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Reappraisal of the anticancer efficacy of quercetin in oral cancer cells
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Journal of the Chinese Medical Association 76 (2013) 146e152 www.jcma-online.com
Original Article
Reappraisal of the anticancer efficacy of quercetin in oral cancer cells Su-Feng Chen a, Shin Nien b, Chien-Hua Wu c, Chia-Lin Liu c, Yun-Ching Chang c, Yaoh-Shiang Lin d,e,f,* a Department of Dental Hygiene, China Medical University, Taichung, Taiwan, ROC Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC c Graduate Institute of Pathology and Parasitology National Defense Medical Center, Taipei, Taiwan, ROC d Department of OtolaryngologydHead and Neck Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ROC e Department of Nutritional Science, Toko University, Chia-Yi County, Taiwan, ROC f Department of OtolaryngologydHead and Neck Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC b
Received May 21, 2012; accepted August 8, 2012
Abstract Background: Despite increased experience in therapy, the overall outcome of oral squamous cell carcinoma (OSCC) has not improved because of the relative resistance to chemotherapeutic drugs in addition to local invasion and frequent regional lymph node metastases. Quercetin (Qu) is a principal flavonoid compound and an excellent free-radical-scavenging antioxidant that promotes apoptosis. Limited reports regarding the molecular or cellular role of Qu in anticancer properties on OSCC have been presented. This study was conducted to clarify the efficacy of Qu on OSCC in vitro and further to evaluate the possible mechanism(s). Methods: Cultured OSCC cells (SCC-25) and human gingival fibroblasts (HGFs) were treated with different concentrations of Qu. Cell viability and cell colony-forming potential were detected with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and colony growth assays. Cell-cycle analysis and apoptosis were measured by flow cytometry. Cell migration and invasion were tested using the micropore chamber assay. Results: Cell viability and colony-forming potential were decreased in a dose-dependent manner following Qu treatment. Qu also dosedependently inhibited the proliferation of SCC-25 cells via both G1 phase cell cycle arrest and mitochondria-mediated apoptosis. In addition, Qu also decreased the abilities of migration and invasion of SCC-25 cells in a dose-dependent manner. Conclusion: Qu effectively inhibits cell growth and invasion/migration of SCC-25 cells in vitro. The cellular and molecular mechanisms are via cell cycle arrest accompanied by mitochondria-mediated apoptosis. Our findings suggest that Qu may have potential as a new chemopreventive agent or serve as a therapeutic adjuvant for OSCC. Copyright Ó 2012 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved. Keywords: apoptosis; invasion; migration; oral squamous cell carcinoma; quercetin
1. Introduction Oral squamous cell carcinoma (OSCC) is one of the most common and lethal head and neck malignancies in Taiwan. OSCC is now a global health problem with increasing * Corresponding author. Dr. Yaoh-Shiang Lin, Department of OtolaryngologydHead and Neck Surgery, Kaohsiung Veterans General Hospital, 386 Ta-Chung 1st Road, Kaohsiung 813, Taiwan, ROC. E-mail address: [email protected] (Y.-S. Lin).
incidence and mortality rates. The major environmental risk factors responsible for the development of OSCC include betel nut chewing, cigarette smoking, alcohol consumption, and exposure to high-risk human papillomavirus.1 OSCC is a very difficult disease to treat because of multidisciplinary and diverse treatment strategies and the varied natural behavior of the cancer. Local invasion and frequent regional lymph node metastases together with relative resistance to chemotherapeutic drugs lead to an unpredictable prognosis. Despite increased experience in therapy, including improvements in
1726-4901/$ - see front matter Copyright Ó 2012 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved. http://dx.doi.org/10.1016/j.jcma.2012.11.008
S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
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surgical technology and adjuvant treatments, the overall outcome of OSCC has not improved, indicating an urgent need for a new strategy for OSCC treatment. Accumulating evidence suggests that natural dietary factors, among which complementary and alternative medicines are consistently represented, may inhibit the process of carcinogenesis and may also effectively reduce the risk of many human cancers.2e4 Quercetin (Qu) is the principal flavonoid compound (3,30,40,5,7-penta hydroxyl flavanone) commonly extracted from cranberries, blueberries, apples, and onions. It possesses a wide spectrum of biopharmacological properties and may offer promising new options for the development of more effective chemopreventive and chemotherapeutic strategies because of its powerful antioxidant and free-radicalscavenging properties.5,6 A review of the literature shows that Qu treatment has been associated with selective antiproliferative effects and induction of cell death, probably through an apoptotic mechanism, in breast or other cancer cell lines but not in normal cells.7,8 The characteristics of the extensive anticarcinogenic and antiproliferative effects of Qu have attracted a great deal of attention and have led to it being considered a potential candidate for cancer prevention and treatment of oral cancer. Few studies of the anticancer properties of Qu in oral cancer cells have been reported. Therefore, the current study was undertaken to test the exact antiproliferative effects of Qu on OSCC in vitro. The study also aimed to evaluate the possible mechanism(s) of Qu, with special emphasis on cellular and molecular changes in the growth and invasion potential of OSCC cells.
mL in 24-well plates, 100 mL/well, and treated with Qu (Sigma-Aldrich, St. Louis, MO, USA) at different dosages for 24 hours. After incubation, the medium was replaced with 50 mL of MTT reagent (2 mg/mL) and incubated in 5% CO2 at 37 C for 1 hour.
2. Methods
2.5. Apoptosis analysis
2.1. Cell lines and culture conditions
Cells that were treated for 24 hours were trypsinized and washed twice with phosphate-buffered saline, then suspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Cells were stained with 5 mL Annexin V conjugated with fluorescein isothiocyanate (FITC) at room temperature in the dark for 30 minutes. They were then analyzed by flow cytometry to measure the fluorescence intensity using the FL-1H channel to detect FITC. Untreated cells served as the negative control.
