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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 10  |  Page : 713-718

Overexpression of Tat-interacting protein 30 inhibits the proliferation, migration, invasion and promotes apoptosis in bladder cancer cells


1 Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515; Department of Urology, The First Affiliated Hospital of Wenzhou Medical University, Zhejiang 325000, China
2 Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

Date of Web Publication24-Sep-2018

Correspondence Address:
Wanlong Tan
Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, 510515
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.206869

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 > Abstract 


Aims: Tat-interacting protein 30 (TIP30), a transcriptional repressor, possesses antitumor effect in different cancer cells. However, little is known about the function of TIP30 in bladder cancer till now.
Materials and Methods: A TIP30-overexpressing plasmid was transfected into the bladder cancer cells (T24). The cell cycle and apoptosis were detected by flow cytometry. The cell proliferation was analyzed using the cell counting kit-8 assay. The migrative and invasive abilities of T24 cells were measured by the transwell assay. The expression of TIP30, cell cycle proteins, migration-related proteins, and cell apoptosis-related proteins was assessed by Western blotting.
Results: The cell proliferation, migration, and invasion of T24 cells were inhibited by overexpression of TIP30. Moreover, the rate of cell apoptosis was increased by the overexpression of TIP30. The expression of cell cycle proteins, phosphorylated EGFR, p-Akt, Bcl-2, cyclin D, cyclin E), migration-related proteins (matrix metalloproteinases 2 [MMP2], MMP6, MMP9), were downregulated, and cell apoptosis-related proteins (bax, cleaved caspase3) were upregulated.
Conclusions: These results suggest that TIP30, as a tumor suppressor in the bladder cancer, might be served as a target in cancer therapies in the future.

Keywords: Bladder cancer, Tat-interacting protein 30, tumor suppressor


How to cite this article:
Li Y, Cai B, Chen S, Fu X, Pang X, Zhu X, Qi H, Tan W. Overexpression of Tat-interacting protein 30 inhibits the proliferation, migration, invasion and promotes apoptosis in bladder cancer cells. J Can Res Ther 2018;14:713-8

How to cite this URL:
Li Y, Cai B, Chen S, Fu X, Pang X, Zhu X, Qi H, Tan W. Overexpression of Tat-interacting protein 30 inhibits the proliferation, migration, invasion and promotes apoptosis in bladder cancer cells. J Can Res Ther [serial online] 2018 [cited 2018 Nov 19];14:713-8. Available from: http://www.cancerjournal.net/text.asp?2018/14/10/713/206869




 > Introduction Top


Bladder cancer is a major public hygienic event and a common cancer in China, which has the highest incidence among urological system cancer.[1] In 2015, the new diagnosed cases and estimated deaths are 74,000 and 16,000, respectively.[2] Tat-interacting protein 30 (TIP30) is a 30-kDa protein originally identified in the highly metastatic human variant small cell lung carcinoma (SCLC),[3] whereas the TIP30 was decreased compared to normal tissues. The expression of TIP30 was decreased in many tumors, such as colon cancer, breast cancer, and lung cancer.[4] Several studies have shown that the TIP30 could affect the cells behaviors.[5] However, there are no studies about the role of TIP30 in bladder cancer cells until recently. The aim of this study is to investigate the function of TIP30 in the bladder cancer cell lines.


 > Materials and Methods Top


Cell culture

The bladder cancer cell line T24 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, People's Republic of China). Cells were cultured in DMEM/F12 (Gibco, Waltham, MA, USA) medium containing 10% fetal bovine serum (Hyclone, Logan, UT, USA). The cells were maintained at 37°C and 5% CO2.

Cell cycle assay

T24 cells were harvested with trypsin and suspended in ice-cold phosphate-buffered saline (PBS). The cells were stained with propidium iodide (PI) reagent according to the manufacturer's instruction. The percentage of PI was determined and analyzed by flow cytometry. The results were indicated as mean values from three independent determinations.

Cell apoptosis assay

T24 cells were harvested with trypsin and suspended in ice-cold PBS and stained with allophycocyanin/PI (APC/PI) reagent according to the manufacturer's instruction. The percentage of APC/PI was determined and analyzed by flow cytometry, which indicates the frequency of total apoptotic cells.

