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Induction of an EMT-like transformation and MET in vitro.

Ding S, Zhang W, Xu Z, Xing C, Xie H, Guo H, Chen K, Song P, Gu Y, Xiao F, Zhou L, Zheng S - J Transl Med (2013)

Bottom Line: We found that Bel-7402 cells co-cultured with HL-7702 or RF/6A cells were induced to undergo MET.We also found that integrin β1 expression was negatively associated with capsular formation, and that integrin β4 expression was negatively associated with CK19 expression.Our findings highlight the strong influences exerted by TM on tumor progression through EMT and MET by impacting the expression of adhesion molecules, including the E-cadherin/catenin complex, laminins and integrins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, Hangzhou, Zhejiang, China.

ABSTRACT

Background: The epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) play pivotal roles in metastasis of epithelial cancers. The distinction between them has shed new light on the molecular mechanisms of tumor metastasis. Recently, tumor microenvironment (TM) has been identified as one of the most potent inducers of EMT and MET. TM is characterized by its complexity and flexibility. The purpose of this study was to ascertain the exact effect of each distinct TM component on the evolution hepatocellular carcinoma (HCC) metastasis.

Methods: Two different cell culture models were used. The HCC cell line Bel-7402 was co-cultured with the normal liver cell line HL-7702 or with the retinal vascular endothelial cell line RF/6A in double-layer six-well plates, imitating the direct interaction between tumor-host cells and tumor cells. Bel-7402 was also cultured in the conditioned medium (CM) of the human lung fibroblast cell line MRC-5, HL-7702 or RF/6A, imitating an indirect interaction. Integrin β1, β3, β4, β7, laminin β3, E-cadherin and Snail levels were measured by quantitative RT-PCR in tumor sepecimens from 42 resected HCC.

Results: We found that Bel-7402 cells co-cultured with HL-7702 or RF/6A cells were induced to undergo MET. The expression of E-cadherin, α-catenin and β-catenin was up-regulated, accompanied with a strengthened E-cadherin/catenin complex on the membrane of co-cultured Bel-7402 cells. Consequently, the invasion and migration ability of cells was declined. Conversely, Bel-7402 cells cultured in conditioned medium from MRC-5 cells underwent an EMT-like transformation as the cells became elongated with increased invasion and migration ability. Furthermore, we demonstrated that HL-7702 cells could generally inhibit the tumorigenicity and viability of Bel-7402 cells. We also found that integrin β1 expression was negatively associated with capsular formation, and that integrin β4 expression was negatively associated with CK19 expression.

Conclusion: Our findings highlight the strong influences exerted by TM on tumor progression through EMT and MET by impacting the expression of adhesion molecules, including the E-cadherin/catenin complex, laminins and integrins.

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Related in: MedlinePlus

Immunofluorescence of epithelial and mesenchymal markers, EMT-related transcription factors and F-actin. (A) Confocal microscopy analysis of Bel-7402 cells co-clutured with RF/6A cells for 72 days. (B) Confocal microscopy analysis of Bel-7402 cells co-clutured with HL-7702 cells for 60 days. The green signal represents corresponding protein staining and the red signal indicates nuclear DNA staining by propidium iodide (PI).
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Figure 4: Immunofluorescence of epithelial and mesenchymal markers, EMT-related transcription factors and F-actin. (A) Confocal microscopy analysis of Bel-7402 cells co-clutured with RF/6A cells for 72 days. (B) Confocal microscopy analysis of Bel-7402 cells co-clutured with HL-7702 cells for 60 days. The green signal represents corresponding protein staining and the red signal indicates nuclear DNA staining by propidium iodide (PI).

Mentions: To elucidate the mechanism underlying impaired invasion and migration capacity of co-cultured Bel-7402, we evaluated the expression profiles of an epithelial marker (E-cadherin/catenin complex), mesenchymal marker (vimentin), EMT-promoted transcription factors (Snail, Slug, Twsit1, Gli-1 and ZEB-2) and MMPs (MMP-1, 3, 7, 9) by Western-blot at days 28 and 44 (Figure 3). The E-cadherin/catenin complex was up-regulated in co-cultured Bel-7402 cells compared to Bel-7402 cells that were not co-cultured. The results also revealed that the expression levels of mesenchymal markers such as Snail, Slug, Twsit1, ZEB-2, MMP-3, MMP-7 and vimentin were higher in co-cultured Bel-7402 cells. Interestingly, expression of the EMT-related transcription factor Gli-1 was decreased. In addition, the expression level trend of MMP-1 and MMP-9 in co-cultured Bel-7402 cells was unstable. A portion of these results was confimed by confocal microscopy, as shown in Figure 4. Moreover, filamentous actin (F-actin), which plays an important role in cell motility, was decreased in co-cultured Bel-7402 cells, suggesting that cellular motility of co-cultured Bel-7402 cells was reduced.


