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

Matei C, Tampa M, Caruntu C, Ion RM, Georgescu SR, Dumitrascu GR, Constantin C, Neagu M - Biol. Res. (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

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.

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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.
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Fig3: 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.

Mentions: In order to prove that the apoptosis mechanisms were induced upon PDT with AlS2Pc we have used flow cytometry evaluation of irradiated non-loaded cells (control) and loaded cells (PDT). From the same cell batches that were subjected to Protein microarray evaluation, at 1.5 hours after irradiation protocol, photosensitizer loaded cells showed a percentage of around 15% cells stained Ann positive and PI negative (early apoptotic events) and nearly 40% cells staining double positive for PI and Ann (late apoptotic events) (Figure 3). The finding that upon PDT mainly late apoptotic events are induced is consistent with previous data[15]. Moreover, the fact that we have found that even cellular membrane is affected upon experimental PDT, as assessed by Ann staining, can either depict the fact that there are still cells that will enter the late apoptotic event and/or as previously published[16]. These types of phthalocyanines can reside in early endosomes thus hindering per se the membrane architecture, hence the positivity of annexin staining.Figure 3


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

Matei C, Tampa M, Caruntu C, Ion RM, Georgescu SR, Dumitrascu GR, Constantin C, Neagu M - Biol. Res. (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4125699&req=5

Fig3: 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.
Mentions: In order to prove that the apoptosis mechanisms were induced upon PDT with AlS2Pc we have used flow cytometry evaluation of irradiated non-loaded cells (control) and loaded cells (PDT). From the same cell batches that were subjected to Protein microarray evaluation, at 1.5 hours after irradiation protocol, photosensitizer loaded cells showed a percentage of around 15% cells stained Ann positive and PI negative (early apoptotic events) and nearly 40% cells staining double positive for PI and Ann (late apoptotic events) (Figure 3). The finding that upon PDT mainly late apoptotic events are induced is consistent with previous data[15]. Moreover, the fact that we have found that even cellular membrane is affected upon experimental PDT, as assessed by Ann staining, can either depict the fact that there are still cells that will enter the late apoptotic event and/or as previously published[16]. These types of phthalocyanines can reside in early endosomes thus hindering per se the membrane architecture, hence the positivity of annexin staining.Figure 3

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus