The GroEL protein of Porphyromonas gingivalis accelerates ...

molecular oral microbiology

molecular oral microbiology

The GroEL protein of Porphyromonas gingivalis accelerates tumor growth by enhancing endothelial progenitor cell function and neovascularization

F-Y. Lin1,2, C-Y. Huang1,2, H-Y. Lu1,2, C-M. Shih1,2, N-W. Tsao3, S-K. Shyue4, C-Y. Lin5, Y-J. Chang6, C-S. Tsai7, Y-W. Lin1,2,8 and S-J. Lin9,10

1 Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 2 Division of Cardiology, Department of Internal Medicine and Cardiovascular Research Center, Taipei Medical University Hospital, Taipei, Taiwan 3 Division of Cardiovascular Surgery, Taipei Medical University Hospital, Taipei, Taiwan 4 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 5 Department of Computer Science and Information Management, Hung Kuang University, Taichung, Taiwan 6 Graduate Institute of Clinical Medicine, Taipei Medical University, Taipei, Taiwan 7 Division of Cardiovascular Surgery, National Defense Medical Center, Taipei, Taiwan 8 Institute of Oral Biology, National Yang-Ming University, Taipei, Taiwan 9 Division of Cardiology, Taipei Veterans General Hospital, Taipei, Taiwan 10 Institute of Clinical Medicine, National Yang-Ming University

Correspondence: Shing-Jong Lin and Yi-Wen Lin, Institute of Clinical Medicine and Institute of Oral Biology, National Yang-Ming University, No.155, Sec.2, Linong Street, Taipei 112, Taiwan Tel.: + 886 2 2826 7000; fax: + 886 2 2820 9310; E-mails: sjlin@.tw (S-JL) and ywlin@ym.edu.tw (Y-WL)

Keywords: GroEL; Porphyromonas gingivalis; tumor Accepted 6 September 2014 DOI: 10.1111/omi.12083

SUMMARY

Porphyromonas gingivalis is a bacterial species that causes destruction of periodontal tissues. Additionally, previous evidence indicates that GroEL from P. gingivalis may possess biological activities involved in systemic inflammation, especially inflammation involved in the progression of periodontal diseases. The literature has established a relationship between periodontal disease and cancer. However, it is unclear whether P. gingivalis GroEL enhances tumor growth. Here, we investigated the effects of P. gingivalis GroEL on neovasculogenesis in C26 carcinoma cell-carrying BALB/c mice and chick eggs in vivo as well as its effect on human endothelial progenitor cells (EPC) in vitro. We found that GroEL treatment accelerated tumor growth (tumor volume and weight) and increased the mortality rate in C26 cell-carrying BALB/c mice. GroEL promoted neovasculogenesis

in chicken embryonic allantois and increased the circulating EPC level in BALB/c mice. Furthermore, GroEL effectively stimulated EPC migration and tube formation and increased E-selectin expression, which is mediated by eNOS production and p38 mitogen-activated protein kinase activation. Additionally, GroEL may enhance resistance against paclitaxel-induced cell cytotoxicity and senescence in EPC. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to the neovasculogenesis of tumor cells and resulting in accelerated tumor growth.

INTRODUCTION

Periodontitis is a bacteria-induced inflammatory disease that destroys periodontal tissues, including the gingiva, cementum, alveolar bone and periodontal

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? 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 198?216

F-Y. Lin et al.

