Activity of a Novel bcl-2/bcl-xL-Bispecific Antisense Oligonucleotide Against Tumors of Diverse Histologic Origins

Background: Increased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL is involved in the development and progression of many tumors. We recently reported that the bcl-2/bcl-xL-bispecific antisense oligonucleotide 4625 induces apoptosis in lung carcinoma cells. To further assess the therapeutic potential of oligonucleotide 4625, we investigated its effect on a series of human tumor cell lines of diverse histologic origins in vitro and in vivo. Methods: Oligonucleotide 4625-mediated inhibition of bcl-2 and bcl-xL expression in vitro was measured in breast carcinoma cells with the use of reverse transcription-polymerase chain reaction (PCR), real-time PCR, and western blotting. Cytotoxicity was assessed in several different cell lines by measurement of tumor cell growth, propidium iodide uptake, and nuclear apoptosis. The in vivo activity of oligonucleotide 4625 was determined by the inhibition of growth of established tumor xenografts in nude mice, immunohistochemical staining of Bcl-2 and Bcl-x proteins in the tumors, and western blotting of tumor lysates. Apoptosis in tumor xenografts was detected with the use of in situ TUNEL (i.e., terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling) staining. All statistical tests are two-sided. Results: In breast carcinoma cells, oligonucleotide 4625 treatment reduced bcl-2 and bcl-xL messenger RNA levels in a dose-dependent manner. At 600 nM, oligonucleotide 4625 reduced Bcl-2 and Bcl-xL protein levels to 25% (95% confidence interval [CI] = 16% to 34%) and 20% (95% CI = 14% to 26%), respectively, of the levels in untreated cells and it decreased viability in all cell lines mainly by inducing apoptosis. In vivo, oligonucleotide 4625 statistically significantly inhibited the growth of breast and colorectal carcinoma xenografts by 51% (95% CI = 28% to 74%) and 59% (95% CI = 44% to 74%), respectively, relative to those treated with control oligonucleotide 4626; it also reduced Bcl-2 and Bcl-xL protein levels and induced tumor cell apoptosis. Conclusion: The bcl-2/bcl-xL-bispecific antisense oligonucleotide 4625 merits further study as a novel compound for cancer therap

Influence of organic diet on the amount of conjugated linoleic acids in breast milk of lactating women in the Netherlands

The aim of the present study was to find out whether the incorporation of organic dairy and meat products in the maternal diet affects the contents of the conjugated linoleic acid isomers (CLA) and trans-vaccenic acid (TVA) in human breast milk. To this purpose, milk samples from 312 breastfeeding mothers participating in the KOALA Birth Cohort Study have been analysed. The participants had documented varying lifestyles in relation to the use of conventional or organic products. Breast milk samples were collected 1month postpartum and analysed for fatty acid composition. The content of rumenic acid (the main CLA) increased in a statistically significant way while going from a conventional diet (no organic dairy/meat products, 0·25 weight % (wt%), n 186) to a moderately organic diet (50-90% organic dairy/meat, 0·29wt%, n 33, P=0·02) and to a strict organic diet (>90% organic dairy/meat, 0·34wt%, n 37, P≤0·001). The levels of TVA were augmented among the participants with a moderately organic diet (0·54wt%) and those with a strict organic diet (0·59wt%, P≤0·001), in comparison with the conventional group (0·48wt%). After adjusting for covariables (recruitment group, maternal age, maternal education, use of supplements and season), statistical significance was retained in the group of the strict organic dairy users (P<0·001 for rumenic acid). Hence, the levels of CLA and TVA in human milk can be modulated if breastfeeding mothers replace conventional dairy and/or meat products by organic ones. A potential contribution of CLA and TVA to health improvement is briefly discusse

Mistletoe lectin is not the only cytotoxic component in fermented preparations of Viscum album from white fir (Abies pectinata)