An OSCC cell line, SCC-25, taken from squamous cell carcinoma of the tongue, was kindly provided by Dr Jenn-Han Chen (from National Defense Medical Center, Taipei, Taiwan). The cell line was cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA). Human gingival fibroblasts (HGFs) were obtained from surgical specimens of patients with head and neck squamous cell carcinomas. The fibroblasts were maintained in Dulbecco’s Modified Eagle Medium and Ham’s Nutrient Mixture-F12 culture medium at a ratio of 3:1. Both SCC-25 and HGFs were kept in a medium supplemented with 10% FBS and 1% penicillin/streptomycin (Biological Industries, Beit Haemek, Israel) at 37 C and 5% CO2. The use of HGFs was approved by the institutional review board of the Tri-Service General Hospital, Taipei, Taiwan (TSGHIRB No.: 1-101-05-052). 2.2. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay Cell viability were determined using the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Sigma). Cells were suspended at a density of 2 104 cells/
2.3. Anchorage-independent colony growth assay Tumor cells were seeded at a density of 3 103 cells/well in a six-well plate and treated with Qu at different dosages then incubated in 5% CO2 at 37 C for 14 days. On day 14, the colonies were stained with 2 mg/mL MTT. Colonies of >50 cells were counted and the colony-forming potential of Qutreated OSCC was expressed as the percentage of colonies of the untreated cells. 2.4. Cell cycle analysis The cells were harvested by Qu treatment for 24 hours and fixed in 70% ethanol at 20 C for 1 hour; next, the cells were collected by centrifugation and reconstituted in phosphate-buffered saline (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Cells were then stained with propidium iodide solution (20 mg/ml propidium iodide and 10 mg/ml RNase A) at 37 C in the dark for 30 minutes. The stained cells were examined by flow cytometry using FL-2A to score the DNA content of cells. The percentages of cells in the G1, S, and G2/M cell-cycle phases were determined using the Modfit software (Verity Software House, Inc., Topsham, ME, USA).
2.6. Migration analysis The migration ability was determined using a micropore chamber assay. Cells (1 104) were seeded onto the top chamber of a 24-well micropore polycarbonate membrane filter with 8-mm pores (Becton Dickinson Labware, Lincoln Park, NJ, USA), and the bottom chamber was filled with RPMI 1640 containing 10% fetal bovine serum as a chemoattractant. After 24 hours of incubation in 5% CO2 at 37 C, cells on the upper surface were carefully removed with a cotton swab, and cells that had migrated to the lower surface of the filter were stained by hematoxylin. Cell migration was quantified by counting the average migrated cells in five random highpowered fields per filter.
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2.7. Invasion analysis Invasion assay was performed in modified Boyden chambers with 8-mm pore size filter inserts for 24-well plates. Filters were coated with 10e12 mL of ice-cold basement membrane extracellular matrix in 8e12 mg of protein/mL. Cells (1 104) were added to the upper chamber in 200 mL of RPMI1640 lacking serum. The lower chamber was filled with 500 mL of RPMI1640 medium supplemented with 10% fetal bovine serum. After 24 hours of incubation, the cells were fixed with methanol and stained by hematoxylin. Cells that remained in the basement membrane or attached to the upper side of the filter were removed with paper towels. Cells on the lower side of the filter were examined using light microscopy and counted. 2.8. Western blotting Cells that had been treated for 24 hours were washed twice with ice-cold phosphate-buffered saline and lysed in 0.5 mL of homogenization buffer (10 mM TriseHCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1:100/v:v proteinase inhibitor cocktail) at 4 C for 30 minutes. Cell lysates were then ultracentrifuged at 100,000 g for 30 minutes at 4 C, and the supernatants were used as the cell extracts. The protein concentration of the cell extract was determined by BCA protein assay, and the extracts were adjusted to 2 mg/mL with homogenization buffer. For immunoblot analysis, the cell extract was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the resolved bands were electrotransferred to PVDF membranes using semidry blot apparatus (Bio-Rad) at 3 mA per cm2 of the gel in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol) at room temperature for 30 minutes. The free protein binding sites on the PVDF membrane were blocked by incubation with 5% nonfat milk in TTBS buffer (20 mM Tris at pH 7.4, 0.15 M NaCl, and 0.2% Tween 20) at 25 C for 2 hours. The membrane was then immunoblotted with 0.1 mg/mL primary antibody. Mouse monoclonal anticaspase 3, B cell lymphoma 2 (Bcl-2), cyclin A, cyclin B1, cyclin D1 and cyclin E antibodies, rabbit polyclonal anti-p27 Kip1, goat polyclonal anti-p21Cip1, poly[ADP-ribose] polymerase (PARP), Bcl-2 associated protein X (Bax), heat shock protein 27 (Hsp27) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), p38MAP kinase (p38MAPK), phosphorylated p38MAP kinase (pp38MAPK) (Cell Signaling), phosphorylated heat shock protein 27 (pHsp27) (R&D Systems, Inc., Minneapolis, MN, USA) in Tris-Buffered Saline Tween-20 buffer containing 3% nonfat milk at 4 C overnight and subsequently with secondary antibody conjugated with peroxidase (1:1000) at 25 C for 1 hour. The immunoblots were developed using an enhanced chemiluminescence system, and the luminescence was visualized on X-ray film. 2.9. Statistical analysis All data are expressed as means SD unless stated otherwise. The differences between groups were calculated using
Student’s unpaired t-test. Dose-dependent effects were calculated using simple linear regression. A p value <0.05 was regarded as statistically significant. All statistical analyses were performed using SPSS version 12.0 (SPSS, Inc., Chicago, IL, USA). 3. Results 3.1. Qu exerted an antiproliferative effect on SCC-25 cells The growth inhibitory effect of Qu on SCC-25 cells was evaluated by cell viability and anchorage-independent colony growth assays. HGF (control) was used to test if Qu has a negative effect on normal cells and was evaluated by cell viability. The survival rate of SCC-25 cells showed a significant dosedependent decrease following treatments with Qu (Fig. 1A). Testing with a dose range of 10e100 mM Qu showed that, expressing cell viability as the percentage of that in untreated cells, a 50% cell survival rate of SCC-25 cells was obtained at a dose of 50 mM. Three doses (25, 50 and 75 mM) were chosen as test concentrations for the remainder of the experiments (Fig. 1B). An anchorage-independent colony growth assay further demonstrated a significant inhibitory effect of Qu, with the number of colonies of SCC-25 cell lines being markedly decreased by Qu treatment in a dose-dependent manner (Fig. 1C). The result was also confirmed by quantitative and statistical analyses (Fig. 1D). The data reported from the above two assays were from three independent experiments expressed as means standard deviation (SD). 