Cisplatin-induced apoptotic cell death in mouse hybrid neurons is blocked by antioxidants through suppression of cisplatin-mediated accumulation of p53 but not of Fas/Fas ligand

Peripheral neuropathy following cisplatin treatment is a major limiting factor in cisplatin chemotherapy of cancer patients. We investigated the pathomechanism underlying cisplatin neuropathy using a mouse dorsal root ganglion neuron-neuroblastoma hybrid cell line (N18D3) developed in our laboratory. DNA fragmentation, a characteristic feature of apoptosis, was induced in hybrid neurons following treatment with cisplatin. Accumulation of p53, Fas, and Fas ligand (Fas-L) was also demonstrated in these neurons. Preincubation with N-acetylcysteine (NAC), a precursor of glutathione, blocked cisplatin-induced apoptosis completely, whereas Trolox, a vitamin E analogue, blocked it partially. Cisplatin-induced p53 accumulation was suppressed by NAC treatment, whereas p53 accumulation was retarded by Trolox treatment. In contrast, neither NAC nor Trolox showed any inhibitory effect on cisplatin-induced Fas/Fas-L accumulation. These results suggest that the neuroprotective effects of antioxidants against cisplatin-induced neurotoxicity in hybrid neurons are mediated mainly through the inhibition of p53 accumulation but not of Fas/Fas-L accumulation by these antioxidants.     

Cisplatin-induced cell death in Saccharomyces cerevisiae is programmed and rescued by proteasome inhibition

Cisplatin is a highly effective chemotherapeutic drug used in the treatment of several tumors. It is a DNA-damaging agent that induces apoptosis of rapidly proliferating cells, an important factor underlying its therapeutic efficacy. Unfortunately, cellular resistance occurs often. A large fraction of tumor cells harbor mutations in p53, contributing to defects in apoptotic pathways and drug resistance. However, cisplatin-induced apoptosis can also occur in p53 deficient cells; thus, elucidation of the molecular mechanism involved will potentially yield new strategies to eliminate tumors that have defects in the p53 pathway. Most of the studies in this field have been conducted in cultured mammalian cells, not amenable to systematic genetic manipulation. Therefore, we aimed to establish a simplified model devoid of a p53 ortholog to study cisplatin-induced programmed cell death (PCD), using the yeast Saccharomyces cerevisiae.Our results indicate cisplatin induces an active form of cell death in yeast, as this process was partially dependent on de novo protein synthesis and did not lead to loss of membrane integrity. Cisplatin also increased DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction other than partial fragmentation. Co-incubation with the proteasome inhibitor MG132 increased resistance to cisplatin and, accordingly, yeast strains deficient in proteasome activity were more resistant to cisplatin than wild-type strains. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Our results indicate inhibition of the proteasome protects budding yeast from cisplatin-induced cell death and validate yeast as a model to study the role of the proteasome in cisplatin-induced PCD. Elucidation of this mechanism will aid in the development of new strategies to increase the efficacy of chemotherapy.     

p53 regulates a non-apoptotic death induced by ROS

DNA damage induced by reactive oxygen species and several chemotherapeutic agents promotes both p53 and poly (ADP-ribose) polymerase (PARP) activation. p53 activation is well known to regulate apoptotic cell death, whereas robust activation of PARP-1 has been shown to promote a necrotic cell death associated with energetic collapse. Here we identify a novel role for p53 in modulating PARP enzymatic activity to regulate necrotic cell death. In mouse embryonic fibroblasts, human colorectal and human breast cancer cell lines, loss of p53 function promotes resistance to necrotic, PARP-mediated cell death. We therefore demonstrate that p53 can regulate both necrotic and apoptotic cell death, mutations or deletions in this tumor-suppressor protein may be selected by cancer cells to provide not only their resistance to apoptosis but also to necrosis, and explain resistance to chemotherapy and radiation even when it kills via non-apoptotic mechanisms.     

