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ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 2  |  Page : 299-304

HMGN2 protein inhibits the growth of infected T24 cells in vitro


State Key laboratory of Oral Diseases, Sichuan University, Chengdu, China

Date of Web Publication14-Jul-2014

Correspondence Address:
Yun Feng
State Key laboratory of Oral Diseases, Sichuan University, No 14, Section 3 South Renming Road, Chengdu, 610041
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.136577

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 > Abstract 

Aims of Study: Natural killer (NK) cells and cytolytic T lymphocytes (CTL) have been implicated as important effectors of antitumor defense. High mobility group nucleosomal-binding domain 2 (HMGN2) may be one of the effector molecules of CTL and NK cells. The antitumor effect and mechanism of HMGN2 was investigated in this study.
Materials and Methods: HMGN2 was isolated and purified from the human monocyte cell line THP-1 and then characterized by Tricine-SDS-PAGE, western blot, and mass spectrum determination. Confluent T24 cells were incubated with Klebsiella pneumoniae for 2 h, after which the extracellular bacteria were killed by the addition of gentamicin. The cells then were treated with a variety of concentrations of HMGN2. The effect of HMGN2 on the proliferation of T24 cells was analyzed with MTT, Hoechst and flow cytometry assays.
Results: Cell growth assay results demonstrated that HMGN2 significantly inhibited the growth of T24 bladder cancer cell lines infected by K. pneumoniae. Furthermore, results of the Hoechst and flow cytometry assays indicated that HMGN2 may promote apoptosis in this experimental model. These results suggest HMGN2 could inhibit the growth of the infected human bladder cancer cells in vitro.
Conclusion: HMGN2 protein could inhibit the growth of infected T24 cells in vitro, and the anti-tumor action of HMGN2 was due to induce apoptosis.

 > Abstract in Chinese 

HMGN2蛋白抑制体外培养的T24细胞增殖
摘要
目的:自然杀伤(NK)细胞和细胞毒性T淋巴细胞(CTL)在肿瘤免疫方面有起重要作用。高迁移率族核小体结合结构域2(HMGN2)可能是CTL和NK细胞的效应分子,本实验主要研究HMGN2的抗肿瘤作用及机制。
材料与方法:从人单核细胞株THP 1分离和纯化HMGN2,并通过聚丙烯酰胺凝胶电泳(Tricine-SDS-PAGE),免疫印迹法,和质谱进行鉴定。融合T24细胞在肺炎克雷伯菌孵育2h后,其胞外菌再用庆大霉素杀死。不同浓度的HMGN2处理细胞。MTT,Hoechst染色和流式细胞仪检测HMGN2对T24cells增殖的影响。
结果:细胞生长测定结果表明,HMGN2有明显抑制受肺炎克雷伯菌感染膀胱癌T24细胞株的作用。此外, Hoechst染色和流式细胞仪检测结果表明,在本实验模型中HMGN2可以促进细胞凋亡。这些结果提示HMGN2可抑制体外感染人膀胱癌细胞的生长。
结论:HMGN2蛋白能够抑制感染T24细胞体外生长,HMGN2的抗肿瘤作用是由于诱导细胞凋亡。
关键词:抗肿瘤活性,细胞凋亡,高迁移率族核小体结合的结构域2,感染t24细胞


Keywords: Antitumor activity, apoptosis, high mobility group nucleosomal-binding domain 2, infected T24 cells


How to cite this article:
Wei D, Zhang P, Zhou M, Feng Y, Chen Q. HMGN2 protein inhibits the growth of infected T24 cells in vitro. J Can Res Ther 2014;10:299-304

How to cite this URL:
Wei D, Zhang P, Zhou M, Feng Y, Chen Q. HMGN2 protein inhibits the growth of infected T24 cells in vitro. J Can Res Ther [serial online] 2014 [cited 2019 Sep 21];10:299-304. Available from: http://www.cancerjournal.net/text.asp?2014/10/2/299/136577