3.2. Qu affected the cell cycle of SCC-25 cells The distribution of Qu-treated cells in the cell cycle was assessed by flow cytometry, and the expression of cell cycle proteins was measured by immunoblotting. Following treatment with different doses of Qu (25e75 mM), the proportion of G1 phase cells was increased with a concomitant decrease in S phase cells compared with untreated controls and a gradual increase in the proportion of G1 phase cells with a concurrent increase of sub-G1 phase cells, indicating the induction of apoptosis (Fig. 2A). Quantitative and statistical analyses showed an obvious increase in G1 phase cells at a Qu dose of 50 mM (Fig. 2B). Analysis of cell cycle proteins showed a dose-dependent decrease in cyclin D1 and increase in p21Cip1 (Fig. 2C). 3.3. Qu induced apoptosis in SCC-25 cells Qu-treated SCC-25 cells were stained with Annexin VeFITC and then analyzed by flow cytometry to assess the proportion of apoptotic cells. When compared with the control groups, there was a significant dose-dependent increase in the proportion of Annexin V-positive OSCC cells following Qu treatment (Fig. 3A), and this increase was also confirmed by quantitative and statistical analyses (Fig. 3B). Immunoblotting analysis showed that the level of the antiapoptotic oncoprotein
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Fig. 1. Growth inhibitory effects of quercetin (Qu) on SCC-25 cells. (A) The growth inhibitory effect of Qu on SCC-25 cells was initially evaluated in a cell viability assay. Qu did not affect human gingival fibroblasts (HGF). (B) Viable cells were viewed directly under a microscope. (C) Anchorage-independent growth of SCC25 cell lines in soft agar under 100 magnification after 2 weeks in culture. (D) The colony growth assay showed a significant inhibitory effect of Qu on SCC-25 cells.
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Cyclin E Cip1/P21 Kip1/P27 α-tubulin Fig. 2. Effects of quercetin (Qu) on the cell cycle of SCC-25 cells. Flow cytometry demonstrated that the percentage of cells in G1 phase was increased and that in S phase, SCC-25 cells treated with Qu were concomitantly decreased compared with controls. (B) Quantitative and statistical analyses of results shown in panel A. (C) Western blot analysis of cell cycle proteins following Qu treatments showed a significant dose-dependent decrease in the expression of cyclin D1, and a significant increase in p21Cip1.
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Bax Bcl-2 CI-Caspase3 CI-PARP α-tublin Fig. 3. Apoptotic effect of quercetin (Qu) on SCC-25 cells. (A) Apoptosis analysis with Annexin VeFITC staining showed that the proportion of fluorescent cells was significantly enhanced after Qu treatment. (B) Quantitative and statistical analyses of results shown in panel A. (C) Immunoblotting of proteins involved in apoptosis showed that following Qu treatment, the level of antiapoptotic oncoprotein Bcl-2 was dramatically decreased, and the level of the proapoptotic protein Bax was increased in SCC-25 cells. The expression of the active form of caspase 3 and PARP were increased.
Bcl-2 decreased dramatically, whereas the level of the proapoptotic protein Bax was increased. Both of the expressions of the active form of caspase 3 (cleaved caspase 3) and the poly ADP-ribose polymerase (cleaved PARP) were increased. 3.4. Qu effectively inhibited the migration and invasion of SCC-25 cells A transwell Boyden chamber migration assay showed that Qu treatment significantly decreased the migration ability of SCC-25 cells in a dose-dependent manner (Fig. 4A). Quantitative and statistical analyses also demonstrated a significant decrease in migration (Fig. 4B). An invasion assay demonstrated that Qu treatment also significantly reduced this invasive ability in a dose-dependent manner (Fig. 4C); this was confirmed by quantitative and statistical analyses (Fig. 4D). 4. Discussion Growing evidence from epidemiological studies and experimental data from animal models indicate that lifestyle modifications, particularly a vegetable- and fruit-rich diet, may substantially reduce the incidence of certain types of cancers.9 Qu, as described earlier, is one of the most abundant flavonoids in vegetables and fruits. This pure compound is a powerful antioxidant and free-radical scavenger. Several studies in vitro and in vivo have further revealed that Qu can inhibit malignant growth and metastasis in a variety of cancer
cells by exhibiting a strong ability to induce apoptosis, potentially making it an additional option for cancer management.5,10 To the best of our knowledge, little is known about the cellular and molecular changes mediating the anticancer properties of Qu or the mechanisms of its effects on the proliferation, apoptosis, invasion, and migration of oral cancer cells. In this study, we used representative SCC-25 cells to clearly demonstrate the effective dose-dependent inhibition of growth and invasion following Qu treatment. The findings represent the first evidence of the cellular and molecular mechanisms of Qu inhibition of SCC-25 cells growth and invasion. As described above, our study showed that Qu treatment of SCC25 cells exerted a marked and dose-dependent inhibitory effect on cell growth. The viability test showed a similar responsiveness of SCC-25 cells to Qu treatment. An anchorageindependent colony growth assay further validated the clear dose-dependent inhibitory effect of Qu on SCC-25 cells. These findings indicate that Qu may have cytotoxic effects on SCC25 cells, similar to those reported in a previous study of oral cancer.11,12 Many oncoproteins and tumor suppressors, such as cyclin A, B, D1, and E, Bcl-2, Bax, p21Cip and p27Kip1, are associated with the cancer cell cycle and tumorigenesis.13 Immunoblotting analysis of the cell cycle proteins in SCC25 cells showed dose-dependent increases of p21Cip1 following Qu treatment, which might facilitate the arrest of SCC-25 in late G1 phase. Using appropriate doses of 50 mM Qu, the increased proportion of G1 phase cells gradually declined
S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
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0 0 25 50 75 Quercetin concentration (μM) Fig. 4. Inhibitory effects of migration and invasion of quercetin (Qu) on SCC-25 cells. (A) Boyden chamber migration assay showed that Qu significantly decreased the migration ability of SCC-25 cells in a dose-dependent manner. (B) Quantitative and statistical analyses of results shown in panel A. (C) Boyden chamber migration assay demonstrated that Qu significantly decreased the invasion ability of SCC-25 cells in a dose-dependent manner. (D) Quantitative and statistical analyses of the results shown in panel C.
