Limits...
TMEPAI/PMEPA1 enhances tumorigenic activities in lung cancer cells.

Vo Nguyen TT, Watanabe Y, Shiba A, Noguchi M, Itoh S, Kato M - Cancer Sci. (2014)

Bottom Line: TMEPAI is constitutively and highly expressed in many types of cancer and is associated with poor prognosis.Knockdown of TMEPAI in Calu3 and NCI-H23 cells enhanced levels of Smad2 phosphorylation and significantly suppressed cell proliferation in the presence of TGF-β, indicating that highly expressed TMEPAI suppresses levels of Smad phosphorylation in these cancer cells and reduces the growth inhibitory effects of TGF-β/Smad signaling.Together, these experiments indicate that TMEPAI promotes tumorigenic activities in lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences and Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

Show MeSH

Related in: MedlinePlus

Calu3-shTMEPAI cells have increased sensitivity to transforming growth factor-β (TGF-β). (a) Generation of TMEPAI-knockdown Calu3 cell lines. The expression of TMEPAI was significantly suppressed by stable expression of two independent shRNAs (#9 and #10) targeting TMEPAI mRNA. Cells were treated with TGF-β for 8 h, and endogenous TMEPAI was detected by immunoblot analysis. β-actin was used as the loading control. (b) Cells were stimulated with TGF-β (1 ng/mL) for 1 h and phosphorylated Smad2 was detected with anti-phospho Smad2 antibody (PS2). β-actin was used as the loading control. (c,d) Cell proliferation assays. (c) TMEPAI-knockdown Calu3 cells (Calu3-sh#9 and Calu3-sh#10) were cultured in 12-well plates without TGF-β (left), with TGF-β (0.1 ng/mL) (middle) or with TGF-β (0.1 ng/mL) and TGF-β neutralizing antibody (1 ng/mL) (right), as indicated. The cell numbers were counted every second day. The means ± SDs are shown. (d) Mean percentage growth inhibition by TGF-β (0.1 ng/mL) and the reversal by inclusion of TGF-β neutralizing antibody (1 ng/mL) was calculated on day 9 in each cell line. Calu3 sh. control, Calu3 sh. control expresses non-targeting shRNA (SHC002, Sigma) in pLKO.1-puro vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4317935&req=5

fig03: Calu3-shTMEPAI cells have increased sensitivity to transforming growth factor-β (TGF-β). (a) Generation of TMEPAI-knockdown Calu3 cell lines. The expression of TMEPAI was significantly suppressed by stable expression of two independent shRNAs (#9 and #10) targeting TMEPAI mRNA. Cells were treated with TGF-β for 8 h, and endogenous TMEPAI was detected by immunoblot analysis. β-actin was used as the loading control. (b) Cells were stimulated with TGF-β (1 ng/mL) for 1 h and phosphorylated Smad2 was detected with anti-phospho Smad2 antibody (PS2). β-actin was used as the loading control. (c,d) Cell proliferation assays. (c) TMEPAI-knockdown Calu3 cells (Calu3-sh#9 and Calu3-sh#10) were cultured in 12-well plates without TGF-β (left), with TGF-β (0.1 ng/mL) (middle) or with TGF-β (0.1 ng/mL) and TGF-β neutralizing antibody (1 ng/mL) (right), as indicated. The cell numbers were counted every second day. The means ± SDs are shown. (d) Mean percentage growth inhibition by TGF-β (0.1 ng/mL) and the reversal by inclusion of TGF-β neutralizing antibody (1 ng/mL) was calculated on day 9 in each cell line. Calu3 sh. control, Calu3 sh. control expresses non-targeting shRNA (SHC002, Sigma) in pLKO.1-puro vector.

Mentions: We next engineered stable knockdown of TMEPAI by two individual shRNAs (shTMEPAI#9 and shTMEPAI#10). These shRNAs significantly reduced TMEPAI expression in Calu3 cells (Calu3-sh#9 and Calu3-sh#10; Fig.3a). As a result, phosphorylated Smad2 levels were clearly enhanced (Fig.3b). Significantly stronger cell growth inhibition was obtained in Calu3-sh#9 and Calu3-sh#10 cells in the presence of 0.1 ng/mL TGF-β, and it was recovered by anti-TGF-β neutralizing antibodies (Fig.3c,d). Similar results were obtained with NCI-H23 cells (Fig. S3a–c).


