Curcumin Functions as a Positive Regulator of miRNA Processing & a Negative Regulator of Cancer Stem Cell Proliferation

Identifying agents that inhibit the proliferation of cancer stem cells is of great importance in cancer biology. Curcumin has been shown to function as an anti-cancer agent. One of the tumor suppressors that appear to be induced in response to curcumin treatment is p53. A recent study suggests that curcumin increases the expression of miRNAs, such as miR-22, miR-181a, b, c, miR-34, miR-103, and miR-21. Intriguingly, miR-34, 181b, c, miR-103, miR-21, and miR-24 have been identified as transcriptional targets of the tumor suppressor p53. This data suggests a possibility that curcumin, by inducing the expression of p53, it could increase the expression of these microRNAs. A number of groups have shown that c-Myc, Sox-2, Klf-4, Oct-4, Sox-2, Nanog, and Lin28 are required for reprogramming of differentiated cells into pluripotent stem cells and for cancer stem cell proliferation. Interestingly, miR-21 has recently been shown to represses stem cell factors such as Oct4, Nanog, sox2, and c-Myc, indicating that curcumin by increasing the expression of miR-21, it could inhibit cancer stem cell proliferation. In addition, curcumin induced p53 may result in increased expression of its target, miR-145. Interestingly, it has recently been shown that miR-145 suppresses the expression of c-myc, klf-4, Oct-4, and Sox-2 in human embryonic stem cells (hESCs) and thereby promotes the differentiation of hESC. This data suggests that p53-dependent miR-145 expression will result in down regulation of Oct-4, Klf-4, Sox-2, Nanog, and c-myc and inhibition of stem cell proliferation. Further, curcumin induced miR-22 appears to inhibit the expression of estrogen receptor α. Remarkably, it has recently been shown that estrogen receptor α inhibits the processing of several microRNAs, including the tumor suppressor miR-145. This data suggests that curcumin, by inhibiting the expression of estrogen receptor α. through its target miR-22, it could increase the processing of miR-145 and down regulate the expression of its target genes (Oct-4, Klf-4, Sox-2, Nanog, and c-myc). Thereby, curcumin could function as a positive regulator of miRNA processing and a negative regulator of cancer stem cell proliferation. 

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Cigarette Smoke Initiates Oxidative Stress-Induced Cellular Phenotypic Modulation Leading to Cerebral Aneurysm Pathogenesis.

OBJECTIVE: Cigarette smoke exposure (CSE) is a risk factor for cerebral aneurysm (CA) formation, but the molecular mechanisms are unclear. Although CSE is known to contribute to excess reactive oxygen species generation, the role of oxidative stress on vascular smooth muscle cell (VSMC) phenotypic modulation and pathogenesis of CAs is unknown. The goal of this study was to investigate whether CSE activates a NOX (NADPH oxidase)-dependent pathway leading to VSMC phenotypic modulation and CA formation and rupture. APPROACH AND RESULTS: In cultured cerebral VSMCs, CSE increased expression of NOX1 and reactive oxygen species which preceded upregulation of proinflammatory/matrix remodeling genes (MCP-1, MMPs [matrix metalloproteinase], TNF-α, IL-1β, NF-κB, KLF4 [Kruppel-like factor 4]) and downregulation of contractile genes (SM-α-actin [smooth muscle α actin], SM-22α [smooth muscle 22α], SM-MHC [smooth muscle myosin heavy chain]) and myocardin. Inhibition of reactive oxygen species production and knockdown of NOX1 with siRNA or antisense decreased CSE-induced upregulation of NOX1 and inflammatory genes and downregulation of VSMC contractile genes and myocardin. p47phox-/- NOX knockout mice, or pretreatment with the NOX inhibitor, apocynin, significantly decreased CA formation and rupture compared with controls. NOX1 protein and mRNA expression were similar in p47phox-/- mice and those pretreated with apocynin but were elevated in unruptured and ruptured CAs. CSE increased CA formation and rupture, which was diminished with apocynin pretreatment. Similarly, NOX1 protein and mRNA and reactive oxygen species were elevated by CSE, and in unruptured and ruptured CAs. CONCLUSIONS: CSE initiates oxidative stress-induced phenotypic modulation of VSMCs and CA formation and rupture. These molecular changes implicate oxidative stress in the pathogenesis of CAs and may provide a potential target for future therapeutic strategies

