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

Acetylation of histones, along with other epigenetic modifications, has been described
as the main epigenetic regulator controlling cell fate and therefore histone deacetylase
inhibitors (HDIs) are being investigated as a new promising group of anticancer drugs
(De Ruijter et al., 2003). HDIs induce hyperacetylation of proteins/histones, thus
decondensing chromatine structure, which subsequently increases the accessibility of
DNA to transcription factors and activates specific genes, tumor suppressor, oncogenes
and non-histones proteins implicated in differentiation, proliferation, cell cycle and
apoptosis. Moreover, HDIs may enhance, through processes described above, cytotoxicity
of drugs targeting DNA. HDIs are of both natural and synthetic origin and they have
demonstrated potent anticancer activity in many pre-clinical and clinical trials, either
as monotherapies or in combination with conventional chemotherapy (Glaser, 2007).
These include derivates of hydroxamic acid: suberoylanilid hydroxamic acid (Vorinostat,
SAHA - treatment of cutaneous T cell lymphoma) and trichostatin A (TSA - antifungal
antibiotic); and a group of short-chain fatty acids HDIs: valproic acid (VPA - epilepsy drug)
and sodium phenylbutyrate (NaPB – disorders of the urea cycle).
Remodeling of chromatin structure may contribute to enhanced sensitivity to
photochemical and photobiological processes caused by PDT, increasing the ability of
oxygen radicals to attack DNA thus influence cell differentiation and cell death. The aim
of this study was therefore to evaluate the impact of HDIs alone and in combination with
hypericin-mediated photodynamic therapy (HY-PDT) on the response of a human HT-29
colon adenocarcinoma cell line.
Based on extensive screening using the MTT assay, one hypericin and two concentrations
for each HDI were chosen for further experiments. Analysis of total cell number
demonstrated that pre-treatment with all HDIs alone and HY-PDT reduced the total cell
number significantly. A more pronounced drop in this parameter was observed when
combined treatment of HDIs with HY-PDT was applied. The impact of HDIs and of HY-PDT
was further characterized using flow cytometry. Lower concentrations of TSA significantly
decreased mitochondrial membrane potential (MMP) in comparison to other HDIs. Pretreatment
with higher concentrations of SAHA and TSA made cells more vulnerable to
damage resulting from HY-PDT and this led to a considerable dissipation of the MMP. A
similar pattern of the effect on MMP between the two groups of HDIs was also observed.
Subsequently, we analysed the effect of HDIs, HY-PDT and their combination on the
viability/metabolic activity of cancer cells. Similarly to MMP, we observed a higher
potential of the hydroxamic acid derivates (SAHA, TSA) to sensitize cancer cells to the
effect of HY-PDT. We detected a significantly higher number of dead cells in combined
treatment than when drugs were applied alone at both time intervals. The analysis of
cell cycle distribution revealed an accumulation of cells in the S phase after treatment
with higher concentrations of SAHA, TSA and VPA in combination with HY-PDT.
In this study we observed that HDIs are able to modulate and enhance the cytotoxic effects
of HY-PDT. However, the details of molecular mechanisms mediating photodynamicsensitization
by HDIs are at present, not elucidated.
Acknowledgements
This work was supported by the Slovak Research and Development Agency under
contract no. APVV-0040-10 and VVCE-0001-07 and the Scientific Grant Agency of the
Ministry of Education of the Slovak Republic under contract No. VEGA 1/0626/11.
102 Analytical Cytometry VII