TMEPAI/PMEPA1 enhances tumorigenic activities in lung cancer cells.

Vo Nguyen TT, Watanabe Y, Shiba A, Noguchi M, Itoh S, Kato M - Cancer Sci. (2014)

Calu3-shTMEPAI cells have increased sensitivity to transforming growth factor-β (TGF-β). (a) Generation of TMEPAI-knockdown Calu3 cell lines. The expression of TMEPAI was significantly suppressed by stable expression of two independent shRNAs (#9 and #10) targeting TMEPAI mRNA. Cells were treated with TGF-β for 8 h, and endogenous TMEPAI was detected by immunoblot analysis. β-actin was used as the loading control. (b) Cells were stimulated with TGF-β (1 ng/mL) for 1 h and phosphorylated Smad2 was detected with anti-phospho Smad2 antibody (PS2). β-actin was used as the loading control. (c,d) Cell proliferation assays. (c) TMEPAI-knockdown Calu3 cells (Calu3-sh#9 and Calu3-sh#10) were cultured in 12-well plates without TGF-β (left), with TGF-β (0.1 ng/mL) (middle) or with TGF-β (0.1 ng/mL) and TGF-β neutralizing antibody (1 ng/mL) (right), as indicated. The cell numbers were counted every second day. The means ± SDs are shown. (d) Mean percentage growth inhibition by TGF-β (0.1 ng/mL) and the reversal by inclusion of TGF-β neutralizing antibody (1 ng/mL) was calculated on day 9 in each cell line. Calu3 sh. control, Calu3 sh. control expresses non-targeting shRNA (SHC002, Sigma) in pLKO.1-puro vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317935&req=5

fig03: Calu3-shTMEPAI cells have increased sensitivity to transforming growth factor-β (TGF-β). (a) Generation of TMEPAI-knockdown Calu3 cell lines. The expression of TMEPAI was significantly suppressed by stable expression of two independent shRNAs (#9 and #10) targeting TMEPAI mRNA. Cells were treated with TGF-β for 8 h, and endogenous TMEPAI was detected by immunoblot analysis. β-actin was used as the loading control. (b) Cells were stimulated with TGF-β (1 ng/mL) for 1 h and phosphorylated Smad2 was detected with anti-phospho Smad2 antibody (PS2). β-actin was used as the loading control. (c,d) Cell proliferation assays. (c) TMEPAI-knockdown Calu3 cells (Calu3-sh#9 and Calu3-sh#10) were cultured in 12-well plates without TGF-β (left), with TGF-β (0.1 ng/mL) (middle) or with TGF-β (0.1 ng/mL) and TGF-β neutralizing antibody (1 ng/mL) (right), as indicated. The cell numbers were counted every second day. The means ± SDs are shown. (d) Mean percentage growth inhibition by TGF-β (0.1 ng/mL) and the reversal by inclusion of TGF-β neutralizing antibody (1 ng/mL) was calculated on day 9 in each cell line. Calu3 sh. control, Calu3 sh. control expresses non-targeting shRNA (SHC002, Sigma) in pLKO.1-puro vector.
Mentions: We next engineered stable knockdown of TMEPAI by two individual shRNAs (shTMEPAI#9 and shTMEPAI#10). These shRNAs significantly reduced TMEPAI expression in Calu3 cells (Calu3-sh#9 and Calu3-sh#10; Fig.3a). As a result, phosphorylated Smad2 levels were clearly enhanced (Fig.3b). Significantly stronger cell growth inhibition was obtained in Calu3-sh#9 and Calu3-sh#10 cells in the presence of 0.1 ng/mL TGF-β, and it was recovered by anti-TGF-β neutralizing antibodies (Fig.3c,d). Similar results were obtained with NCI-H23 cells (Fig. S3a–c).

Bottom Line: TMEPAI is constitutively and highly expressed in many types of cancer and is associated with poor prognosis.Knockdown of TMEPAI in Calu3 and NCI-H23 cells enhanced levels of Smad2 phosphorylation and significantly suppressed cell proliferation in the presence of TGF-β, indicating that highly expressed TMEPAI suppresses levels of Smad phosphorylation in these cancer cells and reduces the growth inhibitory effects of TGF-β/Smad signaling.Together, these experiments indicate that TMEPAI promotes tumorigenic activities in lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences and Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

Show MeSH
Related in: MedlinePlus