Inhibition of Fas expression by RNAi modulates 5-fluorouracil-induced apoptosis in HCT116 cells expressing wild-type p53

Abstract

Drug resistance to 5-fluorouracil (5-FU) is still a major limitation to its clinical use. In addition, the clinical value of p53 as a predictive marker for 5-FU-based chemotherapy remains a matter of debate. Here, we used HCT116 human colorectal cancer cells expressing wild-type p53 and investigated whether inhibition of Fas expression by interference RNA modulates 5-FU-induced apoptosis. Cells were treated with 5-FU (1, 4 or 8 microM) for 8-48 h. Cell viability was evaluated by trypan blue dye exclusion. Apoptosis was assessed by changes in nuclear morphology and caspase activity. The interference RNA technology was used to silence Fas expression. Caspase activation, p53, Fas, cytochrome c, and Bcl-2 family protein expression was evaluated by immunoblotting. 5-FU was cytotoxic in HCT116 cells (p<0.001). Nuclear fragmentation and caspase-3, -8 and -9 activities were also markedly increased in HCT116 cells after 5-FU (p<0.001). In addition, wild-type p53 and Fas expression were 25- and 4-fold increased (p<0.05). Notably, when interference RNA was used to inhibit Fas, 5-FU-mediated nuclear fragmentation and caspase activity were markedly reduced in HCT116 cells. Finally, western blot analysis of mitochondrial extracts from HCT116 cells exposed to 5-FU showed a 6-fold increase in Bax, together with a 3-fold decrease in cytochrome c (p<0.001). In conclusion, 5-FU exerts its cytotoxic effects, in part, through a p53/Fas-dependent apoptotic pathway that involves Bax translocation and mitochondrial permeabilization.

Resveratrol displays converse dose-related effects on 5-fluorouracil-evoked colon cancer cell apoptosis: The roles of caspase-6 and p53

“…7 Several studies have demonstrated that 5-FU elicits apoptosis in colon cancer cells through the activation of intracellular caspases such as caspase-3, 8 and 9. 5 However, there is no conclusive evidence supporting the involvement of caspase-6 activation in 5-FU-evoked apoptosis of colon cancer cells, despite it being a possible major signalling event in colon cancer cell apoptosis. 6 In this study, we showed that 5-FU triggered the cancer cell apoptosis through the activation of caspase-6.…”

“…However, despite advancements in treatment regimens since the inception of 5-FU more than 40 years ago, response rates of colon cancer chemotherapy remain as low as 10-20%, and tumor cell resistance persists as a significant limitation to the clinical use of 5-FU. 5-FU has been shown to inhibit DNA proliferation in colon cancer cells by inhibiting the enzyme thymidylate synthase, leading to apoptosis, 3 a mechanism of active cell death characterized by rapid loss of plasma membrane integrity, DNA fragmentation, and altered expression of numerous genes. 4 Specifically, 5-FU has been shown to cause DNA damage-induced apoptosis in colon cancer cells through expression of Fas ligand and increase in cell surface expression of Fas, triggering the death receptor pathway of apoptosis.…”

“…4 Specifically, 5-FU has been shown to cause DNA damage-induced apoptosis in colon cancer cells through expression of Fas ligand and increase in cell surface expression of Fas, triggering the death receptor pathway of apoptosis. 5 Furthermore, 5-FU exerts its apoptotic effects through bax translocation, mitochondrial permeabilization and cytochrome c release, strongly suggesting a role of the mitochondrial pathway as well. The addition of a modifier of apoptosis signaling mechanisms to augment the chemotherapeutic effect of 5-FU is consequently a candidate approach to overcome 5-FU resistance in tumor cells.…”

5-Fluorouracil signaling through a calcium–calmodulin-dependent pathway is required for p53 activation and apoptosis in colon carcinoma cells

“…However, as shown by immunostaining, in response to 5-FU, both TNF-family receptors DR5 and CD95 are accumulated in the plasma membrane (Figure 1a), indicating that either one of them or both could have a vital role for efficient apoptosis. As conflicting evidences exist in this matter, 12,13,17 we decided to assess the contribution of each individual receptor to initiator caspase-8 and effector caspase-3 activation by means of small interfering RNA (siRNA) technology (Figures 1b and c). This experimental approach clearly demonstrated that DR5 but not CD95 is the sole receptor required for caspase-8 activation and further processing of caspase-2 and effector caspase-3 upon treatment with 5-FU.…”

“…11 Similar data have been provided by using tumor cell lines, although conflicting evidences exist postulating DR4, DR5 or CD95 as the regulatory receptor for caspase-8 activation and apoptosis. [12][13][14] Interestingly, death receptor ligand does not seem to be required for DISC formation in this particular apoptosis pathway. 12 Most DR5 expression studies in clinical tumor sections do not experimentally verify whether the death receptor is induced as a consequence of chemotherapy, or if it is a response to tumorogenesis.…”