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. 2014 May 8;9:15. doi: 10.1186/1749-8546-9-15

Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells: a proteomic study

Hualin Wang 1,2, Jing Zhang 1, Wai-Hung Sit 1, Chung-Yung Jetty Lee 1, Jennifer Man-Fan Wan 1,✉
PMCID: PMC4036300  PMID: 24872842

Abstract

Background

Cordyceps cicadae is a medicinal fungus that is often used for treating cancer. However, the anticancer mechanisms of C. cicadae are largely unknown. This study aims to investigate the anticancer mechanisms of C. cicadae against hepatocellular carcinoma cells in vitro using a proteomic approach.

Methods

Human hepatocellular carcinoma MHCC97H cells were treated with a water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) for 48 h and harvested for cell viability assays. The significant differences in protein expression between control and C. cicadae-treated cells were analyzed by two-dimensional gel-based proteomics coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry. Flow cytometry analysis was employed to investigate the cell cycle and cell death. The anticancer molecular mechanism was analyzed by whole proteome mapping.

Results

The water extract of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) inhibited the growth of MHCC97H cells in a dose-dependent manner via G2/M phase cell cycle arrest with no evidence of apoptosis. Among the identified proteins with upregulated expression were dynactin subunit 2, N-myc downstream-regulated gene 1, heat shock protein beta-1, alpha-enolase isoform 1, phosphatidylinositol transfer protein, and WD repeat-containing protein 1. Meanwhile, the proteins with downregulated expression were 14-3-3 gamma, BUB3, microtubule-associated protein RP/EB family member 1, thioredoxin-like protein, chloride intracellular channel protein 1, ectonucleoside triphosphate diphosphohydrolase 5, xaa-Pro dipeptidase, enoyl-CoA delta isomerase 1, protein-disulfide isomerase-related chaperone Erp29, hnRNP 2H9B, peroxiredoxin 1, WD-40 repeat protein, and serine/threonine kinase receptor-associated protein.

Conclusion

The water extract of C. cicadae reduced the growth of human hepatocellular carcinoma MHCC97H cells via G2/M cell cycle arrest.

Background

Primary liver cancer accounted for 6% of the total cancer cases worldwide in 2008 [1]. The highest incidences of liver cancer were in East Asia (Japan, Korea, and China) [2,3]. In China, liver cancer has the third highest estimated age-standardized cancer incidence rate in men and the fourth in women, and the second and third highest cancer mortality rates in men and women, respectively [1]. The high incidence of liver cancer in China is attributed to consumption of aflatoxin-contaminated grains, liver virus infection, and alcohol drinking [3]. Hepatitis B vaccination can effectively prevent liver cancer, but the treatment of liver cancer is still difficult [3,4].

Chinese medicine (CM) has been widely used in conjunction with chemotherapeutic drugs for liver cancer treatment in China with positive outcomes [5]. Cordyceps is a genus of ascomycete fungi belonging to the Clavicipitaceae family. All Cordyceps species are endo-parasitoids, and most of them parasitize insects and other arthropods. The Cordyceps genus includes nearly 400 species, and some of them have potential anticancer effects. Cordyceps sinensis is a medicinal fungus that has been used for cancer treatment in CM and Traditional Tibetan medicine since the 15th century [6,7]. The anticancer properties of C. sinensis via cancer cell apoptosis induction, proliferation inhibition, or both in various types of cancers, including leukemia, melanoma, Leydig tumor, breast cancer, and human hepatocellular carcinoma (HCC) have been investigated [8-10]. C. sinensis inhibited tumor metastasis in vivo[11,12]. Another well-known species, Cordyceps militaris, showed anticancer effects on leukemia, lung cancer, and breast cancer in vitro and in vivo[13-17], and its bioactive compound, cordycepin, exhibited cytotoxic and reactive oxygen species-generating activity in relation to cancer proliferation inhibition [18-22].

Cordyceps cicadae, another well-known medicinal mushroom that grows on larvae of Cicadae flammata, has been used in CM in China for the prevention and treatment of various diseases, including kidney disease, immune disease, and cancer [23-25]. C. cicadae exhibited immunoregulatory effects on human T lymphocytes and modulated the growth of mononuclear cells [26-29]. C. cicadae inhibited the growth of lung adenocarcinoma and melanoma in vivo and in vitro[30,31]. However, the anticancer mechanism of C. cicadae for liver cancer is still unknown.

