Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells.
It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 μM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.
School of Chemistry, Manchester Interdisciplinary Biocentre, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Dong-Hyun
DH
Kim
Roger M
RM
Jarvis
J William
JW
Allwood
Gavin
G
Batman
Rowan E
RE
Moore
Emma
E
Marsden-Edwards
Lynne
L
Hampson
Ian N
IN
Hampson
Royston
R
Goodacre
D002458:Cell Fractionation; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D052162:Human papillomavirus 16 / Q000302:isolation & purification*; D006801:Humans; D019469:Indinavir / Q000494:pharmacology; D061466:Lopinavir; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D030361:Papillomavirus Infections / Q000188:drug therapy* / Q000378:metabolism / Q000821:virology; D025341:Principal Component Analysis; D011744:Pyrimidinones / Q000494:pharmacology; D012097:Repressor Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000821:virology*
C068651:E6 protein, Human papillomavirus type 16; D017320:HIV Protease Inhibitors; D009856:Oncogene Proteins, Viral; D011744:Pyrimidinones; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D061466:Lopinavir; D019469:Indinavir