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Luteolin Induces Apoptosis in Oral Squamous Cancer Cells
S.-F. Yang1,
W.-E. Yang2,
H.-R. Chang3,
S.-C. Chu4 and
Y.-S. Hsieh2,*
1 Institute of Medicine,
2 Institute of Biochemistry and Biotechnology, Chung Shan Medical University, Taichung 402, Taiwan;
3 Division of Nephrology, Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung 402, Taiwan; and
4 Department of Food Science, Central Taiwan University of Science and Technology, Taichung 406, Taiwan
Correspondence: * corresponding author, csmcysh{at}csmu.edu.tw
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ABSTRACT
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Oral squamous cell carcinoma is the most common malignancy of the oral cavity, and treatment approaches are inadequate. Luteolin, a natural flavonoid compound, has been shown to have anti-tumorigenic properties on various types of tumors. Therefore, we hypothesized that luteolin has anti-tumorigenic properties for oral squamous cell carcinoma, and may provide effective chemotherapy. Results revealed that luteolin reduced the viability of SCC-4 cells and induced apoptosis by decreasing the expression of cyclin-dependent kinase (CDKs), cyclins, and phosphor- retinoblastoma (p-Rb) anti-apoptotic protein, but increased the expression of pro-apoptotic proteins and activated caspase 9 and 3, with a concomitant increase in the levels of cleaved poly-ADP-ribose polymerase (PARP). Combination treatment of luteolin with paclitaxel enhanced the cytotoxic effect of paclitaxel in SCC-4 cells, and continuous administration of luteolin suppressed the growth of xenograft tumors in nude mice. These results suggest that luteolin could be an effective chemotherapeutic agent for the treatment of oral squamous cell carcinoma.
Key Words: oral squamous cell carcinoma • SCC-4 • luteolin • apoptosis
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INTRODUCTION
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Oral squamous cell carcinoma is the most common malignancy of the oral cavity, and causes more deaths than any other oral diseases. Despite significant medical advances over the past 30 years, the five-year survival rate of this disease has remained approximately 50% (Jordan and Daley, 1997; Hsu et al., 2004). Clinically, surgery and radiotherapy are the primary treatments for persons in the early stages of the disease, while for those in late stages, chemotherapy is often used in combination with surgery or radiotherapy (Forastiere et al., 2003; Psyrri et al., 2004).
Previous studies have demonstrated a protective role for vegetables and fruits in treatments for oral cancer, due to their high polyphenol content, particularly of flavonoids (Block et al., 1992; Levi et al., 1998; Sakagami et al., 1999). Flavonoids are complex compounds that provide color and flavor in plants and possess a wide spectrum of pharmacological properties (Havsteen, 1983). Luteolin (3',4',5,7-tetrahydroxyflavone) is a common dietary flavonoid that can be found in large quantities in thyme as well as in many other plants or foods, including beets, cabbage, cauliflower, red wine, olive oil, and tea (Herrmann, 1976; Harnly et al., 2006). The average human daily intake of luteolin is approximately 16 mg per day (Hertog et al., 1993). In cellular studies, luteolin has been shown to have anti-tumorigenic (Yasukawa et al., 1989), anti-inflammatory/anti-allergic (Yamamoto et al., 1998), anti-oxidant, and radical scavenger properties, which reportedly inhibit the development of a series of solid tumor, ascites, and leukemic cell lines (Shimoi et al., 1994; Pettit et al., 1996). A major barrier to the clinical application of luteolin is its extremely low systemic bioavailability, due to extensive metabolism by the liver and intestine (Walle, 2004), making it less likely to be beneficial in combating tumors in organs such as the liver, lung, breast, prostate, and ovaries. However, epithelium of the oral cavity can absorb luteolin directly, and should benefit from high levels of exposure to luteolin or other flavonoids from the diet or health foods (Browning et al., 2005; Walle et al., 2005). Therefore, we used the oral squamous carcinoma SCC-4 cell line as a model to examine the chemotherapeutic effects of luteolin.
