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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 pHeinducesTwist1,Twist2 and Zeb2but notSnail,Slug, and Zeb1mRNA expression. Sub-confluent cells were washed twice with PBS(−), pretreated with serum-free medium at pH 7.4, and incubated in serum-free medium at the indicated pH. After 24 h, total RNA was extracted, reverse-transcribed, and amplified by qPCR with specific primer sets. Data are shown as relative expression compared with their levels in LLCm1 cells cultured at pH 7.4. Representative results were shown from three or more independent experiments. *P < 0.05; ***P < 0.001; NS, not significant.
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Fig7: Acidic pHeinducesTwist1,Twist2 and Zeb2but notSnail,Slug, and Zeb1mRNA expression. Sub-confluent cells were washed twice with PBS(−), pretreated with serum-free medium at pH 7.4, and incubated in serum-free medium at the indicated pH. After 24 h, total RNA was extracted, reverse-transcribed, and amplified by qPCR with specific primer sets. Data are shown as relative expression compared with their levels in LLCm1 cells cultured at pH 7.4. Representative results were shown from three or more independent experiments. *P < 0.05; ***P < 0.001; NS, not significant.

Mentions: Since several repressors of E-cadherin expression have been described, we tested the effects of acidic pHe on the expression of these repressor(s) by RT-qPCR technology. Incubation at acidic pHe induced steady state levels of Twist1, Twist2, and Zeb2 mRNAs (Figure 7), with these levels inversely correlated with E-cadherin expression, suggesting that Twist1, Twist2, and Zeb2 were involved in acidic pHe signaling that repressed E-cadherin expression. Zeb2 mRNA level showed that highest degree of induction, suggesting that Zeb2 was primarily responsible for acidic pHe-induced EMT. In contrast, acidic pHe had little effect on Snail, Slug, and Zeb1 mRNA levels, suggesting that these molecules may be little involved in acidic pHe-induced EMT.Figure 7


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 pHeinducesTwist1,Twist2 and Zeb2but notSnail,Slug, and Zeb1mRNA expression. Sub-confluent cells were washed twice with PBS(−), pretreated with serum-free medium at pH 7.4, and incubated in serum-free medium at the indicated pH. After 24 h, total RNA was extracted, reverse-transcribed, and amplified by qPCR with specific primer sets. Data are shown as relative expression compared with their levels in LLCm1 cells cultured at pH 7.4. Representative results were shown from three or more independent experiments. *P < 0.05; ***P < 0.001; NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Acidic pHeinducesTwist1,Twist2 and Zeb2but notSnail,Slug, and Zeb1mRNA expression. Sub-confluent cells were washed twice with PBS(−), pretreated with serum-free medium at pH 7.4, and incubated in serum-free medium at the indicated pH. After 24 h, total RNA was extracted, reverse-transcribed, and amplified by qPCR with specific primer sets. Data are shown as relative expression compared with their levels in LLCm1 cells cultured at pH 7.4. Representative results were shown from three or more independent experiments. *P < 0.05; ***P < 0.001; NS, not significant.
Mentions: Since several repressors of E-cadherin expression have been described, we tested the effects of acidic pHe on the expression of these repressor(s) by RT-qPCR technology. Incubation at acidic pHe induced steady state levels of Twist1, Twist2, and Zeb2 mRNAs (Figure 7), with these levels inversely correlated with E-cadherin expression, suggesting that Twist1, Twist2, and Zeb2 were involved in acidic pHe signaling that repressed E-cadherin expression. Zeb2 mRNA level showed that highest degree of induction, suggesting that Zeb2 was primarily responsible for acidic pHe-induced EMT. In contrast, acidic pHe had little effect on Snail, Slug, and Zeb1 mRNA levels, suggesting that these molecules may be little involved in acidic pHe-induced EMT.Figure 7

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