Chinese-German Journal of Clinical Oncology

December 2011, Vol. 10, No. 12, P716–P720

DOI 10.1007/s10330-011-0884-6

Influence of (-)-epigallocatechin-3-gallate on the expression of connexin43 and gap junction intercellular communication of the bladder cancer cell lines BIU-87 in vitro* Zhengguo Cao1, Chao Tian1, Maolin Jiang1, Kui Wu2, Xiaojian Zhong1, Jianxin Li1, Yuefu Han1 1

2

Department of Urology, The Affiliated Yuebei People’s Hospital of Shantou University Medical College, Shaoguang 512026, China Department of Urology, Anhui Provincial Hospital Affiliated to Anhui Medical College, Hefei 230001, China Received: 4 August 2011 / Revised: 20 September 2011 / Accepted: 24 October 2011 © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2011 Abstract Objective: The aim of our study was to investigate the effects of (-)-epigallocatechin-3-gallate (EGCG, the major phytochemistry component in green tea) on the expression of connexin43 (Cx43) gene and detect the intercellular communication of the human bladder cancer cell lines BIU-87, and explore its possible mechanisms of prevention and cure for the bladder tumor. Methods: The methyl thiazolyl tetrazolium and Annexin-V/PI double-labeled flow cytometry methods were used to observe the growth inhibitory rate (IR) and apoptosis rate (AR) of BIU-87 cells treated by EGCG at different concentrations (0, 5, 10 and 20 mg/L), respectively. The reverse transcription-polymerase chain reaction (RT-PCR) and Western Blotting analysis were employed to detect the relative expression levels of the Cx43 mRNA and its protein. The scrape-loading fluorescence dye transfer method was used to assess the gap junction intercellular communication (GJIC) under fluorescence microscope. Results: EGCG at concentrations (10 and 20 mg/L) both could significantly inhibit the proliferation and induce the apoptosis of BIU-87 cells. The IR and AR were (15.67 ± 1.15)%, (18.33 ± 1.53)% and (42.00 ± 4.34)%, (27.33 ± 3.21)%, respectively. And compared with the control groups of 0 mg/L and 5 mg/L (P﹤0.05), EGCG could significantly up-regulate the expression of Cx43 mRNA and its protein and enhance the function of BIU-87 cells. The effects had the significant correlation with the dose-dependent of EGCG. Conclusion: EGCG (10, 20 mg/L) could effectively up-regulate Cx43 expression and enhance the GJIC of BIU-87 cells. The results may indicate the effects of EGCG inducing bladder tumor cells apoptosis and inhibiting its growth which provides the experimental evidence for further demonstrating the mechanism of chemical prevention and cure for the bladder tumor by EGCG. Key words

(-)-epigallocatechin-3-gallate; bladder tumor; connexin43; intercellular communication

Recent studies have found the aberrant expression of connexin protein (Cx) gene and defect of gap junction intercellular communication (GJIC) properties which are widespread present in tumor and differentiated cells [1]. The aberrant expression of Cx in tumor cells doesn’t accompany genome DNA loss or mutation, but is confirmed as down-regulation of expression level and can be recovered through medicine, which provides the new target point for tumor treatment. More researches have showed that drinking green tea can prevent tumorigenesis and improve the tumor patient prognosis. Because the major phytochemistry component in green tea polyphenols is Correspondence to: Zhengguo Cao. Email: [email protected] * Supported by a grant from the National Natural Sciences Foundation of China (No. 30801138).

(-)-epigallocatechin-3-gallate (EGCG) and EGCG which acts as active components in prevention of tumor in green tea. At present, however, the mechanisms of prevention have not been clear [2]. The current study is designed to explore its possible mechanisms of prevention and cure for the bladder tumor by investigating the effects of EGCG on the expression of growths, connexin43 (Cx43) gene expression and detecting the intercellular communication of the human bladder cancer cell.

