Comprehensive characterization of naturally occurring antioxidants from the twigs of mulberry (Morus alba) using on-line high-performance liquid chromatography coupled with chemical detection and high-resolution mass spectrometry.

INTRODUCTION: The mulberry tree (Morus alba L.) is a prolific source of biologically active compounds. There is considerable growing interest in probing M. alba twigs as a source of disruptive antioxidant lead candidates for cosmetic skin care product development. OBJECTIVE: An integrated approach using high-performance liquid chromatography (HPLC) coupled with either chemical detection (CD) or high-resolution mass spectrometry (HRMS) was applied to the hydroalcoholic extract of M. alba to detect and identify lead antioxidant compounds, respectively. MATERIAL AND METHODS: The twigs were weighed, powdered and homogenized using a mill and the extract was prepared using 70% aqueous ethanol. The antioxidant metabolites were detected with HPLC coupled with CD (based on the ORAC assay) and their structural identification was carried out using a Q-Exactive Orbitrap MS instrument. RESULTS: Using this approach, 13 peaks were detected as overall contributors to the antioxidant activity of M. alba, i.e. mulberrosides (A & E), oxyresveratrol & its derivatives, moracin & its derivatives and a dihydroxy-octadecadienoic acid, which together accounted for >90% of the antioxidant activity, highlighting the effectiveness of the integrated approach based on HPLC-CD and HPLC-HRMS. Additionally, a (3,4-dimethoxyphenyl-1-O-ß-D-apiofuranosyl-(1″ → 6')-O-ß-D-glucopyranoside was also discovered for the first time from the twig extract and is presented here. CONCLUSION: To our knowledge, this is the first report from M. alba twigs using HPLC-CD and HPLC-HRMS that identifies key compounds responsible for the antioxidant property of this native Chinese medicinal plant.

A new 3D model for genotoxicity assessment: EpiSkin™ Micronucleus Assay.

The European Regulation on Cosmetics (no. 1223/2009) has prohibited the use of animals in safety testing since March 2009 for ingredients used in cosmetics. Irreversible events at the chromosome level (clastogenesis and aneugenesis) are commonly evaluated by scoring either micronuclei or chromosome aberrations using cell-based genotoxicity assays. Like most in vitro genotoxicity assays, the 2D in vitro micronucleus assay exhibits a poor specificity and does not mimic the dermal route. To address these limitations, the current project aims to develop and validate a 3D micronucleus assay using the EpiSkin™ model. This project is scientifically supported by the Cosmetics Europe Genotoxicity Task Force. In a first step, two key criteria for the development of micronucleus assay, namely, the sufficient yield of cells from the EpiSkin™ model and an acceptable proliferation rate of the basal layer, were assessed and demonstrated. Subsequently, six chemicals (vinblastine, n-ethylnitrosourea, ß-butyrolactone, 2-acetylaminofluorene, 2,4-dichlorophenoland d-limonene) were evaluated in the EpiSkin™ Micronucleus Assay. At least two independent experiments using 48- and 72-h incubations were performed for each chemical. Results showed good inter-experimental reproducibility, as well as the correct identification of all six tested chemicals. The metabolism of 2-acetylaminofluorene on the EpiSkin™ model was also investigated and confirmed by the formation of an intermediate metabolite (2-aminofluorene). These preliminary results from the EpiSkin™ Micronucleus Assay indicate that it is a promising in vitro assay for assessing genotoxicity. The availability and suitability of this test method contribute significantly to the development of non-animal testing methods in China and its impact on the worldwide field.

Dichotomy effects of Akt signaling in breast cancer.

