Small Molecule Compound Nerolidol attenuates Hypertension induced hypertrophy in spontaneously hypertensive rats through modulation of Mel-18-IGF-IIR signalling.

BACKGROUND: Cardiovascular diseases are caused by multitudes of stress factors like hypertension and their outcomes are associated with high mortality and morbidity worldwide. Nerolidol, a naturally occurring sesquiterpene found in several plant species, embodies various pharmacological benefits against numerous health disorders. However, their effects on hypertension induced cardiac complications are not completely understood. PURPOSE: The present study is to elucidate the efficacy of nerolidol against hypertension related cardiac hypertrophy in spontaneously hypertensive rats (SHRs). STUDY DESIGN: For preliminary in vitro studies, H9c2 cardiomyoblasts cells were challenged with 200 nM Angiotensin-II (AngII) for 12 h and were then treated with nerolidol for 24 h. The hypertrophic effect in H9c2 cells were analyzed by actin staining and the modulations in hypertrophic protein markers and mediators were determined by Western blotting analysis. For in vivo experiments, sixteen week-old male Wistar Kyoto (WKY) and SHRs were segregated into five groups (n = 9): Control WKY, hypertensive SHRs, SHRs with low dose (75 mg/kg b.w/day) nerolidol, SHRs with high dose (150 mg/kg b.w/day) nerolidol and SHR rats treated with an anti-hypertensive drug captopril (50 mg/kg b.w/day). Nerolidol treatment was given orally for 8 weeks and were analysed through Echocardiography. After euthanasia, hematoxylin and eosin staining, Immunohistochemical analysis and Western blotting was performed on left ventricle tissue. RESULTS: Western blotting analysis revealed that nerolidol significantly attenuates AngII induced expression of hypertrophic markers ANP and BNP in H9c2 cardiomyoblasts. In addition, actin staining further ascertained the potential of nerolidol to ameliorate AngII induced cardiac hypertrophy. Moreover, nerolidol administration suppressed the hypertrophic signalling mediators like calcineurin, GATA4, Mel-18, HSF-2 and IGFIIR in a dose-dependent fashion. In silico studies also ascertained the role of Mel-18 in the ameliorative effects of nerolidol. Further, these intriguing in vitro results were further confirmed in in vivo SHR model. Oral neraolidol in SHRs efficiently reduced blood pressure and ameliorated hypertension induced cardiac hypertrophic effects by effectively reducing the levels of proteins involved in cardiac MeL-18-HSF2-IGF-IIR signalling. CONCLUSION: Collectively, the data reveals that the cardioprotective effect of nerolidol against hypertension induced hypertrophy involves reduction in blood pressure and regulation of the cardiac Mel-18-IGFIIR signalling cascade.

Berberine inhibits chemotherapy-exacerbated ovarian cancer stem cell-like characteristics and metastasis through GLI1.

Despite the remarkable clinical response in ovarian cancer therapy, the distinctively high metastasis rate is still a barrier to achieve satisfying prognosis. Our study aimed to decipher the role of berberine in inhibiting chemotherapy-exacerbated ovarian cancer metastasis. We found that chemotherapy exacerbated the migration and cancer stem cell (CSC)-like characteristics through transcriptional factor GLI1, which regulated the pluripotency-associated gene BMI1 and the epithelial-mesenchymal transition (EMT) markers Vimentin and Snail. Berberine could not only down-regulate CSC-like characteristics but also reverse EMT and migration through inhibiting chemotherapy-activated GLI1/BMI1 signaling pathway. Together, our study revealed the pivotal role of berberine in overcoming chemotherapy-exacerbated ovarian cancer metastasis, thereby provided a potential adjuvant therapeutic agent in combination with chemotherapeutics to prevent metastasis during ovarian cancer chemotherapy.

Effects and mechanism of microRNA­218 against lung cancer.

