Concomitant inactivation of the epidermal growth factor receptor, phosphatidylinositol 3-kinase/Akt and Janus tyrosine kinase 2/signal transducer and activator of transcription 3 signalling pathways in cardiotoxin III-treated A549 cells.
1. Cardiotoxin (CTX) III, a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has potential anticancer therapeutic activity. The aim of the present study was to investigate the apoptotic effect (and the underlying mechanism of action) of CTX III in human adenocarcinoma A549 cells. 2. It was found that CTX III induces apoptosis in A549 cells, as indicated by an increase in the sub-G(1) population, phosphatidylserine externalization, loss of mitochondrial membrane potential (Psi(m)) with cytochrome c release and activation of caspases 9 and 3. These actions were correlated with upregulation of Bax and Bad and downregulation of various anti-apoptotic proteins, including Bcl-2, Bcl-X(L), Mcl-1, X-linked inhibitor of apoptosis protein (XIAP) and p-Bad in CTX III-treated cells. 3. The signal transduction pathways involved in the effects of CTX III in A549 cells were evaluated using 5 micromol/L AG1478, an inhibitor of the epidermal growth factor receptor (EGFR), and exposing cells to the drug for 8 h. The results indicated that CTX III suppresses phosphorylation of EGFR and activation of phosphatidylinositol 3-kinase (PI3-K)/Akt and Janus tyrosine kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3, all of which are downstream molecules in the EGFR signalling pathway. 4. Exposure of cells for 8 h to the PI3-K inhibitor wortmannin (10 micromol/L) blocked JAK2 and STAT3 activation, whereas exposure of cells to the JAK2 inhibitor AG490 (5 micromol/L) decreased levels of phosphorylated (p-) JAK2 and p-STAT3 without affecting PI3-K/Akt activation. These observations suggest that PI3-K is an upstream activator of JAK2/STAT3. Furthermore, 5 micromol/L AG490 and 10 micromol/L wortmannin treatment of A549 cells for 8 h resulted in upregulation of Bax and Bad and downregulation of Bcl-2, Bcl-X(L), XIAP and p-Bad. 5. Together, the results of the present study indicate that CTX III induces apoptosis in A549 cells by inactivating the EGFR, PI3-K/Akt and JAK2/STAT3 signalling pathways.
Su
Jung-Chen
JC
Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Lin
Kuei-Li
KL
Chien
Ching-Ming
CM
Chuang
Pei-Wen
PW
Chang
Long-Sen
LS
Lin
Shinne-Ren
SR
D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004179:Cobra Cardiotoxin Proteins / Q000633:toxicity*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D005434:Flow Cytometry; D006801:Humans; D053614:Janus Kinase 2 / Q000037:antagonists & inhibitors*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects*
D004179:Cobra Cardiotoxin Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C082202:cardiotoxin III, Naja naja atra; D045304:Cytochromes c; D066246:ErbB Receptors; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D051057:Proto-Oncogene Proteins c-akt; D020169:Caspases