (dose similar to the [IC.sub.50] value of the ganoderic acids) or solvent control (0.1% DMSO). For 2-DE, protein sample (150 [micro]g) from control cells or cells treated with ganoderic acid was applied for IEF on a Protein IEF cell (Bio-Rad) using the ReadyStrip IPG Strips, 17 cm, pH 4-7 (Bio-Rad). Then, the strips were loaded onto constant 12% homogeneous SDS-PAGE gels for electrophoresis using a PROTEIN II xi Multi-Cell system (Bio-Rad). Grouped (control and five ganoderic acids-treated) protein samples from 3 independent experiments were used for 2-DE analysis. And, for each group of protein samples, duplicate electrophoreses were performed to ensure reproducibility. The 2-D gels were silver stained using Bio-Rad Silver Stain Plus kit reagents (Bio-Rad). The silver-stained gels were scanned using a Densitometer GS-800 (Bio-Rad) and then analyzed using the PD-Quest Version 7.2 software (Bio-Rad). Comparisons were made between protein profiles of each ganoderic acid-treated group and control group. Quantitative analysis was performed using the Student's t-test and p <0.05 was taken as statistically significant. Protein spots with two fold or more increased or decreased intensity and statistically significant in each ganoderic acid-treated group compared with control were found. Then, among these protein spots, those with similar change tendency in all five ganoderic acid-treated groups compared with control were accepted as possible target-related proteins and were then excised from the gels. After digested with trypsin, the excised spots were used for identification by MALDI-TOF MS/MS using an ABI 4700 Proteomics Analyzer with delayed ion extraction (Applied Biosystems). All data analysis and database Searching were performed using the MASCOT search engine (Matrix Science) against the NCBI protein sequence database.Proteins with protein score more than 62 and best ion score (MS/MS) more than 30 were accepted.

Western blotting analysis

As reported before (Ma et al., 2009), Western blotting analysis was conducted to confirm the change of protein expression found in 2-DE analysis. Briefly, cells were washed three times with cold TBS, harvested using a cell scraper, and lysed in 10 volume of cold lysis buffer (50 mM Tris-HCl, pH 7.2, 250 mM NaCl, 0.1% NP-40, 2 mM EDTA, 10% glycerol, 1 mM PMSF, 5 [micro]g/ml Aprotinin, 5 [micro]g/ml Leupeptin) on ice. Lysates were centrifuged and then the supernatant protein was denatured by mixing with equal volume of 2 x sample loading buffer and then boiling at 100[degrees]C for 5 min. An aliquot (containing 50 [micro]g protein) of the supernatant was loaded onto a 12% SDS gel, separated electrophoretically, and transferred to a PVDF membrane (Bio-Rad). After the PVDF membrane was incubated with 10 mM TBS with 1.0% Tween 20 and 10% dehydrated skim milk to block nonspecific protein binding, the membrane was incubated with primary antibodies overnight at 4[degrees] C. The primary antibodies used were mouse anti-eIF5A monoclonal antibody (1:1000, BD Biosciences, San Diego, CA, USA), rabbit anti-14-3-3 beta/alpha polyclonal antibody (1:1000, Abgent, San Diego, CA, USA) and mouse anti-actin monoclonal antibody (1:2000, Sigma). The secondary antibodies used were HRP-conjugated goat anti-mouse IgG (Sigma) or HRP-conjugated goat anti-rabbit IgG (Sigma). Blots were then incubated with the secondary antibodies for 1h at room temperature at a 1:5000 dilution and then visualized using chemiluminescence (Pierce Biotechnology, Rockford, IL). Three independent experiments were carried out.

Statistical analysis

The significance of difference between groups was determined with the non-paired Student's t-test (GraphPadPrism, GraphPad Software Inc., San Diego, CA, US). For each variable, three independent experiments were carried out. Data were given as the mean [+ or -] SD and p <0.05 was considered significant.

