C-terminal peptides of p53 molecules enhance radiation-induced apoptosis in human mutant p53 cancer cells

We propose here a novel p53-targeting radio-cancer therapy using p53 C-terminal peptides for patients having mutated p53. Hoechst 33342 staining showed that X-ray irradiation alone efficiently induced apoptotic bodies in wild-type p53 (wt p53) human head and neck cancer cells transfected with a neo control vector (SAS/neo cells), but hardly induced apoptotic bodies in mutation-type p53 (m p53) cells transfected with a vector carrying the m p53 gene (SAS/m p53). In contrast, transfection of p53 C-terminal peptides (amino acid residues 361-382 or 353-374) via liposomes caused a remarkable increase of apoptotic bodies in X-ray-irradiated SAS/m p53 cells, but did not enhance apoptotic bodies in X-ray-irradiated SAS/neo cells. In immunocytochemical analysis, positively stained cells for active type caspase-3 were observed at high frequency after X-ray irradiation in the SAS/m p53 cells pre-treated with p53 C-terminal peptides. In SAS/neo cells, positively stained cells for active type caspase-3 were observed with X-ray irradiation alone. Furthermore, protein extracts from X-ray-irradiated SAS/m p53 cells showed higher DNA-binding activity of p53 to p53 consensus sequence when supplemented in vitro with p53 C-terminal peptides than extracts from non-irradiated SAS/m p53 cells. These results suggest that radiation treatment in the presence of p53 C-terminal peptides is more effective for inducing p53 -mediated apoptosis than radiation treatment alone or p53 C-terminal peptide treatment alone, especially in m p53 cancer cells. This novel tool for enhancement of apoptosis induction in m p53 cells might be useful for p53-targeted radio-cancer therapy.     

Curcumin induces apoptosis in human breast cancer cells through p53-dependent Bax induction

The aim of this study was to determine the mechanisms of curcumin-induced human breast cancer cell apoptosis. From quantitative image analysis data showing an increase in the percentage of cells with a sub-G0/G1 DNA content, we demonstrated curcumin-induced apoptosis in the breast cancer cell line MCF-7, in which expression of wild-type p53 could be induced. Apoptosis was accompanied by an increase in p53 level as well as its DNA-binding activity followed by Bax expression at the protein level. Further experiments using p53-null MDAH041 cell as well as low and high p53-expressing TR9-7 cell, in which p53 expression is under tight control of tetracycline, established that curcumin induced apoptosis in tumor cells via a p53-dependent pathway in which Bax is the downstream effector of p53. This property of curcumin suggests that this molecule could have a possible therapeutic potential in breast cancer patients.     

p53 acetylation enhances Taxol-induced apoptosis in human cancer cells

Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and β-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.     

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.     

Tumor-specific induction of apoptosis by a p53-reactivating compound

The tumor suppressor function of p53 is disabled in the majority of tumors, either by a point mutation of the p53 gene, or via MDM2-dependent proteasomal degradation. We have screened a chemical library using a cell-based assay and identified a low molecular weight compound named MITA which induced wild-type p53-dependent cell death in a variety of different types of human tumor cells, such as lung, colon and breast carcinoma cells, as well as in osteosarcoma and fibrosarcoma-derived cells. MITA inhibited p53-MDM2 interaction in vitro and in cells, which in turn prevented MDM2-mediated ubiquitination of p53 and resulted in a prolonged half-life and accumulation of p53 in tumor cells. Notably, p53 induction by MITA resulted in upregulated expression of p53 target genes MDM2, Bax, Gadd45 and PUMA, on protein and mRNA level. Importantly, neither p53 nor these target genes were induced in normal human fibroblasts (HDFs), which correlated with the absence of growth suppression in fibroblasts after treatment with MITA. However, upon activation of oncogenes in fibroblasts an induction and activation of p53 was observed, suggesting that activation of p53 by MITA occurs predominantly in tumor cells.     

