- Email: [email protected]
Effects of tea polyphenols on telomerase activity of a tongue cancer cell line: a preliminary study
Int. J. Oral Maxillofac. Surg. 2006; 35: 352–355 doi:10.1016/j.ijom.2005.07.020, available online at http://www.sciencedirect.com
Research Paper Head and Neck Oncology
Effects of tea polyphenols on telomerase activity of a tongue cancer cell line: a preliminary study
Y. Hua1, L. Jianhua1, W. Qiuliang1, F. Jun2, C. Zhi2 1 Department of Stomatology, First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, PR China; 2Research Institute of Infectious Disease, College of Medicine, Zhejiang University, Hangzhou 310003, PR China
Y. Hua, L. Jianhua, W. Qiuliang, F. Jun, C. Zhi: Effects of tea polyphenols on telomerase activity of a tongue cancer cell line: a preliminary study. Int. J. Oral Maxillofac. Surg. 2006; 35: 352–355. # 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. The aim of this study was to determine, at the mRNA and protein levels, whether tea polyphenols (TPs) affect the expression of the human telomerase reverse transcriptase (hTERT) gene in the Tca8113 cancerous cell line. The expression of this gene was determined at the mRNA level by reverse transcription and polymerase chain reaction and at the protein level by Western blotting. The semi-quantitative scores of hTERT mRNA expression were analyzed by one-way analysis of variance. After 72 h of exposure to TPs, the mean (SD) scores of hTERT mRNA expression in TP 0.1 g/l, TP 0.05 g/l and a control group were 0.32 0.05, 0.41 0.04 and 0.72 0.05, respectively (P < 0.05). The Western blot assay showed that TPs also decreased the expression of hTERT at the protein level. These results indicate that TPs reduce hTERT activity in the human Tca8113 cell line in a time- and dose-dependent manner, disabling telomerase activity and thereby terminating unlimited cancer cell proliferation. These findings suggest a mechanism behind TP’s anticancer activity.
Chinese tea is one of the most consumed beverages in the world. Green tea and black tea are the major types of traditional tea. The tea-leaf extracts contain mainly tea polyphenols (TPs), in the form of dried powder or liquid, which consist of more than 30 products2. The effects of TPs on human health, especially their anti-tumour effect, are becoming a subject of intense study by scientists throughout the world. Chemoprevention is the use of natural or synthetic substances to reduce the risk 0901-5027/040352 + 04 $30.00/0
of cancer or of cancer recurring. Many researchers have confirmed that TPs can inhibit a variety of mutations induced by physical or chemical stimulation, as well as stop the proliferation of cancerous cells, but that they have no inhibiting effect on somatic cells1,4,5. The telomere, which is activated by telomerase, is a repeating segment on the end of the chromosome that is necessary to stabilize the chromosome and for the replication of normal cells. The strong
Key words: tea polyphenols; Tca8113 cell line; telomerase catalytic subunit; hTERT; tongue carcinoma. Accepted for publication 27 July 2005 Available online 8 November 2005
association between telomerase and malignant tumours has led to a great deal of investigation. The human telomerase catalytic subunit (hTCS, also termed hTERT), one of the compositions of telomerase, could activate telomerase and thus lead to cellular immortality8. Some investigations have found that hTERT is associated with oral cancer, as well as other malignant tumours6,9,14. The effects of TPs on oral squamous cell carcinomas have not been fully
# 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Effects of tea polyphenols on telomerase activity of a tongue cancer cell line
353
addressed. In this study, we investigated the expression of the hTERT gene in a cancer cell line of the human tongue (Tca8113), with and without exposure to TPs, at the mRNA and protein levels, using reverse transcription and polymerase chain reaction (RT-PCR) and Western blot methods. Materials and methods Tea polyphenols
Tea polyphenols (90% pure, provided by Institute of Tea Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China) were dissolved in saline to a concentration of 1 g/l and stored at 20 8C. The solution was diluted with RPMI-1640 (Gibco Co., USA) before use. Cancer cell line
The cell line of human tongue carcinoma (Tca8113) was provided by the Pathology Department of the Ninth Hospital of Shanghai and maintained in our laboratory in RPMI-1640 medium supplemented with 10% foetal bovine serum. Experimental design
There were nine experimental groups. Cancer cells were exposed to TPs at 0.1 or 0.05 g/l for 24, 48 or 72 h, and to three untreated control groups for the same time periods. These doses and exposure times are the same as those used in previous research on TPs1,3–5. Gene expression was measured in all nine groups as described below to determine whether the dose of TPs or the duration of exposure had any effect at the gene or protein level. Assay for hTERT gene expression at the mRNA level
The expression of hTERT genes at the mRNA level in the tumour cell line was determined by RT-PCR. The primers used in this study were designed with Primer Designer software, Version 3.1, according to the gene sequences for hTERT mRNA in the Gene Bank. The gene sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers (up and down) and length of PCR products were: hTERT mRNA (NM_003219.1 GI: 4507438): 50 -GAG CCA GTC TCA CCT TCA AC-30 , 50 -GCA GCA GGA GGA TCT TGT AG-30 , 159 bp (nucleotides 2675– 2833); GAPDH mRNA (NM_002046.2 GI: 7669491): 50 -CGA TGC TGG CGC
Fig. 1. Expression of hTERT mRNA and GAPDH by RT-PCR assay at 24 h: (M) DNA marker SM0241, (1) TP 0.1 g/l, (2) TP 0.05 g/l and (3) control.
