Synergistic effects of tea polyphenols and ascorbic acid on human lung adenocarcinoma SPC-A-1 cells

Synergistic effects of tea polyphenols and ascorbic acid on human lung adenocarcinoma SPC-A-1 cells 1 2 first-author 3 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:youytu@zju.edu.cn †E-mail:youytu@zju.edu.cn youytu@zju.edu.cn Department of Tea Science, Zhejiang University, Hangzhou 310029, China Department of Tea Science, Zhejiang University, Hangzhou 310029, China Department of Tea Culture, Zhejiang Shuren University, Hangzhou 310015, China Department of Tea Culture, Zhejiang Shuren University, Hangzhou 310015, China Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China Institute of Biotechnology, Zhejiang University, Hangzhou 310029, China Tea polyphenols have been shown to have ansticancer activity in many studies. In the present study, we investigated effects of theaflavin-3-3′-digallate (TF 3 ), one of the major theaflavin monomers in black tea, in combination with ascorbic acid (AA), a reducing agent, and (−)-epigallocatechin-3-gallate (EGCG), the main polyphenol presented in green tea, in combination with AA on cellular viability and cell cycles of the human lung adenocarcinoma SPC-A-1 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay showed that the 50% inhibition concentrations (IC 50 ) of TF 3 , EGCG, and AA on SPC-A-1 cells were 4.78, 4.90, and 30.62 μmol/L, respectively. The inhibitory rates of TF 3 combined with AA (TF 3 +AA) and EGCG combined with AA (EGCG+AA) at a molar ratio of 1:6 on SPC-A-1 cells were 54.4% and 45.5%, respectively. Flow cytometry analysis showed that TF 3 +AA and EGCG+AA obviously increased the cell population in the G 0 /G 1 phase of the SPC-A-1 cell cycle from 53.9% to 62.8% and 60.0%, respectively. TF 3 -treated cells exhibited 65.3% of the G 0 /G 1 phase at the concentration of its IC 50 . Therefore, TF 3 +AA and EGCG+AA had synergistic inhibition effects on the proliferation of SPC-A-1 cells, and significantly held SPC-A-1 cells in G 0 /G 1 phase. The results suggest that the combination of TF 3 with AA or EGCG with AA enhances their anticancer activity. Theaflavin-3-3′-digallate (TF 3 ) (−)-epigallocatechin-3-gallate (EGCG) Ascorbic acid (AA) Synergism SPC-A-1 cells Cell cycle Introduction 1 Epidemiologic studies have suggested that the consumption of tea is linked to a decreased incidence of various cancers (Yang C.S. et al., 2007 2007 ). The inhibitory action of tea and tea products against carcinogenesis has been demonstrated in animal models, including skin, lung, esophagus, stomach, liver, small intestine, pancreas, prostate, and mammary gland cancers (Conney et al., 1999 1999 ; Tu et al., 2004 2004 ). Both black tea and green tea have monomeric gallated catechins of the flavanol class. The main polyphenol presented in green tea is (−)-epigallo-catechin-3-gallate (EGCG) (Fig. 1a 1a ), which accounts for about 60%–70% of total catechins (Agarwal and Mukhtar, 1996 1996 ). EGCG is a precursor of theaflavin-3-3′-digallate (TF 3 ) (Fig. 1b 1b ). Previous studies showed that TF 3 appears to be the most potent compound in tea on inhibition of proliferation and induction of apoptosis of cancer cells (Yang et al., 1998 1998 ). Black tea, accounting for 78% of the world tea consumption, is the product of tea catechin oxidation and polymerization via fermentation of fresh tea leaves (Yang et al., 2009 2009 ). The fermentation results in the formation of theaflavins. The major theaflavin monomers in black tea are theaflavin (TF 1 ), theaflavin-3-gallate (TF 2 A), theaflavin-3′-gallate (TF 2 B), and theaflavin-3-3′-digallate (TF 3 ). TF 3 has two galloyl groups, TF 2 A and TF 2 B each have one, and TF 1 has none. More specific investigations concerning with theaflavin structure reported that the