Interaction of actinomycin D with promoter element of c-met and its inhibitory effect on the expression of c-Met.
c-Met, the tyrosine kinase receptor for hepatocyte growth factor plays a pivotal role in normal cellular signaling and overexpression of c-Met protein is reported in several human cancers. Thus, transcriptional regulation of c-met appears to be an attractive target for chemotherapy. Therefore, we selected a 24mer GC rich sequence (24R) from the c-met promoter located at -142 to -119 from transcription start site and studied its interaction with anticancer drug, Actinomycin D. Spectroscopic analysis demonstrated a strong complexation between ActD and 24RY as shown by: (i) a high binding constant, K of 4-5 x 10(5) M(-1) with DeltaDeltaG of -47 +/- 1.5 Kcalmol(-1); (ii) marked increase by +10 degrees C in melting temperature of 24RY; and (iii) significant changes in circular dichroic spectra of both ActD and 24RY. Molecular modeling revealed the preference of ActD to the Sp1 binding site, GGCGGG, in 24RY. Expression of the c-Met was checked in HepG2 cells, a human hepatocellular carcinoma cell line by using western blotting and immunocytochemistry. Downregulation of c-Met expression by as much as 50% was observed in the presence of 20ng/ml (IC(50)) of ActD. Taking into account of the binding studies also, we feel that the down regulation of c-Met perhaps involves binding of ActD to the promoter site of c-met. Therefore, c-met could be a challenging and promising target for therapeutic strategies in combating cancer.
Singhal
Garima
G
Department of Biochemistry, All India Institute of Medical Science, New Delhi, India.
Rajeswari
Moganty R
MR
D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002942:Circular Dichroism; D017931:DNA Primers / Q000737:chemistry; D003609:Dactinomycin / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007124:Immunoenzyme Techniques; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011500:Protein Synthesis Inhibitors / Q000378:metabolism*; D019859:Proto-Oncogene Proteins c-met / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D013816:Thermodynamics; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D011500:Protein Synthesis Inhibitors; D003609:Dactinomycin; D019859:Proto-Oncogene Proteins c-met