dc.description.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. | en_US |