antioxidative activities of theaflavin monomers depend on the number and position of hydroxyl groups within their molecules (Yang Z.Y. et al., 2007 2007 ), e.g., the antioxidation activities following the sequence of TF 3 > TF 2 > TF 1 (Leung et al., 2001 2001 ). (a) EGCG; (b) Theaflavins. TF 1 (R 1 =R 2 =OH), TF 2 A (R 1 =gallate, R 2 =OH), TF 2 B (R 1 =OH, R 2 =gallate), TF 3 (R 1 =R 2 =gallate) The antioxidative activities of black tea theaflavins are also studied for their influence on activation of transcription factors such as nuclear factor kappa B (NFκB). Theaflavins, in particular TF 3 , could effectively inhibit the activation of NFκB, preventing the expression of inducible nitric oxide synthase (iNOS) gene or down-regulating the expression of iNOS mRNA, which led to a decrease of nitric oxide synthesis in RAW 264.7 cells (Hong et al., 2001 2001 ). Ascorbic acid (AA) is an essential nutrient involved in many biochemical reactions and is known to protect some flavonoids against oxidative degradation during processing and storage of juices (Kaack and Austed, 1998 1998 ). The biochemical roles of AA are related to its ability to act as an electron donor or reducing agent. As a free radical scavenger, AA can protect cellular biopolymers against the initiation and progression of carcinogenesis (Kim et al., 2006 2006 ). Lung cancer is one of the most common cancers worldwide. It has been suggested that there is an inverse association between tea intake and subsequent lung cancer incident (Yang et al., 2009 2009 ). However, the synergic effects of TF 3 or EGCG with AA on human epithelial lung adenocarcinoma SPC-A-1 cells have not been examined. In this study, we investigated in vitro the combined effects of TF 3 with AA (TF 3 +AA) and EGCG with AA (EGCG+AA) on SPC-A-1 cells. Materials and methods 1 Cell culture 2 Human lung adenocarcinoma SPC-A-1 cells were purchased from the Cell Bank of Chinese Academy of Science (Shanghai, China) and cultured in RPMI-1640 medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 10% (w/v) fetal calf serum (GIBCO BRL) in a humidified atmosphere of 37 °C with 5% CO 2 . Chemicals 2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and AA were purchased from Sigma-Aldrich (St. Louis, MO, USA). EGCG was a generous gift from the Tea Science Department of Zhejiang University, with a purity of above 98% (analyzed by high performance liquid chromatography (HPLC)). Tea polyphenols were provided by Siming Natural Plant Co. (Zhejiang, China). All other chemicals were of analytical grade and were commercially obtained from local chemical suppliers. Preparation and analysis of theaflavin monomers 2 The theaflavins (80% purity) were prepared by the enzymatic oxidation of tea polyphenols using immobilized polyphenol oxidase (PPO) according to the protocol of Tu and Xia ( 2004 2004 ). TF 2 A, TF 2 B, TF 1 , and TF 3 were separated using a Sephadex LH-20 gel (Pharmacia Fine Chemicals Inc., NJ, USA) column (Tu et al., 2004 2004 ). Theaflavin monomer was determined using our previously established method (Yang et al., 2008 2008 ). Briefly, theaflavin monomers (0.4 mg) and EGCG (2.0 mg) were dissolved in 1.0 ml methanol, respectively. The concentrations of theaflavin monomer and EGCG were determined using a Shimadzu LC-2010A (Shimadzu, Kyoto, Japan). Inhibition of tea polyphenols on SPC-A-1 cells 2 Cell viability was measured by quantitative colorimetric assay with MTT, which shows the mitochondrial activity of living cells as described by Mosmann ( 1983 1983 ) and Denizot and Lang ( 1986 1986 ). SPC-A-1 cells were seeded into 96-well plates 0.1 ml/well at a density of 1×10 5 cells/ml. After the cells attached, EGCG, TF 2 B, TF 3 , TF 2 A, or AA at different doses was added. After an exposure period, 10 μl MTT (5 mg/ml) was added to each