siRNA Control Cells Research Articles

The oxido‐reductase enzyme glutathione peroxidase 4 (GPX4) governsSalmonella Typhimurium‐induced neutrophil transepithelial migration

SummaryNeutrophil (polymorphonuclear leucocytes; PMN) transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Using Salmonella enterica serovar Typhimurium (S. Typhimurium) as a prototypic proinflammatory insult, we have previously reported that the eicosanoid hepoxilin A3 (HXA3), an endogenous product of 12-lipoxygenase (12-LOX) activity, is secreted from the apical surface of the intestinal epithelium to establish a chemotactic gradient that guides PMN across the epithelial surface. Since little is known regarding the molecular mechanisms that regulate 12-LOX during S. Typhimurium infection, we investigated this pathway. We found that expression of phospholipid glutathione peroxidase (GPX4), which is known to have an inhibitory effect on 12-LOX activity, is significantly decreased at both the mRNA and protein level during infection with S. Typhimurium. Moreover, employing intestinal epithelial cell monolayers expressing siRNA against GPX4 mRNA, S. Typhimurium-induced PMN migration was significantly increased compared with the non-specific siRNA control cells. Conversely, in cells engineered to overexpress GPX4, S. Typhimurium-induced PMN migration was significantly decreased, which is consistent with the finding that partial depletion of GPX4 by RNAi resulted in a significant increase in HXA3 secretion during S. Typhimurium infection. Mechanistically, although we found Salmonella entry not to be required for the induced decrease in GPX4, the secreted effector, SipA, which is known to induce epithelial responses leading to stimulation of HXA3, governed the decrease in GPX4 in a process that does not lead to an overall increase in the levels of ROS. Taken together, these results suggest that S. Typhimurium induces apical secretion of HXA3 by decreasing the expression of phospholipid GPX, which in turn leads to an increase in 12-LOX activity, and hence HXA3 synthesis.

Serum caveolin-1, a biomarker of drug response and therapeutic target in prostate cancer models

We investigated the effect of dasatinib and sunitinib on tyrosine kinase (TK) signaling, caveolin-1 (Cav-1) expression and secretion and proliferation of PC-3 and DU145 prostate cancer cells in vitro and in vivo. Treatment of both cell lines with either dasatinib or sunitinib reduced phosphorylation of PDGFR, VEGFR2, Akt, FAK, Src (dasatinib only) and Cav-1, and reduced cellular and secreted levels of Cav-1. Both agents dose-dependently inhibited proliferation of these cells. In PC-3 and DU145 subcutaneous xenografts, treatment with dasatinib, sunitinib or anti-Cav-1 antibody (Ab) alone produced significant tumor regression compared with that by vehicle or IgG alone. Combined dasatinib and anti-Cav-1 Ab treatment or sunitinib and anti-Cav-1 Ab produced greater tumor regression than either treatment alone. Serum Cav-1 levels were lower in dasatinib- and sunitinib-treated mice than they were in vehicle-treated mice, and correlated positively with tumor growth in dasatinib- and sunitinib-treated groups (r = 0.48, p = 0.031; r = 0.554, p = 0.0065, respectively), compared with vehicle controls. Cav-1 knockdown, in combination with dasatinib or sunitinib treatment in PC-3 cells, caused a greater reduction in the phosphorylation of PDGFR-β and VEGFR2, and expression and secretion of PDGF-B and VEGF-A than that in PC-3 cells treated with dasatinib or sunitinib alone in control siRNA cells, suggesting that Cav-1 is involved in an autocrine pathway that is affected by these drugs. Overall, our results suggest a role for Cav-1 as a biomarker of response to both dasatinib and sunitinib treatment and as a therapeutic target in prostate cancer.

Partial depletion of gamma‐actin suppresses microtubule dynamics

Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in γ-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of γ-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether γ-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of γ-actin. Partial depletion of γ-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. γ-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of γ-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the γ-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the γ-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the γ-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of γ-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that γ-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between γ-actin and microtubules. © 2013 Wiley Periodicals, Inc

