[High Mobility Group Chromosomal Protein N2 Inhibit Cervical Cancer Transplanted in Nude Mice].

OBJECTIVES: To study the effect of high mobility group chromosomal protein N2 on inhibiting cervical cancer in nude mice. METHODS: Model of cervical cancer were established in nude mice. They were randomly divided into 4 groups including negative control group, HMGN2 group, cisplatin group and HMGN2 with cisplatin group. After 4 injections, the tumor size were calculated and tumor tissues were stained by haematoxylin eosin (HE) staining. RESULTS: Transplated tumor models were established successfully. The tumor sizes of negative control group [(0.38±0.12) cm³] were significantly lower than those of HMGN2 group [(0.14±0.07)cm³, cisplatin group [(0.11±0.06) cm³] and HMGN2 combined with cisplatin group[(0.11±0.07) cm³]. No differences were detected in HMGN2 group, cisplatin group and HMGN2 with cisplatin group in tumor sizes. The tumor inhibition rates of HMGN2 group, cisplatin group and HMGN2 with cisplatin group were 0.62±0.18, 0.71±0.17 and 0.70±0.18, respectively. The necrosis area were smaller in negative control group than in other three groups by HE staining. CONCLUSIONS: HMGN2 has a significant inhibitory effect on transplanted cervical cancer in nude mice.

High-mobility group nucleosome-binding domain 2 protein inhibits the invasion of Klebsiella pneumoniae into mouse lungs in vivo.

Since bacterial invasion into host cells is a critical step in the infection process and the predominance of multiple-antibiotic-resistant Klebsiella (K.) pneumoniae strains, using molecular agents to interfere with K. pneumoniae invasion is an attractive approach for the prevention of infection and suppress the immune inflammatory response. In previous studies by our group, high-mobility group nucleosome-binding domain 2 (HMGN2) protein was shown to exhibit anti-bacterial activity in vitro. The objective of the present study was to investigate the effects of HMGN2 protein on the invasion of K. pneumoniae 03183 in vivo. The results showed that pre-treatment with 128 µg/ml HMGN2 significantly reduced K. pneumoniae 03183 invasion into mouse lungs and increased the mRNA expression of CXCL1 and LCN2 within 2 h. Immunohistochemical staining showed that F-actin expression was significantly decreased, and fluorescence microscopy and western blot analysis further demonstrated that HMGN2 significantly blocked K. pneumoniae 03183-induced actin polymerization. These changes implied that HMGN2 may provide protection against K. pneumoniae 03183 infection in vivo.

HMGN2 protein inhibits the growth of infected T24 cells in vitro.

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.

High-mobility group protein N2 (HMGN2) inhibited the internalization of Klebsiella pneumoniae into cultured bladder epithelial cells.

Since bacterial invasion into host cells is an important step in the infection process, using the agents to interfere with bacterial internalization is an attractive approach to block the infection process. In this work, we describe a new, previously unrecognized role of the human cationic host defense peptide HMGN2 during Klebsiella pneumoniae infections. Our results revealed that the internalization of K. pneumoniae strain 03183 into cultured bladder epithelial cells (T24) was significantly reduced at HMGN2 concentrations that were unable to produce any bacteriostatic or bactericidal effect. Using microarrays and follow-up studies, we demonstrated that HMGN2 affected the internalization of K. pneumoniae strain 03183 by inhibiting the attachment of bacteria, and then decreasing bacteria-induced ERK1/2 activation and actin polymerization, which might contribute to bacterial internalization into T24 cells. This disruption of bacterial internalization implied that HMGN2 could provide protection against K. pneumoniae infections.

Inhibitory effect of HMGN2 protein on human hepatitis B virus expression and replication in the HepG2.2.15 cell line.

Natural killer (NK) cells and cytolytic T lymphocytes (CTL) have been implicated as important effectors of antiviral defense. We previously isolated a novel antibacterial polypeptide, which was identified as high mobility group nucleosomal-binding domain 2 (HMGN2), from human mononuclear leukocytes. This study examined the antiviral activity of HMGN2 against human hepatitis virus B. HMGN2 was isolated and purified from the acid soluble proteins of the human THP-1 cell line, and identified by mass spectrum, Western blot and antibacterial assay. The hepatitis B virus (HBV)-transfected HepG2.2.15 cell line was used in the in vitro assay system. In the range of 1-100 microg/ml HMGN2, no cytotoxicity for HepG2.2.15 cells was detected by MTT assay. When incubated with HMGN2 at 1-100 microg/ml for 72 or 144 h, there was a significant reduction in hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) expression, which were detected by ELISA, and a significant reduction in HBV DNA copies, which was determined by the real time quantitative PCR, in the supernatant of HepG2.2.15 cells. Northern and Southern blot analysis also showed that the levels of the HBV 3.5 kb and the 2.4/2.1 kb mRNA species and HBV replicative intermediate DNA were significantly reduced in the HMGN2-treated HepG2.2.15 cells. These results indicated that HMGN2 protein could markedly inhibit HBV protein expression and replication in vitro.