Lung cancer is the most prevalent and observed type of cancer in Xuanwei County, Yunnan, South China. Lung cancer in this area is called Xuanwei lung cancer. However, its pathogenesis remains largely unknown. To date, a number of studies have shown that microRNA (miR)­218 functions as a tumor suppressor in multiple types of cancer. However, the role of miR­218 and its regulatory gene network in Xuanwei lung cancer have yet to be investigated. The current study identified that the expression levels of miR­218 in XWLC­05 cells were markedly lower compared with those in immortalized lung epithelial BEAS­2B cells. The present study also demonstrated that overexpression of miR­218 could decrease cell proliferation, invasion, viability and migration in Xuanwei lung cancer cell line XWLC­05 and NSCLC cell line NCI­H157. Additionally, the results revealed that overexpression of miR­218 could induce XWLC­05 and NCI­H157 cell apoptosis by arresting the cell cycle at G2/M phase. Finally, the present study demonstrated that overexpression of miR­218 could lead to a significant increase in phosphatase and tensin homolog (PTEN) and YY1 transcription factor (YY1), and a decrease in B­cell lymphoma 2 (BCL­2) and BMI1 proto­oncogene, polycomb ring finger (BMI­1) at the mRNA and protein level in XWLC­05 and NCI­H157 cell lines. However, we did not observe any remarkable difference in the roles of miR­218 and miR­218­mediated regulation of BCL­2, BMI­1, PTEN and YY1 expression in the progression of Xuanwei lung cancer. In conclusion, miR­218 could simultaneously suppress cell proliferation and tumor invasiveness and induce cell apoptosis by increasing PTEN and YY1 expression, while decreasing BCL­2 and BMI­1 in Xuanwei lung cancer. The results demonstrated that miR­218 might serve a vital role in tumorigenesis and progression of Xuanwei lung cancer and overexpression of miR­218 may be a novel approach for the treatment of Xuanwei lung cancer.

Mountain ginseng inhibits skeletal muscle atrophy by decreasing muscle RING finger protein-1 and atrogin1 through forkhead box O3 in L6 myotubes.

ETHNOPHARMACOLOGICAL RELEVANCE: Mountain ginseng (Panax ginseng C.A. Meyer) is a medicinal herb with immune effects, muscle damage protection and energy metabolism effects. However, the pharmacological role of mountain ginseng in dexamethasone (DEXA)-induced muscle atrophy through the forkhead box O (FOXO) family is not understood. Therefore, we hypothesized that mountain ginseng inhibits skeletal muscle atrophy by decreasing muscle RING finger protein-1 (MuRF1) and atrogin1 through FOXO3 in L6 myotubes. METHODS: Rat myoblast (L6) cells or Sprague-Dawley (SD) rats were exposed to DEXA and mountain ginseng. The expressions of muscle atrophy targets such as MuRF1, atrogin1, MyHC (myosin heavy chain), HSP90, p-Akt, Akt, p-ERK1/2, ERK, FOXO3a, FOXO1, myostatin, and follistatin were analyzed by using Western blot analysis or real-time PCR. The diameter of myotubes was measured. Recruitment of glucocorticoid receptor (GR) or FOXO3a was analyzed by performing a chromatin immunoprecipitation (ChIP) assay. RESULTS: Mountain ginseng treatment reduced muscle weight loss and collagen deposition in DEXA-induced rats. Mountain ginseng treatment led to decreases in MuRF1, atrogin1, p-ERK1/2, FOXO3a, FOXO1, and myostatin. Also, mountain ginseng treatment led to increases in the diameter of myotubes, MyHC, HSP90, p-Akt, and follistatin. Treatment with mountain ginseng reduced enrichment of GR, FOXO3a, and RNA polymerase II on the promoters. CONCLUSIONS: These results suggest that mountain ginseng inhibits skeletal muscle atrophy by decreasing MuRF1 and atrogin1 through FOXO3a in L6 myotubes.

Interplay among p21Waf1/Cip1, MUSASHI-1 and Krüppel-like factor 4 in activation of Bmi1-CreER reserve intestinal stem cells after gamma radiation-induced injury.