Role of Mitochondrial NCX on CXCL12-Induced Chemotaxis in A20 B Lymphocytes

Lymphocyte chemotaxis plays important roles in immunological reactions. It has been reported that mitochondria redistribute at the uropod during T lymphocyte migration triggered by a chemokine ligand (CXCL12). However, how mitochondria are involved in lymphocyte chemotaxis has been unclear. We studied roles of mitochondria Ca2+ handling proteins, Na-Ca exchange (NCXm) and Ca2+ uniporter, on CXCL12-induced chemotaxis in A20 B lymphocytes. CXCL12 (100 ng/ml) increased transwell migration of A20 cells from 4.6 ± 0.5 % (non-stimulated cells) to 12.6 ± 0.6 % (P < 0.05). This increase was dose-dependently inhibited by CGP-37157 (an inhibitor of NCXm), but not affected by Ru360 (an inhibitor of mitochondrial Ca2+ uniporter). Knock-down of a gene of NCXm (NCLX) by siRNA reduced NCLX protein expression to 52 ± 9 % and inhibited transwell migration similarly to CGP-37157. In the 8 hrs observation of cell migration under microscope, mean displacement of NCLX siRNA cells without CXCL12 was larger (21.4 ± 1.5 μm) than control siRNA cells (13.5 ± 2.2 μm, P < 0.05), and applying CXCL12 did not increase mean displacement and percentage of directional migration in NCLX siRNA cells. Intracellular Ca2+ measured by fura-2 was higher in NCLX siRNA cells (fura-2 ratio 0.49 ± 0.01) than the control siRNA cells (0.45 ± 0.01, P < 0.05) in the absence of CXCL12. After 2 hrs CXCL12 stimulation, the intracellular Ca2+ increased in control siRNA but not in NCLX siRNA cells. Treating A20 cells with BAPTA-AM (an intracellular Ca2+ chelator) suppressed the cell migration. Our results suggest that NCLX-mediated Ca2+ signaling is associated with CXCL12-induced chemotaxis in B lymphocytes.

Down-regulation of Polη expression leads to increased DNA damage, apoptosis and enhanced S phase arrest in L-02 cells exposed to hydroquinone

DNA polymerase eta (Polη), the product of the xeroderma pigmentosum variant gene, is required for translesion DNA synthesis, and plays a pivotal role in preventing genome instability after DNA damage induced by genotoxic agents. Studies have previously suggested a link between Polη and susceptibility to hydroquinone (HQ)-induced toxicity. To further address the role of Polη in the response of L-02 cells to HQ, we employed RNA interference to silence Polη expression in L-02 cells and examined the susceptibility of these Polη-deficient cells to the toxic effects of HQ. In this study, cell survival rate was determined using the MTT assay, DNA damage was determined by the Comet assay, apoptosis and cell cycle distribution were determined using flow cytometry, the mRNA expression levels of Polη were determined by real-time PCR, and the protein expression levels of Polη and γ-H2AX were determined by Western blot, γ-H2AX foci were visualized by confocal laser scanning fluorescence microscopy after cells were exposed to HQ at various concentrations for 24h in vitro. The results showed that stable Polη-knockdown cells were successfully constructed and more than 80% inhibition of Polη expression was confirmed. The results also showed that down-regulation of Polη led to a decrease in cell proliferation and an enhanced susceptibility to HQ-induced cytotoxicity. Polη-deficient cells were 2-fold more sensitive to HQ when compared with nonspecific siRNA control cells. Moreover, Polη-silenced L-02 cells treated with HQ displayed an increased level of DNA double-strand breaks as measured by olive tail moment, and an elevated DNA damage response as indicated by the induction of γ-H2AX. In addition, knockdown of Polη resulted in more enhanced apoptosis and more pronounced S phase arrest following HQ treatment. Together, these results show that Polη plays an important role in the response of L-02 cells to HQ-induced DNA damage.

P2-11-10: Low Toll-Like Receptor 9 Expression Is Required for the Aggressive Behavior of Triple Negative Breast Cancer Cells in Hypoxia.