 > Introduction Top


Natural killer (NK) cells and cytolytic T lymphocytes (CTL) have been implicated as important effectors of antitumor defense. NK cells and CTL cells are rich in cytoplasmic granules. After the degranulation, they release some biologically active substances, acting on the target cells with cytotoxicity. [1] The granules in the cytoplasm of CTL cells contain perforin, granzyme, granulysin, and other effector molecules involved in the anti-tumor effect, as well as some unidentified components. [2],[3]

In our previous study, an antimicrobial polypeptide was isolated and purified from interleukin (IL)-2-stimulated human peripheral blood mononuclear leukocytes and identified to be high mobility group nucleosomal-binding domain 2 (HMGN2). When cultured mononuclear leukocytes were stimulated with IL-2, HMGN2 expressed in the cytoplasm and then secreted. [4]

HMGN2 is one of the most abundant non-histone nuclear proteins found in the nucleus of the cells of vertebrate and invertebrate organisms. [5] The HMGN2 gene is located in tumor suppressor gene areas and is highly conserved. [6],[7],[8] In addition, experimental evidence showed that the α-helical domain of HMGN2 has the ability of homing to tumors, especially to their vascular endothelium. [9]

In this study, we prepared a bulk of HMGN2 protein from THP-1, a human monocyte cell line, and examined its antitumor activity in a human urinary bladder carcinoma cell line T24. The results demonstrated that HMGN2 significantly inhibited the growth of T24 bladder cancer cell lines infected by K. pneumoniae. Furthermore, results of the Hoechst and flow cytometry assays indicated that HMGN2 may promote apoptosis in this experimental model.


 > Materials and methods Top


HMGN2 protein was isolated and identified of from the human THP-1 cell line. [10] Briefly, THP-1 cells, a monocyte cell line from ATCC, were cultured in RPMI-1640 medium in the presence of lipopolysaccharide (LPS) (Sigma Chemical Co., St.louis, MO) at a concentration of 10 ng/ml for two days, then collected and washed with phosphate-buffered saline. The cell pellet was dissolved in 5% acetic acid solution. The supernatant was collected and dialyzed against water at 4 °C for 48 h, lyophilized, and stored at −70 C. The purification of the HMGN2 proteins was subjected to reverse-phase high-performance liquid chromatography (RP-HPLC). All fractions were collected, lyophilized, reconstituted with 0.01% acetic acid, and stored at −70 C.

Protein concentrations were measured with the bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL). Bovine serum albumin (BSA) was used as the standard for the protein assay. Tricine sodium dodecyl sulfate (SDS)-PAGE was performed in mini-gel format, using the Modular Mini-Protein II electrophoresis system (Bio-Rad, Hercules, CA). The gels were stained with 0.1% Coomassie brilliant blue R-250. Mass spectrometric measurement was performed with an Applied Biosystems Voyager DE-PRO (ABI, Foster City, CA), equipped with a nitrogen laser (337 nm, 3 ns pulse width, 3.0Hz REP rate).

Western blot analysis was performed by using an anti-HMGN2 polyclonal antiserum prepared in Research Unit of Infection and Immunity, West China Medical Center, Sichuan University. [11] The 1:700 dilution of anti-HMGN2 polyclonal antibody in primary antibody buffer was added and incubated for 11 h at room temperature. The membrane then was incubated with goat anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibody at 1:5000-fold dilution in blocking buffer room temperature with gentle agitation. The ECL Detection Kit from Amersham Co. (Buckinghamshire, UK) was used in the experiments.

The T24 cells were cultured in RPMI 1640 (Sigma Chemical Co., St. Louis, MO), supplemented with 10% fetal bovine (Gibco-Invitrogen, Carlsbad, CA) under 5% CO 2 atmosphere at 37 °C. The clinical isolate of Klebsiella pneumoniae 5, was incubated with T24 cells (approximately 100 bacteria per epithelial cell) for 2 h and then treated for another 2 h with fresh medium containing gentamicin (100 μg/ml) to kill extracellular bacteria. Collected the supernatant medium. Then the wells were washed three times with PBS and adherent bacteria were released by the addition of l% TritonX-100. The supernatant medium and adherent bacteria were all quantified by plating appropriate dilutions on Mueller-Hinton (MH) agar plates.

