Silencing Signal Transducer And Activator Of Transcription 3 Research Articles

Errate: Silencing Signal Transducer and Activator of Transcription 3 (STAT3) and Use of Anti-Programmed Cell Death-Ligand 1 (PD-L1) Antibody Induces Immune Response and Anti-Tumor Activity.

It was brought to our attention by the authors that Figures 5A and 6A contained errors. The correct version of Figures 5A and 6A are provided below. The corrected figures do not change the overall findings of the study. Reference: Jiaxing Wang, Fakun Huang, Caiyun Jiang, Pan Chi. Silencing Signal Transducer and Activator of Transcription 3 (STAT3) and Use of Anti-Programmed Cell Death-Ligand 1 (PD-L1) Antibody Induces Immune Response and Anti-Tumor Activity. Med Sci Monit, 2020; 26: e915854. DOI: 10.12659/MSM.915854.

YAP regulates intestinal epithelial cell proliferation through activation of STAT3 in DSS-induced colitis and associated cancer.

Ulcerative colitis (UC) is a chronic, relapsing inflammation of the colon. Impaired epithelial repair is an important biological features of UC. Accelerating intestinal epithelial repair to achieve endoscopic mucosal healing has become a key goal in UC. Yes-associated protein (YAP) is a key transcriptional coactivator that regulates organ size, tissue growth and tumorigenesis. Growing studies have focused on the role of YAP in intestinal epithelial regeneration. This study explore the molecular mechanism for the role YAP in modulating colonic epithelial proliferation, repair, and the development of colitis associated cancer. We constructed the acute colitis mouse model through successive 5 days of 3% dextran sulfate sodium salt (DSS) induction. Then YAP-overexpressed mouse model was constructed by intraperitoneal injection the YAP overexpressed and negative control lentivirus into DSS mice. On the 5th day of DSS induction and the 5th day of normal drinking water after removing DSS (5+5 d), the mice were killed by spinal dislocation. The colon was taken to measure the length, and the bowel 1-2 cm near the anal canal was selected for immunohistochemical and Western blotting. We used YAP over-expressed colonic epithelial cells and small interfering signal transducer and activator of transcription 3 (STAT3) RNA to probe the regulation of YAP on STAT3, using cell counting kit-8 and scratch assays to explore the role of YAP on colonic epithelial cell proliferation. Finally, we conducted co-immunoprecipitation to test the relationship between YAP and STAT3. After DSS treatment, the expression of YAP was dramatically diminished in crypts. Compared with the empty control mice, overexpression of YAP drastically accelerated epithelial regeneration after DSS induced colitis, presenting with more intact of structural integrity in intestinal epithelium and a reduction in the number of inflammatory cells in the mucosa. Further Western blotting, functional experiment and co-immunoprecipitation analyses showed that the expression of YAP in nucleus was significantly increased by 2 h post DSS cessation, accompanied with up-regulated total protein levels of STAT3 and phosphorylated-STAT3 (p-STAT3). Overexpression of YAP enhanced the expression of STAT3, p-STAT3, and their transcriptional targets including c-Myc and Cyclin D1. In addition, it promoted the proliferation and the "wound healing" of colonic cells. However, these effects were reversed when silencing STAT3 on YAP-overexpressed FHC cells. Moreover, protein immunoprecipitation indicated that YAP could directly interact with STAT3 in the nucleus, up-regulatvng the expressvon of STAT3. Finally, during the process of CAC, overexpression of YAP mutant caused the down-regulated expression of STAT3 and inhibited the development and progress of CAC. YAP activates STAT3 signaling in regulation of epithelial cell proliferation and promotes mucosal regeneration after DSS induced colitis, which may serve as a potential therapeutic target in UC. However, persistent and excessive YAP activation may promote CAC development.

Melatonin attenuates low shear stress-induced pyroptosis and endothelial cell dysfunction via the RORα/miR-223/STAT-3 signalling pathway.

