Securinine, a myeloid differentiation agent with therapeutic potential for AML.

As the defining feature of Acute Myeloid Leukemia (AML) is a maturation arrest, a highly desirable therapeutic strategy is to induce leukemic cell maturation. This therapeutic strategy has the potential of avoiding the significant side effects that occur with the traditional AML therapeutics. We identified a natural compound securinine, as a leukemia differentiation-inducing agent. Securinine is a plant-derived alkaloid that has previously been used clinically as a therapeutic for primarily neurological related diseases. Securinine induces monocytic differentiation of a wide range of myeloid leukemia cell lines as well as primary leukemic patient samples. Securinine's clinical potential for AML can be seen from its ability to induce significant growth arrest in cell lines and patient samples as well as its activity in significantly impairing the growth of AML tumors in nude mice. In addition, securinine can synergize with currently employed agents such as ATRA and decitabine to induce differentiation. This study has revealed securinine induces differentiation through the activation of DNA damage signaling. Securinine is a promising new monocytic differentiation inducing agent for AML that has seen previous clinical use for non-related disorders.

p53-Dependent p21-mediated growth arrest pre-empts and protects HCT116 cells from PUMA-mediated apoptosis induced by EGCG.

The tumor suppressor protein p53 plays a key role in regulation of negative cellular growth in response to EGCG. To further explore the role of p53 signaling and elucidate the molecular mechanism, we employed colon cancer HCT116 cell line and its derivatives in which a specific transcriptional target of p53 is knocked down by homologous recombination. Cells expressing p53 and p21 accumulate in G1 upon treatment with EGCG. In contrast, same cells lacking p21 traverse through the cell cycle and eventually undergo apoptosis as revealed by TUNEL staining. Treatment with EGCG leads to induction of p53, p21 and PUMA in p21 wild-type, and p53 and PUMA in p21(-/-) cells. Ablation of p53 by RNAi protects p21(-/-) cells, thus indicating a p53-dependent apoptosis by EGCG. Furthermore, analysis of cells lacking PUMA or Bax with or without p21 but with p53 reveals that all the cells expressing p53 and p21 survived after EGCG treatment. More interestingly, cells lacking both PUMA and p21 survived ECGC treatment whereas those lacking p21 and Bax did not. Taken together, our results present a novel concept wherein p21-dependent growth arrest pre-empts and protects cells from otherwise, in its absence, apoptosis which is mediated by activation of pro-apoptotic protein PUMA. Furthermore, we find that p53-dependent activation of PUMA in response to EGCG directly leads to apoptosis with out requiring Bax as is the case in response to agents that induce DNA damage. p21, thus can be used as a molecular switch for therapeutic intervention of colon cancer.

Securinine induces p73-dependent apoptosis preferentially in p53-deficient colon cancer cells.

The identification of agents that preferentially kill cancer cells while protecting normal cells offers the potential to overcome toxicities found in many existing chemotherapeutic agents. Because p53 is frequently inactivated in cancer, agents that preferentially kill p53-null cells and protect wild-type p53-expressing cells are highly desirable chemotherapeutic agents. By using pairs of isogenic colon cancer cell lines that differ only in p53 expression (RKO and HCT116), securinine was found to exhibit these properties. Securinine (30 microM) induces apoptosis in 73% of p53-null HCT116 cells (LD(50) 17.5 microM) as opposed to 17.6% of HCT116 parental cells (LD(50) 50 microM) at 72 h after treatment. The mechanism of securinine-mediated death in p53-deficient cells involves the induction of the p53 family member, p73. Interestingly, the proapoptotic protein p73 is down-regulated in colon cancer cells expressing p53. This differential regulation of p73 in a p53-dependent fashion reveals a novel pathway for preferentially targeting cancer cells. In contrast to p53-deficient cells, cells expressing p53 are protected from cell death through the p53-mediated up-regulation of p21. These studies reveal a novel approach to specifically target colon cancer cells lacking p53 as well as identify a novel clinically relevant pathway to selectively induce p73 in p53-null cells.