Results

Cytotoxic effects of ganoderic acids

The [IC.sub.50] values of the five ganoderic acids were shown in Table 1. To be noted, the [IC.sub.50] value of GAD was cited from our previous report (Yue et al., 2008a). The results indicated that ganoderic acids exhibited moderate cytotoxicity against HeLa cells.
Table 1

Cytotoxicity of 48 h treatment of ganoderic acids against HeLa cells.

No.  Name                        [IC.sub.50]value ([mu]M)

1    ganoderic acid F (GAF)      19.5 [+ or -] 0.6

2    ganoderic acid K (GAK)      15.1 [+ or -] 0.5

3    ganoderic acid B (GAB)      20.3 [+ or -] 0.4

4    ganoderic acid D (GAD)      17.3 [+ or -] 0.3

5    ganoderic acid AM1 (GAAM1)  19.8 [+ or -] 0.7


2-DE of control and ganoderic acid-treated HeLa cells

Representative group of 2-D gel images for control, GAF, GAK, GAB, GAD, GAAM1 -treated cells are shown in Fig. 2. Each gel resolved up to 700 protein spots. Gel maps of control and each ganoderic acid-treated cells were compared with PDQUEST software to identify the protein spot variations. The number of protein spots differentially expressed in each ganoderic acid-treated group compared with control was shown in Table 2. Among these spots, 12 protein spots showed similar change tendency in all ganoderic acids-treated groups compared with control. Table 3 showed the results of PDQUEST analysis about the average intensity values and their standard deviations of the 12 protein spots, the statistical assay results and the fold differences between control and each ganoderic acid-treated group. The fold difference was represented by the ratio of the intensity value of the ganoderic acid-treated group to the value of the control group. These 12 protein spots were indicated by the arrowed spots in A of Fig. 2 and the expanded plots in B of Fig. 2.

[FIGURE 2 OMITTED]
Table 2

Number of protein spots differentially expressed in ganoderic acid-
treated group compared with control.

No.  Name            Number of       Number of protein  Total number of
                     protein spots   spots with         protein spots
                     with increased  decreased          with changed
                     expression      expression         expression

1    ganoderic acid   6              16                 22
     F (GAF)

2    ganoderic acid  16              21                 37
     K (GAK)

3    ganoderic acid   7              13                 20
     B (GAB)

4    ganoderic acid   9              17                 26
     D (GAD)

5    ganoderic acid   9              23                 32
     AM1 (GAAM1)

Table 3

The intensity values and change fold difference (P <0.05) of the 12
possible target-related proteins in ganoderic acid-treated groups
compared with control.

Spot  Control                                  GAF

      Spot volume (ppm)      Spot volume (ppm)      Fold difference
      (Mean [+ or -] SD)     (Mean [+ or -] SD)     compared with
                                                    control

1      562.8 [+ or -] 60.3    134.8 [+ or -] 24.9   0.24

2     1342.5 [+ or -] 147.6   447.0 [+ or -] 52.0   0.33

3      700.5 [+ or -] 75.0    102.5 [+ or -] 16.5   0.15

4     4275.7 [+ or -] 478.9   975.3 [+ or -] 133.0  0.23

5     1211.8 [+ or -] 220.6   192.2 [+ or -] 26.9   0.16

6     1679.3 [+ or -] 171.2  3476.5 [+ or -] 373.2  2.07

7      136.5 [+ or -] 24.3    469.0 [+ or -] 51.2   3.44

8     1083.5 [+ or -] 217.3   201.0 [+ or -] 48.2   0.19

9     1374.0 [+ or -] 290.5   457.7 [+ or -] 68.6   0.33

10    1118.3 [+ or -] 234.4   492.8 [+ or -] 84.1   0.44

11     651.5 [+ or -] 66.6    127.2 [+ or -] 28.0   0.20

12     680.8 [+ or -] 72.8    120.8 [+ or -] 34.2   0.18

Spot                    GAK                         GAB

      Spot volume (ppm)      Fold difference  Spot volume (ppm)
      (Mean [+ or -] SD)     compared with    (Mean [+ or -] SD)
                             control