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MATERIALS & METHODS
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Cell Culture
SCC-4, a human tongue squamous cell carcinoma cell line obtained from ATCC (Manassas, VA, USA), was cultured in Dulbecco’s modified Eagle’s medium supplemented with a nutrient mixture, F-12 Ham’s medium (Life Technologies, Grand Island, NY, USA), 10% fetal bovine serum (Hyclone Laboratories, Logan, UT, USA), 2 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 400 ng/mL hydrocortisone (Chen et al., 2006). Oral cancer-2 (OC2) cells and gingival fibroblasts (GF) were grown at 37°C, 5% CO2, in RPMI medium containing 10% fetal bovine serum and antibiotics (100 U/mL penicillin, 100 U/mL streptomycin, and 0.25 µg/mL amphotericin B).
Cell Growth and Cell Viability Assays
Cells were plated in 24-well plates and then exposed to luteolin and 7-hydroxyflavanone (Sigma Chemical Co., St. Louis, MO, USA) at various concentrations (0, 20, 40, 60, 80, and 100 µM) for 24, 48, and 72 hrs. Cells were then harvested and counted in duplicate by means of a hemocytometer, and trypan blue exclusion was used to determine viable and dead cells. For the MTT assay, cells were plated in 24-well plates and treated with the indicated concentration of luteolin for 24, 48, and 72 hrs. To examine the adjuvant effect of luteolin in conjunction with an anticancer drug, we treated cells with either paclitaxel (0.3 nM) alone or in combination with luteolin (5 or 10 µM) for 5 and 7 days. At the end of treatment, we added 0.5 mg/mL MTT (Sigma Chemical Co., St. Louis, MO, USA) in fresh medium for an additional four-hour incubation period. We measured the blue formazan crystals, dissolved in isopropanol, by reading the absorbance at a wavelength of 563 nm (Beckman Spectrophotometer DU 640, Beckman Instruments, Fullerton, CA, USA) (Hsiao et al., 2007).
4'-6-Diamidino-2-phenylindole (DAPI) Staining
We quantitated apoptosis by assessing nuclear changes using the nuclear binding dye DAPI (Sigma Chemical Co., St. Louis, MO, USA). After treatment, cells were fixed and permeabilized with 2% (w/v) formaldehyde and 0.4% (w/v) Triton X-100, respectively. Staining was performed with 1 µg/mL DAPI in PBS for 3 min, and chromatin fluorescence was observed by UV light microscopy (Dermitzaki et al., 2002).
Immunoblotting
Lysates of luteolin-treated cells were separated in a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane as previously described (Hsieh et al., 2007). The blot was subsequently treated by standard procedures and probed with cyclins, cyclin-dependent kinases (CDKs), phospo-retinoblastoma (Rb; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), caspases, poly-ADP-ribose polymerase (PARP), and Bcl-2 family antibodies (Cell Signaling Technology Inc., Beverly, MA, USA), and with appropriate peroxidase-conjugated secondary antibodies. The protein expression was detected by chemiluminescence with an ECL Plus detection kit. (Amersham Life Sciences, Inc., Piscataway, NJ, USA).
DNA Fragmentation
Cells were exposed to the indicated concentrations of luteolin for 72 hrs. Adherent cells were incubated in 100 µL of lysis buffer at room temperature for 30 min, followed by 1.5 mg/mL proteinase K and 2.5 mg/mL RNase A at 56°C for 4 hrs. Before cell lysates were loaded on electrophoresis gels, DNA was further extracted by phenol/chloroform/isoamyl alcohol (25:24:1, v/v). After centrifugation (12,000 rpm/5 min), the supernatants were collected and subjected to 2% agarose gel electrophoresis, and then stained with ethidium bromide (EtBr) and photographed under UV light (Shen et al., 2004).
