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Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia.

Tokuda S, Kim YH, Matsumoto H, Muro S, Hirai T, Mishima M, Furuse M - PLoS ONE (2015)

Bottom Line: The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability.The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification.The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

ABSTRACT
The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.

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Effects of hydrostatic pressure on cell proliferation.(A) Effects of hydrostatic pressure on the cell number in MDCK I and MDCK II cells cultured on filters. MDCK I and MDCK II cells were seeded at a density of 2 × 105 cells/cm2 on filters, and the cell number was counted with counting chamber after the trypsinization of the cells at each time point. The density of MDCK I cells under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition. (B) 5-bromo-2’-deoxy-uridine (BrdU) assay under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. BrdU assay was performed as described in Materials and Methods. The upper side is apical side and the lower side is basal side in z axis plane. Scale bars = 20 μm. (C) The ratio of BrdU-positive cells in MDCK I and MDCK II cells cultured on filters. The ratio of the cell number of BrdU-positive cells to that of DAPI-positive cells was calculated. The ratio of BrdU-positive cells under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition in MDCK I cells. (D) The density of BrdU-positive cells in MDCK I and MDCK II cells cultured on filters. The density of BrdU-positive cells under the ‘Basal’ condition was more than four-fold higher than that under the ‘Apical’ condition in MDCK I cells. ** p < 0.01 compared with the ‘Apical’ condition at corresponding days.
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pone.0145522.g007: Effects of hydrostatic pressure on cell proliferation.(A) Effects of hydrostatic pressure on the cell number in MDCK I and MDCK II cells cultured on filters. MDCK I and MDCK II cells were seeded at a density of 2 × 105 cells/cm2 on filters, and the cell number was counted with counting chamber after the trypsinization of the cells at each time point. The density of MDCK I cells under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition. (B) 5-bromo-2’-deoxy-uridine (BrdU) assay under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. BrdU assay was performed as described in Materials and Methods. The upper side is apical side and the lower side is basal side in z axis plane. Scale bars = 20 μm. (C) The ratio of BrdU-positive cells in MDCK I and MDCK II cells cultured on filters. The ratio of the cell number of BrdU-positive cells to that of DAPI-positive cells was calculated. The ratio of BrdU-positive cells under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition in MDCK I cells. (D) The density of BrdU-positive cells in MDCK I and MDCK II cells cultured on filters. The density of BrdU-positive cells under the ‘Basal’ condition was more than four-fold higher than that under the ‘Apical’ condition in MDCK I cells. ** p < 0.01 compared with the ‘Apical’ condition at corresponding days.

Mentions: In the multi-layered epithelia, it is speculated that the total cell number in the filter cup is increased compared to that of the epithelia of single layer. To confirm this possibility, we trypsinized the cells on filters and counted the cell number with counting chamber under the ‘Apical’ and ‘Basal’ conditions in MDCK I and MDCK II cells. MDCK I cells seeded at a density of 2 × 105 cells/cm2 on filters were increased to the density of 6.51 × 105 cells/cm2 at two days after seeding on filters (Fig 7A). Under the ‘Apical’ condition, the density of MDCK I cells was additionally increased by 38% during the four days of the culture (8.95 × 105 cells/cm2). In contrast, the density of MDCK I cells was additionally increased by 232% during the four days of the culture under the ‘Basal’ condition (21.60 × 105 cells/cm2). The density of MDCK I cells at four days after the culture under the ‘Increase’ condition in which there was no difference of the height of the medium surface between the apical and basal sides was comparable to that under the ‘Apical’ condition (8.62 × 105 cells/cm2) (S3 Fig). On the other hand, MDCK II cells seeded at a density of 2 × 105 cells/cm2 on filters were increased to the density of 8.14 × 105 cells/cm2 at two days after seeding, and the density was additionally increased by approximately 10% during the four days of the culture regardless of the ‘Apical’ or ‘Basal’ condition. These results indicate that the cell number show an increase by the hydrostatic pressure from basal to apical side in MDCK I cells but not in MDCK II cells, which is consistent with the hypothesis that the cell number is increased in the multi-layered epithelia.


Effects of Hydrostatic Pressure on Carcinogenic Properties of Epithelia.

