compound 10 was checked by Jasco HPLC analysis possessed more than 99% purity
6.2. Biological evaluation 1. Chemicals and reagents
Benzimidazole (BI), amoxicillin (AMX), clarithromycin (CLR), and metronidazole (MTZ),
gentamicin, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) were
purchased from Sigma–Aldrich (St. Louis, MO). Lipofectamine 2000 and F12 cell culture media
were purchased from Invitrogen (Carlsbad, CA). β-galactosidase expression vector, luciferase
substrate (Promega, Madison, MA), fetal bovine serum (FBS, Gibco BRL, Grand Island, NY) were
obtained from various suppliers. The NF-κB-luc promoter construct was kindly gifted from Dr.
Chih-Hsin Tang (Department of Pharmacology, China Medical University) [30]. All other
chemicals and reagents were of the highest grade commercially available and supplied either by
Sigma-Aldrich or Merck (Whitehouse Station, NJ).
6.2.2. H. pylori strains
H. pylori, strain 26695 (ATCC 700392) was used as a reference strain as described previously
[31]. The antibiotic resistant H. pylori strains, v633 and v1254, were clinical isolates, which were
characterized as resistant to both metronidazole and clarithromycin as previously described [27]. H.
pylori strains were routinely cultured on Brucella blood agar plates (Becton Dickinson, Franklin
Lakes, NJ), containing 10% sheep blood under micro-aerophilic conditions at 37 °C for 48–72 h.
6.2.3. Determination of anti-H. pylori activity
The disk agar diffusion method was used to evaluate the anti-H. pylori activities of BI
derivatives as described previously [31]. Four standard antimicrobial agents were used as positive
controls, namely: benzimidazole (BI, 100 μg/mL), amoxicillin (AMX, 50 μg/mL), clarithromycin
(CLR, 50 μg/mL), and metronidazole (MTZ, 800 50 μg/mL). While DMSO was used as a negative
control.
6.2.4. Determination of minimum bactericidal concentration (MBC)
H. pylori strain 26695 and multidrug-resistant isolates were used to determine the MBC for BI
and FMTMB using micro-dilution analysis at pH 5.0–8.0 as described previously [32]. The MBC is
the lowest concentration of a tested sample that completely inhibits visible H. pylori growth on a
Brucella blood agar plate.
6.2.5. Cell culture and cytotoxicity assay
Human gastric adenocarcinoma epithelial cells (AGS cells, ATCC CRL 1739) obtained from
American Type Culture Collection (ATCC, Rockville, MD), were performed and cultured by a
previously described method [31]. MTT assay was used to test the benzimidazole derivatives
cytotoxicity towards AGS cells as described in our previous report [33].
6.2.6. Assays of H. pylori adhesion to and invasion into AGS cells
H. pylori invasion activity in AGS cells was investigated using a standard gentamicin assay as
previously described [33]. Analyses H. pylori anti-adhesion and anti-invasion of AGS cells were
performed as previously reported [33]. The controls, containing H. pylori infected AGS cells,
without test samples, were used to define 100% adhesion or invasion. Results were expressed as the
percentage of relative inhibition of H. pylori adhesion or invasion, by comparison with the controls.
6.2.7. Analysis of H. pylori-induced vacuolation of AGS cells
The vacuolation activity was determined by using neutral red uptake assay with slightly
modification [19]. Briefly, AGS cells (0.5 × 105 cells) were cultured in 96-well microtiter plates for
24 h followed by infected with H. pylori strain 26695 at an MOI of 100. After 24 h incubation, the
culture medium was removed and stained with 0.05% (w/v) neutral red (Sigma-Aldrich) for 5 min.
The stained cells were then washed three times with PBS, and the neutral red was extracted using
acidified alcohol. The levels of neutral red uptake was measured by a microplate ELISA reader
(Biotek, Winooski, VT) at OD 540 nm. The control was a population of AGS cells without H. pylori
infection or test sample present, and was used to define 100% cellular vacuolation. The results were
expressed as the percentage of relative vacuole formation, by comparison with the control.