Gamma radiation is a commonly used adjuvant treatment for abdominally localized cancer. Since its therapeutic potential is limited due to gastrointestinal (GI) syndrome, elucidation of the regenerative response following radiation-induced gut injury is needed to develop a preventive treatment. Previously, we showed that Krüppel-like factor 4 (KLF4) activates certain quiescent intestinal stem cells (ISCs) marked by Bmi1-CreER to give rise to regenerating crypts following γ irradiation. In the current study, we showed that γ radiation-induced expression of p21Waf1/Cip1 in Bmi1-CreER cells is likely mitigated by MUSASHI-1 (MSI1) acting as a negative regulator of p21Waf1/Cip1 mRNA translation, which promotes exit of the Bmi1-CreER cells from a quiescent state. Additionally, Bmi1-specific Klf4 deletion resulted in decreased numbers of MSI1+ cells in regenerating crypts compared to those of control mice. We showed that KLF4 binds to the Msi1 promoter and activates its expression in vitro. Since MSI1 has been shown to be crucial for crypt regeneration, this finding elucidates a pro-proliferative role of KLF4 during the postirradiation regenerative response. Taken together, our data suggest that the interplay among p21Waf1/Cip1, MSI1 and KLF4 regulates Bmi1-CreER cell survival, exit from quiescence and regenerative potential upon γ radiation-induced injury.

Bmi1 marks gastric stem cells located in the isthmus in mice.

In the mammalian stomach, the isthmus has been considered as a stem cell zone. However, various locations and proliferative activities of gastric stem cells have been reported. We focused here on the stem cell marker Bmi1, a polycomb group protein, aiming to elucidate the characteristics of Bmi1-expressing cells in the stomach and to examine their stem cell potential. We investigated the Bmi1-expressing cell lineage in Bmi1-CreERT; Rosa26-YFP, LacZ or Rosa26-Confetti mice. We examined the in vivo and ex vivo effects of Bmi1-expressing cell ablation by using Bmi1-CreERT; Rosa26-iDTR mice. The Bmi1 lineage was also traced during regeneration after high-dose tamoxifen-, irradiation- and acetic acid-induced mucosal injuries. In the lineage-tracing experiments using low-dose tamoxifen, Bmi1-expressing cells in the isthmus of the gastric antrum and corpus provided progeny bidirectionally, towards both the luminal and basal sides over 6 months. In gastric organoids, Bmi1-expressing cells also provided progeny. Ablation of Bmi1-expressing cells resulted in impaired gastric epithelium in both mouse stomach and organoids. After high-dose tamoxifen-induced gastric mucosal injury, Bmi1-expressing cell lineages expanded and fully occupied all gastric glands of the antrum and the corpus within 7 days after tamoxifen injection. After irradiation- and acetic acid-induced gastric mucosal injuries, Bmi1-expressing cells also contributed to regeneration. In conclusion, Bmi1 is a gastric stem cell marker expressed in the isthmus of the antrum and corpus. Bmi1-expressing cells have stem cell potentials, both under physiological conditions and during regeneration after gastric mucosal injuries. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

RYBP modulates stability and function of Ring1B through targeting UBE3A.

Ring1 and yin yang 1-binding protein (RYBP) are central components of noncanonical polycomb-repressive complex 1 (nc-PRC1), which represses target gene expression and is required for normal organismal development. However, the molecular function of RYBP in this complex is obscure. In this study, we showed that RYBP inhibits the polyubiquitination-mediated proteasomal degradation of Ring1B independently of its ubiquitin (Ub)-protein isopeptide ligase (E3) ligase activity, leading to its stabilization and increased catalytic activity toward monoubiquitination of histone H2A at lysine 119. Mechanistic dissection further disclosed that RYBP directly binds to ubiquitin protein ligase E3A (UBE3A) to promote its ubiquitination and proteasomal degradation in an autoubiquitination-independent manner. The resultant reduction of UBE3A protein level alleviates its effect on ubiquitination-mediated degradation of Ring1B, therefore resulting in increased stability and enhanced transcriptional repressor activity on its target genes. Thus, our current findings lay a foundation for understanding how RYBP functions in nc-PRC1 complexes, which is involved in development, stem cell maintenance, and carcinogenesis.-Li, M., Zhang, S., Zhao, W., Hou, C., Ma, X., Li, X., Huang, B., Chen, H., Chen, D. RYBP modulates stability and function of Ring1B through targeting UBE3A.

BMI-1 Mediates Estrogen-Deficiency-Induced Bone Loss by Inhibiting Reactive Oxygen Species Accumulation and T Cell Activation.