In the last decade, proteomics has been widely used in medical studies for clinical biomarker identification, pathogenesis investigation, new drug discovery, pharmacological research, toxicological examination, and so on [32]. Most biological functions are transmitted via proteins such as enzymes, receptors, and structural components. Therefore, comprehensive proteomic analyses help us to understand the molecular modifications in physiological conditions [33]. This study aims to investigate the anticancer mechanisms of C. cicadae against HCC in vitro by two-dimensional gel-based proteomics coupled with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) mass spectrometry (MS), flow cytometry analysis, and proteome mapping.

Materials and methods

Cell culture and reagents

The MHCC97H cell line was purchased from the Liver Cancer Institute of Fudan University (China). MHCC97H cells were cultured in DMEM (Gibco BRL, USA) supplemented with 10% fetal bovine serum (Gibco BRL) in a humidified incubator containing 5% CO2 in air at 37°C, and subcultured with 0.25% trypsin-0.02% EDTA (Gibco BRL). A lyophilized hot water extract of wild-type C. cicadae (BioAsia Co., China) was dissolved in phosphate-buffered saline (PBS) and adjusted to a final concentration of 10 mg/mL.

Cell proliferation assay

The dose-dependent effect of C. cicadae on cell viability was assessed by the MTT assay. Briefly, suspended MHCC97H cells (1 × 105 cells/mL; 100 μL) were dispersed into the wells of 96-well microtiter plates. After 24 h of incubation, various concentrations of C. cicadae were added to each well and incubated for 48 or 72 h. Next, 10 μL of MTT solution (5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dissolved in c-DMEM) (USB Corporation, USA) was added to each well, and incubated for 3 h at 37°C. The MTT solution was then removed and the insoluble purple formazan crystals formed were dissolved in 50 μL of isopropanol in 0.1 M HCl (MERCK, Germany). The optical density (OD) of each well was measured using a Bio-Rad 550 Microplate Reader (Bio-Rad, USA) at 595 nm with a reference wavelength of 655 nm. The percentage of cell viability was expressed as (Atreatment / Acontrol) × 100%.

Cell cycle analysis

The dose-dependent effect of C. cicadae on the cell cycle distribution was assessed by flow cytometry as described in our previous report [34]. Briefly, MHCC97H cells (1 × 105 cells/mL) were treated with various concentrations of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) for 48 h, and the cells were then harvested, fixed in 70% ethanol (MERCK, Germany), and stored at −20°C for 24 h until further analysis. Next, the cells were washed twice with ice-cold PBS, and incubated with RNase and propidium iodide (PI) (Sigma-Aldrich, USA) for 30 min. The PI-stained cells were excited at a wavelength of 488 nm and emitted at a maximum wavelength of 617 nm. Acquisition of 10,000 events was chosen for measurement of the DNA cell cycle distribution using a COULTER XL Flow Cytometer (Beckman and Coulter, USA). The distribution of cells in the different cell cycle phases shown in the DNA histograms was analyzed using Becton Dickinson Cell Fit Software (BD, USA).

Sample preparation for proteomic analysis

MHCC97H cells were seeded in 100-mm culture dishes at 1 × 106 cells/dish (1 × 105 cells/mL), incubated overnight, and then treated with or without 500 μg/mL C. cicadae for 48 h. The cells were harvested by trypsinization, washed three times with PBS, and centrifuged in a Beckman Spinchron DLX (Beckman and Coulter) at 400 × g for 5 min. The cell pellet was resuspended in lysis buffer [1% Triton X-100 (USB Corporation), 25 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; Sigma-Aldrich), 150 mmol/L NaCl (UNIVAR, USA), 1 mmol/L EDTA disodium salt (Sigma-Aldrich), 1 mmol/L dithiothreitol (DTT; USB Corporation)] with Protease Inhibitor Cocktail Set III (AEBSF, aprotinin, bestatin, E-64, leupeptin hemisulfate, pepstatin A; Bio-Rad). The superfluous salt in the extract was removed by incubation with trichloroacetic acid (TCA)-acetone solution [20% TCA (MERCK, Germany), 20 mmol/L DTT in acetone (MERCK, Germany)] for 4 h at −40°C. The protein pellet was obtained by centrifugation at 15,800 × g for 30 min at 4°C. Excess TCA was removed by three washes with acetone containing 20 mmol/L DTT. The air-dried protein pellet was resuspended in buffer [7 mol/L urea (USB Corporation), 2 mol/L thiourea (Sigma-Aldrich), 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Sigma-Aldrich), 100 mmol/L DTT, 5% glycerol (USB Corporation)], and the protein solution was stored at −80°C until 2-DE analysis. The protein concentration was determined by the Bradford assay (Bio-Rad).