<p>Abstract</p> <p>Background</p> <p>Preparations of mistletoe (<it>Viscum album</it>) are the form of cancer treatment that is most frequently used in the complementary medicine. Previous work has shown that these preparations are able to exert cytotoxic effects on carcinoma cells, the extent of which might be influenced by the host tree species and by the content of mistletoe lectin.</p> <p>Methods</p> <p>Using colorimetric assays, we have now compared the cytotoxic effects of <it>Viscum album </it>preparations (VAPs) obtained from mistletoe growing on oak (<it>Quercus robur </it>and <it>Q. petraea</it>, VAP-Qu), apple tree (<it>Malus domestica</it>,, VAP-M), pine (<it>Pinus sylvestris</it>, VAP-P) or white fir (<it>Abies pectinata</it>, VAP-A), on the <it>in vitro </it>growth of breast and bladder carcinoma cell lines. While MFM-223, KPL-1, MCF-7 and HCC-1937 were the breast carcinoma cell lines chosen, the panel of tested bladder carcinoma cells comprised the T-24, TCC-SUP, UM-UC-3 and J-82 cell lines.</p> <p>Results</p> <p>Each of the VAPs inhibited cell growth, but the extent of this inhibition differed with the preparation and with the cell line. The concentrations of VAP-Qu, VAP-M and VAP-A which led to a 50 % reduction of cell growth (IC₅₀) varied between 0.6 and 0.03 mg/ml. Higher concentrations of VAP-P were required to obtain a comparable effect. Purified mistletoe lectin I (MLI) led to an inhibition of breast carcinoma cell growth at concentrations lower than those of VAPs, but the sensitivity towards purified MLI did not parallel that towards VAPs. Bladder carcinoma cells were in most cases more sensitive to VAPs treatment than breast carcinoma cells. The total mistletoe lectin content was very high in VAP-Qu (54 ng/mg extract), intermediate in VAP-M (25 ng/mg extract), and very low in VAP-P (1.3 ng/mg extract) and in VAP-A (1 ng/mg extract). As to be expected from the low content of mistletoe lectin, VAP-P led to relatively weak cytotoxic effects. Most remarkably, however, the lectin-poor VAP-A revealed a cytotoxic effect comparable to, or even stronger than, that of the lectin-rich VAP-Qu, on all tested bladder and breast carcinoma cell lines.</p> <p>Conclusion</p> <p>The results suggest the existence of cytotoxic components other than mistletoe lectin in VAP-A and reveal an unexpected potential of this preparation for the treatment of breast and bladder cancer.</p

Sleep quality in pregnancy during treatment with Bryophyllum pinnatum: An observational study

INTRODUCTION: Poor sleep quality in pregnancy is frequent. A treatment with sedatives is problematic due to possible adverse effects for mother and embryo/foetus. In the present study, we investigated the sedative effect of Bryophyllum pinnatum, a phytotherapeutic medication used in anthroposophic medicine. In previous clinical studies on its tocolytic effect, B. pinnatum showed a good risk/benefit ratio for mother and child. A recent analysis of the prescribing pattern for B. pinnatum in a network of anthroposophic physicians revealed sleep disorders as one of the most frequent diagnoses for which these preparations are prescribed. MATERIALS AND METHODS: In this prospective, multi-centre, observational study, pregnant women suffering from sleep problems were treated with B. pinnatum (350mg tablets, 50% leaf press juice, Weleda AG, Arlesheim, dosage at physician's consideration). Sleep quality, daily sleepiness and fatigue were assessed with the aid of standardised questionnaires, at the beginning of the treatment and after 2 weeks. Possible adverse drug reactions perceived by the patients during the treatment were recorded. RESULTS: The number of wake-ups, as well as the subjective quality of sleep was significantly improved at the end of the treatment with B. pinnatum. The Epworth Sleeping Scale decreased, indicating a decrease of the tiredness during the day. There was, however, no evidence for prolongation of the sleep duration, reduction in the time to fall asleep, as well as change in the Fatigue Severity Scale after B. pinnatum. No serious adverse drug reactions were detected. CONCLUSION: B. pinnatum is a suitable treatment of sleep problems in pregnancy. The data of this study encourage further clinical investigations on the use of B. pinnatum in sleep disorders

ΔNp73 antisense activates PUMA and induces apoptosis in neuroblastoma cells

The p73 gene codes for various different protein isoforms. They include proteins expressed under the control of the P1 promoter that contain a transactivation domain and are similar in function to p53 (TAp73 isoforms), as well as proteins regulated by the P2 promoter that lack this domain and function as dominant negative inhibitors of TAp73 and p53 (ΔNp73 isoforms). Whereas TAp73 functions as a tumor suppressor with pro-apoptotic function, ΔNp73 is likely to prevent the induction of apoptosis in tumor cells and to participate in oncogenesis. Here we used a loss-of-function strategy to assess the role of ΔNp73 in SH-SY5Y neuroblastoma cells. An antisense oligonucleotide designed to target ΔNp73 mRNA, but not TAp73, was used to effectively downregulate this transcript. ΔNp73 downregulation was accompanied by increased levels of the pro-apoptotic BH3 family member PUMA at the mRNA and protein level, and by conformational activation of BAX which translocated to mitochondria. These ΔNp73 antisense-mediated alterations led to the induction of apoptosis as detected by decreased cell viability, augmented DNA fragmentation and increased caspase-3 activity in cell lysates. Our results demonstrate the cytoprotective role of ΔNp73 in neuroblastoma and suggest its use as a target for molecular intervention therap

