A. B. Rodríguez

Anticancer peptide CIGB-300 binds to nucleophosmin/B23, impairs its CK2-mediated phosphorylation, and leads to apoptosis through its nucleolar disassembly activity

CIGB-300, formerly known as P15-tat, is a proapoptotic peptide with established antiproliferative activity in vitro and antitumoral activity in vivo. This hypothesis-driven peptide was initially selected for its ability to impair the in vitro CK2-mediated phosphorylation in one of its substrates through direct binding to the conserved acidic phosphoaceptor domain. However, the actual in vivo target(s) on human cancer cells among the hundreds of CK2 substrates as well as the subsequent events that lead to apoptosis on tumor cells remains to be determined. In this work, we identified the multifunctional oncoprotein nucleophosmin/B23 as a major target for CIGB-300. In vivo, the CIGB-300–B23 interaction was shown by pull-down experiments and confirmed by the early in situ colocalization of both molecules in the cell nucleolus. Moreover, CIGB-300 inhibits the CK2-mediated phosphorylation of B23 in a dose-dependent fashion both in vitro and in vivo as shown using the recombinant GST fusion protein and the metabolic labeling approach, respectively. Such phosphorylation impairment was correlated with the ability of CIGB-300 to induce nucleolar disassembly as documented by the use of established markers for nucleolar structure. Finally, we showed that such a sequence of events leads to the rapid and massive onset of apoptosis both at the molecular and cellular levels. Collectively, these findings provide important clues by which the CIGB-300 peptide exerts its proapoptotic effect on tumor cells and highlights the suitability of the B23/CK2 pathway for cancer-targeted therapy. [Mol Cancer Ther 2009;8(5):OF1–8]

CIGB-300, a synthetic peptide-based drug that targets the CK2 phosphoaceptor domain. Translational and clinical research

CK2 represents an oncology target scientifically validated. However, clinical research with inhibitors of the CK2-mediated phosphorylation event is still insufficient to recognize it as a clinically validated target. CIGB-300, an investigational peptide-based drug that targets the phosphoaceptor site, binds to a CK2 substrate array in vitro but mainly to B23/nucleophosmin in vivo. The CIGB-300 proapoptotic effect is preceded by its nucleolar localization, inhibition of the CK2-mediated phosphorylation on B23/nucleophosmin and nucleolar disassembly. Importantly, CIGB-300 shifted a protein array linked to apoptosis, ribosome biogenesis, cell proliferation, glycolisis, and cell motility in proteomic studies which helped to understand its mechanism of action. In the clinical ground, CIGB-300 has proved to be safe and well tolerated in a First-in-Human trial in women with cervical malignancies who also experienced signs of clinical benefit. In a second Phase 1 clinical trial in women with cervical cancer stage IB2/II, the MTD and DLT have been also identified in the clinical setting. Interestingly, in cervical tumors the B23/nucleophosmin protein levels were significantly reduced after CIGB-300 treatment at the nucleus compartment. In addition, expanded use of CIGB-300 in case studies has evidenced antitumor activity when administered as compassional option. Collectively, our data outline important clues on translational and clinical research from this novel peptide-based drug reinforcing its perspectives to treat cancer and paving the way to validate CK2 as a promising target in oncology.

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.

L-Tryptophan administered orally at night modifies the melatonin plasma levels, phagocytosis and oxidative metabolism of ringdove (Streptopelia roseogrisea) heterophils

The essential amino acid tryptophan is the precursor in the anabolic pathway of melatonin, a hormone with immunomodulatory properties. The present study shows the in vivo effect of tryptophan on the phagocytic function and oxidative metabolism of heterophils from Streptopelia roseogrisea of < 1 year of age, with a parallel evaluation of the plasma levels of melatonin. The L-tryptophan was administered orally (125 and 300 mg/kg b.w.) at 19:00, before the beginning of the period of darkness, for 7 days. At the end of the tryptophan treatment, determinations were made at 21:00 and 02:00 of the Phagocytosis Index, the Phagocytosis Percentage, the Phagocytic Efficiency and the superoxide anion levels in heterophils isolated from blood and of the plasma levels of melatonin. The results showed, for the determinations at 21:00 in the animals that had received 125 mg L-tryptophan/kg b.w., enhanced heterophil phagocytic function and raised levels of plasma melatonin, with no affect on the oxidative metabolism of the phagocytes. For the administration of the greater concentration of tryptophan (300 mg/kg b.w.), there were raised plasma melatonin levels together with increases in heterophil phagocytic capacity and phagocyte oxidative metabolism at 02:00. The results indicate that tryptophan administered orally at night to diurnal animals of less than 1 year in age affects the circulating levels of melatonin at the same time as inducing stimulation of the innate immune function.