Two-dimensional electrofocusing and polyacrylamide gel electrophoresis

The 2-DE procedures were carried as described in our previous report with modifications [35]. The samples were examined in duplicate and 12 gels (6 for control cells and 6 for C. cicadae-treated cells) were used in total. For the first-dimensional electrophoresis of proteins, protein samples (150 μg) were mixed with 350 μL of rehydration buffer [9.5 mol/L urea, 2% CHAPS, 0.28% DTT, 1% immobilized pH gradient buffer with pH 3–10 (Bio-Rad), 0.002% bromophenol blue (Sigma-Aldrich)] and then rehydrated for 10 h before isoelectric focusing via the following program: (a) linear increase up to 500 V in 1 h; (b) held at 500 V for 2 h; (c) linear increase up to 10,000 V in 4 h; (d) linear increase up to 10,000 V in 3 h; (e) held at 10,000 V to reach a total of 90,000 V × h. Focused immobilized pH gradient (IPG) gel strips were equilibrated for 15 min in a solution [50 mmol/L Tris–HCl, pH 8.8 (Bio-Rad), 6 mol/L urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS; USB Corporation), containing 20 mmol/L DTT], followed by incubation with the same buffer containing 20 mmol/L iodoacetamide (Sigma-Aldrich) for another 15 min. The second-dimensional separation was performed in 1-mm-thick 12.5% polyacrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 15-mA current for 30 min followed by 30-mA current for the rest of the analysis.

Image acquisition and analysis

After the electrophoresis, the gels were stained with SYPRORuby Protein Stain (Bio-Rad), scanned with a Molecular Imager PharosFX Plus System (Bio-Rad), and analyzed using PDQuest 8.0 (Bio-Rad) according to the following procedures: background subtraction, spot detection, and spot matching. The expression levels, expressed as the percentage volume (% vol), were exported. The relative intensities of spots were used for comparisons between the two groups and only those spots with a significant difference (>1.5-fold increase or decrease, P < 0.05) were selected for protein identification.

Protein identification

Spots with differential expression (P < 0.05) after 500 μg/mL C. cicadae treatment were sent to the Genome Research Centre (The University of Hong Kong, Hong Kong) for protein identification by MALDI-TOF/TOF MS (4800 MALDI TOF/TOF Analyzer; Applied Biosystems, USA) analysis after trypsin digestion. The identification was performed by Mascot peptide mass fingerprinting, which can identify proteins from the NCBInr database with taxonomy limited to Homo sapiens. Mascot reported the molecular weight search (MOWSE) score, which was calculated as −10 × log10 (P), where P is the probability that the observed match is a random event. P was limited by the size of the sequence database being searched (limited by taxonomy), with conditions such as peptide fixed modification and variable modification and the settings for trypsin digestion. Each calculated value that fell within a given mass tolerance of an experimental value counted as a match. The threshold was accepted if an event occurred at random with a frequency of < 5%. In this study, a protein match with a score of > 71 was regarded as significant.

Western blot analysis

Western blot analysis was performed with Mighty Small II SE250/260 Cell (Hoefer, USA) and TE 77 PWR Transfer (GE Healthcare, USA) electrophoresis units. Briefly, MHCC97H cells treated with or without various concentrations of C. cicadae (0, 100, 250, and 500 μg/mL) for 48 h were harvested, washed twice with PBS, and lysed in the lysis buffer for sample preparation described above. The lysates were centrifuged at 11000 × g for 30 min. The total protein concentration in the supernatant was determined by the Bradford assay (Bio-Rad). For immunoblotting, each protein extract was mixed with sample buffer [62.5 mmol/L Tris, pH 6.8 (Bio-Rad), 25% glycerol, 2% SDS, 350 mmol/L DTT, 0.01% bromophenol blue] at a ratio of 1:1 (v/v) and then kept in boiling water for 5 min. An aliquot (30 mg protein) of each sample was applied for electrophoresis in 12.5% SDS-PAGE gels with constant voltage (120 V) and transferred to a polyvinylidene difluoride membrane (GE Healthcare). The membranes were blocked with 5% skimmed milk in PBST [PBS, pH 7.4, 0.1% Tween-20 (USB Corporation)] for 3 h at room temperature, followed by incubation for 2 h with the following primary antibodies (ABCAM, UK): anti-CDK1 (ab32384; 1:2000); anti-cyclin B1 (ab32053; 1:2000); anti-HSP27 (ab2790; 1:2000); anti-PRDX1 (ab16805; 1:2000); anti-STRAP (ab46784; 1:2000). The membranes were washed three times with PBST, and incubated with HRP-conjugated goat anti-rabbit or anti-mouse IgG (Bio-Rad; 1:10 000) for 1 h at 37°C. After three washes with PBST, the membranes were developed using Inmun-Star HRP peroxide buffer (Bio-Rad). The relative molecular weight of each protein band was estimated using molecular markers (Precision Plus Protein Standards Dual Color; Bio-Rad). Each sample was measured three times.

