Esha Madan

Gallium compound GaQ3-induced Ca2+ signalling triggers p53-dependent and -independent apoptosis in cancer cells

BACKGROUND AND PURPOSE A novel anti‐neoplastic gallium complex GaQ3 (KP46), earlier developed by us, is currently in phase I clinical trial. GaQ3 induced S‐phase arrest and apoptosis via caspase/PARP cleavage in a variety of cancers. However, the underlying mechanism of apoptosis is unknown. Here, we have explored the mechanism(s) of GaQ3‐induced apoptosis in cancer cells, focusing on p53 and intracellular Ca2+ signalling.

Oxygen cycling in conjunction with stem cell transplantation induces NOS3 expression leading to attenuation of fibrosis and improved cardiac function

AIMS Myocardial infarction (MI) is associated with irreversible loss of viable cardiomyocytes. Cell therapy is a potential option to replace the lost cardiomyocytes and restore cardiac function. However, cell therapy is faced with a number of challenges, including survival of the transplanted cells in the infarct region, which is characterized by abundant levels of oxidants and lack of a pro-survival support mechanism. The goal of the present study was to evaluate the effect of supplemental oxygenation on cell engraftment and functional recovery in a rat model. METHODS AND RESULTS MI was induced in rats by a 60-min occlusion of the coronary artery, followed by restoration of flow. Mesenchymal stem cells (MSCs), isolated from adult rat bone marrow, were transplanted in the MI region. Rats with transplanted MSCs were exposed to hyperbaric oxygen (HBO: 100% O(2), 2 atmospheres absolute) for 90 min, 5 days/week for 4 weeks. The experimental groups were: MI (control), Ox (MI + HBO), MSC (MI + MSC), and MSC + Ox (MI + MSC + HBO). HBO exposure (oxygenation) was started 3 days after induction of MI. MSCs were transplanted 1 week after induction of MI. Echocardiography showed a significant recovery of cardiac function in the MSC + Ox group, when compared with the MI or MSC group. Oxygenation increased the engraftment of MSCs and vascular density in the infarct region. Molecular analysis of infarct tissue showed a four-fold increase in NOS3 expression in the MSC + Ox group compared with the MI group. CONCLUSIONS The results showed that post-MI exposure of rats to daily cycles of hyperoxygenation (oxygen cycling) improved stem cell engraftment, cardiac function, and increased NOS3 expression.

Chaperoning of Mutant p53 Protein by Wild-type p53 Protein Causes Hypoxic Tumor Regression * □

Background: Hypoxia-induced p53 is transcriptionally inactive, and its molecular conformation and functional status in hypoxic tumors are unknown. Results: WT p53 exists in mutant conformation in hypoxic tumors, and its conformation is oxygen-dependent. WT p53 functions as a molecular chaperone. Conclusion: WT p53 chaperones and rescues mutant p53 in hypoxic tumors. Significance: p53 chaperone therapy causes regression of hypoxic tumor xenografts through WT p53 chaperone activity. Mutant (Mt) p53 abrogates tumor suppression functions of wild-type (WT) p53 through mutant-specific, gain-of-function effects, and patients bearing Mt p53 are chemoresistant. The dominant negative effect of p53 mutants results from their aggregation propensity which causes co-aggregation of WT p53. We explored the mechanism of p53 inactivation in hypoxia and hypothesized whether WT p53 could rescue Mt p53 in hypoxic tumors. WT p53 exists in mutant conformation in hypoxic core of MCF-7 solid tumors, and its conformation is oxygen-dependent. Under simulated hypoxia in cells, WT p53 undergoes conformational change in acquiring mutant conformation. An in vivo chaperone assay shows that WT p53 functions as a molecular chaperone in rescuing conformational and structural p53 mutants in cancer cells both at the transcription and proteome levels. WT p53 chaperone therapy is further shown to cause significant regression of tumor xenografts through reconversion of the mutant phenotype to wild-type p53. The chaperone function of WT p53 is directly linked to the induction of apoptosis in both cancer cells and tumor xenografts. As oncogenic p53 mutants are linked to chemoresistance in hypoxic tumors, p53 chaperone therapy will introduce new dimensions to existing cancer therapeutics. We propose that in cancer cells, WT p53 chaperoning may either exist as a cellular event to potentially reverse the dominant negative effect of its oncogenic mutants or to stabilize yet unidentified factors.

p53 Ser15 phosphorylation disrupts the p53-RPA70 complex and induces RPA70-mediated DNA repair in hypoxia.

Cellular stressors are known to inhibit the p53-RPA70 (replication protein A, 70 kDa subunit) complex, and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for the inhibition of apoptosis in hypoxic tumours. We have shown that, in cancer cells, hypoxia disrupts the p53-RPA70 complex, thereby enhancing RPA70-mediated NER (nucleotide excision repair)/NHEJ (non-homologous end-joining) repair. In normal cells, RPA70 binds to the p53-NTD (N-terminal domain), whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD-RPA70 and p53-RPA70 complexes. Serial mutations at serine and threonine residues in the NTD confirm that p53(Ser15) phosphorylation induces dissociation of the p53-RPA70 complex in hypoxia. DNA-PK (DNA-dependent protein kinase) is shown to induce p53(Ser15) phosphorylation, thus enhancing RPA70-mediated NER/NHEJ repair. Furthermore, RPA70 gene silencing induces significant increases in cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53(Ser15) phosphorylation dissociates the p53-RPA70 complex, thus enhancing NER/NHEJ repair, which causes resistance to apoptosis in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics on the basis of the regulation of RPA70-mediated NER/NHEJ repair.