because of the accumulation of sub-G1 phase cells resulting from the early stages of the induction of apoptosis. Induction of cell cycle arrest and apoptosis is a wellestablished method for treating some forms of cancer.14 Based on our data, we propose that cell cycle arrest without repair accompanied by cellular apoptosis may be the main cellular and/or molecular mechanism(s) occurring in SCC-25 cells following Qu treatment. The mitochondrial apoptosis pathway is mediated by the Bcl-2 family and Bax proteins and the expression of Bcl-2. The ratio of Bcl-2 to Bax proteins contributes to the susceptibility of cells to induction of apoptotic cell death. Our immunoblotting analysis revealed that the level of Bcl-2 decreased dramatically in Qu-treated SCC-25 cells, whereas the level of the proapoptotic protein Bax was increased in SCC-25 cells. PARP activity was altered by the upstream apoptosis, the active form of caspase 3, which then induced the downstream apoptosis. Thus, the response of the SCC-25 cells to induction of apoptosis by Qu treatment involved a sustained decrease in expression of the antiapoptotic oncoprotein Bcl-2 along with an elevated level of proapoptotic protein Bax, resulting in an increased Bax/Bcl-2 ratio. These results highlight the importance of the Bax/Bcl-2 ratio in the life and death of oral cancer cells.13,15 Together, these results demonstrate that the cellular and molecular mechanisms of Qu effects on SCC-25 oral cancer cells mainly
depend on cell cycle arrest accompanied by mitochondriamediated apoptosis. Research has revealed that Qu and polyphenol can inhibit the activity of matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9.16 These two key regulators associated with invasion and metastasis can be activated by E2F transcription factor, which plays a pivotal role in cell proliferation by regulating the expression of genes involved in G1eS transition and DNA synthesis.17 Our study also demonstrated that Qu significantly and dose-dependently decreased the invasion and migration abilities of SCC-25 cells. In conclusion, our study demonstrates that Qu treatment inhibits cell growth and invasion/migration on SCC-25 cells in vitro. The cellular and molecular mechanisms of the biopharmacological effects of Qu were mainly via cell cycle arrest accompanied by mitochondria-mediated apoptosis. Our findings clearly suggest that Qu may have potential as a new chemopreventive or therapeutic agent for OSCC. Acknowledgments This study was partly supported by the Tri-Service General Hospital, Grant No. TSGH-C100-007-009-10-S02, TSGHC100161, TSGH-C100-155, ND-I-29 and the Department of Dental Hygiene, China Medical University, Grant No.
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CMU99-N1-04-1, and by the National Science Council, ROC. and Grants No. NSC 99-2320-B-039-028-MY3 and NSC 1002320-B-016-009. References 1. Chen YJ, Chang JT, Liao CT, Wang HM, Yen TC, Chiu CC, et al. Head and neck cancer in the betel quid chewing area: recent advances in molecular carcinogenesis. Cancer Sci 2008;99:1507e14. 2. Kuo SM. Dietary flavonoid and cancer prevention: evidence and potential mechanism. Crit Rev Oncog 1997;8:47e69. 3. Marshall JR. Prevention of colorectal cancer: diet, chemoprevention, and lifestyle. Gastroenterol Clin North Am 2008;37:73e82. 4. Stan SD, Kar S, Stoner GD, Singh SV. Bioactive food components and cancer risk reduction. J Cell Biochem 2008;104:339e56. 5. Tang SN, Singh C, Nall D, Meeker D, Shankar S, Srivastava RK. The dietary bioflavonoid quercetin synergizes with epigallocathechin gallate (EGCG) to inhibit prostate cancer stem cell characteristics, invasion, migration and epithelialemesenchymal transition. J Mol Signal 2010;5:14. 6. Kamaraj S, Vinodhkumar R, Anandakumar P, Jagan S, Ramakrishnan G, Devaki T. The effects of quercetin on antioxidant status and tumor markers in the lung and serum of mice treated with benzo(a)pyrene. Biol Pharm Bull 2007;30:2268e73. 7. Hirpara KV, Aggarwal P, Mukherjee AJ, Joshi N, Buman AC. Quercetin and its derivatives: synthesis, pharmacological uses with special emphasis on anti-tumor properties and prodrug with enhanced bio-availability. Anticancer Agents Med Chem 2009;9:138e61. 8. Seo HS, Ju JH, Jang K, Shin I. Induction of apoptotic cell death by phytoestrogens by up-regulating the levels of phospho-p53 and p21 in