Cell proliferation assay

T24 cells were transfected with siRNA (50 μg/ml) in a 96-well plate and incubated at 37°C for 24 h. Cell proliferation was measured by the Cell Counting Kit-8 (CCK-8) Cell Proliferation Assay Kit (Invitrogen, Waltham, MA, USA). Briefly, CCK-8 reagent (5 μl) was dropped to each well and incubated for 2 h. Cell viability was calculated by absorbance tested at 450 nm. Each condition was repeated in triplicate.

Cell migration assay

Cell migration assay was performed in a 24-well transwell chamber (Corning, Corning, NY, USA) containing a polycarbonate filter (8 μm pore). The T24 cells were seeded as 1 × 105/ml in the transwell chamber. Conditioned medium containing 10% fetal bovine serum was added to the bottom of the chamber. The cells were incubated at 5% CO2 and 37°C for 24 h. After incubation, cells in the upper chamber that were attached but had not migrated were scraped off while the cells on the bottom of the upper chamber were fixed with methanol and stained by hematoxylin. The number of cells was counted in at least 6 random visual fields under a microscope. Results were calculated based on at least three individual experiments.

Cell invasion assay

The invasion assay was performed in a 24-well invasion chamber (Corning, Corning, NY, USA) containing a polycarbonate filter (8 μm pore). The T24 cells were seeded as 1 × 105/ml in the invasion chamber. Conditioned medium with 10% fetal bovine serum was added to the bottom part of the chamber. The cells were incubated in a 5% CO2 and 37°C for 24 h. After incubation, cells in the upper chamber that were attached but had not migrated were scraped off and the migrated cells on upper chamber were fixed with methanol and stained with hematoxylin. The number of cells was counted in at least 6 random visual fields under a microscope. Results were obtained from at least three repeats.

Western blot

The insoluble T24 cell lines protein lysate was removed by centrifugation at 12,000 rpm for 10 min at 4°C. Protein concentration was determined by the bicinchoninic acid protein quantification kit according to the manufacturer's instruction. 30 mg of the protein lysate was resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis, separated proteins were transferred to a nitrocellulose membrane and blocked with 5% nonfat milk in tris-buffered saline Tween-20 for 2 h at room temperature. Membranes were then incubated with specific primary antibodies (1:1000 dilution) in 5% nonfat milk overnight at 4°C with continuous agitation. The primary antibodies were TIP30 (Abgent), phosphorylated epidermal growth factor receptor (pEGFR) (Abcam), p-Akt (CST), Bcl-2 (Abcam), Bax (Proteintech), caspase 3 (CST), p21(Santa), cyclin D (Santa), cyclin E (Santa) matrix metalloproteinases 2 (MMP2) (CST), MMP6 (Abcam), MMP9 (CST). They were then incubated with secondary antibodies conjugated with horseradish peroxidase (1:20,000 dilution) for 2 h at 37°C. The signal was visualized with the enhanced chemiluminescence reagent.


 > Results Top


Overexpression of Tat-interacting protein 30 promoted the apoptosis of the bladder cancer cells

T24 cells were stained with APC/PI after the overexpression of TIP30. We found that overexpression of TIP30 could significantly promote the apoptosis of bladder cancer cells. The percentage of the APC/PI staining in the TIP30 overexpression group was 19.16% while the NC group and control group was 6.28% and 6.89%, respectively [Figure 1].
Figure 1: (a) The percentage of apoptosis cells in each group. (b) The flow cytometry results of apoptosis cells in each group. Overexpression of Tat-interacting protein 30 can promote the apoptosis of bladder cancer cells

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Overexpression of Tat-interacting protein 30 inhibited the proliferation of the bladder cancer cells

The viability of T24 was measured by the CCK-8 assay, and we found that the proliferation of T24 was significantly inhibited after the overexpression of TIP30 [Table 1] and [Figure 2].
Table 1: The percentage of G1, S, M phase on bladder cancer cells

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Figure 2: (a) The quantitative polymerase chain reaction results of examining the effect of overexpression in the T24 cells. (b) The Western blot results of examining the effect of overexpression in the T24 cells. (c) The cell counting kit-8 results of each group. Overexpression of Tat-interacting protein 30 can inhibit the proliferation of bladder cancer cells

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Overexpression of Tat-interacting protein 30 blocked the bladder cancer cells in G0/G1 phase

