Tauroursodeoxycholic acid modulates p53-mediated apoptosis in Alzheimer's disease mutant neuroblastoma cells

Early onset familial Alzheimer's disease (FAD) is linked to autosomal dominant mutations in the amyloid precursor protein (APP) and presenilin 1 and 2 (PS1 and PS2) genes. These are critical mediators of total amyloid beta-peptide (Abeta) production, inducing cell death through uncertain mechanisms. Tauroursodeoxycholic acid (TUDCA) modulates exogenous Abeta-induced apoptosis by interfering with E2F-1/p53/Bax. Here, we used mouse neuroblastoma cells that express either wild-type APP, APP with the Swedish mutation (APPswe), or double-mutated human APP and PS1 (APPswe/DeltaE9), all exhibiting increased Abeta production and aggregation. Cell viability was decreased in APPswe and APPswe/DeltaE9 but was partially reversed by z-VAD.fmk. Nuclear fragmentation and caspase 2, 6 and 8 activation were also readily detected. TUDCA reduced nuclear fragmentation as well as caspase 2 and 6, but not caspase 8 activities. p53 activity, and Bcl-2 and Bax changes, were also modulated by TUDCA. Overexpression of p53, but not mutant p53, in wild-type and mutant neuroblastoma cells was sufficient to induce apoptosis, which, in turn, was reduced by TUDCA. In addition, inhibition of the phosphatidylinositide 3'-OH kinase pathway reduced TUDCA protection against p53-induced apoptosis. In conclusion, FAD mutations are associated with the activation of classical apoptotic pathways. TUDCA reduces p53-induced apoptosis and modulates expression of Bcl-2 family.     

4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (−/−) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs.     

Increased oxidative stress induces apoptosis in human cystic fibrosis cells

Oxidative stress results in deleterious cell function in pathologies associated with inflammation. Here, we investigated the generation of superoxide anion as well as the anti-oxidant defense systems related to the isoforms of superoxide dismutases (SOD) in cystic fibrosis (CF) cells. Pro-apoptotic agents induced apoptosis in CF but not in control cells that was reduced by treatment with SOD mimetic. These effects were associated with increased superoxide anion production, sensitive to the inhibition of IκB-α phosphorylation, in pancreatic but not tracheal CF cells, and reduced upon inhibition of either mitochondrial complex I or NADPH oxidase. CF cells exhibited reduced expression, but not activity, of both Mn-SOD and Cu/Zn-SOD when compared to control cells. Although, expression of EC-SOD was similar in normal and CF cells, its activity was reduced in CF cells. We provide evidence that high levels of oxidative stress are associated with increased apoptosis in CFTR-mutated cells, the sources being different depending on the cell type. These observations underscore a reduced anti-oxidant defense mechanism, at least in part, via diminished EC-SOD activity and regulation of Cu/Zn-SOD and Mn-SOD expressions. These data point to new therapeutic possibilities in targeting anti-oxidant pathways to reduce oxidative stress and apoptosis in CF cells.     

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     

Cadmium induces p53-dependent apoptosis in human prostate epithelial cells

Cadmium, a widespread toxic pollutant of occupational and environmental concern, is a known human carcinogen. The prostate is a potential target for cadmium carcinogenesis, although the underlying mechanisms are still unclear. Furthermore, cadmium may induce cell death by apoptosis in various cell types, and it has been hypothesized that a key factor in cadmium-induced malignant transformation is acquisition of apoptotic resistance. We investigated the in vitro effects produced by cadmium exposure in normal or tumor cells derived from human prostate epithelium, including RWPE-1 and its cadmium-transformed derivative CTPE, the primary adenocarcinoma 22Rv1 and CWR-R1 cells and LNCaP, PC-3 and DU145 metastatic cancer cell lines. Cells were treated for 24 hours with different concentrations of CdCl(2) and apoptosis, cell cycle distribution and expression of tumor suppressor proteins were analyzed. Subsequently, cellular response to cadmium was evaluated after siRNA-mediated p53 silencing in wild type p53-expressing RWPE-1 and LNCaP cells, and after adenoviral p53 overexpression in p53-deficient DU145 and PC-3 cell lines. The cell lines exhibited different sensitivity to cadmium, and 24-hour exposure to different CdCl(2) concentrations induced dose- and cell type-dependent apoptotic response and inhibition of cell proliferation that correlated with accumulation of functional p53 and overexpression of p21 in wild type p53-expressing cell lines. On the other hand, p53 silencing was able to suppress cadmium-induced apoptosis. Our results demonstrate that cadmium can induce p53-dependent apoptosis in human prostate epithelial cells and suggest p53 mutation as a possible contributing factor for the acquisition of apoptotic resistance in cadmium prostatic carcinogenesis.     

