Actin-binding doliculide causes premature senescence in p53 wild type cells

Addressing the actin cytoskeleton as future anticancer target can be an innovative chemotherapeutic approach to combat malignancies. Doliculide is a potent stabilizer of actin filaments and can be used as tool and therapeutic lead in cancer research. Though a variety of molecules are known to bind to actin and lead to either its over- or depolymerization little is known about the pharmacological consequences of these effects within the cancer cell. In this work we used p53 wild-type cells to dissect the reaction of these cells towards subtoxic doses of doliculide. We could show that doliculide leads to a transient change in actin cytoskeleton dynamics that are reversible. The cells react towards the treatment with the induction of premature senescence, an established anti-cancer mechanism, in concentrations that are not cytotoxic. Furthermore, we investigated the signaling pathways that are involved in the induction and maintenance of senescence by a pathway directed mRNA PCR-array. This analysis revealed that under doliculide treatment up to 13% of senescence related genes are altered. Taken together, our data provide evidence for an antitumoral potential of actin binding agents in p53 wild type cells and brings the strategy of targeting the actin cytoskeleton closer to clinical application.     

Identification of NCF2/p67phox as a novel p53 target gene

Analysis of microarrays performed in p53-, TAp63α- and ΔNp63α-inducible SaOs-2 cell lines allowed the identification of NCF2 mRNA upregulation in response to p53 induction. NCF2 gene encodes for p67phox, the cytosolic subunit of the NADPH oxidase enzyme complex. The recruitment of p67phox to the cell membrane causes the activation of the NADPH oxidase complex followed by the generation of NADP+ and superoxide from molecular oxygen. The presence of three putative p53 binding sites on the NCF2 promoter was predicted, and the subsequent luciferase and chromatin immunoprecipitation assays showed the activation of NCF2 promoter by p53 and its direct binding in vivo to at least one of the sites, thus confirming the hypothesis. NCF2 upregulation was also confirmed by real-time PCR in several cell lines after p53 activation. NCF2 knockdown by siRNA results in a significant reduction of ROS production and stimulates cell death, suggesting a protective function of Nox2-generated ROS in cells against apoptosis. These results provide insight into the redox-sensitive signaling mechanism that mediates cell survival involving p53 and its novel target NCF2/p67phox.     

Identification of NCF2/p67phox as a novel p53 target gene

Analysis of microarrays performed in p53-, TAp63α- and ΔNp63α-inducible SaOs-2 cell lines allowed the identification of NCF2 mRNA upregulation in response to p53 induction. NCF2 gene encodes for p67phox, the cytosolic subunit of the NADPH oxidase enzyme complex. The recruitment of p67phox to the cell membrane causes the activation of the NADPH oxidase complex followed by the generation of NADP+ and superoxide from molecular oxygen. The presence of three putative p53 binding sites on the NCF2 promoter was predicted, and the subsequent luciferase and chromatin immunoprecipitation assays showed the activation of NCF2 promoter by p53 and its direct binding in vivo to at least one of the sites, thus confirming the hypothesis. NCF2 upregulation was also confirmed by real-time PCR in several cell lines after p53 activation. NCF2 knockdown by siRNA results in a significant reduction of ROS production and stimulates cell death, suggesting a protective function of Nox2-generated ROS in cells against apoptosis. These results provide insight into the redox-sensitive signaling mechanism that mediates cell survival involving p53 and its novel target NCF2/p67phox.     

Activation of p53-dependent responses in tumor cells treated with a PARC-interacting peptide

We tested the activity of a p53 carboxy-terminal peptide containing the PARC-interacting region in cancer cells with wild type cytoplasmic p53. Peptide delivery was achieved by fusing it to the TAT transduction domain (TAT-p53-C-ter peptide). In a two-hybrid assay, the tetramerization domain (TD) of p53 was necessary and sufficient to bind PARC. The TAT-p53-C-ter peptide disrupted the PARC-p53 complex. Peptide treatment caused p53 nuclear relocation, p53-dependent changes in gene expression and enhancement of etoposide-induced apoptosis. These studies suggest that PARC-interacting peptides are promising candidates for the enhancement of p53-dependent apoptosis in tumors with wt cytoplasmic p53.     

Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status

Sirtuins (SIRT) belonging to the NAD+ dependent histone deacetylase III class of enzymes have emerged as master regulators of metabolism and longevity. However, their role in prevention of organismal aging and cellular senescence still remains controversial. In the present study, we now report upregulation of SIRT2 as a specific feature associated with stress induced premature senescence but not with either quiescence or cell death. Additionally, increase in SIRT2 expression was noted in different types of senescent conditions such as replicative and oncogene induced senescence using multiple cell lines. Induction of SIRT2 expression during senescence was dependent on p53 status as depletion of p53 by shRNA prevented its accumulation. Chromatin immunoprecipitation revealed the presence of p53 binding sites on the SIRT2 promoter suggesting its regulation by p53, which was also corroborated by the SEAP reporter assay. Overexpression or knockdown of SIRT2 had no effect on stress induced premature senescence, thereby indicating that SIRT2 increase is not a cause of senescence; rather it is an effect linked to senescence-associated changes. Overall, our results suggest SIRT2 as a promising marker of cellular senescence at least in cells with wild type p53 status.     

Interaction of the anticancer p28 peptide with p53-DBD as studied by fluorescence,FRET, docking and MD simulations

Background

The p28 peptide, derived from the blue copper protein Azurin, exerts an anticancer action due to interaction with the tumor suppressor p53, likely interfering with its down-regulators. Knowledge of both the kinetics and topological details of the interaction, could greatly help to understand the peptide anticancer mechanism.