Endothelial cells sense changes in blood flow shear stress and affect the progression of atherosclerotic plaques. Pyroptosis is an inflammatory form of cell death and has been implicated in cardiovascular diseases. Melatonin and its nuclear receptor retinoid-related orphan receptor α (RORα) have protective effects on the development of atherosclerosis. To date, whether melatonin can prevent endothelial cell pyroptosis and dysfunction in pathological shear stress remains unclear. In the present study, human umbilical vein endothelial cells (ECs) were cultured under low shear stress conditions (5 dyne/cm2) for 24 h and treated with or without melatonin (2 µmol/l). The binding sites of the microRNA (miR)-223 promoter and RORα were predicted using the JASPAR website. Expression of pyroptosis-related proteins, including cleaved N-terminal gasdermin D, caspase-1, intercellular adhesion molecule 1 (ICAM-1) and nitric oxide (NO) were assessed. The results indicated that low shear stress increased pyroptosis and ICAM-1 expression, whereas it decreased NO levels. Melatonin alleviated pyroptosis and ICAM-1 expression and increased the production of NO in ECs. Further assessment revealed that low-level shear stress decreased RORα protein and mRNA expression, whereas melatonin would bind to RORα and thereby promoted miR-223 transcription in ECs. The present study also identified signal transducer and activator of transcription 3 (STAT-3) as a potential target gene of miR-223-3p. When transfected with miR-223 inhibitor, ECs up-regulated the expression of pyroptosis-related proteins and ICAM-1, and down-regulated NO levels. By contrast, silencing STAT-3 expression diminished the protective effect of miR-223. These results indicated that melatonin prevented ECs from undergoing pyroptosis and alleviated dysfunction via the RORα/miR-223/STAT-3 signalling pathway. This information could aid in the development of novel therapeutic approaches and provide new insights into atherosclerosis.

Stabilization of C/EBPβ through direct interaction with STAT3 in H-Ras transformed human mammary epithelial cells

Signal transducer and activator of transcription 3 (STAT3) plays important roles in cancer-associated inflammation by controlling expression of proinflammatory cytokines and chemokines. Recent studies suggest that C/EBPβ (CCAAT-enhancer binding protein beta) and STAT3 synergistically stimulate cancer cell proliferation and epithelial-mesenchymal transition. C/EBPβ is a leucine-zipper transcription factor that regulates expression of a variety of inflammatory cytokines or chemokines, such as IL-8, G-CSF (granulocyte colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor) which induce neutrophil infiltration and differentiation. However, molecular mechanisms by which STAT3 and C/EBPβ cooperatively interact had not been fully elucidated. In this study, we found that the level of C/EBPβ protein, but not that of its mRNA transcript, was decreased in the absence of STAT3 in H-Ras transformed human mammary epithelial (H-Ras MCF10A) cells. In addition, silencing STAT3 dramatically induced ubiquitination of C/EBPβ for proteasomal degradation. Furthermore, direct interaction between STAT3 and C/EBPβ was confirmed by immunoprecipitation and proximity ligation assays. Taken together, these results suggest that STAT3 stabilizes C/EBPβ, thereby promoting cancer-associated inflammation.

Silencing Signal Transducer and Activator of Transcription 3 (STAT3) and Use of Anti-Programmed Cell Death-Ligand 1 (PD-L1) Antibody Induces Immune Response and Anti-Tumor Activity.

BackgroundThe treatment of cancer is still unable to meet the needs of patients and remains a huge challenge. This study investigated the immune response and anti-cancer effect of silencing STAT3 combined with the use of anti-PD-L1 antibody.Material/MethodsTransfected CT26.WT cells were used to subcutaneously inoculate C57B/L6 mice, which were subsequently injected with anti-PD-L1 antibody. Treated mice were examined for tumor formation and inflammation using HE staining. Tumors were investigated for apoptosis using the TUNEL assay. The expression of STAT3, PD-L1, and C-met was studied immunohistochemistrially and by using PCR and Western blot analysis.ResultsFour weeks after inoculation, tumors were observed in the inoculated mice. HE staining showed obvious inflammation in mice injected with cells that were silenced for STAT3 and injected with PD-L1 antibody. TUNEL assay showed low level of apoptosis in mice injected with cells silenced for STAT3 or injected with PD-L1 antibody, and higher level of apoptosis following combined treatment of STAT3 silencing and PD-L1 antibody injection. Immunohistochemistry, PCR, and Western blot analyses revealed that the expression of C-met, PD-L1, and STAT3 was significantly reduced in tumors following the combined treatment. Compared with treatment of STAT3 silencing or PD-L1 antibody injection, the combined treatment enhanced apoptosis.ConclusionsSilencing STAT3 and PD-L1 antibody injection in combination increased apoptosis in tumor cells and thus offers better anti-cancer activity.

