Joanna Zawacka-Pankau

ROS-dependent activation of JNK converts p53 into an efficient inhibitor of oncogenes leading to robust apoptosis

Rescue of the p53 tumor suppressor is an attractive cancer therapy approach. However, pharmacologically activated p53 can induce diverse responses ranging from cell death to growth arrest and DNA repair, which limits the efficient application of p53-reactivating drugs in clinic. Elucidation of the molecular mechanisms defining the biological outcome upon p53 activation remains a grand challenge in the p53 field. Here, we report that concurrent pharmacological activation of p53 and inhibition of thioredoxin reductase followed by generation of reactive oxygen species (ROS), result in the synthetic lethality in cancer cells. ROS promote the activation of c-Jun N-terminal kinase (JNK) and DNA damage response, which establishes a positive feedback loop with p53. This converts the p53-induced growth arrest/senescence to apoptosis. We identified several survival oncogenes inhibited by p53 in JNK-dependent manner, including Mcl1, PI3K, eIF4E, as well as p53 inhibitors Wip1 and MdmX. Further, we show that Wip1 is one of the crucial executors downstream of JNK whose ablation confers the enhanced and sustained p53 transcriptional response contributing to cell death. Our study provides novel insights for manipulating p53 response in a controlled way. Further, our results may enable new pharmacological strategy to exploit abnormally high ROS level, often linked with higher aggressiveness in cancer, to selectively kill cancer cells upon pharmacological reactivation of p53.

p53-dependent inhibition of TrxR1 contributes to the tumor-specific induction of apoptosis by RITA

Thioredoxin reductase 1 (TrxR1) is a key regulator in many redox-dependent cellular pathways, and is often overexpressed in cancer. Several studies have identified TrxR1 as a potentially important target for anticancer therapy. The low molecular weight compound RITA (NSC 652287) binds p53 and induces p53-dependent apoptosis. Here we found that RITA also targets TrxR1 by non-covalent binding, followed by inhibition of its activity in vitro and by inhibition of TrxR activity in cancer cells. Interestingly, a novel ~130 kDa form of TrxR1, presumably representing a stable covalently linked dimer, and an increased generation of reactive oxygen species (ROS) were induced by RITA in cancer cells in a p53-dependent manner. Similarly, the gold-based TrxR inhibitor auranofin induced apoptosis related to oxidative stress, but independently of p53 and without apparent induction of the ~130 kDa form of TrxR1. In contrast to the effects observed in cancer cells, RITA had no impact on TrxR or ROS formation in normal fibroblasts (NHDF). The inhibition of TrxR1 can sensitize tumor cells to agents that induce oxidative stress and may directly trigger cell death. Thus, our results suggest that a unique p53-dependent effect of RITA on TrxR1 in cancer cells might synergize with p53-dependent induction of pro-apoptotic genes and oxidative stress, thereby leading to a robust induction of cancer cell death, without affecting non-transformed cells.

Inhibition of Glycolytic Enzymes Mediated by Pharmacologically Activated p53: TARGETING WARBURG EFFECT TO FIGHT CANCER*

Background: High dependence of cancer cells on glycolysis is a good target for cancer therapy. Results: Tumor suppressor p53 represses the expression of key regulators of metabolic genes HIF1a and c-Myc and glucose transporters GLUT1 and GLUT12. Conclusion: Blocking ATP production network by pharmacologically activated p53 contributes to cancer cell death. Significance: Tumor-selective killing by reconstituted p53 might be in part due to inhibition of glycolysis. Unique sensitivity of tumor cells to the inhibition of glycolysis is a good target for anticancer therapy. Here, we demonstrate that the pharmacologically activated tumor suppressor p53 mediates the inhibition of glycolytic enzymes in cancer cells in vitro and in vivo. We showed that p53 binds to the promoters of metabolic genes and represses their expression, including glucose transporters SLC2A12 (GLUT12) and SLC2A1 (GLUT1). Furthermore, p53-mediated repression of transcription factors c-Myc and HIF1α, key drivers of ATP-generating pathways in tumors, contributed to ATP production block. Inhibition of c-Myc by p53 mediated the ablation of several glycolytic genes in normoxia, whereas in hypoxia down-regulation of HIF1α contributed to this effect. We identified Sp1 as a transcription cofactor cooperating with p53 in the ablation of metabolic genes. Using different approaches, we demonstrated that glycolysis block contributes to the robust induction of apoptosis by p53 in cancer cells. Taken together, our data suggest that tumor-specific reinstatement of p53 function targets the “Achilles heel” of cancer cells (i.e. their dependence on glycolysis), which could contribute to the tumor-selective killing of cancer cells by pharmacologically activated p53.

p73 tumor suppressor protein: a close relative of p53 not only in structure but also in anti-cancer approach?

The discovery of the p53 tumor suppressor protein in 1979 shed new light on cancer cell biology and introduced a trend in cancer research focusing on p53-like proteins. This in turn led to the discovery of two homologous proteins of p53 - p63 in 1998 and p73 in 1997. The p53 family members are mainly involved in apoptosis induction under cellular stress, but also in early embryonic developmental processes. The p63 and p73 proteins activate the transcription of a number of p53 target genes. The precise role of p63 in cancer cells is not fully revealed yet, unlike that of p53 and p73. The p53 tumor suppressor protein is found inactive in approximately 50% of human cancers. However, p73 is not as often inactivated in tumors. Of importance, transcriptionally active forms of p73 induce apoptosis in cancer cells independent of p53 status. Moreover, the regulatory mechanisms governing p73 stability in cells are well described. These features promoted the research concerning p73-targeted anti-cancer treatment. The p73 protein is subject to sophisticated activatory and inhibitory regulatory mechanisms. The up-to-date anti-cancer compounds targeting p73 protein in vitro inhibit its negative regulators, which leads to the activation of p73 pro-apoptotic function in cancer cells. In the current review we present the recent scientific findings on p73 regulation in cells and the newest anti-cancer strategies concerning its tumor suppressor function.

The p53-mediated cytotoxicity of photodynamic therapy of cancer: Recent advances

Photodynamic therapy (PDT) is a promising modality for the treatment of both pre-malignant and malignant lesions. The mechanism of action converges mainly on the generation of reactive oxygen species which damage cancer cells directly as well as indirectly acting on tumor vasculature. The exact mechanism of PDT action is not fully understood, which is a formidable barrier to its successful clinical application. Elucidation of the mechanisms of cancer cell elimination by PDT might help in establishing highly specific, non-genotoxic anti-cancer treatment of tomorrow. One of the candidate PDT targets is the well-known tumor suppressor p53 protein recognized as the guardian of the genome. Together with its family members, p73 and p63 proteins, p53 is involved in apoptosis induction upon stress stimuli. The wild-type and mutant p53-targeting chemotherapeutics are currently extensively investigated as a promising strategy for highly specific anti-cancer therapy. In photodynamic therapy porphyrinogenic sensitizers are the most widely used compounds due to their potent biophysical and biochemical properties. Recent data suggest that the p53 tumor suppressor protein might play a significant role in porphyrin-PDT-mediated cell death by direct interaction with the drug which leads to its accumulation and induction of p53-dependent cell death both in the dark and upon irradiation. In this review we describe the available evidence on the role of p53 in PDT.

Induction of apoptosis in HL-60 cells through the ROS-mediated mitochondrial pathway by ramentaceone from Drosera aliciae.

Ramentaceone (1) is a naphthoquinone constituent of Drosera aliciae that exhibits potent cytotoxic activity against various tumor cell lines. However, its molecular mechanism of cell death induction has still not been determined. The present study demonstrates that 1 induces apoptosis in human leukemia HL-60 cells. Typical morphological and biochemical features of apoptosis were observed in 1-treated cells. Compound 1 induced a concentration-dependent increase in the sub-G1 fraction of the cell cycle. A decrease in the mitochondrial transmembrane potential (ΔΨm) was also observed. Furthermore, 1 reduced the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax and Bak, induced cytochrome c release, and increased the activity of caspase 3. The generation of reactive oxygen species (ROS) was detected in 1-treated HL-60 cells, which was attenuated by the pretreatment of cells with a free radical scavenger, N-acetylcysteine (NAC). NAC also prevented the increase of the sub-G1 fraction induced by 1. These results indicate that ramentaceone induces cell death through the ROS-mediated mitochondrial pathway.

Plumbagin induces apoptosis in Her2-overexpressing breast cancer cells through the mitochondrial-mediated pathway.