The role of mTOR in SAF-1-mediated VEGF expression and breast cancer progression

Breast cancer is a heterogeneous disease with a huge impact on the community evident from its high indices of morbidity and mortality worldwide. In recent years, research has been extensively directed toward unravelling the molecular basis of the pathogenesis in different molecular subtypes of breast cancer. Better understanding of the different breast cancer cells and the subsequent improvement of diagnostic and therapeutic modalities of the disease resulted in a promising decline in its morbidity and mortality. In addition to surgery, radiotherapy, and conventional chemotherapy, treatment of breast cancer, nowadays, involves more targeted therapeutic options. Treatment options of breast cancer are determined mainly by the tumor stage and molecular subtypes of the tumor cells. Invasive potentials of a tumor involve also its ability to recruit blood supply through a process known as angiogenesis. Angiogenesis is an intricate process mediated by the interaction of many players including the tumor cells and stroma as well as the endothelial cells in adjacent blood vessels. Vascular endothelial growth factor (VEGF) is one of the main proangiogenic molecules secreted by breast cancer cells to promote angiogenesis through binding to the vascular endothelial growth factor receptors (VEGFR) on target cells. Although angiogenesis is a hallmark of invasive solid tumors, it is also required in some normal physiological conditions. Targeting angiogenesis indiscriminately imposes a huge risk of enormous adverse effects. Therefore, the goal of research today is to explore potential mechanisms to counteract angiogenesis selectively in the tumor cells. Secretion of VEGF by breast cancer cells is activated by several stimuli including hypoxia, metabolic stress and inflammatory conditions. Hypoxia is one of the main drivers for both physiological and pathological angiogenesis. Hypoxia induces VEGF expression in hypoxic cells through the action of hypoxia inducible factor-1 (HIF-1). However, evidence indicates that breast cancer cells are capable of secreting VEGF at early stages before hypoxia ensues in the tumor mass. Evidence shows also that breast cancer cells secrete VEGF even under normoxic conditions, which suggests that VEGF expression in breast cancer, particularly at early stages, is mediated by a mechanism(s) other than hypoxia. The goal of our research is to investigate some of these potential mechanism(s). Unravelling these mechanism(s) could pave the road for potentially novel therapeutic modalities in the treatment of breast cancer. Previous work in our lab has identified a role of serum amyloid activating factor-1 (SAF-1), a transcription factor, in overexpression of VEGF in some breast cancer cells. Here, we report that the SAF-1-mediated VEGF expression in breast cancer cells is repressed by Kruppel like factor-4 (KLF-4) transcription factor. Our findings suggest also that KLF-4 is potentially involved in the repression of the VEGF expression in a SAF-1-independent manner. We found that the level of KLF-4 is lower in breast cancer cells compared to normal breast cells. Therefore, we further investigated possible mechanism(s) for upregulation of KLF-4 or downregulation of SAF-1 in breast cancer cells to curtail VEGF expression and counteract angiogenesis and thence progression of breast cancer. There is evidence in literature that inhibition of a serine-threonine kinase called mammalian target of rapamycin (mTOR) exerts antiproliferative effect in vascular smooth muscle cells (VSMC). Evidence showed that the effect of mTOR inhibition on VSMC is mediated by upregulation of KLF-4 through a yet unknown mechanism. Thus, we have explored a potential role of mTOR inhibition in upregulation of KLF-4 in breast cancer cells and inhibition of VEGF expression. VEGF secreted by breast cancer cells not only stimulates proliferation and migration of the endothelial cells, but also promotes proliferation and migration of the secreting breast cancer cells themselves. Mounting evidence substantiates the beneficial role of mTOR inhibition in breast cancer. Our results show a novel mechanism of upregulating KLF-4 and inhibition of VEGF expression in breast cancer cells through inhibition of mTOR. Nonetheless, we noted that the effects of mTOR inhibition on breast cancer cells, including upregulation of KLF-4 and inhibition of proliferation and migration, are variable among different breast cell lines as well as among different mTOR inhibitors. One of the possible explanation of the variable response to mTOR inhibitors is the rebound upregulation of proteins in the mTOR pathway which imposes a risk of emergence of resistance or refractoriness to treatment with mTOR inhibition. The role of active mTOR in the invasiveness of breast cancer is well known, yet relatively little is known about the role and impact of total mTOR protein. We found that total mTOR protein level is higher in breast cancer cells compared to their noncancerous counterparts. mTOR protein is particularly high in the ER+ breast cancer cells which constitute the majority of breast cancer cells subtypes. High mTOR protein level in breast cancer cells could be attributed to decreased mTOR protein degradation, increased mTOR gene expression, or both. Our results indicate a defective degradation of mTOR in breast cancer cells compared to normal cells, which could, at least in part, explain why mTOR protein is high in breast cancer cells. Our results show also that transcription of mTOR gene is elevated in the ER+ breast cancer cells. Our study revealed that promoter region of mTOR in the ER+ breast cancer cells has a truncated dinucleotide tandem repeat region. Tandem repeats are a potential site for regulation of transcription of genes. Shortening of this region could be a possible mechanism of increased transcription of mTOR gene in the ER+ breast cancer cells. Moreover, we have revealed a novel mechanism of increasing mTOR degradation as well as inhibition of MTOR transcription in the ER+ breast cancer cells by treatment with metformin the antidiabetic mTOR inhibitor. In correlation, metformin treatment induced a profound effect on upregulation of KLF-4 and inhibition of proliferation and migration of the breast cancer cells, particularly, the ER+ subtype. These findings could be utilized in the optimization of chemotherapeutic regimens of breast cancer