For the cell growth assay, the cells were seeded in 24-well plates at a density of 5 × 10 4 cells per well. After 48 h later, confluent T24 cell monolayers were incubated with K. pneumonia 5 for 2 h and treated for another 2 h with fresh medium containing gentamicin to kill extracellular bacteria. The cells then were treated with different concentrations of HMGN2 protein (0 μg/ml, 1 μg/ml, 4 μg/ml, 8 μg/ml, or 10 μg/ml). The total cell number was quantified 48 h later.

Apoptosis was measured with the DeadEndTM Fluorometric Hoechst System (Promega Inc., Madison, WI). Cells were cultured on 6-well overnight, treated with K. pneumonia 5 and HMGN2 as indicated previously. After protein treatment, cells were washed with PBS and fixed by 4% methanol-free formaldehyde solution in PBS for 25 min at 4°C, washed with PBS and permeablized by 0.2% Triton X-100 in PBS for 55 min at room temperature. Staining was done according to the manufacturer's instructions. Fluorescence was visualized with an Olympus B × 60 microscope (Olympus Optical Co., Hamburg, Germany).

Apoptotic cell death was assessed with Annexin-V fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining to discriminate apoptotic cells from live cells and necrotic cells. In brief, after the treatment, 2 × 10 5 trypsinized cells of each group were stained according to the instruction of the Annexin-V FITC Apoptosis Detection Kit (R andand D Systems, Abingdon, United Kingdom) and analyzed with a flow cytometer (BD FACSAriaTM). The data were presented as dot plots showing fluorescence intensity of Annexin-V FITC and propidium iodide.

Quantitative results were expressed as the mean ± standard deviation. Student t-test was used for the statistical analysis of data, and P values were determined using a two-tailed t-test assuming unequal variances (Microsoft Excel). Values of P < 0.05 were considered significant.


 > Results Top


Tricine-SDS-PAGE and MS analysis indicated that HMGN2 peptide was isolated and purified from the acid soluble proteins of THP-1 cells. Fraction 20 [Figure 1] was analyzed by SDS-PAGE. Fraction 20 displayed only one protein band with a molecular mass of approximately 14KD [Figure 2]. Mass spectrometric analysis of the HMGN2 protein revealed the same molecular mass (m/z 9256.67) as HMGN2 [Figure 3]. A strong HMGN2 antibody-binding signal migration position was detected by western blot (data not shown). Gel overlay antimicrobial testing showed that the protein had antibacterial activity against E. coli 25922 (data not shown).
Figure 1: HPLC profile of the acid-soluble proteins separated from human THP-1 cells. The acid-soluble protein of human THP-1 cell was subjected to RP-HPLC on a 9.4250 mm Vydac C18 column and eluted with a 0-60% linear gradient of solvent B(0.1%TFA, 60% actonitrile, 40% water) in 60min at a flow rate of 1.0ml/min. Arrow indicates the HMGN2 protein

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Figure 2: Tricine-SDS-PAGE of the isolated HMGN2

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Figure 3: Mass spectrum of the isolated HMGN2. Mass spectrometric analysis of the isolated protein revealed the molecular mass (m/z =9256.67) as HMGN2

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After overnight incubation at 37 C, the plates of plating the released adherent bacteria displayed the K. pneumonia 5 colonies, whereas the supernatant coated was no colonies. This means that urinary bladder epithelial cells were able to contain the growth of intracellular bacteria, moreover, the extracellular bacteria were killed almost totally by using gentamicin at the concentration of 100 μg/ml [Figure 4].
Figure 4: T24 cells infected with Klebsiella pneumoniae was examined by colony assay. After incubation, in plate A, plated with released adherent bacteria, displayed the K. pneumonia 5 colonies. And in B, the extracellular bacteria were killed almost totally by using gentamicin at a certain concentration. The experiments were repeated three times and similar results were obtained

