Sirtinol, a SIRT1 inhibitor, inhibits the EMT and metastasis of 4T1 breast cancer cells and impacts the tumor microenvironment.

INTRODUCTION: The impact of epigenetic drugs on metastasis and the immunological microenvironment is poorly understood. In this study, we looked at how sirtinol, a SIRT1 inhibitor, affected epithelial-mesenchymal transition (EMT), metastasis, and the immune cells. MATERIALS AND METHODS: In vitro experiments were carried out using tumor conditioned medium (TCM). For in vivo experiments, sirtinol was administered i.p. in tumor bearing BALB/c mice at a dose of 2 mg/kg body weight either alone or in combination with cisplatin. Estimation of cytokines was carried out using ELISA or ELIspot. Estimation of different markers was done using flow cytometry or western blot. RESULTS: Sirtinol, a SIRT1 inhibitor, was found to be cytotoxic to 4T1 breast cancer cells with no synergistic effects with cisplatin, both under in vitro and in vivo conditions (p < 0.05). Sirtinol significantly reduced cancer cell metastasis to the spleen which was supported by in vitro findings such as decreased vimentin expression and cell mobility in migration and wound healing assays (p < 0.01). Studies on the effects of 4T1 tumor-conditioned medium on spleen cells indicated changes in T cell proliferation as well as differentiation (p < 0.01). In tumor bearing mice, spleen cells showed elevated IFN-γ secretion, increased CD11b+ cells, and decreased T cells (p < 0.01). This was reversed by sirtinol as well as the combination treatment, which may also have contributed to metastasis inhibition (p < 0.01). CONCLUSION: Sirtinol, a SIRT1 inhibitor inhibits EMT and metastasis of 4T1 breast cancer cells and also has an impact on the immune microenvironment.

TGF-ßR inhibitor SB431542 restores immune suppression induced by regulatory B-T cell axis and decreases tumour burden in murine fibrosarcoma.

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-ß. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses was not abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFß Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFß-mediated pathway and that targeting the Breg-Treg axis can be potentially used as an immunotherapy agent.

Proteomic analysis of radio-resistant breast cancer xenografts: Increased TGF-ß signaling and metabolism.

Our previous studies have shown that MCF-7 breast cancer cell line exposed to 6 Gy and allowed to recover for 7 days (D7-6G) developed radio-resistance. In this study, we have tested the ability of these cells to form tumors in severe combined immunodeficiency (SCID) mice and characterized these tumors by proteomic analyses. Untreated (MCF-C) and D7-6G cells (MCF-R) were injected s.c. in SCID mice and tumor growth monitored. On Day 18, the mice were killed and tumor tissues were fixed in formalin or RNA later. Expression of genes was assessed by reverse transcription-polymerase chain reaction and proteins by enzyme-linked immunosorbent assay/antibody labeling and flow cytometry. Label free proteomic analyses was carried out by liquid chromatography-mass spectrometry. Metabolic analysis was carried out using Seahorse analyzer. MCF-R cells had a shorter latency and formed larger tumors. These tumors were characterized by an increased expression of transforming growth factor ß (TGF-ß) isoforms; its downstream genes pSMAD3, Snail-1, Zeb-1, HMGA2; hybrid epithelial/mesenchymal phenotype; migration, enrichment of cancer stem cells and radioresistance following challenge dose of radiation. Proteomic analysis of MCF-7R tumors resulted in identification of a total of 649 differentially expressed proteins and pathway analyses using protein annotation through evolutionary relationship indicated enrichment of genes involved in metabolism. Data are available via ProteomeXchange with identifier PXD022506. Seahorse analyzer confirmed increased metabolism in these cells with increased oxidative phosphorylation as well as glycolysis. Increased uptake of 2-NBDG further confirmed increased glycolysis. In summary, we demonstrate that radioresistant breast cancer cells had an enrichment of TGF-ß signaling and increased metabolism.

TNF-α and IGF-1 differentially modulate ionizing radiation responses of lung cancer cell lines.