SCO2 Induces p53-Mediated Apoptosis by Thr845 Phosphorylation of ASK-1 and Dissociation of the ASK-1–Trx Complex

ABSTRACT p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr845 residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching “on” an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS–ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

p53 Increases Intra-Cellular Calcium Release by Transcriptional Regulation of Calcium Channel TRPC6 in GaQ3-Treated Cancer Cells

p53 and calcium signaling are inter-dependent and are known to show both synergistic and antagonistic effects on each other in the cellular environment. However, no molecular mechanism or cellular pathway is known which shows direct regulation between these important cellular signaling molecules. Here we have shown that in cancer cells treated with anti-neoplastic drug GaQ3, p53, there is an increase in intracellular calcium levels by transcriptional regulation of a novel calcium channel gene TRPC6. p53 directly binds to a 22 bp response element in the TRPC6 gene promoter and increase its mRNA and protein expression. Over-expression of TRPC6 results in calcium-dependent apoptotic death and activation of apoptotic genes in a variety of cancer cells. This research work shows that p53 and its transcriptional activity is critical in regulation of calcium signaling and an increase in the intracellular calcium level might be one of the anti-cancer strategies to induce apoptosis in cancer cells.

Reactive Oxygen Species-Mediated p53 Core-Domain Modifications Determine Apoptotic or Necrotic Death in Cancer Cells

AIMS p53 is known to induce apoptotic and necrotic cell death in response to stress, although the mechanism of these pathways is unknown. The aim of this study was to determine the molecular mechanism that determines p53s decision to select the apoptotic or necrotic mode of cell death. RESULTS Gold nanoparticles (Au-NPs) induced both apoptosis and necrosis in cancer cells in a p53-dependent manner. In cells undergoing apoptosis and necrosis, differential patterns of reactive oxygen species (ROS) generation were observed that leads to the activation of two different sets of p53-interacting kinases and acetylases. The differential activation of cellular kinases and acetylases caused dissimilar patterns of p53 phosphorylation and acetylation. In apoptotic cells, p53 was post-translationally modified in the core-domain, whereas in necrotic cells, it was modified at both N- and C-terminii, thus displaying differential DNA-binding activity. Au-NP10 and Au-NP80 activated fifty apoptotic and fifty nine necrotic p53-downstream genes, respectively. Both Au-NP10 and Au-NP80 showed HCT (p53+/+) tumor regression in mice xenografts. INNOVATION This study established for the first time that, in cancer cells, Au-NP-mediated apoptosis and necrosis are controlled by differential activation of p53 and its downstream genes. Further, both Au-NP10 and Au-NP80 were shown to regress HCT (p53+/+) tumors via apoptotic and necrotic pathways, respectively. CONCLUSION Au-NP-mediated p53 activation at both transcription and proteome level, through ROS-mediated p53 post-translational modification pattern, is responsible for tumor regression, which may further find wider application of nanoparticles in cancer therapy.

p53's choice of myocardial death or survival: Oxygen protects infarct myocardium by recruiting p53 on NOS3 promoter through regulation of p53-Lys 118 acetylation

Myocardial infarction, an irreversible cardiac tissue damage, involves progressive loss of cardiomyocytes due to p53‐mediated apoptosis. Oxygenation is known to promote cardiac survival through activation of NOS3 gene. We hypothesized a dual role for p53, which, depending on oxygenation, can elicit apoptotic death signals or NOS3‐mediated survival signals in the infarct heart. p53 exhibited a differential DNA‐binding, namely, BAX‐p53RE in the infarct heart or NOS3‐p53RE in the oxygenated heart, which was regulated by oxygen‐induced, post‐translational modification of p53. In the infarct heart, p53 was heavily acetylated at Lys118 residue, which was exclusively reversed in the oxygenated heart, apparently regulated by oxygen‐dependent expression of TIP60. The inhibition of Lys118 acetylation promoted the generation of NOS3‐promoting prosurvival form of p53. Thus, oxygenation switches p53‐DNA interaction by regulating p53 core‐domain acetylation, promoting a prosurvival transcription activity of p53. Understanding this novel oxygen‐p53 survival pathway will open new avenues in cardioprotection molecular therapy.

Cell competition in development: information from flies and vertebrates

Cell competition is a biological mechanism conserved from Drosophila to vertebrates wherein neighboring cells compare their relative fitness status resulting in the elimination of less fit cells by those with higher fitness. This is an active process that is essential for embryonic and organ development, tissue homeostasis, delay of ageing and in various disease models such as cancer. Recent research is beginning to unravel the various mechanisms of cell competition and the sensing of fitness status. Fitness fingerprints, death receptors, mechanical cell competition and a set of unknown genetic or signaling pathways are emerging as important pathways governing the mechanisms for cell to compare their relative fitness levels. In this review we are providing an account of recent advances which help summarize the mechanisms of operation and growing role of cell competition in regulation of oncogenesis in vertebrates.