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After the initial incubation, cell viability had no apparent decline in the K. pneumonia 5 treated group compared with the control groups (P > 0.05) [Figure 5]. Therefore, K. pneumonia 5 was considered as no inhibitory effect on the growth of T24 cells when the bacteria/cell ratio was 100:1. Furthermore, the adherent amount of the K. pneumonia 5 to T24 cells was appropriate.
Figure 5: The effect of K. pneumonia 5 on T24 cells proliferation. After incubation, the cell viability had no apparent decline at 24, 48, or 72 hours later in the K. pneumonia 5 treated group compared with the blank control groups (P>0.05). K. pneumonia 5 was considered as no inhibitory effect on the growth of T24 cells. (A) The cell growth of T24 cells at 24-hr intervals up to 96 hr (×100). (B) The growth curve of T24 cells. Results represent the means ±SE (n=3)

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No significant differences (P > 0.05) were observed in cell growth in only the K. pneumonia 5 treated groups and the 5 mg/ml HMGN2 treated groups as compared with control. Of note, a decline in cell viability was apparent in the HMGN2 treated group compared with the control groups 48 h post-infection with K. pneumonia 5 [Figure 6]. Furthermore, HMGN2 had a dose-dependent inhibitory effect on the growth of infected T24 cells. The minimum inhibitory concentration was 4 μg/ml.
Figure 6: Inhibition of growth by HMGN2 in infected T24cells. Compared with the control group, cell viability was apparent decline in infected T24cells groups by treated with HMGN2 .The data shown represent the mean values (±S.D) based on three independent experiments. (*)P<0.05 and (**) P <0.01vs control at 48 h

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To determine whether the observed inhibition of HMGN2 protein on infected T24 cells was caused by apoptosis, the levels of apoptosis cells in the treatment groups were first measured by Hoechst assay. Our data indicated that in the control group, the cells morphology was rule, the color homogeneous. And in the K. pneumonia 5 treated groups, the morphology and numbers were not significantly different from the control group; however, after infection with K. pneumonia 5, the 8 μg/ml HMGN2 treated group displayed signs of apoptosis including nuclear enrichment, margination, fragmentation, and apoptotic body formation. Therefore, a decline in cell viability was apparent in the HMGN2 treated group as compared to the control groups (P < 0.01). Quantitative data showed that the percentage of Hoechst-positive cells in the control group was as high as 60% in cells, whereas the infected cells treated with 8 μg/ml HMGN2 only reached 49.6% Hoechst-positive [Figure 7].
Figure 7: Examination of apoptosis by Hochest assay. (A) Apoptotic effect was detected by Hochest assay (×100). (B) Hochest-positive cells were presented. The 8 μg/ml HMGN2 treated group displayed signs of apoptosis including nuclear enrichment, fragmentation, and apoptotic body formation compared with the control group . Results represent the means±SE (n=3). (**)P<0.01 vs control at 48 h

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As the Hochest-positive value, represents cells from necrosis and apoptosis, a more sensitive cytometric assay was performed by Annexin-V FITC and PI double staining. The current data indicated that the K. pneumonia 5 and 5 mg/ml HMGN2 treatments had little effect to induce apoptosis (Annexin-V +/PI-2 and Annexin-V -/PI +), whereas 4 μg/ml HMGN2 caused a moderate apoptosis effect on infected T24 cells. Nevertheless, a remarkable increase of apoptosis cells was detected in the 8 μg/ml HMGN2 treated group in infected cells [Figure 8]a. The ratio of apoptotic cells increased in T24 cell infection with K. pneumonia 5 as compared to the control [Figure 8]b.
Figure 8: (a) Flow cytometry analysis of apoptosis by using Annexin-V FITC and PI double staining at 48 h posterior to the treatment. (b) Quantitative analysis of the apoptotic cells from (A). (*)P<0.05 and (**) P<0.01vs control at 48 h

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 > Discussion Top


High mobility group N (HMGN) family contains five chromatin architectural proteins, which are present in higher vertebrates. [12] Of these proteins, HMGN1, 2 and 4 are expressed ubiquitously, [13],[14] while HMGN3 and 5 are expressed in specific tissues. [15],[16] The HMGNs bind specifically to nucleosome core particles, which consist of 147 bp of DNA, wrapped around an octamer of core histones. Each of these classes seems to play roles only in the nucleus. However, now, a variety of experiments have shown that the biologic functions of the HMG family are not so simple.