Then, we asked whether overexpression of TIP30 could affect the cell cycle progress in T24 cells. The result showed that 24 h later, 48.32% cells in the T24 blank group were in G0/G1 phase, 36.9% in S phase, and 14.78% in G2/M phase; 46.81% cells in the T24 NC group were in G0/G1 phase, 36.76% in S phase, and 16.44% in G2/M phase. However, 24 h after the overexpression of TIP30, 60.23% cells were in G0/G1 phase, 25.05% in S phase, and 14.73% in G2/M phase. Forty-eight hour later, 41.46% cells in the T24 blank group were in G0/G1 phase, 48.98% in S phase, and 9.56% in G2/M phase; 43.20% cells in the T24 NC group were in G0/G1 phase, 50.99% in S phase, and 5.81% in G2/M phase; 64.50% cells were in G0/G1 phase, 24.41% in S phase, and 11.10% in G2/M phase in the TIP30 overexpression group [Figure 3].
Figure 3: (a) The proliferation ratio of each group. (b) Percentage of cell cycle in each group

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Overexpression of Tat-interacting protein 30 inhibited the migration and invasion of bladder cancer cells

Then, we asked whether TIP30 affects the migration and invasion of bladder cancer cells. As shown in [Figure 4] and [Figure 5], the migration as well as the invasion of bladder cancer cells was inhibited after the overexpression of TIP30 [Figure 4] and [Figure 5].
Figure 4: Overexpression of Tat-interacting protein 30 can inhibit the migration of bladder cancer cells

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Figure 5: Overexpression of Tat-interacting protein 30 can inhibit the invasion of bladder cancer cells

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Overexpression of Tat-interacting protein 30 inhibited the cell cycle

By overexpressing the TIP30 in the bladder cancer cells, compared with the control group, the EGFR, pEGFR, p-Akt, Bcl-2, cyclin D, cyclin E cell cycle proteins were downregulated and suggested that the TIP30 may inhibit the cell proliferation in this pathway [Figure 6].
Figure 6: Overexpression of Tat-interacting protein 30 affects cell migration, proliferation pathways

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Overexpression of Tat-interacting protein 30 inhibited the cell migration pathway

By overexpressing the TIP30 in the bladder cancer cells, compared with the control group, the migration-related proteins, MMP2, MMP6, MMP9, were downregulated [Figure 6].

Overexpression of Tat-interacting protein 30 promoted the cell apoptosis through caspase pathway

By overexpressing the TIP30 in the bladder cancer cells, compared with the control group, the cell apoptosis-related proteins, bax, cleaved caspase3 were upregulated [Figure 6].


 > Discussion Top


In this article, we investigate the function of TIP30 in bladder cancer cells. We found that overexpression of TIP30 resulted in apoptosis, cell cycle arrest, inhibition of migration, and invasion in bladder cancer cells.

As we all know, the basis on cancer occurring and developing is the change of gene, including the oncogene activation and tumor suppressor gene inactivation. To date, several tumor suppressor genes have been found in cancer, such as deletion and mutation. TIP30, a new identified gene, was found to be related with cancer cells activities, such as proliferation, migration, and invasion.[3] TIP30 was originally identified as a metastasis suppressor gene in variant-SCLC (v-SCLC).[3] Functional studies have found that overexpression of TIP30 could inhibit growth and promote apoptosis in NIH3T3 and v-SCLC cells.[6]

In this study, we used the lentivirus to overexpress the TIP30 in bladder cancer cells and found that proliferation, migration, and invasion had been inhibited in bladder cancer cells when compared with the control group. These findings are similar to the previous study.

To date, the mechanisms by which TIP30 inhibits the development of cancer are still elusive. In some studies, TIP30 was found to affect the p53 pathway to inhibit the proliferation of cancer cells.[7] p53 was a regulator of cell cycle.[8],[9] It can regulate the cell cycle progress from G1 phase to S phase, thereby prolong the phase of cell proliferation and promote apoptosis in cancer cells. One study found that transfection of TIP30 into HepG2 cells lead to the upregulation of p53 gene expression.[10] This result suggested that TIP30 may regulate the proliferation via p53 pathway. In our study, after the overexpression of TIP30, the T24 cells were mostly arrested in the G0 and G1 phase, suggesting that TIP30 may affect the cell proliferation by the same mechanism in lung cancer. p53 pathway was related to the EGFR, p-Akt pathway. In this study, we also found that the EGFR/p-Akt pathway was inhibited by overexpression of TIP30. In this study, we observed a significant suppression of various proteins, which were regulated by the p53, such as EGFR, Bcl-2 cyclin D1, and cyclin E.

The MMPs are family proteins which regulate the cell migration. In this study, MMP2, MMP6, and MMP9 were all downregulated after the overexpression of TIP30. Therefore, TIP30 may inhibit the migration through the modulation of MMPs.

Apoptosis is a program of cell death. There are two pathways which lead to the apoptosis namely intrinsic and extrinsic pathway. In this study, we also found that antiapoptotic protein Bcl-2 was downregulated while proapoptotic protein Bax was upregulated after the overexpression of TIP30. Therefore, TIP30 may trigger apoptosis through the intrinsic pathway. It is well documented that multiple genes and pathways regulate the cellular apoptosis.[11] A report found that TIP30 could combine with importin beta in the nuclear membrane, then block the nuclear import of protein and induce the apoptosis.[12] TIP30 may also induce the expression of Bad and Siva gene to initiate the process of cell apoptosis with the help of p53. Overexpression of TIP30 could increase the level of p53 and then upregulate the apoptosis.[13] Shi et al. found that TIP30 gene could induce the upregulation of p53 and Bax to promote the cell apoptosis.[13] They also found that TIP30 could downregulate the Bcl-xl expression. The overexpression of TIP30 was found to repress the ER-mediated c-myc transcription and deficiency of TIP30 could enhance the c-myc expression.[14] The mutation of TIP30 was also related to the N-cadherin gene, which regulates the metastasis of cancer cells.[15] Several studies have found that transforming growth factor-beta (TGF-β) was regulated by TIP30.[15],[16] Both mitogen-activated protein kinases activation and Smad signaling are essential for TGF-β-induced EMT, which is related to the invasion and metastasis of cancer cells.[17]

TIP30 is a tumor suppressor gene. Overexpression of TIP30 could induce apoptosis and inhibit proliferation, migration, and invasion in the bladder cancer cells T24. However, the mechanism of how the TIP30 was regulated in bladder cancer remains unknown, and several hypotheses were proposed. In the future, we will look into details of the mechanism.

Financial support and sponsorship

This study was supported by the Science Foundation from Health Bureau of Wenzhou City of Zhejiang, China (2014S0236), and Zhejiang Provincial Natural Science Foundation (LY16H050032). We thank Mr. Bing Cai for his expert opinion on data analysis.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

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Zhao J, Ni H, Ma Y, Dong L, Dai J, Zhao F, et al. TIP30/CC3 expression in breast carcinoma: Relation to metastasis, clinicopathologic parameters, and P53 expression. Hum Pathol 2007;38:293-8.  Back to cited text no. 9
    
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Zhang X, Zhao J, Li XD, Yuan CT, Wang HQ, Wu MC, et al. Construction of adenovirus vector expressing TIP30 and its tumor suppressive effect in vitro and in vivo. Zhonghua Zhong Liu Za Zhi 2004;26:85-8.  Back to cited text no. 10
    
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Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70.  Back to cited text no. 11
    
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Shi M, Zhang X, Wang P, Zhang HW, Zhang BH, Wu MC. TIP30 regulates apoptosis-related genes in its apoptotic signal transduction pathway. World J Gastroenterol 2005;11:221-7.  Back to cited text no. 13
    
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Jiang C, Ito M, Piening V, Bruck K, Roeder RG, Xiao H. TIP30 interacts with an estrogen receptor alpha-interacting coactivator CIA and regulates c-myc transcription. J Biol Chem 2004;279:27781-9.  Back to cited text no. 14
    
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Luga V, McLean S, Le Roy C, O'Connor-McCourt M, Wrana JL, Di Guglielmo GM. The extracellular domain of the TGFbeta type II receptor regulates membrane raft partitioning. Biochem J 2009;421:119-31.  Back to cited text no. 15
    
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Ouyang H, Gore J, Deitz S, Korc M. microRNA-10b enhances pancreatic cancer cell invasion by suppressing TIP30 expression and promoting EGF and TGF-ß actions. Oncogene 2014;33:4664-74.  Back to cited text no. 16
    
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Davies M, Robinson M, Smith E, Huntley S, Prime S, Paterson I. Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF-beta1 involves MAPK, Smad and AP-1 signalling pathways. J Cell Biochem 2005;95:918-31.  Back to cited text no. 17
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
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