J Gastric Cancer.  2018 Dec;18(4):356-367. 10.5230/jgc.2018.18.e35.

KLK6 Promotes Growth, Migration, and Invasion of Gastric Cancer Cells

• Zhu S
• Shi J
• Zhang S
• Li Z

• KMID: 2429882
• DOI: http://doi.org/10.5230/jgc.2018.18.e35

Abstract

PURPOSE
Kallikrein (KLK) proteases are hormone-like signaling molecules with critical functions in different cancers. This study investigated the expression of KLK6 in gastric cancer and its potential role in the growth, migration, and invasion of gastric cancer cells.
MATERIALS AND METHODS
In this study, we compared protein levels of KLK6, vascular endothelial growth factor (VEGF), and matrix metallopeptidase (MMP) 9 in normal gastric epithelial and gastric cancer cell lines by western blot. Fluorescence-activated cell sorting was employed to sort 2 clones of SGC-7901 cells with distinct KLK6 expression, namely, KLK6-high (KLK6high) and KLK6-low (KLK6low), which were then expanded. Lastly, immunohistochemical analysis was performed to investigate KLK6 expression in gastric cancer patients.
RESULTS
The expression levels of KLK6, VEGF, and MMP 9, were significantly higher in the gastric cancer cell lines SGC-7901, BGC-823, MKN-28, and MGC-803 than in the normal gastric epithelial cell line GES-1. Compared to KLK6low cells, KLK6high cells showed enhanced viability, colony-forming ability, migration, and invasion potential in vitro. Importantly, immunohistochemical analysis of a human gastric cancer tissue cohort revealed that the staining for KLK6, VEGF, and MMP9 was markedly stronger in the cancerous tissues than in the adjacent normal tissues. KLK6 expression also correlated with that of VEGF and MMP9 expression, as well as several key clinicopathological parameters.
CONCLUSIONS
Together, these results suggest an important role for KLK6 in human gastric cancer progression.

Keyword

Gastric cancer; KLK6 protein, human; MMP9 protein, human; VEGF

MeSH Terms

Blotting, Western
Cell Line
Clone Cells
Cohort Studies
Epithelial Cells
Flow Cytometry
Humans
In Vitro Techniques
Kallikreins
Peptide Hydrolases
Stomach Neoplasms*
Vascular Endothelial Growth Factor A
Kallikreins
Peptide Hydrolases
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Protein expression of KLK6, VEGF, and MMP9 in various gastric cancer and normal gastric epithelial cells. KLK6 = kallikrein 6; VEGF = vascular endothelial growth factor; MMP9 = matrix metallopeptidase 9.

• 

  Fig. 2 (A) Western blot analysis of KLK6 and VEGF levels in KLK6high and KLK6low SGC-7901 cells. (B) The MTT assay was conducted to assess cell viability of KLK6high and KLK6low cells over a duration of 72 hours. (C) Colony formation assay was performed to compare the ability of KLK6high and KLK6low cells to form colonies. The number of colonies was then quantified. KLK6 = kallikrein 6; VEGF = vascular endothelial growth factor; MTT = methyl thiazolyl tetrazolium; KLK6high = kallikrein 6-high; KLK6low = kallikrein 6-low. *Indicated value means statistical significance.

• 

  Fig. 3 (A) Wound healing assay was performed using KLK6low and KLK6high cells. The wound was imaged at 0, 24, and 48 hours after the scratch. (B) Quantification of wound healing area and wound healing ratio (%) in (A). KLK6high = kallikrein 6-high; KLK6low = kallikrein 6-low. *Indicated value means statistical significance.

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  Fig. 4 (A) Transwell invasion assay was performed to assess the invasive potential of KLK6high and KLK6low cells. Cells were allowed to invade for 24 hours, and the inserts were then fixed and stained. (B) Quantification of the invaded cells in (A). KLK6high = kallikrein 6-high; KLK6low = kallikrein 6-low. *Indicated value means statistical significance.

• 

  Fig. 5 (A) qPCR and western blot analyses of KLK6, VEGF, and MMP9 expression in gastric cancer and adjacent normal tissues. (B) Immunohistochemical staining of KLK6, VEGF, and MMP9 in gastric cancer and adjacent normal tissues. The a, c, and e are tumor tissues; b, d, and f are normal tissues. (C) Kaplan–Meier curves suggesting that KLK6− patients had a significantly better chance of survival than KLK6+ patients. The χ2 and P-values are indicated in the plot. qPCR = quantitative polymerase chain reaction; KLK6 = kallikrein 6; VEGF = vascular endothelial growth factor; MMP9 = matrix metallopeptidase 9; mRNA = messenger RNA. *Indicated value means statistical significance.

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