Induction of Apoptotic Signaling Pathways by 3' methyl ATP in Different Malignant Cells: in vitro Study
1Preventive Oncology, Dr. B.R. Ambedkar Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India.
Abstract:
Extracellular ATP is a dynamic signaling molecule that modulates myriad of cellular functions through P2 purinergic receptors activation and is cytotoxic to a variety of cells at high concentration. But the mechanism of this extracellular ATP/ATP analogs- elicited cytotoxicity is not fully understood. In this study we aim to investigate whether there is differential sensitivity towards induction of apoptosis by ATP analogs (2'-Me ATP and 3'-Me ATP) and its effect on receptor mediated or extrinsic and mitochondria mediated or intrinsic apoptotic signaling pathways. Our findings demonstrated that the IC50 values for 2'-Me ATP and 3'-Me ATP were 3mM and 2mM, respectively, in Hep2, and SiHa cells. The downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, along with a significant increase in the expression of the pro-apoptotic protein Bax (p<0.05), indicated the involvement of both pro- and anti-apoptotic factors in HeP2 cells, whereas in SiHa cells, a downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL was observed, whereas the expression level of the pro-apoptotic protein Bax remained unaffected. Furthermore, an upregulation of p53 and apoptosis-inducing factor (AIF) was observed in HeP2 cells (p<0.05) whereas, an upregulation of p53 was observed while no change was seen on the level of apoptosis inducing factor (AIF) was observed in SiHa cells. Additionally, there was a notable rise in caspase-3 and -9 activities, PARP cleavage, and the release of cytochrome c (p<0.05) from the mitochondria to the cytosol in both cells. Collectively, our study suggests that 3'-Me ATP induces apoptosis in Hep2 and SiHa cells through the intrinsic mitochondrial pathway.
Insights
Extracellular ATP analogs, 2' and 3'-Me ATP, induce apoptosis in Hep2 and SiHa cells. The study reveals 3'-Me ATP triggers cell death primarily via the intrinsic mitochondrial pathway, affecting key apoptotic proteins and signaling molecules.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Extracellular ATP (adenosine triphosphate) is a signaling molecule activating P2 purinergic receptors, with high concentrations exhibiting cytotoxicity.
- The precise mechanisms underlying extracellular ATP-induced cytotoxicity, particularly concerning apoptosis, remain incompletely understood.
- Investigating ATP analogs' cytotoxic effects is crucial for elucidating cell death pathways.
Purpose of the Study:
- To investigate the differential sensitivity of Hep2 and SiHa cells to apoptosis induced by ATP analogs (2'-Me ATP and 3'-Me ATP).
- To determine the involvement of receptor-mediated (extrinsic) and mitochondria-mediated (intrinsic) apoptotic signaling pathways in ATP analog-induced cytotoxicity.
- To elucidate the specific pathway through which 3'-Me ATP induces apoptosis in these cell lines.
Main Methods:
- Cell culture of Hep2 and SiHa cell lines.
- Determination of IC50 values for 2'-Me ATP and 3'-Me ATP.
- Analysis of apoptosis-related protein expression (Bcl-2, Bcl-xL, Bax, p53, AIF) using Western blotting or similar techniques.
- Assessment of caspase-3 and -9 activities, PARP cleavage, and cytochrome c release to evaluate mitochondrial pathway activation.
Main Results:
- The IC50 values for 2'-Me ATP and 3'-Me ATP were determined as 3mM and 2mM in Hep2 and SiHa cells, respectively.
- In Hep2 cells, apoptosis involved downregulation of Bcl-2/Bcl-xL, upregulation of Bax, p53, and AIF. In SiHa cells, Bcl-2/Bcl-xL were downregulated, Bax was unaffected, and p53 was upregulated, with no change in AIF.
- Both cell lines exhibited increased caspase-3/-9 activity, PARP cleavage, and cytochrome c release, indicating intrinsic pathway activation.
Conclusions:
- 3'-Me ATP induces apoptosis in Hep2 and SiHa cells, with distinct molecular profiles in each cell line.
- The primary mechanism of 3'-Me ATP-induced apoptosis in these cells involves the intrinsic mitochondrial pathway.
- Findings contribute to understanding extracellular ATP signaling and its role in programmed cell death.
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