Statistical analysis

In cell proliferation assay, experiments were repeated three times, measurements within an experiment were done in six duplicate, and in cell cycle analysis, experiments were repeated three times. Data from three independent experiments were expressed as mean ± standard deviation (SD). The significance of differences in data was determined by Kruskal-Wallis one-way analysis of variance (ANOVA) followed by Dunnets post hoc test for determination of dose–response dependence. In proteomic analysis, six individual samples were prepared and run for 2D gel in each group (i.e., n = 6) the significance of differences in data was determined by a two-tailed Student’s t-test with Levene’s test for equality of variances. All analyses were performed using SPSS 17.0 (IBM, USA) with P < 0.05 as the significance level.

Results

Effects of C. cicadae on MHCC97H cell proliferation

MHCC97H cells were grown in medium containing various concentrations of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) for 48 or 72 h to investigate the effects of C. cicadae on cell proliferation. Dose-dependent inhibition of cell growth was observed with treatment of C. cicadae (P < 0.05) (Figure 1).

Figure 1.

Figure 1

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The effects of C. cicadae on MHCC97H cell viability. The cell viability was tested by MTT assay. Data were expressed as mean ± SD (n = 3 separate experiments). **,***, P < 0.01, 0.001 vs. control in 48 h treatment group, and ##, ###, P < 0.01, 0.001 vs. control in 72 h treatment group, respectively.

Effect of C. cicadae on the cell cycle distribution of MHCC97H cells

Figure 2A shows the effects of C. cicadae (0, 100, 250, 500, and 1000 μg/mL) treatment for 48 h on the cell cycle phase distribution of MHCC97H cells. As shown in Figure 2B, treatment with C. cicadae (100, 250, and 500 μg/mL) for 48 h resulted in cell accumulation in G2/M phase at 23.8%, 30.2%, and 38.6%, respectively, compared with the control cells (19.8%), and decreased the cell percentages in G0/G1 phase by 48.2%, 43.9%, and 35.6%, respectively, compared with the control cells (53.3%). Treatment with C. cicadae at 100, 250, and 500 μg/mL for 48 h did not affect the cell population in S phase (27.9%, 25.8%, and 25.9%, respectively, compared with the control cells (26.91%). Furthermore, most cells (64.74%) were arrested in G2/M phase with the highest dose of 1000 μg/mL C. cicadae. The sub-G1 apoptotic fraction of cells showed no significant changes when C. cicadae (100–500 μg/mL) was added, suggesting that no apoptosis occurred during the period of examination.

Figure 2.

Figure 2

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The effects of C. cicadae on MHCC97H cell cycle. (A) Cellular DNA contents in C. cicadae-treated and control cells were monitored by flow cytometry. The cell cycle distribution of sub-G1, G1, S and G2/M phase cells are presented as DNA histogram. (B) The percentage of cells in each cell cycle phase of MHCC97H after C. cicadae treatment were shown. The data were expressed as mean ± SD and were representative of three independent experiments. *, **, P < 0.05, 0.01, vs. control, respectively.

CDK1/cyclin B complex is involved in the G2/M phase checkpoint by regulating the process of M phase [36]. Figure 3 shows that while the CDK1 protein level was decreased in MHCC97H cells after C. cicadae treatment, the cyclin B1 protein level was unaffected.

Figure 3.

Figure 3

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The effects of C. cicadae on cell cycle checkpoint related proteins.C. cicadae treatment decreased the expression of CDK1 in MHCC97H cells.

