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Acidic extracellular pH promotes epithelial mesenchymal transition in Lewis lung carcinoma model.

Suzuki A, Maeda T, Baba Y, Shimamura K, Kato Y - Cancer Cell Int. (2014)

Bottom Line: Both variants showed decreased levels of E-cadherin and increased levels of vimentin at pH e 6.8.Acidic pH e also induced expression of mRNAs encoding the E-cadherin repressors, Zeb2, Twist1 and Twist2, as well as enhancing cell motility and in vitro invasion through Matrigel®.Acidic pH e can induce EMT in some types of carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Function and Molecular Biology, Ohu University School of Dentistry, Koriyama, Japan ; Department of Oral Physiology and Biochemistry, Ohu University Graduate School of Dentistry, Koriyama, Japan.

ABSTRACT

Background: Epithelial mesenchymal transition (EMT) is thought to be an essential feature of malignant tumor cells when they spread into the stroma. Despite the extracellular acidity of tumor tissues, the effect of acidic extracellular pH (pH e ) on EMT in carcinoma models, including the Lewis lung carcinoma (LLC) model, remains unclear.

Methods: High and low metastatic LLC variants were generated by repeated tail vein injection of metastatic cells. DMEM/F12 medium, which has been supplemented with 15 mM HEPES, 4 mM phosphoric acid, and 1 g/L NaHCO3 and adjusted to the desire pH with HCl or NaOH, was used for cell culture. EMT marker gene expression was determined by quantitative reverse transcription-polymerase chain reaction. Migration and invasion activities were analyzed by wound healing assay and the Boyden chamber assay through Matrigel®, respectively.

Results: Low metastatic variant LLCm1 cells showed a cobble-stone like morphology at pH e 7.4. At pH e 6.8, however, their morphology became fibroblastic, similar in shape to high metastatic variant LLCm4 cells. Steady state levels of matrix metalloproteinase-9 (Mmp9) mRNA were induced by acidic pH e , maximizing at pH 6.8, with the levels of Mmp9 mRNA higher in LLCm4 than in LLCm1 cells. Both variants showed decreased levels of E-cadherin and increased levels of vimentin at pH e 6.8. Acidic pH e also induced expression of mRNAs encoding the E-cadherin repressors, Zeb2, Twist1 and Twist2, as well as enhancing cell motility and in vitro invasion through Matrigel®.

Conclusions: Acidic pH e can induce EMT in some types of carcinoma.

No MeSH data available.


Related in: MedlinePlus

Acidic pHeinduces vimentin but reduces E-cadherin expression. Cultures were pretreated overnight with serum-free DMEM/12 at pH 7.4 and treated for 24 h with acidic serum-free medium at pH 6.8 and 7.4. The cells were subsequently fixed and incubated with antibodies to vimentin (A & B) and E-cadherin (C & D). FITC-labeled signals were detected by fluorescence microscopy. (A & C) Representative results were shown from three or more independent experiments. Immuno-positivity was caliculated and their positivity was shown as relative values compared to LLCm1 cells at pHe 7.4. Bar, 50 μm. *P < 0.05; **P < 0.01; NS, not significant. E. Western blot analysis for E-cadherin. After incubation of cells at pH 6.8, as described above, cells were lysed and analyzed by Western blotting. CM was concentrated and analyzed to detect shedded E-cadherin level in the CM.
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Fig5: Acidic pHeinduces vimentin but reduces E-cadherin expression. Cultures were pretreated overnight with serum-free DMEM/12 at pH 7.4 and treated for 24 h with acidic serum-free medium at pH 6.8 and 7.4. The cells were subsequently fixed and incubated with antibodies to vimentin (A & B) and E-cadherin (C & D). FITC-labeled signals were detected by fluorescence microscopy. (A & C) Representative results were shown from three or more independent experiments. Immuno-positivity was caliculated and their positivity was shown as relative values compared to LLCm1 cells at pHe 7.4. Bar, 50 μm. *P < 0.05; **P < 0.01; NS, not significant. E. Western blot analysis for E-cadherin. After incubation of cells at pH 6.8, as described above, cells were lysed and analyzed by Western blotting. CM was concentrated and analyzed to detect shedded E-cadherin level in the CM.

Mentions: To determine whether acidic pHe induces EMT, we analyzed the expression of E-cadherin and vimentin using immunocytochemistry. In LLCm1 cells, E-cadherin and vimentin were significantly down- and up-regulated, respectively, by acidic pHe, findings typical of EMT (Figure 5). Interestingly, increase in ectodomain of E-cadherin was observed especially in LLCm1 cells (Figure 5E), suggesting that E-cadherin shedding was occurred by acidic pHe. Expression of Cdh1 (E-cadherin) mRNA in both LLCm1 and LLCm4 cells was significantly decreased by incubation at acidic pHe (Figure 6). Moreover, acidic pHe increased the expression of Vim (vimentin) mRNA in both variants, but the difference was significant only in LLCm1 cells. Because LLCm4 is highly metastatic, vimentin expression may have been increased by other stimulatory systems, resulting in acidic pHe having a weak effect on vimentin production by these cells.Figure 5


Acidic extracellular pH promotes epithelial mesenchymal transition in Lewis lung carcinoma model.