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.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

Aronia melanocarpa juice induces a redox-sensitive p73-related caspase 3-dependent apoptosis in human leukemia cells

Polyphenols are natural compounds widely present in fruits and vegetables, which have antimutagenic and anticancer properties. The aim of the present study was to determine the anticancer effect of a polyphenol-rich Aronia melanocarpa juice (AMJ) containing 7.15 g/L of polyphenols in the acute lymphoblastic leukemia Jurkat cell line, and, if so, to clarify the underlying mechanism and to identify the active polyphenols involved. AMJ inhibited cell proliferation, which was associated with cell cycle arrest in G(2)/M phase, and caused the induction of apoptosis. These effects were associated with an upregulation of the expression of tumor suppressor p73 and active caspase 3, and a downregulation of the expression of cyclin B1 and the epigenetic integrator UHRF1. AMJ significantly increased the formation of reactive oxygen species (ROS), decreased the mitochondrial membrane potential and caused the release of cytochrome c into the cytoplasm. Treatment with intracellular ROS scavengers prevented the AMJ-induced apoptosis and upregulation of the expression of p73 and active caspase 3. The fractionation of the AMJ and the use of identified isolated compounds indicated that the anticancer activity was associated predominantly with chlorogenic acids, some cyanidin glycosides, and derivatives of quercetin. AMJ treatment also induced apoptosis of different human lymphoblastic leukemia cells (HSB-2, Molt-4 and CCRF-CEM). In addition, AMJ exerted a strong pro-apoptotic effect in human primary lymphoblastic leukemia cells but not in human normal primary T-lymphocytes. Thus, the present findings indicate that AMJ exhibits strong anticancer activity through a redox-sensitive mechanism in the p53-deficient Jurkat cells and that this effect involves several types of polyphenols. They further suggest that AMJ has chemotherapeutic properties against acute lymphoblastic leukemia by selectively targeting lymphoblast-derived tumor cells.     

TrkA induces apoptosis of neuroblastoma cells and does so via a p53-dependent mechanism

Neuroblastoma (NB) is the most frequent solid extracranial tumor in children. Its clinical prognosis correlates with the expression of members of the Trk neurotrophin receptor family, which includes TrkA and TrkB. TrkA expression is associated with favorable prognosis, whereas TrkB expression is associated with poor prognosis. Here we show that TrkA expression induces the apoptosis of NB cells and does so by modulating the levels or activities of a number of proteins involved in regulating cell survival and apoptosis, including p53, Bcl-2, and caspase-3. TrkA increased the expression of p53 target proteins and failed to induce apoptosis in cells where p53 was inactivated by mutation or via expression of dominant inhibitory p53 or E1B55K, indicating that TrkA mediates apoptosis, at least in part, through p53. Treatment with a caspase inhibitor or overexpression of Bcl-X(L) also prevented TrkA from inducing apoptosis. In contrast, elevated expression of TrkA in non-transformed sympathetic neurons resulted in the suppression of p53 levels and enhanced survival. These results identify apoptosis as a novel biological response of TrkA in NB cells and imply that TrkA is a good prognosis marker for NB due in part to its ability to mediate apoptosis when expressed at sufficient levels.     

p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release

The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.     

