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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 12  |  Page : 964-968

Ganodermanontriol inhibits expression of special AT rich sequence binding protein 1 gene in human hepatocellular carcinoma


1 Department of Epidemiology and Medical Statistics, Guangdong Medical University, Dongguan, Guangdong 523808, China, Dongguan
2 Department of Treatment Center of Liver Diseases, PLA Army General Hospital, Beijing 100086, China
3 Department of Urology, General Hospital of the Chinese People's Armed Police Forces, Beijing 100074, China

Date of Web Publication11-Dec-2018

Correspondence Address:
Dong Pang
Department of Urology, General Hospital of the Chinese People's Armed Police Forces, Beijing 100074
China
Yuanlin Ding
Department of Epidemiology and Medical Statistics, Guangdong Medical University, Dongguan, Guangdong 523808
Dongguan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.203597

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 > Abstract 


Context: The metastasis of liver cancer is a major cause of clinical treatment failure, restrain, and control the cancer metastasis is the major strategy of the treatment and prevention of the disease. Special AT-rich sequence-binding protein 1 (SATB1) gene was overexpressed in many malignant tumors and considered as a potential target of anticancer drug. This study investigated the mechanism how ganodermanontriol effect the expression of SATB1 and thus inhibits the growth and metastasis in hepatocellular carcinoma (HCC).
Aims: This study explored mainly on the mechanism how ganodermanontriol affects the expression of SATB1 and inhibits proliferation of tumor on human hepatoma cell line HepG2.
Settings and Design: The cancer cells were treated with ganodermanontriol. The status of the cells was detected by different methods. The mechanism was checked by various methods.
Materials and Methods: In HepG2 cancer cells treated with various concentrations of ganodermanontriol, the cell proliferation of was detected by MTT assay, cell apoptosis was analyzed by flow cytometry; the mRNA of SATB1, Bcl-2, Bax were detected by reverse transcription-polymerase chain reaction (RT-PCR) and the protein level of SATB1, Bcl-2, Bax, and caspase 3 were analyzed by Western blot.
Statistical Analysis Used: Data are presented as the mean ± standard deviation. The data were analyzed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). A one-way analysis of variance test was used to compare the differences among groups.
Results: This study showed that ganodermanontriol could significantly reduce the expression level of SATB1.
Conclusion: Therefore, downregulate the cascade effect caused by the expression level of Bcl-2 in HCC HepG2 cells.

Keywords: Ganodermanontriol, human hepatocellular carcinoma, special AT-rich sequence-binding protein 1 gene


How to cite this article:
Xu C, Guo H, Kong D, Pang D, Ding Y. Ganodermanontriol inhibits expression of special AT rich sequence binding protein 1 gene in human hepatocellular carcinoma. J Can Res Ther 2018;14, Suppl S5:964-8

How to cite this URL:
Xu C, Guo H, Kong D, Pang D, Ding Y. Ganodermanontriol inhibits expression of special AT rich sequence binding protein 1 gene in human hepatocellular carcinoma. J Can Res Ther [serial online] 2018 [cited 2019 Oct 14];14:964-8. Available from: http://www.cancerjournal.net/text.asp?2018/14/12/964/203597




 > Introduction Top


Hepatocellular carcinoma (HCC) is a very common type of malignancy. Because of its high degree of malignancy and clinically diagnosed in late stage, therefore, it is a serious threat to human health. At present, it is mainly occurred in Africa and South Asia, and also increased year by year in many Western countries.[1],[2] The metastasis of liver cancer is a major cause of clinical treatment failure, also contributed to the high mortality rate of the disease. Therefore, restrain and control the cancer metastasis is the major strategy of the treatment and prevention of the disease.

Special AT-rich sequence-binding protein 1 (SATB1) is one kind of tissue-specific nuclear matrix-bound proteins, specifically binding with the nuclear matrix attachment region (MAR). SATB1 can promote chromatin remodeling, regulate histone acetylation and methylation, thus regulate gene transcription. It plays a key role in normal development of thymocytes and T cells. In recent years, the studies found that the SATB1 gene was overexpressed in many malignant tumors, such as breast cancer, lung cancer, stomach cancer, and liver cancer.[3],[4],[5],[6] It can regulate more than the expression of more than 1000 kinds of tumor-associated gene, which affects tumor growth and metastasis. Therefore, the SATB1 was considered as a potential target of anticancer drug.