Measurement of Tumor Growth in vivo
For the nude mouse xenograft model, we used immunodeficient nude mice (BALB/c nu/nu male mice) (5–6 wks old; weight, 18–22 g). Mice were housed with a regular 12-hour light/12-hour dark cycle, with ad libitum access to standard rodent chow (Laboratory Rodent Diet 5001, LabDiet, St. Louis, MO, USA), and were kept in a pathogen-free environment at the Laboratory Animal Unit. All experimental protocols conducted in the present study were approved by the Committee of Animal Research at Chung Shan Medical University (IACUC Approval No. 429). For xenograft implantation, we injected SCC-4 cells (3 x 106 cells/0.1 mL/mouse) subcutaneously into the right front axilla, and then 5 mice in each group received luteolin (3 and 5 mg/kg body weight), paclitaxel (1 mg/kg body weight), or vehicle (DMSO) by intraperitoneal injection every 2 days for 44 days. The day of cell implantation was designated Day 0. Using vernier calipers, we measured tumors daily to assess long and short dimensions of the tumors. The tumor volumes were calculated according to the following formula: tumor volume = 0.5 x long diameter x short diameter x short diameter (Sigounas et al., 2004). At the end of the experiment, tumor-bearing mice were killed by cervical dislocation, and the tumors were weighed after being separated from the surrounding muscles and dermis (Zi et al., 2005).
Statistical Analysis
Values were expressed as means ± SD. A significant difference from the respective controls was analyzed by Student’s t test (SigmaStat 2.0, Jandel Scientific, San Rafael, CA, USA) for each paired experiment. A P value of < 0.05 or < 0.01 was regarded as a significant difference.
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RESULTS
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Potential Effects of Luteolin on the Viability of SCC-4 Cells
The chemical structure of luteolin has been defined (Fig. 1A ). To evaluate the effect of luteolin on oral squamous cancer cells, we determined the viability and death of SCC-4 cells, with or without luteolin treatment, using an MTT assay and the trypan blue exclusion assay, respectively. After treatment for 24, 48, or 72 hrs with luteolin (0–100 µM), a significantly decreased viability of SCC-4 cells was observed, both dose-and time-dependently (Fig. 1B). Furthermore, luteolin treatment caused a significant increase in the population of dead cells, at higher concentrations (60–100 µM) and at longer treatment times of 48 and 72 hrs, respectively (Fig. 1C). In addition, we also studied the effects of luteolin and 7-hydroxyflavanone on the viability of normal human gingival fibroblasts (GF), OC2 cells, and SCC-4 cells, to exclude the possibility that the addition of luteolin adversely affected the culture medium’s ability to support cell survival. After a 24-hour treatment, luteolin decreased the viability of OC2 cells, while that of GF cells was unchanged. Furthermore, 7-hydroxyflavanone had no effect on the viability of SCC-4 cells (Fig. 1D). Analysis of these data, taken together, suggested that luteolin treatment induced the death of human oral cancer cells.
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Figure 1. The effect of luteolin on viability/number and death of SCC-4 cells. The chemical structure of luteolin (3',4',5,7-tetrahydroxyflavone) (A). Cells were treated with luteolin at a concentration of 0, 20, 40, 60, 80, or 100 µM for 24, 48, and 72 hrs. At the end of treatment, cell viability was determined by MTT assay (B), and dead cells were counted by the trypan blue dye exclusion assay (C), as described in MATERIALS & METHODS. Human gingival fibroblasts and OC2 and SCC-4 cells were treated with luteolin and 7-hydroxyflavanone at a concentration of 0, 20, 40, 60, 80, or 100 µM for 24 hrs. After treatment, cell viability was determined by MTT assay (D). The data are means ± SD of 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001).
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Luteolin-induced G1 Cell-cycle Arrest is Mediated via Inhibition of Cyclin-dependent Kinase (Cdk)2, Cdk4, Cdk6, Cyclin D3, and p-Retinoblastoma (p-RB) Expression
To investigate the underlying mechanisms of luteolin’s anti-proliferative action, we further analyzed the cell-cycle profiles by flow cytometry. The effect of luteolin on cell-cycle progression in SCC-4 cells was determined after treatment with various doses of luteolin (0, 20, 40, 60, 80, and 100 µM) for 24 hrs. Results indicated that luteolin induced a dose-dependent accumulation in the G1 phase (Fig. 2A). Afterward, the effect of luteolin on cell-cycle regulatory molecules involved in the G1 phase of the cell cycle was examined by Western blot analysis, which showed that luteolin treatment caused a dose-dependent decrease of cyclin-dependent kinases (CDKs) 2, 4, and 6, and of cyclin D3 (Fig. 2B). Association of CDK with cyclin forms active kinase complexes, which are inhibited and regulated by binding with cyclin-dependent kinase inhibitor (CDKi). However, luteolin had no effect on the protein level of CDKi, such as Cip1/p21 and Cip2/p27 (Fig. 2C).