Previous studies have shown that estrogen regulates bone homeostasis through regulatory effects on oxidative stress. However, it is unclear how estrogen deficiency triggers reactive oxygen species (ROS) accumulation. Recent studies provide evidence that the B lymphoma Mo-MLV insertion region 1 (BMI-1) plays a critical role in protection against oxidative stress and that this gene is directly regulated by estrogen via estrogen receptor (ER) at the transcriptional level. In this study, ovariectomized mice were given drinking water with/without antioxidant N-acetyl-cysteine (NAC, 1 mg/mL) supplementation, and compared with each other and with sham mice. Results showed that ovariectomy resulted in bone loss with increased osteoclast surface, increased ROS levels, T cell activation, and increased TNF and RANKL levels in serum and in CD4 T cells; NAC supplementation largely prevented these alterations. BMI-1 expression levels were dramatically downregulated in CD4 T cells from ovariectomized mice. We supplemented drinking water to BMI-1-deficient mice with/without NAC and compared them with each other and with wild-type (WT) mice. We found that BMI-1 deficiency mimicked alterations observed in ovariectomy whereas NAC supplementation reversed all alterations induced by BMI-1 deficiency. Because T cells are critical in mediating ovariectomy-induced bone loss, we further assessed whether BMI-1 overexpression in lymphocytes can protect against estrogen deficiency-induced osteoclastogenesis and bone loss by inhibiting oxidative stress, T cell activation, and RANKL production. When WT and Eµ-BMI-1 transgenic mice with BMI-1 specifically overexpressed in lymphocytes were ovariectomized and compared with each other and with WT sham mice, we found that BMI-1 overexpression in lymphocytes clearly reversed all alterations induced by ovariectomy. Results from this study indicate that estrogen deficiency downregulates BMI-1 and subsequently increases ROS, T cell activation, and RANKL production in T cells, thus enhancing osteoclastogenesis and accelerating bone loss. This study clarifies a novel mechanism regulating estrogen deficiency-induced bone loss. © 2016 American Society for Bone and Mineral Research.

RING1 and YY1 binding protein suppresses breast cancer growth and metastasis.

Evidence suggests that RING1 and YY1 binding protein (RYBP) functions as a tumor suppressor. However, its role in breast cancer remains unclear. In the present study, the expression of RYBP was assessed in breast cancer patients and cell lines. Disease-free survival durations of breast cancer patients with high RYBP expression were determined based on the ATCG dataset. The effects of RYBP overexpression on cell growth, migration and invasive potency were also assessed. Nude mouse xenograft and lung metastasis models were also used to confirm the role of RYBP. The involvement of SRRM3 in RYBP-mediated breast cancer suppression was explored using SRRM3 siRNA. The potential relationship between RYBP, SRRM3, and REST-003 was examined by qPCR. The results showed that RYBP was downregulated in breast cancer patients and in several breast cancer cell lines. Breast cancer patients with high expression levels of RYBP displayed better disease-free survival. Overexpression of RYBP in MDA-MB-231 and SK-BR-3 cells significantly decreased cell proliferation, migration, and invasion ability, and increased the proportion of cells arrested in S-phase compared with the negative control cells. Additionally, upregulation of proliferation-related cell cycle proteins (cyclin A and cyclin B1) and E-cadherin, and downregulation of snail were observed in RYBP-overexpressing cells. Overexpression of RYBP reduced tumor volume and weight as well as metastatic foci in the lungs of nude mice. SRRM3 knockdown by siRNA, which is downregulated after RYBP overexpression, suppressed cell growth and metastasis in MDA-MB-231 and SK-BR-3 cells. Furthermore, qPCR analysis revealed that REST-003 ncRNA was downregulated in cells overexpressing RYBP and in SRRM3-inhibited cells. Moreover, cell invasion ability and growth were increased after SRRM3 upregulation in RYBP-overexpressing cells, but they were decreased following si-REST-003 transfection. In conclusion, overexpression of RYBP suppresses breast cancer growth and metastasis both in vitro and in vivo. SRRM3 and REST-003, which are downregulated in cells overexpressing RYBP, may be involved in RYBP-mediated breast cancer progression.

