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 pHeinduces not onlyMmp9but alsoMmp3andMmp13mRNA expressions but not obviousMmp2andMmp14mRNA expressions. Sub-confluent cell cultures were pretreated overnight with serum-free medium at pH 7.4 and stimulated by serum-free media at pH 6.8 or 7.4. 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 LLCm1 cells cultured at pH 7.4. Representative results were shown from three or more independent experiments. **P < 0.01; ***P < 0.001; NS, not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Acidic pHeinduces not onlyMmp9but alsoMmp3andMmp13mRNA expressions but not obviousMmp2andMmp14mRNA expressions. Sub-confluent cell cultures were pretreated overnight with serum-free medium at pH 7.4 and stimulated by serum-free media at pH 6.8 or 7.4. 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 LLCm1 cells cultured at pH 7.4. Representative results were shown from three or more independent experiments. **P < 0.01; ***P < 0.001; NS, not significant.

Mentions: MMP-9 induction by acidic pHe was also confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Accordance with zymographic analysis (Figure 2), the basal level of Mmp9 mRNA at pHe 7.4 was higher in LLCm4 cells than LLCm1 cells. Acid pHe enhanced the steady state levels of Mmp9 mRNA 2-fold in LLCm1 cells and 6-fold in LLCm4 cells (Figure 3). In addition to Mmp9, Mmp3, and Mmp13 mRNA expressions were also stimulated by acidic pHe. Although induction of MMP-2 secretion was observed (see Figure 2), increase in Mmp2 mRNA expression was not statistically significant, suggesting a possibility that acidic pHe affects the efficiency of protein translation. Mmp14 mRNA, whose protein is critical for MMP-2 activation, was higher in LLCm1 cells than LLCm4 cells and was not affected by acidic pHe.Figure 3


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 not onlyMmp9but alsoMmp3andMmp13mRNA expressions but not obviousMmp2andMmp14mRNA expressions. Sub-confluent cell cultures were pretreated overnight with serum-free medium at pH 7.4 and stimulated by serum-free media at pH 6.8 or 7.4. 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 LLCm1 cells cultured at pH 7.4. Representative results were shown from three or more independent experiments. **P < 0.01; ***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

Fig3: Acidic pHeinduces not onlyMmp9but alsoMmp3andMmp13mRNA expressions but not obviousMmp2andMmp14mRNA expressions. Sub-confluent cell cultures were pretreated overnight with serum-free medium at pH 7.4 and stimulated by serum-free media at pH 6.8 or 7.4. 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 LLCm1 cells cultured at pH 7.4. Representative results were shown from three or more independent experiments. **P < 0.01; ***P < 0.001; NS, not significant.
Mentions: MMP-9 induction by acidic pHe was also confirmed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Accordance with zymographic analysis (Figure 2), the basal level of Mmp9 mRNA at pHe 7.4 was higher in LLCm4 cells than LLCm1 cells. Acid pHe enhanced the steady state levels of Mmp9 mRNA 2-fold in LLCm1 cells and 6-fold in LLCm4 cells (Figure 3). In addition to Mmp9, Mmp3, and Mmp13 mRNA expressions were also stimulated by acidic pHe. Although induction of MMP-2 secretion was observed (see Figure 2), increase in Mmp2 mRNA expression was not statistically significant, suggesting a possibility that acidic pHe affects the efficiency of protein translation. Mmp14 mRNA, whose protein is critical for MMP-2 activation, was higher in LLCm1 cells than LLCm4 cells and was not affected by acidic pHe.Figure 3

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