Inactivation of p21WAF1 sensitizes cells to apoptosis via an increase of both p14ARF and p53 levels and an alteration of the Bax/Bcl-2 ratio

p21(WAF1) appears to be a major determinant of the cell fate in response to anticancer therapy. It was shown previously that HCT116 human colon cancer cells growing in vitro enter a stable arrest upon DNA damage, whereas cells with a defective p21(WAF1) response undergo apoptosis. Here we report that the enhanced sensitivity of HCT116/p21(-/-) cells to chemotherapeutic drug-induced apoptosis correlates with an increased expression of p53 and a modification of their Bax/Bcl-2 ratio in favor of the pro-apoptotic protein Bax. Treatment of HCT116/p21(-/-) cells with daunomycin resulted in a reduction of the mitochondrial membrane potential and in activation of caspase-9, whereas no such changes were observed in HCT116/p21(+/+) cells, providing evidence that p21(WAF1) exerts an antagonistic effect on the mitochondrial pathway of apoptosis. Moreover, the role of p53 in activation of this pathway was demonstrated by the fact that inhibition of p53 activity by pifithrin-alpha reduced the sensitivity of HCT116/p21(-/-) cells to daunomycin-induced apoptosis and restored a Bax/Bcl-2 ratio similar to that observed in HCT116p21(+/+) cells. Enhancement of p53 expression after disruption of p21(WAF1) resulted from a stabilization of p53, which correlated with an increased expression of the tumor suppressor p14(ARF), an inhibitor of the ubiquitin ligase activity of Mdm2. In accordance with the role of p14(ARF) in p53 stabilization, overexpression of p14(ARF) in HCT116/p21(+/+) cells resulted in a strong increase in p53 activity. Our results identify a novel mechanism for the anti-apoptotic effect of p21(WAF1) consisting in maintenance of mitochondrial homeostasis that occurs in consequence of a negative control of p14(ARF) expression.     

Anti-cancer effects of JKA97 are associated with its induction of cell apoptosis via a Bax-dependent and p53-independent pathway

p53, one of the most commonly mutated genes in human cancers, is thought to be associated with cancer development. Hence, screening and identifying natural or synthetic compounds with anti-cancer activity via p53-independent pathway is one of the most challenging tasks for scientists in this field. Compound JKA97 (methoxy-1-styryl-9H-pyrid-[3,4-b]-indole) is a small molecule synthetic anti-cancer agent, with unknown mechanism(s). In this study we have demonstrated that the anti-cancer activity of JKA97 is associated with apoptotic induction via p53-independent mechanisms. We found that co-incubation of human colon cancer HCT116 cells with JKA97 inhibited HCT116 cell anchorage-independent growth in vitro and tumorigenicity in nude mice and also induced a cell apoptotic response, both in the cell culture model and in a tumorigenesis nude mouse model. Further studies showed that JKA97-induced apoptosis was dramatically impaired in Bax knock-out (Bax(-/-)) HCT116 cells, whereas the knock-out of p53 or PUMA did not show any inhibitory effects. The p53-independent apoptotic induction by JKA97 was confirmed in other colon cancer and hepatocarcinoma cell lines. In addition, our results showed an induction of Bax translocation and cytochrome c release from the mitochondria to the cytosol in HCT116 cells, demonstrating that the compound induces apoptosis through a Bax-initiated mitochondria-dependent pathway. These studies provide a molecular basis for the therapeutic application of JKA97 against human cancers with p53 mutations.     

Essential roles of the Bcl-2 family of proteins in caspase-2-induced apoptosis

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. Recent studies suggest that it can mediate the death function of tumor suppressor p53 and is activated by a multimeric protein complex, PIDDosome. However, it is not clear how caspase-2 exerts its apoptotic function in cells and whether its enzymatic activity is required for the apoptotic function. In this study, we used both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses to investigate the mechanism by which caspase-2 induces apoptosis. We show that active caspase-2, but neither a catalytically mutated caspase-2 nor active caspase-2 with its inhibitor, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid(-/-) background, and the release could be restored by addition of the wild-type Bid protein, but not by Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax(-/-)Bak(-/-) mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak is essential for apoptosis triggered by caspase-2.     