Abstract Toll-like receptor-9 (TLR9) is a cellular DNA-receptor and a member of the innate immune system which is widely expressed in breast cancers. Although synthetic TLR9-ligands stimulate breast cancer cell invasion in vitro, through down-regulation of tissue inhibitor of matrix metalloproteinase-3 (TIMP-3), the role of this protein in breast or other cancer pathophysiology is unclear. We discovered that low tumor TLR9 expression is associated with a significantly shortened breast cancer specific survival among patients with triple negative tumors, but not among those with ER+ tumors. We further discovered that hypoxia induces TLR9 expression in breast cancer cells in vitro and in vivo. Although the triple negative TLR9 siRNA MDA-MB-231 breast cancer cells (with lower TLR9 expression) were less invasive than control siRNA cells in normoxia, these cells became highly aggressive in hypoxia and they formed significantly larger tumors than corresponding control cells in an orthotopic model in vivo. Further in vitro experiments revealed that the removal of TLR9 expression through siRNA significantly potentiated the hypoxia-induced invasive capacity of the triple negative MDA-MB-231 cells, but not that of the ER+ T47-D cells. Low TLR9 expression in hypoxia resulted also in complete disappearance of TIMP-3 in the MDA-MB-231 cells, but not in the ER+ T47-D cells. Taken together, these studies suggest that low TLR9 expression has different effects on the behavior of triple negative and ER+ breast cancer cells in hypoxia; triple negative cells become more invasive under these conditions, possibly through loss of endogenous invasion inhibitors, such as TIMP-3. Furthermore, these studies suggest that low TLR9 expression in triple negative breast cancers may serve as a novel prognostic factor and help characterize the group of patients that are at a high risk of relapse. Finally, our results suggest that any treatments or factors that lower TLR9 expression or induce hypoxia in residual or metastatic triple negative breast cancer cells, might actually induce their spread. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-11-10.

The Functional Link Between CDK1 and Retinoic Acid Receptor γ (RARγ) in Response to Treatment with All-Trans Retinoic Acid

Abstract 2485Alterations in cell cycle pathways and retinoic acid signaling are implicated in the pathogenesis of leukemia and treatment response. The retinoic acid receptors (RARα, RARβ and RARγ) mediate the differentiation, cell cycle arrest and cellular response to all-trans retinoic acid (ATRA). However, it is largely unknown whether the cyclin dependent kinases (CDK1 and CDK2) mediate the ATRA effects, also whether and how CDK1 and CDK2 are functional linked to RARs. Therefore in the present study, we examined the function of CDKs and their regulation in relationship to the RAR α, β and γ.The use of siRNA mediated knockdown of CDK1 in human leukemic U-937 cells affected cell cycle distribution, resulting in a decreased fraction of G0/G1 cells which coincided with the reduced expression of p27 protein. Treatment of U-937 si-CDK1 cells with ATRA demonstrated reduced terminal differentiation and decreased cell cycle arrest at G0/G1 phases in comparison to U-937 siRNA control cells. We observed a reduced responsiveness to ATRA treatment due to the loss of CDK1 function, and this was accompanied with the alterations in the expression of RARγ and the increased phosphorylation of Akt. However, loss of CDK2 function showed no effects on ATRA-mediated growth arrest and differentiation in U-937 cells.We also found that nuclear CDK1 was actively phosphorylated and was rapidly degraded upon the induction of ATRA, in contrast to its cytoplasmic counterpart in human leukemic U-937 cells. When examining the functional link between RARs and the regulation of CDKs in response to ATRA, we found that RARβ and RARγ, but not RARα, participated in ATRA-mediated protein degradation of CDK1 and CDK2. Moreover, we compared the effects of ATRA on ubiquitin-mediated protein degradation in the F9 teratocarcinoma cells and in F9 cells harboring targeted deletions of the RARα, RARβ and RARγ genes. ATRA induced ubiquitin conjugates showed striking changes in patterns in cells lacking RARγ. This indicates that the blockage in protein degradation of CDK1 may be due to the loss of RARγ function. Among the RAR isoforms, RARγ co-localized with CDK1 predominantly in the nuclear bodies. There was no detectable complex formation between RARβ and CDK1. This suggests that the phosphorylation and degradation of the nuclear CDK1 and its interaction with RARγ may be important events in mediating ATRA-induced effects on cell cycle and differentiation.Our study has revealed a novel mechanism by which CDK1 cooperates with RARγ to mediate the effects of all-trans retinoic acid. Disclosures:No relevant conflicts of interest to declare.

