Experimental immunology
Synergistic anticancer activity of biologicals from green and black tea on DU 145 human prostate cancer cells

Introduction

Recently, considerable interest has been observed in the potential of botanicals preventing and/or alleviating chronic ailments [1, 2]. Among the most studied botanicals are compounds present in green and black teas. Consumed by over two-thirds of the world’s population, tea produced from the leaves of one plant (Camellia sinensis) is the most popular beverage next to water, and the amount of tea consumed is positively correlated to health benefits. Different methods of processing Camellia sinensis leads to green, oolong, or black tea [3, 4]. Dried leaves of tea produce green tea, while partially crushed leaves oxidised in a moist environment for short time produce oolong tea, while longer oxidation results in black tea (Fig. 1). The therapeutic properties and prophylactic properties of green and black tea have been credited to catechins of green tea and theaflavins of black tea. Oolong tea is processed in such a way that it can contain both catechins and theaflavins. Tea polyphenols are potent antioxidants, and they also modulate several signalling pathways and inhibit proteins such as metalloproteinase 9 (MMP-9) or protein plasminogen activator system [1, 5].
An increasing body of evidence suggests that the major catechin (epigallocatechin-3-gallate – EGCG) found in green tea has the capacity to impact a variety of human malignancies. In addition to acting as a powerful antioxidant, EGCG also acts as an antiangiogenic factor, promotes apoptosis, and induces cell growth arrest via cell cycle regulatory proteins; it also activates killer caspases, and suppresses oncogenic transcription factors [6]. It has been shown that EGCG reduces PC3 prostate cancer growth via MEK-independent ERK1/2 activation [7], inhibits proliferation via alteration of expression of CCND1, CDK4, CDKN1A, E2F1, MAPK6 and PCNA genes [8]. In another androgen-independent prostate cancer cell line (DU 145) EGCG alters genes involved in the functions of transcription, RNA processing, protein folding, phosphorylation, protein degradation, cell motility, and ion transport [9], and induces mRNA expression of interleukin (IL)-6, IL-8, CXCL1, IP-10, CCL5, and transforming growth factor  (TGF-) [10].
Research on the influence of theaflavin (TF) on prostate cancer cells is less abundant than for EGCG. Nevertheless, it has been found that theaflavin modifies protein kinase B (PKB) pathways in DU145 and LNCaP (androgen-responsive prostate carcinoma cells) pathways [11]. In a different study it was found that theaflavins stimulate G2/M arrest by modifying expression of p21waf1/cip1, cdc25C, and cyclin B proteins in PC-3 cells [12]. However, it was reported that TF, similarly to EGCG, can affect the protein expression of mitogen-activated protein kinases (MAPK) or nuclear factor B (NF-B) pathways [13].
Theaflavins and catechins seem to act on cancer cells largely by different pathways, so treatment of both could offer synergistic anticancer effects. Literature on the synergistic effects of EGCG and TF is limited to the effects of these two polyphenols on suppression of adhesion and invasion of hepatoma cancer cells [14], protection against nitric oxide toxicity [15], and effects of tea polyphenols and ascorbic acid on human lung adenocarcinoma [16]. Prostate cancer is one of most common cancers among men. Therefore, non-toxic natural agents such as EGCG or TF, used either alone or in combination with conventional therapeutics to prevent tumour progression and/or treatment, can be very helpful. For that reason we have investigated if EGCG in combination with TF can reduce the rate of prostate cancer growth.

Material and methods

Cell line. DU 145 human prostate carcinoma obtained from stock cultures were maintained in Minimum Essential Media (MEM) with L-glutamine, 10% foetal bovine serum (FBS, Life Technologies, Grand Island, NY) with 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich®, St. Louis, MO).
Treatment solutions. Treatment solutions were prepared by dissolving EGCG (Sigma-Aldrich catalogue # E4143) and theaflavin of black tea (TF, Sigma-Aldrich catalogue # E5550) in DMSO.
Cell treatment. Cells were distributed into 24- or 48-well dishes with a density of 50 cells per well and maintained for ~24 hours. Next, cells were treated with EGCG, TF for four hours. Tested doses were: 2.5, 5, 10, 50, 100, and 200 µg/ml of EGCG or TF in the media. Other groups included wells with media only or wells with 0.1% DMSO added. Also, in some experiments cells were treated with mixture of 20 µg/ml EGCG and 20 µg/ml TF to investigate synergist effects. After treatment media was replaced, cells were maintained for seven days followed by fixing with methanol and 0.4% Giemsa stain (Sigma-Aldrich) and colony counts under microscopic (10× magnification) examination.
Statistical analysis was carried out using the Origin 8 program (OriginLab Corporation, Northampton, MA 01060). Statistical significance was set at a p < 0.05, as calculated using ANOVA test.