BACKGROUND: The oncogenic roles contributed by the Akt/PKB kinase family remain controversial and presumably depend on cell context, but are perceived to be modulated by an interplay and net balance between various isoforms. This study is intended to decipher whether distinct Akt kinase isoforms exert either redundant or unique functions in regulating neoplastic features of breast cancer cells, including epithelial-mesenchymal transition (EMT), cell motility, and stem/progenitor cell expansion. RESULTS: We demonstrate that overactivation of Akt signaling in nonmalignant MCF10A cells and in primary cultures of normal human mammary epithelial tissue results in previously unreported inhibitory effects on EMT, cell motility and stem/progenitor cell expansion. Importantly, this effect is largely redundant and independent of Akt isoform types. However, using a series of isogenic cell lines derived from MCF-10A cells but exhibiting varying stages of progressive tumorigenesis, we observe that this inhibition of neoplastic behavior can be reversed in epithelial cells that have advanced to a highly malignant state. In contrast to the tumor suppressive properties of Akt, activated Akt signaling in MCF10A cells can rescue cell viability upon treatment with cytotoxic agents. This feature is regarded as tumor-promoting. CONCLUSION: We demonstrate that Akt signaling conveys novel dichotomy effects in which its oncogenic properties contributes mainly to sustaining cell viability, as opposed to the its tumor suppressing effects, which are mediated by repressing EMT, cell motility, and stem/progenitor cell expansion. While the former exerts a tumor-enhancing effect, the latter merely acts as a safeguard by restraining epithelial cells at the primary sites until metastatic spread can be moved forward, a process that is presumably dictated by the permissive tumor microenvironment or additional oncogenic insults.

Tumor evolution selectively inactivates the core microRNA machinery for immune evasion.

Cancer cells acquire genetic heterogeneity to escape from immune surveillance during tumor evolution, but a systematic approach to distinguish driver from passenger mutations is lacking. Here we investigate the impact of different immune pressure on tumor clonal dynamics and immune evasion mechanism, by combining massive parallel sequencing of immune edited tumors and CRISPR library screens in syngeneic mouse tumor model and co-culture system. We find that the core microRNA (miRNA) biogenesis and targeting machinery maintains the sensitivity of cancer cells to PD-1-independent T cell-mediated cytotoxicity. Genetic inactivation of the machinery or re-introduction of ANKRD52 frequent patient mutations dampens the JAK-STAT-interferon-γ signaling and antigen presentation in cancer cells, largely by abolishing miR-155-targeted silencing of suppressor of cytokine signaling 1 (SOCS1). Expression of each miRNA machinery component strongly correlates with intratumoral T cell infiltration in nearly all human cancer types. Our data indicate that the evolutionarily conserved miRNA pathway can be exploited by cancer cells to escape from T cell-mediated elimination and immunotherapy.

Seed in soil, with an epigenetic view.

It is becoming increasingly evident that discrete genetic alterations in neoplastic cells alone cannot explain multistep carcinogenesis whereby tumor cells are able to express diverse phenotypes during the complex phases of tumor development and progression. The epigenetic model posits that the host microenvironment exerts an initial, inhibitory constraint on tumor growth that is followed by acceleration of tumor progression through complex cell-matrix interactions. This review emphasizes the epigenetic aspects of breast cancer development in light of such interactions between epithelial cells ("seed") and the tumor microenvironment ("soil"). Our recent research findings suggest that epigenetic perturbations induced by the tumor microenvironment may play a causal role in promoting breast cancer development. It is believed that abrogation of these initiators could offer a promising therapeutic strategy.

A genome-scale CRISPR-Cas9 screening in myeloma cells identifies regulators of immunomodulatory drug sensitivity.

Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4CRBN ubiquitin ligase to trigger proteasomal degradation of the essential transcription factors IKZF1 and IKZF3 and multiple myeloma (MM) cytotoxicity. We have shown that CRBN is also targeted for degradation by SCFFbxo7 ubiquitin ligase. In the current study, we explored the mechanisms underlying sensitivity of MM cells to IMiDs using genome-wide CRISPR-Cas9 screening. We validate that CSN9 signalosome complex, a deactivator of Cullin-RING ubiquitin ligase, inhibits SCFFbxo7 E3 ligase-mediated CRBN degradation, thereby conferring sensitivity to IMiDs; conversely, loss of function of CSN9 signalosome activates SCFFbxo7 complex, thereby enhancing degradation of CRBN and conferring IMiD resistance. Finally, we show that pretreatment with either proteasome inhibitors or NEDD8 activating enzyme (NAE) inhibitors can abrogate degradation and maintain levels of CRBN, thereby enhancing sensitivity to IMiDs. These studies therefore demonstrate that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression.