Specific β-Tubulin Isotypes Can Functionally Enhance or Diminish Epothilone B Sensitivity in Non-Small Cell Lung Cancer Cells

Epothilones are a new class of microtubule stabilizing agents with promising preclinical and clinical activity. Their cellular target is β-tubulin and factors influencing intrinsic sensitivity to epothilones are not well understood. In this study, the functional significance of specific β-tubulin isotypes in intrinsic sensitivity to epothilone B was investigated using siRNA gene knockdown against βII-, βIII- or βIVb-tubulins in two independent non-small cell lung cancer (NSCLC) cell lines, NCI-H460 and Calu-6. Drug-treated clonogenic assays showed that sensitivity to epothilone B was not altered following knockdown of βII-tubulin in both NSCLC cell lines. In contrast, knockdown of βIII-tubulin significantly increased sensitivity to epothilone B. Interestingly, βIVb-tubulin knockdowns were significantly less sensitive to epothilone B, compared to mock- and control siRNA cells. Cell cycle analysis of βIII-tubulin knockdown cells showed a higher percentage of cell death with epothilone B concentrations as low as 0.5 nM. In contrast, βIVb-tubulin knockdown cells displayed a decrease in epothilone B-induced G2-M cell cycle accumulation compared to control siRNA cells. Importantly, βIII-tubulin knockdowns displayed a significant dose-dependent increase in the percentage of apoptotic cells upon treatment with epothilone B, as detected using caspase 3/7 activity and Annexin-V staining. Higher concentrations of epothilone B were required to induce apoptosis in the βIVb-tubulin knockdowns compared to control siRNA, highlighting a potential mechanism underlying decreased sensitivity to this agent. This study demonstrates that specific β-tubulin isotypes can influence sensitivity to epothilone B and may influence differential sensitivity to this promising new agent.

Abstract 203: PKR is a doxorubicin sensitivity biomarker for breast cancer

Abstract Breast cancer is the second most frequently diagnosed cancer in U. S. women with &amp;gt;200,000 new cases estimated for 2010. Resistance to standard chemotherapy remains a significant clinical challenge. Thus, the identification of novel molecular biomarkers that predict response to treatment may improve therapeutic decision making and outcomes. Recently it has been reported that the level and activity of the dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and more aggressive primary tumor specimens. Previously, our laboratory has determined that PKR is a key regulator of cell proliferation, apoptosis and transformation. PKR is activated by diverse cellular stresses including growth factor withdrawal from hematopoietic cells, inflammatory cytokine treatment or viral infection to initiate apoptosis presumably as a result eIF2α phosphorylation and inhibition of protein synthesis. However, activated PKR may also promote apoptosis by inhibiting Bcl2 phosphorylation and regulate cell proliferation by mechanisms dependent on p53, STAT1 or NF-κB. In order to gain insight into how PKR signaling may affect breast cancer development and sensitivity to chemotherapy, we measured the level of PKR in primary breast cancer (and paired, normal adjacent tissue) by immunohistochemical staining of a breast tumor tissue microarray containing 50 cases. Increased PKR staining was observed in all tumor types tested (infiltrating ductal carcinoma, mucinous adenocarcinoma, medullary carcinoma, Paget's disease, carcinosarcoma, cystosarcoma phyllodes and benign fibroadenoma) compared to normal adjacent tissue. Furthermore, elevated PKR expression directly correlates with increased tumor grade indicating a more aggressive phenotype. To determine whether PKR may be required for tumor response to doxorubicin, we employed a siRNA strategy to knock-down PKR by &amp;gt;75% in several breast cell lines. Interestingly, basal PKR activity is dramatically higher in those cell lines derived from human mammary carcinoma (i.e. MCF7 and T-47D) compared to a “nontransformed” mammary epithelial cell line (MCF10A). Furthermore, timecourse and dose response studies with doxorubicin, a frequently used and effective chemotherapy, revealed that cells with reduced PKR are significantly less sensitive to the drug compared to control siRNA cells with higher PKR. Interestingly, phosphorylation of eIF2α is unaffected by reduced PKR expression in MCF7 cells even with the highest dose of doxorubicin. This indicates that the mechanism by which PKR promotes doxorubicin sensitivity may be independent of eIF2α phosphorylation and protein synthesis inhibition. Together, these results suggest that increased PKR expression may be a useful biomarker for more aggressive breast tumors and for predicting response to doxorubicin treatment. Based on this model, aggressive tumors that do not display elevated PKR are predicted to be resistant to doxorubicin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 203. doi:10.1158/1538-7445.AM2011-203

Regulation of NF-κB Circuitry by a Component of the Nucleosome Remodeling and Deacetylase Complex Controls Inflammatory Response Homeostasis