Potential role of Shh-Gli1-BMI1 signaling pathway nexus in glioma chemoresistance.

Chemoresistance is a common hurdle for the proper treatment of gliomas. The role of Shh-Gli1 signaling in glioma progression has been reported. However, its role in glioma chemoresistance has not been well studied yet. In this work, we found that Shh-Gli1 signaling regulates the expression of one stem cell marker, BMI1 (B cell-specific Moloney murine leukemia virus), in glioma. Interestingly, we also demonstrated high expression of MRP1 (multi-drug resistance protein 1) in glioma. MRP1 expression was decreased by BMI1 siRNA and Shh-Gli1 cell signaling specific inhibitor GANT61 in our experiments. GANT61 very efficiently inhibited cell colony growth in glioma cell lines, compared to temozolomide. Moreover, a synergic effect of GANT61 and temozolomide drastically decreased the LD50 of temozolomide in the cell colony experiments. Therefore, our results suggest that there is a potential nexus of Shh-Gli1-BMI1 cell signaling to regulate MRP1 and to promote chemoresistance in glioma. Henceforth, our study opens the possibility of facing new targets, Gli1 and BMI1, for the effective treatment of glioma suppression of chemoresistance with adjuvant therapy of GANT61 and temozolomide.

Yin Yang 1 is associated with cancer stem cell transcription factors (SOX2, OCT4, BMI1) and clinical implication.

The transcription factor Yin Yang 1 (YY1) is frequently overexpressed in cancerous tissues compared to normal tissues and has regulatory roles in cell proliferation, cell viability, epithelial-mesenchymal transition, metastasis and drug/immune resistance. YY1 shares many properties with cancer stem cells (CSCs) that drive tumorigenesis, metastasis and drug resistance and are regulated by overexpression of certain transcription factors, including SOX2, OCT4 (POU5F1), BMI1 and NANOG. Based on these similarities, it was expected that YY1 expression would be associated with SOX2, OCT4, BMI1, and NANOG's expressions and activities. Data mining from the proteomic tissue-based datasets from the Human Protein Atlas were used for protein expression patterns of YY1 and the four CSC markers in 17 types of cancer, including both solid and hematological malignancies. A close association was revealed between the frequency of expressions of YY1 and SOX2 as well as SOX2 and OCT4 in all cancers analyzed. Two types of dynamics were identified based on the nature of their association, namely, inverse or direct, between YY1 and SOX2. These two dynamics define distinctive patterns of BMI1 and OCT4 expressions. The relationship between YY1 and SOX2 expressions as well as the expressions of BMI1 and OCT4 resulted in the classification of four groups of cancers with distinct molecular signatures: (1) Prostate, lung, cervical, endometrial, ovarian and glioma cancers (YY1(lo)SOX2(hi)BMI1(hi)OCT4(hi)) (2) Skin, testis and breast cancers (YY1(hi)SOX2(lo)BMI1(hi)OCT4(hi)) (3) Liver, stomach, renal, pancreatic and urothelial cancers (YY1(lo)SOX2(lo)BMI1(hi)OCT4(hi)) and (4) Colorectal cancer, lymphoma and melanoma (YY1(hi)SOX2(hi)BMI1(lo)OCT4(hi)). A regulatory loop is proposed consisting of the cross-talk between the NF-kB/PI3K/AKT pathways and the downstream inter-regulation of target gene products YY1, OCT4, SOX2 and BMI1.

Musculoskeletal Atrophy in an Experimental Model of Knee Osteoarthritis: The Effects of Exercise Training and Low-Level Laser Therapy.

