Abstract
BackgroundSignal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies. Therefore, STAT3 may be a promising target for treatment of tumor cells. To explore the possibility of a double-stranded decoy oligodeoxynucleotide (ODN) targeted blocking STAT3 over-activated tumor cells, we, here, evaluate the efficacy of STAT3 decoy ODN on human lung cancer cells in vitro and in vivo.MethodsA STAT3 decoy ODN was transfected into A549 lung cancer cell line in vitro by using lipofectamine. The flow cytometry and fluorescent microscopy were used to detect the transfection efficiency and the sub-cellular localization of STAT3 decoy ODN in A549 cells. Cell proliferation was determined by counting cell numbers and [3H]-thymidine uptake. Cell apoptosis was examined with Annexin V and propidum iodide by flow cytometry. The expression levels of STAT3 target genes were identified by RT-PCR and immunoblot. For in vivo experiment, A549 lung carcinoma-nude mice xenograft was used as a model to examine the effect of the STAT3 decoy by intratumoral injection. At the end of treatment, TUNEL and immunohistochemistry were used to examine the apoptosis and the expression levels of bcl-xl and cyclin D1 in tumor tissues.ResultsSTAT3 decoy ODN was effectively transfected into A549 lung cancer cells and mainly located in nucleus. STAT3-decoy ODN significantly induced apoptosis and reduced [3H]-thymidine incorporation of A549 cells as well as down-regulated STAT3-target genes in vitro. STAT3 decoy ODN also dramatically inhibited the lung tumor growth in xenografted nude mice and decreased gene expression of bcl-xl and cyclin D1.ConclusionSTAT3 decoy ODN significantly suppressed lung cancer cells in vitro and in vivo, indicating that STAT3 decoy ODN may be a potential therapeutic approach for treatment of lung cancer.
Highlights
Signal transducer and activator of transcription 3 (STAT3) is usually constitutively activated in a variety of malignancies
SiFnTitgoAuTAr3e54w19asceclolsnstitutively activated in A549 human lung cancer cell line and the efficiency of STAT3 decoy ODN transfected STAT3 was constitutively activated in A549 human lung cancer cell line and the efficiency of STAT3 decoy ODN transfected into A549 cells. (A) The A549 lung cancer cells were lysed in lysing buffer and the whole cell extracts (30 μg/lane) were separated by SDS-PAGE and examined by western blotting using anti-STAT3, anti-phospho-specific STAT3 (Tyr 705, Ser727) or β-actin antibodies
STAT3 was constitutively activated in A549 human lung cancer cell line and STAT3 decoy ODN was efficiently transfected into A549 cell lines To examine whether STAT3 is persistently activated in human lung cancer cells, the whole cell lysate from the human non-small-cell-lung carcinoma line A549 was processed for western blotting with anit-STAT3 and antiphospho-specific STAT3 (Tyr705, Ser727) antibodies
Summary
A STAT3 decoy ODN was transfected into A549 lung cancer cell line in vitro by using lipofectamine. The flow cytometry and fluorescent microscopy were used to detect the transfection efficiency and the sub-cellular localization of STAT3 decoy ODN in A549 cells. Cell apoptosis was examined with Annexin V and propidum iodide by flow cytometry. A549 lung carcinoma-nude mice xenograft was used as a model to examine the effect of the STAT3 decoy by intratumoral injection. At the end of treatment, TUNEL and immunohistochemistry were used to examine the apoptosis and the expression levels of bcl-xl and cyclin D1 in tumor tissues. Cell line and cell culture The human non-small-cell-lung cancer line, A549, were grown in RPMI 1640 medium (GIBCO/BRL) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml Ta. Product size β-actin cyclin D1 bcl-xl c-myc mcl-1 survivin cyclinE. 5'-CTC CTT AAT GTC ACG CAC GAT TT-3' 5'-GTG GGG CGC CCC AGG CAC CA-3' 5'-GAG ACC ATC CCC CTG ACG GC-3' 5'-CTC TTC CTC CTC CTC GGC GGC-3' 5'-GGA AAG CGT AGA CAA GGA GAT GC-3' 5'-GGT GGG AGG GTA GAG TGG ATG GT-3' 5'-GGT CTT CCC CTA CCC TCT CAA CGA-3' 5'-GGC AGC AGG ATA GTC CTT CCG AGT-3' 5'-GTG GTG GTG GTG GTT GGT TA-3' 5'-CGG CAG TCG CTG GAG ATT AT-3' 5'-GCA TGG GTG CCC CGA CGT TG-3' 5'-GCT CCG GCC AGA GGC CTC AA-3' 5'-ATA CAG ACC CAC AGA GAC AG-3' 5'-TGC CAT CCA CAG AAA TAC TT-3'
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