Differential expression of proteins in C. cicadae-treated MHCC97H cells

2-DE analyses were performed for each treated sample and repeated six times. Figure 4 shows representative gel images in which more than 1000 spots were detected on each gel. Proteins within the range of 15–225 kDa and having isoelectric points between 3 and 10 were well separated. Table 1 presents the differential expression levels of the identified proteins in MHCC97H cells without (control) and with C. cicadae treatment. Twenty-eight proteins with significant (P < 0.05) changes of > 1.5-fold in volume intensity were selected and further analyzed by MALDI-TOF–MS/MS for peptide identification after trypsin digestion. Among these proteins, the nine upregulated proteins were identified as tubulin beta 2C, dynactin subunit 2 (DCTN2), keratin type II cytoskeletal 7, keratin type I cytoskeletal 10, N-myc downstream-regulated gene 1 (NDRG1), heat shock protein beta-1 (HSPB1, Hsp27), phosphatidylinositol transfer protein beta isoform (PITPNB), alpha-enolase isoform 1 (ENO1), and WD repeat-containing protein 1 isoform 1 (WDR1). The 13 downregulated proteins were identified as 14-3-3 gamma (YWHAG), microtubule-associated protein RP/EB family member 1 (MAPRE1), chloride intracellular channel protein 1 (CLIC1), WD-40 repeat protein (STRAP), thioredoxin-like protein (GLRX3), ACTB protein, xaa-Pro dipeptidase isoform 3 (PEPD), enoyl-CoA delta isomerase 1 mitochondrial (ECI1), protein-disulfide isomerase-related chaperone Erp29 (ERP29), ectonucleoside triphosphate diphosphohydrolase 5 isoform (ENTPD5), hnRNP 2H9B (HNRNPH3), peroxiredoxin 1 (PRDX1), and mitotic checkpoint protein BUB3 isoform A (BUB3). The main properties of the identified proteins were summarized in Table 2.

Figure 4.

Figure 4

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Representative of 2-DE gel proteomic maps of MHCC97H cells with (left) or without (right) C. cicadae treatment. The labelled spots were representative of proteins with significant change.

Table 1.

Differentially expressed proteins between C . cicadae -treated and non-treated (control) MHCC97H cells

Spot No. 1 Protein name GenInfo identifier 2 Protein score 3 Expression quantity (×10 4 ) control 4 Expression quantity (×10 4 ) C . cicadae Expression change ( C . Cicadae /control) P pI 5 Mr (kDa) 5
0111
 14-3-3 Gamma In Complex With A Phosphoserine Peptide
 gi|82407948
 76
 33.0 ± 7.2
 19.0 ± 5.3
 0.58
 0.0004
 4.80
 28.3
1217
 microtubule-associated protein RP/EB family member 1
 gi|6912494
 219
 10.0 ± 3.7
 5.7 ± 2.2
 0.57
 0.026
 5.02
 30.1
1222
 Chloride intracellular channel protein 1
 gi|4588526
 307
 24.8 ± 8.4
 9.3 ± 3.3
 0.38
 0.004112
 5.02
 27.2
1305
 WD-40 repeat protein
 gi|4519417
 221
 42.3 ± 10.2
 12.0 ± 4.1
 0.28
 0.001183
 4.93
 38.8
1427
 Tubulin, beta 2C
 gi|23958133
 188
 3.6 ± 1.7
 7.8 ± 4.2
 2.17
 0.025345
 4.83
 50.2
1514
 Dynactin subunit 2
 gi|22096346
 436
 9.8 ± 2.4
 19.2 ± 5.8
 1.97
 0.000491
 5.10
 44.3
2303
 thioredoxin-like protein
 gi|3646128
 123
 9.3 ± 5.4
 3.4 ± 1.9
 0.37
 0.038567
 5.25
 37.8
2403
 ACTB protein
 gi|15277503
 444
 101.7 ± 33.4
 60.3 ± 18.3
 0.59
 0.001814
 5.55
 40.5
3413
 N-myc downstream regulated gene 1
 gi|119612570
 105
 9.0 ± 3.3
 30.8 ± 11.8
 3.44
 0.00106
 5.98
 34.0
4102
 heat shock protein beta-1
 gi|4504517
 368
 18.1 ± 7.6
 27.7 ± 10.6
 1.53
 0.026641
 5.98
 22.8
4504
 xaa-Pro dipeptidase isoform 3
 gi|260593665
 193
 8.7 ± 2.7
 3.3 ± 1.2
 0.38
 0.027093
 5.7
 48.0
5101
 Enoyl-CoA delta isomerase 1, mitochondrial
 gi|60593479
 67
 11.8 ± 7.8
 5.8 ± 2.9
 0.49
 0.040511
 6.00
 28.9
5106
 The Protein-Disulfide Isomerase Related Chaperone Erp29
 gi|192987144
 247
 16.3 ± 6.8
 9.3 ± 4.3
 0.57
 0.033331
 7.07
 27.2
5218
 phosphatidylinositol transfer protein beta isoform
 gi|6912594
 181
 5.5 ± 1.9
 8.5 ± 2.6
 1.56
 0.004165
 6.41
 31.8
5425
 ectonucleoside triphosphate diphosphohydrolase 5, isoform
 gi|119601555
 181
 5.9 ± 2.1
 2.3 ± 0.8
 0.39
 0.007848
 5.74
 45.7
6334
 hnRNP 2H9B
 gi|7739445
 127
 31.1 ± 12.6
 17.4 ± 6.5
 0.56
 0.023
 6.76
 31.5
6407
 alpha-enolase isoform 1
 gi|4503571
 349
 21.4 ± 7.5
 34.8 ± 12.6
 1.63
 0.028265
 7.01
 47.5
6730
 WD repeat-containing protein 1 isoform 1
 gi|9257257
 217
 9.0 ± 4.6
 13.9 ± 6.3
 1.54
 0.01346
 6.17
 66.8
7122
 peroxiredoxin 1
 gi|55959887
 79
 2.7 ± 1.0
 0.5 ± 0.1
 0.20
 0.024662
 6.41
 19.1
7306 mitotic checkpoint protein BUB3 isoform a gi|4757880 147 37.1 ± 17.4 22.1 ± 10.1 0.60 0.045568 6.36 37.6
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1Spot no.: automatically assigned by the PDQuest software.2GenInfo identifier: sequence identification number assigned by GenBank.