OBJECTIVE: The aim of this study was to evaluate the effects of an exercise training protocol and low-level laser therapy (and the association of both treatments) on musculoskeletal atrophy using an experimental model of knee osteoarthritis (OA). DESIGN: Fifty male Wistar rats were randomly divided into five groups: control group, knee OA control group, OA plus exercise training group, OA plus low-level laser therapy group, and OA plus exercise training associated with low-level laser therapy group. The exercise training and the laser irradiation started 4 wks after the surgery, 3 days per week for 8 wks. The exercise was performed at a speed of 16 m/min, 3 days per week, 50 mins per day, for 8 wks. Laser irradiation was applied at two points of the left knee joint (medial and lateral), for 24 sessions. RESULTS: The results showed that both trained groups (irradiated or not) presented a significant increase in the muscle cross-sectional area and a decrease in muscle fiber density compared with the knee OA control group. Moreover, both trained and laser-irradiated groups demonstrated decreased muscle-specific ring-finger protein 1 and atrogin-1 immunoexpression. CONCLUSIONS: These results suggest that exercise training and low-level laser therapy were effective in preventing musculoskeletal alterations related to atrophy caused by the degenerative process induced by knee OA.

MiR-218-targeting-Bmi-1 mediates the suppressive effect of 1,6,7-trihydroxyxanthone on liver cancer cells.

Traditional Chinese medicine is recently emerged as anti-cancer therapy or adjuvant with reduced side-effects and improved quality of life. In the present study, an active ingredient, 1,6,7-trihydroxyxanthone (THA), derived from Goodyera oblongifolia was found to strongly suppress cell growth and induce apoptosis in liver cancer cells. MicroRNAs are a group of small non-coding RNAs that regulate gene expression at post-transcriptional levels. Our results demonstrated that miR-218 was up-regulated and oncogene Bmi-1 was down-regulated by THA treatment. Further investigation showed that THA-induced-miR-218 up-regulation could lead to activation of tumor suppressor P16(Ink4a) and P14(ARF), the main down-stream targets of Bmi-1. In conclusion, THA might be a potential anti-cancer drug candidate, at least in part, through the activation of miR-218 and suppression of Bmi-1 expression.

H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex.

The bithorax complex (BX-C) in Drosophila melanogaster is a cluster of homeotic genes that determine body segment identity. Expression of these genes is governed by cis-regulatory domains, one for each parasegment. Stable repression of these domains depends on Polycomb Group (PcG) functions, which include trimethylation of lysine 27 of histone H3 (H3K27me3). To search for parasegment-specific signatures that reflect PcG function, chromatin from single parasegments was isolated and profiled. The H3K27me3 profiles across the BX-C in successive parasegments showed a 'stairstep' pattern that revealed sharp boundaries of the BX-C regulatory domains. Acetylated H3K27 was broadly enriched across active domains, in a pattern complementary to H3K27me3. The CCCTC-binding protein (CTCF) bound the borders between H3K27 modification domains; it was retained even in parasegments where adjacent domains lack H3K27me3. These findings provide a molecular definition of the homeotic domains, and implicate precisely positioned H3K27 modifications as a central determinant of segment identity.

Combinatorial interactions are required for the efficient recruitment of pho repressive complex (PhoRC) to polycomb response elements.

Polycomb Group (PcG) proteins are epigenetic repressors that control metazoan development and cell differentiation. In Drosophila, PcG proteins form five distinct complexes targeted to genes by Polycomb Response Elements (PREs). Of all PcG complexes PhoRC is the only one that contains a sequence-specific DNA binding subunit (PHO or PHOL), which led to a model that places PhoRC at the base of the recruitment hierarchy. Here we demonstrate that in vivo PHO is preferred to PHOL as a subunit of PhoRC and that PHO and PHOL associate with PREs and a subset of transcriptionally active promoters. Although the binding to the promoter sites depends on the quality of recognition sequences, the binding to PREs does not. Instead, the efficient recruitment of PhoRC to PREs requires the SFMBT subunit and crosstalk with Polycomb Repressive Complex 1. We find that human YY1 protein, the ortholog of PHO, binds sites at active promoters in the human genome but does not bind most PcG target genes, presumably because the interactions involved in the targeting to Drosophila PREs are lost in the mammalian lineage. We conclude that the recruitment of PhoRC to PREs is based on combinatorial interactions and propose that such a recruitment strategy is important to attenuate the binding of PcG proteins when the target genes are transcriptionally active. Our findings allow the appropriate placement of PhoRC in the PcG recruitment hierarchy and provide a rationale to explain why YY1 is unlikely to serve as a general recruiter of mammalian Polycomb complexes despite its reported ability to participate in PcG repression in flies.

