Small Inhibitory RNA Research Articles

Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis

BackgroundAscites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.MethodsUsing human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.ResultsIn the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.ConclusionsThese results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.

Abstract A78: Broccoli-derived compounds down regulate C-C chemokine ligand 2 expression and monocyte attraction through androgen-dependent pathway in LNCaP prostate cancer cells

Abstract Inflammation has an important role in prostate tumorigenesis. The recruitment of inflammatory monocytes to the tumor site is precipitated by increased levels of the chemo-attractant C-C chemokine ligand 2 (CCL2). In the present study, we test the hypothesis that broccoli-derived cancer protective compounds, indole-3-carbinol (I3C) and 3', 3'-diindolylmethane (DIM), exert anti-inflammatory effects by decreasing CCL2 levels and thereby down regulating CCL2 mediated pathways. Dihydrotestosterone (DHT) induced a time (0-72 hrs) and concentration-dependent (0-1 nM) increase in CCL2 mRNA levels in androgen-responsive LNCaP cells. Consistent with increase in CCL2 mRNA, we also observed increased secretion of CCL2 protein in the media of DHT-treated LNCaP cells. This effect of DHT was mediated through an androgen receptor (AR)-dependent pathway as small inhibitor RNA against AR negated the induction. Conditioned media from androgen-treated cells promoted human monocyte THP-1 cell migration and this effect was blocked by antibody against CCL-2. Both I3C and DIM inhibited promotional effects of DHT on CCL2 expression and monocyte migration in-vitro. These results indicate the effect of I3C and DIM on CCL2 and monocyte migration was androgen dependent. Hence, androgen appeared to stimulate CCL2 secretion and thereby promotes inflammatory micro-environments in prostate tumors. These results suggest that the androgen mediated inflammatory processes are modulated by broccoli-derived compounds. Citation Format: Thomas Wang, Eun-Kyung Kim, John Milner, Young Kim. Broccoli-derived compounds down regulate C-C chemokine ligand 2 expression and monocyte attraction through androgen-dependent pathway in LNCaP prostate cancer cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A78.

Selective Inhibitors of Nuclear Export Block Pancreatic Cancer Cell Proliferation and Reduce Tumor Growth in Mice

Tumor-suppressor proteins are inactivated by many different mechanisms, including nuclear exclusion by chromosome region maintenance (CRM)-1. Increased tumor levels of CRM-1 have been correlated with poor prognosis of patients with pancreatic cancer, making it a therapeutic target. Selective inhibitors of nuclear export (SINEs) bind to CRM-1 to irreversibly inhibit its ability to export proteins; we investigated a new class of SINEs in pancreatic cancer cells. We studied the effects of SINE analogs in a panel of pancreatic cancer cell lines and nontransformed human pancreatic ductal epithelial cells using proliferation, apoptosis, immunoblot, co-immunoprecipitation, small inhibitor RNA, and fluorescence microscopy analyses. The effects of the SINEs also were investigated in mice with subcutaneous and orthotopic tumors. SINEs (KPT-185, KPT-127, KPT-205, and KPT-227) inhibited proliferation and promoted apoptosis of pancreatic cancer cells, but did not affect human pancreatic ductal epithelial cells. The nuclei of cells incubated with KPT-185 accumulated tumor-suppressor proteins (p27, FOXO, p73, and prostate apoptosis response-4 [PAR-4]) and inhibited interactions between CRM-1 and these proteins. Mutations in the region of CRM-1 that bind to SINEs (Cys-528), or small inhibitor RNA knockdown of PAR-4, prevented the ability of KPT-185 to block proliferation and induce apoptosis of pancreatic cancer cells. Oral administration of KPT-330 to mice reduced growth of subcutaneous and orthotopic xenograft tumors without major toxicity. Analysis of tumor remnants showed that KPT-330 disrupted the interaction between CRM-1 and PAR-4, activated PAR-4 signaling, and reduced proliferation of tumor cells. We identified SINEs that inhibit CRM-1 and promote nuclear accumulation of tumor-suppressor proteins in pancreatic cancer cells. Oral administration of the drug to mice reduces growth of xenograft tumors.

Abstract B76: Circumventing chemoresistance as a strategy to reduce the mortality disparities associated with advanced prostate cancer.