well. The final concentrations for each of AA and TF 2 A added to SPC-A-1 were 5, 10, 50, 100, and 200 μmol/L, and the final concentrations for each of TF 3 , EGCG, and the compound of TF 2 B and TF 3 were 0.5, 1, 5, 10, and 20 μmol/L. Six replicates were set for each treatment. Analysis of synergistic effects 2 Using the median-effect method developed by Chou and Talalay ( 1984 1984 ), the dose-response curve was plotted for each drug, and the multiple dose of a fixed-ratio combination was plotted by f a / f u =( D / D m ) m , where D is the dose administered, D m is the dose required for 50% inhibition of growth (IC 50 ), f a is the fraction affected by dose D , f u is the unaffected fraction, and m is a coefficient signifying the sigmoidicity of the dose-response curve. The dose-response curve was plotted using a logarithmic conversion of the equation to determine the m and D m values. D x required for x percent effect ( f a ) x was then calculated by D x = D m [( f a ) x /( f u ) x ] 1/ m . Thus, the combination index (CI) can be defined by the isobologram CI=( D ) 1 /( D x ) 1 +( D ) 2 /( D x ) 2 + α ( D ) 1 ( D ) 2 /[( D x ) 1 ( D x ) 2 ], where ( D x ) 1 is the dose of drug-1 for producing x percent effect and ( D ) 1 for producing the same x percent effect in combination with drug-2; ( D x ) 2 and ( D ) 2 correspond to drug-2. Consequently, CI < 1 indicates synergism, CI > 1 shows antagonism, and CI=1 points to additive effects. The CI values obtained from both the mutually non-excusive ( α =1) and mutually exclusive ( α =0) isobologram equations are presented in this work. Detection of cell cycle by flow cytometry 2 Cell cycle analysis for the cell distribution in the sub-G 1 region was determined by flow cytometric analysis after propidium iodide (PI) staining. Cells were treated with: AA (30.62 μmol/L), TF 3 (4.78 μmol/L), EGCG (4.90 μmol/L), TF 3 (4.78 μmol/L)+AA (30.62 μmol/L), and EGCG (4.90 μmol/L)+AA (30.62 μmol/L) according to their IC 50 for 48 h. The cells in suspension (5×10 4 cells/ml) with or without drugs were fixed with 80% ethanol at 4 °C for 24 h and then stained with 25 μg/ml PI. After staining, the population of cells in each cell cycle phase was determined using the FAC Star flow cytometry (Becton-Dickinson, San Jose, California, USA). Statistical analysis 2 All experiments were performed in triplicate. One-way analysis of variance (ANOVA) was used to estimate overall significance, followed by the post-hoc Tukey’s tests corrected for multiple comparisons. Data are presented as mean±standard deviation (SD). A probability level less than 5% ( P < 0.05) was considered significant. Results 1 Effects of theaflavin monomers, AA, and EGCG on the proliferation of SPC-A-1 cells 2 Firstly, the effects of tea polyphenols (TF 2 A, TF 2 B+TF 3 , TF 3 , and EGCG) and AA on cell viability of SPC-A-1 were measured. The highest inhibition rate of TF 2 A on the proliferation of SPC-A-1 cells was 40.39% at 100 μmol/L, so we were not able to calculate the IC 50 of TF 2 A. Thus, the TF 2 A was not considered in the following study. Cells were continuously exposed to various drug treatments for 48 h and then tested for the cell viability using MTT assay. The lower the IC 50 value, the higher the inhibition on the SPC-A-1 cells. TF 2 B+TF 3 , TF 3 , and EGCG showed the most potent inhibition on the SPC-A-1 cells, with the IC 50 values of 6.70, 4.78, and 4.90 μmol/L, respectively. The IC 50 value of AA was 30.6 μmol/L, which was significantly higher than that of tea polyphenols alone. Cytotoxicities of EGCG+AA and TF 3 +AA on SPC-A-1 cells 2 The interactions of EGCG with AA and TF 3 with AA on SPC-A-1 were analyzed by the method of Chou and Talalay ( 1984 1984 ). The molar ratios of TF 3 to AA and EGCG to AA were both set at 1:6 based on their IC 