Downregulation of signal transduction and STAT3 expression exacerbates oxidative stress mediated by NLRP3 inflammasome.

Activated nucleotide binding to the oligonucleotide receptor protein 3 (NLRP3) inflammasome is possibly involved in the pathogenesis of Alzheimer's disease through oxidative stress and neurogenic inflammation. Low expression of the signal transducer and activator of transcription 3 (STAT3) gene may promote the occurrence of neurodegenerative diseases to some extent. To clarify the roles of the NLRP3 inflammasome and STAT3 expression in oxidative stress, (1) SHSY5Y cells were incubated with 1 mM H2O2 to induce oxidative stress injury, and the expression of human-cell-specific signal transduction, STAT3-shRNA silencing signal transduction and STAT3 were detected. Cells were pretreated with Ca2+ chelator BAPATA-AM (0.1 mM) for 30 minutes as a control. (2) Western blot assay was used to analyze the expression of caspase-1, NLRP3, signal transduction and STAT3. Enzyme-linked immunosorbent assay was used to analyze interleukin-1β levels. Flow cytometry was carried out to calculate the number of apoptotic cells. We found that H2O2 treatment activated NLRP3 inflammasomes and decreased phosphorylation of signal transduction and STAT3 serine 727. BAPTA-AM pretreatment abolished the H2O2-induced activation of NLRP3 inflammasomes, caspase-1 expression, interleukin-1β expression and apoptosis in SHSY5Y cells, and had no effect in cells with downregulated STAT3 expression by RNAi. The findings suggest that downregulation of signal transduction and STAT3 expression may enhance the oxidative stress mediated by NLRP3, which may not depend on the Ca2+ signaling pathway.

CircRNA_100782 regulates pancreatic carcinoma proliferation through the IL6-STAT3 pathway.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs that play an important role in cancer. However, the mechanisms by which circRNAs regulate gene expression in pancreatic ductal adenocarcinoma (PDAC) remain unclear. This study seeks to elucidate the role that circRNAs play in the proliferation of PDAC cells. On the basis of previous studies of circRNA expression profiles in PDAC, we found that the circRNA_100782 was markedly upregulated in PDAC tissue. Functional experiments revealed that circRNA_100782 down-regulation inhibited BxPC3 cell proliferation and colony formation. Loss-of-function studies showed that knockdown of circRNA_100782 inhibited cell proliferation by downregulating the microRNA-124 (miR-124) target genes interleukin-6 receptor (IL6R) and signal transducer and activator of transcription 3 (STAT3). Overexpression of miR-124 also inhibited BxPC3 cell proliferation by reducing the expression of IL6R and STAT3, which was consistent with the result of silencing circRNA_100782. In addition, luciferase assay revealed that miR-124 was a direct target of circRNA_100782. Silencing STAT3 inhibited BxPC3 cell proliferation and colony formation. Cell viability was reduced in BxPC3 cells treated with si-circRNA_100782 and miR-124 mimic, and this effect could be attenuated by activating STAT3. In vivo study validated that circRNA_100782 knockdown suppressed BxPC3 xenografts in nude mice. Taken together, these results suggest that circRNA_100782 regulates BxPC3 cell proliferation by acting as miR-124 sponge through the IL6–STAT3 pathway.

