title: Chemotherapy regimen GOLF induces apoptosis in colon cancer cells through multi-chaperone complex inactivation and increased Raf-1 ubiquitin-dependent degradation.
abstract: The multi-drug combination of oxaliplatin (OXA), 5-Fluorouracil (5-FU) and leucovorin (LF) is currently considered as the gold standard treatment for metastatic colorectal carcinoma. In previous studies, we have studied a chemotherapy regimen containing gemcitabine (GEM), OXA, LF, and 5-FU (named GOLF regimen) that has shown a good safety profile and highly significant anti-tumor activity. In the present study, we have investigated on the anti-tumour mechanisms of GOLF in human colon cancer HT-29 and WiDr cell lines. We have found that GOLF induced growth inhibition that was largely caused by apoptosis differently from other combinations. Moreover, the different drugs composing GOLF were highly synergistic in inducing growth inhibition. Apoptosis induced by GOLF combination was paralleled by PARP cleavage and caspase 9 and 3 activation that were not recorded in the other combinations. An about 85% decrease of the activity of Erk and Akt was found in GOLF-treated cells. These effects were likely due to decreased expression of the upstream activator Raf-1 and of Akt itself, respectively. The intracellular levels of these signalling components can be post-translationally regulated by ubiquitin-dependent degradation through proteasome. Therefore, we have evaluated the expression of some chaperone components and we have found that GOLF did not affect the expression of both heat shock protein (HSP) 90 and 27 but induced an about 90% increase of HSP70 levels suggesting the inactivation of the multi-chaperone complex. Moreover, an about 4-fold increase of the ubiquitination of Raf-1 was also found and the addition for 12 h of 10 microM proteasome inhibitor lactacystin caused an accumulation of the ubiquitinated isoforms of Raf-1. In conclusions, GOLF was a combination highly synergistic in inducing both growth inhibition and apoptosis of colon cancer cells. These effects likely occurred through the disruption of critical survival pathways and the inactivation of multi-chaperone complex.
authors.affiliation: Experimental Oncology Department, National Cancer Institute Fondazione G. Pascale, Naples, Italy. michele.caraglia@fondazionepascale.it
authors.forename: Michele
authors.initials: M
authors.lastname: Caraglia
authors.forename: Monica
authors.initials: M
authors.lastname: Marra
authors.forename: Alfredo
authors.initials: A
authors.lastname: Budillon
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Meo
authors.forename: Filippo
authors.initials: F
authors.lastname: Ricciardiello
authors.forename: Ettore
authors.initials: E
authors.lastname: Bismuto
authors.forename: Giovanni
authors.initials: G
authors.lastname: Brachelente
authors.forename: Guido
authors.initials: G
authors.lastname: Francini
authors.forename: Antonio
authors.initials: A
authors.lastname: Giordano
authors.forename: Pierpaolo
authors.initials: P
authors.lastname: Correale
authors.forename: Alberto
authors.initials: A
authors.lastname: Abbruzzese
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects*; D002277:Carcinoma / Q000473:pathology; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D019073:HT29 Cells; D006801:Humans; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors*; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; D025801:Ubiquitin; D019908:Proto-Oncogene Proteins c-raf