The mechanism by which tumor microenvironment derived cytokine network modulates therapy response is of great concern in lung cancer but is not completely understood. In this study, we evaluated the effects of tumor necrosis factor α (TNF-α) and insulin-like growth factor 1 (IGF-1) on response of lung cancer cell lines to ionizing radiation (IR). While TNF-α increased radio sensitivity and inhibited cell migration, treatment with IGF-1 promoted cell growth and increased migration. These effects of TNF- α were mediated by increased immediate activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) and p38. IR induced DNA damage was increased by TNF- α and not altered by IGF-1. However, in IGF-1 treated cells, there was decreased γ- H2AX along with an increase in mitotic index, resulting in abnormal chromosomal segregation in the cells. Bio informatics analysis of 982 lung cancer patients revealed that higher expression of TNF- α was associated with low risk of cancer progression while overexpression of IGF-1 was correlated with high risk. Collectively, these results reveal that the cytokines in the tumor microenvironment differentially modulate radiation therapy through a variety of signaling mechanisms.

Significant alterations of the novel 15 gene signature identified from macrophage-tumor interactions in breast cancer.

BACKGROUND: Tumor microenvironment is composed of a largely altered extracellular matrix with different cell types. The complex interplay between macrophages and tumor cells through several soluble factors and signaling is an important factor in breast cancer progression. METHODS: We have extended our earlier studies on monocyte and macrophage conditioned medium (MϕCM) and have carried out proteomic analysis to identify its constituents as well as validation. The 8-gene signature identified through macrophage-breast cancer cell interactions was queried in cBioportal for bioinformatic analyses. RESULTS: Proteomic analysis (MALDI-TOF and LC-MS/MS) revealed integrin and matrix metalloproteinases in MϕCM which activated TGF-ß1, IL-6, TGF- ßRII and EGFR as well as its downstream STAT and SMAD signaling in breast cancer cells. Neutralization of pro-inflammatory cytokines (TNF-α. Il-1ß, IL-6) abrogated the MϕCM induced migration but invasion to lesser extent. The 8- gene signature identified by macrophage-tumor interactions (TNF-α, IL-1ß, IL-6, MMP1, MMP9, TGF-ß1, TGF-ßRII, EGFR) significantly co-occurred with TP53 mutation, WTAPP1 deletion and SLC12A5 amplification along with differential expression of PSAT1 and ESR1 at the mRNA level and TPD52and PRKCD at the protein level in TCGA (cBioportal). Together these genes form a novel 15 gene signature which is altered in 63.6% of TCGA (1105 samples) data and was associated with high risk and poor survival (p<0.05) in many breast cancer datasets (SurvExpress). CONCLUSIONS: These results highlight the importance of macrophage signaling in breast cancer and the prognostic role of the15-gene signature. GENERAL SIGNIFICANCE: Our study may facilitate novel prognostic markers based on tumor-macrophage interaction.

PGE2 induced miR365/IL-6/STAT3 signaling mediates dendritic cell dysfunction in cancer.

AIM: To understand the mechanism of prostaglandin E2 (PGE2)-mediated immunosuppression in dendritic cells (DCs). MAIN METHODS: In vivo experiments were conducted on 4T1 tumor bearing mice (TBM). In vitro experiments were performed in bone marrow-derived DCs (BMDCs), or spleen cells. Cytokines were monitored by ELISA/ELIspot. Gene expression was monitored by RT-PCR/flow cytometry. KEY FINDINGS: In silico, in vitro, and in vivo experiments in 4T1 TBM revealed that PGE2 induced IL-6/pSTAT3 signaling through EP4 receptors in DCs, resulting in their dysfunction. These effects were reversed by EP4 antibody neutralization, EP4 antagonist, and STAT3 inhibitory peptides. PGE2 induced IL-6 was regulated by miR-365, as its mimic inhibited PGE2 induced IL-6 and the inhibitor increased lL-6 levels in DC. Bio-informatic analysis in human mammary cancers also revealed a strong compared co-relation between PGE2 and IL-6 (Correlation AnalyzeR) (R = 0.94). Mice bearing PTGS-2 KD 4T1 tumors had decreased tumor burden, PGE2, EP4, IL-6, and pSTAT3 signaling, along with improved DCs and T cell functions. Treatment of mice with a cyclooxygenase-2 (COX-2) inhibitor or EP4 antagonist decreased tumor burden, and this effect of EP4 antagonist was abrogated upon in vivo depletion of CD11c cells, indicating the crucial role of PGE2 signaling in DCs in tumor progression. SIGNIFICANCE: In summary, our data highlights the importance of dendritic cells in mediating PGE2-mediated immunosuppression and the use of EP4 or STAT3 inhibitors or miR365 mimics can restore immunogenicity in cancer.