Functional proteomics of resveratrol-induced colon cancer cell apoptosis: caspase-6-mediated cleavage of lamin A is a major signaling loop

The study investigated the molecular basis of resveratrol (RSV)-evoked apoptosis in four (Bax+/-, Bax-/-, p53+/+, and p53-/-) HCT116 colon cancer cell lines. RSV induced apoptosis in all the cells in a dose-dependent manner; however, Bax+/- and p53+/+ cells were more susceptible than their knockout counterparts (Bax-/- and p53-/-, respectively). Using Bax+/- cells as a model, proteomic analysis revealed four RSV-responsive events: fragmentation of lamin A/C protein; increase in concentration of a more basic isoelectric variant of the ribosomal protein P0; and decrease in concentration of dUTPase as well as stathmin 1. Lamin A cleavage in response to RSV treatment was confirmed using Western blot analysis. Caspase-6 was activated, which was evidenced by cleavage and accumulation in active form of caspase-6 as well as upregulation of the protease activity. RSV-elicited lamin A cleavage and apoptosis were entirely abrogated by the peptide inhibitors of caspase-6. Likewise, partial knockdown of caspase-6 expression using small interfering RNA resulted in significant inhibition of RSV-elicited lamin A cleavage and apoptosis. Furthermore, the lower apoptosis sensitivity of the knockout cells (Bax-/- and p53-/-) correlated with the relatively reduced processing of caspase-6 and lamin A cleavage. Taken together, these data highlight the critical role of caspase-6 and its cleavage of lamin A in apoptotic signaling triggered by RSV in the colon carcinoma cells, which can be activated in the absence of Bax or p53.     

Bax-dependent regulation of Bak by voltage-dependent anion channel 2

Many studies have demonstrated a critical role of Bax in mediating apoptosis, but the role of Bak in regulating cancer cell apoptotic sensitivities in the presence or absence of Bax remains incompletely understood. Using isogenic cells with defined genetic deficiencies, here we show that in response to intrinsic, extrinsic, and endoplasmic reticulum stress stimuli, HCT116 cells show clear-cut apoptotic sensitivities in the order of Bax+/Bak+ > Bax+/Bak- > Bax-/Bak+ > Bax-/Bak-. Small interference RNA-mediated knockdown of Bak in Bax-deficient cells renders HCT116 cells completely resistant to apoptosis induction. Surprisingly, however, Bak knockdown in Bax-expressing cells only slightly affects the apoptotic sensitivities. Bak, like Bax, undergoes the N terminus exposure upon apoptotic stimulation in both Bax-expressing and Bax-deficient cells. Gel filtration, chemical cross-linking, and co-immunoprecipitation experiments reveal that different from Bax, which normally exists as monomers in unstimulated cells and is oligomerized by apoptotic stimulation, most Bak in unstimulated HCT116 cells exists in two distinct protein complexes, one of which contains voltage-dependent anion channel (VDAC) 2. During apoptosis, Bak and Bax form both homo- and hetero-oligomeric complexes that still retain some VDAC-2. However, the oligomeric VDAC-2 complexes are diminished, and Bak does not interact with VDAC-2 in Bax-deficient HCT116 cells. These results highlight VDAC-2 as a critical inhibitor of Bak-mediated apoptotic responses.     

Polo-like kinase 1 inhibitors,mitotic stress and the tumor suppressor p53

Polo-like kinase 1 has been established as one of the most attractive targets for molecular cancer therapy. In fact, multiple small-molecule inhibitors targeting this kinase have been developed and intensively investigated. Recently, it has been reported that the cytotoxicity induced by Plk1 inhibition is elevated in cancer cells with inactive p53, leading to the hypothesis that inactive p53 is a predictive marker for the response of Plk1 inhibition. In our previous study based on different cancer cell lines, we showed that cancer cells with wild type p53 were more sensitive to Plk1 inhibition by inducing more apoptosis, compared with cancer cells depleted of p53. In the present work, we further demonstrate that in the presence of mitotic stress induced by different agents, Plk1 inhibitors strongly induced apoptosis in HCT116 p53^+/+ cells, whereas HCT116 p53^−/− cells arrested in mitosis with less apoptosis. Depletion of p53 in HCT116 p53^+/+ or U2OS cells reduced the induction of apoptosis. Moreover, the surviving HCT116 p53^−/− cells showed DNA damage and a strong capability of colony formation. Plk1 inhibition in combination with other anti-mitotic agents inhibited proliferation of tumor cells more strongly than Plk1 inhibition alone. Taken together, the data underscore that functional p53 strengthens the efficacy of Plk1 inhibition alone or in combination by strongly activating cell death signaling pathways. Further studies are required to investigate if the long-term outcomes of losing p53, such as low differential grade of tumor cells or defective DNA damage checkpoint, are responsible for the cytotoxicity of Plk1 inhibition.     

