Thymoquinone inhibits tumor growth and induces apoptosis in a breast cancer xenograft mouse model: The role of p38 MAPK and ROS

Due to narrow therapeutic window of cancer therapeutic agents and the development of resistance against these agents, there is a need to discover novel agents to treat breast cancer. The antitumor activities of thymoquinone (TQ), a compound isolated from Nigella sativa oil, were investigated in breast carcinoma in vitro and in vivo. Cell responses after TQ treatment were assessed by using different assays including MTT assay, annexin V-propidium iodide staining, Mitosox staining and Western blot. The antitumor effect was studied by breast tumor xenograft mouse model, and the tumor tissues were examined by histology and immunohistochemistry. The level of antioxidant enzymes/molecules in mouse liver tissues was measured by commercial kits. Here, we show that TQ induced p38 phosphorylation and ROS production in breast cancer cells. These inductions were found to be responsible for TQ’s anti-proliferative and pro-apoptotic effects. Moreover, TQ-induced ROS production regulated p38 phosphorylation but not vice versa. TQ treatment was found to suppress the tumor growth and this effect was further enhanced by combination with doxorubicin. TQ also inhibited the protein expression of anti-apoptotic genes, such as XIAP, survivin, Bcl-xL and Bcl-2, in breast cancer cells and breast tumor xenograft. Reduced Ki67 and increased TUNEL staining were observed in TQ-treated tumors. TQ was also found to increase the level of catalase, superoxide dismutase and glutathione in mouse liver tissues. Overall, our results demonstrated that the antiproliferative and pro-apoptotic effects of TQ in breast cancer are mediated through p38 phosphorylation via ROS generation

Increased Expression of Bcl11b Leads to Chemoresistance Accompanied by G1 Accumulation

BACKGROUND: The expression of BCL11B was reported in T-cells, neurons and keratinocytes. Aberrations of BCL11B locus leading to abnormal gene transcription were identified in human hematological disorders and corresponding animal models. Recently, the elevated levels of Bcl11b protein have been described in a subset of squameous cell carcinoma cases. Despite the rapidly accumulating knowledge concerning Bcl11b biology, the contribution of this protein to normal or transformed cell homeostasis remains open. METHODOLOGY/PRINCIPAL FINDINGS: Here, by employing an overexpression strategy we revealed formerly unidentified features of Bcl11b. Two different T-cell lines were forced to express BCL11B at levels similar to those observed in primary T-cell leukemias. This resulted in markedly increased resistance to radiomimetic drugs while no influence on death-receptor apoptotic pathway was observed. Apoptosis resistance triggered by BCL11B overexpression was accompanied by a cell cycle delay caused by accumulation of cells at G1. This cell cycle restriction was associated with upregulation of CDKN1C (p57) and CDKN2C (p18) cyclin dependent kinase inhibitors. Moreover, p27 and p130 proteins accumulated and the SKP2 gene encoding a protein of the ubiquitin-binding complex responsible for their degradation was repressed. Furthermore, the expression of the MYCN oncogene was silenced which resulted in significant depletion of the protein in cells expressing high BCL11B levels. Both cell cycle restriction and resistance to DNA-damage-induced apoptosis coincided and required the histone deacetylase binding N-terminal domain of Bcl11b. The sensitivity to genotoxic stress could be restored by the histone deacetylase inhibitor trichostatine A. CONCLUSIONS: The data presented here suggest a potential role of BCL11B in tumor survival and encourage developing Bcl11b-inhibitory approaches as a potential tool to specifically target chemoresistant tumor cells

Gata4 Is Required for Formation of the Genital Ridge in Mice

In mammals, both testis and ovary arise from a sexually undifferentiated precursor, the genital ridge, which first appears during mid-gestation as a thickening of the coelomic epithelium on the ventromedial surface of the mesonephros. At least four genes (Lhx9, Sf1, Wt1, and Emx2) have been demonstrated to be required for subsequent growth and maintenance of the genital ridge. However, no gene has been shown to be required for the initial thickening of the coelomic epithelium during genital ridge formation. We report that the transcription factor GATA4 is expressed in the coelomic epithelium of the genital ridge, progressing in an anterior-to-posterior (A-P) direction, immediately preceding an A-P wave of epithelial thickening. Mouse embryos conditionally deficient in Gata4 show no signs of gonadal initiation, as their coelomic epithelium remains a morphologically undifferentiated monolayer. The failure of genital ridge formation in Gata4-deficient embryos is corroborated by the absence of the early gonadal markers LHX9 and SF1. Our data indicate that GATA4 is required to initiate formation of the genital ridge in both XX and XY fetuses, prior to its previously reported role in testicular differentiation of the XY gonadHoward Hughes Medical Institut