3Protein score: generated by the MS identification system.

4Expression Quantity were calculated by the PDQuest software, data expressed as mean ± SD.

5Mr and pI: relative molecular mass (Mr) and isoelectric point (pI) generated by the MS system.

Table 2.

The major bio-functions of identified proteins

Protein name Protein symbol Subcellular location Protein functions Up/down regulation
Cell growth and cell cycle regulation
14-3-3 Gamma, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma
 14-3-3γ
 Cytoplasm
 G2 DNA damage checkpoint [37]
 ↓
mitotic checkpoint protein BUB3 isoform A
 BUB3
 Nucleus
 Spindle-assembly checkpoint signalling and establishment of correct kinetochore-microtubule attachments [38]
 ↓
microtubule-associated protein RP/EB family member 1
 MAPRE1
 Cytoplasm
 Microtubule polymerization [39]
 ↓
Dynactin subunit 2
 DCTN2
 Cytoplasm
 Modulates cytoplasmic dynein binding to an organelle, and prometaphase chromosome alignment and spindle organization during mitosis [40]
 ↑
thioredoxin-like protein
 GLRX3
 Cytoplasm
 Human cancer cell growth and metastasis regulation [41]
 ↓
Chloride intracellular channel protein 1
 CLIC1
 Nucleus membrane, cell membrane
 Formation of chloride ion channels, and regulation of the cell cycle [42]
 ↓
Anti-cancer effects
ectonucleoside triphosphate diphosphohydrolase 5, isoform
 ENTPD5
 Endoplasmic reticulum lumen
 Promoting glycolysis in proliferating cells in response to phosphoinositide 3-kinase signalling in the AKT1-PTEN signalling pathway [43]
 ↓
N-myc downstream regulated gene 1
 NDRG1
 Cytoplasm, nucleus, cell membrane
 Metastasis suppression [44]
 ↑
heat shock protein beta-1
 HSPB1
 Cytoplasm, nucleus
 Stress resistance and actin organization [45]
 ↑
alpha-enolase isoform 1
 ENO1
 Cytoplasm, cell membrane
 Multifunctional enzyme in various processes such as growth control, hypoxia tolerance and allergic responses [46]
 ↑
Other functions
xaa-Pro dipeptidase isoform 3
 PEPD
 Cytoplasm
 Collagen and GSH metabolism [47]
 ↓
Enoyl-CoA delta isomerase 1, mitochondrial
 ECI1
 Mitochondrion matrix
  