Suzuki A, Maeda T, Baba Y, Shimamura K, Kato Y - Cancer Cell Int. (2014)

Acidic pHeinduces vimentin but reduces E-cadherin expression. Cultures were pretreated overnight with serum-free DMEM/12 at pH 7.4 and treated for 24 h with acidic serum-free medium at pH 6.8 and 7.4. The cells were subsequently fixed and incubated with antibodies to vimentin (A & B) and E-cadherin (C & D). FITC-labeled signals were detected by fluorescence microscopy. (A & C) Representative results were shown from three or more independent experiments. Immuno-positivity was caliculated and their positivity was shown as relative values compared to LLCm1 cells at pHe 7.4. Bar, 50 μm. *P < 0.05; **P < 0.01; NS, not significant. E. Western blot analysis for E-cadherin. After incubation of cells at pH 6.8, as described above, cells were lysed and analyzed by Western blotting. CM was concentrated and analyzed to detect shedded E-cadherin level in the CM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4260188&req=5

Fig5: Acidic pHeinduces vimentin but reduces E-cadherin expression. Cultures were pretreated overnight with serum-free DMEM/12 at pH 7.4 and treated for 24 h with acidic serum-free medium at pH 6.8 and 7.4. The cells were subsequently fixed and incubated with antibodies to vimentin (A & B) and E-cadherin (C & D). FITC-labeled signals were detected by fluorescence microscopy. (A & C) Representative results were shown from three or more independent experiments. Immuno-positivity was caliculated and their positivity was shown as relative values compared to LLCm1 cells at pHe 7.4. Bar, 50 μm. *P < 0.05; **P < 0.01; NS, not significant. E. Western blot analysis for E-cadherin. After incubation of cells at pH 6.8, as described above, cells were lysed and analyzed by Western blotting. CM was concentrated and analyzed to detect shedded E-cadherin level in the CM.
Mentions: To determine whether acidic pHe induces EMT, we analyzed the expression of E-cadherin and vimentin using immunocytochemistry. In LLCm1 cells, E-cadherin and vimentin were significantly down- and up-regulated, respectively, by acidic pHe, findings typical of EMT (Figure 5). Interestingly, increase in ectodomain of E-cadherin was observed especially in LLCm1 cells (Figure 5E), suggesting that E-cadherin shedding was occurred by acidic pHe. Expression of Cdh1 (E-cadherin) mRNA in both LLCm1 and LLCm4 cells was significantly decreased by incubation at acidic pHe (Figure 6). Moreover, acidic pHe increased the expression of Vim (vimentin) mRNA in both variants, but the difference was significant only in LLCm1 cells. Because LLCm4 is highly metastatic, vimentin expression may have been increased by other stimulatory systems, resulting in acidic pHe having a weak effect on vimentin production by these cells.Figure 5

Bottom Line: Both variants showed decreased levels of E-cadherin and increased levels of vimentin at pH e 6.8.Acidic pH e also induced expression of mRNAs encoding the E-cadherin repressors, Zeb2, Twist1 and Twist2, as well as enhancing cell motility and in vitro invasion through Matrigel®.Acidic pH e can induce EMT in some types of carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Function and Molecular Biology, Ohu University School of Dentistry, Koriyama, Japan ; Department of Oral Physiology and Biochemistry, Ohu University Graduate School of Dentistry, Koriyama, Japan.

ABSTRACT

Background: Epithelial mesenchymal transition (EMT) is thought to be an essential feature of malignant tumor cells when they spread into the stroma. Despite the extracellular acidity of tumor tissues, the effect of acidic extracellular pH (pH e ) on EMT in carcinoma models, including the Lewis lung carcinoma (LLC) model, remains unclear.

Methods: High and low metastatic LLC variants were generated by repeated tail vein injection of metastatic cells. DMEM/F12 medium, which has been supplemented with 15 mM HEPES, 4 mM phosphoric acid, and 1 g/L NaHCO3 and adjusted to the desire pH with HCl or NaOH, was used for cell culture. EMT marker gene expression was determined by quantitative reverse transcription-polymerase chain reaction. Migration and invasion activities were analyzed by wound healing assay and the Boyden chamber assay through Matrigel®, respectively.

Results: Low metastatic variant LLCm1 cells showed a cobble-stone like morphology at pH e 7.4. At pH e 6.8, however, their morphology became fibroblastic, similar in shape to high metastatic variant LLCm4 cells. Steady state levels of matrix metalloproteinase-9 (Mmp9) mRNA were induced by acidic pH e , maximizing at pH 6.8, with the levels of Mmp9 mRNA higher in LLCm4 than in LLCm1 cells. Both variants showed decreased levels of E-cadherin and increased levels of vimentin at pH e 6.8. Acidic pH e also induced expression of mRNAs encoding the E-cadherin repressors, Zeb2, Twist1 and Twist2, as well as enhancing cell motility and in vitro invasion through Matrigel®.

Conclusions: Acidic pH e can induce EMT in some types of carcinoma.

No MeSH data available.


Related in: MedlinePlus