TGA GTA CG-30 , 50 -CAG GAG GCA TTG CTG ATG AT-30 , 192 bp (nucleotides 339–530). All primers were synthesized by Shanghai Sangon Bioengineering Ltd. Total cellular RNA was extracted from the above samples by Trizol extraction (Gibco). The content and purity of the RNA sample was determined from assays of o.d.260 nm/o.d.280 nm with the Gene Quant II RNA/DNA Calculator. The RNA quality was checked by agarose gel electrophoresis. Single-stranded cDNA was synthesized from 5 mg of total RNA with oligodT, and reverse transcriptase (Molony Murine Leukemia Virus Reverse Transcriptase, MBI, Canada) was extinguished at 70 8C for 15 min. PCR was carried out by amplifying hTERT and GAPDH with specific oligonucleotide primers as described above. In all, 25 ml of reaction mixture containing 2 ml of cDNA, 1 ml of dNTP mix, 1 ml of each primer and 2.5 units of Taq polymerase (Promega, USA) were amplified on a
thermal cycler (PTC-200 DNA Engine, MJ Research, USA). Reaction mixtures underwent 32 amplification cycles (predenaturation at 94 8C for 4 min, denaturation at 94 8C for 45 s, annealing at 60 8C for 1 min, and extension at 72 8C for 1 min). A final extension step was performed at 72 8C for 10 min, and the mixture was stored at 4 8C. The PCR products were then electrophoresed on 2% agarose gels and stained by ethidium bromide for viewing under ultraviolet radiation. hTERT and GAPDH were considered to be expressed when a band of the appropriate size for each gene was seen (159 bp for hTERT and 192 bp for GAPDH). Western blot assay for hTERT gene expression at the protein level
The effects of TPs on the protein expression of hTERT in Tca8113 cells were determined by the Western blot method. Whole cell extracts were prepared in chilled lysis buffer (0.15 M NaCl, 5 mM
Fig. 2. Expression of hTERT mRNA and GAPDH by RT-PCR assay at 72 h: (M) DNA marker SM0241, (1) TP 0.1 g/l, (2) TP 0.05 g/l, and (3) control.
354
Hua et al.
Fig. 3. Expression of hTERT protein and GAPDH for the three groups (TP 0.1 g/l, TP 0.05 g/l, control) at 24, 48 and 72 h, by the Western blot method.
