GCS overexpression is associated with multidrug resistance of human HCT-8 colon cancer cells

Abstract

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.

Figure 1

Knocking down GCS inhibits mRNA expression of MDR1 and protein level of P-pg. A, the mRNA level are higher in HCT-8/VCR cells compared with HCT-8 cells. The GCS mRNA level decreased when transfected with shGCS plasmids. The MDR1 gene expressin increased in HCT-8/VCR cells compared with HCT-8 cells. The MDR1 mRNA level also decreased when knocking down GCS. B, the protein level of P-pg decreased when knocking down GCS. Protein level of β-actin was set as 100%. *Ρ < 0.01 compared with the HCT-8/VCR and HCT-8/VCR-sh-mock cells.

Figure 2

Knocking down GCS suppresses HCT-8/VCR cell proliferation. HCT-8 cell (2 × 10³) were seeded in 96-well in 100 ul PRMI-1640 medium. Cell proliferation was determined at 24-h intervals up to 96 h in sh-mock or sh-GCS stably transfected cells. Data are shown as means ± S.D.

Figure 3

Knocking down GCS causes HCT-8/VCR more sensitive to cisplatin induced cell death. HCT-8, HCT-8/VCR, HCT-8/VCR sh-mock or sh-GCS stably transfected cells (5 × 10³) were seeded in 96-well in 100 ul PRMI-1640 medium. Cells were treated with different concentaration of cisplatin for 48 h. The relative drug resistance folds were analyzed by compared with IC50. The cell survival rate of cells without cisplatin complexes was set as 100%. *Ρ < 0.01 compared with the HCT-8/VCR and HCT-8/VCR-sh-mock cells.

Figure 4

Knocking down GCS affects Caspase-3 protein level. The Caspase-3 protein level decreased when transfected with shGCS plasmids.

Figure 5

Knocking down GCS affects HCT-8/VCR cells apoptosis. The apoptosis of HCT-8, HCT-8/VCR, HCT-8/VCR sh-mock or sh-GCS stably transfected cells were measured with flow cytometry (A, HCT-8, B, HCT-8/VCR, C, HCT-8/VCR-sh-mock and D, HCT-8/VCR-sh-GCS).