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normal and malignant estrogen receptor alpha-negative breast cells. Nutr Res. 2011;31:139e46. Danaei G, Vander Hoorn S, Lopez AD, Murray CJ, Ezzati M. Causes of cancer in the world: comparative risk assessment of nine behavioural and environmental risk factors. Lancet 2005;366:1784e93. Zhou W, Kallifatidis G, Baumann B, Rausch V, Mattern J, Giadkich J, et al. Dietary polyphenol quercetin targets pancreatic cancer stem cells. Int J Oncol 2010;37:551e61. ElAttar TM, Virji AS. Modulating effect of resveratrol and quercetin on oral cancer cell growth and proliferation. Anticancer Drugs 1999;10:187e93. Kang JW, Kim JH, Song K, Kim SH, Yoon JH, Kim KS. Kaempferol and quercetin, components of Ginkgo biloba extract (EGb 761), induce caspase-3-dependent apoptosis in oral cavity cancer cells. Phytother Res 2010;24:S77e82. Reed JC. Balancing cell life and death: bax, apoptosis, and breast cancer. J Clin Invest 1996;97:2403e4. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998;281:1309e12. Reed JC, Miyashita T, Takayama S, Wang HG, Sato T, Krajewski S, et al. BCL-2 family proteins: regulators of cell death involved in the pathogenesis of cancer and resistance to therapy. J Cell Biochem 1996;60:23e32. Vijayababu MR, Arunkumar A, Kanagaraj P, Ilangovan R, Dharmarajan A, Arunakaran J. Quercetin downregulates matrix metalloproteinases 2 and 9 proteins expression in prostate cancer cells (PC-3). Mol Cell Biochem 2006;287:109e16. La VD, Bergeron C, Gafner S, Grenier D. Grape seed extract suppresses lipopolysaccharide-induced matrix metalloproteinase (MMP) secretion by macrophages and inhibits human MMP-1 and -9 activities. J Periodontol 2009;80:1875e82.
Journal of the Chinese Medical Association 76 (2013) 146e152 www.jcma-online.com
Original Article
Reappraisal of the anticancer efficacy of quercetin in oral cancer cells Su-Feng Chen a, Shin Nien b, Chien-Hua Wu c, Chia-Lin Liu c, Yun-Ching Chang c, Yaoh-Shiang Lin d,e,f,* a Department of Dental Hygiene, China Medical University, Taichung, Taiwan, ROC Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC c Graduate Institute of Pathology and Parasitology National Defense Medical Center, Taipei, Taiwan, ROC d Department of OtolaryngologydHead and Neck Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, ROC e Department of Nutritional Science, Toko University, Chia-Yi County, Taiwan, ROC f Department of OtolaryngologydHead and Neck Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, ROC b
Received May 21, 2012; accepted August 8, 2012
Abstract Background: Despite increased experience in therapy, the overall outcome of oral squamous cell carcinoma (OSCC) has not improved because of the relative resistance to chemotherapeutic drugs in addition to local invasion and frequent regional lymph node metastases. Quercetin (Qu) is a principal flavonoid compound and an excellent free-radical-scavenging antioxidant that promotes apoptosis. Limited reports regarding the molecular or cellular role of Qu in anticancer properties on OSCC have been presented. This study was conducted to clarify the efficacy of Qu on OSCC in vitro and further to evaluate the possible mechanism(s). Methods: Cultured OSCC cells (SCC-25) and human gingival fibroblasts (HGFs) were treated with different concentrations of Qu. Cell viability and cell colony-forming potential were detected with the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and colony growth assays. Cell-cycle analysis and apoptosis were measured by flow cytometry. Cell migration and invasion were tested using the micropore chamber assay. Results: Cell viability and colony-forming potential were decreased in a dose-dependent manner following Qu treatment. Qu also dosedependently inhibited the proliferation of SCC-25 cells via both G1 phase cell cycle arrest and mitochondria-mediated apoptosis. In addition, Qu also decreased the abilities of migration and invasion of SCC-25 cells in a dose-dependent manner. Conclusion: Qu effectively inhibits cell growth and invasion/migration of SCC-25 cells in vitro. The cellular and molecular mechanisms are via cell cycle arrest accompanied by mitochondria-mediated apoptosis. Our findings suggest that Qu may have potential as a new chemopreventive agent or serve as a therapeutic adjuvant for OSCC. Copyright Ó 2012 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved. Keywords: apoptosis; invasion; migration; oral squamous cell carcinoma; quercetin
1. Introduction Oral squamous cell carcinoma (OSCC) is one of the most common and lethal head and neck malignancies in Taiwan. OSCC is now a global health problem with increasing * Corresponding author. Dr. Yaoh-Shiang Lin, Department of OtolaryngologydHead and Neck Surgery, Kaohsiung Veterans General Hospital, 386 Ta-Chung 1st Road, Kaohsiung 813, Taiwan, ROC. E-mail address: [email protected] (Y.-S. Lin).