Breast cancer is the leading cause of death-related cancers in women. Approximately 30% of breast cancers overexpress the Her2 oncogene, which is associated with a poor prognosis and increased resistance to chemotherapy. Plumbagin (1), a constituent of species in the plant genera Drosera and Plumbago, displays antineoplastic activity toward various cancers. The present study was aimed at determining the anticancer potential of 1 toward Her2-overexpressing breast cancer cells and defining the mode of cell death induced in these cells. The results showed that 1 exhibited high antiproliferative activity toward the Her2-overexpressing cell lines SKBR3 and BT474. The antiproliferative activity of 1 was associated with apoptosis-mediated cell death, as revealed by caspase activation and an increase in the sub-G1 fraction of the cell cycle. Compound 1 increased the levels of the proapoptotic Bcl-2 family of proteins and decreased the level of the antiapoptotic Bcl-2 protein in SKBR3 and BT474 cells. Thus, these findings indicate that 1 induces apoptosis in Her2-overexpressing breast cancers through the mitochondrial-mediated pathway and suggest its potential for further investigation for the treatment of Her2-overexpressing breast cancer.

Pharmacological reactivation of p53 as a strategy to treat cancer

It has been confirmed through studies using the technique of unbiased sequencing that the TP53 tumour suppressor is the most frequently inactivated gene in cancer. This finding, together with results from earlier studies, provides compelling evidence for the idea that p53 ablation is required for the development and maintenance of tumours. Genetic reconstitution of the function of p53 leads to the suppression of established tumours as shown in mouse models. This strongly supports the notion that p53 reactivation by small molecules could provide an efficient strategy to treat cancer. In this review, we summarize recent advances in the development of small molecules that restore the function of mutant p53 by different mechanisms, including stabilization of its folding by Apr‐246, which is currently being tested in a Phase II clinical trial. We discuss several classes of compounds that reactivate wild‐type p53, such as Mdm2 inhibitors, which are currently undergoing clinical testing, MdmX inhibitors and molecules targeting factors upstream of Mdm2/X or p53 itself. Finally, we consider the clinical applications of compounds targeting p53 and the p53 pathway.

Protoporphyrin IX Interacts with Wild-type p53 Protein in Vitro and Induces Cell Death of Human Colon Cancer Cells in a p53-dependent and -independent Manner

Photodynamic therapy (PDT) of cancer is an alternative treatment for tumors resistant to chemo- and radiotherapy. It induces cancer cell death mainly through generation of reactive oxygen species by a laser light-activated photosensitizer. It has been suggested that the p53 tumor suppressor protein sensitizes some human cancer cells to PDT. However, there is still no direct evidence for this. We have demonstrated here for the first time that the photosensitizer protoporphyrin IX (PpIX) binds to p53 and disrupts the interaction between p53 tumor suppressor protein and its negative regulator HDM2 in vitro and in cells. Moreover, HCT116 colon cancer cells exhibited a p53-dependent sensitivity to PpIX in a dose-dependent manner, as was demonstrated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and fluorescence-activated cell sorter (FACS) analysis of cell cycle profiles. We have also observed induction of p53 target pro-apoptotic genes, e.g. puma (p53-up-regulated modulator of apoptosis), and bak in PpIX-treated cells. In addition, p53-independent growth suppression by PpIX was detected in p53-negative cells. PDT treatment (2 J/cm2) of HCT116 cells induced p53-dependent activation of pro-apoptotic gene expression followed by growth suppression and induction of apoptosis.

Evaluation of the Role of the Pharmacological Inhibition of Staphylococcus aureus Multidrug Resistance Pumps and the Variable Levels of the Uptake of the Sensitizer in the Strain-Dependent Response of Staphylococcus aureus to PPArg2-Based Photodynamic Inactivation

The emergence of antibiotic resistance among pathogenic bacteria has caused an urgent need for the development of alternative therapeutics. One possibility is a combination of nontoxic photosensitizers (PS) and visible light, recognized as photodynamic therapy. Although it is known that Staphylococcus aureus is susceptible to photodynamic inactivation (PDI), the factors that determine the emerging variation among strains in the response to the treatment remain unclear. Some data indicate that cationic photosensitizing dyes such as phenothiaziniums which vary a lot in the chemical structure might target multidrug resistance pumps. In this study, we analyzed whether the uptake and activity of the multidrug resistance pumps might influence the previously observed variations among the clinical strains to protoporphyrin‐derived, amphipilic protoporphyrin diarginate‐mediated photodynamic treatment (12 J cm−2). Using a new set of four additionally selected methicillin‐resistant and methicillin‐susceptible clinical as well as ATCC S. aureus strains we confirmed that the bactericidal effect of the PDI is strain‐dependent as it ranged from 0 to 5 log10‐unit reduction in viable counts. However, neither the variable levels of the uptaken PS nor the pharmacological inhibition of NorA efflux pump explained such a phenomenon.