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.

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.

Sjogren's syndrome: Review of the aetiology, Pathophysiology & Potential therapeutic interventions.

BACKGROUND: Sjogren's syndrome (SS) is an autoimmune disorder characterised by lymphocytic infiltration of exocrine glands, resulting in glandular dysfunction. Objectives: This study aims to review the aetiology of Sjogren's syndrome, highlight aspects that contribute to the pathophysiology of the disease and explore treatment options that target different mediators of pathogenesis. MATERIAL AND METHODS: The MEDLINE/PubMed and Google Scholar databases were searched systematically with the terms "Sjogren's syndrome"; "clinical"; "treatment"; "management". Eligible studies had to meet a predefined inclusion criteria. RESULTS: 912 identified studies were evaluated against the inclusion criteria. 25 eligible studies were included for review. Sjogren's syndrome is a multifactorial condition with genetic, environmental and hormonal factors playing a role in establishing the condition. B-cell activating factor (BAFF) is an important mediator in the induction and perpetuation of this condition. Elevated BAFF levels, found in patients with SS, promote growth of B-cells and subsequent production of autoantibody; anti-SSA/Ro. BAFF inhibitors are important potential therapeutic drugs that may be effective in patients with Sjogren's syndrome. Other potential targets include CD20 and CD22 that cause B-cell depletion. CONCLUSIONS: The pathophysiology of this exocrinopathy has not fully been elucidated. Potential therapeutic interventions include BAFF inhibitors and anti-CD20 and anti-CD22 therapy. However, no clinical trials have been conducted on subjects with Sjogren's syndrome to support existing research. Key words:Sjogren's syndrome, autoimmune, rheumatology.