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[p14ARF enhances cisplatin-induced apoptosis in human osteosarcoma cells in p53-independent pathway]
Xiufang Huang1, Xiangwei Yuan2, Zhongxian Chen1
1Department of Pathology, the Affiliated Jiangmen Hospital, Sun Yat-sen University, Jiangmen 529030, China.
Objective:
To study effect of tumor suppressor p14ARF on cisplatin-induced apoptosis in human osteosarcoma cells with its molecular mechanisms to provide evidences for increasing chemosensitivity of osteosarcoma.
Methods:
pcDNA3.1-p14ARF plasmid was stable transfected into MG63 cells lack of p14ARF expression. Expression of p14ARF on mRNA and protein level was evaluated with RT-PCR and Western blot. MG63, MG63-vec and MG63-ARF cells were treated with cisplatin. Cell growth inhibition and IC50 were determined through MTT assay. Apoptosis was detected using fluorescence-activated cell sorting and Hoechst33258 staining. The expression of p53, Bax, p21, Mdm2, Fas, Caspase-3, caspase-9 and PARP was detected with Western blot. RNAi was used to silence p53. Cells were pre-treated with Caspase-9 specific inhibitor Z-LEHD-FMK to determine whether the effect was Caspase-9-dependent.
Results:
There was no expression of p14ARF in MG63 and MG63-vec cells but obvious expression in MG63-ARF cells on mRNA and protein level. Cell viability was 84.2%±4.3%, 80.8%±4.3% and 58.9%±5.4% in MG63, MG63-vec, and MG63-ARF cells after treatment of cisplatin for 72 h. IC50 was (11.1±0.6), (10.7±0.9) and (7.2±0.7) µmol/L. The apoptotic rate was 13.6%, 18.5% and 35.9% in groups, There were more obvious apoptotic more changes in MG63-ARF cells than MG63 and MG63-vec cells, and activation of Caspase-3, 9 and PARP on higher level in U2OS-ARF cells after stimulation with cisplatin for 72 h. The expression of p53, Bax, p21, Mdm2 and Fas, in MG63-vec and MG63-ARF cells did not changed (P>0.05). The expression of p53 was effectively and continuously suppressed by p53-siRNA in U2OS-vec and U2OS-ARF cells. The p53 silencing did not alter the cytotoxicity mediated by cisplatin treatment for 72 h (P>0.05). Cell viability was 96.8%±3.6%, 54.1%±5.7% and 89.5%±5.1% in Z-LEHD-FMK, cisplatin and Z-LEHD-FMK+cisplatin groups.
Conclusion:
p14ARF enhances cisplatin-induced apoptosis in human osteosarcoma MG63 cells in p53-independent caspase-9-dependent pathway, in which the intrinsic mitochondrial apoptotic pathway is involved.
Insights
The tumor suppressor p14ARF enhances cisplatin-induced apoptosis in human osteosarcoma cells. This occurs through a p53-independent, caspase-9-dependent pathway involving the intrinsic mitochondrial apoptotic pathway.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Osteosarcoma is a challenging bone cancer with limited treatment options.
- Chemotherapy resistance remains a significant obstacle in treating osteosarcoma.
- The tumor suppressor p14ARF plays a role in cell cycle regulation and apoptosis.
Purpose of the Study:
- To investigate the effect of p14ARF on cisplatin-induced apoptosis in human osteosarcoma cells.
- To elucidate the molecular mechanisms underlying p14ARF's influence on chemosensitivity.
- To provide evidence for enhancing osteosarcoma chemosensitivity through p14ARF modulation.
Main Methods:
- Stable transfection of MG63 osteosarcoma cells with pcDNA3.1-p14ARF plasmid.
- Assessment of p14ARF expression via RT-PCR and Western blot.
- Treatment with cisplatin and evaluation of cell viability (MTT assay), IC50, and apoptosis (FACS, Hoechst staining).
- Western blot analysis of apoptosis-related proteins (p53, Bax, p21, Mdm2, Fas, Caspase-3, caspase-9, PARP).
- p53 silencing using RNAi and assessment of cisplatin cytotoxicity.
- Inhibition of Caspase-9 to determine pathway dependency.
Main Results:
- p14ARF expression was successfully established in MG63-ARF cells.
- MG63-ARF cells exhibited significantly reduced viability and lower IC50 values after cisplatin treatment compared to control cells.
- Cisplatin treatment led to a higher apoptotic rate in MG63-ARF cells.
- Activation of Caspase-3, caspase-9, and PARP was elevated in MG63-ARF cells post-cisplatin stimulation.
- p53 silencing did not affect cisplatin-induced cytotoxicity, indicating a p53-independent mechanism.
- Caspase-9 inhibition partially reversed the effect of cisplatin, confirming its crucial role.
Conclusions:
- p14ARF significantly enhances cisplatin-induced apoptosis in human osteosarcoma MG63 cells.
- The mechanism is p53-independent and caspase-9-dependent.
- The intrinsic mitochondrial apoptotic pathway is implicated in p14ARF-mediated chemosensitization.
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