The MTA1 coregulator (metastatic tumor antigen 1), a component of the nucleosome remodeling and deacetylase (NuRD) complex, has been intimately linked with human cancer, but its role in inflammatory responses remains unknown. Here, we discovered that MTA1 is a target of inflammation, and stimulation of macrophages with Escherichia coli lipopolysaccharide (LPS) stimulates MTA1 transcription via the NF-kappaB pathway. Unexpectedly, we found that MTA1 depletion in LPS-stimulated macrophages impairs NF-kappaB signaling and expression of inflammatory molecules. MTA1 itself acts as a transcriptional coactivator of inflammatory cytokines in LPS-stimulated macrophages, and in contrast, it acts as a corepressor in resting primary macrophages as its depletion induced cytokine expression. LPS stimulates S-nitrosylation of histone deacetylase 2 (HDAC2) and interferes with its binding to MTA1, which, in turn, resulted in the loss of corepressor behavior of MTA1.HDAC complex in activated macrophages. Consequently, the net levels of inflammatory cytokines in LPS-stimulated macrophages from MTA1(-/-) mice were high compared with wild-type mice. Accordingly, MTA1(-/-) mice were much more susceptible than control mice to septic shock induced by LPS, revealing that MTA1 protects mice from deregulated host inflammatory response. These findings reveal a previously unrecognized, critical homeostatic role of MTA1, both as a target and as a component of the NF-kappaB circuitry, in the regulation of inflammatory responses.

Requirements for Protein Phosphorylation and the Kinase Activity of Polo-like Kinase 1 (Plk1) for the Kinetochore Function of Mitotic Arrest Deficiency Protein 1 (Mad1)

Mitotic arrest deficiency protein 1 (Mad1) is associated with microtubule-unattached kinetochores in mitotic cells and is a component of the spindle assembly checkpoint (SAC). Here, we have studied the phosphorylation of Mad1 and mapped using liquid chromatography-tandem mass spectrometry several phosphorylated amino acids in this protein. One phosphorylated residue, Thr680, was characterized to be important for the kinetochore localization of Mad1 and its SAC function. We also found that in mitotic cells Mad1 co-immunoprecipitated with Plk1. Depletion of cellular Plk1 using small interfering RNAs and inhibition of the kinase activity of Plk1 using a kinase-dead mutant or a small molecule inhibitor attenuated Mad1 phosphorylation and its association with kinetochores. Collectively, these findings indicate mechanistic roles contributed by protein phosphorylation and Plk1 to the SAC activity of Mad1.

Inhibition of epidermal growth factor receptor signaling pathway by delphinidin, an anthocyanidin in pigmented fruits and vegetables

Inhibitors of the epidermal growth factor receptor (EGFR) have generated considerable hope for cancer treatment, specifically for lung and breast cancers. Therefore, identification of a natural, nontoxic agent(s) as an inhibitor of EGFR is of considerable importance. Delphinidin, an anthocyanidin present in pigmented fruits and vegetables, possesses potent antioxidant and antiproliferative properties. In our study, employing EGFR positive breast cancer AU-565 cells and immortalized MCF-10A cells, we evaluated the effect of delphinidin on EGFR and its downstream signaling pathways. Delphinidin (5-40 microM; 3 hr) treatment of both AU-565 cells and MCF-10A cells inhibited the (i) phosphorylation of EGFR, (ii) activation of PI3K, (iii) phosphorylation of AKT and MAPK. Further, delphinidin treatment of AU-565 cells inhibited EGF-induced autophosphorylation of EGFR, AKT and MAPK, activation of PI3K and cell invasion. We then compared the growth inhibitory effects of delphinidin (5-40 microM; 48 hr), and found that it resulted in a decrease in cell growth of AU-565 and MCF-10A cells but had only minimal effects on normal mammary epithelial 184A1 cells. Treatment of AU-565 cells with delphinidin resulted in (i) induction of apoptosis, (ii) cleavage of PARP protein, (iii) activation of caspase-3 and (iv) downregulation of Bcl-2 with an increase in the expression of Bax. In summary, our study identifies a naturally occurring dietary agent delphinidin as an effective inhibitor of EGFR signaling in breast cancer cells. We suggest that delphinidin could be developed as an agent for the management of EGFR positive human cancers.

