title: Identification and quantitative analysis of cellular proteins affected by treatment with withaferin a using a SILAC-based proteomics approach.
abstract: BACKGROUND
  Withaferin A (WA) is a major bioactive compound isolated from the medicinal plant Withania somnifera Dunal, also known as "Ashwagandha". A number of published reports suggest various uses for WA including its function as an anti-inflammatory and anti-angiogenic drug molecule. The effects of WA at the molecular level in a cellular environment are not well understood. Knowledge of the molecular mechanism of action of WA could enhance its therapeutic value and may reveal novel pathways it may modulate.
  
  
  METHODS
  In order to identify and characterize proteins affected by treatment with WA, we used SILAC- based proteomics analysis on a mouse microglial cell line (N9), which replicates phenotypic characteristics of primary microglial cells.
  
  
  RESULTS
  Using stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry (MS), a total of 2300 unique protein groups were identified from three biological replicates, with significant expression changes in 32 non-redundant proteins. The top biological functions associated with these differentially expressed proteins include cell death and survival, free radical scavenging, and carbohydrate metabolism. Specifically, several heat shock proteins (Hsps) were found to be upregulated, which suggests that the chaperonic machinery might be regulated by WA. Furthermore, our study revealed several novel protein molecules that were not previously reported to be affected by WA. Among them, annexin A1, a key anti-inflammatory molecule in microglial cells was found to be downregulated. Hsc70, Hsp90α and Hsp105 were found to be upregulated. We also found sequestosome1/p62 (p62) to be upregulated. We performed Ingenuity Pathway Analysis (IPA) and found a number of pathways that were affected by WA treatment.
  
  
  CONCLUSIONS
  SILAC-based proteomics analysis of a microglial cell model revealed several novel proteins whose expression is regulated by WA and probable pathways regulated by WA.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Byrd Alzheimer's Institute, University of South Florida-Health, 4001 E. Fletcher Ave, MDC36, Tampa, FL 33613, United States.
authors.forename: Malathi
authors.initials: M
authors.lastname: Narayan
authors.affiliation: Florida Center of Excellence for Drug Discovery & Innovation at the University of South Florida, 3720 Spectrum Blvd., Suite 303, IDR Building, Tampa, FL 33612, United States.
authors.forename: Kent W
authors.initials: KW
authors.lastname: Seeley
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, Byrd Alzheimer's Institute, University of South Florida-Health, 4001 E. Fletcher Ave, MDC36, Tampa, FL 33613, United States. Electronic address: UJinwal@health.usf.edu.
authors.forename: Umesh K
authors.initials: UK
authors.lastname: Jinwal
mesh_terms: D000818:Animals; D017305:Annexin A1 / Q000378:metabolism; D050260:Carbohydrate Metabolism; D016923:Cell Death; D002460:Cell Line; D002470:Cell Survival; D006360:Heat-Shock Proteins / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D051379:Mice; D017628:Microglia / Q000187:drug effects* / Q000378:metabolism; D040901:Proteomics; D054358:Withanolides / Q000494:pharmacology*
chemical_list: D017305:Annexin A1; D006360:Heat-Shock Proteins; D054358:Withanolides; C009684:withaferin A
keywords: Ashwagandha; Microglial cell; SILAC-based proteomics analysis; Withaferin A; Withania somnifera