1      127.2 [+ or -] 26.0   0.23              252.7 [+ or -] 38.2

2      367.3 [+ or -] 49.6   0.27              666.2 [+ or -] 76.4

3       95.5 [+ or -] 14.5   0.14              220.0 [+ or -] 40.0

4     1385.8 [+ or -] 170.2  0.32             2085.5 [+ or -] 306.2

5      406.0 [+ or -] 53.5   0.34              589.0 [+ or -] 67.0

6     3910.3 [+ or -] 425.8  2.33             3385.0 [+ or -] 386.0

7      814.0 [+ or -] 83.0   5.96              286.5 [+ or -] 48.9

8      464.3 [+ or -] 61.2   0.43              333.5 [+ or -] 67.5

9      683.8 [+ or -] 70.2   0.50              662.2 [+ or -] 136.9

10     275.3 [+ or -] 57.3   0.25              468.0 [+ or -] 55.6

11     244.0 [+ or -] 57.6   0.37              223.5 [+ or -] 42.9

12      92.2 [+ or -] 16.1   0.14               89.0 [+ or -] 18.0

Spot       GAB                            GAD

      Fold difference   Spot volume (ppm)      Fold difference compared
      compared with     (Mean [+ or -] SD)     with control
      control

1     0.45              119.7 [+ or -] 28.9    0.21

2     0.50              212.0 [+ or -] 30.5    0.16

3     0.31               71.7 [+ or -] 15.0    0.10

4     0.49             1867.5 [+ or -] 313.1   0.44

5     0.49              394.7 [+ or -] 45.4    0.33

6     2.02             3606.3 [+ or -] 498.1   2.15

7     2.10              995.5 [+ or -] 154.4   7.29

8     0.31              269.5 [+ or -] 57.1    0.25

9     0.48              473.5 [+ or -] 63.2    0.34

10    0.42              286.7 [+ or -] 57.3    0.26

11    0.34              232.5 [+ or -] 51.9    0.36

12    0.13              130.0 [+ or -] 30.6    0.19

Spot                    GAAM1

      Spot volume (ppm)      Fold difference compared with control
      (Mean [+ or -] SD)

1      257.7 [+ or -] 52.4   0.46

2      435.2 [+ or -] 52.5   0.32

3      120.0 [+ or -] 22.8   0.17

4     1939.8 [+ or -] 326.0  0.45

5      278.5 [+ or -] 39.4   0.23

6     3490.0 [+ or -] 390.4  2.08

7      452.0 [+ or -] 48.6   3.31

8      484.0 [+ or -] 57.4   0.45

9      677.0 [+ or -] 102.6  0.49

10     247.0 [+ or -] 51.6   0.22

11     252.0 [+ or -] 56.4   0.39

12      84.8 [+ or -] 15.2   0.12


Identification of differentially expressed proteins

Results of MS/MS identification of the 12 differentially expressed protein spots were summarized in Table 4. For each protein identified, detailed identification data including accession number, theoretical molecular mass, theoretical pl, protein score, best ion score, percent of sequence coverage and number of unique peptides were all shown in Table 4. The MALDI-TOF MS/MS analysis result of spot 9, which was identified as nucleobindin-1, was shown in Fig. 3 as an example.

[FIGURE 3 OMITTED]
Table 4

The results of protein identifications of differentially expressed
proteins using MALDI-TOF MS/MS.