Abstract Prostate cancer (PCa) is the most commonly diagnosed cancer and the second leading cause of cancer death among men in the U.S., with African American men presenting a disproportionately high incidence and mortality from this disease. Although these disparities result from the combination of multiple factors (socioeconomic, health care, lifestyle, biological, etc.), there is growing consensus that PCa in African-American men is in general more aggressive than in other ethnic groups. Reducing PCa mortality disparities will therefore require novel strategies for the effective treatment of advanced, aggressive tumors characterized by chemotherapy resistance. Currently, chemotherapy with docetaxel (DTX), the standard of care for advanced PCa, is limited because most patients treated with the drug ultimately develop chemoresistance and succumb to the disease. Circumventing this resistance is therefore essential for increasing the effectiveness of DTX and other emerging anti-PCa drugs, and reducing the high mortality associated with advanced PCa. We established previously that lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival oncoprotein, is overexpressed in clinical prostate tumors, and that its overexpression in PCa cell models promotes resistance to DTX treatment via inhibition of lysosomal cell death. In the present study we evaluated if overexpression of LEDGF/p75 in PCa cells also promotes resistance to other chemotherapeutic drugs, and if its depletion sensitizes chemoresistant PCa cells to drug-induced cell death. We treated PC-3 cells (with and without stable LEDGF/p75 overexpression) with increasing concentrations of the anti-cancer drugs DTX, paclitaxel, doxorubicin, and TRAIL for up to 48 hrs. We then assessed cell viability by MTT assays and morphological examination. LEDGF/p75 overexpressing PC-3 cells showed higher viability than control cells in response to DTX and doxorubicin. However, LEDGF/p75 did not protect against paclitaxel and TRAIL, indicating selectivity in its ability to promote chemoresistance. We also observed by immunoblotting and quantitative PCR that metastatic PCa cell lines selected for DTX resistance, designated PC3-DR and DU-145DR, naturally express high levels of endogenous LEDGF/p75 relative to their sensitive parental cell lines. To determine if the elevated expression of LEDGF/p75 in these two cell lines contribute to DTX resistance, we knocked down this protein with a specific small inhibitory RNA (si-p75). Cell viability significantly decreased in both cell lines in response to combined treatment with DTX and si-p75, relative to siRNA controls. We also observed that these chemoresistant PCa cell lines express high levels of proteins associated with inflammation and cellular response to oxidative stress, such as IL-6, IL-6R, Hsp27, PRDX6, and cytoglobin, and that the levels of these proteins decreased upon LEDGF/p75 knockdown. Our preliminary results suggest that LEDGF/p75 overexpression promotes selective chemoresistance in cellular models of advanced PCa, and that its inhibition increases sensitization to DTX associated with downregulation of inflammation/oxidative stress-related genes. These promising results have implications for the development of novel therapeutic strategies for chemoresistant PCa. Studies are in progress to assess the expression levels of LEDGF/p75 in sera and prostate tissues from African-American and Caucasian PCa patients, and correlate these levels with advanced disease and chemotherapy resistance in these patients. Citation Format: Leslimar Ríos-Colón, Anamika Basu, Christina Cajigas, Catherine Elix, Carlos A. Casiano. Circumventing chemoresistance as a strategy to reduce the mortality disparities associated with advanced prostate cancer. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B76.

Nod2-Induced Autocrine Interleukin-1 Alters Signaling by ERK and p38 to Differentially Regulate Secretion of Inflammatory Cytokines

