Journal of Cancer Prevention 2013; 18(4): 330-336
Published online December 31, 2013
© Korean Society of Cancer Prevention
Ki-Rim Kim1, Kwang-Kyun Park1,2, Won-Yoon Chung1,2, and Young Sun Hwang3
1Oral Cancer Research Institute, Yonsei University College of Dentistry, Seoul, 2Department of Oral Biology and BK21 PLUS Project, Yonsei University College of Dentistry, Seoul, 3Department of Dental Hygiene, College of Health Science, Eulji University, Seongnam, Korea
Correspondence to :
Young Sun Hwang, Department of Dental Hygiene, College of Health Science, Eulji University, 212 Yangji-dong, Sujeong-gu, Seongnam 461-713, Korea, Tel: +82-31-740-7493, Fax:+ 82-2-364-7113, E-mail: email@example.com
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Buddlejasaponin IV (BS-IV), a triterpene saponin isolated from
YD-10B cells were treated with various concentrations of BS-IV, and the cell viability was evaluated by MTT assay. Flow cytometry was conducted to examine cell phase distribution and DAPI staining was performed to observe apoptotic morphological changes in BS-IV-treated YD-10B cells. Western blot analysis was used to investigate the expression of proteins associated with cell cycle arrest and apoptosis.
BS-IV treatment significantly reduced the viability of YD-10B cells and partially arrested cell cycle progression at the G2/M phase. Treatment with BS-IV substantially decreased the levels of cyclin B1 and stimulated the phosphorylation of checkpoint kinase 2 (Chk2). The expression of p21 was increased but the phosphorylation of Akt was inhibited in BS-IV-treated YD-10B cells. Furthermore, BS-IV induced release of cytochrome
The treatment with BS-IV inhibits the growth of YD-10B cells by inducing p21-dependent cell cycle arrest at G2/M phase and apoptosis through both mitochondrial-dependent and death receptor-mediated pathways. Thus, BS-IV is an excellent candidate for a chemopreventive agent to block the progression of human OSCC.
Keywords: Oral squamous cell carcinoma, Buddlejasaponin IV, Cell cycle arrest, Apoptosis
Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity worldwide and consistently increasing in developing countries. OSCC is characterized by a high degree of local invasiveness and highly metastasize to the cervical lymph nodes.1,2 Despite recent improved clinical surgery and development of new anticancer drugs, OSCC has high mortality rate in patients with distant metastases. Therefore, the early diagnosis and standard treatments is extremely important to inhibit the onset and progression of oral carcinomas.3 Furthermore, alternative cancer therapy has been demanded for control OSCC and phytochemicals has been suggested as the major source for anticancer agents against a various malignant tumors.
Apoptosis, the programmed cell death, is critical for cellular homeostasis and is also one of the important mechanisms for the treatment of various cancers.4 The apoptotic process is induced by both death receptor (extrinsic) pathway and mitochondrial (intrinsic) pathway. The death receptor-mediated pathway involves the Fas, FasL and procaspase-8. The mitochondria-dependent pathway is controlled by members of Bcl-2 family, causing the release of cytochrome c from mitochondria and the activation of caspase cascade. Two main death pathways converge at caspase-3 activation, and then activated caspase-3 cleaves many substrate proteins including the DNA repair enzyme PARP.5
Buddlejasaponin IV (BS-IV) (Fig. 1) is a major component of the aerial part of
The present study aimed to estimate the chemopreventive potential of BS-IV against the highly invasive human OSCC cell line. We examined whether BS-IV could induce cell cycle arrest and apoptosis in YD-10B cells, and further explored the mechanism underlying its activity.
BS-IV was generously provided by Professor Hee-Juhn Park in Sangji University,6 dissolved in dimethyl sulfoxide (DMSO), stored at ?20°C and further diluted with the culture medium. Highly invasive YD-10B human OSCC cells, which have been derived from tongue cancer tissues of patients, were obtained by Professor Jin Kim in Yonsei University College of Dentistry.10 DMEM: nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), antibiotic-antimycotic (10,000 units/ml penicillin G sodium, 10,000
2. Cell culture
YD-10B OSCC cells were grown in DMEM/F12 containing 10% FBS, 1×10?10 M cholera toxin, 0.4 mg/ml hydro-cortisone, 5
3. MTT assay
YD-10B cells (5×103 cells/well) were plated into a 96-well culture plate, and left overnight to adhere. The attached cells were treated with various concentrations of BS-IV for 24 and 48 h, respectively. Viable cells were detected using a 5 mg/ml MTT solution for an additional 4 h at 37°C, followed by dissolving the produced formazan product in cells with 200
4. Cell cycle analysis
YD-10B cells (4×105 cells/well) were treated with 10
5. Western blot analysis
YD-10B cells were treated with different concentrations of BS-IV for 24 h. The harvested cells were suspended in lysis buffer containing 50 mM Tris (pH 7.5), 1% NP-40, 2 mM EDTA, 10 mM NaCl, 20
6. DAPI staining
YD-10B cells (1×103 cells/well) were treated with 10
7. Cytochrome c release
YD-10B cells, treated with BS-IV, were harvested and suspended in ice-cold buffer A (20 mM HEPES, 250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10
8. Statistical analysis
Data were expressed as the means±standard deviation (SD) of three independent experiments and analyzed via one-way ANOVA with multiple comparisons using InStatTM statistical software (GraphPad Software, Inc., San Diego, CA, USA). P values of less than 0.05 were considered statistically significant.