Hypoxia-induced cell death of HepG2 cells involves a necrotic cell death mediated by calpain

To elucidate mechanism of cell death in response to hypoxia, we attempted to compare hypoxia-induced cell death of HepG2 cells with cisplatin-induced cell death, which has been well characterized as a typical apoptosis. Cell death induced by hypoxia turned out to be different from cisplatin-mediated apoptosis in cell viability and cleavage patterns of caspases. Hypoxia-induced cell death was not associated with the activation of p53 while cisplatin-induced apoptosis is p53 dependent. In order to explain these differences, we tested involvement of μ-calpain and m-calpain in hypoxia-induced cell death. Calpains, especially μ-calpain, were initially cleaved by hypoxia, but not by cisplatin. Interestingly, the treatment of a calpain inhibitor restored PARP cleavage that was absent during hypoxia, indicating the recovery of activated caspase-3. The inhibition of calpains prevented proteolysis induced by hypoxia. In addition, hypoxia resulted in a necrosis-like morphology while cisplatin induced an apoptotic morphology. The calpain inhibitor prevented necrotic morphology induced by hypoxia and converted partially to apoptotic morphology with nuclear segmentation. Our result suggests that calpains are involved in hypoxia-induced cell death that is likely to be necrotic in nature and the inhibition of calpain switches hypoxia-induced cell death to apoptotic cell death without affecting cell viability.     

Proteasome inhibition prevents cell death induced by the chemotherapeutic agent cisplatin downstream of DNA damage

Cisplatin is a highly effective chemotherapeutic drug acting as a DNA-damaging agent that induces apoptosis of rapidly proliferating cells. Unfortunately, cellular resistance still occurs. Mutations in p53 in a large fraction of tumor cells contribute to defects in apoptotic pathways and drug resistance. To uncover new strategies to eliminate tumors through a p53-independent pathway, we established a simplified model devoid of p53 to study cisplatin-induced regulated cell death, using the yeast Saccharomyces cerevisiae. We previously showed that cisplatin induces an active form of cell death accompanied by DNA condensation and fragmentation/degradation, but no significant mitochondrial dysfunction. We further demonstrated that proteasome inhibition, either with MG132 or genetically, increased resistance to cisplatin. In this study, we sought to determine how proteasome inhibition is important for cisplatin resistance by analyzing how it affects several phenotypes associated with the DNA damage response. We found MG132 does not seem to affect the activation of the DNA damage response or increase damage tolerance. Moreover, central modulators of the DNA damage response are not required for cisplatin resistance imparted by MG132. These results suggest the proteasome is involved in modulation of cisplatin toxicity downstream of DNA damage. Proteasome inhibitors can sensitize tumor cells to cisplatin, but protect others from cisplatin-induced cell death. Elucidation of this mechanism will therefore aid in the development of new strategies to increase the efficacy of chemotherapy.     

Resveratrol- induced apoptosis is mediated by p53-dependent pathway in Hep G2 cells

Resveratrol, a phytoalexin found in many plants, has been reported to possess a wide range of pharmacological properties and is one of the promising chemopreventive agents for cancer. Here, we examined the antiproliferation effect of resveratrol in two human liver cancer cell lines, Hep G2 and Hep 3B. Our results showed that resveratrol inhibited cell growth in p53-positive Hep G2 cells only. This anticancer effect was a result of cellular apoptotic death induced by resveratrol via the p53-dependent pathway. Here we demonstrated that the resveratrol-treated cells were arrested in G1 phase and were associated with the increase of p21 expression. In addition, we also illustrated that the resveratrol-treated cells had enhanced Bax expression but they were not involved in Fas/APO-1 apoptotic signal pathway. In contrast, the p53-negative Hep 3B cells treated with resveratrol did not show the antiproliferation effect neither did they show significant changes in p21 nor Fas/APO-1 levels. In summary, our study demonstrated that the resveratrol effectively inhibited cell growth and induced programmed cell death in Hepatoma cells on a molecular basis. Furthermore, these results implied that resveratrol might also be a new potent chemopreventive drug candidate for liver cancer as it played an important role to trigger p53-mediated molecules involved in the mechanism of p53-dependent apoptotic signal pathway.     

p73-dependent expression of DAN during cisplatin-induced cell death and osteoblast differentiation

We previously reported that DAN, a founding member of the DAN family of secreted proteins, acts as an inhibitor of cell cycle progression and is closely involved in retinoic acid-induced neuroblastoma differentiation. In this study, we found that DAN as well as p73, the recently identified p53 family member, was up-regulated during osteoblast differentiation. Additionally, the expression of DAN was increased in response to cisplatin-induced cell death of neuroblastoma SH-SY5Y cells. Consistent with the previous reports, p73 was accumulated after the treatment with cisplatin. Intriguingly, we found a putative p53/p73-binding site in the 5'-upstream region of the human DAN gene. A luciferase reporter assay and an in vitro DNA-binding experiment revealed that this canonical p53/p73-binding site was a functional responsive element and was specific for p73. Our results suggest that there exists a functional association between DAN and p73 during osteoblast differentiation as well as cisplatin-induced cell death.     