Cytokines from the tumor microenvironment modulate sirtinol cytotoxicity in A549 lung carcinoma cells.

Cytokines in tumor microenvironment play an important role in the success or failure of molecular targeted therapies. We have chosen tumor necrosis factor α (TNF-α), TNF related apoptosis inducing ligand (TRAIL), insulin-like growth factor 1 (IGF-1) and transforming growth factor ß (TGF-ß) as representative pro-inflammatory, pro-apoptotic, anti-apoptotic and anti-inflammatory tumor derived cytokines. Analysis of Oncomine database revealed the differential expression of these cytokines in a subset of cancer patients. The effects of these cytokines on cytotoxicity of FDA approved drugs - cisplatin and taxol and inhibitors of epidermal growth factor receptor - AG658, Janus kinase - AG490 and SIRT1 - sirtinol were assessed in A549 lung cancer cells. TRAIL augmented cytotoxicity of sirtinol and IGF-1 had a sparing effect. Since TRAIL and IGF-1 differentially modulated sirtinol cytotoxicity, further studies were carried out to identify the mechanisms. Sirtinol or knockdown of SIRT1 increased the expression of death receptors DR4 and DR5 and sensitized A549 cells to TRAIL. Increased cell death in presence of TRAIL and sirtinol was caspase independent and demonstrated classical features of necroptosis. Inhibition of iNOS increased caspase activity and switched the mode of cell death to caspase mediated apoptosis. Interestingly, sirtinol or SIRT1 knockdown did not increase IGF-1R expression. Instead, it abrogated ligand induced downregulation of IGF-1R and increased cell survival through PI3K-AKT pathway. In conclusion, these findings reveal that the tumor microenvironment contributes to modulation of cytotoxicity of drugs and that combination therapy, with agents that increase TRAIL signaling and suppress IGF-1 pathway may potentiate anticancer effect.

Arabinogalactan G1-4A isolated from Tinospora cordifolia induces PKC/mTOR mediated direct activation of natural killer cells and through dendritic cell cross-talk.

BACKGROUND: Tinospora cordifolia polysaccharide G1-4A activates antigen-presenting cells, but its effect on natural killer (NK) cells is not known. The objective of this study is to assess the effect of G1-4A on NK cells; direct effects as well as through dendritic cell (DC) cross-talk. METHODS: NK cell phenotype and function were assessed in spleen cells treated in vitro with G1-4A or isolated from mice administered with G1-4A. Following treatment with G1-4A in vitro or in cells isolated from G1-4A treated mice (in vivo), activated NK cell phenotype was characterized as CD3-NKp46+CD69+ cells by flow cytometry; NK cell function was evaluated by IFN-γ secretion (ELISA) and cytotoxicity assay (calcein release by target cells in effector: target cells co-culture assay). RESULTS: Both in vitro as well as in vivoG1-4A treatment increased phenotypic and functional activation of NK cells. So, we wanted to determine if this was through NK-DC crosstalk or direct activation of NK cells. There was increased NK cell activation following co-culture with bone marrow derived DC matured withG1-4A in vitro or splenic DC isolated from G1-4A administered mice indicating crosstalk. G1-4A also increased activation of NK cells in (a) CD11c depleted splenic cells that was contact dependent and (b) purified NKp46+ cells that was abrogated by PKC/mTOR inhibitors indicating direct effects on NK cells. CONCLUSION: In summary, treatment with G1-4A results in phenotypic and functional activation of NK cells directly as well as through NK-DC cross talk and has the potential to be used as an immunotherapeutic agent.

Macrophage induced ERK-TGF-ß1 signaling in MCF7 breast cancer cells result in reversible cancer stem cell plasticity and epithelial mesenchymal transition.