6.2.8. Analysis of cytotoxin-associated gene A (CagA) translocation and phosphorylation of AGS
cells
The preparation of immunoprecipitates for analysis of H. pylori translocated and phosphorylated
CagA was described in our previous report [34]. The immunoprecipitates were then subjected to
6.5% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF)membrane (Pall, East Hills,
NY) for immunoblot analysis. CagA was probed with mouse anti-CagA antibody (Santa Cruz
Biotechnology); tyrosine-phosphorylated CagA was probed with mouse anti-phosphotyrosine
antibody (4G10) (Upstate Biotechnology, Billerica, MA). To ensure equal loading of each prepared
sample, actin from whole-cell lysates, was stained using goat anti-actin antibody (Santa Cruz
Biotechnology). The proteins of interestwere visualized using enhanced chemiluminescence reagents
(GE Healthcare, Buckinghamshire, UK) and weredetected by exposure to X-ray film (Kodak, Boca
Raton, FL, USA).
6.2.9. Assessment of H. pylori-induced hummingbird phenotype of AGS cells
AGS cells (1 × 106 cells) were cultured in 12-well plates at 37 °C for 24 h followed by infection
with H. pylori strain 26695at a MOI of 50 for 6 h. Elongatedcells (hummingbird phenotype) were
defined as cells that had thin needlelike protrusionsthat were greater than 20 µm long, and presented
a typical elongated shape as described by our previous report [34]. All samples were determined in
duplicate from three independent experiments. The proportion of elongatedcells was calculated from
the numbers of H. pylori-infected cells having the hummingbird phenotypes.
6.2.10. Transient transfection of NF-κB reporter and measurement of IL-8
The analysis of NF-κB reporter luciferase activity and measurement of IL-8 expression have
been described in our previous reports [32, 33].
6.2.11. Statistical analysis
The Student's t-test was used to calculate the statistical significance of experimental results
between two groups. Differences in data values were considered significant at *P < 0.05.
Acknowledgements
This study was supported by the National Science Council of Taiwan
(NSC98-2320-B-039-007-MY3, NSC99-2815-C-039-050-B, NSC100-2313-B-039-003), by China
Medical University (CMU97-346, CMU98-S-09, CMU98-S-38), and by Tomorrow Medical
Foundation. We thank Yen-Ru Chen (National Chung Hsing University) for expert technical
assistance. This study was supported in part by the Taiwan Department of Health Clinical Trial and
Research Center of Excellence (DOH100-TD-B-111-004).
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Figure captions
Figure 1. Effects of FMTMB and standard antimicrobial agents on H. pylori adhesion (A) and invasion (B) to gastric epithelial cells. AGS cells were treated with FMTMB and antimicrobial agents, followed by incubation with H. pylori at MOI 50 for 6 h. Amoxicillin (AMX),
clarithromycin (CLR), metronidazole (MTZ), and benzimidazole (BI) were used as positive control
antimicrobial agents. Each experiment result shown represents mean values ± the standard deviation
of three independent experiments. Statistical analysis was calculated using the Student’s t-test when
compared to DMSO treated cells. *P < 0.05 was considered as statistically significant.
Figure 2. FMTMB attenuates VacA-induced vacuolation of H. pylori-infected AGS cells.
Pretreatment of H. pylori-infected AGS cells with BI, and various concentrations (5, 10, and 20 μg/mL) of FMTMB; the cells were cultured for 24 h and stained with 0.05% neutral red. The net
OD for each well, indicating the accumulation of neutral red dye in cells, was evaluated. The
control of AGS cells without H. pylori infection and without test sample present was used as a
baseline for 100% cellular vacuolation. Results are shown as mean values ± standard deviations
from three independent experiments. *P < 0.05 was considered as significantly different.