Stimulation of nucleotide-binding oligomerization domain-containing (Nod)2 and other pattern recognition receptors (PRR) in human monocyte-derived macrophages induces interleukin (IL)-1, which increases mitogen-activated protein kinase (MAPK) activation and cytokine secretion. Activation of MAPK by PRR has varied effects on inflammatory cytokine secretion. We investigated whether different levels of autocrine IL-1 mediate these varied effects. Macrophage responses to PRR ligands were analyzed by enzyme-linked immunosorbent assay and flow cytometry. We overexpressed or reduced MAPK levels (using small inhibitory RNA). Nod2 and other PRR activated signaling via extracellular signal-related kinase (ERK) and p38 that inhibited inflammatory cytokine production by human monocyte-derived macrophages; autocrine IL-1 production prevented this inhibition. ERK and p38 inhibited inflammatory cytokine production by human macrophages that produce low levels of IL-1 (such as M2, endotoxin-tolerant, and intestinal macrophages); adding exogenous IL-1 caused ERK and p38 to stimulate production of inflammatory cytokines in these cells. In mouse macrophages, which do not produce IL-1 in response to PRR stimulation alone, addition of exogenous IL-1 reversed the ERK-mediated inhibition of IL-12p40. Increasing activation of c-Jun N-terminal kinase in Nod2-stimulated human monocyte-derived macrophages, in the absence of autocrine IL-1 signaling, caused ERK and p38 to stimulate inflammatory cytokines secretion. Importantly, infection of human intestinal macrophages with pathogens that induce IL-1 production reversed the inhibition of inflammatory cytokine production by ERK and p38. In response to PRR stimulation of macrophages, the level of MAPK signaling is regulated by autocrine IL-1 and determines whether production of inflammatory cytokines is inhibited or stimulated. This mechanism could account for reported differences in MAPK regulation of inflammatory cytokines and propagate the inflammatory response to pathogens.

SiSPOTR: a tool for designing highly specific and potent siRNAs for human and mouse

RNA interference (RNAi) serves as a powerful and widely used gene silencing tool for basic biological research and is being developed as a therapeutic avenue to suppress disease-causing genes. However, the specificity and safety of RNAi strategies remains under scrutiny because small inhibitory RNAs (siRNAs) induce off-target silencing. Currently, the tools available for designing siRNAs are biased toward efficacy as opposed to specificity. Prior work from our laboratory and others’ supports the potential to design highly specific siRNAs by limiting the promiscuity of their seed sequences (positions 2–8 of the small RNA), the primary determinant of off-targeting. Here, a bioinformatic approach to predict off-targeting potentials was established using publically available siRNA data from more than 50 microarray experiments. With this, we developed a specificity-focused siRNA design algorithm and accompanying online tool which, upon validation, identifies candidate sequences with minimal off-targeting potentials and potent silencing capacities. This tool offers researchers unique functionality and output compared with currently available siRNA design programs. Furthermore, this approach can greatly improve genome-wide RNAi libraries and, most notably, provides the only broadly applicable means to limit off-targeting from RNAi expression vectors.

Cigarette Smoke–Induced CXCR3 Receptor Up-Regulation Mediates Endothelial Apoptosis

Endothelial monocyte-activating polypeptide II (EMAP II) and interferon-inducible protein (IP)-10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke-exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke-induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II-induced and IP-10-induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II-induced and IP-10-induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II-induced and IP-10-induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II-induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema.

Generation of PVY Coat Protein siRNAs in Transgenic Potatoes Resistant to PVY

Global climate change has the potential to quickly alter the distribution and profile of crop pests and pathogens. Ready tools for mobilization of resistance into elite crops will be part of an integrated strategy to maintain agricultural productivity. Engineered virus resistance through expression of coat protein is well established. The mechanisms of virus suppression by small RNAs (sRNAs) have only recently been elucidated. Understanding the role of sRNAs in gene regulation and development is rapidly evolving. High throughput sequencing (HTS) can generate large data sets of sRNA sequences which can be analyzed to recognize the abundant sRNAs that result from transcript degradation and to identify the regulatory micro RNAs (miRNA) involved in gene regulation and small inhibitory RNAs (siRNA) that confer virus resistance. Transgenic Solanum tuberosum that are glass house or field grown and that express the potato virus Y coat protein (PVY-CP) inverted hairpin RNA (ihRNA) from the 409s promoter with GBSS6 intron as a spacer exhibited resistance to PVY. Southern blots of genomic DNA indicated one or two copies of the transgene and analysis of HTS data of the small RNA population indicated high levels of siRNA production from the transgenic hairpin construct. Up to 47 of the top 200 most frequent sRNA reads were attributable to the PVY-CP transgenic construct in Ranger Russet line #5. This combination of transgene and transcription strength is functional for constructing efficient resistance cassettes for crop protection. Up to 95 of the top 200 sRNA sequences were 21 nucleotides in length with as many as 19 sequences corresponding to known miRNA transcripts. Additional endogenous sRNAs with homology to resistance gene analogs may be indicative of additional or complimentary resistance mechanisms produced in concert with the PVY siRNAs arising from PVY-CP RNA transcription.