Restoration of p53 functions protects cells from concanavalin A-induced apoptosis.

A great majority of human cancers encounter disruption of the p53 network. Identification and characterization of molecular components important in both p53-dependent and p53-independent apoptosis might be useful in developing novel therapies. Previously, we reported that concanavalin A (Con A) induced p73-dependent apoptosis of cells lacking functional p53. In the present study, we investigated the mechanism and role of p53 in protection from apoptosis induced by Con A. Treatment with Con A resulted in apoptosis of p53-null ovarian cancer, SKOV3, or Li-Fraumeni syndrome, MDAH041 (041), cells. However, their isogenic pairs, SKP53 and TR9-7, expressing wild-type p53 were much less sensitive and were protected by G(1) arrest. Inhibition of p53 function rendered these cells sensitive to Con A. Con A-induced apoptosis was accompanied by upregulation of forkhead box O1a (FOXO1a) and Bcl-2-interacting mediator (Bim), which were strongly inhibited after p53 expression and rescued after p53 ablation. Moreover, ablation of Bim by short hairpin RNA protected cells from apoptosis. Taken together, our study suggests that Con A induces apoptosis of cells lacking p53 by activating FOXO1a-Bim signaling and that expression of p53 protects these cells by inducing G(1) arrest and by downregulating the expression of both FOXO1a and Bim, identifying a novel cross-talk between FOXO1a and p53 transcription factors.

Identification of 6-benzylthioinosine as a myeloid leukemia differentiation-inducing compound.

As the pathophysiology of acute myelogenous leukemia (AML) involves a block of myeloid maturation, a desirable therapeutic strategy is to induce leukemic cell maturation to increase the efficacy and to avoid the side effects of traditional chemotherapeutics. Through a compound library screen, 6-benzylthioinosine (6BT) was identified as a promising differentiation-inducing agent. 6BT induces monocytic differentiation of myeloid leukemia cell lines such as HL-60 and OCI-AML3, as well as primary patient samples as evidenced by morphology, immunophenotyping, and nitroblue tetrazolium reduction. Not only can 6BT induce differentiation but a subset of AML cell lines such as MV4-11 and HNT34 instead undergo 6BT-mediated cell death. Despite inducing cell death in some leukemic cells, 6BT exhibits extremely low toxicity on several nonmalignant cells such as fibroblasts, normal bone marrow, and endothelial cells. This toxicity profile may relate to the function of 6BT as an inhibitor of the nucleoside transporter, ent1, which is thought to prevent it from entering many cell types. In contrast, 6BT likely enters at least some leukemic cell lines as shown by its requirement for phosphorylation for its differentiation activity. 6BT is also able to synergize with currently used myeloid differentiation agents such as ATRA and decitabine. Early studies indicate that the mechanism of action of this compound may involve ATP depletion that leads to growth inhibition and subsequent differentiation. Besides in vitro activity, 6BT also shows the ability to impair HL-60 and MV4-11 tumor growth in nude mice. 6BT is a promising new monocytic differentiation agent with apparent leukemic cell-specific activity.

DNA synthesis from unbalanced nucleotide pools causes limited DNA damage that triggers ATR-CHK1-dependent p53 activation.