Cisplatin-induced non-apoptotic death of pancreatic cancer cells requires mitochondrial cyclophilin-D-p53 signaling

The pancreatic cancer remains a fatal disease for the majority of patients. Cisplatin has displayed significant cytotoxic effects against the pancreatic cancer cells, however the underlying mechanisms remain inconclusive. Here, we found that cisplatin mainly induced non-apoptotic death of the pancreatic cancer cells (AsPC-1 and Capan-2), which was associated with a significant p53 activation (phosphorylation and accumulation). Further, activated p53 was found to translocate to mitochondria where it formed a complex with cyclophilin D (Cyp-D). We provided evidences to support that mitochondrial Cyp-D/p53 complexation might be critical for cisplatin-induced non-apoptotic death of pancreatic cancer cells. Inhibition of Cyp-D by its inhibitor cyclosporine A (CsA), or by shRNA-mediated knockdown suppressed cisplatin-induced pancreatic cancer cell death. Both CsA and Cyp-D knockdown also disrupted the Cyp-D/p53 complex formation in mitochondria. Meanwhile, the pancreatic cancer cells with p53 knockdown were resistant to cisplatin. On the other hand, HEK-293 over-expressing Cyp-D were hyper-sensitive to cisplatin. Interestingly, camptothecin (CMT)-induced pancreatic cancer cell apoptotic death was not affected CsA or Cyp-D knockdown. Together, these data suggested that cisplatin-induced non-apoptotic death requires mitochondria Cyp-D-p53 signaling in pancreatic cancer cells.     

Tauroursodeoxycholic acid partially prevents apoptosis induced by 3-nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition

Ursodeoxycholic acid (UDCA) has been shown to be a strong modulator of the apoptotic threshold in both hepatic and nonhepatic cells. 3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, appears to cause apoptotic neuronal cell death in the striatum, reminiscent of the neurochemical and anatomical changes associated with Huntington's disease (HD). This study was undertaken (a) to characterize further the mechanism by which 3-NP induces apoptosis in rat neuronal RN33B cells and (b) to determine if and how the taurine-conjugated UDCA, tauroursodeoxycholic acid (TUDCA), inhibits apoptosis induced by 3-NP. Our results indicate that coincubation of cells with TUDCA and 3-NP was associated with an approximately 80% reduction in apoptosis (p < 0.001), whereas neither taurine nor cyclosporin A, a potent inhibitor of the mitochondrial permeability transition (MPT), inhibited cell death. Moreover, TUDCA, as well as UDCA and its glycine-conjugated form, glycoursodeoxycholic acid, prevented mitochondrial release of cytochrome c (p < 0.001), which probably accounts for the observed inhibition of DEVD-specific caspase activity and poly(ADP-ribose) polymerase cleavage. 3-NP decreased mitochondrial transmembrane potential (p < 0.001) and increased mitochondrial-associated Bax protein levels (p < 0.001). Coincubation with TUDCA was associated with significant inhibition of these mitochondrial membrane alterations (p < 0.01). The results suggest that TUDCA inhibits 3-NP-induced apoptosis via direct inhibition of mitochondrial depolarization and outer membrane disruption, together with modulation of Bax translocation from cytosol to mitochondria. In addition, cell death by 3-NP apparently occurs through pathways that are independent of the MPT.     

SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53

Nephrotoxicity is one of the important dose-limiting factors during cisplatin treatment. There is a growing body of evidence that activation of p53 has a critical role in cisplatin-induced renal apoptotic injury. The nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 decreases apoptosis through deacetylating of p53, and resveratrol is known as an activator of SIRT1. To study the role of SIRT1 in cisplatin-induced renal injury through interaction with p53, mouse proximal tubular cells (MPT) were treated with cisplatin and examined the expression level of SIRT1, acetylation of p53, PUMA-α, Bax, the cytosolic/mitochondrial cytochrome c ratio, and active caspase-3. The expression of SIRT1 was decreased by cisplatin. Resveratrol, a SIRT1 activator, ameliorated cisplatin-induced acetylation of p53, apoptosis, and cytotoxicity in MPT cells. In addition, resveratrol remarkably blocked cisplatin-induced decrease of Bcl-xL in MPT cells. Further specific SIRT1 inhibition with EX 527 or small interference RNA specific to SIRT1 reversed the effect of resveratrol on cisplatin-induced toxicity. Inhibition of p53 by pifithrin-α reversed the effect of EX527 in protein expression of PUMA-α, Bcl-xL, and caspase-3 and cytotoxicity in MPT cells. SIRT1 protein expression after cisplatin treatment was significantly decreased in the kidney. SIRT1 activation by resveratrol decreased cisplatin-induced apoptosis while improving the glomerular filtration rate. Taken together, our findings suggest that the modulation of p53 by SIRT1 could be a possible target to attenuate cisplatin-induced kidney injury.     

Apoptosis-inducing factor is involved in the regulation of caspase-independent neuronal cell death

Caspase-independent death mechanisms have been shown to execute apoptosis in many types of neuronal injury. P53 has been identified as a key regulator of neuronal cell death after acute injury such as DNA damage, ischemia, and excitotoxicity. Here, we demonstrate that p53 can induce neuronal cell death via a caspase-mediated process activated by apoptotic activating factor-1 (Apaf1) and via a delayed onset caspase-independent mechanism. In contrast to wild-type cells, Apaf1-deficient neurons exhibit delayed DNA fragmentation and only peripheral chromatin condensation. More importantly, we demonstrate that apoptosis-inducing factor (AIF) is an important factor involved in the regulation of this caspase-independent neuronal cell death. Immunofluorescence studies demonstrate that AIF is released from the mitochondria by a mechanism distinct from that of cytochrome-c in neurons undergoing p53-mediated cell death. The Bcl-2 family regulates this release of AIF and subsequent caspase-independent cell death. In addition, we show that enforced expression of AIF can induce neuronal cell death in a Bax- and caspase-independent manner. Microinjection of neutralizing antibodies against AIF significantly decreased injury-induced neuronal cell death in Apaf1-deficient neurons, indicating its importance in caspase-independent apoptosis. Taken together, our results suggest that AIF may be an important therapeutic target for the treatment of neuronal injury.     

Ursodeoxycholic acid modulates the ubiquitin-proteasome degradation pathway of p53

p53/Mdm-2 interaction is a prime target of ursodeoxycholic acid (UDCA) for regulating apoptosis in primary rat hepatocytes. Here, we further explored the role of UDCA in downregulating p53 by Mdm-2. UDCA reduced the stability of p53 by decreasing protein half-life. Although proteasomal activity was slightly increased with UDCA, the effect was also observed for other bile acids. More importantly, immunoprecipitation assays revealed that UDCA promoted p53 ubiquitination, therefore leading to increased p53 degradation. In this regard, proteasome inhibition after UDCA pre-treatment resulted in accumulation of ubiquitinated p53, which in turn was prevented in cells overexpressing a mutated form of p53 that does not undergo Mdm-2 ubiquitination. The involvement of Mdm-2 in UDCA-mediated response was further confirmed by siRNA-mediated gene silencing experiments. Finally, the protective effect of UDCA against p53-induced apoptosis was abolished after inhibition of proteasome activity and prevention of p53 ubiquitination by Mdm-2. These findings suggest that UDCA protects cells from p53-mediated apoptosis by promoting its degradation via the Mdm-2-ubiquitin-proteasome pathway.     