Basidiomycetes fungus belongs to the genus Ganoderma fungus, its fruiting body is precious Chinese medicine, is a traditional Chinese tonic herbs and has been considered a treasure to prolong life. Numerous studies have shown that fruiting bodies of Ganoderma lucidum have activities of immunomodulatory, antitumor, liver protection, anti-aging, etc.[7] Triterpenes is a large class of anticancer compound found in G. lucidum, its function of anti-tumor becomes a hotspot of research in recent years. However, there is no study on anticancer metastasis. This study investigated the mechanism how ganodermanontriol effect the expression of SATB1 and thus inhibits the growth and metastasis in HCC.


 > Materials and Methods Top


Cell culture

The human hepatoma cell line HepG2 cells (Shanghai Institute of life sciences, Shanghai, PRC) are cultured in DMEM medium (GIBCO, USA), with 10% of fetal bovine serum (GIBCO, USA), 100 U/ml of penicillin and 100 μg/mL streptomycin at 37°C in the incubator with 5% CO2 and 70–75% humidity. The medium was changed every 72 h.

MTT assay

The 1 × 104 HepG2 cells were suspended into each well in 96-well plates, various concentrations of ganodermanontriol was added (99% purity; Sigma-Aldrich, St. Louis, MO, USA) and incubated for 24, 48 and 72 h at 37°C incubator, normal cultured cells as negative control. After incubation, the medium was replaced with fresh medium containing 20 μl of MTT (5 mg/mL; Sigma-Aldrich) and incubated for another 4 h. The 96-well plate was centrifuged in 1200 g for 10 min at 4°C, carefully aspirated the supernatant, then add 50 μl of DMSO into 96-well plate to bleach in the orbital Shaker for 10 min. MTT was measured by enzyme-linked immunosorbent assay apparatus under 490 nm absorbance and formulas was used to calculate effects on cell proliferation. Inhibition (inhibition ratio) = (1-average optical density [OD] value of test group/average OD value of control group) × 100%. The IC50 value of ganodermanontriol was calculated using the inhibitory rate of cell in 48 h.

Apoptosis detection

Forty-eight hours posttreatment in HepG2 cells, cells were collected and washed once with PBS, then resuspended with 500 μl binding buffer, 5 μl Annexin V-FITC, and 5 μl PI, mixed gently, avoiding light and incubated for 15 min at room temperature. Stained cells were Followed by flow cytometry detection (BD), using the FL1 channel (FITC green), FL2 (PI red fluorescence) to get scatter chart with these two parameters, fluorescence compensation was adjusted based on control, combined quadrants ratios in the top right and bottom right as the rate of apoptosis. The experiment was repeated three times.

Reverse transcription polymerase chain reaction

Forty-eight hours treatment in HepG2 cells, total RNA was extracted as described in TRIzol Reagent Kit, reverse transcripted into cDNA, cDNA was used as a template for SATB1, Bcl-2, caspase 3 and Bax gene amplification. Gene and reference sequence is in [Table 1]. RT-PCR reaction conditions: Denaturation temperature 95°C for 5 min, denaturation temperature 94°C for 20 s, annealing temperature 60°C for 20 s, extension temperature 72°C for 40 s and repeated for total 40 cycles.
Table 1: The nucleotide sequence of primers for RT-PCR

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Western blot

Forty-eight hours posttreatment in HepG2 cells, total protein was extracted as described in extraction Kit, protein concentration was determined by BCA method (Beyotime Institute of biotechnology, Shanghai, China). The 40 μg protein was loaded in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel to separate protein. Semi-dry method was used to transfer protein onto a polyvinylidene difluoride (PVDF) membrane (0.45 μm; Millipore Corporation, Bedford, MA, USA). The PVDF membrane was incubated with 5% skim milk powder in Tris Buffered Saline with Tween 20 (TBST) for 90 min, antibodies against SATB1, Bax, Bcl-2, caspase 3, and GAPDH were added and incubated for 4°C overnight (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Next, membrane washed with TBST three times; horseradish peroxidase conjugated sheep anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was added and incubated at 37°C for 2 h. Finally, the membrane was washed with TBST three times, developed with electrochemiluminescence developer, and detected by ChemiDoc XRS imaging system (Bio-Rad USA), the gray values of the protein was determined with Quantity One software. The experiment was repeated three times.

Statistical analysis

Data are presented as the mean ± standard deviation. The data were analyzed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). A one-way analysis of variance test was used to compare the differences among groups.


