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STAT1 modification improves therapeutic effects of interferons on lung cancer cells.

Chen J, Zhao J, Chen L, Dong N, Ying Z, Cai Z, Ji D, Zhang Y, Dong L, Li Y, Jiang L, Holtzman MJ, Chen C - J Transl Med (2015)

Bottom Line: We found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness.STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.The results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang, China. jiestory414@163.com.

ABSTRACT

Background: Interferons (IFNs) have potent anti-proliferative, pro-apoptotic, and immunomodulatory activities against cancer. However, the clinical utility of IFNs is limited by toxicity and pharmacokinetics making it difficult to achieve sustained therapeutic levels especially in solid tumors.

Methods: Signal Transducer and Activator of Transcription 1 (STAT1) or a modified STAT1 (designated STAT1-CC) that is hyper-responsive to IFN were overexpressed in lung cancer SPC-A-1 and H1299 cells using lentiviral vectors. Transduction efficiency was monitored using enhanced green fluorescent protein (EGFP) expression. After transduction, cells were treated with interferon-gamma (IFN-γ) or interferon-beta (IFN-β) and monitored for cell proliferation, migration, and invasiveness using Cell Counting Kit-8 and transwell chamber assays and for apoptosis using Annexin V detection by flow cytometry. In addition, levels of STAT1, STAT1 Tyr-701 phosphorylation (pSTAT1), fibronectin, and β-catenin were determined using western blotting. In the case of IFN-γ stimulation, levels of S100A4, proliferating cell nuclear antigen (PCNA), and c-fos expression were also determined.

Results: We found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness. Moreover, STAT1 and STAT1-CC expression caused increases in pSTAT1 and decreases in fibronectin and β-catenin levels. STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.

Conclusion: The results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells. Results from this study suggest that combined treatment of IFNs and STAT1-CC might be a feasible approach for the clinical management of lung cancer in the future.

No MeSH data available.


Related in: MedlinePlus

STAT1-CC increases STAT1 phosphorylation in lung cancer cells. a Western blot for pSTAT1, fibronectin and β-catenin in STAT1- or STAT1-CC-expressing SPC-A-1 cells. d Western blot for pSTAT1, S100A4, PCNA and c-fos in STAT1- or STAT1-CC-expressing SPC-A-1 cells treated with IFN-γ (1000 U/ml) for 72 h. β-actin was used as a loading control. b, c, e–i Western blot levels of indicated proteins were quantified with the ChemiDoc ™ XRS+ scanner and Image Lab Software imaging system in the SPC-A-1 cells. The densities of each sample were normalized to the β-actin
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Fig6: STAT1-CC increases STAT1 phosphorylation in lung cancer cells. a Western blot for pSTAT1, fibronectin and β-catenin in STAT1- or STAT1-CC-expressing SPC-A-1 cells. d Western blot for pSTAT1, S100A4, PCNA and c-fos in STAT1- or STAT1-CC-expressing SPC-A-1 cells treated with IFN-γ (1000 U/ml) for 72 h. β-actin was used as a loading control. b, c, e–i Western blot levels of indicated proteins were quantified with the ChemiDoc ™ XRS+ scanner and Image Lab Software imaging system in the SPC-A-1 cells. The densities of each sample were normalized to the β-actin

Mentions: STAT1 Tyr-701 phosphorylation is an essential step for STAT1 activation. As shown in Fig. 6a, overexpression of STAT1 or STAT1-CC in SPC-A-1 cells promoted STAT1 tyrosine phosphorylation under basal conditions. STAT1-CC-expressing cells have a higher pSTAT1 level, although the protein level is 2.2- to 2.5-fold lower than that in STAT1-expressing cells. To better understand the decrease in migration and invasiveness of STAT1-CC cells, we examined several factors involved in cell growth, inhibition, migration and invasion. Expression of fibronectin was reduced at similar levels in STAT1- and STAT1-CC-expressing cells, while there was a greater decrease of β-catenin expression in STAT1-CC cells compared to STAT1-expressing cells (Fig. 6a, b). In the case of IFN-γ stimulation, the level of pSTAT1 in STAT1-CC-expressing cells is increased 12.6-fold whereas the pSTAT1 level in STAT1-expressing cells is only increased 2.2-fold compared to that of EGFP-expressing cells (Fig. 6d, f). Moreover, overexpression of STAT1 or STAT1-CC reduced S100A4, PCNA, and c-fos expressions when treated with IFN-γ, greater decreased levels of these proteins were observed in STAT1-CC cells compared to STAT1-expressing cells (Fig. 6d). The quantified data are shown in Fig. 6b, c and e–i.Fig. 6


STAT1 modification improves therapeutic effects of interferons on lung cancer cells.

