Nutr Res Pract.  2015 Apr;9(2):111-116. 10.4162/nrp.2015.9.2.111.

Ethanol extract of Innotus obliquus (Chaga mushroom) induces G1 cell cycle arrest in HT-29 human colon cancer cells

• Lee HS
• Kim EJ
• Kim SH

• KMID: 2313817
• DOI: http://doi.org/10.4162/nrp.2015.9.2.111

Abstract

BACKGROUND/OBJECTIVES
Inonotus obliquus (I. obliquus, Chaga mushroom) has long been used as a folk medicine to treat cancer. In the present study, we examined whether or not ethanol extract of I. obliquus (EEIO) inhibits cell cycle progression in HT-29 human colon cancer cells, in addition to its mechanism of action.
MATERIALS/METHODS
To examine the effects of Inonotus obliquus on the cell cycle progression and the molecular mechanism in colon cancer cells, HT-29 human colon cancer cells were cultured in the presence of 2.5 - 10 microg/mL of EEIO, and analyzed the cell cycle arrest by flow cytometry and the cell cycle controlling protein expression by Western blotting.
RESULTS
Treatment cells with 2.5 - 10 microg/mL of EEIO reduced viable HT-29 cell numbers and DNA synthesis, increased the percentage of cells in G1 phase, decreased protein expression of CDK2, CDK4, and cyclin D1, increased expression of p21, p27, and p53, and inhibited phosphorylation of Rb and E2F1 expression. Among I. obliquus fractions, fraction 2 (fractionated by dichloromethane from EEIO) showed the same effect as EEIO treatment on cell proliferation and cell cycle-related protein levels.
CONCLUSIONS
These results demonstrate that fraction 2 is the major fraction that induces G1 arrest and inhibits cell proliferation, suggesting I. obliquus could be used as a natural anti-cancer ingredient in the food and/or pharmaceutical industry.

Keyword

Inonotus obliquus; cell cycle; Rb; colon cancer; anti-cancer

MeSH Terms

Blotting, Western
Cell Cycle
Cell Cycle Checkpoints
Cell Proliferation
Colonic Neoplasms*
Cyclin D1
DNA
Drug Industry
Ethanol*
Flow Cytometry
G1 Phase
G1 Phase Cell Cycle Checkpoints*
HT29 Cells
Humans
Medicine, Traditional
Methylene Chloride
Phosphorylation
Cyclin D1
DNA
Ethanol
Methylene Chloride

Figure

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  Fig. 1 Preparation of extract and various fractions of Inonotus obliquus

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  Fig. 2 Effect of EEIO on HT-29 cell growth. HT-29 cells were plated in 24-well plates at a density of 50,000 cells/well in DMEM/F12 supplemented with 10% FBS. One day later, the monolayers were serum-starved with serum-free DMEM/F12 supplemented with 5 mg/L transferrin, 0.1 g/L BSA, and 5 µg/mL selenium for 24 h. After serum starvation, cells were incubated in serum-free medium in the absence or presence of various concentrations of EEIO. Cell numbers were estimated by MTT assay. Each bar represents the mean ± SEM (n = 6). Bars with different letters are significantly different at P < 0.05 by Duncan's multiple range test at each time point.

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  Fig. 3 Effect of EEIO on 5-bromo-2'-deoxyuridine (BrdU) incorporation in HT-29 cells. HT-29 cells were plated and treated with EEIO for 2 days as described in Fig. 2. After BrdU was added, incubation was continued for another 5 h to measure incorporation into DNA. Each bar represents the mean ± SEM (n = 6). *Different from 0 µg/mL, P < 0.05.

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  Fig. 4 Effect of EEIO on cell cycle progression in HT-29 cells. Cells were plated and treated with EEIO for 2 days as described in Fig. 2. Cells were trypsinized and collected. Cells were then fixed and digested with RNase, after which cellular DNA was stained with propidium iodide and analyzed by flow cytometry. Each bar represents the mean ± SEM (n = 6). *Different from 0 µg/mL, P < 0.05.

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  Fig. 5 Effect of EEIO on expression of various cell cycle-regulating proteins in HT-29 cells. Cells were cultured and treated with EEIO for 2 days as described in Fig. 2. Cell lysates were analyzed by immunoblotting with an antibody against p21, p27, p53, CDK2, CDK4, cyclin D1, Rb, pRb, E2F1, or β-actin. A photograph of chemiluminescent detection of a blot, which is representative of 3 independent experiments, is shown.

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  Fig. 6 Effects of various fractions of EEIO on HT-29 cell growth. HT-29 cells were plated and treated with 0 or 10 µg/mL of various fractions for 2 days as described in Fig. 2. Cell numbers were estimated by MTT assay. Each bar represents the mean ± SEM (n = 6). Bars with different letters are significantly different at P < 0.05 by Duncan's multiple range test at each time point.

• 

  Fig. 7 Effects of various fractions of EEIO on 5-bromo-2'-deoxyuridine (BrdU) incorporation in HT-29 cells. HT-29 cells were plated and treated with 0 or 10 µg/mL various fractions for 2 days as described in Fig. 2. BrdU was added and incubation was continued for another 5 h to measure incorporation into DNA. Each bar represents the mean ± SEM (n = 6). Bars with different letters are significantly different at P < 0.05 by Duncan's multiple range test.

• 

  Fig. 8 Effects of various fractions of EEIO on cell cycle progression in HT-29 cells. Cells were plated and treated with various fractions for 2 days as described in Fig. 2. Cells were trypsinized and collected. Cells were then fixed and digested with RNase, after which cellular DNA was stained with propidium iodide and analyzed by flow cytometry. Each bar represents the mean ± SEM (n = 6). Bars with different letters are significantly different at P < 0.05 by Duncan's multiple range tests.

• 

  Fig. 9 Effects of various fractions of EEIO on expression of various cell cycle-regulating proteins in HT-29 cells. Cells were cultured and treated with various fractions for 2 days as described in Fig. 2. Cell lysates were analyzed by immunoblotting with an antibody against p21, p27, p53, CDK2, CDK4, Cyclin D1, Rb, pRb, E2F1,or β-actin. A photograph of chemiluminescent detection of a blot, which is representative of 3 independent experiments, is shown.

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