Spot  Target protein     NCBI       TheoreticalMr (kDa)/pl  Protein
                         accession                          score
                         number

1     interleukin-17E     3355455   11.3/7.03                65
      (IL-17E)

2     translation        54037409   16.7/5.08               131
      initiation factor
      5A (eIF5A)

3     peroxiredoxin 2    77744389   22.0/5.66                98

4     ubiquilin 2        16753207   65.7/5.15                72

5     Cu/Zn-superoxide    1237406   15.9/5.86                69
      dismutase

6     14-3-3 beta/alpha   1345590   28.1/4.76               123

7     TPM4-ALK fusion    10441386   27.5/4.77               355
      oncoprotein type
      2

8     PP2A, subunit A,   21361399   65.2/5.00                90
      PR65-alpha
      isoform

9     nucleobindin-1      1144316   53.8/5.15               252

10    heterogeneous      55958543   34.0/5.54                66
      nuclear
      ribonucleoprotein
      K

11    reticulocalbin 1    4506455   38.9/4.86               102

12    chain A, DJ-1      42543006   19.8/6.33                75
      protein

Spot  Target protein     Sequence coverage  Unique     Best ion score
                         (%)                peptides

1     interleukin-17E    19                 2           36
      (IL-17E)

2     translation        42                 3          120
      initiation factor
      5A (eIF5A)

3     peroxiredoxin 2    25                 4           62

4     ubiquilin 2        33                 3           45

5     Cu/Zn-superoxide   21                 2           37
      dismutase

6     14-3-3 beta/alpha  65                 3           92

7     TPM4-ALK fusion    52                 3           41
      oncoprotein type
      2

8     PP2A, subunit A,   20                 4           46
      PR65-alpha
      isoform

9     nucleobindin-1     52                 3           54

10    heterogeneous      26                 2           50
      nuclear
      ribonucleoprotein
      K

11    reticulocalbin 1   15                 4           36

12    chain A, DJ-1      29                 2           40
      protein


The 12 differentially expressed protein spots could be considered as the possible target-related proteins of ganodeirc acids. Briefly, based on their biological functions, these 12 proteins could be generally classified into one of the following four categories: (1) cell proliferation and/or cell death, (2) carcinogenosis, (3) oxidative stress and (4) calcium signaling and endoplasmic reticulum (ER) stress.

(1) Proteins including eIF5A, ubiquilin 2, 14-3-3 beta/alpha and PP2A subunit A PR65-alpha isoform were related to cell proliferation and/or cell death. eIF5A is an important protein in translation initiation and is fundamental to cell survival and proliferation (Jao and Chen, 2002). Ubiquilin 2 is suggested to increase the half-life of proteins destined to be degraded by the proteasome thus modulate proteasome-mediated protein degradation. 14-3-3 beta/alpha belongs to the 14-3-3 family that is involved in many different cellular processes, including mitogenesis, cell cycle control, and apoptosis (Tzivion et al., 2006). PP2A subunit A PR65-alpha isoform is a subunit of PP2A, a large family of highly conserved heterotrimeric enzymes essential for cell survival, cell cycle regulation and DNA damage response (Janssens and Goris, 2001). Recent studies showed that PP2A was closely related to regulation of cell apoptosis by affecting the degradation of BCL-2 (Lin et al., 2006).

(2) Proteins including IL-17E, TPM4-ALK fusion oncoprotein type 2 and heterogeneous nuclear ribonucleoprotein K are carcinogenosis-related proteins. These proteins were differentially expressed in carcinoma cells or tumors compared with normal cells or tissues. They might also play roles in cell proliferation and/or cell death though their roles were not clearly clarified. IL-17E belongs to the IL-17 family, which had been shown to play a potential role in T-cell mediated angiogenesis and promote tumourigenicity of human cervical cancer (Wagsater et al., 2006). The expression of TPM4-ALK fusion oncoprotein type 2 was significantly changed in esophageal squamous tumors compared with adjacent normal esophageal tissues (Du et al., 2007). Report about heterogeneous nuclear ribonucleoprotein K showed that it was overexpressed in oral squamous cell carcinoma (Roychoudhury and Chaudhuri, 2007).

