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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

Lentiviral vector constructs for STAT1 and STAT1-CC. Diagram illustrates the target sequence of the sites for double-cysteine mutations in the SH2 domain of STAT1 and subsequent orientation into STAT1 and then into lentiviral vectors expressing EGFP with intervening IRES. The abbreviation of STAT1 domains: ND N-terminal domain, CCD coiled coil domain, DBD DNA-binding domain, LD linker domain, SH2D SH2 domain, TD transactivation domain
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Fig1: Lentiviral vector constructs for STAT1 and STAT1-CC. Diagram illustrates the target sequence of the sites for double-cysteine mutations in the SH2 domain of STAT1 and subsequent orientation into STAT1 and then into lentiviral vectors expressing EGFP with intervening IRES. The abbreviation of STAT1 domains: ND N-terminal domain, CCD coiled coil domain, DBD DNA-binding domain, LD linker domain, SH2D SH2 domain, TD transactivation domain

Mentions: The full-length cDNA of STAT1 or STAT1-CC with Ala-656 to Cys-656 and Asn-658 to Cys-658 substitutions were amplified by PCR from plasmid Mx-STAT1-Flag-IRES-EGFP-neo and Mx-STAT1-CC-Flag-IRES-EGFP-neo that were generated as described previously [11]. The PCR products were then inserted into a lentiviral vector and confirmed by sequencing. The lentivirus particles were prepared in 293T cells as described previously [13, 14]. Lentiviral vectors encoding STAT1-IRES-EGFP and STAT1-CC-IRES-EGFP (Fig. 1) were transduced into lung cancer SPC-A-1 and H1299 cells to obtain stable STAT1- and STAT1-CC-expressing cells. Flow cytometry analysis of enhanced green fluorescent protein (EGFP) expression was used to detect the relative transduction efficiencies of the cell lines.Fig. 1


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)

Lentiviral vector constructs for STAT1 and STAT1-CC. Diagram illustrates the target sequence of the sites for double-cysteine mutations in the SH2 domain of STAT1 and subsequent orientation into STAT1 and then into lentiviral vectors expressing EGFP with intervening IRES. The abbreviation of STAT1 domains: ND N-terminal domain, CCD coiled coil domain, DBD DNA-binding domain, LD linker domain, SH2D SH2 domain, TD transactivation domain
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Lentiviral vector constructs for STAT1 and STAT1-CC. Diagram illustrates the target sequence of the sites for double-cysteine mutations in the SH2 domain of STAT1 and subsequent orientation into STAT1 and then into lentiviral vectors expressing EGFP with intervening IRES. The abbreviation of STAT1 domains: ND N-terminal domain, CCD coiled coil domain, DBD DNA-binding domain, LD linker domain, SH2D SH2 domain, TD transactivation domain
Mentions: The full-length cDNA of STAT1 or STAT1-CC with Ala-656 to Cys-656 and Asn-658 to Cys-658 substitutions were amplified by PCR from plasmid Mx-STAT1-Flag-IRES-EGFP-neo and Mx-STAT1-CC-Flag-IRES-EGFP-neo that were generated as described previously [11]. The PCR products were then inserted into a lentiviral vector and confirmed by sequencing. The lentivirus particles were prepared in 293T cells as described previously [13, 14]. Lentiviral vectors encoding STAT1-IRES-EGFP and STAT1-CC-IRES-EGFP (Fig. 1) were transduced into lung cancer SPC-A-1 and H1299 cells to obtain stable STAT1- and STAT1-CC-expressing cells. Flow cytometry analysis of enhanced green fluorescent protein (EGFP) expression was used to detect the relative transduction efficiencies of the cell lines.Fig. 1

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