p53-dependent G(1) and G(2) cell cycle checkpoints are activated in response DNA damage that help to maintain genomic stability. p53 also helps to protect cells from damage that occurs during S phase, for example, when the cells are starved for DNA precursors or irradiated with a low dose of UV. p53 is activated in normal cells starved for pyrimidine nucleotides by treatment with N-(phosphonacetyl)-l-aspartate (PALA). The treated cells progress through a first S phase with kinetics similar to those of untreated cells. However, the DNA of the treated cells begins to become damaged rapidly, within 12 h, as revealed by a comet assay, which detects broken DNA, and by staining for phosphorylated histone H2AX, which accumulates at sites of DNA damage. Because the cells survive, the damage must be reversible, suggesting single-strand breaks or gaps as the most likely possibility. The transiently damaged DNA stimulates activation of ATR and CHK1, which in turn catalyze the phosphorylation and accumulation of p53. Although PALA-induced DNA damage occurs only in dividing cells, the p53 that is activated is only competent to transcribe genes such as p21 and macrophage inhibitory cytokine 1 (whose products regulate G(2) and G(1) or S phase checkpoints, respectively) after the cells have exited the S phase during which damage occurs. We propose that p53 is activated by stimulation of mismatch repair in response to the misincorporation of deoxynucleotides into newly synthesized DNA, long before the lack of pyrimidine nucleoside triphosphates causes the rate of DNA synthesis to slow appreciably.

A role for SHPS-1/SIRPalpha in Concanavalin A-dependent production of MMP-9.

SHPS-1/SIRPalpha1 is a transmembrane glycoprotein that belongs to the immunoglobulin (Ig) super family. In the present study, we show that SHPS-1 strongly associates with Concanavalin A (Con A), a plant lectin obtained from jack beans. Further studies with SHPS-1 mutants reveal that the extracellular domain of SHPS-1 containing the Ig sequence is responsible for its association with Con A. Con A treatment induces cross-linking and multimerization of the SHPS-1 protein in the plasma membrane, accompanied by its tyrosine phosphorylation and recruitment of SHP-2. In contrast, Ricinus communis agglutinin (RCA), another lectin obtained from castor bean, does not bind or activate tyrosine phosphorylation of SHPS-1. Moreover, Con A activates Akt in a SHP-2-dependent manner. Treatment of mouse embryonic fibroblasts (MEFs) with Con A induces secretion of matrix metalloproteinase (MMP)-9, a phenomenon that is inhibited in cells expressing YF mutant of SHPS-1, a dominant negative form of Akt or in cells pre-treated with an Akt inhibitor, LY294002 or extracellular-signal regulated kinase (Erk) inhibitor, U0126. In addition, expression of the YF mutant of SHPS-1 inhibits Con A-dependent activation of Akt and Erk kinases. Taken together, our results suggest that SHPS-1 is a receptor for Con A that mediates Con A-dependent MMP-9 secretion through SHP-2-promoted activation of both Akt and Erk pathways.

A novel role for p73 in the regulation of Akt-Foxo1a-Bim signaling and apoptosis induced by the plant lectin, Concanavalin A.

Virtually all human cancers encounter disruption of the "p53 network." From a therapeutic point of view, it is important to devise strategies that eliminate cancer cells, which are often defective in functional p53 and protect p53-expressing normal cells. By comparing the response of a pair of isogenic cell lines, we identify a plant-derived compound, Concanavalin A (Con A), which differentially kills p53-null cells. Further, we find that p53 family member, p73, plays a critical role that is unmasked in the absence of p53. Con A treatment leads to induction of p73 and several others that are important mediators of apoptosis and act downstream, such as p21, Bax, Foxo1a, and Bim. Inactivation of p73 reverses the expression of these proteins and apoptosis. Inhibition of Akt activation sensitizes otherwise resistant cells. These observations thus reveal a novel role for p73 in the regulation of Akt-Foxo1a-Bim signaling and apoptosis especially when p53 is absent.

SHP-2 tyrosine phosphatase inhibits p73-dependent apoptosis and expression of a subset of p53 target genes induced by EGCG.