Toluene downregulates filaggrin expression via the extracellular signal-regulated kinase and signal transducer and activator of transcription–dependent pathways

Toluene downregulates filaggrin expression via the extracellular signal-regulated kinase and signal transducer and activator of transcription–dependent pathways

Abstract P5-07-01: Granulin, a novel STAT3-interacting protein, promotes breast cancer tumorigenicity

Abstract Since the neoplastic phenotype of a cell is largely driven by its gene expression patterns, increasing attention is focused on transcription factors that regulate critical mediators of tumor formation and metastatic progression like the oncogenic transcription factor, signal transducer and activator of transcription 3 (STAT3). Whereas normal cells have transient activation of STAT3 due to tight control by negative regulators, cancer cells frequently have inappropriate constitutive activation of STAT3 which drives increased expression of genes involved in tumorigenesis. However, little is known about proteins that interact with STAT3 to modulate its function. To identify novel STAT3-interacting proteins, we performed liquid chromatography tandem mass spectrometry-based profiling of STAT3-containing complexes immunoprecipitated from the triple-negative breast cancer cell lines MDA-MB-468 and SUM159PT, which have constitutively active STAT3. We identified granulin (GRN) as a novel STAT3-interacting protein and validated the STAT3-GRN interaction in breast cancer cells by co-immunoprecipitation. To investigate the functional effect of GRN on STAT3 activity, we silenced GRN using small interfering RNA. We found that GRN was necessary for constitutive and maximal cytokine-induced STAT3 transcriptional activity in breast cancer cells. GRN modulated cytokine-induced STAT3 function by enhancing STAT3 DNA binding and increasing the time-integrated amount of STAT3 activation and nuclear translocation. Silencing GRN mirrored the effect of silencing STAT3 on reducing the viability, clonogenesis, and migratory capacity of triple-negative breast cancer cells. Furthermore, GRN mRNA levels were significantly and positively correlated with STAT3 gene expression signatures indicative of STAT3 activation as well as with reduced overall survival in breast cancer patients. These studies used a proteomics approach to identify GRN as a novel STAT3 interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer. Citation Format: Jennifer E Yeh, Simion Kreimer, Sarah R Walker, Andrea Richardson, Alexander R Ivanov, David A Frank. Granulin, a novel STAT3-interacting protein, promotes breast cancer tumorigenicity [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-01.

The Chance of Small Interfering RNAs as Eligible Candidates for a Personalized Treatment of Prostate Cancer

Prostate cancer is one of the leading malignant tumors in men. Current therapies are associated with severe side effects making it problematic for many multi-morbid patients to receive treatment. Prostate specific antigen, serum response factor (SRF), signal transducer and activator of transcription-3 (STAT3), hypoxia-inducible factor-1α (HIF-1α), HIF-2α, E2F1 and Survivin are well known proteins being overexpressed in cancer cells, expediting cell growth and also demonstrated in prostate cancer cells. Targeting these genes using the RNA-Interference pathway could be a new approach for prostate cancer therapy with fewer side effects. Three prostate cancer cell lines were cultured under standard conditions and transfected with three different concentrations (25 nM, 50 nM, 100 nM) of specific small interfering RNAs (siRNAs) targeting SRF, STAT3, HIF1α, HIF2α, E2F1 and Survivin in a non-viral manner. Cells treated with non-specific siRNA (SCR-siRNA) served as control. Changes of messenger RNA (mRNA) levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The analysis of the effect of siRNA on the number of cells was detected using CASY cell counter system. Transfections of the PC-3 cell line with specific siRNA especially against Survivin, E2F1, HIF1α- and HIF2α-siRNA resulted in a significant reduction of intracellular mRNA concentration together with a significant decreased number of cells. In the LnCAP and DU-145 cell lines Survivin and E2F1 showed similar effects. The impact of silencing STAT3 or SRF showed little influence on the amount of cells in all three cell lines. This study shows that RNAi succeeds in silencing gene expression and reducing the number of cells in differing dimensions depending on the transfected cell line and used siRNA.