Endoglin Is Essential for the Maintenance of Self-Renewal and Chemoresistance in Renal Cancer Stem Cells.

Renal cell carcinoma (RCC) is a deadly malignancy due to its tendency to metastasize and resistance to chemotherapy. Stem-like tumor cells often confer these aggressive behaviors. We discovered an endoglin (CD105)-expressing subpopulation in human RCC xenografts and patient samples with a greater capability to form spheres in vitro and tumors in mice at low dilutions than parental cells. Knockdown of CD105 by short hairpin RNA and CRISPR/cas9 reduced stemness markers and sphere-formation ability while accelerating senescence in vitro. Importantly, downregulation of CD105 significantly decreased the tumorigenicity and gemcitabine resistance. This loss of stem-like properties can be rescued by CDA, MYC, or NANOG, and CDA might act as a demethylase maintaining MYC and NANOG. In this study, we showed that Endoglin (CD105) expression not only demarcates a cancer stem cell subpopulation but also confers self-renewal ability and contributes to chemoresistance in RCC

Computational modeling of shear forces and experimental validation of endothelial cell responses in an orbital well shaker system

Vascular endothelial cells are continuously exposed to hemodynamic shear stress. Intensity and type of shear stress are highly relevant to vascular physiology and pathology. Here, we modeled shear stress distribution in a tissue culture well (R = 17.5 mm, fill volume 2 ml) under orbital translation using computational fluid dynamics with the finite element method. Free surface distribution, wall shear stress, inclination angle, drag force, and oscillatory index on the bottom surface were modeled. Obtained results predict nonuniform shear stress distribution during cycle, with higher oscillatory shear index, higher drag force values, higher circular component, and larger inclination angle of the shear stress at the periphery of the well compared with the center of the well. The oscillatory index, inclination angle, and drag force are new quantitative parameters modeled in this system, which provide a better understanding of the hydrodynamic conditions experienced and reflect the pulsatile character of blood flow in vivo. Validation experiments revealed that endothelial cells at the well periphery aligned under flow and increased Kruppel-like Factor 4 (KLF-4), cyclooxygenase-2 (COX-2) expression and endothelial nitric oxide synthase (eNOS) phosphorylation. In contrast, endothelial cells at the center of the well did not show clear directional alignment, did not induce the expression of KLF-4 and COX-2 nor increased eNOS phosphorylation. In conclusion, this improved computational modeling predicts that the orbital shaker model generates different hydrodynamic conditions at the periphery versus the center of the well eliciting divergent endothelial cell responses. The possibility of generating different hydrodynamic conditions in the same well makes this model highly attractive to study responses of distinct regions of the same endothelial monolayer to different types of shear stresses thereby better reflecting in vivo conditions

Reprogramming Primordial Germ Cells into Pluripotent Stem Cells

