The Molecular Mechanisms of 5-Fluorouracil-Induced Apoptosis in Human Colorectal Cancer Cells and the Use of 5-FU as a Sensitising Agent to TRAIL

Abstract

Colorectal cancer is the third most common cause of death worldwide and despite many advances in recent years resistance to apoptosis induced by chemotherapeutic drugs, such as 5-fluorouracil (5-FU), remains a serious issue. A better understanding of how chemotherapeutic drugs induce apoptosis in colon cancer cells will better inform us on how to develop novel therapeutic strategies to overcome this resistance. We investigated cell death induced by 5-FU in the colon cancer cell line HCT116, and found that not only cell death, but also cytochrome-c release was dependent on an upstream caspase activity. Upon further investigation, we discovered a large (1-2 MDa) apoptosis-inducing complex forming following 5-FU-mediated RNA stress. This complex could be purified and identified by sucrose gradient density fractionation and was found to contain the core components caspase-8, FADD and RIP1 with interaction from Bid. In the absence of caspase-8 and FADD, complex formation and apoptosis was abolished. This complex forms without involvement of death receptors such as CD95 or the TRAIL receptors DR4 and DR5 despite a clear upregulation of DR5 protein levels following 5-FU. Futhermore, knocking down DR5 had no effect on initial caspase-8 cleavage and did not prevent complex formation. This complex forms upstream of the mitochondria, evidenced by overexpression of Bcl-2 or knocking down Bax or Bid. In addition we could demonstrate the inducible interaction between the complex members caspase-8 and FADD as well as FADD with RIP1 by co-immunoprecipitation, and the inducible interaction of FADD molecules following 5-FU stimulation. We also explored the contribution of the 5-FU-induced DR5 upregulation to Tumour necrosis-factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis in HCT116 cells. TRAIL selectively induces rapid apoptosis in most tumour cell types, and represents a promising anti-cancer agent. Subtoxic doses of TRAIL-induced apoptosis could be enhanced by co-treatment with low doses of 5-FU in a caspase dependent manner. Sensitisation to TRAIL involved enhanced DR5 expression and activation of the caspase cascade. Our results demonstrate that caspase-8 expression is necessary for this 5-FU-mediated TRAIL sensitivity. Finally, we demonstrated that low-dose 5-FU pre-treatment sensitises HCT116 cells to Mesenchymal stem cellmediated delivery of sTRAIL in vitro.