BACKGROUND: Breast cancer is a heterogenous disease composed of multiple clonal populations and the mechanism by which the tumor microenvironment induces cancer stem cell plasticity is not fully understood. METHODS: MCF7 breast cancer cells were treated with macrophage conditioned medium (MɸCM). PD98059 and SB431542 were used for ERK and TGF-ßR inhibition respectively. Epithelial-mesenchymal transition (EMT) and cancer stem cell markers (CSC) were studied using qRT-PCR and flowcytometry. SCID mice were used for animal experiments. RESULTS: MɸCM- induced ERK/TGF-ß1 signaling led to enrichment of CSC and EMT in MCF7 cells and mammospheres. These effects were abrogated by both MEK inhibitor PD98059 (TGF-ß1 synthesis) and SB431542 (TGF-ß1 signaling). The increase in CSC was both hybrid (ALDH1+) and mesenchymal (CD44+ CD24- cells). Increase in hybrid E/M state was at a single cell level as confirmed by the increase in both claudin-1 (E) and vimentin (M). This did not have any growth advantage in SCID mice and monitoring of CSC and EMT markers before and after growth in SCID mice indicated reversal of these markers in tumor cells recovered from mice. Removal of MɸCM and neutralization of TNF-α, IL-6 and IL-1ß in MɸCM abrogated ERK phosphorylation, TGF-ß and CSC enrichment indicating the requirement of continuous signaling for maintenance. CONCLUSIONS: ERK signaling plays an important role in MɸCM- induced EMT and CSC plasticity which is completely reversible upon withdrawal of signals. GENERAL SIGNIFICANCE: Our experimental observations support the semi-independent nature of EMT-stemness connection which is very dynamic and reversible depending on the microenvironment.

Identification of EPZ004777 and FG2216 as inhibitors of TGF-ß1 induced Treg cells by screening a library of epigenetic compounds.

AIMS: Regulatory T cells play an essential role in immune tolerance and homeostasis. Their long-term stability depends on epigenetic modifications and identification of small molecule modulators of Treg differentiation therefore has many applications. In this study, we performed a novel functional screen of an epigenetic compound library to identify compounds that can modulate generation of TGF-ß1 induced T regulatory cells. MAIN METHODS: A screening strategy based on IFN-γ ELISA was designed to screen epigenetic compound library. Effect of hit compounds on Treg phenotype and function was assessed by RT-PCR, flow cytometry and suppression assays. TGF-ß signalling proteins were assayed by western blotting. Chromatin Immunoprecipitation (ChIP) assay was used to assess epigenetic modifications at Foxp3 gene locus. KEY FINDINGS: We screened 160 compounds to identify hits capable of reversing TGF-ß induced inhibition of IFN-γ production in activated spleen cells and CD4+ T cells. Two compounds EPZ004777 and FG-2216 consistently reversed TGF-ß1 iTregs in terms of (a) differentiation of naïve T cells into CD4+CD25+Foxp3+Treg cells, (b) Foxp3 target gene expression and (c) Treg suppressive function without affecting TGF-ß downstream signalling. ChIP assay revealed that the compounds were able to reverse - TGF- ß mediated decrease in epigenetic marks H3K27me3 and 5-mC and an increase in epigenetic marks H3K4me3 and H3K27Ac in the promoter and conserved non coding sequence (CNS1) regions of the Foxp3 gene. SIGNIFICANCE: EPZ004777 and FG-2216 have been identified as potent epigenetic modulators that can reverse TGF-ß1 induced T regulatory cells and may be used to treat diverse immune disorders.

Effects of prolonged treatment of TGF-ßR inhibitor SB431542 on radiation-induced signaling in breast cancer cells.