(3) Proteins including peroxiredoxin 2, Cu/Zn-superoxide dismutase and chain A of DJ-1 protein are antioxidant proteins and play important roles in oxidative stress. Peroxiredoxin 2 belongs to the peroxiredoxins family, which control the constitutive level of [H.sub.2][O.sub.2] in the cell and thus protect cell against ROS-induced damage (Chevallet et al., 2003). Cu/Zn-superoxide dismutase is a well-known anti-oxidant protein. DJ-1 is an atypical peroxiredoxin-like peroxidase and it could function as a redox-sensitive chaperone and as a sensor for oxidative stress (Andres-Mateos et al., 2007).

(4) Proteins including nucleobindin-1 and reticulocalbin 1 are related to calcium signaling and ER stress. Nucleobindin-1 is the major Golgi calcium-binding protein though it is present both in the cytosol and Golgi. It plays role in ER stress by regulating the function of activating transcription factor 6, an ER membrane-anchored transcription factor (Tsukumo et al., 2007). Reticulocalbin 1 is an endoplasmic reticulum resident Ca(2+)-binding protein with multiple EF-hand motifs and a carboxyl-terminal HDEL sequence. It is involved in the regulation of calcium-dependent activities in the endoplasmic reticulum lumen or post-ER compartment (Ozawa and Muramastsu, 1993).

Confirmation of differentially expressed proteins by Western blotting

Consistent with the proteomic results, eIF5A was found to be down-regulated while 14-3-3 beta/alpha was found to be up-regulated in all five ganoderic acids-treated HeLa cells (Fig. 4).

Discussion

Ganoderma lucidum is a well-known medicinal fungus that has been used as a traditional drug in China, Korea, Japan and other Asian countries for a long time. Nowadays, it is still widely prescribed by traditional Chinese medical doctors for the prevention and treatment of various types of illnesses such as cancer, hepatopathy, chronic bronchitis, arthritis, hypertension, neurasthenia, hyperglycemia, insomnia, cardiovascular diseases, debility and weakness, etc (Lin, 2001). Among the active components of Ganoderma lucidum, Ganoderma triterpenes have received considerable attention owing to their conspicuous pharmacological activities. Ganoderma triterpenes were shown to have cytotoxic activity on cancer cells, anti-human immunodeficiency virus type 1 activity, antihistamine activity, antinociceptive activity, anticholesterol activity, and inhibitive activity on angiotensin converting enzyme and glucosyltransferase (El-Mekkawy et al., 1998; Hada et al., 1989; Kin et al., 1998; Kohda et al., 1985; Lin et al., 1991; Morigiwa et al., 1986; Sliva, 2006; Toth et al., 1983; Yeung et al., 2004).