Results and discussion

Epigallocatechin-3-gallate and TF inhibit proliferation of many cancer cell lines, as has been reported by numerous papers [17-19]. Our initial experiment showed that cell proliferation was reduced in concentrations similar to those published by other authors (Table 1).
Albrecht et al. tested the potency of EGCG for the inhibition of cell proliferation in PC-3 prostate cancer cells and reported similar concentration values [7, 13, 20-22] to ours. It is of great importance that EGCG had no effect on cell proliferation in the RWPE-1 non-tumorigenic prostate epithelial cell line. The authors suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signalling pathway is accountable for the inhibition of proliferation in PC-3 cells [7]. Work done on DU 145 showed inhibition of proliferation mediated via inhibition of phosphorylation of ERK1/2 and p38 pathways [23]. They also observed inhibition of two metalloproteinases (MMP-2, MMP-9) critical in cancer cell invasion and metastasis. No work was done with TF on DU145 prostate cancer cells. However, as we stated in the introduction, TFs were used to limit proliferation in different prostate cancer cells in similar concentrations as those used in our experiments [13, 17, 22].
One of the criticisms of prophylactic or therapeutic use of green or black tea polyphenols was presumably poor bioavailability of these chemicals. Work done by Henning et al. showed that EGCG and TF can be detected in the small and large intestine, liver, and prostate of C57BL mice after consumption of tea extracts. The prostate bioavailability of TF was 70% higher than that of EGCG. They also performed a study in which 20 men scheduled for prostatectomy were randomly assigned to consume green or black tea for five days before surgery. Tea polyphenols were detected in prostate samples from men consuming both green and black teas. This pioneering human study showed that polyphenols and theaflavins are bioavailable in the human prostate [21]. This fact supports the feasibility of providing EGCG and TF polyphenols found in green and black tea as prophylactic and therapeutic treatments.
Usually the combination of different drugs used in cancer therapy is justified by high toxicity or resistance of cancer cells to one or the other drugs (for example platinum-based drugs). Utilising the combination of EGCG and TF is different. These two polyphenols are not toxic and have diverse and mostly independent effects on cancer cells. Therefore, EGCG and TF would be perfect candidates for this type of treatment. Despite the enormous number of studies done on cancer therapy using EGCG [20, 24-26] or TF [17, 27, 28], surprisingly there are no reports on the synergistic effects of these polyphenols on cancer cells. In our research we chose the concentration of 20 µg/ml for both polyphenols as this concentration had a weak anticancer effect for both compounds individually. As seen in Table 2, combining EGCG and TF produced greater cancer cell death than EGCG or TF separately.

Conclusions

Combining lower concentrations of EGCG and TF results in greater cell death compared to either extract alone at the same concentration. Also, increased cell death rates were statistically significant compared to controls.
The authors declare no conflict of interest.
This study was supported in part by grants from the Frank Stranahan Endowed Chair and Children Miracle Network.