Green tea polyphenol, epigallocatechin-3-gallate (EGCG) differentially regulates the cellular growth of cancer cells in a p53-dependent manner through apoptosis and/or cell cycle arrest. In an effort to further elucidate the mechanism of differential growth regulation by EGCG, we have investigated the role of the tyrosine phosphatase, SHP-2. Comparing the responses of mouse embryonic fibroblasts (MEFs), expressing either WT or functionally inactive/truncated SHP-2, we find that inactivation of SHP-2 remarkably sensitizes cells to EGCG-mediated killing. MEFs lacking functional SHP-2 undergo massive apoptosis upon treatment with EGCG. By comparing gene expression profiles, we have identified a set of transcriptional targets of p53 that are differentially modulated in cells undergoing apoptosis. Western blot and real-time PCR analyses of a select group of genes further confirm that the expression is SHP-2-dependent. Similar observations were made in MEFs lacking p53, confirming that the expression of these "p53 target genes" is p53-independent. In addition, EGCG treatment induced the expression of p73 mRNA and protein in both cell types, but not p63. Inactivation of p73 in cells expressing nonfunctional SHP-2 markedly inhibited apoptosis and p53 target gene expression. Although phosphorylation of JNK is differentially regulated by SHP2, it was found to be dispensable for EGCG-induced apoptosis and p53 target gene expression. Our results have identified SHP-2 as a negative regulator of EGCG-induced-apoptosis and have identified a subset of p53 target genes whose expression is paradoxically not mediated by p53 but by one of its family members, p73.

DNA replication licensing factor minichromosome maintenance deficient 5 rescues p53-mediated growth arrest.

Inactivation of p53 signaling by mutation of p53 itself or abrogation of its normal function by other transfactors, such as MDM2, is a key event in the development of most human cancers. To identify novel regulators of p53, we have used a phenotype-based selection in which a total cDNA library in a retroviral vector has been introduced into TR9-7ER cells, which arrest when p53 is expressed from a tetracycline-regulated promoter. We have isolated several clones derived from cells that are not growth-arrested when p53 is overexpressed. In one clone, the levels of p53, p21, and MDM2 are comparable with those in TR9-7ER cells and, therefore, the abrogation of growth arrest by an exogenous cDNA is likely to be distal to p21. Using reverse transcription-PCR, we were able to isolate a cDNA of approximately 2.2 kb, which was found to have 99% identity to the nucleotides between about 80 and 2,288 of the open reading frame of a gene encoding DNA replication licensing factor. It encodes complete peptide of 734 residues of this protein also called minichromosome maintenance deficient 5 (MCM5) or cell division cycle 46 (Saccharomyces cerevisiae). Northern and Western blot analyses revealed that the expression of MCM5 and its transcriptional regulator, E2F1, is negatively regulated by p53. When MCM5 cDNA was reintroduced into fresh TR9-7ER cells, numerous colonies that grow in the absence of tetracycline were formed. This novel observation establishes a role for MCM5 in negating the growth arrest function of p53.

Macrophage inhibitory cytokine 1 mediates a p53-dependent protective arrest in S phase in response to starvation for DNA precursors.

p53 is essential for the cellular responses to DNA damage that help to maintain genomic stability. Protective p53-dependent cell-cycle checkpoints are activated in response to a wide variety of stresses, including not only DNA damage but also arrest of DNA synthesis and of mitosis. In addition to its role in activating the G(1) and G(2) checkpoints, p53 also helps to protect cells in S phase when they are starved for DNA precursors by treatment with the specific aspartate transcarbamylase inhibitor N-phosphonacetyl-l-aspartate (PALA), which blocks the synthesis of pyrimidine nucleotides. Even though p53 is activated, PALA-treated cells expressing low levels of p53 or lacking expression of p21 do not arrest in G(1) or G(2) but are blocked in S phase instead. In the complete absence of p53, PALA-treated cells continue to synthesize DNA slowly and eventually progress through S phase, suffering severe DNA damage that in turn triggers apoptosis. Expression of the secreted protein macrophage inhibitory cytokine 1 (MIC-1), a member of the TGF-beta superfamily, increases substantially after PALA treatment, and application of exogenous MIC-1 or its constitutive expression from a cDNA provides remarkable protection of p53-null cells from PALA-mediated apoptosis, arguing that the p53-dependent secretion of MIC-1 provides a major part of such protection. Stimulation of MIC-1-dependent S phase arrest in normal gut epithelial cells might help to revitalize the clinical use of PALA, which has been limited by gut toxicity.

p130/p107/p105Rb-dependent transcriptional repression during DNA-damage-induced cell-cycle exit at G2.