Materials and methods Major reagents and instruments EGCG, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT), fluorescence yellow dye (LY) and the polyclonal antibody against Cx43 protein of hu-

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man were purchased from Sigma Co., USA. Annexin-V/ Propidium Iodide (PI) FITC double-labeled kit was from Shenzhen Jingmei BioTech Co., Ltd (China). The primers of Cx43 and human β-actin were all synthesized by Shanghai Sangon BioTech Co., Ltd (China). The TaKaRa RNA PCR Kit 3.0 was purchased from TaKaRa Co., Ltd (China). Western Blotting and enhanced chemiluminescence (ECL) kits were purchased from Pierce Co., USA. BIU-87 cells, a human bladder cancer cell line was obtained from Wuhan University Center for Type Culture Collection. Cell culture and intervention BIU-87 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum and seeded into 6well microplate at a density of 1 × 105/well in a humidified chamber at 37 ℃ and 5% CO2. When the cells were grown to logarithm growth phase, cells were washed by pure RPMI-1640 and used as study. The dropwise addition of EGCG was added into each well to prepare the different final concentration at 0, 5, 10, 20 mg/L, respectively. After BIU-87 cells had been interfered for 24 h, the procedure was as follows. BIU-87 cells proliferation inhibitory rate assay MTT method was applied in this assay. BIU-87 cells were collected, adjusted and seeded into 96-well microplate at a density of 1 × 104/well. Each group had 3 wells and different final concentration EGCG (0, 5, 10, 20 mg/L) which were added according to designated groups. Blank control group was also established. 20 μL (5 g/L) of MTT was added into each well and the cells were incubated for another 4 h. The culture medium was removed and 150 μL DMSO was added into each well. After shaking thoroughly for 10 min, absorbance (A) in each well was read on enzyme labeling-instrument using a test wavelength of 570 nm. The growth inhibitory rate (IR) of BIU-87 cells in each group was calculated according to the equation as follows: IR% = (Acontrol – Aexperiment)/Acontrol × 100%. Detection of apoptosis by Annexin-V/PI flow cytometry (FCM) BIU-87 cells were treated in the same manner as above and seeded into well microplate. Then cells concentration was adjusted by 0.25% trypsin and collected by centrifugation and washed by ice-cold PBS. Cells were resuspended in 250 μL prediluted binding buffer and cells density was adjusted to about from 1 × 105/mL to 1 × 106/mL. 5 μL Annexin V-FITC was added into 195 μL cell suspension and mixed for 10 min in the dark at room temperature. Cells were washed and resuspended again in 190 μL prediluted binding buffer. 10 μL of 20 μg/mL propidium iodide stock solution was added. The foregoing experiment was performed three times and cells apoptosis was detected by

FCM analysis in 1 h. Scrape-loading fluorescence dye transfer method When the cells glass slide was prepared, the BIU-87 cells were washed by PBS three times. The cells surface scrape blotting formed after cell was scraped by a sharp-pointed knife. 1 mL of LY (0.05%) was added into and labeled for 3 min. Then, cells were washed by pure RPMI-1640. The intercellular fluorescence dye transfer was observed under fluorescence microscope at a test stimulation wavelength of 490 nm. The negative criterion was assessed when the LY was only located in single layer cells close to scrape blotting. On the contrary, the LY transferred beyond two layers around cells. Reverse transcription polymerase chain reaction (RT-PCR) The total RNA of was isolated by using the Trizol Reagent kit according to the manufacturer’s instructions. The primer sequence of Cx43 gene was designed on the basis of software of Primer Design 5.0 version and the homology analysis of BLAST as follows: 5’-CTATGTGAT GCGAAAGGA-3’ (sense), 5’-AGGAAACAGTCCACCT GA-3’ (antisense), resulting in a 309 bp product. The βactin of interior reference control was also established. RT-PCR was performed using TaKaRa RNA PCR kits according to the protocols provided by the manufacturer. Prior to establishing the RT reaction, the mRNA template and random primers were mixed and pre-incubated. The RT reaction was performed when Oligo-dT was used as primer. PCR consisted of 32 cycles. Each cycle consisted of 20 s of denaturation at 94 ℃, 20 s of annealing at 55 ℃, 3 min of extension at 72 ℃. After PCR reaction ending, 5 μL PCR products were mixed with loading buffer and applied directly to electrophoresis on 1.5% agarose gels containing ethidum bromide. The gels were scanned and the intensity of each PCR fragment was measured using an image analyzer. To quantify the relative changes of Cx43 mRNA expression, the ratio of Cx43 and β-actin band intensity was analyzed. Western blot BIU-87 cells were collected and washed twice by precooling PBS. The tissue protein lysate whose volume was five times as much as the volume of cell suspension added into the cell suspension. Aggregates were removed by centrifugation for 2 min at a centrifuge of 12 000 r/min. 10 μL supernatant was taken out. Then 2 × buffer solution of application of sample at the same volume was mixed with supernatant. The mixture was heated to boil for 10 min. The supernatant was taken again after centrifuge. 100 μg denaturalizated total protein was applied directly to electrophoresis on polyacrylamide gel and transferred