Porphyromonas gingivalis accelerates tumor growth

ligament. To date, several bacterial species have been reported to be associated with periodontitis. Periodontopathogenic bacteria may contribute to tissue and bone destruction in periodontitis through their capacity to release a number of virulence factors, such as lipopolysaccharide, gingipains (Malek et al., 1994; Reife et al., 1995), and GroEL (Tabeta et al., 2000; Chung et al., 2003). GroEL, belonging to the heat-shock protein 60 family, has an important role in the folding of newly synthesized proteins and prevents their aggregation. However, GroEL is also widely recognized as an important molecule in various bacterial infections and autoimmune diseases (Maeda et al., 2000; Ueki et al., 2002). Several studies have reported that some bacterial GroELs stimulate the production of proinflammatory cytokines in human monocytes (Zhang et al., 1993; Retzlaff et al., 1994; Tabona et al., 1998) and also upregulate the expression of adhesion molecules (Verdegaal et al., 1996; Galdiero et al., 1997). On the other hand, there is evidence demonstrating that the salivary GroEL-associated immunoglobulin A is related to the severity of periodontal disease (Fukui et al., 2006). GroEL from Aggregatibacter actinomycetemcomitans and Campylobacter rectus may stimulate the production of interleukin-6 or interleukin-8 by human gingival epithelial cells or gingival fibroblasts (Goulhen et al., 1998; Hinode et al., 1998; Tanabe et al., 2003); therefore, periodontal treatments may effectively decrease the serum level of anti-GroEL antibody (Yamazaki et al., 2004), and a vaccine directed against the Porphyromonas gingivalis GroEL protein may reduce bacteria-induced alveolar bone loss (Lee et al., 2006). Previous evidence strongly indicates that the GroEL from periodontopathogenic bacteria may possesses biological activity and may be involved in the progression of periodontal diseases. Porphyromonas gingivalis is an anaerobic gram-negative bacterium that is strongly associated with disease progression (Lamont & Jenkinson, 2000; Ezzo & Cutler, 2003). In many periodontitis patients, sera were positively reactive with the GroEL of P. gingivalis and the titer of anti-GroEL antibody was higher, indicating the presence of an immune response to GroEL in patients with periodontitis (Tabeta et al., 2000; Chung et al., 2003). The P. gingivalis GroEL is also able to stimulate the transcriptional activation of nuclear factor-jB and the inflammatory response, which may be reversed by

the inhibition of Toll-like receptors 2 and 4 in THP-1 cells (Argueta et al., 2006). GroEL from P. gingivalis is a critical immunodominant antigen in patients with periodontitis and may contribute to pathogenic processes and inflammation.

There is strong evidence indicating that periodontitis is associated with the occurrence of systemic complications such as atherosclerosis, diabetes, pulmonary disease, preterm delivery and cancer (Michaud et al., 2008; Hayashi et al., 2010). More recently, several studies have revealed an association between periodontal diseases and cancers, such as metastatic pancreatic cancer (Michaud et al., 2007), osteogenic sarcoma (Solomon et al., 1975), esophageal cancer (Fitzpatrick & Katz, 2010), gastric cancer (Abnet et al., 2005; Michaud et al., 2008; Fitzpatrick & Katz, 2010), lung cancer (Hujoel et al., 2003) and nonHodgkin lymphoma (Michaud et al., 2008). Porphyromonas gingivalis was especially abundant in the malignant oral epithelium, indicating a critical relationship between P. gingivalis and carcinoma (Katz et al., 2011). However, P. gingivalis has the capability to prevent the apoptosis of epithelial cells and protect cancerous processes (Yilmaz, 2008; Hajishengallis, 2009) via the manipulation of an intercellular signaling pathway (Kuboniwa et al., 2008). Although GroEL from P. gingivalis was suggested to be a potent stimulator of inflammatory cytokines in periodontal disease and systemic inflammation, many potential confounding effects of GroEL on tumorigenesis still need to be elucidated. Therefore, in this study, we used a tumor cell model in mice to explore the effects of GroEL from P. gingivalis on tumorigenesis. Additionally, neovascularization is required for the growth, survival and metastasis of tumors, which is mediated by endothelial progenitor cell (EPC) function in the majority of tumors. We also investigated the effects of GroEL on neovascularization in animals in vivo and explored the underlying mechanisms using human EPC in vitro.

METHODS

Construction of the P. gingivalis GroEL expression vectors

The genomic DNA of P. gingivalis (ATCC No. 33277) was extracted using the EasyPure Genomic DNA mini kit (Bioman Scientific Co., Taipei, Taiwan). The open reading frame of GroEL was originally amplified

? 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 30 (2015) 198?216

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by polymerase chain reaction (PCR) using 100 ng P. gingivalis genomic DNA as a template, 0.2 mM dNTPs, 1 lM each of the gene specific primers and 1 U Pfu DNA polymerase (Promega, Madison, WI, USA) with the following program: one cycle of 95?C for 5 min; 38 cycles of 95?C for 45 s, 68?C for 45 s, and 72?C for 2 min; and one cycle of 68?C for 45 s and 72?C for 10 min with a final incubation at 72?C for 10 min with 1 U Taq DNA polymerase. The GroEL-specific forward and reverse primers used in the PCR are shown in Table 1. The amplified GroEL cDNA fragment was then cloned into the pCR2.1TOPO vector (Invitrogen, Carlsbad, CA, USA) and subsequently cloned in-frame into the EcoRI sites of the pGEX-5X-1 expression vector (GE Healthcare Amersham Biosciences, Carlsbad, CA, USA) for expression in Escherichia coli (DH5a).