Efficient inhibition of intraperitoneal ovarian cancer growth in nude mice by liposomal delivery of short hairpin RNA against STAT3

Signal transducer and activator of transcription 3 (STAT3) plays an important role in the tumor formation, prognosis and chemoresistance of ovarian cancer. Our goal was to investigate the effect of silencing STAT3 on ovarian cancer cell apoptosis, proliferation, angiogenesis and expression of key targets in vitro and in vivo. The ovarian cancer cell lines A2780CP and A2780s were used. STAT3 was knocked down by the plasmid-based short hairpin RNA (shRNA) expression system. In vitro, a colony formation assay and Hoechst staining were used to examine cell proliferation and apoptosis. The expression level of STAT3 and apoptosis-related proteins were determined by Western blot. The A2780CP intraperitoneal model was used to evaluate the effect of shSTAT3 on tumor growth in mice. Proliferation, apoptosis, and angiogenesis in tumor tissues were measured by proliferating cell nuclear antigen, TUNEL and CD31 immunostaining, respectively. Treatment with shSTAT3 resulted in apoptosis and inhibition of cell proliferation in vitro. Western blot analysis demonstrated that shSTAT3 induced the expression of cleaved caspase-3 and reduced the expression of survivin, Bcl-2 and vascular endothelial growth factor. In vivo, the tumor weight was reduced to 13.46% of 5% glucose by shSTAT3/lipoplexes (P < 0.01), accompanied with apoptosis induction (P < 0.01), proliferation inhibition (P < 0.01) and angiogenesis inhibition (P < 0.01). We find that treatment with shSTAT3 inhibits tumor growth in vitro and in vivo by inducing apoptosis and inhibiting cell proliferation. This work should provide the scientific foundation for future investigation of shSTAT3 as a strategy for ovarian cancer gene therapy and the combination of gene therapy with chemotherapy.

STAT3 silencing enhances the efficacy of the HSV.tk suicide gene in gastrointestinal cancer therapy

Aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling has been shown to be associated with uncontrolled cell proliferation and suppression of host-immune surveillance. Conversely, silencing STAT3 can have the dual effects of inhibiting cancer cell proliferation and inducing anti-tumor immune responses. Here, we report on the effects of STAT3 silencing on suicide gene therapy with thymidine kinase (tk). STAT3 silencing by siRNA inhibited the proliferation of AGS human gastric cancer cells through G1 cell cycle arrest, decreased levels of immune-suppressive cytokines, and increased levels of immune-activating cytokines. CT26 mouse colon adenocarcinoma cells, in which STAT3 expression was knocked-down by a STAT3 shRNA-containing lentivirus, grew more slowly in syngenic model Balb/c mice than control CT26 cells. Moreover, we found that STAT3 silencing augmented the efficacy of suicide gene therapy in CT26 cell xenografted mice. When we administrated adenoviruses harboring the herpes simplex virus thymidine kinase gene (Ad5.CMV.HSV.tk) into STAT3-silenced CT26 cell tumors, extensive apoptosis was observed and there was a significant reduction in the size of CT26 cell tumors. STAT3 silencing also enhanced the recruitment and cytotoxic activity of CD3(+)CD8(+) T-cells, and changed the cytokine expression pattern of CT26 cell tumors, reflecting augmentation of anti-cancer immune responses. We conclude that combining suicide gene therapy with STAT3 silencing can result in enhanced anti-cancer effects.

P3-16-02: Targeting Tumor Initiating Cells with siRNA/Nanotherapy in Triple Negative Breast Cancer.