[Application of HMGN2-tag constructs to analysis of HMGN2 distribution in HeLa cells].

This study sought to clarify the distribution of HMGN2 in HeLa cells. The recombinant eukaryotic expression vectors pcDNA3. 1-myc-his-HMGN2 and pEGFP-N1-HMGN2 were constructed, and then were transfected into HeLa cells. immunocytochemistry staining indicated that HMGN2 were present not only in HeLa nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant by ELISA with rabbit anti-serum against HMGN2 and mouse anti-His6 monoclonal antibodies. The confocal microscope observation showed the same subcellular localization as that of immunocytochemistry staining. There results suggested that HMGN2 could be present in the nucleus and cytoplasm of HeLa cell as well as in the extracellular environment.

HMGN2: a novel antimicrobial effector molecule of human mononuclear leukocytes?

Leukocytes are a central cellular element of innate-immune defense in mammals. In addition to the generation of toxic oxygen radicals and nitric oxide, leukocytes express and secrete a broad array of antimicrobial proteins and peptides. In the study, an antimicrobial polypeptide was isolated and purified from human peripheral blood mononuclear leukocytes in the presence of interleukin (IL)-2. Microsequencing provided that its N-terminal amino sequence was PKRKAEGDAK, which was identical to high mobility group nucleosomal-binding domain 2 (HMGN2). Mass spectrometric value and Western blot also indicated its individual character of HMGN2. The antimicrobial assays showed that the Escherichia coli-based production of HMGN2 had a potent antimicrobial activity against E. coli ML-35p, Pseudomonas aeruginosa ATCC 27853, and to some extent, against Candida albicans ATCC 10231. The HMGN2 alpha-helical domain had the same antimicrobial activity as HMGN2. The immunocytochemistry staining, enzyme-linked immunosorbent assay, and Western blot revealed that HMGN2 was present in the cytoplasm of mononuclear leukocytes and released to the extracellular environment when stimulated with IL-2. These results suggest that HMGN2 would be a novel antimicrobial effector molecule of human mononuclear leukocyte.

[E. coli-based production of recombinant HMG-17 and its antibacterial domain].

Total RNA was extracted from human LAK cell, and a cDNA encoding mature peptide HMG-17 and its alpha helix domain was amplified by RT-PCR. The recombinant prokaryotic expression vector pGEX-1lambdaT-HMG-17 and pGEX-1lambdaT HMG-17alpha helix was constructed. Using affinity chromatography, thrombin cleaving and AU-PAGE elution, we obtained the purified HMG-17. Analyses of MIC, MEC and MBC indicated that HMG-17 and HMG-17alpha had strong antibacterial activity. MIC of the alpha-helic domain was almost the same as that of HMG17, suggesting that the alpha-helic structure would be essential for the antibacterial activity of HMG-17.

Alpha-helical domain is essential for antimicrobial activity of high mobility group nucleosomal binding domain 2 (HMGN2).

AIM: To examine the antimicrobial spectrum and functional structure of high mobility group nucleosomal binding domain 2 (HMGN2). METHODS: OMIGA protein structure software was used to analyze the two-dimensional structure of HMGN2. Synthetic short peptides were generated for studying the relationship between function and structure. Prokaryotic expression vectors were constructed for the holo-HMGN2 and its helical domain. Their E coli-based products were also prepared for antimicrobial testing. The antimicrobial assay included minimal effective concentration, minimal inhibitory concentration, and minimal bactericidal concentration. RESULTS: OMIGA protein structure software analysis revealed a transmembrane alpha-helical structure (the putative antimicrobial domain) located from position 18 to 48 of the HMGN2 protein sequence. The antimicrobial assay showed that the MIC of the recombinant holo-HMGN2 against E coli ML-35p (an ampicillin-resistance strain), Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231 were 12.5, 25, and 100 mg/L, respectively. Against the same microorganisms, the MIC of the synthetic HMGN2 alpha-helical domain were 12.5, 25, and 100 mg/L, respectively, that is, the same as with the recombinant form of HMGN2. In contrast, recombinant holo-HMGN2 was inactive against Staphylococcus aureus ATCC 25923. The synthetic N-terminal and C-terminal fragments of HMGN2 had no antimicrobial activity against E coli ML-35p, P aeruginosa ATCC 27853 or C albicans ATCC 10231. CONCLUSION: HMGN2 showed potent antimicrobial activity against E coli ML-35p, P aeruginosa ATCC 27853 and, to some extent, against C albicans ATCC 10231, but was inactive against S aureus ATCC 25923 in these assay systems. Itos alpha-helical structure may be essential for the antimicrobial activity of HMGN2.