Curcumin induced apoptosis is mediated through oxidative stress in mutated p53 and wild type p53 colon adenocarcinoma cell lines

Curcumin has anti‐oxidant, anti‐cancer and anti‐carcinogen property. Our laboratory had previously reported that, curcumin treatment induces reactive oxygen species (ROS) generation in HT‐29 cell line, an effect contradictory to its anti‐oxidant property. This study evaluates the role of p53 in curcumin mediated ROS generation and cell death. Curcumin induced ROS was determined by 2’,7’‐dichlorofluorescein and apoptosis by Hoechst33342/PI staining in HT‐29 and HCT‐116 cell lines. ROS generation occurs within 1 hour of 40 µM curcumin treatment and a reduction was observed by third hour in HCT‐116 insinuating p53 involvement. N‐acetyl cysteine (NAC) pre‐treatment effectively quenched ROS and inhibited membrane potential loss in HT‐29, but less effective in HCT‐116. Mitochondrial membrane potential loss is evident with 10 and 40 µM curcumin in HCT‐116 and at 40 µM curcumin in HT‐29. Total p53 protein level increase was observed by 24 hours in HCT‐116 upon NAC pre‐treatment. Our results indicate that curcumin induces ROS mediated cell death in colon adenocarcinoma cell lines and may be mediated via p53.     

Gene expression profiling of p53-sensitive and -resistant tumor cells using DNA microarray

Overexpression of wild-type p53 in ECV-304 tumor cells induced extensive apoptosis and the eventual death of nearly all of the cells. We generated ECV-304 cells resistant to p53-induced apoptosis as a strategy to identify novel genes that might be relevant to p53-mediated apoptosis. ECV-304 cells resistant to p53 were isolated by repeated infections with a recombinant p53 adenovirus and were designated as DECV. The expression of 5,730 genes in p53-resistant (DECV) and p53-sensitive ECV-304 cells were profiled by DNA microarray analysis. We report here the expression of 80 genes that differed by 2-fold or more between sensitive and resistant cells upregulated for p53. Many of these differentially expressed genes are regulated by p53 in ECV-304 and H1299 p53-null cells. Our analysis identifies many new potential targets for p53 that play roles in cell cycle regulation, DNA repair, redox control, cell adhesion, apoptosis, and differentiation.     

p53在诱导性多潜能干细胞向肿瘤细胞转化中的作用

p53基因是抑制肿瘤形成的抑癌基因,p53通过阻断细胞周期、诱导衰老细胞凋亡和修复受损的DNA从而抑制肿瘤的形成。而突变的p53则具有相反的功能,被认为是促进细胞重编程的关键。这些发现表明p53在去分化过程中具有重要的作用,而肿瘤的形成与细胞重编程存在高度的相似性,所以探讨p53与诱导性多能干细胞(induced pluripotent stem cell,iPSC)和肿瘤形成之间的联系具有一定的意义。     

Differential regulation of signal transduction pathways in wild type and mutated p53 breast cancer epithelial cells by copper and zinc

Previous studies have suggested that cells may differ in their response to metal stress. This study was undertaken to investigate the role of PI3K/Akt signaling pathway in metal resistance in human breast cancer epithelial cells with different p53 and estrogen receptor status. Exposure to copper and zinc increased Akt phosphorylation with its nuclear localization only in MDA-MB-231 cells with no estrogen receptor and mutated p53. Cyclin D1 expression and cell-cycle progression followed the metal-induced Akt phosphorylation. Treatment with LY294002 abrogated these effects, suggesting the essential role of PI3-kinase. In contrast, in MCF-7 cells with wild type p53 and estrogen receptor, there was no change in Akt activation, while suppression of p53 activity by pifithrin-alpha increased phosphorylation of Akt after the treatment with copper. In MCF-7 cells, the metal treatment increased the phosphorylation of p53 at serine 15, up-regulated p21 expression, and resulted in cell-cycle arrest in G1 phase with apoptosis. These results demonstrate that copper-induced apoptosis in MCF-7 cells is p53 dependent, whereas the metal resistance in MDA-MB-231 cells may be due to activation of Akt in the absence of a functional p53.     

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

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

Depletion of the novel p53-target gene carnitine palmitoyltransferase 1C delays tumor growth in the neurofibromatosis type I tumor model

Despite the prominent pro-apoptotic role of p53, this protein has also been shown to promote cell survival in response to metabolic stress. However, the specific mechanism by which p53 protects cells from metabolic stress-induced death is unknown. Earlier we reported that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific member of a family of mitochondria-associated enzymes that have a central role in fatty acid metabolism promotes cell survival and tumor growth. Unlike other members of the CPT family, the subcellular localization of CPT1C and its cellular function remains elusive. Here, we report that CPT1C is a novel p53-target gene with a bona fide p53-responsive element within the first intron. CPT1C is upregulated in vitro and in vivo in a p53-dependent manner. Interestingly, expression of CPT1C is induced by metabolic stress factors such as hypoxia and glucose deprivation in a p53 and AMP activated kinase-dependent manner. Furthermore, in a murine tumor model, depletion of Cpt1c leads to delayed tumor development and a striking increase in survival. Taken together, our results indicate that p53 protects cells from metabolic stress via induction of CPT1C and that CPT1C may have a crucial role in carcinogenesis. CPT1C may therefore represent an exciting new therapeutic target for the treatment of hypoxic and otherwise treatment-resistant tumors.