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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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Establishment of the conditioned medium (CM) models. CM of MRC-5 cells induced Bel-7402 cells to undergo an EMT-like transformation, while CM of HL-7702 cells or RF/6A cells did not induce such a transformation at day 14. (A) Bel-7402 cells cultured in CM of MRC-5 cells became elongated. (B) and (C) The invasion and migration ability of Bel-7402 cells cultured in CM of MRC-5 cells was increased (p < 0.05). (D) Bel-7402 cells cultured in CM of MRC-5 cells had more actin-rich protrusions.
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Figure 5: Establishment of the conditioned medium (CM) models. CM of MRC-5 cells induced Bel-7402 cells to undergo an EMT-like transformation, while CM of HL-7702 cells or RF/6A cells did not induce such a transformation at day 14. (A) Bel-7402 cells cultured in CM of MRC-5 cells became elongated. (B) and (C) The invasion and migration ability of Bel-7402 cells cultured in CM of MRC-5 cells was increased (p < 0.05). (D) Bel-7402 cells cultured in CM of MRC-5 cells had more actin-rich protrusions.

Mentions: We used CM from MRC-5, HL-7702 and RF/6A cells to culture Bel-7402 cells for 28 days. Bel-7402-(MRC-5)-CM cells were unrecognizable at day 14 (Figure 5), with an elongated and spindle-like morphology containing actin-rich protrusions. We further evaluated the invasion and migration ability of Bel-7402-(MRC-5)-CM cells. The results in Figure 5 show that the invasion and migration ability of Bel-7402-(MRC-5)-CM cells was increased significantly (p < 0.05). In contrast, the morphology and motility of Bel-7402-(HL-7702)-CM and Bel-7402-(RF/6A)-CM cells did not change significantly compared to Bel-7402 control cells at day 14 (p > 0.05). These results indicate that MRC-5 cells induced Bel-7402 cells to undergo an EMT-like transformation while the CM of HL-7702 or RF/6A cells failed to induce Bel-7402 cells to undergo MET.


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)

Establishment of the conditioned medium (CM) models. CM of MRC-5 cells induced Bel-7402 cells to undergo an EMT-like transformation, while CM of HL-7702 cells or RF/6A cells did not induce such a transformation at day 14. (A) Bel-7402 cells cultured in CM of MRC-5 cells became elongated. (B) and (C) The invasion and migration ability of Bel-7402 cells cultured in CM of MRC-5 cells was increased (p < 0.05). (D) Bel-7402 cells cultured in CM of MRC-5 cells had more actin-rich protrusions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Establishment of the conditioned medium (CM) models. CM of MRC-5 cells induced Bel-7402 cells to undergo an EMT-like transformation, while CM of HL-7702 cells or RF/6A cells did not induce such a transformation at day 14. (A) Bel-7402 cells cultured in CM of MRC-5 cells became elongated. (B) and (C) The invasion and migration ability of Bel-7402 cells cultured in CM of MRC-5 cells was increased (p < 0.05). (D) Bel-7402 cells cultured in CM of MRC-5 cells had more actin-rich protrusions.
Mentions: We used CM from MRC-5, HL-7702 and RF/6A cells to culture Bel-7402 cells for 28 days. Bel-7402-(MRC-5)-CM cells were unrecognizable at day 14 (Figure 5), with an elongated and spindle-like morphology containing actin-rich protrusions. We further evaluated the invasion and migration ability of Bel-7402-(MRC-5)-CM cells. The results in Figure 5 show that the invasion and migration ability of Bel-7402-(MRC-5)-CM cells was increased significantly (p < 0.05). In contrast, the morphology and motility of Bel-7402-(HL-7702)-CM and Bel-7402-(RF/6A)-CM cells did not change significantly compared to Bel-7402 control cells at day 14 (p > 0.05). These results indicate that MRC-5 cells induced Bel-7402 cells to undergo an EMT-like transformation while the CM of HL-7702 or RF/6A cells failed to induce Bel-7402 cells to undergo MET.

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