Rescue of platinum-damaged oocytes from programmed cell death through inactivation of the p53 family signaling network

Non-proliferating oocytes within avascular regions of the ovary are exquisitely susceptible to chemotherapy. Early menopause and sterility are unintended consequences of chemotherapy, and efforts to understand the oocyte apoptotic pathway may provide new targets for mitigating this outcome. Recently, the c-Abl kinase inhibitor imatinib mesylate (imatinib) has become the focus of research as a fertoprotective drug against cisplatin. However, the mechanism by which imatinib protects oocytes is not fully understood, and reports of the drug''s efficacy have been contradictory. Using in vitro culture and subrenal grafting of mouse ovaries, we demonstrated that imatinib inhibits the cisplatin-induced apoptosis of oocytes within primordial follicles. We found that, before apoptosis, cisplatin induces c-Abl and TAp73 expression in the oocyte. Oocytes undergoing apoptosis showed downregulation of TAp63 and upregulation of Bax. While imatinib was unable to block cisplatin-induced DNA damage and damage response, such as the upregulation of p53, imatinib inhibited the cisplatin-induced nuclear accumulation of c-Abl/TAp73 and the subsequent downregulation of TAp63 and upregulation of Bax, thereby abrogating oocyte cell death. Surprisingly, the conditional deletion of Trp63, but not ΔNp63, in oocytes inhibited apoptosis, as well as the accumulation of c-Abl and TAp73 caused by cisplatin. These data suggest that TAp63 is the master regulator of cisplatin-induced oocyte death. The expression kinetics of TAp63, c-Abl and TAp73 suggest that cisplatin activates TAp63-dependent expression of c-Abl and TAp73 and, in turn, the activation of TAp73 by c-Abl-induced BAX expression. Our findings indicate that imatinib protects oocytes from cisplatin-induced cell death by inhibiting c-Abl kinase, which would otherwise activate TAp73-BAX-mediated apoptosis. Thus, imatinib and other c-Abl kinase inhibitors provide an intriguing new way to halt cisplatin-induced oocyte death in early follicles and perhaps conserve the endocrine function of the ovary against chemotherapy.     

A novel in vivo post-translational modification of p53 by PARP-1 in MPTP-induced parkinsonism

Sporadic Parkinson's disease (PD) affects primarily dopaminergic neurons of the substantia nigra pars compacta. There is evidence of necrotic and apoptotic neuronal death in PD, but the mechanisms behind selected dopaminergic neuronal death remain unknown. The tumor suppressor protein p53 functions to selectively destroy stressed or abnormal cells during life and development by means of necrosis and apoptosis. Activation of p53 leads to death in a variety of cells including neurons. p53 is a target of the nuclear enzyme Poly(ADP-ribose)polymerase (PARP), and PARP is activated following DNA damage that occurs following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP is the favored in vivo model of PD, and reproduces the pathophysiology, anatomy and biochemistry of PD. p53 protein normally exhibits a fleeting half-life, and regulation of p53 stability and activation is achieved mainly by post-translational modification. We find that p53 is heavily poly(ADP-ribosyl)ated by PARP-1 following MPTP intoxication. This post-translational modification serves to stabilize p53 and alters its transactivation of downstream genes. These influences of PARP-1 on p53 may underlie the mechanisms of MPTP-induced parkinsonism and other models of neuronal death.     

Alpha-lipoic acid protects against cisplatin-induced ototoxicity via the regulation of MAPKs and proinflammatory cytokines

Cisplatin is an effective antineoplastic drug that is widely used to treat various cancers; however, it causes side effects such as ototoxicity via the induction of apoptosis of hair cells in the cochlea. Alpha-lipoic acid (ALA) has been reported to exert a protective effect against both antibiotic-induced and cisplatin-induced hearing loss. Therefore, this study was conducted to (1) elucidate the mechanism of the protective effects of ALA against cisplatin-induced ototoxicity using in vitro and ex vivo culture systems of HEI-OC1 auditory cells and rat cochlear explants and (2) to gain additional insight into the apoptotic mechanism of cisplatin-induced ototoxicity. ALA pretreatment significantly reduced apoptotic cell death of the inner and outer hair cells in cisplatin-treated organ of Corti explants and attenuated ototoxicity via marked inhibition of the increase in the expression of IL-1β and IL-6, the phosphorylation of ERK and p38, the degradation of IκBα, the increase in intracellular levels of ROS, and the activation of caspase-3 in cisplatin-treated HEI-OC1 cells. This study represents the first histological evaluation of the organ of Corti following treatment with ALA, and these results indicate that the protective effects of ALA against cisplatin-induced ototoxicity are mediated via the regulation of MAPKs and proinflammatory cytokines.