Inhibition of CD44 Expression by Small Interfering RNA to Suppress the Growth and Metastasis of Ovarian Cancer Cells in Vitro and in Vivo

Since ovarian cancer cells express CD44, which causes very strong cell adhesion to peritoneal mesothelium and an unfavourable prognosis, we designed small interfering RNA (siRNA) targeting the CD44 gene to analyse the functional consequences of this inhibition in human ovarian cancer. We transfected ovarian cancer cell line SKOV-3 with well-designed CD44 siRNA or control siRNA. Western blot analysis was used to assess the CD44 expression. Following stable transfection, significant inhibition of CD44 expression with 66.13 ± 4.21 % (P &lt; 0.05) in CD44 siRNA1 cells and 62.01 ± 3.97 % (P &lt; 0.05) in CD44 siRNA2 cells was detected. We performed in vitro experiments including cellular adhesion to hyaluronan and human peritoneal mesothelial cells, etoposide-induced apoptosis, and Boyden chamber invasion assays. The adhesion percentages of CD44 siRNA1 and CD44 siRNA2 cells were significantly lower than those of the control siRNA cells in adhesion both to hyaluronan and to human peritoneal mesothelium. The CD44 siRNA transfectants showed significant inhibition of in vitro invasion and loss of resistance to apoptosis than the control siRNA cells. In vivo study with BALB/c mice was applied to compare the tumour growth and peritoneal dissemination. Nude mice treated with CD44 siRNA cells revealed significantly lower tumour volume and less peritoneal dissemination compared to mice treated with the control siRNA cells. In conclusion, down-regulation of CD44 expression by siRNA inhibits the in vitro adhesion, invasion and resistance to apoptosis of ovarian cancer cells, suppresses tumour growth and peritoneal dissemination of human ovarian cancer xenograft in nude mice.

Down-regulation of CXCR4 expression by siRNA inhibits invasive ability of breast cancer cells

OBJECTIVE To investigate the efficiency of gene silencing by CXCR4-siRNAs (small interfering RNA), and to examine the invasive ability and the expression of other metastatic associated genes in siRNA-treated breast cancer cells. METHODS Three siRNAs were designed and cloned into the pSilenc™ 3.1-H1 neo vector. The reconstructed plasmids were purified and trans-fected into the T47D breast cancer cell line, which highly expressed CXCR4. The amount of CXCR4 expression in the transfected cells was measured by flow cytometry and Real-time PCR. Cell invasive ability was evaluated us -ing 24-well Matrigel invasion chambers. In addition, the expression of other metastatic-associated genes, such as E-cad, IGFBP-5, FN and MMP-2, was assessed by Real-time PCR. RESULTS The suppression rates of CXCR4 mRNA expression reached 95.7%, 85.9% and 98.3%compared with control-siRNA cells in the 3 CXCR4-siRNA T47D cells respectively. FCM assays for CXCR4 protein expression showed a similar inhibitory effect. The invasion indexes of these CXCR4-siRNA cells were 0.037, 0.290 and 0.188respectively compared with control-siRNA cells. After treatment of the cells with CXCR4-siRNA, the expression of E-cad showed an upward tendency and that of IGFBP-5 had a downward trend, while alteration in expression of FN and MMP2 varied without a con -sistant effect. CONCLUSION CXCR4 plays an important role in modulating migra-tion of human breast cancer cells. Small interfering RNA can significantly silence the CXCR4 gene in the human T47D breast cancer cell line. The results of this study strengthen the need for further research on novel gene therapy against breast cancer metastasis.

The Anti-Inflammatory Effect of Ciglitazone is Partially Mediated by Glucocorticoid Induced Leucine Zipper in Human Airway Epithelial Cells

RATIONALE: PPARγ activation has been shown to inhibit airway inflammation but the mechanisms are not fully understood; we therefore assessed whether the anti-inflammatory protein Glucocorticoid-Induced Leucine Zipper (GILZ), which we have shown is upregulated by the PPARγ agonist ciglitazone, is involved in this process. METHODS: The expression of GILZ was assessed by immunoblotting in A549 and normal human bronchial epithelial cells (NHBE) at baseline and following ciglitazone treatment. RNA silencing was used to knock down GILZ protein levels in A549 cells in order to determine whether GILZ mediates the anti-inflammatory effects of ciglitazone. GILZ siRNA- and Control siRNA-treated A549 cells were stimulated for 20 hours with ciglitazone followed by IL-1beta for 4 hours. Total RNA was isolated and IL-8 mRNA levels were assessed by quantitative real-time PCR. RESULTS: GILZ levels were upregulated by ciglitazone in A549 and NHBE cells. Ciglitazone potently inhibited the induction of IL-8 mRNA by IL-1beta in control siRNA cells, whereas in GILZ siRNA cells this effect was partially abrogated. CONCLUSION: Here we demonstrate the induction of GILZ, an anti-inflammatory protein known to inhibit NF-κB and AP-1, by ciglitazone in human airway epithelial cells. Further we demonstrate that GILZ in part mediates the anti-inflammatory effects of agonist-bound PPARγ. These findings help elucidate a mechanism via which PPARγ agonists reduce airway inflammation, and suggest the potential of the thiazolidinediones in the treatment of asthma.