 ↓
The Protein-Disulfide Isomerase Related Chaperone Erp29
 ERP29
 Endoplasmic reticulum lumen
 Processing of secretory proteins within the endoplasmic reticulum, possibly by participating in the folding of proteins in the ER [48]
 ↓
phosphatidylinositol transfer protein beta isoform
 PITPNB
 Cytoplasm, Golgi apparatus
 Catalyzes the transfer of PtdIns and phosphatidylcholine between membranes [49]
 ↑
hnRNP 2H9B
 HNRNPH3
 Nucleus
 Involved in the splicing process and participates in early heat shock-induced splicing arrest [50]
 ↓
WD repeat-containing protein 1 isoform 1
 WDR1
 Cytoplasm
 Induces disassembly of actin filaments in conjunction with ADF/cofilin family proteins [51]
 ↑
peroxiredoxin 1
 PRDX1
 Cytoplasm
 Involved in redox regulation of the cell. Reduces peroxides with reducing equivalents provided through the thioredoxin system but not from glutaredoxin [52]
 ↓
WD-40 repeat protein, serine/threonine kinase receptor associated protein STRAP Cytoplasm, nucleus The SMN complex plays an essential role in spliceosomal snRNP assembly in the cytoplasm and is required for pre-mRNA splicing in the nucleus [53,54] ↓
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Western blot analyses of a few randomly selected proteins were performed for proteomic data validation. In agreement with the proteome changes identified by the proteomic analysis, the expression levels of Hsp27, peroxiredoxin 1, and WD 40 were upregulated in the cancer cells with C. cicadae treatment at 500 μg/mL compared with the control cells (Figure 5). These results were similar to those obtained in the proteomic analysis.

Figure 5.

Figure 5

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Western blotting analysis of differential expressed proteins. With 500 μg/mL of C. cicadae treatment, the expression of Hsp27 was upregulated and Peroxiredoxin 1 and WD 40 were up-regulated in the cancer cells as compared to control (500 μg/mL of C. cicadae).

Discussion

Species in the genus Cordyceps can reduce cancer cell proliferation [14,15,55-58], but only limited studies on liver cancer are available. Several polysaccharides isolated from C. cicadae exhibited antitumor and immunomodulatory properties [14,15,55-58] but the mechanisms have not been fully explored. In the present study, we showed that C. cicadae inhibited the growth of the human HCC cell line MHCC97H via G2/M cell cycle arrest. In support of the cell cycle measurements, several differentially expressed proteins for cell cycle regulation, mitosis, and protein synthesis were identified by the proteomic analysis.

Western blot analyses revealed a decrease in the level of CDK1, which is a G2/M checkpoint cyclin. The cell cycle measurements also indicated accumulation of cells in G2/M phase rather than induction of apoptosis with C. cicadae. To our knowledge, this is the first study to demonstrate that the anticancer mechanisms of C. cicadae involve G2/M arrest. Other studies showed that C. militaris could induce G2/M cell cycle arrest in human colon cancer HT-29 cells [37].

Figure 6 summarizes the magnitude changes in expression of the identified proteins with reference to the control cells. 14-3-3 gamma, BUB3, DCTN2, MAPRE1, GLRX3, and CLIC1 are thought to be involved in cell cycle regulation, G2/M checkpoint control, microtubule polymerization, and spindle organization during mitosis etc. Although their mechanisms related to the G2/M cell cycle arrest induced by C. cicadae were not clear, 14-3-3 gamma is important for cell cycle regulation and 14-3-3 gamma expression change-induced cell cycle deregulation has been implicated in cancer formation [37]. 14-3-3 gamma blocked premature mitotic entry into G2/M phase after DNA damage [59,60], while 14-3-3 gamma-knockdown cells showed inhibition of cdc25C function, which induced overriding of both the incomplete S phase and the G2 DNA damage checkpoint [61]. Overexpression of 14-3-3 gamma in HCC patients was associated with extrahepatic metastasis and worse survival [62,63]. Our proteomic data showed that C. cicadae treatment reduced the expression of 14-3-3 gamma in the human HCC cell line MHCC97H, suggesting that the anticancer and G2/M phase arrest effects of C. cicadae could be attributed to 14-3-3 gamma expression inhibition.

Figure 6.

Figure 6

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Summary of the differential expressed proteins in response to C. cicadae treatment in MHCC97H. The proteins with increased expression level after 500 μg/mL C. cicadae treatment were plotted on the right hand side of axis, and proteins with decreased expression level were plotted on the left hand side.

BUB3, DCTN2, and MAPRE1 are involved in spindle checkpoint and mitosis regulation. BUB3 is a mitosis checkpoint protein for early stages of mitosis to prevent premature sister chromatid separation, missegregation, and aneuploidy via regulation of anaphase-promoting complex/cyclosome (APC/C) [38,64]. DCTN2 is involved in functions for the microtubule anchor dynactin complex, inactivation of the spindle checkpoint through kinetochore disassembly, and recruitment of cell cycle regulators to centrosomes [40,65]. MAPRE1 is involved in microtubule polymerization, microtubule dynamics control [39], and mitotic spindle function [66], and is dominantly expressed in poorly differentiated HCCs [67]. The expression changes of BUB3, DCTN2, and MAPRE1 in C. cicadae-treated MHCC97H cells indicate a mitosis-regulatory effect of C. cicadae, which may contribute to G2/M phase arrest.