Abstract Residual human breast tumor cells after conventional therapies are enriched in tumor-initiating cells (TIC) characterized by CD44+/ CD24-/low/lineage- with self-renewal capacities. Our gene expression analyses in those cells and in breast cancer cells propagated as mammospheres (MSs) reveal an epithelial-mesenchymal-transition (EMT) signature (400 genes) mainly found in claudin-low molecular subtype human breast tumors. We performed lentiviral shRNA knockdown of that signature in MS. Critical shRNAs were found to decrease the TIC subpopulation. Among those genes, we found STAT3 (signal transducer and activator of transcription 3), NTN4 (netrin 4), and RPL39L (ribosomal protein L39-like). Here, we used a multiscale siRNA delivery system targeting those three genes in order to clarify the effect of silencing those genes over the self-renewal capacity on MSs with claudin-low features. To this end, duplex small interfering RNA (siRNA) against STAT3, NTN4, RPL39L, and scrambled siRNA as control, were introduced into neutral nanoliposomes (dioleoyl phosphatidylcholine, DOPC), and using mesoporous microscale biodegradable silicon particles as carriers. This multistage siRNA delivery system has been reported as a good approach for sustained gene silencing. Claudin-low-like human breast cancer cell lines (SUM159 and BT549) were plated in 24-well ultralow attachment plates with mammary epithelial growth medium (MEGM) (5,000 cells/well). Both cell lines were then treated with 1μg/well/6 wells of silicon particles loaded with DOPC nanoliposomes/siRNA. The primary MSs were allowed to grow for 3 days. MFs were counted by day 3 with a GelCount colony counter (Oxford Optronix, Oxford, UK). Mammosphere-Forming Efficiency (MSFE) was calculated by dividing the number of MSs by the number of seeded cells. In addition, established MSs were serially passaged by dissociation, and single cells were replated on fresh 24-well ultra-low attachment plates to form secondary mammospheres, which were counted after 3 days. One-way ANOVA and Tukey test were performed. A p value less than 0.05 was considered as significant. Our results show that silencing STAT3, NTN4, and RPL39L significantly reduces the MSFE in both primary (1%, 0.9%, and 1.7% respectively) (Figure 1) and secondary MSs (0.49%, 0.51%, and 0.45% respectively) (Figure 2) when compared to the scrambled control (2.4% and 1 % respectively) in BT549 cells. For SUM159 cells, we did not find any change in primary MSs for STAT3 (2.2%) and NTN4 (2.1%), even we found a higher percentage of MSFE in those cells treated with RPL39L siRNA (3.9%) when compared to the scrambled control (2.1%) (Figure 3). Nevertheless, a lesser MSFE were observed in those cells treated with STAT3 (0.9%, not significant), NTN4 (0.8%) and RPL39L (0.78%) in comparison to control (1.1%) (Figure 4). In conclusion, knocking-down of EMT-related genes (STAT3, NTN4, and RPL39L) decreases significantly the self-renewal capacity in mammospheres derived from claudin-low-like human breast tumor cells, being BT549 cells more sensitive than SUM159 cells to that silencing with siRNA loaded in DOPC nanoliposomes into silicon particles as carriers. Acknowledgments: We thank Fundacion Alfonso Martin Escudero for the personal support of Dr. Sergio Granados-Principal. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-16-02.

Signal Transducer and Activator of Transcription 3 (STAT3) and Suppressor of Cytokine Signaling (SOCS3) Balance Controls Cytotoxicity and IL-10 Expression in Decidual-Like Natural Killer Cell Line NK-92

OBJECTIVES In previous studies, we have shown that sHLA-G reduces cytotoxicity of decidual NK cells, which was dependent upon reduction in signal transducer and activator of transcription 3 (STAT3) and perforin. In this study, we aimed to confirm the role of STAT3 for induction of cytotoxicity and to analyze the regulative role of its antagonist suppressor of cytokine signaling 3 (SOCS3). Furthermore, the influence of both factors on cytokine expression should be analyzed. METHODS All experiments were performed on NK-92 cells. STAT3 and SOCS3 have been silenced using two different small interfering RNA sequences each. Silencing efficiency and STAT3 tyrosine phosphorylation have been analyzed by Western blotting. Cytotoxicity to K562 target cells has been assessed by flow cytometry. Expression of IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α, and TNF-β has been measured using cytometric bead arrays for flow cytometry. RESULTS STAT3 and SOCS3 have been successfully silenced. STAT3 silencing reduced cytotoxicity. SOCS3 silencing induced increase in STAT3 tyrosine phosphorylation and cytotoxicity. STAT3 silencing reduced IL-10 expression significantly, while SOCS3 silencing induced, also significantly, the opposite effect. The other cytokines were expressed at very low concentration or not constantly affected. CONCLUSION STAT3 and SOCS3 are involved in regulation of NK cell cytotoxicity and IL-10 expression.