EDTA pH 8.0, 1% Triton X100, 10 mM Tris–Cl pH 7.4; just before use, 1:1000 5 M DTT, 1:1000 100 mM PMSF in isopropanol, 1:1000 5 M aminocaproic acid were added). Cell debris was removed by centrifugation (4 8C, 12,000 rpm for 5 min), and the supernatants were stored at 20 8C. The protein concentration of each sample was determined by the Brandford method, and then adjusted to 2 mg/ml. The samples were heated for 3 min at 100 8C and then electro-transferred (SDS–PAGE) to a pyroxylin membrane (2 mg/ml 20 ml). The membrane was blocked in 3% casein, and subjected to immunoblot analysis with a 1:1000 dilution of monoclonal antibody (sheepanti-human TERT) overnight at 4 8C and then with the secondary antibody of mouse-peroxidase-linked anti-sheep IgG antibody (1:500 dilution). Proteins were visualized by enhanced chemiluminescence (Amersham, UK), and bands were quantitated using Image Quant software. Statistical methods
The data were analyzed by one-way analysis of variance with the SPSS 11.0 for windows statistical software package. Results
The results are shown in Figs 1 and 2. All samples were positive for GAPDH, confirming that the RT-PCR products had not degenerated. TPs reduced the expression of hTERT mRNA (Table 1). The semi-quantitative scores of hTERT mRNA in Tca8113 cells after exposure to TPs indicated that higher concentrations of TPs and longer exposures were associated with
greater inhibition of hTERT mRNA expression. The expression of hTERT protein became weaker at the higher TP dose, as well as at the longer TP exposure (Fig. 3). Discussion
The chemoprevention of human cancers has long been the subject of research. The chemopreventive effects of TPs have been demonstrated in animal models for cancers of the lung, skin, oesophagus, colon, and mammary glands10,11, but in cancers of the human tongue they are not well characterized. Telomerase activity appears to stabilize telomeres and thus leads to cellular immortality. Telomerase is active in 80–90% of malignant tissues and many immortal cell lines8,12,15. Of the telomerase subunits, reverse transcriptase (hTERT) determines enzyme activity7. In the present study, expression of the hTERT gene was reduced, suggesting that the transcription of telomerase was also reduced. The possible anticancer mechanisms of TPs accepted by most researchers are as follows: (1) inhibition of gene mutation; (2) inhibition of cancer-cell proliferation; (3) induction of cancer-cell apoptosis; (4) termination of metastasis1,3,13. The present study confirms that TPs can inhibit hTERT gene transcription and expression in a timeand dose-dependent manner, consequently disabling telomerase activity and thereby terminating unlimited cancer-cell proliferation. The results also further support the other above mechanisms. The results presented here provide important information for exploring new methods of treating patients with tongue cancer. This study is, however, only pre-
Table 1. Semi-quantitative scores of hTERT mRNA expression in Tca8113 cell lines after exposure to TPs by RT-PCR Experimental group Exposure time (h) 24 48 72
Control, mean (SD)
TP 0.05 g/l, mean (SD)
TP 0.1 g/l, mean (SD)
0.70 (0.05) 0.72 (0.04) 0.72 (0.05)
0.63 (0.04) 0.50 (0.08)* 0.41 (0.04)*
0.56 (0.05)* 0.42 (0.04)* 0.32 (0.05)*,#
P values are from one-way ANOVA. * P < 0.05 vs. control. # P < 0.05 vs. TP 0.05 g/l.
liminary, and future work in this area is required to corroborate the apparent relationship between TPs and hTERT, ideally using primers designed to amplify only the functional variants of hTERT mRNA. Our data are not adequate to conclude that TPs actually slow the growth of cancer. Further studies aimed at illustrating the significance of TPs in treating squamous cell carcinoma of human tongue, as well as other malignant tumours, are necessary. References 1. Conney AH, Lu YP, Lou YR, Huang MT. Inhibitory effects of tea and caffeine on UV-induced carcinogenesis: relationship to enhanced apoptosis and decreased tissue fat. Eur J Cancer Prev 2002: 2(Suppl.):S28–S36. 2. Huang H, Xu XQ. Anticancer activity of tea: evidence from recent animal experiments and human studies. J Tea Sci 2004: 24: 1–11. 