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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 5  |  Page : 829-832

Effects of exogenous hydrogen sulfide on the proliferation and invasion of human Bladder cancer cells


Department of Urology Surgery, Huaihe Hospital, Henan University, Henan, Kaifeng 475000, PR China

Date of Web Publication13-Dec-2017

Correspondence Address:
Yang Li
Department of Urology Surgery, Huaihe Hospital, Henan University, Henan, Kaifeng 475000
PR China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_423_17

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 > Abstract 


Objective: The objective of this study is to evaluate the effects of exogenous hydrogen sulfide (H2S) on the proliferation and invasion of human bladder cancer cells.
Methods: Human bladder cancer EJ cells were cultured and then treated with sodium bisulfide (NaHS) (100 μmol/L for low dosage, 200 μmol/L for moderate dosage, 400 μmol/L for high dosage). The differences on proliferation and invasion of EJ cells were detected among different groups with MTT and transwell invasion assays. The differences in the expression levels of matrix metalloproteinase 2 (MMP2) and MMP9 among the groups were detected with Western blot.
Results: Cell proliferation activity was elevated by exogenous NaHS, with significantly statistical difference compared to the blank control group (P < 0.05). With the increased NaHS concentration, the EJ cell proliferation activity presented a statistically significant trend of increase, showing comparative differences among different groups (P < 0.05). Exogenous NaHS could also improve the invasion ability of bladder cancer EJ cells, which was significantly enhanced with increased NaHS dose (P < 0.001). The levels of MMP2 and MMP9 in EJ cells were significantly upregulated with the increased NaHS dose (P < 0.001).
Conclusion: Exogenous H2S may promote cell proliferation and invasion by upregulating the expression level of MMP2 and MMP9 in human bladder cancer EJ cells.

Keywords: Bladder cancer, hydrogen sulfide, invasion, proliferation


How to cite this article:
Liu H, Chang J, Zhao Z, Li Y, Hou J. Effects of exogenous hydrogen sulfide on the proliferation and invasion of human Bladder cancer cells. J Can Res Ther 2017;13:829-32

How to cite this URL:
Liu H, Chang J, Zhao Z, Li Y, Hou J. Effects of exogenous hydrogen sulfide on the proliferation and invasion of human Bladder cancer cells. J Can Res Ther [serial online] 2017 [cited 2019 Nov 19];13:829-32. Available from: http://www.cancerjournal.net/text.asp?2017/13/5/829/220476

Hui Liu and Junkai Chang contributed equally to this work.





 > Introduction Top


Bladder cancer has been one of the most common solid malignancies.[1] In China, the incidence of bladder cancer has been of the first place in the male genitourinary malignancies, accounting for 2.5% of the incidence of systemic malignancy.[2] Bladder cancer is a transitional epithelial tumor with the characteristics of easy recurrence, infiltration, metastasis, and other biological characteristics. Recurrence after the surgery operation has been an important cause for clinical treatment failure.[3],[4]

Hydrogen sulfide (H2S) is generally observed in the human body and participates in a variety of pathophysiological processes, which is considered to be a new gas signaling molecule in the human body.[5],[6],[7] Studies[8],[9],[10] have also shown that H2S and its synthetases were involved in the pathophysiological processes of tumors, but little research has been performed on the relationship between H2S and human bladder cancer proliferation, invasion and metastasis, and other biological behaviors. In this study, different concentrations of exogenous H2S were applied to treat human bladder cancer EJ cells. The effects of exogenous H2S on the proliferation and invasion abilities of bladder cancer cells and its possible mechanism were explored.


 > Methods Top


Cell culture and treatment

Human bladder cancer EJ cells were cultured in RPMI-1640 medium (life technology) in humidified air at 37°C with 5% CO2. The media was supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The EJ cells were treated with NaHS (there were three groups according to NaHS concentrations, 100 μmol/L for low dosage, 200 μmol/L for moderate dosage, and 400 μmol/L for high dosage).

MTT assay

Human bladder EJ cells were seeded into a 96-well plate and settled overnight. Twenty microliter of MTT solution was added into each well, and the plates were subsequently incubated for 4h in the incubator. Then, the liquid was removed from the plate, and 150 μl of dimethyl sulfoxide was added into each well. All plates were read at 490 nm.

Transwell invasion assays

The cells were treated with NaHS for 24 h. The cells (2 × 104/well) after treatment were seeded into the upper well of transwell chamber coated with Matrigel (with the pore size of 8 μm, BD Bioscience, USA) and allowed to invade toward the medium containing 10% FBS in bottom chamber for 24 h. The cells on the bottom of the chamber were stained with 0.1% crystal violet and photographed.

Western blot

Human bladder cancer EJ cells were lysed in protein radioimmunoprecipitation assay buffer and were cleared by centrifugation at 12,000 rpm for 40 min at 4°C. Protein expression was analyzed by Western blot with primary antibodies against matrix metalloproteinase 2 (MMP2) (Sigma, 1:1000), MMP9 (Sigma, 1:1000) or GAPDH (Santa Cruz, 1:1000), and followed by incubation with enzyme labeled secondary antibodies. Proteins were visualized by chemiluminescence detection.