Overexpression of Apoptosis-Inducing Factor (AIF) and Pro-Survival Function in AML.

Apoptosis inducing factor (AIF) is a bifunctional flavoprotein with NADH oxidase activity with a vital function in bioenergetics and redox metabolism while also inducing apoptosis in cancer cells. The gene is localized on chromosome human X (q25–26). AIF is synthesized in the cytosol as a 67KDa precursor molecule and then imported into the intermembrane space of mitochondria. In response to pro-apoptotic stimuli, the change of mitochondrial membrane potential (MMP) results in release of mature AIF (57KDa) to the cytoplasm and translocation into the nuclei where AIF can induce caspase-independent large scale DNA fragmentation (~ 50 Kbp) and chromatin condensation. The mechanism of induction of cell death by AIF remains poorly understood. We have previously reported that AIF induced cell death is both caspase-dependent and independent and related to mitochondrial membrane depolarization in leukemic cells treated with chemotherapeutic agents. In this study, we investigatethe expression level and function of AIF and its relationship to Bcl-2 and Bcl-2 family members in leukemia cells; andthe expression levels of AIF in AML patient's samples.To study the function of AIF, we introduced AIF si-RNA into K562 CML cell line using lentiviral vectors and established permanent AIF knockdown cell lines. Western blot analysis showed that the expression levels of XIAP, MCL-1, Bcl-xl and suvivin were increased while the AIF expression level was reduced. Cell growth was diminished two-fold (siAIF-clone 2, cell doubling time ~52-hr) to four-fold (siAIF-clone 1, cell doubling time ~99-hr) in AIF knockdown cells compared to the parental cells (24-hr). This suggests that AIF is supporting cell growth.Next, AIF knockdown and scrambled siRNA control cells were treated with Ara-C (10μM) or STI571 (Gleeevec) (2 μM). The frequency of apoptotic cells was determined by flow cytometry (table).24hr48hrControlAra-CSTI571ControlAra-CSTI-571Control17.0430.5630.2320.2148.4876.12siAIF-13.093.3617.037.510.1244.92siAIF-26.776.647.7311.9510.7341.85AIF-siRNA in K562 cells showed increased sensitivity to Ara-C and STI-571 (Gleevec). The results support again a pro-survival function of AIF in K562 cells.To examine the expression levels of AIF in primary AML patients, 148 samples (newly diagnosed n=32, relapsed n=27, refractory n=18, CR n=5, and normal-donors n=31) were analyzed by western blot and 40 samples of newly diagnosed AML were analyzed by RT-PCR. Results show that in comparison with normal CD34+ cells, AIF mRNA levels in newly diagnosed AML patients were significantly higher (95.0 copies AIF per hundred copies ABL1 in AML, 57.4 copies AIF per hundred copies in NBM CD34+, p = 0.02). The AIF/actin protein ratios in AML (0.43±0.05) was increased compared with normal BM-CD34+ (0.12±0.02) (p=0.01). Comparison of AIF expression levels in normal CD34+ cells vs. AML with good (0.5±0.05), bad (0.34±0.05), and intermediate (0.39±0.06) cytogenetics respectively, were significantly different (p<0.002). The transcription levels of AIF also showed significant differences (p<0.04) in patients with different cytogenetics abnormalities.Taken together, we conclude thatAIF transcript and protein levels are significantly higher in AML compared to normal CD34 cells.AIF in K562 cells functions as pro-survival factor for drug sensitivity.These results question the previously assumed predominant pro-apoptotic function of AIF.