Nitrogen and protein content analysis of human milk, diurnality vs nocturnality

Breast milk is changing with the progression of lactation and during a 24-h period. To determine the effect of diurnality or nocturnality on total nitrogen and protein content of the breast milk. We collected human milk samples from health mothers living throughout Community of Extremadura (Spain) from January 2008 to December 2008 with less than two months of lactation. We divided the samples in three groups: calostral group (1-5 days postpartum), transitional group (6-15 days postpartum) and mature group (> 15 days postpartum). All samples were stored in a freezer at -80 ºC. We considered as day period between 08:00-20:00 h and night period 20:00-08:00 h. Analysis of the human milk samples was based on the Kjeldahl method. Protein contents were calculated from total nitrogen x 6,25. The statistical analysis of the data was descriptive (mean ± standard deviation) and inferential (T-Student test). No differences (P > 0,05) were found to exist among the contents of individual human milk samples. The mean contents of each component were as follows: Total nitrogen of calostral, transitional and mature group was 0,30 ± 0,06 g/dL (night period), 0,29 ± 0,05 g/dL (day period); 0,26 ± 0,04 g/dL (night period), 0,25 ± 0,04 g/dL (day period); 0,22 ± 0,05 g/dL (night period), 0,20 ± 0,04 g/dL (day period) respectively, in this mature group with a statistical variation (P < 0,05). Protein content of calostral, transitional and mature group was 1,88 ± 0,4 g/dL (night period), 1,81 ± 0,3 g/dL (day period); 1,62 ± 0,3 g/dL (night period), 1,59 ± 0,3 g/dL (day period); 1,35 ± 0,3 g/dL (night period), 1,26 ± 0,3 g/dL (day period) respectively, in this mature group with a statistical variation (P < 0,05). Although we observed differences in the nitrogen and protein content during the individual stages of lactation, it is just in the population of mature lactating women, where the components analyzed varied significantly between day and night.

CIGB-300: A Promising Anti-Casein Kinase 2 (CK2) Peptide for Cancer Targeted Therapy

Over the past few years, the development of CK2 inhibitors using small molecules has emerged as a paradigmatic approach for blocking the enzymatic activity. However, despite successful experimental validation, so far only one of such chemical compounds has entered into clinical trials. Using a different rationale to inhibit CK2, we have developed CIGB-300 as a novel hypothesis-driven peptide targeting the CK2 phosphoacceptor domain instead of the ATP-binding site. Data from in vitro studies have revealed that at least in human cell lines from solid tumors, CIGB-300 binds mainly to and inhibits CK2-mediated phosphorylation of B23/npm. Studies of the molecular and cellular events downstream this interaction have demonstrated that CIGB-300 induces apoptosis in vitro and in vivo, modulating a wide array of proteins involved in cell proliferation, apoptosis, ribosome biogenesis, drug resistance, cell motility, and adhesion among other processes. Accordingly, CIGB-300 has shown synergistic interaction with anticancer drugs, suppressing angiogenesis and exhibiting antimetastatic properties. The pharmacology of this peptide-based drug has already been investigated in cancer patients. Different Phase 1 clinical trials have shown CIGB-300 to be safe and well tolerated and have studied its pharmacokinetics after either local or systemic administration. Remarkably, during a dose-finding Phase 2 trial in women with cervical cancer, cohorts receiving CIGB-300 and chemoradiotherapy concomitantly had a higher frequency of complete response than those receiving chemoradiotherapy alone. Taken together, the data presented here summarize all relevant preclinical and clinical findings that make CIGB-300 a promising peptide-based drug for the treatment of cancer patients.

Components in formula milks that improve sleep

Human milk is the nonpareil food for the first six months of life. Breast milk is a non-static biological fluid and so it is a dynamic fluid that changes not only over the course of the period of lactation but also during the day. As a result, the design of formula milk has to be as similar as possible to breast milk because this contains nutrients for sleep and for awakening according to the time of day. For several years already, some manufacturers of formula milks have been trying to improve sleep in babies who suffer from sleep problems by adding substances that promote sleep, for instance the essential amino acid tryptophan or vegetable extracts like chamomile or valerian among others. Other vegetable extracts have also been proposed, which are reported to be able to enhance sleep in children. Therefore, by applying chrononutrition – a field of chronobiology that establishes the principle of consuming foodstuffs at times of the day when they are most useful for health – to infant formula milks, we can increase sleep in babies.