3. Ioannides C, Yoxall V. Antimutagenic activity of tea: role of polyphenols. Curr Opin Clin Nutr Metab Care 2003: 6: 649– 656. 4. Jia X, Han C, Chen J. Effects of tea on preneoplastic lesions and cell cycle regulators in rat liver. Cancer Epidemiol Biomarkers Prev 2002: 11: 1663–1667. 5. Kemberling JK, Hampton JA, Keck RW, Gomez MA, Selman SH. Inhibition of bladder tumor growth by the green tea derivative epigallocatechin-3-gallate. J Urol 2003: 170: 773–776. 6. Kirkpatrick KL, Clark G, Ghilchick M, Newbold RF, Mokbel K. hTERT mRNA expression correlates with telomerase activity in human breast cancer. Eur J Surg Oncol 2003: 9: 321– 326. 7. Kirkpatrick KL, Mokbel K. The significance of human telomerase reverse transcriptase in human cancer. Eur J Surg Oncol 2001: 27: 754–760. 8. Kirkpatrick KL, Newbold RF, Mokbel K. The mRNA expression of hTERT in human breast carcinomas correlates with VEGF expression. J Carcinogen 2004: 3: 1–9. 9. Lippman SM, Sudbo J, Hong WK. Oral cancer prevention and the evolution of molecular-targeted drug development. J Clin Oncol 2005: 23: 346–356. 10. Lu YP, Lou YR, Xie JG, Yen P, Huang MT, Conney AH. Inhibitory effect of
Effects of tea polyphenols on telomerase activity of a tongue cancer cell line black tea on the growth of established skin tumors in mice: effects on tumor size, apoptosis, mitosis and bromodeoxyuridine incorporation into DNA. Carcinogenesis 1997: 18: 2163–2169. 11. Mimoto J, Kiura K, Matsuo K, Yoshino T, Takata I, Ueoka H, Kataoka M, Harada M. Epigallocatechin gallate can prevent cisplatin induced lung tumorigenesis in A/J mice. Carcinogenesis 2000: 21: 915–919. 12. Mokbel K. The role of telomerase in breast cancer. Eur J Surg Oncol 2000: 26: 509–514.
13. Park AM, Dong Z. Signal transduction pathways: targets for green and black tea polyphenols. J Biochem Mol Biol 2003: 36: 66–77. 14. Wu A, Ichihashi M, Ueda M. Correlation of the expression of human telomerase subunits with telomerase activity in normal skin and skin tumors. Cancer 1999: 86: 2038–2044. 15. Xu D, Gruber A, Peterson C, Pisa P. Telomerase activity and the expression of telomerase components in acute myelogenous leukaemia. Br J Haematol 1998: 102: 1367–1375.
Address: Liu Jianhua Department of Stomatology First Affiliated Hospital College of Medicine Zhejiang University 79 Qingchun Road Hangzhou 310003 PR China Tel: +86 571 85984797 Fax: +86 571 87217089 E-mail: [email protected]
355
Research Paper Head and Neck Oncology
Effects of tea polyphenols on telomerase activity of a tongue cancer cell line: a preliminary study
Y. Hua1, L. Jianhua1, W. Qiuliang1, F. Jun2, C. Zhi2 1 Department of Stomatology, First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, PR China; 2Research Institute of Infectious Disease, College of Medicine, Zhejiang University, Hangzhou 310003, PR China
Y. Hua, L. Jianhua, W. Qiuliang, F. Jun, C. Zhi: Effects of tea polyphenols on telomerase activity of a tongue cancer cell line: a preliminary study. Int. J. Oral Maxillofac. Surg. 2006; 35: 352–355. # 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. The aim of this study was to determine, at the mRNA and protein levels, whether tea polyphenols (TPs) affect the expression of the human telomerase reverse transcriptase (hTERT) gene in the Tca8113 cancerous cell line. The expression of this gene was determined at the mRNA level by reverse transcription and polymerase chain reaction and at the protein level by Western blotting. The semi-quantitative scores of hTERT mRNA expression were analyzed by one-way analysis of variance. After 72 h of exposure to TPs, the mean (SD) scores of hTERT mRNA expression in TP 0.1 g/l, TP 0.05 g/l and a control group were 0.32 0.05, 0.41 0.04 and 0.72 0.05, respectively (P < 0.05). The Western blot assay showed that TPs also decreased the expression of hTERT at the protein level. These results indicate that TPs reduce hTERT activity in the human Tca8113 cell line in a time- and dose-dependent manner, disabling telomerase activity and thereby terminating unlimited cancer cell proliferation. These findings suggest a mechanism behind TP’s anticancer activity.