incidence and mortality rates. The major environmental risk factors responsible for the development of OSCC include betel nut chewing, cigarette smoking, alcohol consumption, and exposure to high-risk human papillomavirus.1 OSCC is a very difficult disease to treat because of multidisciplinary and diverse treatment strategies and the varied natural behavior of the cancer. Local invasion and frequent regional lymph node metastases together with relative resistance to chemotherapeutic drugs lead to an unpredictable prognosis. Despite increased experience in therapy, including improvements in
1726-4901/$ - see front matter Copyright Ó 2012 Elsevier Taiwan LLC and the Chinese Medical Association. All rights reserved. http://dx.doi.org/10.1016/j.jcma.2012.11.008
S.-F. Chen et al. / Journal of the Chinese Medical Association 76 (2013) 146e152
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surgical technology and adjuvant treatments, the overall outcome of OSCC has not improved, indicating an urgent need for a new strategy for OSCC treatment. Accumulating evidence suggests that natural dietary factors, among which complementary and alternative medicines are consistently represented, may inhibit the process of carcinogenesis and may also effectively reduce the risk of many human cancers.2e4 Quercetin (Qu) is the principal flavonoid compound (3,30,40,5,7-penta hydroxyl flavanone) commonly extracted from cranberries, blueberries, apples, and onions. It possesses a wide spectrum of biopharmacological properties and may offer promising new options for the development of more effective chemopreventive and chemotherapeutic strategies because of its powerful antioxidant and free-radicalscavenging properties.5,6 A review of the literature shows that Qu treatment has been associated with selective antiproliferative effects and induction of cell death, probably through an apoptotic mechanism, in breast or other cancer cell lines but not in normal cells.7,8 The characteristics of the extensive anticarcinogenic and antiproliferative effects of Qu have attracted a great deal of attention and have led to it being considered a potential candidate for cancer prevention and treatment of oral cancer. Few studies of the anticancer properties of Qu in oral cancer cells have been reported. Therefore, the current study was undertaken to test the exact antiproliferative effects of Qu on OSCC in vitro. The study also aimed to evaluate the possible mechanism(s) of Qu, with special emphasis on cellular and molecular changes in the growth and invasion potential of OSCC cells.
mL in 24-well plates, 100 mL/well, and treated with Qu (Sigma-Aldrich, St. Louis, MO, USA) at different dosages for 24 hours. After incubation, the medium was replaced with 50 mL of MTT reagent (2 mg/mL) and incubated in 5% CO2 at 37 C for 1 hour.
2. Methods
2.5. Apoptosis analysis
2.1. Cell lines and culture conditions
Cells that were treated for 24 hours were trypsinized and washed twice with phosphate-buffered saline, then suspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Cells were stained with 5 mL Annexin V conjugated with fluorescein isothiocyanate (FITC) at room temperature in the dark for 30 minutes. They were then analyzed by flow cytometry to measure the fluorescence intensity using the FL-1H channel to detect FITC. Untreated cells served as the negative control.
An OSCC cell line, SCC-25, taken from squamous cell carcinoma of the tongue, was kindly provided by Dr Jenn-Han Chen (from National Defense Medical Center, Taipei, Taiwan). The cell line was cultured in RPMI 1640 medium (Gibco, Grand Island, NY, USA). Human gingival fibroblasts (HGFs) were obtained from surgical specimens of patients with head and neck squamous cell carcinomas. The fibroblasts were maintained in Dulbecco’s Modified Eagle Medium and Ham’s Nutrient Mixture-F12 culture medium at a ratio of 3:1. Both SCC-25 and HGFs were kept in a medium supplemented with 10% FBS and 1% penicillin/streptomycin (Biological Industries, Beit Haemek, Israel) at 37 C and 5% CO2. The use of HGFs was approved by the institutional review board of the Tri-Service General Hospital, Taipei, Taiwan (TSGHIRB No.: 1-101-05-052). 2.2. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay Cell viability were determined using the MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Sigma). Cells were suspended at a density of 2 104 cells/
2.3. Anchorage-independent colony growth assay Tumor cells were seeded at a density of 3 103 cells/well in a six-well plate and treated with Qu at different dosages then incubated in 5% CO2 at 37 C for 14 days. On day 14, the colonies were stained with 2 mg/mL MTT. Colonies of >50 cells were counted and the colony-forming potential of Qutreated OSCC was expressed as the percentage of colonies of the untreated cells. 2.4. Cell cycle analysis The cells were harvested by Qu treatment for 24 hours and fixed in 70% ethanol at 20 C for 1 hour; next, the cells were collected by centrifugation and reconstituted in phosphate-buffered saline (10 mM HEPES, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). Cells were then stained with propidium iodide solution (20 mg/ml propidium iodide and 10 mg/ml RNase A) at 37 C in the dark for 30 minutes. The stained cells were examined by flow cytometry using FL-2A to score the DNA content of cells. The percentages of cells in the G1, S, and G2/M cell-cycle phases were determined using the Modfit software (Verity Software House, Inc., Topsham, ME, USA).
2.6. Migration analysis The migration ability was determined using a micropore chamber assay. Cells (1 104) were seeded onto the top chamber of a 24-well micropore polycarbonate membrane filter with 8-mm pores (Becton Dickinson Labware, Lincoln Park, NJ, USA), and the bottom chamber was filled with RPMI 1640 containing 10% fetal bovine serum as a chemoattractant. After 24 hours of incubation in 5% CO2 at 37 C, cells on the upper surface were carefully removed with a cotton swab, and cells that had migrated to the lower surface of the filter were stained by hematoxylin. Cell migration was quantified by counting the average migrated cells in five random highpowered fields per filter.