The progression of normal cells from G2 into mitosis is stably blocked when their DNA is damaged. Tumor cells lacking p53 arrest only transiently in G2, but eventually enter mitosis. We show that an important component of the stable G2 arrest in normal cells is the transcriptional repression of more than 20 genes encoding proteins needed to enter into and progress through mitosis. Studies from a number of labs including our own have shown that, by inducing p53 and p21/WAF1, DNA damage can trigger RB-family-dependent transcriptional repression. Our studies reported here show that p130 and p107 play a key role in transcriptional repression of genes required for G2 and M in response to DNA damage. For plk1, repression is partially abrogated by loss of p130 and p107, and is completely abrogated by loss of all three RB-family proteins. Mouse cells lacking RB-family proteins do not accumulate with a 4N content of DNA when exposed to adriamycin, suggesting that all three RB-family proteins contribute to G2 arrest in response to DNA damage. Stable arrest in the presence of functional p53-to-RB signaling is probably due to the ability of cells to exit the cell cycle from G2, a conclusion supported by our observation that KI67, a marker of cell-cycle entry, is downregulated in both G1 and G2 in a p53-dependent manner.

Ablation of either p21 or Bax prevents p53-dependent apoptosis induced by green tea polyphenol epigallocatechin-3-gallate.

Treatment with epigallocatechin-3-gallate (EGCG), a polyphenolic compound of green tea, results in activation of p53 and induction of apoptosis in prostate cancer LnCaP cells. However, no direct evidence has delineated the role of p53 and p53-dependent pathways in EGCG-mediated apoptosis. To understand the mechanism of negative growth regulation of prostate cancer cells by EGCG we undertook a genetic approach and generated an isogenic pair of prostate carcinoma cells PC3 (p53-/-) by stably introducing a cDNA encoding wild-type p53. Treatment of the resultant cells, PC3-p53, with EGCG led to, as reported earlier in LnCaP cells, an increase in p53 protein, which exacerbated both G1 arrest and apoptosis. This response was accompanied by an increase in the levels of p21 and Bax. The cells lacking p53 continued to cycle and did not undergo apoptosis upon treatment with similar concentrations of EGCG, thus establishing the action of EGCG in a p53-dependent manner. This observation was revalidated in another prostate cancer LNCaP cells harboring wild-type p53. Inactivation of p53 using small interfering RNA (siRNA) rendered these cells resistant to EGCG-mediated apoptosis. Because p53 activation led to increase in p21 and Bax, we investigated whether these two proteins are important in this process. Ablation of p21 protein by siRNA prevented G1 arrest and apoptosis in PC3-p53 cells. The p53-dependent increase in Bax expression altered the Bax/Bcl-2 ratio and paralleled the activation of caspase 9 and 3 and cleavage of PARP. Transfection of cells with Bax siRNA abolished these effects and inhibited apoptosis but did not affect the accumulation of the cells in G1. In summary, using isogenic cell lines and siRNA, we have clearly demonstrated that EGCG activates growth arrest and apoptosis primarily via p53-dependent pathway that involves the function of both p21 and Bax such that down-regulation of either molecule confers a growth advantage to the cells.

In vivo recombinant adenovirus-mediated p53 gene therapy in a syngeneic rat model for colorectal cancer.