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www.springerlink.com/content/1613-9089 Results of the growth inhibiting rate (IR), apoptosis rate (AR) and Cx43 relative expression levels of BIU-87 cells in four groups (n = 3 )

Groups

IR (%)

AR (%)

Control 5 mg/L 10 mg/L 20 mg/L

2.13 ± 0.31 6.93 ± 1.22** 15.67 ± 1.15** 42.00 ± 4.34**

3.83 ± 0.31 7.69 ± 0.51* 18.33 ± 1.53* 27.33 ± 3.21**

Cx43 mRNA level 0.25 ± 0.02 0.29 ± 0.07 0.57 ± 0.06** 0.70 ± 0.09**

Cx43 protein level 0.22 ± 0.04 0.25 ± 0.04 0.48 ± 0.05** 0.68 ± 0.05**

* P < 0.05, compared with control group; ** P < 0.01, compared with control group

to PVDF membrane. Then, protein was blocked by confining liquid including defatted milk powder for 2 h. The diluted (1:500) polyclonal antibody against Cx43 protein of human and peroxidase labeling IgG against rabbit were successively followed on dropping after the confining liquid added to block and incubate for 1 h at room temperature. ECL developing reagent was exposed in the dark room. The each protein strap was scanned and optical density value was measured using an image analyzer to calculate relative expression of Cx43 protein. Statistical analysis Data were expressed as mean ± SD. One-Way ANOVA method was applied for total comparison difference. The difference between groups was analyzed by LSD and SNKq tests and the statistical package of SPSS 11.5 version. A level of P < 0.05 was considered statistically significant.

Results Effects of EGCG on proliferation inhibition of BIU-87 cells (Table 1) The MTT results showed that EGCG of three different concentrations could significantly inhibit BIU-87 cells growth (P < 0.05). Moreover, when the concentration increased, the inhibition effects could also enhance and directly depend on the concentration. Among the different concentration EGCG, the greatest growth inhibitory rate was (42.00 ± 4.34)% by EGCG of 20 mg/L on BIU-87 cells which significantly was higher than those in other groups (P < 0.01), as shown in Table 1. Effects of EGCG on apoptosis of BIU-87 cells The Annexin-V/PI double-labeled FCM showed that EGCG of three different concentration could significantly induce BIU-87 cells apoptosis (P < 0.05) and the effects on apoptosis also reflected dose-dependent relationship. The greatest growth inhibitory rate was (27.33 ± 3.21)% by EGCG of 20 mg/L on BIU-87 cells which was significantly higher than those in other groups, as shown in Table 1. Detection of GJIC by scrape-loading fluorescence dye transfer method Under fluorescence microscope, LY fluorescence label only localized in single BIU-87 cell between blank con-