Chinese tea is one of the most consumed beverages in the world. Green tea and black tea are the major types of traditional tea. The tea-leaf extracts contain mainly tea polyphenols (TPs), in the form of dried powder or liquid, which consist of more than 30 products2. The effects of TPs on human health, especially their anti-tumour effect, are becoming a subject of intense study by scientists throughout the world. Chemoprevention is the use of natural or synthetic substances to reduce the risk 0901-5027/040352 + 04 $30.00/0
of cancer or of cancer recurring. Many researchers have confirmed that TPs can inhibit a variety of mutations induced by physical or chemical stimulation, as well as stop the proliferation of cancerous cells, but that they have no inhibiting effect on somatic cells1,4,5. The telomere, which is activated by telomerase, is a repeating segment on the end of the chromosome that is necessary to stabilize the chromosome and for the replication of normal cells. The strong
Key words: tea polyphenols; Tca8113 cell line; telomerase catalytic subunit; hTERT; tongue carcinoma. Accepted for publication 27 July 2005 Available online 8 November 2005
association between telomerase and malignant tumours has led to a great deal of investigation. The human telomerase catalytic subunit (hTCS, also termed hTERT), one of the compositions of telomerase, could activate telomerase and thus lead to cellular immortality8. Some investigations have found that hTERT is associated with oral cancer, as well as other malignant tumours6,9,14. The effects of TPs on oral squamous cell carcinomas have not been fully
# 2005 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Effects of tea polyphenols on telomerase activity of a tongue cancer cell line
353
addressed. In this study, we investigated the expression of the hTERT gene in a cancer cell line of the human tongue (Tca8113), with and without exposure to TPs, at the mRNA and protein levels, using reverse transcription and polymerase chain reaction (RT-PCR) and Western blot methods. Materials and methods Tea polyphenols
Tea polyphenols (90% pure, provided by Institute of Tea Sciences, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China) were dissolved in saline to a concentration of 1 g/l and stored at 20 8C. The solution was diluted with RPMI-1640 (Gibco Co., USA) before use. Cancer cell line
The cell line of human tongue carcinoma (Tca8113) was provided by the Pathology Department of the Ninth Hospital of Shanghai and maintained in our laboratory in RPMI-1640 medium supplemented with 10% foetal bovine serum. Experimental design
There were nine experimental groups. Cancer cells were exposed to TPs at 0.1 or 0.05 g/l for 24, 48 or 72 h, and to three untreated control groups for the same time periods. These doses and exposure times are the same as those used in previous research on TPs1,3–5. Gene expression was measured in all nine groups as described below to determine whether the dose of TPs or the duration of exposure had any effect at the gene or protein level. Assay for hTERT gene expression at the mRNA level
The expression of hTERT genes at the mRNA level in the tumour cell line was determined by RT-PCR. The primers used in this study were designed with Primer Designer software, Version 3.1, according to the gene sequences for hTERT mRNA in the Gene Bank. The gene sequence of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primers (up and down) and length of PCR products were: hTERT mRNA (NM_003219.1 GI: 4507438): 50 -GAG CCA GTC TCA CCT TCA AC-30 , 50 -GCA GCA GGA GGA TCT TGT AG-30 , 159 bp (nucleotides 2675– 2833); GAPDH mRNA (NM_002046.2 GI: 7669491): 50 -CGA TGC TGG CGC
Fig. 1. Expression of hTERT mRNA and GAPDH by RT-PCR assay at 24 h: (M) DNA marker SM0241, (1) TP 0.1 g/l, (2) TP 0.05 g/l and (3) control.