PURPOSE: We have earlier characterized increased TGF-ß signaling in radioresistant breast cancer cells. In this study, we wanted to determine the effect of prolonged treatment of TGF-ßR inhibitor SB431542 on radiation-induced signaling, viz., genes regulating apoptosis, EMT, anti and pro-inflammatory cytokines. MATERIALS AND METHODS: Breast cancer cells were pretreated with TGF-ßR inhibitor (SB 431542) followed by exposure to 6 Gy and recovery period of 7 days (D7-6G). We assessed cell survival by MTT assay, cytokines by ELISA and expression analysis by RT-PCR, flow cytometry, and western blot. We carried out migration assays using trans well inserts. We performed bioinformatics analyses of human cancer database through cBioportal. RESULTS: There was an upregulation of TGF-ß1 and 3 and downregulation of TGF-ß2, TGF-ßR1, and TGF-ßR2 in invasive breast carcinoma samples compared to normal tissue. TGF-ß1 and TNF-α was higher in radioresistant D7-6G cells with upregulation of pSMAD3, pNF-kB, and ERK signaling. Pretreatment of D7-6G cells with TGF-ßR inhibitor SB431542 abrogated pSMAD3, increased proliferation, and migration along with an increase in apoptosis and pro-apoptotic genes. This was associated with hybrid E/M phenotype and downregulation of TGF-ß downstream genes, HMGA2 and Snail. There was complete agreement in the expression of mRNA and protein data in genes like vimentin, Snail and HMGA2 in different treatment groups. However, there was disagreement in expression of mRNA and protein in genes like Bax, Bcl-2, E-cadherin, Zeb-1 among the different treatment groups indicating post-transcriptional and post-translational processing of these proteins. Treatment of cells with only SB431542 also increased expression of some E/M genes indicating TGF-ß independent effects. Increased IL-6 and IL-10 secretion by SB431542 along with increase in pSTAT3 and pCREB1 could probably explain these TGF-ß/Smad3 independent effects. CONCLUSION: These results highlight that TGF-ß-pSMAD3 and TNF-α-pNF-kB are the predominant signaling pathways in radioresistant cells and possibility of some TGF-ß/Smad3 independent effects on prolonged treatment with the drug SB431542.

Spray drying as an efficient route for synthesis of silica nanoparticles-sodium alginate biohybrid drug carrier of doxorubicin.

Biohybrids (a combination of biological material and inorganic nanoparticles) offer a number of advantages like improved functionality over conventional materials.Thus, to understand the practical application of biohybrids as drug carriers, a biohybrid drug carrier of colloidal silica nanoparticles (NP)-sodium alginate loaded with doxorubicin (Dox-biohybrid) was synthesized by evaporation induced self-assembly (EISA) using spray drying technique. Further, the morphology, size and interactions between various components of the biohybrid were studied through SEM, DLS and FTIR techniques. The drug loading efficiency, release profile, cellular uptake and cytotoxicity of Dox-biohybrid was investigated and compared with free Dox. The drug loading efficiencies of Dox-biohybrid, Dox-silica NP and Dox-sodium alginate were 93.7 %, 96.4 % and 88.3 % respectively. In vitro release study showed a slow release of entrapped Dox from Dox-biohybrid as compared to other carriers. This release was also pH-responsive with significantly higher cumulative drug release at pH 5.5 (cancer microenvironment) in comparison to pH 7.4 (physiological conditions). The empty biohybrid carrier did not show cytotoxicity to normal mouse lymphocytes upto a concentration of 25 µg/mL which was used further. The uptake of Dox from Dox-biohybrid by A549 cells was more than 2fold as compared to uptake from free Dox. in vitro viability assay revealed that treatment of lung carcinoma A549 cells with Dox-biohybrid resulted in 50 % loss of cell viability at 500 nM, compared to only 12 % loss with free Dox. Thus, we report the synthesis of a novel biohybrid drug delivery system by means of spray drying process that has promising applications in cancer treatment.

Radiation-induced augmentation in dendritic cell function is mediated by apoptotic bodies/STAT5/Zbtb46 signaling.

Purpose: To evaluate the effect of ionizing radiation (IR) exposure on differentiation and maturation of dendritic cells (DC).Materials and methods: Bone marrow progenitor cells irradiated in vitro or isolated from mice exposed to whole body or localized tumor irradiation were differentiated into DC. Phenotypic maturation of DC was characterized by labeling with specific antibodies and flow cytometry analysis. Cytokines were estimated by ELISA.Results: Splenic and bone marrow-derived DC (BMDC) from tumor-bearing mice exposed to localized irradiation showed abrogation of tumor-induced immunosuppression. This was not due to the effect of radiation on tumor cells as DC derived from normal mice exposed to whole-body irradiation (WBI) also showed increase in immune-activating potential of DC. This was observed in terms of increased phenotypic and functional activation of DCs. This phenomenon was also recapitulated if DC were differentiated from in vitro irradiated progenitor cells and was found to be due to STAT5/Zbtb46 signaling mediated by the irradiation-induced apoptotic bodies (ABs). When these ABs were depleted using annexin-beads, these effects were reversed confirming the involvement of this pathway. The role of ABs was further validated in DC derived from mice exposed to WBI in adaptive response experiments with 0.1 Gy priming dose prior to 2 Gy challenge dose. A corresponding reduction in DC maturation markers was observed with decrease in apoptosis in vivo. Further, these DCs derived from irradiated progenitors (IP) could resist the suppressive effects of tumor conditioned medium (TCM) and had increased immune-activating potential as seen in the tumor-bearing mice.Conclusions: Though radiation is the most commonly used therapeutic modality for cancer, its effects on dendritic cell differentiation is not completely understood. We demonstrate here for the first time that exposure to select doses of IR can increase immune-activating potential of DC through ABs. This can have implications in selection of appropriate doses of IR during radiotherapy of cancer patients.