Characterization of siRNA-Mediated SBDS-Knockdown Cells: Specific Hypersensitivity to Fas Stimulation.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by bone marrow failure, pancreatic insufficiency, and a marked propensity for myelodysplastic syndrome and leukemia. Approximately 90% of the patients have mutations in the SBDS gene. Recent studies suggested a role of the yeast SBDS homologue, YLR022C, in RNA processing and ribosomal biogenesis. However, the function of the human SBDS has not been clarified yet. We previously showed that marrow cells from SDS patients are characterized by accelerated apoptosis, overexpression of Fas and hypersensitivity to Fas stimulation. To study the function of SBDS and determine whether the above abnormalities are caused by deficiency of SBDS, we established stably transfected Hela cell lines expressing two different siRNAs against SBDS and lines expressing scrambled siRNA control. SBDS-knockdown was confirmed by Western blotting using polyclonal chicken anti-human SBDS antibody. The SBDS expression in the scrambled siRNA control cells was comparable to that of wild-type Hela cells. DNA content analysis by propidium iodide staining showed a prominent increase in sub-G1 population in asynchronous, non-treated SBDS-knockdown cells, suggesting that these cells are prone to cell death, however, no cell cycle arrest was noted. To further characterize the SBDS-knockdown cells, we examined their sensitivity to four groups of cell death inducers: DNA damaging agents (etoposide, cisplatin, and doxorubicin), transcriptional inhibitors (actinomycin D and α-amanitin), translation blocker (cycloheximide), and Fas pathway activator (agonistic anti-Fas antibody CH-11). Dose-response curves were obtained by MTT assay performed 48 hrs after treatment of the cells with the reagents. Interestingly, SBDS-deleted cells showed marked hypersensitivity to CH-11; while 3 μ g/ml of CH-11 reduced the survival fraction to 50% in wild-type and control cells, a similar effect was obtained at 0.02 μ g/ml in the SBDS-deleted cells. The hypersensitivity to Fas stimulation was also demonstrated by DNA content analysis. Based on the possible role of the yeast SBDS orthologue in RNA metabolism, we anticipated that the SBDS-deficient cells would be hypersensitive to the transcription inhibitors. However, even at concentrations which completely abolished RNA polymerase I or RNA polymerase II activity as determined by BrUTP labeling, the sensitivity of the SBDS-knockdown cells to the transciptional inhibitors was not remarkably different from that of the control or wild type cells. Similarly, the sensitivity to the genotoxic agents and protein synthesis blocker was not obviously different between the SBDS-deficient and proficient cells. To study the mechanism for Fas hypersensitivity, we analyzed Fas expression by flow cytometry using Cy5-conjugated anti-CD95 antibody and found overexpression of Fas in the SBDS-deleted cells in comparison with the Fas expression in the wild-type and control cells. Although further investigation is needed, these results suggest that the SBDS protein might be involved in cell death pathway, especially in the regulation of Fas-mediated apoptosis. The siRNA-mediated SBDS knock-down Hela cells duplicate important features of SDS cells, and may serve as a useful model to investigate the function of the human SBDS protein.

The chemokine stromal cell derived factor-1 (CXCL12) promotes glioma invasiveness through MT2-matrix metalloproteinase

Chemokines have been found to alter tumor growth and metastasis. We have described previously that a particular chemokine receptor, CXCR4, was predominantly expressed on various glioma cell lines and in resected glioblastoma specimens. Herein, we have tested the ligand of CXCR4, stromal cell derived factor-1alpha (SDF-1alpha, CXCL12), on the response of human glioma cells. We found that SDF-1alpha increased the expression of membrane type-2 matrix metalloproteinase (MT2-MMP), but not the other MT-MMPs, MMP-2 or MMP-9. The SDF-1alpha enhanced MT2-MMP expression was blocked by a CXCR4 antagonist, AMD3100. Functional invasion assays showed that SDF-1alpha stimulated glioma cells to invade through matrigel-coated chambers and this effect was inhibited in glioma cells by the stable downregulation of MT2-MMP expression using small interfering RNA (siRNA). In vivo and at asymptomatic stages following intracerebral implant of cells, mice harboring MT2-MMP siRNA downregulated clones had smaller and less invasive tumors compared with mice implanted with non-specific siRNA control cells. Analyses at symptomatic stages demonstrate that mice with MT2-MMP siRNA clones survive longer than mice harboring control cells. These results highlight MT2-MMP as an effector of CXCR4 signaling in glioma cells, and they reveal the novel role of MT2-MMP in modulating tumor activity.