Limits...
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 pHeinducesVim(vimentin) but reducesCdh1(E-cadherin) mRNA expression. Sub-confluent cultures were pretreated with serum-free medium at pH 7.4 and incubated in serum-free medium at pH 6.8 and 7.4. After 24 h, total RNA was extracted, reverse-transcribed, and amplified by qPCR with specific primer pairs. Data are shown as relative expression compared with their levels in LLCm1 cells cultured at pH 7.4. Representative results were shown from three independent experiments. *P < 0.05; NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Acidic pHeinducesVim(vimentin) but reducesCdh1(E-cadherin) mRNA expression. Sub-confluent cultures were pretreated with serum-free medium at pH 7.4 and incubated in serum-free medium at pH 6.8 and 7.4. After 24 h, total RNA was extracted, reverse-transcribed, and amplified by qPCR with specific primer pairs. Data are shown as relative expression compared with their levels in LLCm1 cells cultured at pH 7.4. Representative results were shown from three independent experiments. *P < 0.05; NS, not significant.

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 pHeinducesVim(vimentin) but reducesCdh1(E-cadherin) mRNA expression. Sub-confluent cultures were pretreated with serum-free medium at pH 7.4 and incubated in serum-free medium at pH 6.8 and 7.4. After 24 h, total RNA was extracted, reverse-transcribed, and amplified by qPCR with specific primer pairs. Data are shown as relative expression compared with their levels in LLCm1 cells cultured at pH 7.4. Representative results were shown from three independent experiments. *P < 0.05; NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Acidic pHeinducesVim(vimentin) but reducesCdh1(E-cadherin) mRNA expression. Sub-confluent cultures were pretreated with serum-free medium at pH 7.4 and incubated in serum-free medium at pH 6.8 and 7.4. After 24 h, total RNA was extracted, reverse-transcribed, and amplified by qPCR with specific primer pairs. Data are shown as relative expression compared with their levels in LLCm1 cells cultured at pH 7.4. Representative results were shown from three independent experiments. *P < 0.05; NS, not significant.
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