GC-Rich Sequence Elements Recruit PRC2 in Mammalian ES Cells

Polycomb proteins are epigenetic regulators that localize to developmental loci in the early embryo where they mediate lineage-specific gene repression. In Drosophila, these repressors are recruited to sequence elements by DNA binding proteins associated with Polycomb repressive complex 2 (PRC2). However, the sequences that recruit PRC2 in mammalian cells have remained obscure. To address this, we integrated a series of engineered bacterial artificial chromosomes into embryonic stem (ES) cells and examined their chromatin. We found that a 44 kb region corresponding to the Zfpm2 locus initiates de novo recruitment of PRC2. We then pinpointed a CpG island within this locus as both necessary and sufficient for PRC2 recruitment. Based on this causal demonstration and prior genomic analyses, we hypothesized that large GC-rich elements depleted of activating transcription factor motifs mediate PRC2 recruitment in mammals. We validated this model in two ways. First, we showed that a constitutively active CpG island is able to recruit PRC2 after excision of a cluster of activating motifs. Second, we showed that two 1 kb sequence intervals from the Escherichia coli genome with GC-contents comparable to a mammalian CpG island are both capable of recruiting PRC2 when integrated into the ES cell genome. Our findings demonstrate a causal role for GC-rich sequences in PRC2 recruitment and implicate a specific subset of CpG islands depleted of activating motifs as instrumental for the initial localization of this key regulator in mammalian genomes.Burroughs Wellcome FundCharles E. Culpeper FoundationMassachusetts General HospitalBroad Institute of MIT and Harvar

Global gene expression analysis in time series following N-acetyl L-cysteine induced epithelial differentiation of human normal and cancer cells in vitro

BACKGROUND: Cancer prevention trials using different types of antioxidant supplements have been carried out at several occasions and one of the investigated compounds has been the antioxidant N-acetyl-L-cysteine (NAC). Studies at the cellular level have previously demonstrated that a single supplementation of NAC induces a ten-fold more rapid differentiation in normal primary human keratinocytes as well as a reversion of a colon carcinoma cell line from neoplastic proliferation to apical-basolateral differentiation [1]. The investigated cells showed an early change in the organization of the cytoskeleton, several newly established adherens junctions with E-cadherin/β-catenin complexes and increased focal adhesions, all features characterizing the differentiation process. METHODS: In order to investigate the molecular mechanisms underlying the proliferation arrest and accelerated differentiation induced by NAC treatment of NHEK and Caco-2 cells in vitro, we performed global gene expression analysis of NAC treated cells in a time series (1, 12 and 24 hours post NAC treatment) using the Affymetrix GeneChip™ Human Genome U95Av2 chip, which contains approximately 12,000 previously characterized sequences. The treated samples were compared to the corresponding untreated culture at the same time point. RESULTS: Microarray data analysis revealed an increasing number of differentially expressed transcripts over time upon NAC treatment. The early response (1 hour) was transient, while a constitutive trend was commonly found among genes differentially regulated at later time points (12 and 24 hours). Connections to the induction of differentiation and inhibition of growth were identified for a majority of up- and down-regulated genes. All of the observed transcriptional changes, except for seven genes, were unique to either cell line. Only one gene, ID-1, was mutually regulated at 1 hour post treatment and might represent a common mediator of early NAC action. The detection of several genes that previously have been identified as stimulated or repressed during the differentiation of NHEK and Caco-2 provided validation of results. In addition, real-time kinetic PCR analysis of selected genes also verified the differential regulation as identified by the microarray platform. CONCLUSION: NAC induces a limited and transient early response followed by a more consistent and extensively different expression at later time points in both the normal and cancer cell lines investigated. The responses are largely related to inhibition of proliferation and stimulation of differentiation in both cell types but are almost completely lineage specific. ID-1 is indicated as an early mediator of NAC action

Mammalian sex determination—insights from humans and mice

Disorders of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. Many of the genes required for gonad development have been identified by analysis of DSD patients. However, the use of knockout and transgenic mouse strains have contributed enormously to the study of gonad gene function and interactions within the development network. Although the genetic basis of mammalian sex determination and differentiation has advanced considerably in recent years, a majority of 46,XY gonadal dysgenesis patients still cannot be provided with an accurate diagnosis. Some of these unexplained DSD cases may be due to mutations in novel DSD genes or genomic rearrangements affecting regulatory regions that lead to atypical gene expression. Here, we review our current knowledge of mammalian sex determination drawing on insights from human DSD patients and mouse models