STAT3 Silencing in Dendritic Cells by siRNA Polyplexes Encapsulated in PLGA Nanoparticles for the Modulation of Anticancer Immune Response

In dendritic cells (DCs), the induction of signal transducer and activator of transcription 3 (STAT3) by tumor-derived factors (TDFs) renders DCs tolerogenic and suppresses their antitumor activity. Therefore, silencing STAT3 in DCs is beneficial for cancer immunotherapy. We have shown that STAT3 knockdown in B16 murine melanoma by siRNA polyplexes of polyethylenimine (PEI) or its stearic acid derivative (PEI-StA) induces B16 cell death in vitro and in vivo. Here, we investigated the physical encapsulation of siRNA/PEI and PEI-StA polyplexes in poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) for STAT3 knockdown in DCs. PLGA NPs containing siRNA polyplexes of PEI (PLGA-P) and PEI-StA (PLGA-PS) had an average diameter of ~350 to 390 nm and a zeta potential of ∼-13 to -19 mV, respectively. The encapsulation efficiency (E.E.) of siRNA in PLGA-P and PLGA-PS was 26% and 43%, respectively. In both NP types, siRNA release followed a triphasic pattern, but it was faster in PLGA-PS. Our uptake study by fluorescence microscopy confirmed DC uptake and endosomal localization of both NP types. After exposure to B16.F10 conditioned medium, DCs showed high STAT3 and low CD86 expression indicating impaired function. STAT3 silencing by PLGA-P and PLGA-PS of STAT3 siRNA restored DC maturation and functionality as evidenced by the upregulation of CD86 expression, high secretion of TNF-α and significant allogenic T cell proliferation. Moreover, encapsulation in PLGA NPs significantly reduced PEI-associated toxicity on DCs. We propose this formulation as a strategy for targeted siRNA delivery to DCs. The potential of this approach is not limited to STAT3 downregulation in DCs but can be used to target the expression of other proteins as well. Moreover, it can be combined with other means for cancer immunotherapy like cancer vaccine strategies.

Abstract 2950: Silencing of STAT3 and Src gene by RNA interference enhances the cytotoxic effect of cisplatin in cervical cancer cell lines

Abstract Purpose: Signal transducer and activator of transcription-3 (STAT3) is well known to regulate oncogenesis, angiogenesis and immunosuppression in cancer cells. The Src family consists of nonreceptor tyrosine kinases and participates in cell cycle progression, adhesion, spreading, migration, and differentiation. Activation of STAT3 by constitutively activated Src pathway in cancer cells results in cancer progression. We investigated the cytotoxic effect of cisplatin in cervical cancer cell lines according to the presence of RNA interference-mediated STAT3 and Src gene silencing. Experimental procedures: We prepared two cervical cancer cell lines of C4I and HeLa. First of all, reverse transcription with an RT-PCR kit was performed in order to confirm the increased STAT3 expression in cervical cancer cells. We synthesized STAT3-specific siRNA, using six different sequences of oligonucleotides encoding STAT3 siRNA. After ligation and transformation into bacteria, the positive colonies were selected by antibiotic resistance. HeLa cells were transfected with the plasmids with luciferase reagent according to the established methods. As the same methods, we obtained the HeLa cells transfected with c-Src and v-Src-targeting siRNAs. We checked the reduction rate of each mRNAs by RT-PCR after 48 hours. HeLa and C4I cells transfected with indicated siRNAs, which showed the highest reduction rate at silencing STAT3, c-Src and v-Src, were cultured and mock transfection without any DNA was always included as experimental controls. At 36 hours after starting culture, cisplatin was administered as indicated concentrations (0, 100, 250, 500, 1000 ng/mL). At 96 hours after starting culture, the viability of HeLa and C4I cells was determined by the MTT assay. Results: We confirmed the increased expression of STAT3 in HeLa cell lines. STAT3 mRNAs were reduced very effectively in all cases of STAT3-targeting siRNAs with different 6 sequences to HeLa (80.1%∼93.4%) and the following sequence of oligonucleotides encoding STAT3 siRNA showed the highest reduction rate; 5′-GCGTCCAGTTCACTACTAAAGTCAG-3′ (upper strand) and 5′-CTGACTTTAGTAGTGAACTGGACGC-3′ (lower strand). The reduction rate at silencing c-Src with different 5 sequences siRNAs was high (73.8%∼88.9%). The reduction rate at silencing v-Src with different 5 siRNAs was lower than other two cases (40.5%∼66.6%). The enhancement of cytotoxicity of cisplatin by gene silencing of STAT3 and c-Src, but not of v-Src, was confirmed in HeLa and C4I cells. Conclusion: In this study, we confirmed that STAT3/c-Src targeting siRNAs down-regulated the expression of STAT3 and c-Src and they could enhance the cytotoxic effect of cisplatin in cervical cancer cell lines. We will continue the upgraded study to investigate methods for enhancement of drug susceptibility, including the effect of co-silencing STAT3 and c-Src and their in vivo responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2950.

