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Paclitaxel induces apoptosis in Saos-2 cells with CD95L upregulation and Bcl-2 phosphorylation
B Pucci1, L Bellincampi, M Tafani
1Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Abstract:
We examined the effect of paclitaxel on human osteoblastic cells Saos-2 to determine if paclitaxel can affect proliferation and apoptosis. We used a p53-negative cell line in order to mimic the loss of function frequently observed at the clinical level. Paclitaxel induced cell death in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were observed by Hoechst 33258 stain, DNA ladder formation, electron microscopy, and flow cytometry at concentrations as low as 100 nM, a concentration which can be achieved by infusion in human plasma. At 100 nM, paclitaxel induced a G2 arrest at 8 h of treatment. The cells then continued to accumulate in G2 until 72 h when the percentage of apoptotic events reached 54%. At the molecular level, Bcl-2 protein was phosphorylated at 16 h and PARP protein was cleaved, indicating the activation of caspase-3-like proteases. Caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK rescued Saos-2 cells from paclitaxel-induced apoptosis. CD95 expression was constantly high, while CD95L showed a threefold increase in expression. This suggests that, following the G2 arrest, apoptosis is induced through the CD95/CD95L system.
Insights
Paclitaxel induces apoptosis in human osteoblastic cells (Saos-2) by causing G2 cell cycle arrest and activating the CD95/CD95L pathway. This cell death occurs in a dose- and time-dependent manner, even at low concentrations.
Area of Science:
- Cell Biology
- Molecular Biology
- Pharmacology
Background:
- Osteoblastic cells are crucial for bone formation and remodeling.
- Paclitaxel is a chemotherapy drug known to induce apoptosis.
- Understanding paclitaxel's effects on osteoblasts is important for managing bone health during cancer treatment.
Purpose of the Study:
- To investigate the effects of paclitaxel on human osteoblastic Saos-2 cell proliferation and apoptosis.
- To elucidate the molecular mechanisms underlying paclitaxel-induced cell death in a p53-negative osteoblastic cell line.
Main Methods:
- Saos-2 cells were treated with varying concentrations of paclitaxel.
- Cell death, nuclear morphology, cell cycle progression, and protein expression were analyzed.
- Techniques included Hoechst staining, DNA laddering, electron microscopy, flow cytometry, and Western blotting.
- Caspase inhibitors were used to assess the role of caspases in apoptosis.
Main Results:
- Paclitaxel induced dose- and time-dependent cell death in Saos-2 cells.
- Apoptosis was characterized by nuclear condensation, chromatin fragmentation, and DNA ladder formation.
- Paclitaxel caused a G2 cell cycle arrest at 100 nM, leading to significant apoptosis by 72 hours.
- Bcl-2 phosphorylation, PARP cleavage, and increased CD95/CD95L expression indicated caspase activation and involvement of the CD95 pathway.
Conclusions:
- Paclitaxel induces apoptosis in human osteoblastic Saos-2 cells via G2 arrest and activation of the CD95/CD95L signaling pathway.
- The findings highlight paclitaxel's potential impact on bone cells and suggest therapeutic strategies involving caspase and CD95 inhibition.