Discovery of Pyridopyrimidinones as Potent and Orally Active Dual Inhibitors of PI3K/mTOR.

The identification and lead optimization of a series of pyridopyrimidinone derivatives are described as a novel class of efficacious dual PI3K/mTOR inhibitors, resulting in the discovery of 31. Compound 31 exhibited high enzyme activity against PI3K and mTOR, potent suppression of Akt and p70s6k phosphorylation in cell assays, and good pharmacokinetic profile. Furthermore, compound 31 demonstrated in vivo efficacy in a PC-3M tumor xenograft model.

Hypoxia inducible factor-1alpha and leukemic cell differentiation.

Arsenic trioxide (As2O3, ATO) is a recently developed drug for the effective treatment of acute promyelocytic leukemia (APL). Experimental studies showed that in vitro differentiation-inducing ability on APL cells of this drug is not significant compared with its in vivo activity. We unexpectedly found recently that hypoxia-mimetic agents and moderate real hypoxia triggered acute myeloid leukemic cells to undergo differentiation. Furthermore, intermittent hypoxia significantly prolonged the survival of the transplanted leukemic mice with inhibition of infiltration and induction of differentiation of leukemic cells. In the following works, molecular mechanisms of hypoxia-induced differentiation were investigated and some interesting results have been obtained. This review will shortly summarize the related progresses and discuss the questions remained to be further investigated.

Hypoxia-simulating agents and selective stimulation of arsenic trioxide-induced growth arrest and cell differentiation in acute promyelocytic leukemic cells.

BACKGROUND AND OBJECTIVES: We recently reported that hypoxia-mimetic agents cobalt chloride (CoCl2 CoCl2 ) and desferrioxamine (DFO) could induce differentiation of acute myeloid leukemic (AML) cells. Here, we investigate whether these two agents influence the in vitro differentiation-inducing effect of arsenic trioxide (As2O3) on AML cells, an effective drug for the treatment of acute promyelocytic leukemia (APL) that is a unique subtype of AML with a specific fusion protein, PML-RARalpha. DESIGN AND METHODS: The APL cell line NB4 and non-APL promonocytic leukemic cell line U937 were treated with As2O3 (0.5 microM) combined with CoCl2 (50 microM) or DFO (10 microM). The U937/PR9 subclone, whose expression of PML-RARalpha protein can be induced by Zn2+, was also investigated. Cellular differentiation was evaluated by morphological criteria and myeloid differentiation-related antigens and marker gene expression. The hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA and protein were detected, respectively, by semi-quantitative/real-time quantitative reverse transcription polymerase chain reaction and immunoblots. PML-RARalpha protein was also analyzed. RESULTS: CoCl2 and DFO potentiated the growth-inhibiting and differentiation-inducing effects of low-dose As2O3, the latter enhancing CoCl2 and DFO-induced accumulation of HIF-1alpha protein in NB4 cells but not in U937 cells. These two hypoxia-mimetic agents also accelerated As2O3-induced modulation and degradation of PML-RARalpha protein in NB4 cells. Furthermore, inducible expression of the fusion gene restored the co-operative effects of As2O3 and CoCl2/DFO on U937/PR9 cells in terms of growth arrest, differentiation induction and HIF-1alpha protein accumulation. INTERPRETATION AND CONCLUSIONS: Mimicked hypoxia enhanced As2O3-induced differentiation, in which HIF-1alpha and PML/RARalpha proteins played an important role. These data provide new insights into the understanding of the mechanisms of the action of As2O3 in the treatment of patients with APL.

Evaluation of STAT3 signaling in ALDH+ and ALDH+/CD44+/CD24- subpopulations of breast cancer cells.