Alpha-melanocyte-stimulating hormone suppresses oxidative stress through a p53-mediated signaling pathway in human melanocytes

Epidermal melanocytes are skin cells specialized in melanin production. Activation of the melanocortin 1 receptor (MC1R) on melanocytes by α-melanocyte-stimulating hormone (α-MSH) induces synthesis of the brown/black pigment eumelanin that confers photoprotection from solar UV radiation (UVR). Contrary to keratinocytes, melanocytes are slow proliferating cells that persist in the skin for decades, in an environment with high levels of UVR-induced reactive oxygen species (ROS). We previously reported that in addition to its role in pigmentation, α-MSH also reduces oxidative stress and enhances the repair of DNA photoproducts in melanocytes, independent of melanin synthesis. Given the significance of ROS in carcinogenesis, here we investigated the mechanisms by which α-MSH exerts antioxidant effects in melanocytes. We show that activation of the MC1R by α-MSH contributes to phosphorylation of p53 on serine 15, a known requirement for stabilization and activation of p53, a major sensor of DNA damage. This effect is mediated by the cAMP/PKA pathway and by the activation of phosphoinositide 3-kinase (PI3K) ATR and DNA protein kinase (DNA-PK). α-MSH increases the levels of 8-oxoguanine DNA glycosylase (OGG1) and apurinic apyrimidinic endonuclease 1 (APE-1/Ref-1), enzymes essential for base excision repair. Nutlin-3, an HDM2 inhibitor, mimicked the effects of α-MSH resulting in reduced phosphorylation of H2AX (γ-H2AX), a marker of DNA damage. Conversely, the p53 inhibitor pifithrin-α or silencing of p53 abolished the effects of α-MSH and augmented oxidative stress. These results show that p53 is an important target of the downstream MC1R signaling that reduces oxidative stress and possibly malignant transformation of melanocytes.     

NSC-87877 inhibits DUSP26 function in neuroblastoma resulting in p53-mediated apoptosis