GLRX3 maintains redox homeostasis in living cells [41]. In GLRX3-knockout mice, embryonic fibroblasts were arrested at G2/M phase [68]. CLIC1 is a nuclear chloride ion channel and involved in G2/M cell cycle regulation [42]. These findings may suggest a reason why the expression levels of GLRX3 and CLIC1 were changed in C. cicadae-treated MHCC97H cells.

ENTPD5 is an endoplasmic reticulum (ER) enzyme that hydrolyzes purine nucleoside diphosphates and is essential for liver functions, and ENTPD5-deficiency resulted in hepatocellular neoplasia [69]. Overexpression of ENTPD5 was observed in prostate cancer [43,70]. NDRG1 is a member of the NDRG gene family, and exhibited anticancer and metastasis-suppression effects in pancreatic cancer, colon cancer, cervical and ovarian cancer, prostate cancer, and breast cancer [44,71-73]. MHCC97H cells exhibited an 80% pulmonary metastatic rate when injected into nude mice [74]. HSPB1 is a subunit of HSP27, and suppressed cell apoptosis in MHCC97H cells via an NF-kappaB pathway [45]. The elevation of this protein indicated some resistance of the cancer cells, and could be responsible for the absence of apoptosis in the C. cicadae-treated cancer cells. ENO1, a glycolysis module protein, was overexpressed in HCC cells [46,75], and its increased level suggests that C. cicadae treatment increased the glycolytic activity of the cancer cells.

A reduction in PEPD is involved in collagen and GSH metabolism of C. cicadae. PEPD is a homodimeric iminodipeptidase that releases carboxy-terminal proline or hydroxyproline residues from oligopeptides, and is used as a marker of non-alcoholic liver fibrosis [47,76]. ERP29 works in the early secretory pathway in the ER, and overexpression of ERP29 resulted in higher expression of HSP27 in breast cancer cells [48]. The reduced expression of ERP29 is likely to be attributed to reduced ER stress with treatment of C. cicadae.

STRAP is involved in spliceosomal mRNP assembly in the cytoplasm and pre-mRNA splicing in the nucleus [53]. Overexpression of STRAP was reported in several cancers [53,77]. A STRAP-activated p53-related apoptosis-induction effect and higher expression of STRAP in drug-treated HCC cells were observed [54,78]. In the present study, the expression of STRAP was reduced, which may be related to the non-apoptotic effect of C. cicadae.

PRDX1 is a member of the peroxiredoxin family involved in redox regulation in cells, regulation of hydrogen peroxide signaling through its peroxidase activity, and a protein chaperone function. The aspect of whether the reduced expression of PRDX1 contributes to reduced survival of MHCC97H cells via failure in redox regulation against reactive oxygen species remains to be determined. Elevated expression of PRDX1 was found in various cancers [52,79,80].

The present study has identified novel molecular signatures in C. cicadae-induced cell cycle arrest and cell death in MHCC97H cells using proteomics. Among the differentially expressed proteins identified, 14-3-3 gamma, BUB3, DCTN2, MAPRE1, GLRX3, and CLIC1 may play some significant roles in the C. cicadae-induced G2/M phase arrest in MHCC97H cells. The identified proteins will further enhance a molecular explanation for the cell cycle arrest process induced by C. cicadae. Further validation of these markers in gene knockout studies would greatly improve our understanding of the molecular mechanisms behind the liver cancer therapy potential of C. cicadae.

Conclusions

The water extract of C. cicadae reduced the growth of the human HCC cell line MHCC97H via G2/M cell cycle arrest.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

HW and JMFW conceived and designed the study. HLW, ZJ and WHS performed experiments. HLW performed data analysis. HW, CYJL and JMFW wrote the manuscript. All authors read and approved the final version of the manuscript.

Contributor Information

Hualin Wang, Email: wanghualin313@gmail.com.

Jing Zhang, Email: icelemon716@126.com.

Wai-Hung Sit, Email: whsit@hku.hk.

Chung-Yung Jetty Lee, Email: jettylee@hku.hk.

Jennifer Man-Fan Wan, Email: jmfwan@hku.hk.

Acknowledgments

This work was supported by the Chinese Medicine Research Fund funded by BioAsia (Institute of Life Sciences Shanghai Pan Asia, Shanghai, China] and partially funded by Seed Funding Programme for Basic Research funded by the University of Hong Kong.

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