Correlation between hydrophobic properties and efficiency of carrier-mediated membrane transduction and apoptosis of a p53 C-terminal peptide

Two membrane transporters, the 17 amino acid (aa) oligopeptide penetratin derived from the homeodomain of Antennapedia (Ant) and an analogue of the basic domain of TAT (aa 47-57) (TAT-a) from HIV-1, were tested as carriers for a p53 C-terminal peptide (aa 361-382) into human breast cancer cells. The studies were performed to determine whether the membrane-transduction efficiency of membrane carriers: Ant, TAT or TAT analogue (TAT-a) correlated with peptide hydrophobic features. Peptide-sequence analysis clearly demonstrated that the Ant sequence and p53 peptide sequence (p53p) together created a peptide with enhanced hydrophobic characteristics; while the TAT or TAT analogue (TAT-a) and p53p sequence together created a peptide with significantly less hydrophobic qualities. The degree of hydrophobic moment and helical wheel plots for these peptides correlated directly with their ability to transduce the p53 peptide. Western blot analysis revealed that Ant was able to transduce p53 C-terminal peptide into human breast cancer cells as a highly efficient membrane transporter. Compared to Ant, TAT-a fused to the C-terminus of p53 peptide (p53p-TAT-a) was a less efficient carrier into these cells under the conditions of our study. Additionally, N-terminal linked TAT-a to p53p (TAT-a-p53p) showed even lower efficiency as a transporter than p53-TAT-a. Apoptosis assays showed that the p53 peptide, fused at its C-terminus to Ant (p53p-Ant), induced a higher percentage of apoptotic cells in human breast cancer cell lines expressing mutant or wild-type p53 as compared to p53 peptide fused at its C-terminus to the TAT-a sequence (p53p-TAT-a) or when fused at the N-terminus to TAT-a (TAT-a-p53p). These data suggested a direct correlation between hydrophobic characteristics and efficiency as a transporter. Sequence study, using hydrophobic moment and helical wheel analyses, may be useful predictive tools for choosing the best carrier for a peptide.     

Evidence for a G2 checkpoint in p53-independent apoptosis induction by X-irradiation.

The p53 tumor suppressor gene is thought to be required for the induction of programmed cell death (apoptosis) initiated by DNA damage. We show here, however, that the human promyelocytic leukemia cell line HL-60, which is known to be deficient in p53 because of large deletions in the p53 gene, can be induced to undergo apoptosis following X-irradiation. We demonstrate that the decision to undergo apoptosis in this cell line appears to be made at a G2 checkpoint. In addition, we characterize an HL-60 variant, HCW-2, which is radioresistant. HCW-2 cells display DNA damage induction and repair capabilities identical to those of the parental HL-60 cell line. Thus, the difference between the two cell lines appears to be that X-irradiation induces apoptosis in HL-60, but not in HCW-2, cells. Paradoxically, HCW-2 cells display high levels of expression of bax, which enhances apoptosis, and no longer express bcl-2, which blocks apoptosis. HCW-2 cells' resistance to apoptosis may be due to the acquisition of expression of bcl-XL, a bcl-2-related inhibitor of apoptosis. In summary, apoptosis can be induced in X-irradiated HL-60 cells by a p53-independent mechanism at a G2 checkpoint, despite the presence of endogenous bcl-2. The resistance shown by HCW-2 cells suggests that bcl-XL can block this process.     

Glycerol restores heat-induced p53-dependent apoptosis of human glioblastoma cells bearing mutant p53

Background  

We have previously reported that glycerol acts as a chemical chaperone to restore the expression of WAF1 in some human cancer cell lines bearing mutant p53. Since the expression of WAF1 is up-regulated by activated wildtype p53, glycerol appears to restore wtp53 function. The aim of the present study is to examine the restoration of heat-induced p53-dependent apoptosis by glycerol in human glioblastoma cells (A-172) transfected with a vector carrying a mutant p53 gene (A-172/mp53 cells) or neo control vector (A-172/neo cells).     

