Apoptosis induction preceded by mitochondrial depolarization in multiple myeloma cell line U266 by
Ken Shirato1, Kazuhiko Imaizumi, Keisuke Miyazawa
1Laboratory of Physiological Sciences, Faculty of Human Sciences, Waseda University, Tokorozawa, Saitama 359-1192, Japan.
Abstract:
The aim of the present study was to investigate the mechanism of apoptosis in human multiple myeloma cell line, U266, caused by 2-aminophenoxazine-3-one (Phx-3). Flow-cytometrical and morphological analyses showed that Phx-3 increased the population of annexin V-positive cells including early stage apoptotic cells and late stage apoptotic cells and induced DNA fragmentation or apoptotic body formation in U266 cells, indicating that Phx-3 induced the apoptosis of U266 cells. Activity of caspase-3 was extensively increased in U266 cells treated with Phx-3 time-dependently within 24 h, but this Phx-3-stimulated activity of the enzyme in the cells was completely cancelled by the addition of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. The addition of z-VAD-fmk almost blocked the apoptotic effect of Phx-3 against U266 cells, indicating that Phx-3-induced apoptosis of U266 cells was dependent on a caspase signaling pathway. Moreover, the apoptosis of U266 cells occurred after the induction of cell cycle arrest of the cells in the S and G(2)/M phase, the loss of mitochondrial membrane potential, and activation of caspase-3 reached maximum, which were caused by Phx-3 within 24 h. These results support the views that the apoptosis of U266 cells caused by Phx-3 may be preceded by the cell cycle arrest, depolarization of mitochondria and activation of caspase-3. These results support the view that Phx-3 may be utilized in future as chemotherapeutic agent against multiple myeloma which is extremely refractory to chemotherapy.
Insights
2-aminophenoxazine-3-one (Phx-3) induces apoptosis in human multiple myeloma cells by activating caspase-3 and causing cell cycle arrest. This suggests Phx-3
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- Multiple myeloma is a hematological malignancy characterized by uncontrolled proliferation of plasma cells.
- Existing chemotherapies for multiple myeloma often face challenges due to drug resistance.
- Investigating novel therapeutic agents is crucial for improving treatment outcomes.
Purpose of the Study:
- To elucidate the mechanism by which 2-aminophenoxazine-3-one (Phx-3) induces apoptosis in the U266 human multiple myeloma cell line.
- To determine the role of caspase signaling pathways in Phx-3-mediated apoptosis.
- To assess the potential of Phx-3 as a chemotherapeutic agent for multiple myeloma.
Main Methods:
- Flow cytometry was used to analyze annexin V-positive cells and DNA fragmentation.
- Morphological assessments were performed to identify apoptotic bodies.
- Caspase-3 activity was measured in Phx-3-treated U266 cells.
- The effect of a pan-caspase inhibitor (z-VAD-fmk) on Phx-3-induced apoptosis was evaluated.
- Mitochondrial membrane potential and cell cycle progression were analyzed.
Main Results:
- Phx-3 treatment significantly increased the population of annexin V-positive cells and induced DNA fragmentation in U266 cells.
- Caspase-3 activity was time-dependently elevated in Phx-3-treated cells, and this activation was inhibited by z-VAD-fmk.
- z-VAD-fmk partially blocked the apoptotic effects of Phx-3, confirming caspase pathway involvement.
- Phx-3 induced cell cycle arrest in the S and G(2)/M phases and decreased mitochondrial membrane potential prior to maximal caspase-3 activation.
- Apoptosis in U266 cells was preceded by cell cycle arrest, mitochondrial depolarization, and caspase-3 activation.
Conclusions:
- Phx-3 induces apoptosis in U266 multiple myeloma cells through a caspase-dependent signaling pathway.
- The apoptotic process initiated by Phx-3 involves cell cycle arrest and mitochondrial membrane potential loss.
- Phx-3 demonstrates potential as a chemotherapeutic agent for refractory multiple myeloma.
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