HMGNs are chromatin architectural proteins, which until lately were considered to be transcription co-regulators. However, in recent years their role in DNA repair and cancer progression has been established primarily by using HMGN1 knock-out mice. [17],[18],[19] These studies suggest that the archetype of HMGN1, has characteristics of a tumor suppressor gene. In addition to HMGN1, recently, the expression of HMGN5 (previously termed NSBP1) [20] was found to be elevated by 4-fold in highly metastatic breast cancer cells compared to low metastatic cells. [21] In mice, over-expression of HMGN5 in the uterus was associated with the development of uterine adenocarcinoma. [22] So HMGN5 may be involved in cancer progression also.

The HMGN2 gene is located in chromosome 1p36.1 and contains six exons, with an extremely high GC content and an "HpaII tiny fragment" island, indicative of a housekeeping gene that could be crucial for the regular functioning of cells. [23],[24] However, until now, the biological role of this protein has not been fully defined. A variety of experiments have shown that HMGN2 is preferentially associated with chromatin subunits. Furthermore, the abnormal gene or protein expression of HMGN2 is related to diseases such as neoplasms [9],[25] and autoimmune diseases. [26],[27],[28] Interestingly, in the search for tumor invasion-inhibiting factors in bovine liver extracts a peptide of 21 amino acids, which is identical to the C'-terminal part of HMGN2, was identified. [29],[30] Screening of phage-displayed cDNA libraries for peptides that selectively bind tumor cells, identified a peptide identical to the N'-terminal part of HMGN2. This 31 amino acids peptide, which corresponds to the NBD of HMGN2, selectively binds tumor cells both in vitro and in vivo. Upon binding the tumor cells the peptide is internalized and accumulated in the nucleus of the cells. [9]

CTL and NK cells are rich in cytoplasmic granules. After the degranulation, they release some biologically active substances, acting on the target cells with cytotoxicity. [1] The granules in the cytoplasm of CTL contain perforin, granzyme, granulysin, and other effector molecules involved in the anti-tumor effect, as well as some unidentified components. [2],[3] In our previous study, [4] we found that HMGN2 is released by human peripheral blood mononuclear leukocytes in the presence of IL-2. HMGN2 may be one of the effector molecules of CTL or NK cells.

The present study investigated the role of HMGN2 in bladder carcinoma using infected human urinary bladder transitional cell carcinoma cell lines (T24). The results showed that HMGN2 isolated from THP-1 cell lines inhibited the growth and induced the apoptosis of T24 cells infected by Klebsiella Pneumonia in vitro. As shown in [Figure 8], HMGN2 protein had no significant effect on the growth of normal T24 cell lines; however, HMGN2 could induce the apoptosis of Klebsiella pneumonia infected T24 cells. Therefore, infection is a critical factor in the inhibitory effect of HMGN2 protein on T24 cells. Chronic bacterial infection can contribute to tumor development, and a relationship may exist between this type of tumor and HMGN2. Klebsiella is gram-negative bacteria; in addition to E. coli, Klebsiella is considered as the most important opportunistic pathogen in urinary bladder infection. The current study suggests that in cancer cell lines, the activity of HMGN2 could be affected by the invasion of Klebsiella. Further studies are underway to determine whether the same is true for other species of cancer cell and bacteria, to delineate the role of HMGN2 as an effector molecule of anti-tumor activity of CTL cells, and to elucidate the anti-tumor mechanism of this protein.

In summary, the current study demonstrates that HMGN2 could inhibit the growth and induce the apoptosis of T24 cells infected by Klebsiella Pneumonia. This may be a novel strategy for the treatment of human urinary bladder transitional cell cancer.

 
 > References Top

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