title: Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C.
abstract: Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor Gö-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.
authors.affiliation: Department of Life Science and Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan, 300, Republic of China.
authors.forename: Chih-Wen
authors.initials: CW
authors.lastname: Shu
authors.forename: Nai-Lin
authors.initials: NL
authors.lastname: Cheng
authors.forename: Whei-meih
authors.initials: WM
authors.lastname: Chang
authors.forename: Tzu-Ling
authors.initials: TL
authors.lastname: Tseng
authors.forename: Yiu-Kay
authors.initials: YK
authors.lastname: Lai
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D016227:Benzoquinones; D002118:Calcium / Q000378:metabolism*; D002289:Carcinoma, Non-Small-Cell Lung / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D047029:Lactams, Macrocyclic; D008175:Lung Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology*; D015533:Transcriptional Activation
chemical_list: D016227:Benzoquinones; D017931:DNA Primers; D047029:Lactams, Macrocyclic; D011809:Quinones; D011493:Protein Kinase C; D002118:Calcium; C001277:geldanamycin