The p53 gene has a significant role in controlling genomic stability of cancer. The purpose of this study was to evaluate the tumor response of allograft colorectal tumor treated with Ad5CMV-p53 in a syngeneic rat model. Two weeks after the inoculation of WB-2054-M5 tumor cells in the flank of rats, rats were randomly assigned by tumor size to one of three groups (n=18 in each): phosphate buffered saline (PBS), Ad5CMV, and Ad5CMV-p53. Recombinant adenovirus or PBS was administered through intratumoral injection at three divided doses every other day for 4 weeks. Apoptosis of the tumors was evaluated using TUNEL assay. After 2 and 4 weeks of treatment, the volume (cm(3)) of tumors in PBS, Ad5CMV, and Ad5CMV-p53 was as follows: 2 week: 1.66 +/-0.43, 1.40 +/-0.47, 0.75 +/-0.26 (p<0.001), 4 week: 4.41 +/-0.88, 3.93 +/-1.86, 2.33 +/-0.51 (p<0.001). Tumor growth showed no statistically significant difference between the PBS and Ad5CMV groups (6-week vol. p=0.32). The TUNEL assay results revealed more apparent apoptotic cells in Ad5CMV-p53-treated tumors than in other groups. Growth of allograft colorectal cancer in the syngeneic rat model was significantly suppressed by intratumoral Ad5CMV-p53 gene therapy. These results demonstrate that gene replacement therapy with p53 may provide a novel modality of treatment in conjunction with other present treatments for metastatic colorectal cancer.

The Wilms tumor suppressor-1 target gene podocalyxin is transcriptionally repressed by p53.

Wilms tumors are a heterogeneous class of tumors in which Wilms tumor suppressor-1 (WT1) and the p53 tumor suppressor may be variously inactivated by mutation, reduced in expression, or even overexpressed in the wild-type state. The downstream transcriptional targets of WT1 and p53 that are critical for mediating their roles in Wilms tumorigenesis are not well defined. The WiT49 cell line is characteristic of anaplastic Wilms tumors that are refractory to treatment and expresses wild-type WT1 and mutant p53. We have used the small molecule compound CP-31398 (Pfizer) to restore wild-type p53 function to the codon 248 mutant p53 present in WiT49 cells. In these cells, CP-31398 activated transcription of p53-regulated promoters and enhanced UV light-induced apoptosis without altering the overall p53 protein level. These phenotypes were accompanied by restored binding of the p53 protein to promoter sequences in vivo. Gene expression profiling of CP-31398-treated WiT49 cells revealed subsets of putative p53 target genes that were up- or down-regulated. A preferred target of p53-mediated repression in this system is the podocalyxin (PODXL) gene. PODXL is also transcriptionally regulated by WT1 and has roles in cell adhesion and anti-adhesion. Our results show that PODXL is a bona fide target of p53-mediated transcriptional repression while being positively regulated by WT1. We propose that inappropriate expression of PODXL due to changes in WT1 and/or p53 activity may contribute to Wilms tumorigenesis.

Limited role of N-terminal phosphoserine residues in the activation of transcription by p53.

The p53 tumor suppressor is phosphorylated in response to various cellular stress signals, such as DNA damage, leading to its release from MDM2 and consequent stabilization and activation as a transcription factor. In human U2OS cells, treatment with adriamycin causes p53 to be phosphorylated on all six serine residues tested, leading to the dissociation of p53 from MDM2 and transcription of the p21 and mdm2 genes. In contrast, in these cells, IPTG-dependent induction of p14ARF, which sequesters MDM2 away from p53, does not lead to detectable phosphorylation of any of the five N-terminal serine residues tested (6, 9, 15, 20, 37). Only C-terminal serine 392 is phosphorylated. However, the increase of p21 and mdm2 mRNAs was indistinguishable following treatment with adriamycin or induction of p14ARF. By using cDNA arrays to examine global p53-dependent gene expression in response to adriamycin or p14ARF, we found that most genes were regulated similarly by the two treatments. However, a subset of p53-regulated genes whose products have proliferative roles or regulate VEGF activity, newly described here, are repressed by p14ARF much more than by adriamycin. We conclude that the phosphorylation of p53 on N-terminal serine residues is not required for increased transcription of the great majority of p53-responsive genes and that the induction of p53 by p14ARF, with little phosphorylation, leads to substantial repression of genes whose products have roles in proliferation.