Targeting colorectal cancer stem cells with inducible caspase-9

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133⁺ CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase 3 activation, which is required for apoptosis as confirmed using the pan-caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role for caspase 8 in curcumin-induced caspase 3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase 3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation, both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of pro-apoptotic proteins, such as Bax, Bak, Bid and Bim. Cross-linking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid and Bim causes Bax channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation and apoptosis. Importantly, p21 deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement for p21 in Apaf-1-dependent caspase activation and apoptosis. Together, our findings identify Apaf-1, Bax and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.Key words: curcumin, mitochondria, cytochrome c, Apaf-1, caspase, p21     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase-3 activation, which is required for apoptosis as confirmed using the pan caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role of caspase-8 in curcumin-induced caspase-3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase-3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of proapoptotic proteins such as Bax, Bak, Bid, and Bim. Crosslinking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid, and Bim causes Bax-channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation, and apoptosis. Importantly, p21-deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement of p21 in Apaf-1 dependent caspase activation and apoptosis. Together, our findings demonstrate that Apaf-1, Bax, and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.     

The relative contribution of pro-apoptotic p53-target genes in the triggering of apoptosis following DNA damage in vitro and in vivo

The p53 pathway displays a large degree of redundancy in the expression of a number of pro-apoptotic mechanisms following DNA damage that, among others, involves increased expression of several pro-apoptotic genes through transactivation. Spatial and temporal cellular contexts contribute to the complexity of the regulation of apoptosis, hence different genes may show a cell- and tissue-dependent specificity with regard to the regulation of cell death and act in concert or show redundancy with one and another. We used siRNA technology to assess the effect of multiple ablations of documented pro-apoptotic p53 target genes (PPG) in the colorectal cancer cell line HCT116 and generated mice deficient in both of the extrinsic and intrinsic PPGs genes Dr5 and Puma following treatment with chemotherapeutics and ionizing radiation. DR5, Fas, Bax, Bad, Puma and Bnip3L were induced by 5-FU and adriamycin (ADR) in HCT116 cells in a p53-dependent manner. The resulting caspase 3/7 activity in HCT116 cells following treatment were suppressed by ablated expression of the PPGs in the extrinsic as well as the intrinsic pathway. To our surprise, knocking-down any of the PPGs concomitantly with DR5 did not further inhibit caspase 3/7 activity whereas inhibiting DR5-expression in HCT116Bax knockdown (kd) and HCT116Fas kd did, suggesting that these genes act downstream or in synergy with DR5. This was supported by our in vivo observations, since Puma and Dr5 were equally efficient in protecting cells of the spleen from sub-lethal radiation-induced apoptosis but less effective compared with irradiated p53^-/- mice. To our surprise, Dr5^-/-; Puma^-/- mice did not show additive protection from radiation-induced apoptosis in any of the investigated organs. Our data indicates that the intrinsic pathway may rely on extrinsic signals to promote cell death in a cell- and tissue-dependent manner following DNA damage. Furthermore, p53 must rely on mechanisms independent of DR5 and PUMA to initiate apoptosis following γ-radiation in the spleen and thymus in vivo.     

Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway

Nbk/Bik (natural born killer/Bcl-2-interacting killer) is a tissue-specific BH3-only protein whose molecular function is still largely unknown. To investigate the mechanism of Nbk action, we established a single- vector adenoviral system based on the Tet-off conditional expression of Nbk. Upon Nbk expression, only Bax-positive, but not Bax-deficient cells were found to undergo apoptosis. Interestingly, Nbk failed to induce apoptosis in the absence of Bax, even despite expression of the related molecule Bak. Re-expression of Bax restored the sensitivity to Nbk. Similarly, Bax wild-type HCT116 cells were highly susceptible, whereas HCT116 Bax knock-out cells remained resistant to Nbk-induced apoptosis. In Bax-positive cells, Nbk induced a conformational switch in the Bax N-terminus coinciding with cytochrome c release, mitochondrial permeability transition and caspase-9 processing. Immunoprecipitation studies revealed that Nbk interacts with Bcl-x(L) and Bcl-2 but not with Bax. Since, in addition, Nbk did not localize to the mitochondria, our data suggest a model in which Nbk acts as an indirect killer to trigger Bax-dependent apoptosis, whereas Bak is not sufficient to confer sensitivity to Nbk.     

Nanosecond pulsed electric fields induce apoptosis in p53-wildtype and p53-null HCT116 colon carcinoma cells

