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Detection of the Calcium and ATP Role in Apoptosis of Retinoblastoma Culture Cells through Caspase-3 Expression


Affiliations
1 Department of Ophthalmology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
2 Department of Neurology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
3 Department of Phisiology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
4 Department of Hematology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
5 Department of Clinical Pathology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
     

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Retinoblastoma is a malignant retinal tumor associated with apoptotic deregulation. Retinoblastoma cells are sensitive to NK cells. These cells can stimulate apoptosis. Apoptosis in retinoblastoma occurs in the early phase and is paradoxical. The increasing amounts of Bcl-2 and Caspase-3 as apoptotic executors are inversely proportional to apoptosis. Calcium and ATP as second messengers and signaling molecules play role in mediating cell responses including retinoblastoma cell development and death. Apoptosis requires sufficient energy from ATP and its mediated by Calcium. This study aimed to perceive the effects of Calcium and ATP in the process of retinoblastoma cell death through Caspase-3 pathway. The subjects were poorly differentiated retinoblastoma cell cultures treated with NK cells (treatment group) compared to those which were not exposed to NK cells (control group). Through the cell flowcytometry test that expresses Caspase-3 and apoptosis is calculated, meanwhile the levels of Calcium and ATP activity produced during the apoptosis process are quantitatively calculated. Examination of ATP activity uses a colorimetric method while the calcium content is calculated using a clinical chemistry system. Calcium and ATP were negatively correlated at 27.4% (p <0.05). ATP also showed a very significant negative correlation of 75.8% (p <0.01) against Caspase-3 and is significantly positively correlated with apoptosis of 46.8% (p,0.05). Whereas Caspase-3 is negatively correlated with apoptosis by 46.6% (p <0.05). In the Caspase-3 pathway, allogeneic NK cell administration in retinoblastoma cells increases the level of Calcium which plays a role in the early phase apoptosis process, whereas ATP which was formed is insufficient to cause maximal apoptosis due to extracellular calcium entry into NK cells. Extracellular ATP does not play a role in the induction of apoptosis in retinoblastoma cells treated with NK cells.

Keywords

Allogeneic NK Cells, Caspase-3, Apoptosis, Calcium, ATP.
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  • Detection of the Calcium and ATP Role in Apoptosis of Retinoblastoma Culture Cells through Caspase-3 Expression

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Authors

Hendrian D. Soebagjo
Department of Ophthalmology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
Susy Fatmariyanti
Department of Ophthalmology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
Paulus Sugianto
Department of Neurology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
Bambang Purwanto
Department of Phisiology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
Ugroseno Y. Bintoro
Department of Hematology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia
Endang R. Kusumowidagdo
Department of Clinical Pathology, University of Airlangga, Dr. Soetomo General Hospital Surabaya, Surabaya, East Java, Indonesia

Abstract


Retinoblastoma is a malignant retinal tumor associated with apoptotic deregulation. Retinoblastoma cells are sensitive to NK cells. These cells can stimulate apoptosis. Apoptosis in retinoblastoma occurs in the early phase and is paradoxical. The increasing amounts of Bcl-2 and Caspase-3 as apoptotic executors are inversely proportional to apoptosis. Calcium and ATP as second messengers and signaling molecules play role in mediating cell responses including retinoblastoma cell development and death. Apoptosis requires sufficient energy from ATP and its mediated by Calcium. This study aimed to perceive the effects of Calcium and ATP in the process of retinoblastoma cell death through Caspase-3 pathway. The subjects were poorly differentiated retinoblastoma cell cultures treated with NK cells (treatment group) compared to those which were not exposed to NK cells (control group). Through the cell flowcytometry test that expresses Caspase-3 and apoptosis is calculated, meanwhile the levels of Calcium and ATP activity produced during the apoptosis process are quantitatively calculated. Examination of ATP activity uses a colorimetric method while the calcium content is calculated using a clinical chemistry system. Calcium and ATP were negatively correlated at 27.4% (p <0.05). ATP also showed a very significant negative correlation of 75.8% (p <0.01) against Caspase-3 and is significantly positively correlated with apoptosis of 46.8% (p,0.05). Whereas Caspase-3 is negatively correlated with apoptosis by 46.6% (p <0.05). In the Caspase-3 pathway, allogeneic NK cell administration in retinoblastoma cells increases the level of Calcium which plays a role in the early phase apoptosis process, whereas ATP which was formed is insufficient to cause maximal apoptosis due to extracellular calcium entry into NK cells. Extracellular ATP does not play a role in the induction of apoptosis in retinoblastoma cells treated with NK cells.

Keywords


Allogeneic NK Cells, Caspase-3, Apoptosis, Calcium, ATP.

References