1. BS-IV inhibits the viability of YD-10B OSCC cells
When YD-10B human OSCC cells were cultured in serum-free medium with various concentrations of BS-IV, cell viability was dose- and time-dependently decreased (Data not shown). Its half maximal inhibitory concentration (IC50) values were 22.5
BS-IV induces cell cycle arrest at G2/M phase in YD-10B OSCC cells
We determined whether BS-IV could block cell cycle progression of YD-10B cells by flow cytometric analysis. When YD-10B cells were treated with 10
BS-IV induces apoptosis in YD-10B OSCC cells
We further studied whether BS-IV could induce apoptosis in YD-10B cells. BS-IV treatment for 24 h induced morphological changes and apoptotic morphological changes were confirmed by DAPI staining (Fig. 4). The expression of Bax was increased and Bcl-2 level was reduced in BS-IV-treated cells (Fig. 5A). Consequently, BS-IV treatment resulted in an increase in the Bax/Bcl-2 ratio, leading to the release of cytochrome c from mitochondria (Fig. 5B). Moreover, treatment with BS-IV elevated the level of active caspase-3 and induced the cleavage of PARP (Fig. 5B). Furthermore, BS-IV stimulation caused the increased expression of Fas and FasL (Fig. 5C).
OSCC is particularly dangerous because of a high risk of producing secondary tumors and captures about 90% of overall oral cancers.2 Combination therapy of surgical and non-surgical approaches has been developed, but there has been no significant decline in the mortality rate over 20 years due to a lack of markers for early prognosis and the failure of advanced tumors to respond to chemotherapy. In this study, we estimate whether BS-IV can be a promising candidate as a chemoprotective agent on OSCC. Human YD-10B cells are moderately differentiated squamouse cell carcinoma originated from tongue. YD-10B was characterized as OSCC cells showing a sheet of polygonal cells scattered with dyskeratotic cells and has high tumorigenicity and invasiveness. YD-10B cells typically expressed E-cadherin and EGFR, but did not express p53.15
Cell growth is controlled by the rates of cell proliferation and cell death. In most cancer cells, abnormal proliferation is induced, but cell death and differentiation are inhibited. Therefore, the manipulation of the apoptotic process in cancer cells is a critical point for the removal of transformed cells and preventing carcinogenesis. To elucidate YD-10B cell growth inhibition by BS-IV treatment, we investigated its effect on cell cycle progression and apoptosis. As a result, cell cycle was arrested at G2/M phase in BS-IV-treated YD-10B cells.
Next, we determined the molecular mechanism underlying cell cycle arrest and induction of apoptosis by BS-IV. Of cyclins and Cdks initiating entrance into M phase, BS-IV reduced expression of cyclin B1 and also increased the activation of Chk2, which are activated by ATM/ATR in response to DNA damage.16 Cyclin-Cdk complexes are regulated by Cdk inhibitors. Thus, the deregulated actions of the Cdk inhibitors contribute importantly to cancer development. In particular, p21 is targeted specifically to Cdc2 and Cdk2 complexes and regulated by Akt.17 Akt phosphorylates p21 Cdk inhibitor in the nucleus and exports them into the cytoplasm. Cytoplasmic p21 proteins do not influence cyclin-Cdk complexes.14 BS-IV treatment induced p21 expression and suppressed the phosphorylation of Akt. These findings demonstrate that treatment with BS-IV may at least inhibit cell growth by cell cycle arrest at G2/M phase due to the reduced expression of cyclin B1 and Chk2 activation. BS-IV-induced cell cycle arrest is closely associated with p53-independent p21 expression.
Apoptosis can be induced via the activation of the pro-apoptotic members of Bcl-2 family of proteins. In mitochondrial-dependent pathway, the increased proapoptotic Bcl-2-related proteins, particularly Bax, which antagonize the anti-apoptotic Bcl-2, favor cytochrome c release into cytosol through opening of the outer mitochondrial membrane.18 The released cytochrome c results in the activation of the caspase cascade and resultant cleavage of PARP for DNA repair.19 We found that BS-IV induced release of cytochrome c from mitochondria by reducing anti-apoptotic Bcl-2 level and increasing pro-apoptotic Bax level. In turn, active caspase-3 and PARP cleavage were significantly increased in BS-IVtreated YD-10B cells. Furthermore, BS-IV suppressed the expression of Fas death receptor and FasL. These results suggest that BS-IV may induce apoptosis by two distinct signaling pathways via mitochondria and death receptor in YD-10B cells.
Collectively, the treatment with BS-IV inhibits the growth of YD-10B cells by inducing p21-dependent G2/M cell cycle arrest and apoptosis through both mitochondrial- dependent and death receptor-mediated pathways. BS-IV is an excellent candidate for a chemopreventive agent to block the progression of highly invasive OSCC. In addition, BS-IV has anti-inflammatory, anti-viral and anti-hepatotoxic activities as active compound of P. kamtschaticum traditionally used to treat various kinds of disorders such as colds, arthritis, and impotence. Thus, BS-IV and the extracts with it may serve as beneficial supplement for health promotion. Furthermore, the evaluation on bioavailability of BS-IV is determined.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2009 -0094027).
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