The mode of action of the HIV protease inhibitor lopinavir against HPV

Abstract

Human papillomavirus (HPV) related cervical cancer is still the most commongynaecological malignancy in developing countries and, as yet, there is noalternative to surgery for the treatment of HPV-associated pre-malignant lesions.HPV 'hijack's' the host-cell ubiquitin-proteasome system to degrade the p53 andRb tumour suppressor proteins which in turn, leads to the development of cancer.Previous studies have shown that the HIV protease inhibitor lopinavir selectivelyinhibits the chymotryptic-like activity of the 26S proteasome which stabilises p53and induces the apoptosis of HPV positive cervical carcinoma cells. Based on thisit was hypothesised that lopinavir treatment of HPV positive cervical carcinomacells would produce changes in the levels of a wide range of cellular proteins thatare dis-regulated by HPV-related activation of the proteasome. In order to addressthis, antibody microarray screening was carried out on lopinavir treated and controluntreated HPV positive SiHa cervical carcinoma cells. This showed lopinavirinduced alterations in 51 proteins including the cellular antiviral defence proteinRNase L. Lopinavir induced both a dose and time dependent increase in RNase Lwhich was subsequently confirmed by western blotting. Transient siRNA silencingof RNase L expression reduced the lopinavir-dependent toxicity in SiHa cells,suggesting an important role for this protein in the toxicity of lopinavir in HPVinfected cells. SiHa cells were much more sensitive to lopinavir than CaSKicervical carcinoma cells which had much higher levels of the E6 protein and didnot up regulate RNase L. Furthermore, lopinavir treated HPV16 E6/E7immortalised keratinocytes were also shown to up regulate RNase L proteinexpression and these cells were much more sensitive to lopinavir inducedapoptosis than mortal control keratinocytes. In addition, transient expression ofRNase L in RNase L-deficient C33A cells and the same cells stably transfectedwith HPV16 E6 (C33AE6) demonstrated that E6 protected these cells from RNaseL-induced cell death. Surprisingly, analysis of RNase L protein levels in these cellsdemonstrated that E6 did not induce the degradation of the RNase L protein.Instead it was found that E6 stabilised the interaction between RNase L and itsendogenous inhibitor protein, ABCE1, and that lopinavir de-stabilised thisinteraction. Given that C33A tumour cells, E6/E7 immortalised keratinocytes andhTert immortalised keratinocytes are all sensitive to lopinavir, this implies that thiscompound does not specifically target HPV immortalised cells but rather targetsimmortalised cells in general, regardless of how this was achieved. The optimumconcentration of lopinavir for all these effects was 25 μM, which is 15-fold higherthan is observed in cervico-vaginal secretions following oral dosing with the drugKaletra. In conclusion these results have confirmed the potential of lopinavir totreat HPV related pre-cancerous cervical lesions and provided at least part of themode-of-action. Indeed they strongly support the use of lopinavir as a low-cost,self-applied topical alternative to surgery for this disease which will be of particularbenefit in low-resource countries. Finally, the ability of lopinavir to induceapoptosis of non-HPV related immortalised cells merits further investigation sincethis indicates this drug may be useful for the treatment of other non HPV relatedpre-malignant conditions.

Date of Award	1 Aug 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Lynne Hampson (Supervisor) & Ian Hampson (Supervisor)