Figure 3. The effects of FMTMB on H. pylori CagA translocation and phosphorylation. (A) AGS cells were treated with various concentrations of FMTMB (5, 10, and 20 μg/mL) after prior
infection by H. pylori at MOI 100 for 6 h. After washing three times, whole-cell lysates were
immunoprecipitated for CagA, and subjected to immunoblot analysis. Translocated CagA and
phosphorylated CagA were stained using mouse anti-CagA and mouse anti-phosphotyrosine antibodies, respectively. β-actin was detected using goat anti-actin antibody to represent an internal
control for equal loading. The quantitative results of (B) CagA translocation and (C) CagA
phosphorylation were determined using densitometric analysis. Results are shown as mean values ±
standard deviations from three independent experiments. *P < 0.05 was considered as statistically
significant.
Figure 4. Inhibitory effects of FMTMB on H. pylori CagA-induced pathogenesis of AGS cells.
The hummingbird phenotype of H. pylori CagA-induced AGS cells was reduced after treatment
with FMTMB. (A) Mock infection, (B) H. pylori-infected AGS cells, (C) pretreatment of H.
pylori-infected AGS cells with FMTMB (20 μg/mL), followed by incubation for a further 6 h, (D)
the proportion of elongated (hummingbird phenotype) cells was evaluated. Results are shown as
mean values ± standard deviations from three independent experiments. Differences were
considered significant for *P < 0.05. Scale bar, 20 μm.
Figure 5. The effects of FMTMB on H. pylori-induced AGS cells inflammatory response.
FMTMB inhibits (A) NF-κB activation and (B) IL-8 expression. Benzimidazole (BI) (20 μg/mL)
was used as a control standard antimicrobial agent. We determined luciferase activity and IL-8
secretion in the culture supernatants. Results are shown as mean values ± standard deviations from
three independent experiments. Statistical significance was calculated using the Student’s t-test
when compared to the DMSO control. *P < 0.05 was considered as statistically significant.
Tables
Table 1. Chemical structures of benzimidazole derivatives and their inhibitory effect on H. pylori 26695 (ATCC 700392) growth during the disk agar diffusion test
N
23 C25H26N2O4 CH3 H 2-OCH3-Ph 21
aFMTMB: 2-fluorophenyl-5-methyl-1-(3,4,5-trimethoxybenzyl)benzimidazole, BI: Benzimidazole, AMX: amoxicillin, CLR: clarithromycin, and MTZ: metronidazole. Concentration of compounds 7–34 was at 100 μg/mL, BI at 100 μg/mL, AMX and CLR at 50 μg/mL, and MTZ at 800 μg/mL.
DMSO was used as a negative control.
Table 2. Effect of pH on minimum bactericidal concentration (MBC) of FMTMB against H. pylori 26695, and multidrug-resistant strains v633 and v1354.
Tested compounda
H. pylori strains
MTZ BI FMTMB
pH pH pH
5.0 7.0 8.0 5.0 7.0 8.0 5.0 7.0 8.0
26695 6.25 12.5 12.5 > 800 400 > 800 25 25 25
v633 50 50 50 > 800 400 400 12.5 12.5 12.5
v1254 100 100 100 > 800 400 400 12.5 12.5 12.5
aAMX: amoxicillin, BI: Benzimidazole, FMTMB: 2-fluorophenyl-5-methyl-1-(3,4,5-
trimethoxybenzyl)benzimidazole. Concentrations of FMTMB and standard antimicrobial agents
were in μg/mL.
Figures
Scheme
Scheme 1. Synthesis of trimethoxybenzylbenzimidazole derivatives
(i) (ii)
reflux; (iii) 3,4,5-trimethoxybenzaldehyde, MeOH, rt; (iv) NaBH4, MeOH; (v) MeOH/4N HCl (2:1),
50 oC.
Compounds 7-20
Click here to download Mol Files: compounds 7-20.mol
Compounds 21-29
Click here to download Mol Files: compounds 21-29.mol
Compounds 30-34
Click here to download Mol Files: Compounds 30-34.mol