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Figure 2. Effects of luteolin on cell-cycle progression and protein levels of cell-cycle regulators. Cells were treated with 0, 20, 40, 60, 80, or 100 µM of luteolin for 24 hrs. The cell-cycle distribution was analyzed by flow cytometry (A). For protein levels, cells were subjected to Western blot for analysis of the expression of CDK2, 4, and 6, cyclin D1, D3, and E (B), Cip1/p21, Cip2/p27, and phospho-Rb (C), together with β-actin as an internal control. The data are means SD of 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001).
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Down-regulation of CDK4 and CDK6 has been shown to be associated with a decrease in the expression of retinoblastoma (pRb) tumor suppressor protein, a key regulator of the G1-to-S-phase transition in the cell cycle. Therefore, the effect of luteolin on protein expression of pRb was subsequently investigated, and results indicated that luteolin treatment caused a significant decrease in the expression of pRb in SCC-4 cells (Fig. 2C ).
Luteolin-induced Apoptosis in SCC-4 Cells
Luteolin increased the number of cells exhibiting chromatin condensation by DAPI nuclear staining (Fig. 3A). Likewise, DNA fragmentation was observed in luteolin-treated SCC-4 cells examined by agarose gel electrophoresis (Fig. 3B). Since Bcl-2 and Bax are vital in the induction of apoptosis and the activation of the caspase cascade leading to poly-ADP-ribose polymerase (PARP) cleavage, regarded as a major pathway in apoptosis induction, the expression levels of Bcl-2 and Bax after a 48-hour luteolin treatment were assayed. Western blot analysis indicated that the level of the pro-apoptotic protein Bax was increased in luteolin-treated cells (Fig 3C), whereas the expression of the anti-apoptotic protein Bcl-2 was decreased. Similarly, the levels of cleaved caspase-9, caspase-3, and PARP were elevated in luteolin-treated cells (Fig. 3C).
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Figure 3. Apoptotic effect of luteolin on SCC-4 cells. After a 48-hour luteolin treatment with 0, 20, 40, 60, 80, and 100 µM, DNA condensation and fragmentation were analyzed by DAPI staining (A) and DNA fragmentation assay (B). Meanwhile, treated cell lysates were subjected to SDS-PAGE, followed by Western blotting with antibodies against Bcl-2 and Bax, cleaved forms of caspase 3, 9, and PARP, with β-actin as an internal control (C). The data are means ± SD of 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001).
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Luteolin Augmented the Cytotoxic Activity of Paclitaxel and Suppressed Tumor Growth in SCC-4 Xenograft Nude Mice
To assess if luteolin affected the anticancer effect of paclitaxel, we treated SCC-4 cells with either paclitaxel (0.3 nM) alone or a combination with an indicated concentration of luteolin (5 or 10 µM) for 4 and 7 days. Result showed that luteolin significantly enhanced the cytotoxic effects of paclitaxel in SCC-4 cells from Day 7 and Day 4 for 5 µM and 10 µM of luteolin, respectively (Fig.4A).
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Figure 4. The improved effect of luteolin with paclitaxel on SCC-4 cells, and its anticancer effects in vitro and in vivo. (A) Cells were cultured and treated with paclitaxel (0.3 nM) alone or in combination with luteolin (5, 10 µM), and cell viability was determined at the end of Days 4 and 7 by MTT assay. (B) After subcutaneous implantation of SCC-4 cells, BALB/c nu/nu mice received vehicle (DMSO as control; n = 5), luteolin (3 or 5 mg/kg body weight; n = 5), paclitaxel (1 mg/kg body weight; n = 5), or combination treatment with luteolin (3 mg/kg body weight) and paclitaxel (1 mg/kg body weight; n = 5), then were analyzed for tumor growth. The data are means ± SD of 3 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001).