Activity of a Novel bcl-2/bcl-xL-Bispecific Antisense Oligonucleotide Against Tumors of Diverse Histologic Origins

Background: Increased expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL is involved in the development and progression of many tumors. We recently reported that the bcl-2/bcl-xL-bispecific antisense oligonucleotide 4625 induces apoptosis in lung carcinoma cells. To further assess the therapeutic potential of oligonucleotide 4625, we investigated its effect on a series of human tumor cell lines of diverse histologic origins in vitro and in vivo. Methods: Oligonucleotide 4625-mediated inhibition of bcl-2 and bcl-xL expression in vitro was measured in breast carcinoma cells with the use of reverse transcription-polymerase chain reaction (PCR), real-time PCR, and western blotting. Cytotoxicity was assessed in several different cell lines by measurement of tumor cell growth, propidium iodide uptake, and nuclear apoptosis. The in vivo activity of oligonucleotide 4625 was determined by the inhibition of growth of established tumor xenografts in nude mice, immunohistochemical staining of Bcl-2 and Bcl-x proteins in the tumors, and western blotting of tumor lysates. Apoptosis in tumor xenografts was detected with the use of in situ TUNEL (i.e., terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling) staining. All statistical tests are two-sided. Results: In breast carcinoma cells, oligonucleotide 4625 treatment reduced bcl-2 and bcl-xL messenger RNA levels in a dose-dependent manner. At 600 nM, oligonucleotide 4625 reduced Bcl-2 and Bcl-xL protein levels to 25% (95% confidence interval [CI] = 16% to 34%) and 20% (95% CI = 14% to 26%), respectively, of the levels in untreated cells and it decreased viability in all cell lines mainly by inducing apoptosis. In vivo, oligonucleotide 4625 statistically significantly inhibited the growth of breast and colorectal carcinoma xenografts by 51% (95% CI = 28% to 74%) and 59% (95% CI = 44% to 74%), respectively, relative to those treated with control oligonucleotide 4626; it also reduced Bcl-2 and Bcl-xL protein levels and induced tumor cell apoptosis. Conclusion: The bcl-2/bcl-xL-bispecific antisense oligonucleotide 4625 merits further study as a novel compound for cancer therap

Use of complementary and alternative medicine by patients presenting to a Paediatric Emergency Department

Although the popularity of complementary and alternative medicine (CAM) has risen in the last decade, information about its use by paediatric patients presenting to an Emergency Department is still sparse. We report here the results of a cross-sectional survey of paediatric patients presenting to an urban, tertiary paediatric Emergency Department between October 2006 and March 2007. In total, 1143 questionnaires (68% of those distributed) were completed and available for analysis. Of these, 58% (n = 665) of all respondents admitted that their child had received some form of CAM therapy, while 25% (n = 291) admitted that their child was receiving CAM for the present illness. In 31% of the respondents (n = 354), CAM had been prescribed by a physician, while 50% (n = 575) used CAM as self-medication. Patients presented to the Emergency Department mostly because of an infection (42% of total; 29% of these used CAM) or a trauma (38% of total; 19% of these used CAM). Parents of CAM-users were significantly older, more often born in Switzerland and had significantly higher school education than those of the non-users. Nearly two-thirds of the administered CAM therapies were not prescribed by a physician, and 50% of the families using CAM did not discuss this with their general practitioner. Parental requirements implied that medical professionals on a paediatric Emergency Department should know the effects and side-effects of CAM therapies and even be able to recommend them. The study population, even trauma patients, frequently used CAM. The use of CAM is characterised by a high rate of self-medication and the exclusion of the physicians from the decision-making process. The parents of paediatric patients frequently demand that CAM be considered as a possible treatment option and wish to have an open discussion with the medical professionals on this topic