Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells
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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.
KeywordsHPV Indinavir Lopinavir Raman chemical mapping LC-MS
This research was funded by an ORS award to D-H.K. R.M.J. is very thankful to the UK BBSRC for funding. J.W.A., E.M.-E. and R.G. are grateful to the EU-funded project, Metabolomics for Plants Health and OutReach (META-PHOR: FOOD-CT-2006-036220), for financial support. R.G. is grateful to both the UK BBSRC and EPSRC for financial support of the MCISB. The authors also acknowledge the support of the Humane Research Trust, the Caring Cancer Research Trust and Cancer Research UK.
The authors have no conflict of interest
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