Targeting radiation-resistant hypoxic tumour cells through ATR inhibition.

BACKGROUND: Most solid tumours contain regions of sub-optimal oxygen concentration (hypoxia). Hypoxic cancer cells are more resistant to radiotherapy and represent the most aggressive fraction of a tumour. It is therefore essential that strategies continue to be developed to target hypoxic cancer cells. Inhibition of the DNA damage response (DDR) might be an effective way of sensitising hypoxic tumour cells to radiotherapy. METHODS: Here, we describe the cellular effects of pharmacological inhibition of the apical DDR kinase ATR (Ataxia Telangiectasia and Rad 3 related) with a highly selective inhibitor, VE-821, in hypoxic conditions and its potential as a radiosensitiser. RESULTS: VE-821 was shown to inhibit ATR-mediated signalling in response to replication arrest induced by severe hypoxia. In these same conditions, VE-821 induced DNA damage and consequently increased Ataxia Telangiectasia Mutated-mediated phosphorylation of H2AX and KAP1. Consistently, ATR inhibition sensitised tumour cell lines to a range of oxygen tensions. Most importantly, VE-821 increased radiation-induced loss of viability in hypoxic conditions. Using this inhibitor we have also demonstrated for the first time a link between ATR and the key regulator of the hypoxic response, HIF-1. HIF-1 stabilisation and transcriptional activity were both decreased in response to ATR inhibition. CONCLUSION: These findings suggest that ATR inhibition represents a novel strategy to target tumour cells in conditions relevant to pathophysiology and enhance the efficacy of radiotherapy.

Anticancer Properties of Essential Oils and Other Natural Products.

Essential oils are secondary metabolites with a key-role in plants protection, consisting primarily of terpenes with a volatile nature and a diverse array of chemical structures. Essential oils exhibit a wide range of bioactivities, especially antimicrobial activity, and have long been utilized for treating various human ailments and diseases. Cancer cell prevention and cytotoxicity are exhibited through a wide range of mechanisms of action, with more recent research focusing on synergistic and antagonistic activity between specific essential oils major and minor components. Essential oils have been shown to possess cancer cell targeting activity and are able to increase the efficacy of commonly used chemotherapy drugs including paclitaxel and docetaxel, having also shown proimmune functions when administered to the cancer patient. The present review represents a state-of-the-art review of the research behind the application of EOs as anticancer agents both in vitro and in vivo. Cancer cell target specificity and the use of EOs in combination with conventional chemotherapeutic strategies are also explored.

Avaliação da técnica de vitrificação de ovários de camundongos da linhagem B6D2F1, pertencentes ao Instituto de Ciência e Tecnologia em Biomodelos - ICTB/Fiocruz-RJ / Evaluation of ovary vitrification technique from B6D2F1 strain belonging to the Institute of Biomodels Science and Technology ICTB / Fiocruz-RJ

Objetivou-se testar a vitrificação de ovários de camundongos do ICTB/Fiocruz. Inicialmente, fez-se coleta e maturação in vitro dos oócitos de ovários a fresco e vitrificados, bem como avaliação de estruturas no cultivo embrionário, pós-fertilização in vitro. Fêmeas B6D2F1 foram eutanasiadas para remoção dos ovários (n=60) e divididas em três grupos: grupo 1 (n=30 animais) - oócito de ovários vitrificados, maturados e fertilizados in vitro (120 fragmentos); grupo 2 (n=15) (controle 1) - oócitos coletados a fresco, maturados e fertilizados in vitro; e grupo 3 (n=15) (controle 2) - oócitos maturados in vivo e fertilizados in vitro. A técnica foi verificada no desenvolvimento embrionário in vitro, que foi avaliado pelo teste de qui-quadrado (BioStat 5.0). Recuperaram-se 123, 224 e 328 oócitos nos G1, G2 e G3, respectivamente. Observaram-se diferenças significativas nas taxas de clivagem às 24 horas (embriões ≥ 2 células) entre G1 (8%) e G2 (32%) (P<0,1) e G1 e G3 (49%) (P<0,05), mas não entre G2 e G3 (P>0,05). Para blastocistos, às 96 horas, os grupos G1, G2 e G3 apresentaram, respectivamente, 6%, 11% e 46%, diferindo significativamente entre eles (P<0,05). A vitrificação de ovários, a maturação oocitária e a fertilização in vitro são alternativas para a produção de embriões de camundongos in vitro.(AU)