Tocotrienol-rich fraction of palm oil activates p53, modulates Bax/Bcl2 ratio and induces apoptosis independent of cell cycle association.

Anti-cancer properties of palm oil have been attributed to the presence of tocotrienols and carotenoids. Studies from various laboratories have shown that tocotrienol-rich fraction (TRF) of palm oil inhibits cell growth and induces apoptosis in both preneoplastic and neoplastic cells. However, the mechanism by which TRF induces apoptosis remains largely unknown. Since several chemopreventive agents have been shown to utilize p53 pathway in negative regulation of cell growth, using human colon carcinoma RKO cells which express wild type p53, we investigated the effect of TRF on components of p53 signaling network. Treatment of cells with TRF resulted in a dose- and time- dependent inhibition of growth and colony formation. Further, TRF treatment of RKO cells resulted in the induction of WAF1/p21 which appears to be independent of cell cycle regulation and is transcriptionally upregulated in p53 dependent fashion. These results were further confirmed by using cells that express luciferase from a p53 responsive promoter where TRF treatment leads to activation of p53 reporter activity. TRF treatment also resulted in alteration in Bax/Bcl2 ratio in favor of apoptosis, which was associated with the release of cytochrome c and induction of apoptotic protease-activating factor-1. This altered expression of Bcl2 family members triggered the activation of initiator caspase-9 followed by activation of effector caspase-3. These signaling cascades lead to condensed chromatin, DNA fragmentation and shrinkage of cell membrane resulting into apoptosis. Our data suggest that TRF-induced apoptosis in colon carcinoma cells is mediated by p53 signaling network which appears to be independent of cell cycle association.

Role of p53 and NF-kappaB in epigallocatechin-3-gallate-induced apoptosis of LNCaP cells.

We have recently shown that oral consumption of green tea polyphenols inhibits prostate carcinogenesis in transgenic mouse model of prostate cancer and suggested that induction of apoptosis in prostate cancer cells is responsible for these effects. Much of the chemopreventive effects of green tea are attributed to its major polyphenolic constituent (-) epigallocatechin-3-gallate (EGCG). In the present study, we report that EGCG-induced apoptosis in human prostate carcinoma LNCaP cells is mediated via modulation of two related pathways: (a) stabilization of p53 by phosphorylation on critical serine residues and p14ARF-mediated downregulation of murine double minute 2(MDM2) protein, and (b) negative regulation of NF-kappaB activity, thereby decreasing the expression of the proapoptotic protein Bcl-2. EGCG-induced stabilization of p53 caused an upregulation in its transcriptional activity, thereby resulting in activation of its downstream targets p21/WAF1 and Bax. Thus, EGCG had a concurrent effect on two important transcription factors p53 and NF-kappaB, causing a change in the ratio of Bax/Bcl-2 in a manner that favors apoptosis. This altered expression of Bcl-2 family members triggered the activation of initiator capsases 9 and 8 followed by activation of effector caspase 3. Activation of the caspases was followed by poly (ADP-ribose) polymerase cleavage and induction of apoptosis. Taken together, the data indicate that EGCG induces apoptosis in human prostate carcinoma cells by shifting the balance between pro- and antiapoptotic proteins in favor of apoptosis.

Stat1-dependent, p53-independent expression of p21(waf1) modulates oxysterol-induced apoptosis.

7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21(waf1), or Stat1, we found that optimal 7kchol-induced apoptosis requires p21(waf1) and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser(727) but not Tyr(701) was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-beta) blunted Ser(727) phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-beta receptor subunit exhibited blunted apoptosis. IFN-beta alone did not induce apoptosis; thus, 7kchol-induced release of IFN-beta was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21(waf1) was much less than in wild-type cells; introducing transient expression of p21(waf1) restored apoptosis. Stat1 and p21(waf1) were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.