Purification of the GroEL recombinant protein

BL21 cells were transformed with the pGEX-5X-1GroEL expression vector, and the GroEL recombinant protein was purified. Briefly, the BL21 cells (RBC Bioscience, New Taipei City, Taiwan) containing the pGEX-5X-1-GroEL plasmid were grown overnight at 37?C in 2 ml LB medium supplemented with 100 lg ml?1 ampicillin. Then, 1.25 ml overnight culture was transferred into 100 ml LB/ampicillin medium and grown at 37?C to an A600 of 0.6?0.8 (approximately 2 h). Expression of the fusion protein was then induced by adding Isopropyl b-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM at 30?C for 6 h. The bacteria were pelleted by centrifugation for 10 min at 9800g, and recombinant GroEL was extracted under

Table 1 Primers used for polymerase chain reactions

Gene Integrin a1 Integrin a2 Integrin b1 Integrin b3 E-selectin GAPDH

Sequence

Forward: 50-tgc cat tat ggg tca tcc tgc tg-30 Reverse: 50-cac ata ttt gag gca aac ctg agg-30 Forward: 50-cat caa cgt tcc aga cag tac agc-30 Reverse: 50-gct aac agc aaa agg att cca gc-30 Forward: 50-ctg gtg tgg ttg ctg gaa ttg ttc-30 Reverse: 50-cct cat act tcg gat tga cca cag-30 Forward: 50-cct gct cat ctg gaa act cct ca-30 Reverse: 50-cgg tac gtg ata ttg gtg aag gta g-30 Forward: 50-ttg gta gct gga ctt tct gct gc-30 Reverse: 50-gta aga agg ctt ttg gta gct tcc-30 Forward: 50-tgc ccc ctc tgc tga tgc c-30 Reverse: 50-cct ccg acg cct gct tca cca c-30

GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

native conditions using the GST Gene Fusion System according to the manufacturer's instructions (GE Healthcare Amersham Biosciences). Finally, the recombinant GroEL protein was purified with elution buffer containing 50 mM Tris?HCl and 10 mM reduced glutathione (pH 8.0). The quantity of recombinant GroEL protein was measured using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). The fusion protein was detected by sodium dodecyl sulfate gel electrophoresis and identified by immunoblotting with a GST antibody (GE Healthcare Amersham Biosciences). The endotoxin levels in the recombinant GroEL protein were measured using a Limulus Amebocyte Lysate kit from Cambrex Inc. (Walkersville, MD, USA). Lipopolysaccharide levels were below 1 pg ml?1.

Cultivation of C26 mouse colon carcinoma cells

BALB/c mouse-derived colon carcinoma C26 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum, 2 mM glutamine, 4.5 g l?1 glucose, 10 mmol l?1 HEPES, 1.0 mmol l?1 sodium pyruvate and 1% antibiotic/antimycotic mixture. The medium was refreshed every 2?3 days, and the cells were subcultured when the cell density reached 70?80% confluence. Before the injection of C26 cells for the murine tumorigenesis study, the cells were freshly prepared as single-cell suspensions in phosphatebuffered saline (PBS; 106 cells in 100 ll PBS).

Murine tumorigenesis model

All 6- to 8-week-old male BALB/c mice were purchased from BioLASCO Taiwan Co., Ltd. All the animals were treated according to protocols approved by the Institutional Animal Care Committee of Taipei Medical University (Taipei, Taiwan). The experimental procedures and animal care conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All mice were kept in microisolator cages on a 12-h day/night cycle and fed a commercial mouse chow diet (Scientific Diet Services, Essex, UK) with water provided ad libitum. Fifty-four BALB/c mice were used and randomly divided into nine groups. Group 1 (naive) was the naive control group; mice in group 2 (C26 cell injection control) received 106 C26 cells via subcutaneous injection into their shaved back at the beginning of week 2