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2.7. Invasion analysis Invasion assay was performed in modified Boyden chambers with 8-mm pore size filter inserts for 24-well plates. Filters were coated with 10e12 mL of ice-cold basement membrane extracellular matrix in 8e12 mg of protein/mL. Cells (1 104) were added to the upper chamber in 200 mL of RPMI1640 lacking serum. The lower chamber was filled with 500 mL of RPMI1640 medium supplemented with 10% fetal bovine serum. After 24 hours of incubation, the cells were fixed with methanol and stained by hematoxylin. Cells that remained in the basement membrane or attached to the upper side of the filter were removed with paper towels. Cells on the lower side of the filter were examined using light microscopy and counted. 2.8. Western blotting Cells that had been treated for 24 hours were washed twice with ice-cold phosphate-buffered saline and lysed in 0.5 mL of homogenization buffer (10 mM TriseHCl at pH 7.4, 2 mM EDTA, 1 mM EGTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1:100/v:v proteinase inhibitor cocktail) at 4 C for 30 minutes. Cell lysates were then ultracentrifuged at 100,000 g for 30 minutes at 4 C, and the supernatants were used as the cell extracts. The protein concentration of the cell extract was determined by BCA protein assay, and the extracts were adjusted to 2 mg/mL with homogenization buffer. For immunoblot analysis, the cell extract was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the resolved bands were electrotransferred to PVDF membranes using semidry blot apparatus (Bio-Rad) at 3 mA per cm2 of the gel in transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine, and 20% methanol) at room temperature for 30 minutes. The free protein binding sites on the PVDF membrane were blocked by incubation with 5% nonfat milk in TTBS buffer (20 mM Tris at pH 7.4, 0.15 M NaCl, and 0.2% Tween 20) at 25 C for 2 hours. The membrane was then immunoblotted with 0.1 mg/mL primary antibody. Mouse monoclonal anticaspase 3, B cell lymphoma 2 (Bcl-2), cyclin A, cyclin B1, cyclin D1 and cyclin E antibodies, rabbit polyclonal anti-p27 Kip1, goat polyclonal anti-p21Cip1, poly[ADP-ribose] polymerase (PARP), Bcl-2 associated protein X (Bax), heat shock protein 27 (Hsp27) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), p38MAP kinase (p38MAPK), phosphorylated p38MAP kinase (pp38MAPK) (Cell Signaling), phosphorylated heat shock protein 27 (pHsp27) (R&D Systems, Inc., Minneapolis, MN, USA) in Tris-Buffered Saline Tween-20 buffer containing 3% nonfat milk at 4 C overnight and subsequently with secondary antibody conjugated with peroxidase (1:1000) at 25 C for 1 hour. The immunoblots were developed using an enhanced chemiluminescence system, and the luminescence was visualized on X-ray film. 2.9. Statistical analysis All data are expressed as means SD unless stated otherwise. The differences between groups were calculated using
Student’s unpaired t-test. Dose-dependent effects were calculated using simple linear regression. A p value <0.05 was regarded as statistically significant. All statistical analyses were performed using SPSS version 12.0 (SPSS, Inc., Chicago, IL, USA). 3. Results 3.1. Qu exerted an antiproliferative effect on SCC-25 cells The growth inhibitory effect of Qu on SCC-25 cells was evaluated by cell viability and anchorage-independent colony growth assays. HGF (control) was used to test if Qu has a negative effect on normal cells and was evaluated by cell viability. The survival rate of SCC-25 cells showed a significant dosedependent decrease following treatments with Qu (Fig. 1A). Testing with a dose range of 10e100 mM Qu showed that, expressing cell viability as the percentage of that in untreated cells, a 50% cell survival rate of SCC-25 cells was obtained at a dose of 50 mM. Three doses (25, 50 and 75 mM) were chosen as test concentrations for the remainder of the experiments (Fig. 1B). An anchorage-independent colony growth assay further demonstrated a significant inhibitory effect of Qu, with the number of colonies of SCC-25 cell lines being markedly decreased by Qu treatment in a dose-dependent manner (Fig. 1C). The result was also confirmed by quantitative and statistical analyses (Fig. 1D). The data reported from the above two assays were from three independent experiments expressed as means standard deviation (SD). 3.2. Qu affected the cell cycle of SCC-25 cells The distribution of Qu-treated cells in the cell cycle was assessed by flow cytometry, and the expression of cell cycle proteins was measured by immunoblotting. Following treatment with different doses of Qu (25e75 mM), the proportion of G1 phase cells was increased with a concomitant decrease in S phase cells compared with untreated controls and a gradual increase in the proportion of G1 phase cells with a concurrent increase of sub-G1 phase cells, indicating the induction of apoptosis (Fig. 2A). Quantitative and statistical analyses showed an obvious increase in G1 phase cells at a Qu dose of 50 mM (Fig. 2B). Analysis of cell cycle proteins showed a dose-dependent decrease in cyclin D1 and increase in p21Cip1 (Fig. 2C). 3.3. Qu induced apoptosis in SCC-25 cells Qu-treated SCC-25 cells were stained with Annexin VeFITC and then analyzed by flow cytometry to assess the proportion of apoptotic cells. When compared with the control groups, there was a significant dose-dependent increase in the proportion of Annexin V-positive OSCC cells following Qu treatment (Fig. 3A), and this increase was also confirmed by quantitative and statistical analyses (Fig. 3B). Immunoblotting analysis showed that the level of the antiapoptotic oncoprotein
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Fig. 1. Growth inhibitory effects of quercetin (Qu) on SCC-25 cells. (A) The growth inhibitory effect of Qu on SCC-25 cells was initially evaluated in a cell viability assay. Qu did not affect human gingival fibroblasts (HGF). (B) Viable cells were viewed directly under a microscope. (C) Anchorage-independent growth of SCC25 cell lines in soft agar under 100 magnification after 2 weeks in culture. (D) The colony growth assay showed a significant inhibitory effect of Qu on SCC-25 cells.
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Cyclin E Cip1/P21 Kip1/P27 α-tubulin Fig. 2. Effects of quercetin (Qu) on the cell cycle of SCC-25 cells. Flow cytometry demonstrated that the percentage of cells in G1 phase was increased and that in S phase, SCC-25 cells treated with Qu were concomitantly decreased compared with controls. (B) Quantitative and statistical analyses of results shown in panel A. (C) Western blot analysis of cell cycle proteins following Qu treatments showed a significant dose-dependent decrease in the expression of cyclin D1, and a significant increase in p21Cip1.
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Bax Bcl-2 CI-Caspase3 CI-PARP α-tublin Fig. 3. Apoptotic effect of quercetin (Qu) on SCC-25 cells. (A) Apoptosis analysis with Annexin VeFITC staining showed that the proportion of fluorescent cells was significantly enhanced after Qu treatment. (B) Quantitative and statistical analyses of results shown in panel A. (C) Immunoblotting of proteins involved in apoptosis showed that following Qu treatment, the level of antiapoptotic oncoprotein Bcl-2 was dramatically decreased, and the level of the proapoptotic protein Bax was increased in SCC-25 cells. The expression of the active form of caspase 3 and PARP were increased.
