Effect of Fluid Shear Stress on the Cellular Morphology and Tight Junction of Laryngeal Squamous Carcinoma Hep2 Cells_Journal of Biomedical Engineering

Authors：

ZHOUFating ¹ , YINHongmei ² , LIUShuangfeng ¹ , SHENYang ¹ , HONGJinyong ¹ , XIAQing ¹ ,  LIUXiaoheng ¹

1. Institute of Biomedical Engineering, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China;
2. West China School of Pharmacy, Sichuan University, Chengdu 610041, China;

Corresponding author：

LIUXiaoheng, Email: liuxiaohg@scu.edu.cn

Keywords：

fluid shear stress; laryngeal squamous carcinoma; tight junction

DOI：

10.7507/1001-5515.20150019

Video：

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This paper is aimed to investigate the effect of fluid shear stress on the tight junction of laryngeal squamous carcinoma (Hep2) cells and to explore the potential molecular mechanism. Hep2 cells were selected and subjected to the fluid shear stress of 1.4 dyn/cm² for different time, respectively. The morphological changes of Hep2 cells under shear stress were observed using inverted microscope. The cell-cell junctions were examined by transmission electron microscope (TEM). The expressions of tight junction proteins (including Occludin, Claudin-5 and ZO-1) and the distribution of Claudin-5 were examined by Western blot assay and laser scanning confocal microscope, respectively. The results indicated that Hep2 cells turned to spindle-like shapes after exposed to shear stress, and showed the trend of the recovering to original shapes when the shear stress was cancelled. The cell-cell junctions were tight under the shear flow condition, and the permeability was reduced under the condition of 1.4 dyn/cm² shear flow. The expressions of tight junction proteins were enhanced with increased duration of shear flow, but reduced after removing shear flow. The result of Claudin-5 expression by immufluorescence assay was consistent with that by Western blot. The Claudin-5 mainly distributed in the cytoplasm under static condition, while it located at the intercellular after shear flow stimulation, and it appeared intercellular and cytoplasm after stopping shear flow stimulation. Therefore, it can be concluded that shear stress changes the morphology of laryngeal squamous carcinoma Hep2 cells, and upregulates the tight junction.

Citation： ZHOUFating, YINHongmei, LIUShuangfeng, SHENYang, HONGJinyong, XIAQing, LIUXiaoheng. Effect of Fluid Shear Stress on the Cellular Morphology and Tight Junction of Laryngeal Squamous Carcinoma Hep2 Cells. Journal of Biomedical Engineering, 2015, 32(1): 104-109. doi: 10.7507/1001-5515.20150019 Copy

1.	SANCHEZ-TILLO E, LIU Yongqing, DE BARRIOS O, et al. EMT-activating transcription factors in Cancer: beyond EMT and tumor invasiveness[J]. Cellular and Molecular Life Sciences, 2012, 69(20): 3429-3456.
2.	KALLURI R, WEINBERG R A. The basics of epithelial-mesenchymal transition[J]. J Clin Invest, 2009, 119(6): 1420-1428.
3.	YILMAZ M, CHISTOFORI G E, EMT, the cytoskeleton. and cancer cell invasion[J]. Cancer and Metastasis Reviews, 2009, 28(1/2): 15-33.
4.	YAMAGUCHI T, SHISHIDO T, HARA T, et al. Significance of sucrose permeability test in detecting early gastric Cancer and changes of permeability after endoscopic mucosal resection[J]. Hepatogastroenterology, 2009, 56(90): 561-564.
5.	RUNKLE E A, MU D. Tight junction proteins: From barrier to tumorigenesis[J]. Cancer Lett, 2013, 337(1): 41-48.
6.	TAKAHASHI A, KONDOH M, KODAKA M, et al. Peptides as tight junction modulators[J]. Curr Pharm Des, 2011, 17(25): 2699-2703.
7.	MINETA K, YAMAMOTO Y, YAMAZAKI Y, et al. Predicted expansion of the claudin multigene family[J]. FEBS Lett, 2011, 585(4): 606-612.
8.	YU A S, MCCARTHY K M, FRANCIS S A, et al. Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells[J]. Am J Physiol Cell Physiol, 2005, 288(6): C1231-C1241.
9.	PHILLIPS B E, CANCEL L, TARBELL J M. Occludin independently regulates permeability under hydrostatic pressure and cell division in retinal pigment epithelial cells[J]. Invest Ophthalmol Vis Sci, 2008, 49(6): 2568-2576.
10.	KATSUNO T, UMEDA K, MATSUI T, et al. Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells[J]. Mol Biol Cell, 2008, 19(6): 2465-2475.
11.	POLLARD T D, BLANCHOIN L, MULLINS R D. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells[J]. Annu Rev Biophys Biomol Struct, 2000, 29(1): 545-576.
12.	MITCHELL M J, KING M R. Computational and experimental models of Cancer cell response to fluid shear stress[J]. Front Oncol, 2013, (3): 44.
13.	EGORVA A D, DERUITER M C, VANDEPAS S, et al. Shear stress modulates TGF-beta signaling and EMT in endothelial cells[C], 2009, 23:830.8.
14.	FERNANDO R I, CASTILLO M D, LITZINGER M A, et al. IL-8 signaling plays a critical role in the Epithelial-Mesenchymal transition of human carcinoma cells[J]. Cancer Res, 2011, 71(15): 5296-5306.
15.	DICK M, JONAK P, LEASK R L. Statin therapy influences endothelial cell morphology and F-actin cytoskeleton structure when exposed to static and laminar shear stress conditions[J]. Life Sci, 2013, 92(14/16): 859-865.
16.	GONZALEZ-MARISCAL L, TAPIA R, CHAMORRO D. Crosstalk of tight junction components with signaling pathways[J]. Biochimica et Biophysica Acta-Biomembranes, 2008, 1778(3): 729-756.
17.	CONKLIN B S, VITO R P, CHEN C. Effect of low shear stress on permeability and occludin expression in porcine artery endothelial cells[J]. World J Surg, 2007, 31(4): 733-743.
18.	YU H C, HUANG X L, MA Y L, et al. Interleukin-8 regulates endothelial permeability by down-regulation of tight junction but not dependent on integrins induced focal adhesions[J]. Int J Biol Sci, 2013, 9(9): 966-979.
19.	DEMAIO L, CHANG Y S, GARDNER T W, et al. Shear stress regulates occludin content and phosphorylation[J]. Am J Physiol Heart Circ Physiol, 2001, 281(1): H105-H113.