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Porphyromonas gingivalis accelerates tumor growth

of the experiment; mice in group 3 [GST 400 lg kg?1 body weight (BW) + C26 cell injection] received a tail vein injection of 400 lg kg?1 BW of GST protein twice a week throughout the experiment (5 weeks) and received an injection of C26 cells at the beginning of week 2 of the experiment; mice in group 4 (GroEL 100 lg kg?1 BW + C26 cell injection) received a tail vein injection of 100 lg kg?1 BW of GroEL twice a week throughout the experiment and received an injection of C26 cells at the beginning of week 2 of the experiment; mice in group 5 (GroEL 200 lg kg?1 BW + C26 cell injection) received a tail vein injection of 200 lg kg?1 BW of GroEL twice a week throughout the experiment and received an injection of C26 cells at the beginning of week 2 of the experiment; mice in group 6 (GroEL 400 lg kg?1 BW + C26 cell injection) received a tail vein injection of 400 lg kg?1 BW of GroEL twice a week throughout the experiment and received an injection of C26 cells at the beginning of week 2 of the experiment; mice in group 7 (GST 400 lg kg?1 BW) received a tail vein injection of 400 lg kg?1 BW of GST twice a week throughout the experiment; mice in group 8 (GroEL 200 lg kg?1 BW) received a tail vein injection of 200 lg kg?1 BW of GroEL twice a week throughout the experiment; and mice in group 9 (GroEL 400 lg kg?1 BW) received a tail vein injection of 400 lg kg BW of GroEL twice a week throughout the experiment. Additionally, groups 3?9 began to receive tail vein injections of the indicated GroEL or GST twice during the first week of the experiment. The length and width of mass were examined twice per week. At the end of experiment (day 35), the mice were sacrificed and whole tumors were removed. The tumor masses were determined and the tumor tissue was analysed by immunohistochemistry.

Laser scanning confocal microscopy for blood vessel visualization

At the end of the experiment, the mice were anesthetized by intraperitoneal injection of xylocaine (2 mg kg?1 of BW) plus Zoletil (containing the dissociative anesthetic Tiletamine/Zolazepam at a ratio of 1 : 1; 5 mg kg?1 BW) and live perfused by direct intracardiac injection of the lipophilic carbocyanine dye (7 ml per mouse) 1,10-dioctadecyl-3,3,30,30-tetramethylindo-carbocyanine perchlorate (DiI) (Life Technology, Carlsbad, CA, USA) as previously reported (Li et al., 2008; Liu et al., 2010). After the

DiI infusion was complete, 4% paraformaldehyde was injected for fixation, and the whole tumor mass was harvested. Tumor masses were embedded with paraffin and cross-sectioned in 5-lm-thick sections. The slides were observed using laser scanning confocal microscopy (LSM510, Zeiss Inc., Oberkochen, Germany). The DiI-stained signal was counted in high-power field (9 200).

Immunohistochemical analysis of tumor masses

The whole tumor masses were harvested, the adhering tissues were carefully removed, and serial 5-lm-thick paraffin-embedded sections were immersion-fixed with 4% buffered paraformaldehyde. Immunohistochemical staining was performed on mouse tumors using the goat anti-Von Willebrand factor (vWF; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) antibody (eNOS; Cell Signaling, Danvers, MA, USA) followed by counterstaining with hematoxylin. The stained slides were observed using light microscopy. After staining, the brown-stained vessel-like structure was calculated in high-power field (9 200).

Flow cytometry of mice circulating EPC

To investigate the mobilization of EPC, a fluorescence-activated cell sorting (FACS) Caliber flow cytometer (Becton Dickinson, San Jose, CA, USA) was used. Total leukocytes were isolated from 100 to 200 ll of peripheral blood by incubation with 200? 400 ll of red blood cell lysis buffer and washing twice in PBS. Then, the total leukocytes were incubated with fluorescein isothiocyanate-conjugated anti-mouse CXCR4 (eBioscience, San Diego, CA, USA), allophycocyanin-conjugated anti-mouse Flk-1 (VEGFR-2, eBioscience, San Diego, CA, USA), and phycoerythrinconjugated anti-mouse Sca-1 antibodies (eBioscience, San Diego, CA, USA). Isotype-identical antibodies served as controls (Becton Dickinson, Franklin Lakes, NJ, USA). Each analysis included 150,000~300,000 total leukocytes. Circulating EPC were considered to be derived from the monocyte population and were gated with triple positivity for CXCR4, Sca-1 and Flk-1.

