ABSTRACTS – ORAL PRESENTATIONS - AMCA, spol. s r.o.

P8. PROADIFEN (SKF-525A) ATTENUATES CISPLATIN RESISTANCE IN A2780CIS
OVARIAN CARCINOMA CELLS
Rastislav Jendželovský, Zuzana Papčová, Lucia Hiľovská, Jaromír Mikeš, Ján Kovaľ, Peter
Fedoročko
P. J. Šafárik University in Košice, Faculty of Science, Institute of Biology and Ecology,
Košice, Slovak Republic; rastislav.jendzelovsky@upjs.sk
Cytochrome P450 monooxygenases represent a large family of proteins involved primarily
in phase I metabolism of xenobiotics. Some of these enzymes catalyze the conversion of
arachidonic acid to epoxyeicosatrienoic acids (EETs), hydroxyeicosatrienoic acids (HETEs)
and diols. Proadifen is known as a broad-spectrum inhibitor of P450 monooxygenases.
Because of this ability proadifen should be included in the large class of non-steroidal
anti-inflammatory drugs (NSAIDs) together with inhibitors of cyclooxygenases and
lipoxygenases. Analogous to most of these NSAIDs proadifen possess also antiproliferative
and pro-apoptotic activities in cancer cells originating from different tissues
(Hoferová et al., 2004; Jendželovský et al., 2012).
In our previous study, we have found that pre-treatment of HT-29 colon adenocarcinoma
cells with proadifen administered prior to hypericin-mediated photodynamic therapy
(HY-PDT) increased hypericin content and enhanced oxidative stress in the cells, which
led to the onset of apoptosis. We also found that proadifen as P450 monooxygenase
inhibitor affected the activity of transport proteins MRP1 and BCRP (Jendželovský et
al., 2009). Based on these results we asked, whether proadifen is able to increase the
toxicity of well-known chemotherapeutic agent, cisplatin, whose anticancer effect may
be affected among other mechanisms either by enhanced efflux or increased inactivation
(Materna et al., 2005; Wang et al., 2013).
In this study IC20 values of proadifen in combination with IC20 values of cisplatin were
used to treat both cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian
carcinoma cells. Estimated IC20 values were extrapolated from an exponential (cisplatin)
and polynomial fit (proadifen) to the metabolic activity data. Metabolic activity was
established by MTT assay. Expression of drug efflux transporters (MRP1, MRP2, BCRP),
CYP3A4 and proteins engaged in apoptosis and/or tumour progression was analysed by
Western blot. Phosphatidylserine externalization and cell viability analysis, mitochondrial
membrane depolarization, histone H2AX phosphorylation (pS139) and transport activity
of ABC proteins were examined by flow cytometry.
Combined proadifen and cisplatin treatment inhibited metabolic activity of A2780cis
cells, which was accompanied by significant onset of cell death. Moreover, flow cytometry
analyses revealed that proadifen affected mainly the function of drug efflux protein
MRP2 but also the MRP1 in smaller extend. In agreement with these results, histone
H2AX phosphorylation was also reversed by proadifen pre-treatment. Downregulation
of MRP1, MRP2, CYP3A4 and anti-apoptotic proteins with most pronounced effect on
survivin expression was also observed.
Our findings demonstrate that downregulation of MRP2 and survivin by proadifen
should be primarily responsible for the enhanced cytotoxic effect of cisplatin in A2780cis
96 Analytical Cytometry VII