Early-life Disruption of Epigenetic Marks May Contribute to the Origins of Mental Illness

Epigenetic marks, including DNA methyl ation (DNAm), histone modifications and small inhibitory RNAs, are heritable and provide critical layers of gene regulatory control; however, they also are dynamic and can be influenced by environmental exposures [1–3]. Studies primarily focused on cancer and late-onset diseases have revealed that a growing number of human diseases are linked to epigenetic disruptions. For example, DNAm became a hallmark of human cancer, a link that was first recognized in 1983, when tumor cells were found to be aberrantly methylated compared with their normal cell counterparts due to a loss of methylation from repetitive regions of the genome [4]. Together with the finding that healthy monozygotic twins show indistinguishable childhood DNAm [5], epigenetic mechanisms have long been thought to be highly coordinated throughout early development and more susceptible to drift in adulthood. In this article, we will discuss the impact that recent findings, of dramatic DNAm changes throughout childhood, will have on future epigenetic studies conducted in children and adults.

Effects of 18-kDa translocator protein knockdown on gene expression of glutamate receptors, transporters, and metabolism, and on cell viability affected by glutamate

Previously, several important roles for glutamate have been described for the biology of primary brain tumors. For example, glutamate has been suggested to promote glioma cell proliferation by the activation of the 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) subtype of glutamate receptors. In the present study, we determined the potential regulatory roles of the 18-kDa translocator protein (TSPO) in the glutamatergic system in relation to cell death of brain tumor cells through knockdown of the TSPO by genetic manipulation. With microarray analysis and validation of gene expression of particular genes using real-time PCR, we found effects because of small inhibitory RNA knockdown of the TSPO in human U118MG glioblastoma cells on gene expression of glutamate receptors, glutamate transporters, and enzymes for glutamate metabolism. We also applied antisense RNA to silence TSPO in rat C6 glioblastoma cells and assayed the effects on DNA fragmentation, indicative of apoptosis, because of glutamate exposure. In particular, the effects of TSPO silencing in human U118MG cells related to glutamate metabolism indicate a net effect of a reduction in glutamate levels, which may potentially protect the cells in question from cell death. The TSPO knockdown in C6 cells showed that TSPO is required for the induction of apoptosis because of glutamate exposure. These findings show that interactions between the TSPO and the glutamatergic system may play a role in tumor development of glioblastoma cells. This may also have implications for our understanding of the involvement of the TSPO in secondary brain damage and neurodegenerative diseases.

Melanocortin 1 Receptor Regulates Melanoma Cell Migration by Controlling Syndecan-2 Expression

The melanocortin 1 receptor (MC1R), a key regulator of melanogenesis, is known to control inflammation, acting in concert with the MC1R ligand α-melanocyte-stimulating hormone. Although cell migration is a key event in inflammation, few studies have addressed the function of MC1R in this context. Using highly motile melanoma cells, we found that the expression level of MC1R was associated with the extent of migration of mouse melanoma cells, suggesting that MC1R plays a functional role in controlling this migration. Overexpression of MC1R enhanced melanoma cell migration, whereas the opposite was true when MC1R levels were knocked down using small inhibitory RNAs. Interestingly, MC1R expression enhanced the synthesis of syndecan-2, a cell surface heparan sulfate proteoglycan known to be involved in melanoma cell migration. Knockdown of syndecan-2 expression decreased MC1R-mediated cell migration. Further, MC1R inhibited the activation of p38 MAPK, subsequently enhancing expression of sydnecan-2, in parallel with an increase in the extent of cell migration. Consistently, activation of p38 by H(2)O(2) inhibited syndecan-2 expression and cell migration, whereas inhibition of p38 activation enhanced syndecan-2 expression and cell migration. Finally, we found that α-melanocyte-stimulating hormone inhibited MC1R-mediated cell migration via activation of p38 and inhibition of syndecan-2 expression. Together, the data strongly suggest that MC1R regulates melanoma cell migration via inhibition of syndecan-2 expression.

