title: Thiol dependent NF-κB suppression and inhibition of T-cell mediated adaptive immune responses by a naturally occurring steroidal lactone Withaferin A.
abstract: Withaferin A (WA), a steroidal lactone isolated from ayurvedic medicinal plant Withania somnifera, was shown to inhibit tumor growth by inducing oxidative stress and suppressing NF-κB pathway. However, its effect on T-cell mediated adaptive immune responses and the underlying mechanism has not been investigated. Since both T-cell responses and NF-κB pathway are known to be redox sensitive, the present study was undertaken to elucidate the effect of WA on adaptive immune responses in vitro and in vivo. WA inhibited mitogen induced T-cell and B-cell proliferation in vitro without inducing any cell death. It inhibited upregulation of T-cell (CD25, CD69, CD71 and CD54) and B-cell (CD80, CD86 and MHC-II) activation markers and secretion of Th1 and Th2 cytokines. WA induced oxidative stress by increasing the basal ROS levels and the immunosuppressive effects of WA were abrogated only by thiol anti-oxidants. The redox modulatory effects of WA in T-cells were attributed to its ability to directly interact with free thiols. WA inhibited NF-κB nuclear translocation in lymphocytes and prevented the direct binding of nuclear NF-κB to its consensus sequence. MALDI-TOF analysis using a synthetic NF-κB-p50 peptide containing Cys-62 residue suggested that WA can modify the cysteine residue of NF-κB. The pharmacokinetic studies for WA were also carried out and in vivo efficacy of WA was studied using mouse model of Graft-versus-host disease. In conclusion, WA is a potent inhibitor of T-cell responses and acts via a novel thiol dependent mechanism and inhibition of NF-κB pathway.
authors.affiliation: Radiation Biology & Health Sciences Division, Bio-science Group, Bhabha Atomic Research Centre, Mumbai, India.
authors.forename: Lokesh
authors.initials: L
authors.lastname: Gambhir
authors.affiliation: Radiation Biology & Health Sciences Division, Bio-science Group, Bhabha Atomic Research Centre, Mumbai, India.
authors.forename: Rahul
authors.initials: R
authors.lastname: Checker
authors.affiliation: Radiation Biology & Health Sciences Division, Bio-science Group, Bhabha Atomic Research Centre, Mumbai, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Sharma
authors.affiliation: Radiation Biology & Health Sciences Division, Bio-science Group, Bhabha Atomic Research Centre, Mumbai, India.
authors.forename: M
authors.initials: M
authors.lastname: Thoh
authors.affiliation: Advanced Centre for Treatment Research and Education in Cancer, Kharghar, Navi Mumbai, India.
authors.forename: Anand
authors.initials: A
authors.lastname: Patil
authors.affiliation: Institute of Chemical Technology, Matunga, Mumbai, India.
authors.forename: M
authors.initials: M
authors.lastname: Degani
authors.affiliation: Advanced Centre for Treatment Research and Education in Cancer, Kharghar, Navi Mumbai, India.
authors.forename: Vikram
authors.initials: V
authors.lastname: Gota
authors.affiliation: Radiation Biology & Health Sciences Division, Bio-science Group, Bhabha Atomic Research Centre, Mumbai, India. Electronic address: sskumar@barc.gov.in.
authors.forename: Santosh K
authors.initials: SK
authors.lastname: Sandur
mesh_terms: D021581:Active Transport, Cell Nucleus; D056704:Adaptive Immunity / Q000187:drug effects*; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D001402:B-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D049109:Cell Proliferation / Q000187:drug effects; D016207:Cytokines / Q000378:metabolism; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D006086:Graft vs Host Disease / Q000188:drug therapy / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007111:Immunity, Cellular / Q000187:drug effects*; D007166:Immunosuppressive Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000187:drug effects*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D011401:Promoter Regions, Genetic; D000067996:RAW 264.7 Cells; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D013997:Time Factors; D054358:Withanolides / Q000493:pharmacokinetics / Q000494:pharmacology*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D000975:Antioxidants; D016207:Cytokines; D007166:Immunosuppressive Agents; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D054358:Withanolides; C009684:withaferin A
keywords: Anti-inflammatory; Cytokines; Graft-versus-host disease; Pharmacokinetics; ROS