References

1. Darvesh AS, Bishayee A (2013): Chemopreventive and therapeutic potential of tea polyphenols in hepatocellular cancer. Nutr Cancer 65: 329-344.
2. Ferguson LR, Philpott M (2007): Cancer prevention by dietary bioactive components that target the immune response. Curr Cancer Drug Targets 7: 459-464.
3. Gupta S, Saha B, Giri AK (2002): Comparative antimutagenic and anticlastogenic effects of green tea and black tea: a review. Mutat Res 512: 37-65.
4. Luczaj W, Skrzydlewska E (2005): Antioxidative properties of black tea. Prev Med 40: 910-918.
5. Skrzypczak-Jankun E, Jankun J (2010): Theaflavin digallate inactivates plasminogen activator inhibitor: could tea help in Alzheimer’s disease and obesity? Int J Mol Med 26: 45-50.
6. Singh BN, Shankar S, Srivastava RK (2011): Green tea catechin, epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and clinical applications. Biochem Pharmacol 82: 1807-1821.
7. Albrecht DS, Clubbs EA, Ferruzzi M, et al. (2008): Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation. Chem Biol Interact 171: 89-95.
8. Jiang J, Eliaz I, Sliva D (2011): Suppression of growth and invasive behavior of human prostate cancer cells by ProstaCaid: mechanism of activity. Int J Oncol 38: 1675-1682.
9. Luo KL, Luo JH, Yu YP (2010): (-)-Epigallocatechin-3-gallate induces Du145 prostate cancer cell death via downregulation of inhibitor of DNA binding 2, a dominant negative helix-loop-helix protein. Cancer Sci 101: 707-712.
10. Mukherjee S, Siddiqui MA, Dayal S, et al. (2014): Epigallocatechin-3-gallate suppresses proinflammatory cytokines and chemokines induced by Toll-like receptor 9 agonists in prostate cancer cells. J Inflamm Res 7: 89-101.
11. Siddiqui IA, Adhami VM, Afaq F, et al. (2004): Modulation of phosphatidylinositol-3-kinase/protein kinase B- and mitogen-activated protein kinase-pathways by tea polyphenols in human prostate cancer cells. J Cell Biochem 91: 232-242.
12. Prasad S, Kaur J, Roy P, et al. (2007): Theaflavins induce G2/M arrest by modulating expression of p21waf1/cip1, cdc25C and cyclin B in human prostate carcinoma PC-3 cells. Life Sci 81: 1323-1331.
13. Kalra N, Seth K, Prasad S, et al. (2007): Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways. Life Sci 80: 2137-2146.
14. Zhang G, Miura Y, Yagasaki K (2000): Suppression of adhesion and invasion of hepatoma cells in culture by tea compounds through antioxidative activity. Cancer Lett 159: 169-173.
15. Paquay JB, Haenen GR, Stender G, et al. (2000): Protection against nitric oxide toxicity by tea. J Agric Food Chem 48: 5768-5772.
16. Li W, Wu JX, Tu YY (2010): Synergistic effects of tea polyphenols and ascorbic acid on human lung adenocarcinoma SPC-A-1 cells. J Zhejiang Univ Sci B 11: 458-464.
17. Bode AM, Dong Z (2006): Molecular and cellular targets. Mol Carcinog 45: 422-430.
18. Sang S, Lambert JD, Ho CT, et al. (2011): The chemistry and biotransformation of tea constituents. Pharmacol Res 64: 87-99.
19. Weisburger JH (1999): Mechanisms of action of antioxidants as exemplified in vegetables, tomatoes and tea. Food Chem Toxicol 37: 943-948.
20. Butt MS, Ahmad RS, Sultan MT, et al. (2015): Green tea and anticancer perspectives: Updates from last decade. Crit Rev Food Sci Nutr 55: 792-805
21. Henning SM, Aronson W, Niu Y, et al. (2006): Tea polyphenols and theaflavins are present in prostate tissue of humans and mice after green and black tea consumption. J Nutr 136: 1839-1843.
22. Sun S, Pan S, Miao A, et al. (2013): Active extracts of black tea (Camellia Sinensis) induce apoptosis of PC-3 prostate cancer cells via mitochondrial dysfunction. Oncol Rep 30: 763-772.
23. Vayalil PK, Katiyar SK (2004): Treatment of epigallocatechin-3-gallate inhibits matrix metalloproteinases-2 and -9 via inhibition of activation of mitogen-activated protein kinases, c-jun and NF-kappaB in human prostate carcinoma DU-145 cells. Prostate 59: 33-42.
24. Jankun J, Skrzypczak-Jankun E (2009): Yin and yang of the plasminogen activator inhibitor. Pol Arch Med Wewn 119: 410-417.
25. Narotzki B, Reznick AZ, Aizenbud D, et al. (2012): Green tea: a promising natural product in oral health. Arch Oral Biol 57: 429-435.
26. Zlotogorski A, Dayan A, Dayan D, et al. (2013): Nutraceuticals as new treatment approaches for oral cancer: II. Green tea extracts and resveratrol. Oral Oncol 49: 502-506.
27. Butt MS, Imran A, Sharif MK, et al. (2014): Black tea polyphenols: a mechanistic treatise. Crit Rev Food Sci Nutr 54: 1002-1011.
28. Lin JK (2002): Cancer chemoprevention by tea polyphenols through modulating signal transduction pathways. Arch Pharm Res 25: 561-571.