 > Results Top


Ganodermanontriol inhibits the proliferation of HepG2 cells

When HepG2 cells were treated with ganodermanontriol, cell proliferation was inhibited with varying degrees. Compared with the control group with ganodermanontriol in different concentrations (5, 15, 25, 50, 100, and 200 μm), ganodermanontriol had significantly inhibition effects in HepG2 cells, either at 48 h or 72 h (P < 0.05). Even at 24 h, with increasing drug concentrations, inhibition rates also increased, but there was no significant difference. With the increase of drug action, time and an increase in the concentration of ganodermanontriol, the proliferation inhibition of HepG2 cells significantly increased (P < 0.05). This increase in inhibition is dose and time dependent [Figure 1].
Figure 1: The inhibition of ganodermanontriol on the HepG2 cells growth. Inhibition of HepG2 cell proliferation by ganodermanontriol in a time- and dose-dependent manner. Results are expressed as the mean ± standard deviation (n = 6) (***P < 0.01)

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Ganodermanontriol induced apoptosis in HepG2 cells

Ganodermanontriol showed killing effect in HepG2 cells. Compared with the control group, and 48 h after treatment with different concentrations (5, 15, 25, 50, 100, and 200 μm) of ganodermanontriol, HepG2 cells were significantly killed (P < 0.05) [Figure 2].
Figure 2: The apoptosis rate of HepG2 cells after treatment with ganodermanontriol in different concentration detected by flow cytometry. (a) The apoptosis of HepG2 cells were analyzed in 25, 50 and 100 μm/ml with ganodermanontriol, untreated cells as control. 10,000 events were collected for this analysis. Bars correspond to mean ± standard deviation. **P < 0.01 versus control group. (b) Data were collected from four independent experiments

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Effects of ganodermanontriol on SATB1 and its downstream related genes in HepG2 cells

Compared with the control group, post 48 h treatment with different concentrations (5, 15, 25, 50, 100, and 200 μm) ganodermanontriol, the SATB1 gene and protein expression in HepG2 cells were significantly downregulated (P < 0.01). Meanwhile, compared with control, in drug-treated cells, the gene and protein expression of Bcl-2 were down-regulated, while the gene and protein expression of Bax were up-regulated and the protein of caspase 3 was up-regulated. These results suggested that ganodermanontriol reduces the expression of SATB1, thereby affecting the SATB1, downstream related gene expression, to achieve the inhibition effect of tumor. The results of RT-PCR and Western blot were as shown in [Figure 3].
Figure 3: Ganodermanontriol inhibits special AT rich sequence binding protein 1 expression and its downstream related genes and protein. (a) The special AT rich sequence binding protein 1, Bcl-2 and Bax mRNA expression level in HepG2 cells treated by different concentration drugs were analyzed by quantitative reverse transcription polymerase chain reactio analysis. (b) The expression levels of special AT rich sequence binding protein 1, Bcl-2, Bax and caspase 3 protein in HepG2 cells after treatment with different concentration drugs were detected by Western blotting. (c) Quantification of the western blot assay. *P < 0.05, **P < 0.01, ***P < 0.001 versus control (one-way analysis of variance)

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 > Discussion Top


Liver cancer is one of the most common malignant tumors of the world; its incidence is on the rise in most countries, is called King of cancer in China. Although there are surgery, radiation, and chemotherapy treatments, but mortality rate of liver cancer has been very high in recent decades, extensive metastasis and diagnostic delays are the main causes of the high death rate. The urgent need to resolve the problem is to look for medicine which not only can prevent tumor metastasis with low side effects, so as to improve the survival rate and quality of life for patients. Current research results show that the effect of ganodermanontriol is an effective inducer of apoptosis of tumor cells can induce apoptosis in tumor cells and reach the goal of anticancer treatment through cytotoxicity. Applanoxidic acid A, C, F and G extracted from the Southern G. lucidum (Ganoderma australe) can inhibit the reproduction and growth of human promyelocytic leukemia cell line (HL-60 cell line).[8] Ganoderic acid T can significantly inhibit metastasis lung cancer cell lines (95-D), mitochondrial dysfunction and protein expression of P53 mediated apoptosis of these cells, through induction of apoptosis and cell cycle arrest in the G1 phase.[9] However, its specific anti-tumor mechanism is still not very clear.