Chick chorioallantoic assay for angiogenesis

The chorioallantoic assay was modified according to previous reports (Liu et al., 2011a). Eight-day-hatched

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fertile chicken eggs were randomly divided into six groups. At the beginning of the studies, the eggs were sterilized with 70% ethanol then opened in an air chamber (at the blunt pole of the egg) to expose the crust endomembrane. Autoclaved 5-mm diameter circular chromatography paper (Whatman, Maidstone, Kent, UK) was moistened and saturated with experimental GST, GroEL, or phorbol myristate acetate. The conditioned circular paper was transplanted onto the chicken embryonic allantois carefully, and then, the hole of the crust was sealed with UV-irradiated adhesive tape. The eggs were incubated in a chamber at 37?C with 80% humidity under normoxia. The crust hole was reopened on the 11th breeding day. The neo-angiogenic vessels were observed and photographed. According to a previous report, the vessels were divided into two classes. Vessel diameter 1 mm was defined as Class I; vessel diameter < 1 mm was defined as Class II (Liu et al., 2011a).

Isolation and cultivation of human EPC

Total mononuclear cells were isolated from 40 ml peripheral blood from healthy young male volunteers by density-gradient centrifugation with Histopaq-1077 (density, 1.077 g ml?1; Sigma, St Louis, CA, USA). The Institutional Review Board (Taipei Medical University-Joint Institutional Review Board) approved this study, and all volunteers gave written informed consent before all procedures. Mononuclear cells (1 9 107 cells) were plated in 2 ml of endothelial growth medium with supplements (EGM-2 MV; Cambrex, Charles, IA, USA) on fibronectin-coated six-well plates and incubated at 37?C. After 4 days of culture, the medium was changed, and non-adherent cells were removed; the medium was replaced every 3 days, and each colony/cluster was observed. A certain number of early EPC continued to grow into colonies of late EPC, which emerged 2?4 weeks after the start of the mononuclear cell culture. The characterization of the EPC was described previously (Chen et al., 2007).

Late EPC tube formation assay

A tube formation assay was performed on EPC to assess their angiogenic capacity, which is involved in new vessel formation (Hiroki et al., 2004). The in vitro tube formation assay was performed using the Angio-

genesis Assay Kit (Chemicon, Temecula, CA, USA) according to the manufacturer's protocol. In brief, ECMatrix gel solution was thawed at 4?C overnight, mixed with ECMatrix diluent buffer, and placed in a 96-well plate at 37?C for 1 h to allow the matrix solution to solidify. EPC were treated with GroEL and then harvested. A total of 104 cells were placed on the matrix solution with GroEL, and the samples were incubated at 37?C for 18 h. Tubule formation was inspected under an inverted light microscope. Four representative fields were observed, and the average of the total area of the completed tubes formed by the cells was compared using IMAGE-PRO PLUS computer software.

Wound-healing assay for late EPC migration

The migratory function of late EPC, which is essential for vasculogenesis, was evaluated using a woundhealing assay. Late EPC were cultured in a 12-well plate. The confluent cells (approximately 2 9 105 cells per well) were wounded by scraping with a 100-ll pipette tip, which denuded a strip of the monolayer that was 300 lm in diameter. The cells were supplied with medium containing 5% fetal bovine serum, and the rate of wound closure was observed after 24 h. The distance of the gap was measured under the 4 9 phase objective of a light microscope (Olympus IX71, Tokyo, Japan), monitored with a CCD camera (Macro FIRE 2.3A), and captured with a video graphic system (PICTURE FRAME APPLICATION 2.3 software).

Western blot analysis

Membrane fractions and total cell lysates were extracted from the EPC. The proteins were separated by sodium dodecyl sulfate?polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were probed with the mouse anti-eNOS antibody (Millipore, Billerica, MA, USA), anti-phospho-eNOS antibody (Millipore), rabbit anti-p38 antibody, rabbit anti-phospho-p38 antibody, rabbit anti-c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) antibody, rabbit antiphospho-JNK/SAPK antibody, rabbit anti-p44/p42 mitogen-activated protein kinase (MAPK) antibody, or mouse anti-phospho-p44/p42 MAPK antibody (all MAPK antibodies were purchased from Cell Signaling Technology). The proteins were visualized with an

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