Background: Specification of primordial germ cells (PGCs) results in the conversion of pluripotent epiblast cells into monopotent germ cell lineage. Blimp1/Prmt5 complex plays a critical role in the specification and maintenance of the early germ cell lineage. However, PGCs can be induced to dedifferentiate back to a pluripotent state as embryonic germ (EG) cells when exposed to exogenous signaling molecules, FGF-2, LIF and SCF. Methodology and Principal Findings: Here we show that Trichostatin A (TSA), an inhibitor of histone deacetylases, is a highly potent agent that can replace FGF-2 to induce dedifferentiation of PGCs into EG cells. A key early event during dedifferentiation of PGCs in response to FGF-2 or TSA is the down-regulation of Blimp1, which reverses and apparently relieves the cell fate restriction imposed by it. Notably, the targets of Blimp1, which include c-Myc and Klf-4, which represent two of the key factors known to promote reprogramming of somatic cells to pluripotent state, are up-regulated. We also found early activation of the LIF/Stat-3 signaling pathway with the translocation of Stat-3 into the nucleus. By contrast, while Prmt5 is retained in EG cells, it translocates from the nucleus to the cytoplasm where it probably has an independent role in regulating pluripotency. Conclusions/Significance: We propose that dedifferentiation of PGCs into EG cells may provide significant mechanistic insights on early events associated with reprogramming of committed cells to a pluripotent state

Hematopoietic progenitors express embryonic stem cell and germ layer genes

Cell therapy for tissue regeneration requires cells with high self-renewal potential and with the capacity to differentiate into multiple differentiated cell lineages, like embryonic stem cells (ESCs) and adult somatic cells induced to pluripotency (iPSCs) by genetic manipulation. Here we report that normal adult mammalian bone marrow contains cells, expressing the cell surface antigen CD34, that naturally express the genes that are characteristic of ESCs and that are required to generate iPSCs. In addition, these CD34+ cells spontaneously express, without genetic manipulation, genes characteristic of the three embryonic germ layers ectoderm, mesoderm and endoderm. In addition to the neural lineage genes we previously reported in these CD34+ cells, we found that they express genes of the mesodermal cardiac muscle lineage and of the endodermal pancreatic lineage as well as intestinal lineage genes. Thus, these normal cells in the adult spontaneously exhibit the characteristics of embryonic-like stem cells

Experimental Limitations Using Reprogrammed Cells for Hematopoietic Differentiation

We review here our experiences with the in vitro reprogramming of somatic cells to induced pluripotent stem cells (iPSC) and subsequent in vitro development of hematopoietic cells from these iPSC and from embryonic stem cells (ESC). While, in principle, the in vitro reprogramming and subsequent differentiation can generate hematopoietic cell from any somatic cells, it is evident that many of the steps in this process need to be significantly improved before it can be applied to human cells and used in clinical settings of hematopoietic stem cell (HSC) transplantations