1. SANCHEZ-TILLO E, LIU Yongqing, DE BARRIOS O, et al. EMT-activating transcription factors in Cancer: beyond EMT and tumor invasiveness[J]. Cellular and Molecular Life Sciences, 2012, 69(20): 3429-3456.
2. KALLURI R, WEINBERG R A. The basics of epithelial-mesenchymal transition[J]. J Clin Invest, 2009, 119(6): 1420-1428.
3. YILMAZ M, CHISTOFORI G E, EMT, the cytoskeleton. and cancer cell invasion[J]. Cancer and Metastasis Reviews, 2009, 28(1/2): 15-33.
4. YAMAGUCHI T, SHISHIDO T, HARA T, et al. Significance of sucrose permeability test in detecting early gastric Cancer and changes of permeability after endoscopic mucosal resection[J]. Hepatogastroenterology, 2009, 56(90): 561-564.
5. RUNKLE E A, MU D. Tight junction proteins: From barrier to tumorigenesis[J]. Cancer Lett, 2013, 337(1): 41-48.
6. TAKAHASHI A, KONDOH M, KODAKA M, et al. Peptides as tight junction modulators[J]. Curr Pharm Des, 2011, 17(25): 2699-2703.
7. MINETA K, YAMAMOTO Y, YAMAZAKI Y, et al. Predicted expansion of the claudin multigene family[J]. FEBS Lett, 2011, 585(4): 606-612.
8. YU A S, MCCARTHY K M, FRANCIS S A, et al. Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells[J]. Am J Physiol Cell Physiol, 2005, 288(6): C1231-C1241.
9. PHILLIPS B E, CANCEL L, TARBELL J M. Occludin independently regulates permeability under hydrostatic pressure and cell division in retinal pigment epithelial cells[J]. Invest Ophthalmol Vis Sci, 2008, 49(6): 2568-2576.
10. KATSUNO T, UMEDA K, MATSUI T, et al. Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells[J]. Mol Biol Cell, 2008, 19(6): 2465-2475.
11. POLLARD T D, BLANCHOIN L, MULLINS R D. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells[J]. Annu Rev Biophys Biomol Struct, 2000, 29(1): 545-576.
12. MITCHELL M J, KING M R. Computational and experimental models of Cancer cell response to fluid shear stress[J]. Front Oncol, 2013, (3): 44.
13. EGORVA A D, DERUITER M C, VANDEPAS S, et al. Shear stress modulates TGF-beta signaling and EMT in endothelial cells[C], 2009, 23:830.8.
14. FERNANDO R I, CASTILLO M D, LITZINGER M A, et al. IL-8 signaling plays a critical role in the Epithelial-Mesenchymal transition of human carcinoma cells[J]. Cancer Res, 2011, 71(15): 5296-5306.
15. DICK M, JONAK P, LEASK R L. Statin therapy influences endothelial cell morphology and F-actin cytoskeleton structure when exposed to static and laminar shear stress conditions[J]. Life Sci, 2013, 92(14/16): 859-865.
16. GONZALEZ-MARISCAL L, TAPIA R, CHAMORRO D. Crosstalk of tight junction components with signaling pathways[J]. Biochimica et Biophysica Acta-Biomembranes, 2008, 1778(3): 729-756.
17. CONKLIN B S, VITO R P, CHEN C. Effect of low shear stress on permeability and occludin expression in porcine artery endothelial cells[J]. World J Surg, 2007, 31(4): 733-743.
18. YU H C, HUANG X L, MA Y L, et al. Interleukin-8 regulates endothelial permeability by down-regulation of tight junction but not dependent on integrins induced focal adhesions[J]. Int J Biol Sci, 2013, 9(9): 966-979.
19. DEMAIO L, CHANG Y S, GARDNER T W, et al. Shear stress regulates occludin content and phosphorylation[J]. Am J Physiol Heart Circ Physiol, 2001, 281(1): H105-H113.

Journal of Biomedical Engineering

Effect of Fluid Shear Stress on the Cellular Morphology and Tight Junction of Laryngeal Squamous Carcinoma Hep2 Cells

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