Abstract 775: Targeting the transcriptional coactivator LEDGF/p75 to overcome chemoresistance in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer death among men in the US, with African American men presenting a disproportionately high incidence and mortality. Reducing this mortality disparity requires developing novel strategies for overcoming prostate tumor chemoresistance. Currently, chemotherapy with Docetaxel (DTX), the standard of care for advanced PCa, is limited because most patients treated with the drug ultimately develop chemoresistance and succumb to the disease. Circumventing this resistance is therefore essential for increasing the effectiveness of DTX and other anti-PCa drugs. Our group has established previously that lens ephitelium-derived growth factor p75 (LEDGF/p75), a stress survival oncoprotein, is overexpressed in human prostate tumors, and that its overexpression in PCa cell models promotes resistance to DTX via inhibition of lysosomal cell death. The present study was designed to evaluate if overexpression of LEDGF/p75 in PCa cells also promotes resistance to other chemotheurapeutic drugs, and if its depletion sensitizes chemoresistant PCa cells to drug-induced cell death. First, we treated PC-3 cells (with and without stable LEDGF/p75 overexpression) with increasing concentrations of DTX, paclitaxel, doxorubicin, and TRAIL for up to 48 hrs. We then assessed cell viability by MTT assays and morphological examination. LEDGF/p75 overexpressing PC-3 cells showed higher viability than parental PC-3 cells in response to DTX, paclitaxel, and doxorubicin. However, LEDGF/p75 did not protect against TRAIL-induced cell death, indicating selectivity in the ability of this stress oncoprotein to induce chemoresistance. We also observed by immunoblotting and quantitative PCR that metastatic PCa cell lines selected for DTX resistance, designated PC3-DR and DU-145DR, naturally express high levels of endogenous LEDGF/p75 relative to their sensitive parental cell lines. To assess the contribution of elevated LEDGF/p75 expression to DTX resistance in these two cell lines, we knocked down this protein with a specific small inhibitory RNA (si-p75). Immunoblots showed that si-p75 depleted cells of LEDGF/p75. Cell viability significantly decreased in both PC3-DR and DU-145DR cell lines in response to combined treatment with DTX and si-p75, relative to scrambled siRNA control. Our preliminary results suggest that LEDGF/p75 overexpression promotes chemoresistance in cellular models of advanced PCa, and that its inhibition increases sensitization to DTX. These promising results have implications for the development of novel theraupetic strategies for chemoresistant PCa. Further studies are in progress to determine the spectrum of anti-PCa drugs antagonized by LEDGF/p75, and if depletion of this protein sensitizes PCa cells to a wide variety of antitumor drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 775. doi:1538-7445.AM2012-775

Impaired Inflammatory Responses in Murine Lrrk2-Knockdown Brain Microglia

LRRK2, a Parkinson's disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation.

Broccoli‐derived compounds modulate androgen up‐regulation of C‐C chemokine ligand 2 and enhancement of monocyte attraction to prostate cancer cells

Inflammation plays a key role in prostate tumorigenesis. Recruitment of inflammatory monocytes to the tumor site is mediated by C‐C chemokine ligand 2 (CCL2) through binding to its receptor CCR2. We hypothesized that androgen could modulate CCL2 expression in hormone‐responsive prostate cancer cells and thereby promote recruitment of monocytes. Given the inhibitory effect of broccoli‐derived compounds indole‐3‐carbinol (I3C) and 3,3′‐diindolylmethane (DIM) on androgen‐dependent pathway, we also reasoned that I3C and DIM could modulate the effect of androgen on CCL2‐mediated pathways. We found that androgen dihydrotestosterone (DHT) induced time (0–72 hrs) and concentration‐dependent (0–1 nM) increases in CCL2 mRNA levels in the androgen‐responsive human prostate cancer cell LNCaP. The increase in CCL2 mRNA corresponded to increased secretion of CCL2 protein by LNCaP cells. This effect of DHT was mediated through an androgen receptor (AR)‐dependent pathway as small inhibitor RNA against AR negated the induction of CCL2 mRNA by DHT. Moreover, conditioned media from androgen‐treated cells promoted human monocyte THP‐1 cell migration. Both I3C and DIM inhibited promotional effects of DHT on CCL2 and migration. These results suggested that androgen regulates CCL2 and promotes inflammatory micro‐environments in prostate tumors, and this process can be regulated by broccoli‐derived compounds.