p53 is required for etoposide-induced apoptosis of human embryonic stem cells

The molecular mechanisms controlling DNA-damage-induced apoptosis of human embryonic stem cells (hESC) are poorly understood. Here we investigate the role of p53 in etoposide-induced apoptosis. We show that p53 is constitutively expressed at high levels in the cytoplasm of hESC. Etoposide treatment results in a rapid and extensive induction of apoptosis and leads to a further increase in p53 and PUMA expression as well as Bax processing. p53 both translocates to the nucleus and associates with the mitochondria, accompanied by colocalization of Bax with Mcl1. hESC stably transduced with p53 shRNA display 80% reduction of endogenous p53 and exhibit an 80% reduction in etoposide-induced apoptosis accompanied by constitutive downregulation of Bax and an attenuated upregulation of PUMA. Our data further show that undifferentiated hESC that express Oct4 are much more sensitive to etoposide-induced apoptosis than their more differentiated progeny. Our study demonstrates that p53 is required for etoposide-induced apoptosis of hESC and reveals, at least in part, the molecular mechanism of DNA-damage-induced apoptosis in hESC.     

Tumor suppressor p53 plays a key role in induction of both tristetraprolin and let-7 in human cancer cells

Tristetraprolin (TTP) and let-7 microRNA exhibit suppressive effects on cell growth through down-regulation of oncogenes. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. However, the precise mechanism of this repression is unknown. We here demonstrate that p53 stimulated by the DNA-damaging agent doxorubicin (DOX) induced the expression of TTP in cancer cells. TTP in turn increased let-7 levels through down-regulation of Lin28a. Correspondingly, cancer cells with mutations or inhibition of p53 failed to induce the expression of both TTP and let-7 on treatment with DOX. Down-regulation of TTP by small interfering RNAs attenuated the inhibitory effect of DOX on let-7 expression and cell growth. Therefore, TTP provides an important link between p53 activation induced by DNA damage and let-7 biogenesis. These novel findings provide a mechanism for the widespread decrease in TTP and let-7 and chemoresistance observed in human cancers.     

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.     

Emodin-induced apoptosis through p53-dependent pathway in human hepatoma cells

Most of the commonly used cytotoxic anticancer drugs have been shown to induce apoptosis in susceptible cells. However, the signaling pathway of their apoptotic effects remains undefined. In this study, the cytotoxic effect of emodin on various human hepatoma cell lines was investigated. Results demonstrated that emodin exhibited strongly suppressing effect on HepG2/C3A, PLC/PRF/5, and SK-HEP-1 cells, with the IC(50) value of 42.5, 46.6, and 53.1 microM, respectively. Furthermore, emodin induced apoptosis in HepG2/C3A cells was clearly verified by the appearance of DNA fragmentation and sub-G(1) accumulation. Besides, HepG2/C3A cells were found to be arrested in G(2)/M phase after the cells were treated with 60 microM emodin for 48 h. Moreover, significant increase in the levels of apoptosis-related signals such as p53 (419.3 pg/ml), p21 (437.4 units/ml), Fas (6.6 units/ml), and caspase-3 (35.4 pmol/min) were observed in emodin treated HepG2/C3A cells. Taken together, emodin displays effective inhibitory effects on the growth of various human hepatoma cell lines and stimulates the expression of p53 and p21 that resulted in the cell cycle arrest of HepG2/C3A cells at G(2)/M phase. Results also suggest that emodin-induced apoptosis in HepG2/C3A cells were mediated through the activation of p53, p21, Fas/APO-1, and caspase-3. It implies that emodin could be a useful chemotherapeutical agent for treatment of hepatocellular carcinoma (HCC).     

翻译后修饰——p53功能调节的分子基础

p53的稳定与活化是细胞应对癌基因激活或DNA损伤等刺激的关键早期事件。可逆的翻译后修饰可严密调控p53的总蛋白质水平和反式激活能力,对维持正常的细胞生长、抑制细胞的早期癌变及肿瘤的发生至关重要。最新研究发现,除了磷酸化、泛素化和乙酰化修饰外,p53还能发生多个位点的甲基化、类泛素化和糖基化等修饰。这些翻译后修饰之间彼此联系,构成一个复杂的调控网络,对p53的稳定及其功能产生深远影响。     

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.     

Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53

Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.     

Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53

Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.