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, 15 (1), e0227408
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Pepsin Promotes Laryngopharyngeal Neoplasia by Modulating Signaling Pathways to Induce Cell Proliferation

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Pepsin Promotes Laryngopharyngeal Neoplasia by Modulating Signaling Pathways to Induce Cell Proliferation

Kai Niu et al. PLoS One.

Abstract

Pepsin plays an important role in laryngopharyngeal reflux (LPR), a risk factor for the development of hypopharyngeal squamous cell carcinomas (HPSCC). However, the role of pepsin in HPSCC is not clear. We show by immunohistochemistry that pepsin positivity occurs in a significant proportion of human primary HPSCC specimens, and in many cases matched adjacent uninvolved epithelia are negative for pepsin. Pepsin positivity is associated with nodal involvement, suggesting that pepsin may have a role in metastasis. Treatment of FaDu cancer cells with pepsin increased cell proliferation, possibly by inducing G1/S transition. We also observed significant changes in expression of genes involved in NF-kappaB, TRAIL and Notch signaling. Our data suggest that pepsin plays an important role in HPSCC and that targeting pepsin could have potential therapeutic benefits.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Pepsin staining in primary HPSCC tumors and matched adjacent tissues.
Pepsin stained positively in HPSCC tumor 1a and matched adjacent tissue 1b. There was no pepsin in HPSCC tumor 3a and matched adjacent tissue 3b. Tumor 2a was positive but adjacent tissues 2b was negative, for pepsin staining. Gastric oxyntic mucosa (4) showed strong pepsin staining while tonsil (5) showed negative pepsin staining.
Fig 2
Fig 2. Pepsin increases the growth and proliferation of FaDu cells.
FaDu cells were treated with pepsin for the indicated length of time and cultured in fresh complete growth media for 24 hours before analysis. (a) FaDu cells treated with pepsin at a concentration of 0.2mg/ml at pH 7 for 30 min and incubated in fresh media for 24 hours at 37°C. (b) Treatment with pepsin at a concentration of 0.4 mg/mL. (c, d) Treatment with irreversibly inactivated pepsin. Data are from five biological replicates. Bar graphs show mean ± standard deviation. Dose-response data were analyzed by one-way analysis of variance and Tukey multiple comparisons post-test. Time-response data were analyzed by two-way analysis of variance and the Bonferroni multiple comparisons post-test. *P < 0.05.
Fig 3
Fig 3. Cell cycle distribution of FaDu cells in response to pepsin treatment.
Cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. (a) control, (b) pepsin (0.2mg/ml) treatment for 30 min, and (c) pepsin treatment (0.2mg/ml) for 1 hour at 37°C. (d) Summary of data. Data are from five biological replicates and presented as mean ± standard error of the mean. Statistical analyses were performed by one-way analysis of variance and Tukey multiple comparisons post-test. *P<0.05.
Fig 4
Fig 4. Localization of pepsin to the lysosomes of FaDu.
FaDu cells were treated with Pepsin-Cy3 for 0.5 h, washed and incubated for 24 hours or 36 hours. After labeling with Lyso-tracker red, cells were stained with DAPI and visualized under a confocal microscope. Pepsin taken up by cells mainly localized to the lysosomes at 36 hours post-treatment.
Fig 5
Fig 5. P21, C-Myc, and p65 expression in FaDu cells treated with pepsin.
Cells were visualized by confocal microscopy (left panel, magnification = 400X). Quantifications are presented on the right panel. Control and pepsin treated cells were analyzed for expression of p21 (a, b), c-Myc (c, d), and NF-κB p65 (e, f). Arrows in (e) indicate nuclear translocation of NF-κB p65 protein. Hoechst 33342 was used to identify nuclei. Triplicate samples of 100 cells were scored, and data are presented as mean percentage ± SD. * P < 0.05, ** P <0.01.
Fig 6
Fig 6. Pepsin induced phosphorylation of P65 and IκB.
Protein levels were semi-quantitatively analyzed by scanning densitometry. Ratios of phosphorylated to total proteins are presented on the right panel. **P < 0.05, ##P < 0.05.

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Grant support

This work received a grant from Jilin Provincial Department of Health, China (Grant number 20132016) to KN and WY. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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