Published in:
Open Access 01-12-2012 | Research
GCS overexpression is associated with multidrug resistance of human HCT-8 colon cancer cells
Published in: Journal of Experimental & Clinical Cancer Research | Issue 1/2012
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Purpose
Multidrug resistance is one of the main impediments to the successful treatment of colon cancer. Glucosylceramide synthase (GCS) which is related to multidrug resistance (MDR) can reduce the level of ceramide and can help cells escape from the ceramide-induced cell apoptosis. However, the underlying mechanism is still unclear.
Methods
The cell proliferation and cell toxicity were measured with Cell Counting Kit-8 (CCK-8). The mRNA levels of GCS and MDR1 were detected by semiquantitative reverse transcription-PCR amplification, the protein levels of GCS, caspase-3 and P-gp proteins were indicated by Western blotting. The apoptosis rates of cells were measured with flow cytometry.
Results
The relative mRNA levels of GCS in HCT-8, HCT-8/VCR, HCT-8/VCR- sh-mock and HCT-8/VCR-sh-GCS were 71.4 ± 1.1%, 95.1 ± 1.2%, 98.2 ± 1.5%, and 66.6 ± 2.1% respectively. The mRNA levels of MDR1 were respectively 61.3 ± 1.1%, 90.5 ± 1.4%, 97.6 ± 2.2% and 56.1 ± 1.2%. The IC50 of Cisplatin complexes were respectively 69.070 ± 0.253 μg/ml, 312.050 ± 1.46 μg/ml, 328.741 ± 5.648 μg/ml, 150.792 ± 0.967 μg/ml in HCT-8, HCT-8/VCR, HCT-8/VCR-sh-mock and HCT-8/VCR-sh-GCS. The protein levels of caspase-3 were 34.2 ± o.6%, 93.0 ± 0.7%, 109.09 ± 0.7%, 42.7 ± 1.3% respectively. The apoptosis rates of cells were 8.77 ± 0.14%, 12.75 ± 0.54%, 15.39 ± 0.41% and 8.49 ± 0.23% respectively.
Conclusion
In conclusion, our research indicated that suppression of GCS restores the sensitivity of multidrug resistance colon cancer cells to drug treatment.