Tokuda S, Kim YH, Matsumoto H, Muro S, Hirai T, Mishima M, Furuse M - PLoS ONE (2015)

Effects of hydrostatic pressure on cell proliferation.(A) Effects of hydrostatic pressure on the cell number in MDCK I and MDCK II cells cultured on filters. MDCK I and MDCK II cells were seeded at a density of 2 × 105 cells/cm2 on filters, and the cell number was counted with counting chamber after the trypsinization of the cells at each time point. The density of MDCK I cells under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition. (B) 5-bromo-2’-deoxy-uridine (BrdU) assay under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. BrdU assay was performed as described in Materials and Methods. The upper side is apical side and the lower side is basal side in z axis plane. Scale bars = 20 μm. (C) The ratio of BrdU-positive cells in MDCK I and MDCK II cells cultured on filters. The ratio of the cell number of BrdU-positive cells to that of DAPI-positive cells was calculated. The ratio of BrdU-positive cells under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition in MDCK I cells. (D) The density of BrdU-positive cells in MDCK I and MDCK II cells cultured on filters. The density of BrdU-positive cells under the ‘Basal’ condition was more than four-fold higher than that under the ‘Apical’ condition in MDCK I cells. ** p < 0.01 compared with the ‘Apical’ condition at corresponding days.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4696811&req=5

pone.0145522.g007: Effects of hydrostatic pressure on cell proliferation.(A) Effects of hydrostatic pressure on the cell number in MDCK I and MDCK II cells cultured on filters. MDCK I and MDCK II cells were seeded at a density of 2 × 105 cells/cm2 on filters, and the cell number was counted with counting chamber after the trypsinization of the cells at each time point. The density of MDCK I cells under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition. (B) 5-bromo-2’-deoxy-uridine (BrdU) assay under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. BrdU assay was performed as described in Materials and Methods. The upper side is apical side and the lower side is basal side in z axis plane. Scale bars = 20 μm. (C) The ratio of BrdU-positive cells in MDCK I and MDCK II cells cultured on filters. The ratio of the cell number of BrdU-positive cells to that of DAPI-positive cells was calculated. The ratio of BrdU-positive cells under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition in MDCK I cells. (D) The density of BrdU-positive cells in MDCK I and MDCK II cells cultured on filters. The density of BrdU-positive cells under the ‘Basal’ condition was more than four-fold higher than that under the ‘Apical’ condition in MDCK I cells. ** p < 0.01 compared with the ‘Apical’ condition at corresponding days.
Mentions: In the multi-layered epithelia, it is speculated that the total cell number in the filter cup is increased compared to that of the epithelia of single layer. To confirm this possibility, we trypsinized the cells on filters and counted the cell number with counting chamber under the ‘Apical’ and ‘Basal’ conditions in MDCK I and MDCK II cells. MDCK I cells seeded at a density of 2 × 105 cells/cm2 on filters were increased to the density of 6.51 × 105 cells/cm2 at two days after seeding on filters (Fig 7A). Under the ‘Apical’ condition, the density of MDCK I cells was additionally increased by 38% during the four days of the culture (8.95 × 105 cells/cm2). In contrast, the density of MDCK I cells was additionally increased by 232% during the four days of the culture under the ‘Basal’ condition (21.60 × 105 cells/cm2). The density of MDCK I cells at four days after the culture under the ‘Increase’ condition in which there was no difference of the height of the medium surface between the apical and basal sides was comparable to that under the ‘Apical’ condition (8.62 × 105 cells/cm2) (S3 Fig). On the other hand, MDCK II cells seeded at a density of 2 × 105 cells/cm2 on filters were increased to the density of 8.14 × 105 cells/cm2 at two days after seeding, and the density was additionally increased by approximately 10% during the four days of the culture regardless of the ‘Apical’ or ‘Basal’ condition. These results indicate that the cell number show an increase by the hydrostatic pressure from basal to apical side in MDCK I cells but not in MDCK II cells, which is consistent with the hypothesis that the cell number is increased in the multi-layered epithelia.

Bottom Line: The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability.The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification.The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

ABSTRACT
The relationship between chronic inflammation and cancer is well known. The inflammation increases the permeability of blood vessels and consequently elevates pressure in the interstitial tissues. However, there have been only a few reports on the effects of hydrostatic pressure on cultured cells, and the relationship between elevated hydrostatic pressure and cell properties related to malignant tumors is less well understood. Therefore, we investigated the effects of hydrostatic pressure on the cultured epithelial cells seeded on permeable filters. Surprisingly, hydrostatic pressure from basal to apical side induced epithelial stratification in Madin-Darby canine kidney (MDCK) I and Caco-2 cells, and cavities with microvilli and tight junctions around their surfaces were formed within the multi-layered epithelia. The hydrostatic pressure gradient also promoted cell proliferation, suppressed cell apoptosis, and increased transepithelial ion permeability. The inhibition of protein kinase A (PKA) promoted epithelial stratification by the hydrostatic pressure whereas the activation of PKA led to suppressed epithelial stratification. These results indicate the role of the hydrostatic pressure gradient in the regulation of various epithelial cell functions. The findings in this study may provide clues for the development of a novel strategy for the treatment of the carcinoma.

Show MeSH
Related in: MedlinePlus