Radio resistance in breast cancer cells is mediated through TGF-ß signalling, hybrid epithelial-mesenchymal phenotype and cancer stem cells.

AIMS: A major obstacle for effective cancer treatment by radiation therapy is the development of radio-resistance and identification of underlying mechanisms and activated pathways will lead to better combination therapies. MAIN METHODS: Irradiated MCF-7 and MDA-MB-231 breast cancer cell lines were characterised following different recovery periods. Proliferation was assessed by MTT, BrdU and clonogenic assays and apoptosis by Annexin V/ propidium iodide staining and flow cytometry. Gene expression was monitored by real time PCR/ELISA/antibody labelling and migration using transwell inserts. KEY FINDINGS: Breast cancer cell lines exposed to 6 Gy followed by recovery period for 7 days (D7-6 G) had increased ability for proliferation as well as apoptosis. D7-6 G from both cell lines had increased expression of transforming growth factor isoforms (TGF)-ß1, ß2 and ß3, their receptors TGF-ßR1 and TGF-ßR2 which are known for such dual effects. The expression of downstream transcription factors Snail, Zeb-1 and HMGA2 also showed a differential pattern in D7-6 G cells with upregulation of at least two of these transcription factors. D7-6 G cells from both cell lines displayed hybrid epithelial-mesenchymal (E/M) phenotype with increased expression of E/M markers and migration. D7-6 G cells had increased expression of cancer stem cells markers Oct4, Sox2, and Nanog; aldehyde dehydrogenase expression and activity; proportion of CD44+CD24-cells. This was accompanied by radio resistance when exposed to a challenge dose of radiation. Treatment with TGF-ßRI inhibitor abrogated the increase in proliferation of D7-6 G cells. SIGNIFICANCE: Blocking of TGF-ß signalling may therefore be an effective strategy for overcoming radio resistance induced by radiation exposure.

Differential radio-adaptive responses in BALB/c and C57BL/6 mice: pivotal role of calcium and nitric oxide signalling.