BACKGROUND: STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH(+)), or cell surface molecule CD44-positive (CD44(+)) but CD24-negative (CD24(-)) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells is unknown. METHODS AND RESULTS: We examined STAT3 activation in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH(+)) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH(-)) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH(+) subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH(+) subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH(+) subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH(+) breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH(+) cells were further selected for the stem cell markers CD44(+) and CD24(-). CONCLUSION: These studies demonstrate an important role for STAT3 signaling in ALDH(+) and ALDH(+)/CD44(+)/CD24(-) subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.

Epigenetic repression of RARRES1 is mediated by methylation of a proximal promoter and a loss of CTCF binding.

BACKGROUND: The cis-acting promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We first compared methylation occurring at the sequences (-664~+420) flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region (-664~-86), while the downstream element (-85~+420) proximal to the transcriptional start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter bound with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effect. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores. CONCLUSION/SIGNIFICANCE: This report delineated silencing of RARRES1 by hypermethylation is occurring at a proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread.

STAT3 is necessary for proliferation and survival in colon cancer-initiating cells.

STAT3 is constitutively activated in colon cancer but its contributions in cancer-initiating cells have not been explored. In this study, we characterized STAT3 in aldehyde dehydrogenase (ALDH)-positive (ALDH(+)) and CD133-positive (CD133(+)) subpopulations of human colon tumor cells that exhibited more potent tumor-initiating ability than ALDH(-)/CD133(-) cells in tumor xenograft assays in mice. We found that ALDH(+)/CD133(+) cells expressed higher levels of the active phosphorylated form of STAT3 than either ALDH(-)/CD133(-) or unfractionated colon cancer cells. STAT3 inhibition by RNA interference-mediated knockdown or small-molecule inhibitors LLL12 or Stattic blocked downstream target gene expression, cell viability, and tumorsphere-forming capacity in cancer-initiating cells. Similarly, treatment of mouse tumor xenografts with STAT3 short hairpin RNA (shRNA), interleukin 6 shRNA, or LLL12 inhibited tumor growth. Our results establish that STAT3 is constitutively activated in colon cancer-initiating cells and that these cells are sensitive to STAT3 inhibition. These findings establish a powerful rationale to develop STAT3 inhibitory strategies for treating advanced colorectal cancers.

Protein kinase Cdelta mediates retinoic acid and phorbol myristate acetate-induced phospholipid scramblase 1 gene expression: its role in leukemic cell differentiation.

Although phospholipid scramblase 1 (PLSCR1) was originally identified based on its capacity to promote transbilayer movement of membrane phospholipids, subsequent studies also provided evidence for its role in cell proliferation, maturation, and apoptosis. In this report, we investigate the potential role of PLSCR1 in leukemic cell differentiation. We show that all-trans retinoic acid (ATRA), an effective differentiation-inducing agent of acute promyelocytic leukemic (APL) cells, can elevate PLSCR1 expression in ATRA-sensitive APL cells NB4 and HL60, but not in maturation-resistant NB4-LR1 cells. ATRA- and phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation is accompanied by increased PLSCR1 expression, whereas only a slight or no elevation of PLSCR1 expression is observed in U937 cells differentiated with dimethyl sulfoxide (DMSO), sodium butyrate, or vitamin D3. Cell differentiation with ATRA and PMA, but not with vitamin D3 or DMSO, results in phosphorylation of protein kinase Cdelta (PKCdelta), and the PKCdelta-specific inhibitor rottlerin nearly eliminates the ATRA- and PMA-induced expression of PLSCR1, while ectopic expression of a constitutively active form of PKCdelta directly increases PLSCR1 expression. Finally, decreasing PLSCR1 expression with small interfering RNA inhibits ATRA/PMA-induced differentiation. Taken together, these results suggest that as a protein induced upon PKCdelta activation, PLSCR1 is required for ATRA- and PMA-triggered leukemic cell differentiation.