50 values. CI plots were constructed by computer analysis using Eq. ( 3 3 ); and both the mutually non-exclusive ( α =1) and mutually exclusive ( α =0) isobologram equations were used. Biphasic effects of a drug combination were shown for TF 3 :AA and EGCG:AA at the molar ratio of 1:6 against SPC-A-1 cells, with a turning point appearing at 54.4% and 45.5% of f a values (Fig. 2 2 ). The combinations of AA with EGCG and AA with TF 3 both enhanced their inhibition on SPC-A-1 cells at a higher concentration, which is indicated by CI < 1. SPC-A-1 cells were treated with tea polyphenol monomers and AA at a fixed molar ratio: (a) TF 3 :AA =1:6 and (b) EGCG:AA=1:6. Using the mutually exclusive or mutually non-exclusive isobologram equation, the affected fraction ( f a )-CI plots for SPC-A-1 cells were constructed by computer analysis of the data generated from the median effect analysis. CI < 1 occurred over a wide range of inhibition levels, indicating synergism Effects of polyphenols and AA on the growth inhibition of SPC-A-1 cells and G 1 -phase arrest 2 Cell cycle arrest is a characteristic of many anticancer drugs. Cells in G 0 /G 1 phase arrest might go into apoptosis, or recover from the G 0 /G 1 phase and enter into S phase. The effects of EGCG and TF 3 on distribution of cells in the cell cycle were analyzed by the flow cytometry. The results show that while all treatments led to an increase of cell population in G 0 /G 1 phase, TF 3 promoted the biggest increase (Fig. 3c 3c ). TF 3 +AA and EGCG+AA obviously increased the cell population in G 0 /G 1 phase of the SPC-A-1 cell cycle from 53.9% to 62.8% and 60.0%, respectively (Figs. 3a, 3e and 3f 3a, 3e and 3f ). TF 3 +AA reduced the cell population in S phase from 46.1% to 23.8% (Figs. 3a and 3e 3a and 3e ), and increased apoptosis from 1.0% to 22.3%. Interestingly, TF 3 -treated cells exhibited 65.3% of G 0 /G 1 phase at the IC 50 . After cells were fixed and stained with propidium iodide, and the DNA content was measured by flow cytometry. Cell cycle distribution was analyzed using the FAC Star flow cytometry. (a) Control; (b) Ascorbic acid (30.62 μmol/L); (c) TF 3 (4.78 μmol/L); (d) EGCG (4.90 μmol/L); (e) TF 3 (4.78 μmol/L)+AA (30.62 μmol/L); (f) EGCG (4.90 μmol/L)+AA (30.62 μmol/L) Discussion 1 EGCG displayed strong growth inhibitory effects against lung cancer lines H661 and H1299, with an estimated IC 50 value of 22 μmol/L (Yang et al., 1998 1998 ), which is much higher than that of TF 3 or EGCG in lung cancer lines SPC-A-1 in the present study. Moon et al. ( 2007 2007 ) demonstrated that EGCG was a strong inducer of early growth response gene-1 expression and mediated early growth response gene-1 nuclear translocation via extracellular signal-regulated protein kinase signaling pathway in A549 cells. TF 3 had stronger inhibition on I kappa B (IκB) kinase (IKK) activity in activated macrophages than other polyphenols. TF 3 strongly inhibited both IKK 1 and IKK 2 activities and prevented the degradation of IκBα and IκBβ in activated macrophage cells (Pan et al., 2000 2000 ). This suggests that the inhibition of IKK activity by TF 3 could occur by a direct effect on IKKs or on upstream events in the signal transduction pathway. Growth inhibitory activities of EGCG have been demonstrated by Yang et al. ( 2009 2009 ), which is also confirmed in this study. Whereas green tea contains EGCG, black tea contains theaflavins. In the present study, we showed that TF 3 was as effective as EGCG. We extended our studies to the synergistic inhibition of TF 3 in combination with AA and EGCG with AA on human lung cancer SPC-A-1 cells, and found that AA enhanced the inhibitory abilities of TF 3 and EGCG on lung cancer cells. When EGCG or TF 3 was combined with AA at a lower concentration, the combination significantly