Nerve growth factor modulates human rhinovirus infection in airway epithelial cells by controlling ICAM-1 expression

Human rhinoviruses (HRV) are the most common agent of upper respiratory infections and an important cause of lower respiratory tract symptoms. Our previous research with other viral pathogens has shown that virus-induced airway inflammation and hyperreactivity involve neurotrophic pathways that also affect tropism and severity of the infection. The goals of this study were to analyze systematically the expression of key neurotrophic factors and receptors during HRV-16 infection of human airway epithelial cells and to test the hypothesis that neurotrophins modulate HRV infection by controlling the expression of a major cellular receptor for this virus, the intercellular adhesion molecule 1 (ICAM-1). Neurotrophins and ICAM-1 expression were analyzed at the mRNA level by real-time PCR and at the protein level by flow cytometry and immunocytochemistry. A small inhibitory RNA (siRNA) or a specific blocking antibody was utilized to suppress nerve growth factor (NGF) expression and measure its effects on viral replication and virus-induced cell death. Nasal and bronchial epithelial cells were most susceptible to HRV-16 infection at 33°C and 37°C, respectively, and a significant positive relationship was noted between expression of NGF and tropomyosin-related kinase A (TrkA) and virus copy number. ICAM-1 expression was dose dependently upregulated by exogenous NGF and significantly downregulated by NGF inhibition with corresponding decrease in HRV-16 replication. NGF inhibition also increased apoptotic death of infected cells. Our results suggest that HRV upregulates the NGF-TrkA pathway in airway epithelial cells, which in turn amplifies viral replication by increasing HRV entry via ICAM-1 receptors and by limiting apoptosis.

Inhibition of Interleukin-17 Promotes Differentiation of CD25 Cells Into Stable T Regulatory Cells in Patients With Autoimmune Hepatitis

Patients with autoimmune hepatitis (AIH) have reduced numbers and function of CD4+CD25(high)FOXP3+ T regulatory cells (Tregs). Tregs can be generated from CD25⁻ (ngTreg) cells, which suppress the immune response less efficiently than Tregs. We investigated whether their differentiation into T-helper (Th)17 cells, an effector subset that has the same CD4+ progenitors as Tregs, accounts for the reduced suppressive functions of ngTregs. We investigated whether blocking interleukin (IL)-17 increased the immunosuppressive activity of Tregs. ngTregs were generated from 36 patients with AIH and 23 healthy subjects (controls). During Treg differentiation, expression of IL-17 was inhibited by physical removal of IL-17-secreting cells, exposure to recombinant transforming growth factor β or neutralizing antibodies against IL-6 and IL-1β (to promote differentiation of ngTregs vs Th17 cells), small inhibitory RNAs specific for the Th17 transcription factor RORC, or a combination of all these approaches. ngTregs from patients with AIH contained greater proportions of IL-17+ and RORC+ cells than Tregs from controls. All approaches to inhibit IL-17 increased expression of FOXP3 by ngTregs and their suppressive functions. Inhibition of IL-17 led to development of ngTregs that were phenotypically stable and did not acquire proinflammatory properties after exposure to IL-6 and IL-1β. Blocking Th17 allows ngTregs to differentiate into functionally stable immune inhibitory cells; this approach might be developed for therapy of patients with AIH.