Non-ionizing radiation produced by nanosecond pulsed electric fields (nsPEFs) is an alternative to ionizing radiation for cancer treatment. NsPEFs are high power, low energy (non-thermal) pulses that, unlike plasma membrane electroporation, modulate intracellular structures and functions. To determine functions for p53 in nsPEF-induced apoptosis, HCT116p53^+/+ and HCT116p53^−/− colon carcinoma cells were exposed to multiple pulses of 60 kV/cm with either 60 ns or 300 ns durations and analyzed for apoptotic markers. Several apoptosis markers were observed including cell shrinkage and increased percentages of cells positive for cytochrome c, active caspases, fragmented DNA, and Bax, but not Bcl-2. Unlike nsPEF-induced apoptosis in Jurkat cells (Beebe et al. 2003a) active caspases were observed before increases in cytochrome c, which occurred in the presence and absence of Bax. Cell shrinkage occurred only in cells with increased levels of Bax or cytochrome c. NsPEFs induced apoptosis equally in HCT116p53^+/+ and HCT116p53^−/− cells. These results demonstrate that non-ionizing radiation produced by nsPEFs can act as a non-ligand agonist with therapeutic potential to induce apoptosis utilizing mitochondrial-independent mechanisms in HCT116 cells that lead to caspase activation and cell death in the presence or absence of p-53 and Bax. This work was supported by the U.S. Air Force Office of Scientific Research/DOD MURI grant on Subcellular Responses to Narrow Band and Wide Band Radio Frequency Radiation, administered by Old Dominion University, and the American Cancer Society.     

Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin

Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53(-/-) HCT116 and p21(-/-) HCT116 cell lines with a high concentration (250 nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nm) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53(-/-) HCT116 and p21(-/-) HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nm, but not 250 nm CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21(-/-) HCT116 cells following treatment with 20 nm CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.     

Comparison of growth inhibition profiles and mechanisms of apoptosis induction in human colon cancer cell lines by isothiocyanates and indoles from Brassicaceae

The isothiocyanates sulforaphane and PEITC (beta-phenethyl isothiocyanate) as well as the indoles indole-3-carbinol and its condensation product 3,3'-diindolylmethane are known to inhibit cancer cell proliferation and induce apoptosis. In this study, we compared the cell growth inhibitory potential of the four compounds on the p53 wild type human colon cancer cell line 40-16 (p53(+/+)) and its p53 knockout derivative 379.2 (p53(-/-)) (both derived from HCT116). Using sulforhodamin B staining to assess cell proliferation, we found that the isothiocyanates were strongly cytotoxic, whereas the indoles inhibited cell growth in a cytostatic manner. Half-maximal inhibitory concentrations of all four compounds in both cell lines ranged from 5-15 microM after 24, 48 and 72 h of treatment. Apoptosis induction was analyzed by immunoblotting of poly(ADP-ribose)polymerase (PARP). Treatment with sulforaphane (15 microM), PEITC (10 microM), indole-3-carbinol (10 microM) and 3,3'-diindolylmethane (10 microM) induced PARP cleavage after 24 and 48 h in both 40-16 and the 379.2 cell lines, suggestive of a p53-independent mechanism of apoptosis induction. In cultured 40-16 cells, activation of caspase-9 and -7 detected by Western blotting indicated involvement of the mitochondrial pathway. We detected time- and concentration-dependent changes in protein expression of anti-apoptotic Bcl-x(L) as well as pro-apoptotic Bax and Bak proteins. Of note is that for sulforaphane only, ratios of pro- to anti-apoptotic Bcl-2 family protein levels directly correlated with apoptosis induction measured by PARP cleavage. Taken together, we demonstrated that the glucosinolate breakdown products investigated in this study have distinct profiles of cell growth inhibition, potential to induce p53-independent apoptosis and to modulate Bcl-2 family protein expression in human colon cancer cell lines.     

Nanosecond pulsed electric fields (nsPEF) induce direct electric field effects and biological effects on human colon carcinoma cells

Nanosecond pulsed electric fields (nsPEFs) are ultrashort pulses with high electric field intensity (kV/cm) and high power (megawatts), but low energy density (mJ/cc). To determine roles for p53 in response to nsPEFs, HCT116 cells (p53+/+ and p53-/-) were exposed to nsPEF and analyzed for membrane integrity, phosphatidylserine externalization, caspase activation, and cell survival. Decreasing plasma membrane effects were observed in both HCT116p53+/+ and p53-/- cells with decreasing pulse durations and/or decreasing electric fields. However, addition of ethidium homodimer-1 and Annexin-V-FITC post-pulse demonstrated greater fluorescence in p53-/- versus p53+/+ cells, suggesting a postpulse p53-dependent biological effect at the plasma membrane. Caspase activity was significantly higher than nonpulsed cells only in the p53-/- cells. HCT116 cells exhibited greater survival in response to nsPEFs than HL-60 and Jurkat cells, but survival was more evident for HCT116p53+/+ cells than for HCT116p53-/- cells. These results indicate that nsPEF effects on HCT116 cells include (1) apparent direct electric field effects, (2) biological effects that are p53-dependent and p53-independent, (3) actions on mechanisms that originate at the plasma membranes and at intracellular structures, and (4) an apparent p53 protective effect. NsPEF applications provide a means to explore intracellular structures and functions that can reveal mechanisms in health and disease.     

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

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.