During the last two decades, more than 140 triterpenes have been isolated from the fruiting bodies, spores and cultured mycelia of Ganoderma lucidum (Hirotani et al., 1993; Lin et al., 1988; Min et al., 2004, 2005). The five ganoderic acids used in the present study belong to major constituents of Ganoderma triterpenens. According to our previous study, the contents of these ganoderic acids in the dried fruit bodies of Ganoderma lucidum were 20-1323 [micro]g/g (unpublished data), 19-1191 [micro]g/g, 19-1759 [micro]g/g, 100-1452 [micro].g/g and 55-829 [micro]g/g for GAF, GAK, GAB, GAD and GAAM1, respectively (Wang et al., 2006). Furthermore, our previous work checking the pharmacokinetics characteristics of GAB and GAK in rat indicated that, after oral administration, the two ganoderic acids were quickly absorbed into the body fluid from the gastrointestinal tract and distributed widely in the organs. At 6.26 min and 32.10 min after oral administration of 1.2g/kg Ganoderma lucidum extract, the plasma concentrations of GAB and GAK reached the maximum level of 13.15 [micro]g/ml (25.5 [micro]M) and 2.86 [micro]g/ml (5 [micro]M), respectively (Wang et al., 2007a). And, results checking the urinary excretion of these ganoderic acids indicated that, within 72 h, the cumulative urinary excretion of GAB and GAK was 0.23 and 0.13, respectively (Wang et al., 2007b). In the present study, ganoderic acids including GAB and GAK exhibited cytotoxicity on HeLa human cervical carcinoma cells with [IC.sub.50] values of 15-20 [micro].M. By using proteomic method, the protein expression profiles of HeLa cells after treatment of ganoderic acids at 15 [micro]M were checked and 12 proteins that might be target-related proteins of ganoderic acids were found. These 12 proteins were reported to play important roles in cell proliferation and/or cell death, carcinogenosis, oxidative stress, calcium signaling and ER stress. Furthermore, result of the present study was consistent with our previous proteomic study result using only GAD (Yue et al., 2008a) and result using Ganoderma lucidum extract (Yue et al., 2008b). Three kinds of proteins, i.e. elF5A, 14-3-3 protein and peroxiredoxin protein, were found in all these studies. The results suggested that elF5A, 14-3-3 protein and peroxiredoxin protein might be the most important target-related proteins of ganoderic acids. The regulation of these proteins might contribute to the cytotoxicity of ganoderic acids. 14-3-3 proteins are important regulators in many different cellular processes such as mitogenesis, cell cycle control, and apoptosis (Tzivion et al., 2006). And, regulation of elF5A, an important protein in translation initiation, might contribute to the growth inhibition of HeLa cells induced by ganoderic acids. Furthermore, decrease of peroxiredoxin protein, a regulator of intracellular ROS level, suggested that ganoderic aicds might increase ROS level of HeLa cells. Our previous study did show that Ganoderma triterpenes extract caused increase in ROS level of HeLa cells (Yue et al., 2008b).

[FIGURE 4 OMITTED]

Though there was no clinical report about using Ganoderma triterpenes in cancer therapy, a clinical study was reported checking the effect of using Ganoderma triterpenes in men with lower urinary tract symptoms (Noguchi et al., 2008). The clinical study indicates that Ganoderma triterpenes were well tolerated and could improve International Prostate Symptom Score. Presently, we are cooperating with another laboratory to check in an animal study the potential of Ganoderma triterpenes extract for complementary cancer therapy. Though further study is necessary, the results of this study shed light on the anti-cancer mechanism of ganoderic acids as well as Ganoderma triterpenes from a molecular perspective point of view and provides useful information for the possible use of Ganoderma triterpenes in clinic for complementary cancer therapy.

Acknowledgments

This work was supported in part by grants from the National Natural Science Foundation of China (30701077) and the Science and Technology Commission of Shanghai Municipality (2008DFA30350).

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Q.-X. Yue (a), (b), (1), X.-Y. Song (a), (b), (1), C. Ma (a), L.-X. Feng (a), S.-H. Guan (a), W.-Y. Wu (a), M. Yang (a), B.-H. Jiang (a), X. Liu (a), Y.-J. Cui (b), *, D.-A. Guo (a), (b), *

(a) Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, PR China

(b) College of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, PR China

Abbreviations: GAF, Ganoderic acid F; GAK, Ganoderic acid K; GAB, Ganoderic acid B; GAD, Ganoderic acid D; GAAM1, Ganoderic acid AM1; 2-DE, two-dimensional electrophoresis: MTT, 3, [4,5-dimethylthiazol-2-yl-] diphenyltetra-zolium bromide; IL-17E, interleukin-17E; eIF5A, eukaryotic translation initiation factor 5A; ER, endoplasmic reticulum

* Corresponding authors. Tel.: +86 21 50271516; fax: +86 21 50272223.

E-mail addresses: Janney808@sina.com (Y.-J. Cui), gda5958@163.com (D.-A. Guo).

(1) Both authors contributed equally to the work.

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doi: 10.1016/j.phymed.2009.12.013
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Author:Yue, Q.-X.; Song, X.-Y.; Ma, C.; Feng, L.-X.; Guan, S.-H.; Wu, W.-Y.; Yang, M.; Jiang, B.-H.; Liu, X
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jul 1, 2010
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