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Efficient Stable Cell Line Generation of Survivin as an In Vitro Model for Specific Functional Analysis in Apoptosis
Rashid Houshdarpour1, Farangis Ataei2, Saman Hosseinkhani1
1Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Abstract:
Recognizing proteins that lead to a decreased efficiency of treatment in cancer cells constitutes a main goal for biomedical and biotechnological research and applications. Establishing recombinant cells that overexpress a gene of interest stably is important for treatment studies and drug/compound screening. Survivin is an anti-apoptotic protein which can be a potential candidate for regulating cell death and survival. To investigate the association between survivin increment and apoptosis rate, survivin-reconstituted HEK (HEK-S) cell was developed as in vitro model. RT-PCR and Western blot demonstrated that survivin was constitutively overexpressed in HEK-S cells. Both morphological observation and survival assay showed that HEK-S cells were significantly resistant to apoptotic stimuli. Survivin overexpression led to a decrease in caspase 3/7 activity, whereas YM155 led to a corresponding enhance of caspase activity. ROS level was decreased but ATP content increased in HEK-S cells. Also, HEK-S showed less red- fluorescence and reduced cell proliferation compared to HEK after stimulation. Resistance to laser irradiation was clearly visible as compared with control. Moreover, scratching analysis demonstrated the ability of survivin to cause neighboring cells to increase resistance to drug, whereas YM155 enhanced apoptotic rate and declined invasion in HEK-S cells.
Insights
Overexpressing survivin, an anti-apoptotic protein, in HEK cells enhances resistance to cancer treatments and reduces apoptosis. This finding is crucial for developing novel cancer therapies and drug screening methods.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Identifying proteins affecting cancer treatment efficiency is vital.
- Survivin, an anti-apoptotic protein, regulates cell death and survival.
- Stable overexpression of genes in recombinant cells aids treatment studies and drug screening.
Purpose of the Study:
- To investigate the link between survivin levels and apoptosis rate.
- To develop an in vitro model for studying survivin's role in apoptosis resistance.
- To analyze the effects of survivin overexpression on cellular responses to stimuli.
Main Methods:
- Development of survivin-reconstituted HEK (HEK-S) cells.
- Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western blot for survivin expression analysis.
- Morphological observation, survival assays, caspase activity assays, ROS level, ATP content measurement, and scratching analysis.
Main Results:
- Survivin was constitutively overexpressed in HEK-S cells.
- HEK-S cells exhibited significant resistance to apoptotic stimuli, decreased caspase 3/7 activity, increased ATP content, and reduced ROS levels.
- Survivin overexpression conferred resistance to laser irradiation and drug treatment in neighboring cells, while YM155 (a survivin inhibitor) enhanced apoptosis and reduced invasion.
Conclusions:
- Survivin overexpression confers significant resistance to apoptosis and various cellular stresses.
- Survivin plays a critical role in regulating cell survival and resistance to cancer treatments.
- Targeting survivin may offer a therapeutic strategy for overcoming treatment resistance in cancer.
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