Chen J, Zhao J, Chen L, Dong N, Ying Z, Cai Z, Ji D, Zhang Y, Dong L, Li Y, Jiang L, Holtzman MJ, Chen C - J Transl Med (2015)

STAT1-CC increases STAT1 phosphorylation in lung cancer cells. a Western blot for pSTAT1, fibronectin and β-catenin in STAT1- or STAT1-CC-expressing SPC-A-1 cells. d Western blot for pSTAT1, S100A4, PCNA and c-fos in STAT1- or STAT1-CC-expressing SPC-A-1 cells treated with IFN-γ (1000 U/ml) for 72 h. β-actin was used as a loading control. b, c, e–i Western blot levels of indicated proteins were quantified with the ChemiDoc ™ XRS+ scanner and Image Lab Software imaging system in the SPC-A-1 cells. The densities of each sample were normalized to the β-actin
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4562290&req=5

Fig6: STAT1-CC increases STAT1 phosphorylation in lung cancer cells. a Western blot for pSTAT1, fibronectin and β-catenin in STAT1- or STAT1-CC-expressing SPC-A-1 cells. d Western blot for pSTAT1, S100A4, PCNA and c-fos in STAT1- or STAT1-CC-expressing SPC-A-1 cells treated with IFN-γ (1000 U/ml) for 72 h. β-actin was used as a loading control. b, c, e–i Western blot levels of indicated proteins were quantified with the ChemiDoc ™ XRS+ scanner and Image Lab Software imaging system in the SPC-A-1 cells. The densities of each sample were normalized to the β-actin
Mentions: STAT1 Tyr-701 phosphorylation is an essential step for STAT1 activation. As shown in Fig. 6a, overexpression of STAT1 or STAT1-CC in SPC-A-1 cells promoted STAT1 tyrosine phosphorylation under basal conditions. STAT1-CC-expressing cells have a higher pSTAT1 level, although the protein level is 2.2- to 2.5-fold lower than that in STAT1-expressing cells. To better understand the decrease in migration and invasiveness of STAT1-CC cells, we examined several factors involved in cell growth, inhibition, migration and invasion. Expression of fibronectin was reduced at similar levels in STAT1- and STAT1-CC-expressing cells, while there was a greater decrease of β-catenin expression in STAT1-CC cells compared to STAT1-expressing cells (Fig. 6a, b). In the case of IFN-γ stimulation, the level of pSTAT1 in STAT1-CC-expressing cells is increased 12.6-fold whereas the pSTAT1 level in STAT1-expressing cells is only increased 2.2-fold compared to that of EGFP-expressing cells (Fig. 6d, f). Moreover, overexpression of STAT1 or STAT1-CC reduced S100A4, PCNA, and c-fos expressions when treated with IFN-γ, greater decreased levels of these proteins were observed in STAT1-CC cells compared to STAT1-expressing cells (Fig. 6d). The quantified data are shown in Fig. 6b, c and e–i.Fig. 6

Bottom Line: We found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness.STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.The results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang, China. jiestory414@163.com.

ABSTRACT

Background: Interferons (IFNs) have potent anti-proliferative, pro-apoptotic, and immunomodulatory activities against cancer. However, the clinical utility of IFNs is limited by toxicity and pharmacokinetics making it difficult to achieve sustained therapeutic levels especially in solid tumors.

Methods: Signal Transducer and Activator of Transcription 1 (STAT1) or a modified STAT1 (designated STAT1-CC) that is hyper-responsive to IFN were overexpressed in lung cancer SPC-A-1 and H1299 cells using lentiviral vectors. Transduction efficiency was monitored using enhanced green fluorescent protein (EGFP) expression. After transduction, cells were treated with interferon-gamma (IFN-γ) or interferon-beta (IFN-β) and monitored for cell proliferation, migration, and invasiveness using Cell Counting Kit-8 and transwell chamber assays and for apoptosis using Annexin V detection by flow cytometry. In addition, levels of STAT1, STAT1 Tyr-701 phosphorylation (pSTAT1), fibronectin, and β-catenin were determined using western blotting. In the case of IFN-γ stimulation, levels of S100A4, proliferating cell nuclear antigen (PCNA), and c-fos expression were also determined.

Results: We found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness. Moreover, STAT1 and STAT1-CC expression caused increases in pSTAT1 and decreases in fibronectin and β-catenin levels. STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.

Conclusion: The results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells. Results from this study suggest that combined treatment of IFNs and STAT1-CC might be a feasible approach for the clinical management of lung cancer in the future.

No MeSH data available.


Related in: MedlinePlus