trol group and 5 mg/L group and no obvious fluorescence dye transfer was observed, which showed BIU-87 cells had significantly functional defect of GJIC. However, LY fluorescence dye could transfer to close cells from two layers to three layers by passing through gap junction intercellular after cells were treated by EGCG of large dose including 10 mg/L and 20 mg/L which showed the GJIC function had improved. Detection of Cx43 mRNA expression by RT-PCR (Fig. 1) The results of RT-PCR electrophoresis of Cx43 mRNA and relative expression level were shown in Fig. 1 and Table 1, respectively. As shown in Fig. 1, Cx43 product length was 309 bp. As shown in Table 1, the up-regulation effect of EGCG on expression of Cx43 mRNA depended on dose ranging from 10 mg/L to 20 mg/L and the relative expression levels were significantly higher than those of control group. But EGCG of 5 mg/L had no significant effect on the expression of Cx43 mRNA (P﹥0.05). Detection of Cx43 protein expression by western Blot (Fig. 2) The results of Western Blot electrophoresis of Cx43 protein and relative expression level were shown in Fig. 2 and Table 1, respectively. As shown in Table 1, the Cx43 protein expression levels in 10 mg/L group and 20 mg/ L group were all significantly higher than those of control group and 5 mg/L group, respectively which showed dose-dependent relationship. And there was also no difference between control group and 5 mg/L group.

Discussion Gap junction is one of intercellular communication which has been widespread present in zoogenic tissue and cells and mainly made up of Cx. The exchange of intercellular information, energy and material has been completed through gap junction intercellular communication (GJIC) which is mediated by GJ, which plays a key regulation role in the physiological procedure, such as growth, differentiation, and apoptosis, et al [1]. Cx43 was the widely present and most investigated in the family of Cx. Along with thorough research of Cx and its family, more studies and researches have showed that Cx43 has

Chinese-German J Clin Oncol, December 2011, Vol. 10, No. 12

Fig. 1 Expression of Cx43 mRNA in four groups of RT-PCR electrophoresis. Lane 1: 20 mg/L EGCG; Lane 2 and 3: 10 mg/L EGCG; Lane 4: 5 mg/L EGCG; Lane 5: 0 mg/L EGCG

Fig. 2 Expression of Cx43 protein in four groups of Western Blot electrophoresis. Lane 1: 20 mg/L EGCG; Lane 2: 10 mg/L EGCG; Lane 3: 5 mg/L EGCG; Lane 4: 0 mg/L EGCG

close relation with genesis, development, invasion and metastasis potency of malignant tumor, which has provided the new target point for tumor diagnosis and treatment. Transitional cell carcinoma of the bladder (BTCC) is the most commonly urological malignant tumor. Our early studies have indicated that the down-regulated expression of Cx43 gene is closely associated with the genesis, development, invasion and metastasis of BTCC and Cx43 can be used as an early diagnosis and prognostic indicator for BTCC [3]. Therefore, Cx43 was considered as a non-mutation tumor inhibitory gene other than p53 gene and is also called as the second group non-mutation antioncogene. To promote the tumor cell to express Cx43 and to induce and improve the function of GJIC will become a new target for tumor treatment. The mechanisms of green tea anti-tumor have not been determined now. Many studies have showed that EGCG can inhibit many tumors cells in vitro. The research of anti-cancer mechanisms of EGCG mainly concentrated on promoting apoptosis signal transmission path, regulating cell cycle and inhibiting cell excess proliferation [4, 5] . Investigations found that after cancer inductor had blocked expression of Cx43 and weaken function of GJIC, signal regulation transmission of cell proliferation and apoptosis was out of control, which finally resulted in making tumor cell growth. However, up-regulation of Cx43 expression could reconstruct function of GJIC so as to inhibit tumor cell growth and malignant transformation [6]. But there was no report about the effects of EGCG