Induction of an EMT-like transformation and MET in vitro.

Ding S, Zhang W, Xu Z, Xing C, Xie H, Guo H, Chen K, Song P, Gu Y, Xiao F, Zhou L, Zheng S - J Transl Med (2013)

Immunofluorescence of epithelial and mesenchymal markers, EMT-related transcription factors and F-actin. (A) Confocal microscopy analysis of Bel-7402 cells co-clutured with RF/6A cells for 72 days. (B) Confocal microscopy analysis of Bel-7402 cells co-clutured with HL-7702 cells for 60 days. The green signal represents corresponding protein staining and the red signal indicates nuclear DNA staining by propidium iodide (PI).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716679&req=5

Figure 4: Immunofluorescence of epithelial and mesenchymal markers, EMT-related transcription factors and F-actin. (A) Confocal microscopy analysis of Bel-7402 cells co-clutured with RF/6A cells for 72 days. (B) Confocal microscopy analysis of Bel-7402 cells co-clutured with HL-7702 cells for 60 days. The green signal represents corresponding protein staining and the red signal indicates nuclear DNA staining by propidium iodide (PI).
Mentions: To elucidate the mechanism underlying impaired invasion and migration capacity of co-cultured Bel-7402, we evaluated the expression profiles of an epithelial marker (E-cadherin/catenin complex), mesenchymal marker (vimentin), EMT-promoted transcription factors (Snail, Slug, Twsit1, Gli-1 and ZEB-2) and MMPs (MMP-1, 3, 7, 9) by Western-blot at days 28 and 44 (Figure 3). The E-cadherin/catenin complex was up-regulated in co-cultured Bel-7402 cells compared to Bel-7402 cells that were not co-cultured. The results also revealed that the expression levels of mesenchymal markers such as Snail, Slug, Twsit1, ZEB-2, MMP-3, MMP-7 and vimentin were higher in co-cultured Bel-7402 cells. Interestingly, expression of the EMT-related transcription factor Gli-1 was decreased. In addition, the expression level trend of MMP-1 and MMP-9 in co-cultured Bel-7402 cells was unstable. A portion of these results was confimed by confocal microscopy, as shown in Figure 4. Moreover, filamentous actin (F-actin), which plays an important role in cell motility, was decreased in co-cultured Bel-7402 cells, suggesting that cellular motility of co-cultured Bel-7402 cells was reduced.

Bottom Line: We found that Bel-7402 cells co-cultured with HL-7702 or RF/6A cells were induced to undergo MET.We also found that integrin β1 expression was negatively associated with capsular formation, and that integrin β4 expression was negatively associated with CK19 expression.Our findings highlight the strong influences exerted by TM on tumor progression through EMT and MET by impacting the expression of adhesion molecules, including the E-cadherin/catenin complex, laminins and integrins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health, Hangzhou, Zhejiang, China.

ABSTRACT

Background: The epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) play pivotal roles in metastasis of epithelial cancers. The distinction between them has shed new light on the molecular mechanisms of tumor metastasis. Recently, tumor microenvironment (TM) has been identified as one of the most potent inducers of EMT and MET. TM is characterized by its complexity and flexibility. The purpose of this study was to ascertain the exact effect of each distinct TM component on the evolution hepatocellular carcinoma (HCC) metastasis.

Methods: Two different cell culture models were used. The HCC cell line Bel-7402 was co-cultured with the normal liver cell line HL-7702 or with the retinal vascular endothelial cell line RF/6A in double-layer six-well plates, imitating the direct interaction between tumor-host cells and tumor cells. Bel-7402 was also cultured in the conditioned medium (CM) of the human lung fibroblast cell line MRC-5, HL-7702 or RF/6A, imitating an indirect interaction. Integrin β1, β3, β4, β7, laminin β3, E-cadherin and Snail levels were measured by quantitative RT-PCR in tumor sepecimens from 42 resected HCC.

Results: We found that Bel-7402 cells co-cultured with HL-7702 or RF/6A cells were induced to undergo MET. The expression of E-cadherin, α-catenin and β-catenin was up-regulated, accompanied with a strengthened E-cadherin/catenin complex on the membrane of co-cultured Bel-7402 cells. Consequently, the invasion and migration ability of cells was declined. Conversely, Bel-7402 cells cultured in conditioned medium from MRC-5 cells underwent an EMT-like transformation as the cells became elongated with increased invasion and migration ability. Furthermore, we demonstrated that HL-7702 cells could generally inhibit the tumorigenicity and viability of Bel-7402 cells. We also found that integrin β1 expression was negatively associated with capsular formation, and that integrin β4 expression was negatively associated with CK19 expression.

Conclusion: Our findings highlight the strong influences exerted by TM on tumor progression through EMT and MET by impacting the expression of adhesion molecules, including the E-cadherin/catenin complex, laminins and integrins.

Show MeSH
Related in: MedlinePlus