Purpose: Our earlier studies demonstrated that transient radio-adaptive responses (RAR) in BALB/c mice were due to MAPK hyperactivation. The objective of this study was to determine the time duration of this low dose induced MAPK activation in BALB/c mice and to find out if similar adaptive responses are observed in C57BL/6 mice. Materials and methods: Mice were irradiated with 0.1 Gy priming dose (PD), 2 Gy challenge dose (CD) with an interval of 4 h (P + CD) and radiation induced immunosuppression in splenic lymphocytes was monitored as the endpoint for RAR. Results: Time kinetics following 0.1 Gy demonstrated persistence of MAPK hyperactivation till 48 h. Similar experiments in C57BL/6 mice indicated absence of RAR at 24 h following CD, in spite of MAPK activation which was also confirmed by time kinetics. Therefore, upstream activators of MAPK, viz., reactive oxygen and nitrogen species (ROS, RNS) and calcium levels were estimated. There was increased intracellular calcium (Ca2+) and nitric oxide (NO) in BALB/c and an increase in intracellular ROS in C57BL/6 mice 24 h after PD. Inhibition of NO and calcium chelation abrogated RAR in BALB/c mice. In vitro treatment of spleen cells with combination of NO donor and Ca2+ ionophore mimicked the effect of PD and induced adaptive response after 2 Gy not only in BALB/c but also in C57BL/6 mice confirming their crucial role in RAR. Conclusions: These results suggest that low dose induced differential induction of Ca2+ and NO signaling along with MAPK was responsible for contrasting RAR with respect to immune system of BALB/c and C57BL/6 mice. Abbreviations [3H]-TdR: 3H-methyl-thymidine; BAPTA: 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; CD: Challenge Dose; CFSE: Carboxy Fluorescein Succinamidyl Ester; on A: Concanavalin A; DAF-FM: 4-amino-5-methylamino-2',7'-difluorescein; DCF-DA: 2',7'-dichlorofluorescein diacetate; DSB: Double Strand Break; ELISA: Enzyme Linked ImmunoSorbent Assay; ERK: Extracellular signal-Regulated protein Kinase; FBS: Fetal Bovine Serum; HIF-1A: Hypoxia-Inducible Factor 1-alpha; LDR: Low Dose Radiation; MAPK: Mitogen Activated Protein Kinase; MAPKK/MKK: MAPK Kinase; MAPKKK: MAPK Kinase Kinase; NO: Nitric Oxide; NOS: Nitric Oxide Synthase; P + CD: Priming + Challenge dose; PBS: Phosphate Buffered Saline; PBST: Phosphate Buffered Saline-Tween 20; PD: Priming Dose; PI3K: Phosphatidyl Inositol 3-Kinase; PKC: Protein Kinase C; RAR: Radio Adaptive Response; RNS: Reactive Nitrogen Species; ROS: Reactive Oxygen Species; RPMI-1640: Roswell Park Memorial Institute-1640 medium; SAPK/JNK: Stress-Activated Protein Kinase/ c-Jun NH2-terminal Kinase; SEM: Standard Error of Mean; SNAP: S-nitro amino penicillamine; TP53: Tumor Protein 53; γ-H2AX: Gamma- H2A histone family member X; Th1: Type 1 helper T cell responses; Th2: Type 2 helper T cell responses.

COX-2 inhibitor prevents tumor induced down regulation of classical DC lineage specific transcription factor Zbtb46 resulting in immunocompetent DC and decreased tumor burden.

The interaction between the immune and tumor cells in the microenvironment is an important factor deciding the progression of cancer. Though many of the soluble mediators in the microenvironment that mediate immunosuppression are known, the mechanism by which the tumor affects the distal progenitors is not known. We report that the tumor derived prostanoids down regulated classical dendritic cells DC (cDC) lineage specific transcription factor Zbtb46 in the progenitor cells which affects its differentiation. Prostanoids also induced ERK/CREB/IL-10 signaling pathway in DC that is more important for maturation of DC. This was observed under in vitro as well as in vivo conditions leading to phenotypic and functional impairment of DC. siRNA mediated knockdown of Zbtb46 and not exogenous IL-10 mimicked the effects of tumor conditioned medium (TCM) on suppression of maturation markers. Treatment of tumor cells with COX-2 inhibitor NS-398 averted TCM induced phenotypic impairment of DC in vitro. Treatment of tumor bearing mice with NS-398 prevented tumor induced down regulation of Zbtb46 resulting in immunocompetent DC which in turn led to a decrease in tumor burden. The effects of NS-398 was indeed through immunomodulation was corroborated by no such response in SCID mice. Our study provides novel insight into the distal regulation of progenitor cells by tumor and the importance of Zbtb46 expression in anti-tumor immunity. These results identify Zbtb46 expression as an indicator of immunocompetent DC in tumor and also highlights that COX-2 inhibitors could be useful in cancer immunotherapy.

FcepsilonRI cross-linking activates a type II phosphatidylinositol 4-kinase in RBL 2H3 cells.

Crosslinking of FcepsilonRI on rat basophilic leukemia (RBL 2H3) cells leads to an increase in Phosphatidylinositol 4-kinase activity. This increase in Ptdlns 4-kinase activity is strongly correlated with its tyrosyl phosphorylation state. Characterization of the enzyme activity in anti phosphotyrosine immunoprecipitates suggests it as a type II Ptdlns 4-kinase. Membrane cholesterol depletion studies showed a reduction in type II Ptdlns 4-kinase activity suggesting that lipid rafts play an important role in activation of the enzyme. The enzyme activity was inhibited by resveratrol. In situ inhibition of type II Ptdlns 4-kinase activity showed a reduction in beta-hexosaminidase release upon FcepsilonRI cross-linking. These studies suggest that a type II Ptdlns 4-kinase is an integral component of FcepsilonRI mediated signal transduction mechanisms.