enhanced the antagonism (CI > 1). Polyphenols are generally unstable under oxidative conditions, so EGCG levels decrease rapidly, even in solutions of neutral pH (Hatano et al., 2003 2003 ). The synergism in this study suggests that AA may protect EGCG from oxidation. The chemical structure of TF 3 is bulkier and has two A-rings linked by a fused seven-member ring (Yang Z.Y. et al., 2007 2007 ). Theaflavin monomers are transformed from tea catechins through oxidation catalyzed by enzyme and chemical condensation, which makes theaflavin monomers more stable than EGCG. Some experiments demonstrated that the theaflavins presented in black tea have at least the same antioxidant potential as catechins presented in green tea, and that the conversion of catechins to theaflavins during black tea production does not alter significantly their free radical-scavenging activity (Leung et al., 2001 2001 ). More studies indicated that the synergistic anticancer effects of AA and green tea extract on several cancer cell lines in tissue culture were higher than those of the individual nutrients (Roomi et al., 2006 2006 ). AA as a well-known antioxidant can protect tea polyphenols from oxidation. We also found that the synergistic effects of TF 3 +AA and EGCG+AA on SPC-A-1 cells were through cell cycle arrest, but the EGCG or TF 3 alone can lead to more apoptosis. In synergistic cultures, the relative number of apoptotic cells did not increase; a maximum of 2.1% of apoptotic cells were observed. TF 3 can increase the cell cycle at the G 0 /G 1 phase. When AA is added, it can produce a large amount of H 2 O 2 by reducing the concentration of peroxidase (Zheng et al., 2002 2002 ). TF 3 shows the most potent hydroxyl radicals-scavenging ability (Yang et al., 2008 2008 ). Using human lymphoma cells, Saeki et al. ( 1999 1999 ) revealed that TF 3 and TF 1 had equivalent apoptosis-inducing activities in vitro. Especially, Tu et al. ( 2004 2004 ) suggested that TF 2 B was a more powerful inhibitor to the growth of human liver cancer cells, gastric cancer cells, and leukemia cells than TF 3 . Apparently, these findings suggest that multiple mechanisms may be responsible for the anticancer effects of theaflavins on different types of cancers. In this study, we found that AA with EGCG or TF 3 acts synergistically to suppress cell proliferation of SPC-A-1 in vitro. Presently, it is not clearly defined how black tea theaflavins exert their cytotoxic effects (Lung et al., 2004 2004 ), cell cycle arrest (Chung et al., 1999 1999 ), and inducing of apoptosis (Lu et al., 2000 2000 ). Studies show that cancer cells are more sensitive than normal cells to the in vitro anti-proliferation effects of EGCG (Mittal et al., 2004 2004 ) and black tea polyphenol extracts (Weisburg et al., 2004 2004 ). After entering the cells, EGCG may produce reactive oxygen species (ROS) by an unknown mechanism (Yang et al., 2009 2009 ). In conclusion, this paper showed that TF 3 , one of the major theaflavin monomers in black tea, in combination with AA, a reducing agent, and EGCG, the main polyphenol presented in green tea, in combination with AA could synergistically inhibited the proliferation of SPC-A-1 cells, and increased its cell population in G 0 /G 1 phase of cell cycle. 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Ascorbic acid induces redifferentiation and growth inhibition in human hepatoma cells by increasing endogenous hydrogen peroxide 10.1631/jzus.B0900355 Project supported by the Key Program of Science and Technology of Zhejiang Province (No. 2007C12068) of China and the National Natural Science Foundation of China (No. 30901002) 2009-11-11 2010-04-28 2010-03-28 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2010 2010 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 11