Statistical analysis

The data were expressed as mean ± standard deviation and analyzed by two-sided Student's t-test (SPSS 17.0 software, http://publisher.brothersoft.com/spss-inc..html). In all statistical analyses, P value <0.05 was considered statistically significant.


 > Results Top


Effects of exogenous NaHS on the proliferation of EJ cells

The cell proliferation activity of EJ cells can be elevated with exogenous NaHS. NaHS was applied to treat EJ cells and generate intracellular H2S. Compared with the blank control group, the difference was statistically significant (P < 0.05); [Figure 1], and the cell proliferation activity was increased with the increased NaHS dose. The comparative differences were statistically significant among different concentrations of NaSH groups (P < 0.05); [Figure 1].
Figure 1: Proliferation activity of EJ cells after treatment with different dosages of NaHS. EJ cells were seeded in 96-well plates. After treating with different dosages of NaHS for 24 h or 48 h, MTT assay was performed. The experiments were repeated for three times

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Effects of exogenous NaHS on the invasion of EJ cells

The invasion ability of bladder cancer EJ cells can be improved with exogenous NaHS. With the increase of NaHS dose, the invasion of bladder cancer EJ cells was synchronously increased with statistical significance (P < 0.001); [Figure 2].
Figure 2: Effects of exogenous NaHS on invasion of EJ cells. (a) EJ cells were treated with different dosages of NaHS. Transwell invasion assay was performed after 24 h. The panel B showed the bar graph of cell number per field of the four groups

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Effects of NaHS on matrix metalloproteinase 2 and matrix metalloproteinase 9 expression levels

The levels of MMP2 and MMP9 in the bladder cancer EJ cells were significantly increased with the increased NaHS dosage (P < 0.001); [Figure 3].
Figure 3: Effects of exogenous NaHS on matrix metalloproteinase 2 and matrix metalloproteinase 9 expression level. (a) EJ cells were treated with different dosages of NaHS. Western blot assay was performed after 24 h and the protein level of matrix metalloproteinase 2 and matrix metalloproteinase 9 were detected. (b) Bar graph of matrix metalloproteinase 2 and matrix metalloproteinase 9 expression levels was quantitative analysis from (a)

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 > Discussion Top


H2S is the third gas signaling molecule observed in the human body, which is generated by essential amino-acid cysteine (L-cysteine, L-cys) in vivo through one carbon unit metabolism and transfer-sulfur pathway with enzyme catalysis. The included enzymes were mainly cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase.[11],[12],[13],[14] Endogenous H2S has been involved in a variety of pathophysiological processes in vivo. It reported that MAPK-Erk1/2 pathway, cyclin, cell death-related genes, ion channel, and endoplasmic reticulum stress were all linked with H2S signaling.[15],[16]

Bladder cancer has been one of the most common tumors in the urinary system. It is known that one carbon unit metabolism and transfer sulfur pathway that participated in the production of endogenous H2S may increase the risk of bladder cancer.[17],[18]

The content of H2S in 76 cases of bladder cancer tissues and 15 cases of normal bladder tissues was determined with sensitive sulfur electrode.[19] The differences of H2S content in each group were compared as well as the synthetases. Moreover, the relationship was explored between H2S and its synthetases, as well as the malignant progression of bladder cancer. The results showed that H2S content was associated with the depth of tumor infiltration and H2S content in muscle-invasive bladder cancer would beelevated. The results also showed that the content of endogenous H2S in bladder cancer was significantly higher than that of in normal bladder tissues. In addition, the H2S content was significantly different among different stages and grades of bladder cancer.

In the present study, different concentrations of exogenous NaHS were applied to treat human bladder cancer EJ cells and to explore the effects of exogenous NaHS on the proliferation and invasion abilities of bladder cancer cells, as well as the possible mechanism. The results showed that exogenous NaHS could promote both the cell proliferation and invasion abilities, both of which were increased synchronously with the increased NaHS concentrations. We also found that exogenous NaHS treatment significantly increased the expression levels of MMP2 and MMP9 in EJ cells, both of which were increased along with the increasd NaHS dose. Therefore, we speculated that exogenous H2S could promote cell proliferation and invasion by upregulating the expression levels of MMP2 and MMP9 in human bladder cancer EJ cells.

In general, MMPs expression stayed at low level or even absent in normal tissues, while it would become abnormally high in malignant tumors. Specifically, the relationship between IV-type collagenase (MMP2, MMP9) and the tumor was even more close.[20] Invasion has been one of the most important processes linked to tumor metastasis. In the process of invasion, the tumor must enable to hydrolyze the extracellular matrix to get through the extracellular matrix into new colonization site. MMP2 and MMP9 are two most essential enzyme systems for hydrolyzing IV-type collagen. In this study, with the increase of exogenous NaHS concentrations, the invasion ability of EJ cells was increased, and the expression levels of MMP2 and MMP9 were synchronously increased. Therefore, the increase of MMP2 and MMP9 may be important factors for enhancing EJ cell invasion. However, the molecular mechanism of H2S promotion on human bladder cancer EJ cell proliferation and invasion has still not been clear, and further, research is necessary.

Financial support and sponsorship

This study was financially supported by the Key project of science and technology research of Henan Provincial Department of Education (No. 15A320045).

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3]



 

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