TGA GTA CG-30 , 50 -CAG GAG GCA TTG CTG ATG AT-30 , 192 bp (nucleotides 339–530). All primers were synthesized by Shanghai Sangon Bioengineering Ltd. Total cellular RNA was extracted from the above samples by Trizol extraction (Gibco). The content and purity of the RNA sample was determined from assays of o.d.260 nm/o.d.280 nm with the Gene Quant II RNA/DNA Calculator. The RNA quality was checked by agarose gel electrophoresis. Single-stranded cDNA was synthesized from 5 mg of total RNA with oligodT, and reverse transcriptase (Molony Murine Leukemia Virus Reverse Transcriptase, MBI, Canada) was extinguished at 70 8C for 15 min. PCR was carried out by amplifying hTERT and GAPDH with specific oligonucleotide primers as described above. In all, 25 ml of reaction mixture containing 2 ml of cDNA, 1 ml of dNTP mix, 1 ml of each primer and 2.5 units of Taq polymerase (Promega, USA) were amplified on a
thermal cycler (PTC-200 DNA Engine, MJ Research, USA). Reaction mixtures underwent 32 amplification cycles (predenaturation at 94 8C for 4 min, denaturation at 94 8C for 45 s, annealing at 60 8C for 1 min, and extension at 72 8C for 1 min). A final extension step was performed at 72 8C for 10 min, and the mixture was stored at 4 8C. The PCR products were then electrophoresed on 2% agarose gels and stained by ethidium bromide for viewing under ultraviolet radiation. hTERT and GAPDH were considered to be expressed when a band of the appropriate size for each gene was seen (159 bp for hTERT and 192 bp for GAPDH). Western blot assay for hTERT gene expression at the protein level
The effects of TPs on the protein expression of hTERT in Tca8113 cells were determined by the Western blot method. Whole cell extracts were prepared in chilled lysis buffer (0.15 M NaCl, 5 mM
Fig. 2. Expression of hTERT mRNA and GAPDH by RT-PCR assay at 72 h: (M) DNA marker SM0241, (1) TP 0.1 g/l, (2) TP 0.05 g/l, and (3) control.
354
Hua et al.
Fig. 3. Expression of hTERT protein and GAPDH for the three groups (TP 0.1 g/l, TP 0.05 g/l, control) at 24, 48 and 72 h, by the Western blot method.
EDTA pH 8.0, 1% Triton X100, 10 mM Tris–Cl pH 7.4; just before use, 1:1000 5 M DTT, 1:1000 100 mM PMSF in isopropanol, 1:1000 5 M aminocaproic acid were added). Cell debris was removed by centrifugation (4 8C, 12,000 rpm for 5 min), and the supernatants were stored at 20 8C. The protein concentration of each sample was determined by the Brandford method, and then adjusted to 2 mg/ml. The samples were heated for 3 min at 100 8C and then electro-transferred (SDS–PAGE) to a pyroxylin membrane (2 mg/ml 20 ml). The membrane was blocked in 3% casein, and subjected to immunoblot analysis with a 1:1000 dilution of monoclonal antibody (sheepanti-human TERT) overnight at 4 8C and then with the secondary antibody of mouse-peroxidase-linked anti-sheep IgG antibody (1:500 dilution). Proteins were visualized by enhanced chemiluminescence (Amersham, UK), and bands were quantitated using Image Quant software. Statistical methods
The data were analyzed by one-way analysis of variance with the SPSS 11.0 for windows statistical software package. Results
The results are shown in Figs 1 and 2. All samples were positive for GAPDH, confirming that the RT-PCR products had not degenerated. TPs reduced the expression of hTERT mRNA (Table 1). The semi-quantitative scores of hTERT mRNA in Tca8113 cells after exposure to TPs indicated that higher concentrations of TPs and longer exposures were associated with
greater inhibition of hTERT mRNA expression. The expression of hTERT protein became weaker at the higher TP dose, as well as at the longer TP exposure (Fig. 3). Discussion
The chemoprevention of human cancers has long been the subject of research. The chemopreventive effects of TPs have been demonstrated in animal models for cancers of the lung, skin, oesophagus, colon, and mammary glands10,11, but in cancers of the human tongue they are not well characterized. Telomerase activity appears to stabilize telomeres and thus leads to cellular immortality. Telomerase is active in 80–90% of malignant tissues and many immortal cell lines8,12,15. Of the telomerase subunits, reverse transcriptase (hTERT) determines enzyme activity7. In the present study, expression of the hTERT gene was reduced, suggesting that the transcription of telomerase was also reduced. The possible anticancer mechanisms of TPs accepted by most researchers are as follows: (1) inhibition of gene mutation; (2) inhibition of cancer-cell proliferation; (3) induction of cancer-cell apoptosis; (4) termination of metastasis1,3,13. The present study confirms that TPs can inhibit hTERT gene transcription and expression in a timeand dose-dependent manner, consequently disabling telomerase activity and thereby terminating unlimited cancer-cell proliferation. The results also further support the other above mechanisms. The results presented here provide important information for exploring new methods of treating patients with tongue cancer. This study is, however, only pre-
Table 1. Semi-quantitative scores of hTERT mRNA expression in Tca8113 cell lines after exposure to TPs by RT-PCR Experimental group Exposure time (h) 24 48 72
Control, mean (SD)
TP 0.05 g/l, mean (SD)
TP 0.1 g/l, mean (SD)
0.70 (0.05) 0.72 (0.04) 0.72 (0.05)
0.63 (0.04) 0.50 (0.08)* 0.41 (0.04)*
0.56 (0.05)* 0.42 (0.04)* 0.32 (0.05)*,#
P values are from one-way ANOVA. * P < 0.05 vs. control. # P < 0.05 vs. TP 0.05 g/l.
