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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 MMP-9 production. Sub-confluent cells were cultured overnight in serum-free medium at pH 7.4 and stimulated with serum-free medium at the indicated pH. After 24 h, the conditioned medium was collected, concentrated, and analyzed by gelatin zymography. A. A clear zone indicates gelatinolytic activity. B. Intensity of zymogram was quantified by Scion Image (Scion corp., Frederick, MD, USA). Representative results were shown from three independent experiments. *P < 0.01; **P < 0.01; ***P < 0.001; NS, not significant.
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Fig2: Acidic pHeinduces MMP-9 production. Sub-confluent cells were cultured overnight in serum-free medium at pH 7.4 and stimulated with serum-free medium at the indicated pH. After 24 h, the conditioned medium was collected, concentrated, and analyzed by gelatin zymography. A. A clear zone indicates gelatinolytic activity. B. Intensity of zymogram was quantified by Scion Image (Scion corp., Frederick, MD, USA). Representative results were shown from three independent experiments. *P < 0.01; **P < 0.01; ***P < 0.001; NS, not significant.

Mentions: As shown previously, MMP-9 is an acidic pHe-signal target gene, with the acid-induced level of MMP-9, but not MMP-2, expression positively correlated with the metastatic potential of mouse B16 melanoma variants [6]. We therefore assessed whether acidic pHe induces MMP-9 production, and whether its level correlated with the metastatic potential of the two LLC variants. MMP-9 production by both variants was clearly pHe dependent (Figure 2), with maximal production at pHe 6.8. The acid pHe stimulated expression of MMP-9 was much higher by LLCm4 than by LLCm1 cells, while LLCm4 cells produced constitutively higher level of MMP-9 as the basal level at pHe 7.4. Although MMP-2 level was also slightly induced by acidic pHe, the highly metastatic LLCm4 cells produced much less MMP-2 than LLCm1 cells. This finding was in accordance with the mouse B16 melanoma model, in that MMP-2 levels are high in parental B16 cells but negligible in highly metastatic B16-F10 and B16-BL6 cells [6].Figure 2


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 MMP-9 production. Sub-confluent cells were cultured overnight in serum-free medium at pH 7.4 and stimulated with serum-free medium at the indicated pH. After 24 h, the conditioned medium was collected, concentrated, and analyzed by gelatin zymography. A. A clear zone indicates gelatinolytic activity. B. Intensity of zymogram was quantified by Scion Image (Scion corp., Frederick, MD, USA). Representative results were shown from three independent experiments. *P < 0.01; **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

Fig2: Acidic pHeinduces MMP-9 production. Sub-confluent cells were cultured overnight in serum-free medium at pH 7.4 and stimulated with serum-free medium at the indicated pH. After 24 h, the conditioned medium was collected, concentrated, and analyzed by gelatin zymography. A. A clear zone indicates gelatinolytic activity. B. Intensity of zymogram was quantified by Scion Image (Scion corp., Frederick, MD, USA). Representative results were shown from three independent experiments. *P < 0.01; **P < 0.01; ***P < 0.001; NS, not significant.
Mentions: As shown previously, MMP-9 is an acidic pHe-signal target gene, with the acid-induced level of MMP-9, but not MMP-2, expression positively correlated with the metastatic potential of mouse B16 melanoma variants [6]. We therefore assessed whether acidic pHe induces MMP-9 production, and whether its level correlated with the metastatic potential of the two LLC variants. MMP-9 production by both variants was clearly pHe dependent (Figure 2), with maximal production at pHe 6.8. The acid pHe stimulated expression of MMP-9 was much higher by LLCm4 than by LLCm1 cells, while LLCm4 cells produced constitutively higher level of MMP-9 as the basal level at pHe 7.4. Although MMP-2 level was also slightly induced by acidic pHe, the highly metastatic LLCm4 cells produced much less MMP-2 than LLCm1 cells. This finding was in accordance with the mouse B16 melanoma model, in that MMP-2 levels are high in parental B16 cells but negligible in highly metastatic B16-F10 and B16-BL6 cells [6].Figure 2

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