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To evaluate the anti-tumor effects of luteolin and paclitaxel, we performed an in vivo anti-tumor study, using a nude mouse xenograft model with subcutaneous inoculation of SCC-4 cells. After solid tumors were established (larger than 100 mm3 at Day 8, following cell inoculation), luteolin or paclitaxel was administered daily. Results on Day 44 showed that the growth of SCC-4 xenograft tumors was significantly affected by luteolin (3 or 5 mg/kg body weight) or paclitaxel (1 mg/kg body weight), showing reductions of 68.8%, 92.65%, and 94.92%, compared with tumors in control animals (Fig. 4B). Furthermore, a combination treatment with luteolin (3 mg/kg body weight) and paclitaxel (1 mg/kg body weight) also showed a synergistic effect (Fig. 4B).
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DISCUSSION
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Oral squamous cell carcinoma is among the most malignant neoplasms occurring in the oral cavity. Treatment for oral cancer has relied on surgical resection, radiation, and chemotherapy, or a combination of these methods (Miller et al., 1981). However, chemotherapy is relatively ineffective and has numerous side-effects, making it crucial to identify chemopreventive tools for traditional therapy that can enhance the chances of survival for oral cancer patients (Hsu et al., 2004). In this study, we demonstrated that luteolin significantly induces cell-cycle arrest and apoptotic cell death, reducing the viability and inducing the death of SCC-4 cells. These results provide evidence of the anti-neoplastic effect of luteolin in oral cancer. To our knowledge, this is the first report on the tumor-suppressive function of luteolin in oral cancer cells via inducing apoptosis and cell-cycle arrest.
The anti-tumor properties and anti-proliferative activity of luteolin have been demonstrated in various types of human cancer cell lines (Post and Varma, 1992; Fotsis et al., 1997; Huang et al., 1999; Shi et al., 2005). However, the anticancer activities of luteolin in an oral cancer cell line had not previously been well-studied. In our studies, a SCC-4 cell line was used as a model to provide in vitro evidence, and we used nude mouse xenograft as the in vivo model, to show that luteolin can induce G1 phase arrest of cell-cycle progression and apoptotic cell death, thus demonstrating the effect of luteolin in reducing cell viability and inducing cell death. In recent years, the cell-cycle components and apoptosis have been emphasized as targets for anti-cancer intervention. Inhibition of proliferation, growth arrest at one or more checkpoints of the cell cycle, and modulation of signal transduction pathways have been related to altered expressions of key enzymes (Graña et al., 1995; Singh et al., 2002). We have demonstrated that luteolin decreased the expression of CDK, cyclins, pRb, and the activated caspase cascade. Our study in a nude mouse xenograft model also provided evidence that luteolin exerts an in vivo inhibitory effect on tumor growth of human oral cancer cells. Despite the five-fold difference in concentrations used, the anti-tumor efficacy of luteolin (5 mg/kg) was found to be equivalent to that of paclitaxel (1 mg/kg) in the nude mouse xenograft model in vivo, indicating that luteolin may have inhibitory potential for oral cancer development in vivo, similar to that of paclitaxel, a well-known anti-cancer agent.
In conclusion, our study indicates that luteolin inhibits cellular growth, and induces G1 arrest and apoptotic cell death of SCC-4 cells. We also provide evidence that luteolin supplementation can improve the activity of paclitaxel and suppress tumor growth in vivo. Therefore, luteolin may act as a potential candidate for cancer chemoprevention.
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ACKNOWLEDGMENTS
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This study was supported by grants from the National Science Council, Taiwan (NSC 95- 2311-B-040-002 & NSC 94-2313-B-166-004).
Received for publication June 24, 2007.
Revision received November 7, 2007.
Accepted for publication January 18, 2008.
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Journal of Dental Research, Vol. 87, No. 4,
401-406 (2008)
DOI: 10.1177/154405910808700413
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ABSTRACT
INTRODUCTION