Docosahexaenoic acid mediates peroxisomal elongation, a prerequisite for peroxisome division

Peroxisome division is regulated by several factors, termed fission factors, as well as the conditions of the cellular environment. Over the past decade, the idea of metabolic control of peroxisomal morphogenesis has been postulated, but remains largely undefined to date. In the current study, docosahexaenoic acid (DHA, C22:6n-3) was identified as an inducer of peroxisome division. In fibroblasts isolated from patients that carry defects in peroxisomal fatty acid β-oxidation, peroxisomes are much less abundant than normal cells. Treatment of these patient fibroblasts with DHA induced the proliferation of peroxisomes to the level seen in normal fibroblasts. DHA-induced peroxisomal proliferation was abrogated by treatment with a small inhibitory RNA (siRNA) targeting dynamin-like protein 1 and with dynasore, an inhibitor of dynamin-like protein 1, which suggested that DHA stimulates peroxisome division. DHA augmented the hyper-oligomerization of Pex11pβ and the formation of Pex11pβ-enriched regions on elongated peroxisomes. Time-lapse imaging analysis of peroxisomal morphogenesis revealed a sequence of steps involved in peroxisome division, including elongation in one direction followed by peroxisomal fission. DHA enhanced peroxisomal division in a microtubule-independent manner. These results suggest that DHA is a crucial signal for peroxisomal elongation, a prerequisite for subsequent fission and peroxisome division.

Blockade of ataxia telangiectasia mutated sensitizes hepatoma cell lines to sorafenib by interfering with Akt signaling

Sorafenib is a multi-kinase inhibitor applicable to hepatocellular carcinoma (HCC), but its limited therapeutic effects are a major problem to be solved. Here, we show that blockade of ataxia telangiectasia mutated (ATM) improves the antitumor effects of sorafenib. When hepatoma cell lines HepG2 and PLC/PRF/5 were treated with sorafenib plus ATM small inhibitory RNAs, ATM inhibitor KU55933 or caffeine, Akt signaling was suppressed and the cytotoxic effects were significantly potentiated. Moreover, ATM inhibition effectively suppressed the sorafenib-induced cell migration. Taken together, manipulation of ATM activity might be a useful strategy for improving sorafenib treatment of HCC.

Flotillin-2 is an acrosome-related protein involved in mouse spermiogenesis

Spermatogenesis is a complex process of terminal differentiation by which mature sperms are generated, and it can be divided into three phases: mitosis, meiosis and spermiogenesis. In a previous study, we established a series of proteomic profiles for spermatogenesis to understand the regulation of male fertility and infertility. Here, we further investigated the localization and the role of flotillin-2 in spermiogenesis. Flotillin-2 expression was inves-tigated in the testis of male CD1 mice at various developmental stages of spermatogenesis by using Western blot-ting, immunohistochemistry and immunofluorescence. Flotillin-2 was knocked down in vivo in three-week-old male mice using intratesticular injection of small inhibitory RNA (siRNA), and sperm abnormalities were assessed three weeks later. Flotillin-2 was expressed at high levels in male germ cells during spermatogenesis. Flotillin-2 immunoreactivity was observed in pachytene spermatocytes as a strong dot-shaped signal and in round spermatids as a sickle-shaped distribution ahead of the acrosome. Immunofluorescence confirmed flotillin-2 was localized in front of the acrosome in round spermatids, indicating that flotillin-2 was localized to the Golgi apparatus. Knock-down of flotillin-2 in vivo led to a significant increase in head sperm abnormalities isolated from the cauda epidi-dymis, compared with control siRNA-injected testes. This study indicates that flotillin-2 is a novel Golgi-related protein involved in sperm acrosome biogenesis

The matrix metalloproteinase-7 regulates the extracellular shedding of syndecan-2 from colon cancer cells

The cell surface heparan sulfate proteoglycan syndecan-2 regulates the activation of matrix metalloproteinase-7 (MMP-7) as a docking receptor. Here, we demonstrate the role of MMP-7 on syndecan-2 shedding in colon cancer cells. Western blot analysis showed that shed syndecan-2 was found in the culture media from various colon cancer cells. Overexpression of MMP-7 enhanced syndecan-2 shedding, whereas the opposite was true when MMP-7 levels were knocked-down using small inhibitory RNAs. Consistently, HT29 cells treated with MMP-7, but neither MMP-2 nor MMP-9, showed increased shed syndecan-2 in a time- and concentration-dependent manner. Furthermore, MALDI-TOF MS analysis and N-terminal amino acid sequencing revealed that MMP-7 cleaved both recombinant syndecan-2 and an endogenously glycosylated syndecan-2 ectodomain in the N-terminus at Leu149 residue in vitro. Taken together, the data suggest that MMP-7 directly mediates shedding of syndecan-2 from colon cancer cells.