Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy

Abstract

Background: Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential.

Results: The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude.

Conclusions: Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.

Figure 1

The structure of metallo-sulphonated phthalocyanines (M = metal; R = SO3-/H).

Figure 2

Protein microarray technology principle.

Figure 3

Apoptosis evaluation by flow-cytometry of DOK cell line subjected to PDT. A) Apoptosis evaluation for triplicates repeated in four identical experiments. Annexin V-FITC and PI (red) quantified early, late apoptosis and necrosis: An-Pi- viable cells; An + Pi- early apoptotic cells; An + Pi + late apoptotic/necrotic cells. Cell samples are from the same batches that were subjected post-irradiation to Protein microarray analysis. B) Example of flow cytometry registration of un-loaded (control) and loaded cells with AlS₂Pc and subjected to PDT.

Figure 4

Results of protein microarray analysis of the main proteins involved in apoptosis in DOK cells following PDT using AlS2Pc; mean values and standard deviations are reported, measured in arbitrary fluorescent units. Control cells: un-loaded irradiated DOK cells; AlS2Pc cells: loaded irradiated DOK cells.

Figure 5

Molecular mechanisms activated by photodynamic therapy using AlS2Pc in DOK cell line – delineative representation resulted from protein microarray analysis.