Metallothionein 3 Is a Hypoxia-Upregulated Oncogene Enhancing Cell Invasion and Tumorigenesis in Human Bladder Carcinoma Cells.

Ke-Hung Tsui,Yi-Syuan Shin,Chiu-Chun Chen,Tsui-Hsia Feng,Chen-Pang Hou,Horng-Heng Juang,Kang-Shuo Chang,Yu-Hsiang Lin

doi:10.3390/ijms20040980

Abstract

Metallothioneins have been viewed as modulators in a number of biological regulations regarding cancerous development; however, the function of metallothionein 3 (MT3) in bladder cancer is unexplored. We determined the regulatory mechanisms and potential function of MT3 in bladder carcinoma cells. Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-qPCR) assays revealed that TSGH-8301 cells expressed more MT3 levels than RT-4, HT1376, and T24 cells. Immunoblot and RT-qPCR assays showed that arsenic (AS2O3) treatments enhanced the gene expression of MT3. Hypoxia induced HIF-1α, HIF-2α, and MT3 expression; furthermore, HIF-2α-knockdown attenuated hypoxic activation on MT3 expression. Ectopic overexpression of MT3 increased cell proliferation, invasion, and tumorigenesis significantly in T24 and HT1376 cells in vitro and in vivo; however, MT3-knockdown in TSGH-8301 cells had the reverse effect. Moreover, knockdown of MT3 enhanced arsenic-induced apoptosis determined by the Annexin V-FITC apoptosis assay. MT3-overexpression downregulated the gene expressions of N-myc downstream regulated gene 1 (NDRG1), N-myc downstream regulated gene 2 (NDRG2), and the mammary serine protease inhibitor (MASPIN) in HT1376 and T24 cells, whereas MT3-knockdown in TSGH-8301 cells had the opposite effect. The experiments indicated that MT3 is an arsenic- and hypoxia-upregulated oncogene that promotes cell growth and invasion of bladder carcinoma cells via downregulation of NDRG1, NDRG2, and MASPIN expressions.

Highlights

Bladder cancer has become the ninth most common worldwide cancer and the sixth most seen malignancy in the United States, according to an epidemiologic statistics report published in 2017 [1,2]. many tumor markers associated with the development of bladder cancer have been well recognized, the rates of mortality and recurrence of bladder cancer are still high [3,4].Metallothioneins (MTs) are a class of low-molecular weight metal-binding proteins, which were first identified from the cortex of a horse kidney in 1957
Results of reporter assays showed that transient overexpression of HIF-1α and HIF-2α induced promoter activity of the human metallothionein 3 (MT3) gene (Figure 1H); in addition, 5 -delation report assays showed that HIF-1α and HIF-2α induced MT3 promoter activity was dependent on the 5 -flanking DNA fragment (−1 to −480) (Figure 1I)
We found that ectopic MT3 overexpression in HT1376 and T24 cells blocked gene expressions of N-myc downstream regulated gene 1 (NDRG1), N-myc downstream regulated gene 2 (NDRG2), and mammary serine protease inhibitor (MASPIN), but not NDRG3

Summary

Introduction

Bladder cancer has become the ninth most common worldwide cancer and the sixth most seen malignancy in the United States, according to an epidemiologic statistics report published in 2017 [1,2]. many tumor markers associated with the development of bladder cancer have been well recognized, the rates of mortality and recurrence of bladder cancer are still high [3,4].Metallothioneins (MTs) are a class of low-molecular weight metal-binding proteins, which were first identified from the cortex of a horse kidney in 1957. Bladder cancer has become the ninth most common worldwide cancer and the sixth most seen malignancy in the United States, according to an epidemiologic statistics report published in 2017 [1,2]. MTs were regarded as modulators, regulating several biological processes including cellular proliferation, differentiation, invasion, and carcinogenesis [5,6,7], with four main isoforms: Metallothionein 1 (MT1) (subtypes A, B, E, F, G, H, L, M, X), metallothionein 2 (MT2), metallothionein 3 (MT3), and metallothionein 4 (MT4). Several in vivo studies found that trace elements and hypoxia modulated the expressions of MTs in mammalian cells [9,10,11]. Unlike MT1 and MT2, the MT3 isoform has been a subject of limited understanding

Methods

Results

Discussion

Conclusion