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on Cx43 expression and GJIC function of bladder tumor in worldwide. The results of MTT have indicated that different concentration EGCG all can inhibit the growth and induce the apoptosis of BIU-87 cells. And the inhibitory effects increase with EGCG concentration showing the dosedependent relationship, which is also similar to Chen et al’ studies [5]. Takahashi et al [7] held that green tea could strongly increase the function of GJIC in renal carcinoma cell, but the mechanism was not clear. Moreover, the results of scrape-loading fluorescence dye transfer method also showed that fluorescence dye transfection function was significantly improved in BIU-87 cells treated by EGCG of 10 mg/L and 20 mg/L. In other words, EGCG can improve the function of GJIC. Meanwhile, the quantitative analysis results of RT-PCR and Western Blot all demonstrated EGCG of 5 mg/L had no influence on Cx43 mRNA transcription and its protein expression, but the larger dose EGCG of 10 mg/L and 20 mg/L could obviously promote Cx43 mRNA transcription and protein expression which made it clear that EGCG could reconstruct GJIC of BIU-87 cells with use of promoting the expression of Cx43. It further demonstrated that the Cx43 gene alteration and GJIC function defect were possible genetic events in carcinogenesis and development process. However, the mechanism of Cx43 gene alteration was not clear. Ruch et al [8] found the alteration had relationship with the aberrant elaboration and modification of Cx43 protein after transcription and translation. The phosphorylation procedure accounted for the aberrant expression of Cx43 protein. Furthermore, many studies have not found Cx43 gene mutation. Even more scholars agreed to the opinion of Cx43 gene expression aberrance. Other vitro studies also demonstrated that glioma cell could inhibit some important autocrine growth factors by transferring Cx43 gene [9]. For this reason, the renewing expression of Cx43 has played an important role in reversing tumor cell phaenotype. With help of up-regulation of Cx43, cell can again obtain differentiation function and normal cell can again get control of the tumor growth. In conclusion, the results of the present study indicated that EGCG (10, 20 mg/L) could effectively up-regulate Cx43 expression, produce gap junction, restore GJIC, induce BIU-87 cells apoptosis and promote normal cells to be transformed so as to inhibit bladder tumor cells growth which provides the experimental evidence for further demonstrating the mechanisms of chemical prevention and cure for the bladder tumor by green tea. However, the effects of EGCG on expression of other Cx gene and the molecule mechanisms of up-regulation of Cx43 are not clear, some work need to be done in future.

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References 1. Krysko DV, Mussche S, Leybaert L, et al. Gap junctional communication and connexin43 expression in relation to apoptotic cell death and survival of granulosa cells. J Histochem Cytochem, 2004, 52: 1119–1207. 2. Beltz LA, Bayer DK, Moss AL, et al. Mechanisms of cancer prevention by green and black tea polyphenols. Anticancer Agents Med Chem, 2006, 6: 389–406. 3. Cao ZG, Qi L, Fang LC, et al. Expression of connexin43 and its correlation with the expression of bcl-2 and bax protein in transitional cell carcinoma of the bladder. Tumor (Chinese), 2007, 27: 398–401. 4. Rieger-Christ KM, Hanley R, Lodowsky C, et al. The green tea compound, (-)-epigallocatechin-3-gallate downregulates N-cadherin and suppresses migration of bladder carcinoma cells. J Cell Biochem, 2007, 102: 377–378.

5. Chen JJ, Ye ZQ, Koo MW. Growth inhibition and cell cycle arrest effects of epigallocatechin gallate in the NBT-II bladder tumour cell line. BJU Int, 2004, 93: 1082–1086. 6. Ogawa T, Hayashi T, Tokunou M, et al. Suberoylanilide hydroxamic acid enhances gap junctional intercellular communication via acetylation of histone containing connexin 43 gene locus. Cancer Res, 2005, 65: 9771–9778. 7. Takahashi H, Nomata K, Mori K, et al. The preventive effect of green tea on the gap junction intercellular communication in renal epithelial cells treated with a renal carcinogen. Anticancer Res, 2004, 24: 3757–3762. 8. Ruch RJ, Trosko JE, Madhukar BV. Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation. J Cell Biochem, 2001, 83: 163–169. 9. Xia ZB, Pu PY, Huang Q, et al. Preliminary study on the mechanism of connexin 43 gene transfection in the control of glioma cell proliferation. Chin J Oncol (Chinese), 2003, 25: 4–8.

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