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[Potential of cdc25A-Fas chimeric expression vector in inducing apoptosis of Tca8113 cells in vitro]
Chong-Jin Feng1, Chun-Yang Li, Yun Hong
1Department of Stomatology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, 510080, PR China.
Background & Objective:
Previous studies have revealed the close relationship between Fas/FasL pathway and carcinogenesis of oral squamous cell carcinoma (OSCC). This study was designed to explore the potential of cdc25A-Fas chimeric expression vector in inducing apoptosis of human OSCC cell line Tca8113.
Methods:
The 2 chimeric expression vector pAdTrack-CMV-cdc25A-Fas (pCCF), and pAdTrack-cdc25A-Fas (pCF) were constructed by gene engineering, pCCF, pCF, and the control plasmid pAdTrack-CMV were transfected into Tca8113 cells by liposome, respectively. The transfection efficiency was presented by the expression of the report gene, green fluorescence protein (GFP). The mRNA and protein levels of Fas were determined by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemistry methods. The apoptosis of transfected Tca8113 cells was analyzed by techniques of DNA agarose gel electrophoresis, TUNEL, Annexin V label, and flow cytometry (FCM).
Results:
(1) The chimeric expression vectors pCCF, and pCF were successfully transfected into Tca8113 cells and the maximum transfection (15%) was observed at the 5th-7th day after transfection. (2) Up-regulation of Fas expression was detected at the 3rd day after transfection in pCCF, and pCF transfection groups. At 3rd, 5th, and 7th day after transfection, Fas protein was found to express on membrane and in plasma of transfected Tca8113 cells with distinct morphology of partial apoptosis. (3) From 2.5 days after transfection, early apoptosis had been observed in pCCF, and pCF transfection groups. At 3rd day, the increased apoptosis index (AI,=25%) of pCCF, and pCF transfection groups was significant higher than that of control group (P< 0.05), whereas there was no significant difference in AI between pCCF transfection group and pCF transfection group (P >0.05). (4) FCM analysis showed that the peak of GFP-expression cells was identical to that of the apoptotic cells.
Conclusion:
The cdc25A-Fas chimeric expression vector was able to initiate the apoptosis of Tca8113 cells by up-regulating Fas expression. This result indicated that the 27 bp cdc25A fragment can be designed as a cis-regulatory element to modulate the effects of C-Myc/Max in cellular proliferation and apoptosis.
Insights
The cdc25A-Fas chimeric expression vector successfully induced apoptosis in oral squamous cell carcinoma (OSCC) cells by upregulating Fas expression. This vector shows potential for targeting OSCC proliferation and apoptosis.
Area of Science:
- Molecular Biology
- Cancer Research
- Cell Biology
Background:
- The Fas/FasL pathway is closely linked to oral squamous cell carcinoma (OSCC) development.
- Understanding this pathway is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To investigate the efficacy of a cdc25A-Fas chimeric expression vector in inducing apoptosis in the human OSCC cell line Tca8113.
- To explore the vector's potential in modulating cancer cell proliferation and apoptosis.
Main Methods:
- Construction of cdc25A-Fas chimeric expression vectors (pCCF, pCF).
- Transfection of Tca8113 cells with vectors and assessment of transfection efficiency via GFP expression.
- Analysis of Fas mRNA and protein levels using Northern blot, RT-PCR, Western blot, and immunohistochemistry.
- Evaluation of apoptosis using DNA gel electrophoresis, TUNEL, Annexin V, and flow cytometry (FCM).
Main Results:
- Successful transfection of Tca8113 cells with pCCF and pCF vectors, with peak transfection efficiency observed 5-7 days post-transfection.
- Significant upregulation of Fas expression (mRNA and protein) in transfected cells, correlating with observed partial apoptosis.
- Early apoptosis detected from 2.5 days post-transfection, with a significantly increased apoptosis index in pCCF and pCF groups compared to controls.
- Flow cytometry analysis confirmed a correlation between GFP expression and apoptotic cells.
Conclusions:
- The cdc25A-Fas chimeric expression vector effectively induces apoptosis in Tca8113 OSCC cells by upregulating Fas expression.
- The 27 bp cdc25A fragment can function as a cis-regulatory element to influence C-Myc/Max-mediated cellular proliferation and apoptosis.