This work aimed test ovarian vitrification of hybrid mouse from ICTB/Fiocruz. Protocol collection and oocyte in vitro maturation from fresh and vitrified ovaries was established and embryos were evaluated after fertilization. B6D2F1 females were euthanized for ovarian removal (n= 60) and divided into 3 groups: G1 (n= 30) - ovaries fragmented (n= 120), vitrified, matured and fertilized; G2 (n= 15) - in vitro fertilization of oocytes matured in vitro from fresh ovaries; G3 (n= 15) - ampulla region oocytes in vitro fertilizated. Viability was verified by thawing, oocyte in vitro maturation and fertilization. In vitro embryo development of each group was evaluated by Chi-square test (BioStat 5.0). 123, 224 and 328 oocytes were recovered from G1, G2 and G3, respectively. Significant differences were observed in cleavage rates at 24 hours (embryos with 2 cells or more) between G1 (8%) and G2 (32%) (P< 0.1) and G1 and G3 (49%) (P< 0.05) but not between G2 and G3 (P> 0.05). Blastocysts at 96 hours presented 6%, 11% and 46%, respectively for G1, G2 and G3, differing significantly (P< 0.05). Ovary vitrification, oocyte in vitro maturation and in vitro fertilization were available for the production of in vitro mouse embryos.(AU)

Hypoxia-driven cell motility reflects the interplay between JMY and HIF-1α.

Junction-mediating and regulatory protein (JMY) is a novel p53 cofactor that regulates p53 activity during stress. JMY interacts with p300/CBP, which are ubiquitous transcriptional co-activators that interact with a variety of sequence-specific transcription factors, including hypoxia-inducible factor-1α (HIF-1α). In addition, JMY is an actin-nucleating protein, which, through its WH2 domains, stimulates cell motility. In this study, we show that JMY is upregulated during hypoxia in a HIF-1α-dependent manner. The JMY gene contains HIF-responsive elements in its promoter region and HIF-1α is recruited to its promoter during hypoxia. HIF-1α drives transcription of JMY, which accounts for its induction under hypoxia. Moreover, the enhanced cell motility and invasion that occurs during hypoxia requires JMY, as depleting JMY under hypoxic conditions causes decreased cell motility. Our results establish the interplay between JMY and HIF-1α as a new mechanism that controls cell motility under hypoxic stress.

Oxaliplatin responses in colorectal cancer cells are modulated by CHK2 kinase inhibitors.

BACKGROUND AND PURPOSE: Checkpoint kinase 2 (CHK2) is activated by DNA damage and can contribute to p53 stabilization, modulating growth arrest and/or apoptosis. We investigated the contribution of CHK2 to oxaliplatin-mediated toxicity in a colorectal cancer model. EXPERIMENTAL APPROACH: We evaluated the ability of CHK2 small molecule inhibitors to potentiate oxaliplatin-induced toxicity. The role of CHK2 in oxaliplatin-induced apoptosis was investigated in HCT116 cells that were wild-type (WT) or KO for CHK2. Small molecule inhibitors of CHK2 were used in combination studies with oxaliplatin in this cell model. KEY RESULTS: In oxaliplatin-treated CHK2 KO cells, accelerated apoptosis was accompanied by attenuated p53 stabilization and p21(WAF-1) up-regulation correlating with increased Bax expression, cytochrome c release and elevated caspase activity. The higher levels of apoptosis in CHK2 KO cells were restored to control (WT) levels when CHK2 was re-introduced. This 'uncoupling' of p53 stabilization and Bax up-regulation in CHK2 KO cells suggested oxaliplatin-induced apoptosis was due to a p53-independent response. Combination studies revealed that CHK2 inhibitor II or debromohymenialdisine antagonized the responses to oxaliplatin. This inhibitory effect correlated with decreases in apoptosis, p53 stabilization and DNA inter-strand cross-link formation, and was dependent on the presence (but not activity) of CHK2. CONCLUSIONS AND IMPLICATIONS: Combinations of CHK2 inhibitors with oxaliplatin should further sensitize cells to oxaliplatin treatment. However, these inhibitors produced an antagonistic effect on the response to oxaliplatin, which was reversed on the re-introduction of CHK2. These observations may have implications for the use of oxaliplatin in colorectal cancer therapy in combination with therapies targeting CHK2.