liminary, and future work in this area is required to corroborate the apparent relationship between TPs and hTERT, ideally using primers designed to amplify only the functional variants of hTERT mRNA. Our data are not adequate to conclude that TPs actually slow the growth of cancer. Further studies aimed at illustrating the significance of TPs in treating squamous cell carcinoma of human tongue, as well as other malignant tumours, are necessary. References 1. Conney AH, Lu YP, Lou YR, Huang MT. Inhibitory effects of tea and caffeine on UV-induced carcinogenesis: relationship to enhanced apoptosis and decreased tissue fat. Eur J Cancer Prev 2002: 2(Suppl.):S28–S36. 2. Huang H, Xu XQ. Anticancer activity of tea: evidence from recent animal experiments and human studies. J Tea Sci 2004: 24: 1–11. 3. Ioannides C, Yoxall V. Antimutagenic activity of tea: role of polyphenols. Curr Opin Clin Nutr Metab Care 2003: 6: 649– 656. 4. Jia X, Han C, Chen J. Effects of tea on preneoplastic lesions and cell cycle regulators in rat liver. Cancer Epidemiol Biomarkers Prev 2002: 11: 1663–1667. 5. Kemberling JK, Hampton JA, Keck RW, Gomez MA, Selman SH. Inhibition of bladder tumor growth by the green tea derivative epigallocatechin-3-gallate. J Urol 2003: 170: 773–776. 6. Kirkpatrick KL, Clark G, Ghilchick M, Newbold RF, Mokbel K. hTERT mRNA expression correlates with telomerase activity in human breast cancer. Eur J Surg Oncol 2003: 9: 321– 326. 7. Kirkpatrick KL, Mokbel K. The significance of human telomerase reverse transcriptase in human cancer. Eur J Surg Oncol 2001: 27: 754–760. 8. Kirkpatrick KL, Newbold RF, Mokbel K. The mRNA expression of hTERT in human breast carcinomas correlates with VEGF expression. J Carcinogen 2004: 3: 1–9. 9. Lippman SM, Sudbo J, Hong WK. Oral cancer prevention and the evolution of molecular-targeted drug development. J Clin Oncol 2005: 23: 346–356. 10. Lu YP, Lou YR, Xie JG, Yen P, Huang MT, Conney AH. Inhibitory effect of
Effects of tea polyphenols on telomerase activity of a tongue cancer cell line black tea on the growth of established skin tumors in mice: effects on tumor size, apoptosis, mitosis and bromodeoxyuridine incorporation into DNA. Carcinogenesis 1997: 18: 2163–2169. 11. Mimoto J, Kiura K, Matsuo K, Yoshino T, Takata I, Ueoka H, Kataoka M, Harada M. Epigallocatechin gallate can prevent cisplatin induced lung tumorigenesis in A/J mice. Carcinogenesis 2000: 21: 915–919. 12. Mokbel K. The role of telomerase in breast cancer. Eur J Surg Oncol 2000: 26: 509–514.
13. Park AM, Dong Z. Signal transduction pathways: targets for green and black tea polyphenols. J Biochem Mol Biol 2003: 36: 66–77. 14. Wu A, Ichihashi M, Ueda M. Correlation of the expression of human telomerase subunits with telomerase activity in normal skin and skin tumors. Cancer 1999: 86: 2038–2044. 15. Xu D, Gruber A, Peterson C, Pisa P. Telomerase activity and the expression of telomerase components in acute myelogenous leukaemia. Br J Haematol 1998: 102: 1367–1375.
Address: Liu Jianhua Department of Stomatology First Affiliated Hospital College of Medicine Zhejiang University 79 Qingchun Road Hangzhou 310003 PR China Tel: +86 571 85984797 Fax: +86 571 87217089 E-mail: [email protected]
355