Involvement of MAPK signalling in radioadaptive response in BALB/c mice exposed to low dose ionizing radiation.

To investigate low dose ionizing radiation (LDIR)-induced adaptive response in lymphocytes of BALB/c mice and to elucidate related molecular mechanisms. Mice were exposed to a priming dose (PD) of 0.1 Gy and challenge dose (CD) of 2 Gy ionizing radiation. Proliferation response to mitogen concanavalin A was assessed using (3)H thymidine incorporation and carboxyfluoresceinsuccinamidylester (CFSE) dye dilution. Early activation markers were assessed by flow cytometry, cytokines by ELISA, DNA damage by comet assay and mitogen activated protein kinase (MAPK) signaling by Western blotting. Radioadaptive response was observed in lymphocytes of mice exposed to PD prior to CD of ionizing radiation in terms of DNA damage, early activation markers CD69, CD71, cytokines IL-2, IFN-γ as well as proliferation. This effect was transient and observed 24 h after CD and not after 0 h or 72 h. Hyper activation of MAPK signaling pathways in lymphocytes from LDIR-exposed mice and abrogation by ERK and p38 inhibitors suggests the involvement of MAPK signaling in radioadaptive response. Our study demonstrates that LDIR-induced transient adaptive response was due to hyper activation of MAPK signaling. Our findings contribute towards the understanding of LDIR-induced adaptive response.

Potential Role of TRAIL in Metastasis of Mutant KRAS Expressing Lung Adenocarcinoma.

Apo2L/tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL, TNFSF10) is an important cytokine in the tumor microenvironment and plays a major role in the balance of cell survival/death pathways. Bioinformatic analyses of 839 adenocarcinoma (AC) and 356 squamous cell lung carcinoma patient data (SCC) by cBioPortal (genomic analyses) shows that TRAIL expression leads to differential outcomes of disease free survival in AC and SCC. Oncomine datamining (transcript analyses) reveal that TRAIL is upregulated in 167 SCC as compared to 350 AC patients from six data sets. Genomic analyses using cBioPortal revealed high rates of KRAS mutation in AC accompanied by higher incidence of metastasis and increased amplifications of TRAIL gene in SCC. Bioinformatic analyses of an additional lung cancer patient database also showed that risk of disease progression was significantly increased with high TRAIL expression in AC (461 samples). In vitro studies demonstrated that TRAIL increased phosphorylation of ERK only in adenocarcinoma cell lines with mutant KRAS. This was associated with increased migration that was abrogated by MEK inhibitor PD98059. Effects of increased migration induced by TRAIL persisted even after exposure to ionizing radiation with suppression of DNA damage response. These results help understand the role of TRAIL signaling in metastasis which is essential to develop strategies to revert these signals into pro-apoptotic pathways.

Low dose radiation induced immunomodulation: effect on macrophages and CD8+ T cells.

PURPOSE: The aim of the present investigation was to study the effect of fractionated whole body low dose ionizing radiation (LDR) on the functional responses of T lymphocytes, their subpopulations and macrophages. MATERIALS AND METHODS: C57BL/6 mice were exposed to 4 cGy from a (60)Co source, at 0.31 cGy/min, at 24 h intervals for 5 days (total dose 20 cGy). Phagocytic activity was measured by flow cytometry using Bioparticles and nitric oxide generation was estimated by spectrophotometry. Proliferation of lymphocytes in response to concanavalin A (con A) and alloantigens was measured by (3)H thymidine incorporation. Expression of cell surface markers was assessed by flow cytometric analysis of antibody labeled cells. Target cell killing by cytotoxic T cells (CTL) generated against allogenic cells was assessed by flow cytometry using PKH26 labeled target cells. Cytokines were estimated by enzyme linked immunosorbent assay. RESULTS: Exposure to LDR enhanced nitric oxide secretion and phagocytosis. The expression of early activation antigen, CD69, was enhanced in CD8(+) T lymphocytes concomitant with enhanced proliferation in response to con A. In addition, mixed lymphocyte reaction (MLR) and CTL response were augmented and secretion of interferon gamma (IFN-gamma) was suppressed following LDR exposure. CONCLUSIONS: LDR exposure enhanced the function of macrophages and responses of CD8(+) T cells in C57BL/6 mice.