PS-068 Analysis and improvement of prescription and administration in hospital transitions

Background Anticholinergic drugs (AchD) are often prescribed in elderly patients. They may increase cognitive and functional disorders, decrease the effects of anticholinesterase drugs (AcsD) and cause other adverse reactions, especially in Alzheimer’s disease (AD). Purpose To analyse atropinic burden (AB) using various scales in order to quantify the associated risk and to identify the most prescribed AchD in hospitalised patients with AD. Material and methods Bibliographic search in Pubmed using as the main terms ‘atropinic’, ‘anticholinergic’ and ‘Alzheimer’. This was a retrospective study performed from May 2015 to July 2016. APD prescription software and DIRAYA database were used to obtain prescription, demographic and patient data. 9 scales (7 of them validated) were chosen to identify drugs with AB and to quantify them. Results 49 hospitalised patients were studied, 26 men and 23 women, with an average age of 81.82±7.13 years. There were 6 deaths during hospitalisation (12.25%). All patients were receiving 1 or more AcsD to treat AD. 4 patients (8%) were not receiving AchD during hospitalisation while the rest were taking 1 or more AchD: 16 patients 1 drug (32.65%), 19 patients 2 drugs (38.77%), 5 patients 3 drugs (10.20%) and 3 patients were taking 4 AchD (6.12%). Only 1 patient was not at risk, 4 were at intermediate–low risk and 44 were at high risk according at least to 1 scale (89.79%). 28 patients were at high risk in 5 or more scales (57.14%). Psychotropic drugs with anticholinergic effects were prescribed in 28 patient (57%). Most prescribed drugs were haloperidol (11 patients, 22.45%), quetiapine (14 patients, 28.57%) and ipratropium (7 patients, 14.28%). Conclusion It is common practice to prescribe AchD in elderly hospitalised patients with AD. Cumulative administration of these drugs makes this fragile type of patients especially vulnerable to anticholinergic adverse effects. References and/or acknowledgements Villalba-Moreno AM, et al. Systematic review on the use of anticholinergic scales in poly pathological patients. Arch Gerontol Geriatr2015;62:1–8. Montrastuc F, et al. Atropinic burden of prescriptions forms in patients with Alzheimer disease: a cross-sectional study in a French pharmacovigilance database. EJC Pharmacology 2015;71./:891–5. Grey SL, et al. Cumulative use of strong anticholinergic medications and incident dementia. JAMA Intern Med 2015;175:401–7. No conflict of interest

Human milk nucleotides improve sleep: a focus on circadian profiles

Human milk and the milk of several mammalian species contain nutrients, anti-infectious and immunocompetent substances and nucleotides into the non-protein fraction. These compounds are involved in several different functions, and although changes in the concentrations of some breast milk components over the course of the day are already well described, until now little is known about the nature and underlying cause of the circadian pattern. This chapter will explain the roles of the nucleotides in the sleep/wake cycle and will discuss the circadian oscillations of the concentration of these substances in human breast milk.

Abstract 1376: Antiangiogenic properties of the peptide CIGB-300

CK2 is a highly conserved protein serine/threonine kinase that is ubiquitously distributed in eukaryotes, constitutively active and has been implicated in multiple cellular functions, as well as in tumorigenesis and transformation. Elevated CK2 activity has been associated with the malignant transformation of several tissues and is associated with aggressive tumor behavior. Previously, we have described a peptide CIGB-300 (also named P15-Tat) targeting the acidic phophorylation domain of the CK2 substrates. Different groups around the world have tried to manipulate this biochemical event by targeting the ATP-binding site of CK2 or its gene transcription using antisense oligonucleotides. Otherwise, CIGB-300 peptide was developed following the innovative approach of targeting the phosphoaceptor site on the CK2 substrates rather than the enzyme per se. We demonstrated that CIGB-300 induced apoptosis on tumor cells and tumor regression when injected directly into solid tumors or by systemic administration in mice. In this work we have evaluated the CIGB-300 peptide on the angiogenic process, both in vitro and in vivo. We have focused in the exploration of CIGB-300 properties to inhibit proliferation, tube formation and RNA pattern expression on endothelial cells (HUVEC). Likewise we used the chicken chorioallatoic membrane (CAM) assay as a model in vivo to asses the CIGB-300 activity on angiogenesis. Others authors have studied the role of different CK2 inhibitors on angiogenesis finding that CK2 is involved in endothelial cell proliferation, survival, migration and tube formation. Intraperitoneally administered CK2 inhibitors significantly reduced preretinal neovascularization in a mouse model of proliferative retinopathy. Solid tumors require the growth of new blood vessels (angiogenesis) to grow. Tumor angiogenesis utilizes at least some of the angiogenic signalling pathways that are required during vascular development. Tumor angiogenesis has become an important target for antitumor therapy, with most current therapies aimed at blocking the VEGF pathway. However, not all tumors are responsive to VEGF blockers, and some of them that are responsive initially may become resistant during the course of treatment, thus there is a need to explore other angiogenesis signalling pathways. In this paper we have examined the antiangiogenic properties of CIGB-300 peptide in vitro and in vivo. The data suggests that CK2 is involved in angiogenic processes and CIGB-300 could act as promising antiangiogenic inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1376.