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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 invasive activity. Confluent cultures were serum-starved and treated with serum-free medium at pH 7.4 or pH 6.8 for 18 h. The conditioned medium was collected and the cells were harvested by trypsinization. The cells were subsequently incubated with 10% FBS for 30 min to inhibit trypsin activity, washed twice with PBS(−) and resuspended in their own conditioned medium. The chemoattractant was 20% FBS. A. Phase contrast micrograph. B. Relative numbers of invasive cells compared with LLCm1 cells at pH 7.4. Representative results were shown from three independent experiments. *P < 0.05; ***P < 0.001.
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Fig9: Acidic pHeinduces invasive activity. Confluent cultures were serum-starved and treated with serum-free medium at pH 7.4 or pH 6.8 for 18 h. The conditioned medium was collected and the cells were harvested by trypsinization. The cells were subsequently incubated with 10% FBS for 30 min to inhibit trypsin activity, washed twice with PBS(−) and resuspended in their own conditioned medium. The chemoattractant was 20% FBS. A. Phase contrast micrograph. B. Relative numbers of invasive cells compared with LLCm1 cells at pH 7.4. Representative results were shown from three independent experiments. *P < 0.05; ***P < 0.001.

Mentions: Migration activity was determined using wound healing assays. Cells that formed a confluent sheet were scratched with a plastic tip, and the distance between the wound edge and the migration front were estimated. As expected, LLCm4 cells showed higher migration (Figure 8) and in vitro invasion (Figure 9) activities than LLCm1 cells. Both LLCm1 and LLCm4 cells migrated significantly further under acidic than neutral conditions. Furthermore, acidic pHe stimulated the in vitro invasion activity of both cell lines.Figure 8


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 invasive activity. Confluent cultures were serum-starved and treated with serum-free medium at pH 7.4 or pH 6.8 for 18 h. The conditioned medium was collected and the cells were harvested by trypsinization. The cells were subsequently incubated with 10% FBS for 30 min to inhibit trypsin activity, washed twice with PBS(−) and resuspended in their own conditioned medium. The chemoattractant was 20% FBS. A. Phase contrast micrograph. B. Relative numbers of invasive cells compared with LLCm1 cells at pH 7.4. Representative results were shown from three independent experiments. *P < 0.05; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig9: Acidic pHeinduces invasive activity. Confluent cultures were serum-starved and treated with serum-free medium at pH 7.4 or pH 6.8 for 18 h. The conditioned medium was collected and the cells were harvested by trypsinization. The cells were subsequently incubated with 10% FBS for 30 min to inhibit trypsin activity, washed twice with PBS(−) and resuspended in their own conditioned medium. The chemoattractant was 20% FBS. A. Phase contrast micrograph. B. Relative numbers of invasive cells compared with LLCm1 cells at pH 7.4. Representative results were shown from three independent experiments. *P < 0.05; ***P < 0.001.
Mentions: Migration activity was determined using wound healing assays. Cells that formed a confluent sheet were scratched with a plastic tip, and the distance between the wound edge and the migration front were estimated. As expected, LLCm4 cells showed higher migration (Figure 8) and in vitro invasion (Figure 9) activities than LLCm1 cells. Both LLCm1 and LLCm4 cells migrated significantly further under acidic than neutral conditions. Furthermore, acidic pHe stimulated the in vitro invasion activity of both cell lines.Figure 8

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