The SATB1 gene is located in 3p23 on chromosome three, the protein encoded by 763 amino acids in length, containing 2 CUT Motif, which can form dimer by similar PDZ-like domains, combined with MAR sequences with very high affinity. The SATB1 works in chromatin remodeling and histone acetylation and methylation processes, changes the packaging of chromatin structure, regulates the expression of related genes.[10] SATB1 is overexpressed in many malignant tumors, regulates the expression of more than 1000 gene expression and promotes cells to grow over normal proliferation restriction and induces cancer.[3],[4],[5],[6],[10],[11] During research on thymocyte apoptosis, Galande et al.[10] discovered nuclear matrix MAR combines the SATB1 protein to induce degradation, followed by DNA degradation. SATB1 monomer dissociation from chromosome formed after degradation lost the ability to combine MAR and regulate gene expression while keeping the original structural domains of MAR identification.

Bcl-2 is proto-oncogene to inhibit cell apoptosis, the encoded protein is a membrane-bound, mainly located in the mitochondria, endoplasmic reticulum, and nuclear membrane, expressed in normal cell development and activation process, but expression level is low in mature cells. In malignant tumors, Bcl-2 is often overexpressed, by reducing the production of oxygen free radicals and lipid peroxides, inhibits cell apoptosis and promotes immortalization of tumor cell.[12] In recent years, studies have found that SATB1 binds with main breakpoint region of the Bcl-2 gene to regulate Bcl-2 gene expression positively.[13],[14] Studies found that SATB1 binds with sequence of SB1 and SB2 in Bcl-2 transcription start site antagonizes the sequence of SB1 and SB2 which negatively regulates Bcl-2 transcription, thus promotes the expression of Bcl-2 genes and reduces apoptosis in Jurkat cells.[15],[16]

Bax is a member of the Bcl-2 family, located in the cytoplasm. When DNA is damaged, Bax can form homologous or heterologous dimmer with Bcl-2, antagonize the anti-apoptotic function of Bcl-2 protein and promote apoptosis.[17],[18] There is a fixed proportion of Bcl-2 and Bax in normal cells. When internal and external environment changed, cells can change the proportion of Bcl-2 and Bax and cause a series of caspase cascade eventually leading to apoptosis.[18] Caspase 3 protein was a member of the caspase family in Bcl-2/Bax cascade downstream, through the degradation of the structural protein and functional protein in the cell to induce apoptosis, together with caspase-6 and caspase-7 known as the real performer apoptosis.[19]

Human hepatoma HepG2 cell lines are highly aggressive, and SATB1 is over expressed in HepG2 cells. We found ganodermanontriol have a strong inhibitory effect on tumor cell proliferation, and this effect is time and dose-dependent. We also found that, compared with the control group, ganodermanontriol could significantly reduce the mRNA and protein expression levels of SATB1. At the same time, the expression levels of Bcl-2 mRNA and protein in cells were down-regulated, the expression of mRNA and protein levels of downstream Bax were increased and the protein level of and caspase 3 was increased. The results of this study showed that ganodermanontriol promotes tumor cell apoptosis through the inhibition of SATB1 gene expression, which may be related to down regulation of Bcl-2, and up regulation of Bax and caspase 3. However, current research work is carried out at the cellular level; there is still a lot of work to do in the future, including animal studies.

To sum up, ganodermanontriol can inhibit cell proliferation and metastasis of liver cancer cells significantly. The mechanism may be by inhibiting the SATB1 gene expression, and then expression regulation of Bcl-2, Bax, and caspase 3, ganodermanontriol induces cell apoptosis and promotes tumor suppression.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Ramakrishnan M, Liu WM, DiCroce PA, Posner A, Zheng J, Kohwi-Shigematsu T, et al. Modulated binding of SATB1, a matrix attachment region protein, to the AT-rich sequence flanking the major breakpoint region of BCL2. Mol Cell Biol 2000;20:868-77.  Back to cited text no. 13
    
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Ma C, Zhang J, Durrin LK, Lv J, Zhu D, Han X, et al. The BCL2 major breakpoint region (mbr) regulates gene expression. Oncogene 2007;26:2649-57.  Back to cited text no. 14
    
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Gong F, Sun L, Sun Y. A novel SATB1 binding site in the BCL2 promoter region possesses transcriptional regulatory function. J Biomed Res 2010;24:452-9.  Back to cited text no. 15
    
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Gong F, Sun L, Wang Z, Shi J, Li W, Wang S, et al. The BCL2 gene is regulated by a special AT-rich sequence binding protein 1-mediated long range chromosomal interaction between the promoter and the distal element located within the 3'-UTR. Nucleic Acids Res 2011;39:4640-52.  Back to cited text no. 16
    
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Slee EA, Adrain C, Martin SJ. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem 2001;276:7320-6.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
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