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 µM 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. ABBREVIATIONS

5-Fluorouracil induces apoptosis in rat cardiocytes through intracellular oxidative stress

“…We have found that the antiproliferative activity of 5-FU ± LF was more pronounced in colon cancer cells than on cardiocytes and this effect was not surprising since this was on line with previous data demonstrating the in vitro activity of these drugs in colon cancer cell lines [35,36]. Thereafter, we have characterized the mechanism of cell death caused by the different treatments and we have found that cardiocytes were more sensitive to apoptosis induced by 5-FU and LF than colon cancer cells.…”

New frontiers in the treatment of colorectal cancer: Autophagy and the unfolded protein response as promising targets

“…After treatment with 5-FU and leucovorin, OS increased to 12 mo 43 . The GOLF regimen (gemcitabine, oxaliplatin, leucovorin, 5-FU) is another combination highly synergistic in inducing both growth inhibition and apoptosis of colon cancer cells 44 . Introduction of infusion regimens, such as FOLFIRI, has raised OS to a median of about 20 mo 45 .…”

Immunity Feedback and Clinical Outcome in Colon Cancer Patients Undergoing Chemoimmunotherapy with Gemcitabine + FOLFOX followed by Subcutaneous Granulocyte Macrophage Colony-Stimulating Factor and Aldesleukin (GOLFIG-1 Trial)

“…5-FU (GOLF) showed synergistic cytotoxic and proapoptotic effects in human colon cancer cell lines in vitro, and also led to molecular and antigenic changes (11,12), which could have promising immunotherapeutic implications. This regimen, in fact, showed the ability to convert the tumor cells into a powerful source of antigens, making them a potential vaccine source (12).…”