Silencing STAT3 with short hairpin RNA enhances radiosensitivity of human laryngeal squamous cell carcinoma xenografts in vivo

Short hairpin RNA (shRNA) targeting signal transducer and activator of transcription 3 (STAT3) potentiate the radiosensitivity of human laryngeal squamous carcinoma cells in vitro. In the present study, we investigated the inhibitory effect of STAT3 shRNA plus radiotherapy on nude mouse laryngeal squamous cell carcinoma xenografts. The xenotransplanted tumors were treated with STAT3 shRNA, with or without radiation, following a planned scheme. The inhibition rate for tumor growth was calculated and the tumor growth curve was plotted. In addition, the expression of p-STAT3, B cell lymphoma 2 (Bcl-2), p53, vascular endothelial growth factor (VEGF) protein and intratumoral microvessel density (MVD) was determined by immunohistochemistry. Flow cytometry was used to detect the rate of cell apoptosis. The results revealed that STAT3 shRNA transfection plus radiotherapy significantly minimized tumor volume and increased the rate of tumor inhibition. p-STAT3 protein expression and intratumoral MVD were observed to be down-regulated, whereas apoptosis was increased. There was a positive correlation between the expression of p-STAT3 and Bcl-2, and also between the expression of p53 and VEGF, and MVD. These findings indicate that STAT3 shRNA potentiate the radiosensitivity of laryngeal carcinoma xenografts in vivo by regulating downstream signaling proteins in the STAT3 pathway.

NOD2 receptors in adenopituitary folliculostellate cells: expression and function

Folliculostellate cells (FS cells) are non-endocrine cells from the pituitary gland that respond to bacterial endotoxins by producing cytokines. In immune cells, an important component of bacterial recognition are the toll-like receptors (TLRs). Previously, we showed that FS cells express TLR4. The TLR4 ligand lipopolysaccharide (LPS) stimulates interleukin-6 (IL6) production through nuclear factor kappaB (NFKB) induction. Binding of IL6 to gp130 receptor activates signal transducer and activator of transcription 3 (STAT3), an important mediator of inflammatory response. Another family involved in innate immune response following bacterial infection is the nucleotide-binding oligomerisation domain (NOD) intracellular receptor family. Herein, we describe for the first time the expression and function of NOD receptors in human pituitary and FS TtT/GF cell line. The NOD2 agonist muramyl dipeptide (MDP) increased Nf kappa b1-transcriptional activity, -protein expression and IL6 secretion in TtT/GF cells. Furthermore, these effects were potentiated by the combination of MDP and LPS. Silencing NOD2 abolished the action of LPS on NFKB transcriptional activity and IL6 production, indicating that, in TtT/GF cells, TLR4 transduces its signal through NOD2 receptor. We show here that in TtT/GF cells, Nod2 overexpression or stimulation by MDP increased STAT3 transcriptional activity. Furthermore, silencing STAT3 inhibited basal, LPS and MDP stimulated NFKB protein expression and overexpression of protein inhibitor of activated STAT3 (Pias3) markedly decreased basal NFKB activity. These data suggest that in TtT/GF cells, STAT3 acting upstream to NFKB mediates NOD2 receptor signalling pathway. In conclusion, the present study demonstrates that NOD molecules play a modulatory role in the pituitary by regulating the function and activation of FS cells in response to bacterial components.