Mammary cancer chemoprevention by withaferin A is accompanied by in vivo suppression of self-renewal of cancer stem cells.

Current dogma favors elimination of therapy-resistant cancer stem cells for chemoprevention of breast cancer. We showed recently that mammary cancer development in a transgenic mouse model (mouse mammary tumor virus-neu; MMTV-neu) was inhibited significantly upon treatment with withaferin A (WA), a steroidal lactone derived from a medicinal plant. Herein, we demonstrate that the mammary cancer prevention by WA is accompanied by in vivo suppression of breast cancer stem cells (bCSC). In vitro mammosphere formation was dose-dependently inhibited by WA treatment in MCF-7 and SUM159 human breast cancer cells. Other markers of bCSC, including aldehyde dehydrogenase 1 (ALDH1) activity and CD44(high)/CD24(low)/epithelial-specific antigen-positive (ESA+) fraction, were also decreased significantly in the presence of plasma achievable doses of WA. However, WA exposure resulted in cell line-specific changes in Oct4, SOX-2, and Nanog mRNA expression. WA administration to MMTV-neu mice (0.1 mg/mouse, 3 times/week for 28 weeks) resulted in inhibition of mammosphere number and ALDH1 activity in vivo. Mechanistic studies revealed that although urokinase-type plasminogen activator receptor overexpression conferred partial protection against bCSC inhibition by WA, Notch4 was largely dispensable for this response. WA treatment also resulted in sustained (MCF-7) or transient (SUM159) downregulation of Bmi-1 (B-cell-specific Moloney murine leukemia virus insertion region-1) protein. Ectopic expression of Bmi-1 conferred partial but significant protection against ALDH1 activity inhibition by WA. Interestingly, WA treatment caused induction of Kruppel-like factor 4 (KLF4) and its knockdown augmented bCSC inhibition by WA. In conclusion, this study shows in vivo effectiveness of WA against bCSC.

YY1 DNA binding and interaction with YAF2 is essential for Polycomb recruitment.

Polycomb Group (PcG) proteins are crucial for epigenetic inheritance of cell identity and are functionally conserved from Drosophila to humans. PcG proteins regulate expression of homeotic genes and are essential for axial body patterning during development. Earlier we showed that transcription factor YY1 functions as a PcG protein. YY1 also physically interacts with YAF2, a homolog of RYBP. Here we characterize the mechanism and physiologic relevance of this interaction. We found phenotypic and biochemical correction of dRYBP mutant flies by mouse YAF2 demonstrating functional conservation across species. Further biochemical analysis revealed that YAF2 bridges interaction between YY1 and the PRC1 complex. ChIP assays in HeLa cells showed that YAF2 is responsible for PcG recruitment to DNA, which is mediated by YY1 DNA binding. Knock-down of YY1 abrogated PcG recruitment, which was not compensated by exogenous YAF2 demonstrating that YY1 DNA binding is a priori necessary for Polycomb assembly on chromatin. Finally, we found that although YAF2 and RYBP regulate a similar number of Polycomb target genes, there are very few genes that are regulated by both implying functional distinction between the two proteins. We present a model of YAF2-dependent and independent PcG DNA recruitment by YY1.

Synergistic effects of curcumin with emodin against the proliferation and invasion of breast cancer cells through upregulation of miR-34a.

Curcumin, a biphenyl compound derived from rhizome, is a powerful anti-cancer agent. Emodin is an active component isolated from the root and rhizome of Rheum palmatum that has been widely used in traditional Chinese medicine for the treatment of various diseases. Currently, there are no studies examining the effect of curcumin in combination with emodin on tumor cell growth. In this study, we report for the first time that combined curcumin and emodin administration synergistically inhibits proliferation (MTT assay), survival (flow cytometry), and invasion (transwell migration assay) of breast cancer cells. Synergism is determined by the Chou-Talalay method. Moreover, we demonstrate that miR-34a is upregulated by curcumin and emodin. This microRNA helps mediate the anti-tumor effects of curcumin and emodin by downregulating Bcl-2 and Bmi-1. Our results not only provide insight into the mechanism of synergy between curcumin and emodin in breast cancer cells, but also suggest a new and potentially useful approach for breast cancer therapy.