Dual specificity protein phosphatase 26 (DUSP26) is overexpressed in high-risk neuroblastoma (NB) and contributes to chemoresistance by inhibiting p53 function. In vitro, DUSP26 has also been shown to effectively inhibit p38 MAP kinase. We hypothesize that inhibiting DUSP26 will result in decreased NB cell growth in a p53 and/or p38-mediated manner. NSC-87877 (8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid), a novel DUSP26 small molecule inhibitor, shows effective growth inhibition and induction of apoptosis in NB cell lines. NB cell lines treated with small hairpin RNA (shRNA) targeting DUSP26 also exhibit a proliferation defect both in vitro and in vivo. Treatment of NB cell lines with NSC-87877 results in increased p53 phosphorylation (Ser37 and Ser46) and activation, increased activation of downstream p38 effector proteins (heat shock protein 27 (HSP27) and MAP kinase-activated protein kinase 2 (MAPKAPK2)) and poly ADP ribose polymerase/caspase-3 cleavage. The cytotoxicity resulting from DUSP26 inhibition is partially reversed by knocking down p53 expression with shRNA and also by inhibiting p38 activity with SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine). In an intrarenal mouse model of NB, NSC-87877 treatment results in decreased tumor growth and increased p53 and p38 activity. Together, these results suggest that DUSP26 inhibition with NSC-87877 is an effective strategy to induce NB cell cytotoxicity in vitro and in vivo through activation of the p53 and p38 mitogen-activated protein kinase (MAPK) tumor-suppressor pathways.Neuroblastoma (NB) remains the most common extracranial solid tumor in children and is associated with a very poor prognosis in high-risk patients.¹ Current treatment strategy for this subgroup of patients includes intense myeloablative chemotherapy, radical surgical resection of primary tumor, radiation therapy and stem cell rescue. In spite of these therapies, survival in high-risk NB patients is <50% at 5 years from diagnosis.² These therapies confer major long-term toxicities in over 90% of long-term survivors; therefore, efforts are underway to identify more specific biologic therapies with less toxicity and better efficacy at targeting NB.A therapeutic strategy that is gaining much interest is utilizing small molecule inhibitors to activate innate, non-mutated cell senescence and death pathways, such as the p53 tumor suppressor. Mutations in the p53 gene are seen in over 60% of adult cancers; however, pediatric solid tumors, particularly NB, do not exhibit frequent p53 mutations and actually have an intact pathway that is suppressed by other mechanisms.³ Mouse double minute 2 (MDM2) inhibition is a strategy to activate p53 using compounds such as Nutlin-3a, RITA and RG7112, which has already been tested in a phase I clinical trial in adults.^{4, 5, 6} The p38 stress kinase, MAP kinase, pathway is another tumor-suppressive pathway that is upstream from p53 and can function through p53-dependent and -independent mechanisms to induce apoptosis. Although described as oncogenic in some cancers, there is evidence that p38 activation leads to tumor cell apoptosis in NB.^{7, 8, 9, 10} Both of these tumor-suppressive pathways are regulated through phosphorylation and dephosphorylation events by an array of kinases and phosphatases.Phosphatase targeting in NB has had very limited application because of the limited number of phosphatases found to have an oncogenic role. Protein phosphatase 2A (PP2A), protein tyrosine phosphatase receptor delta (PTPRD) and dual specificity protein phosphatase 12 (DUSP12) have been found to be involved in NB cell differentiation and tumor suppression.^{11, 12, 13, 14} First discovered in breast cancer, PPM1D, or Wip-1 phosphatase, is active in NB, and small molecule inhibition results in p53 activation and chemosensitivity.^{15, 16, 17} In this report, we show DUSP26 functions by inhibiting p53 and p38 function to promote growth of NB tumor cells.DUSP26 (MKP-8, LDP-4) was originally described as a dual specifity phosphatase with enzymatic activity against p38 MAP kinase resulting in dephosphorlyation of the primary p38 activation sites, Thr180/Tyr182.^{18, 19} Song et al.²⁰ showed that NSC-87877 (8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid), a small molecule phosphatase inhibitor of SHP-1 (src homology region 2 domain-containing phosphatase), specifically inhibits DUSP26 phosphatase activity at a much lower IC₅₀ than other phosphatases resulting in increased p38 phosphorylation in vitro. Yu et al.²¹ have shown that DUSP26 is overexpressed in anaplastic thyroid cancer tissue samples and functions by inhibiting the p38 MAP kinase pathway.A novel DUSP26 function shown in NB is enzymatic regulation of the p53 tumor suppressor.²² We demonstrated that DUSP26 physically binds p53, dephosphorylates p53 at Ser20 and Ser37, and causes inhibition of downstream p53 signaling. DUSP26 activity leads to increased chemoresistance to doxorubicin and VP-16 (etoposide) treatment, and overexpression was seen in high-risk NB tumor tissue samples correlating with a worse prognosis in these patients. Here, we show how DUSP26 has pro-proliferative effects in NB by inhibiting the p53 tumor-suppressor pathway, as well as the p38 mitogen-activated protein kinase (MAPK) pathway. Inhibition with small hairpin RNA (shRNA) targeting DUSP26 or NSC-87877 results in decreased proliferation and cell viability in NB cell lines in vitro and in vivo. Inhibiting the p53 or p38 pathways reverses this phenotype seen with inhibition of DUSP26. These data establish DUSP26 inhibition as a promising novel therapeutic approach for NB.     

Human 8-oxoguanine DNA glycosylase suppresses the oxidative stress induced apoptosis through a p53-mediated signaling pathway in human fibroblasts

Human 8-oxoguanine DNA glycosylase (hOGG1) is the main defense enzyme against mutagenic effects of cellular 7,8-dihydro-8-oxoguanine. In this study, we investigated the biological role of hOGG1 in DNA damage-related apoptosis induced by hydrogen peroxide (H(2)O(2))-derived oxidative stress. The down-regulated expression of hOGG1 by its small interfering RNA prominently triggers the H(2)O(2)-induced apoptosis in human fibroblasts GM00637 and human lung carcinoma H1299 cells via the p53-mediated apoptotic pathway. However, the apoptotic responses were specifically inhibited by hOGG1 overexpression. The p53-small interfering RNA transfection into the hOGG1-deficient GM00637 markedly inhibited the H(2)O(2)-induced activation of p53-downstream target proteins such as p21, Noxa, and caspase-3/7, which eventually resulted in the increased cell viability. Although the cell viability of hOGG1-knockdown H1299 p53 null cells was similar to that of the hOGG1 wild-type H1299, after the overexpression of p53 the hOGG1-knockdown H1299 showed the significantly decreased cell viability compared with that of the hOGG1 wild-type H1299 at the same experimental condition. Moreover, the array comparative genome hybridization analyses revealed that the hOGG1-deficient GM00637 showed more significant changes in the copy number of large regions of their chromosomes in response to H(2)O(2) treatment. Therefore, we suggest that although p53 is a major modulator of apoptosis, hOGG1 also plays a pivotal role in protecting cells against the H(2)O(2)-induced apoptosis at the upstream of the p53-dependent pathway to confer a survival advantage to human fibroblasts and human lung carcinomas through maintaining their genomic stability.     

Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells

Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer.     

Celecoxib induces p53-PUMA pathway for apoptosis in human colorectal cancer cells

Celecoxib, a clinical non-steroidal anti-inflammatory drug, displays anticarcinogenic and chemopreventive activities in human colorectal cancers, although the mechanisms of apoptosis by celecoxib are poorly understood. The existence of functional p53 but not securin in colorectal cancer cells was higher on the induction of cytotoxicity than the p53-mutational colorectal cancer cells following celecoxib treatment. The p53-wild type HCT116 cells were more susceptible to increase ∼25% cell death than the p53-null HCT116 cells after treatment with 100 μM celecoxib for 24 h. Transfection with a small interfering RNA of p53 reduced the celecoxib-induced cytotoxicity in the RKO (p53-wild type) colorectal cancer cells. Celecoxib (80-100 μM for 24 h) significantly increased total p53 proteins and the phosphorylated p53 proteins at serine-15, -20, -46, and -392 in RKO cells. However, the phospho-p53 (serine-15, -20, and -392) proteins were presented on the nuclei of cells but the phospho-p53 (serine-46) protein was located on the cytoplasma of apoptotic cells following treatment with celecoxib. Interestingly, the p53 up-regulated modulator of apoptosis (PUMA) protein, which located on the mitochondria, was induced by celecoxib in the p53-functional colorectal cancer cells but not in the p53-mutational cells. Together, this study provides the first time that celecoxib induces the various phosphorylated sites of p53 and activates p53-PUMA pathway, which potentiates the apoptosis induction in human colorectal cancer cells.     

Iron deprivation induces apoptosis independently of p53 in human and murine tumour cells

Iron deprivation induces apoptosis in some sensitive cultured tumour cells, while other cells are resistant. In order to elucidate the mechanisms involved in apoptosis induction by iron deprivation, we studied the expression of p53 and the expression of selected p53-regulated genes. To discriminate between changes coupled only with iron deprivation and changes involved in apoptosis induction by iron deprivation, we compared the expression of the genes in sensitive (human Raji, mouse 38C13) versus resistant (human HeLa, mouse EL4) cells under iron deprivation. Iron deprivation was achieved by incubation in a defined iron-free medium. The level of p53 mRNA decreased significantly under iron deprivation in sensitive cells, but it did not change in resistant cells. On the contrary, the level of the p53 protein under iron deprivation was slightly increased in sensitive cells while it was not changed in resistant cells. The activity of p53 was assessed by the expression of selected p53-regulated targets, i.e. p21(WAF1/CIP1) gene, mdm2, bcl-2 and bax. We did not detect any relevant change in mRNA levels as well as in protein levels of these genes under iron deprivation with the exception of p21(WAF1/CIP1). We detected a significant increase in the level of p21 mRNA in both (sensitive and resistant) mouse cell lines tested, however, we did not find any change in both (sensitive and resistant) human cell lines. Moreover, the p21(WAF1/CIP1) protein was accumulated in mouse-sensitive 38C13 cells under iron deprivation while all other cell lines tested, including human-sensitive cell line Raji, did not show any accumulation of p21(WAF1/CIP1) protein. It seems that the p21(WAF1/CIP1) mRNA, as well as protein accumulation, is not specifically coupled with apoptosis induction by iron deprivation and that it is rather cell-line specific. Taken together, we suggest that iron deprivation induces apoptosis at least in some cell types independently of the p53 pathway.