Bcl-2 decreased dramatically, whereas the level of the proapoptotic protein Bax was increased. Both of the expressions of the active form of caspase 3 (cleaved caspase 3) and the poly ADP-ribose polymerase (cleaved PARP) were increased. 3.4. Qu effectively inhibited the migration and invasion of SCC-25 cells A transwell Boyden chamber migration assay showed that Qu treatment significantly decreased the migration ability of SCC-25 cells in a dose-dependent manner (Fig. 4A). Quantitative and statistical analyses also demonstrated a significant decrease in migration (Fig. 4B). An invasion assay demonstrated that Qu treatment also significantly reduced this invasive ability in a dose-dependent manner (Fig. 4C); this was confirmed by quantitative and statistical analyses (Fig. 4D). 4. Discussion Growing evidence from epidemiological studies and experimental data from animal models indicate that lifestyle modifications, particularly a vegetable- and fruit-rich diet, may substantially reduce the incidence of certain types of cancers.9 Qu, as described earlier, is one of the most abundant flavonoids in vegetables and fruits. This pure compound is a powerful antioxidant and free-radical scavenger. Several studies in vitro and in vivo have further revealed that Qu can inhibit malignant growth and metastasis in a variety of cancer
cells by exhibiting a strong ability to induce apoptosis, potentially making it an additional option for cancer management.5,10 To the best of our knowledge, little is known about the cellular and molecular changes mediating the anticancer properties of Qu or the mechanisms of its effects on the proliferation, apoptosis, invasion, and migration of oral cancer cells. In this study, we used representative SCC-25 cells to clearly demonstrate the effective dose-dependent inhibition of growth and invasion following Qu treatment. The findings represent the first evidence of the cellular and molecular mechanisms of Qu inhibition of SCC-25 cells growth and invasion. As described above, our study showed that Qu treatment of SCC25 cells exerted a marked and dose-dependent inhibitory effect on cell growth. The viability test showed a similar responsiveness of SCC-25 cells to Qu treatment. An anchorageindependent colony growth assay further validated the clear dose-dependent inhibitory effect of Qu on SCC-25 cells. These findings indicate that Qu may have cytotoxic effects on SCC25 cells, similar to those reported in a previous study of oral cancer.11,12 Many oncoproteins and tumor suppressors, such as cyclin A, B, D1, and E, Bcl-2, Bax, p21Cip and p27Kip1, are associated with the cancer cell cycle and tumorigenesis.13 Immunoblotting analysis of the cell cycle proteins in SCC25 cells showed dose-dependent increases of p21Cip1 following Qu treatment, which might facilitate the arrest of SCC-25 in late G1 phase. Using appropriate doses of 50 mM Qu, the increased proportion of G1 phase cells gradually declined
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0 0 25 50 75 Quercetin concentration (μM) Fig. 4. Inhibitory effects of migration and invasion of quercetin (Qu) on SCC-25 cells. (A) Boyden chamber migration assay showed that Qu significantly decreased the migration ability of SCC-25 cells in a dose-dependent manner. (B) Quantitative and statistical analyses of results shown in panel A. (C) Boyden chamber migration assay demonstrated that Qu significantly decreased the invasion ability of SCC-25 cells in a dose-dependent manner. (D) Quantitative and statistical analyses of the results shown in panel C.
because of the accumulation of sub-G1 phase cells resulting from the early stages of the induction of apoptosis. Induction of cell cycle arrest and apoptosis is a wellestablished method for treating some forms of cancer.14 Based on our data, we propose that cell cycle arrest without repair accompanied by cellular apoptosis may be the main cellular and/or molecular mechanism(s) occurring in SCC-25 cells following Qu treatment. The mitochondrial apoptosis pathway is mediated by the Bcl-2 family and Bax proteins and the expression of Bcl-2. The ratio of Bcl-2 to Bax proteins contributes to the susceptibility of cells to induction of apoptotic cell death. Our immunoblotting analysis revealed that the level of Bcl-2 decreased dramatically in Qu-treated SCC-25 cells, whereas the level of the proapoptotic protein Bax was increased in SCC-25 cells. PARP activity was altered by the upstream apoptosis, the active form of caspase 3, which then induced the downstream apoptosis. Thus, the response of the SCC-25 cells to induction of apoptosis by Qu treatment involved a sustained decrease in expression of the antiapoptotic oncoprotein Bcl-2 along with an elevated level of proapoptotic protein Bax, resulting in an increased Bax/Bcl-2 ratio. These results highlight the importance of the Bax/Bcl-2 ratio in the life and death of oral cancer cells.13,15 Together, these results demonstrate that the cellular and molecular mechanisms of Qu effects on SCC-25 oral cancer cells mainly
depend on cell cycle arrest accompanied by mitochondriamediated apoptosis. Research has revealed that Qu and polyphenol can inhibit the activity of matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9.16 These two key regulators associated with invasion and metastasis can be activated by E2F transcription factor, which plays a pivotal role in cell proliferation by regulating the expression of genes involved in G1eS transition and DNA synthesis.17 Our study also demonstrated that Qu significantly and dose-dependently decreased the invasion and migration abilities of SCC-25 cells. In conclusion, our study demonstrates that Qu treatment inhibits cell growth and invasion/migration on SCC-25 cells in vitro. The cellular and molecular mechanisms of the biopharmacological effects of Qu were mainly via cell cycle arrest accompanied by mitochondria-mediated apoptosis. Our findings clearly suggest that Qu may have potential as a new chemopreventive or therapeutic agent for OSCC. Acknowledgments This study was partly supported by the Tri-Service General Hospital, Grant No. TSGH-C100-007-009-10-S02, TSGHC100161, TSGH-C100-155, ND-I-29 and the Department of Dental Hygiene, China Medical University, Grant No.
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