Figure 1.
Knocking down KRAS expression in KRAS-mutant (G13D) CRC cells confers oxaliplatin resistance and ERCC1 upregulation.
(A) KRAS-knocked-down DLD-1G13D cells were more resistant to oxaliplatin, but have the same sensitivity to irinotecan, 5FU, and doxorubicin than parental DLD-1G13D cells, as demonstrated by MTT assay. (B) The protein level of ERCC1, but not those of TOPO1 or TS, was upregulated after DLD-1G13D cells were knocked-down by KRAS siRNA. (C) The mRNA level of ERCC1, but not those of TOPO1 or TS, was upregulated after DLD-1G13D cells were knocked-down by KRAS siRNA. ***: p<0.001.
Figure 2.
Knocking down KRAS expression in other KRAS-mutant subtype (G12V) CRC cells results in oxaliplatin resistance and ERCC1 upregulation.
(A) KRAS-knocked-down SW480G12V cells were more resistant to oxaliplatin, but have the same sensitivity to irinotecan, 5FU, and doxorubicin than parental SW480G12V cells, as demonstrated by MTT assay. (B) The protein level of ERCC1, but not those of TOPO1 or TS, was upregulated after SW480G12V cells were knocked-down by KRAS siRNA. (C) The mRNA level of ERCC1, but not those of TOPO1 or TS, was upregulated after SW480G12V cells were knocked-down by KRAS siRNA. ***: p<0.001.
Figure 3.
Overexpressing KRAS in KRAS wild-type CRC cells leads to oxaliplatin sensitivity and ERCC1 downregulation.
(A) KRASG13D-DDK-myc-COLO320DM cells were more sensitive to oxaliplatin, but have the same sensitivity to irinotecan, 5FU, and doxorubicin than parental COLO320DM cells, as demonstrated by MTT assay. (B) The protein level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRASG13D mutant vector. (C) The mRNA level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRASG13D mutant vector. **: p<0.01.
Figure 4.
Overexpressing KRAS by another KRAS overexpression vector (G12V) in KRAS wild-type CRC cells leads to oxaliplatin sensitivity and ERCC1 downregulation.
(A) KRASG12V-DDK-myc-COLO320DM cells were more sensitive to oxaliplatin, but have the same sensitivity to irinotecan, 5FU, and doxorubicin than parental COLO320DM cells, as demonstrated by MTT assay. (B) The protein level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRASG12V mutant vector. (C) The mRNA level of ERCC1, but not those of TOPO1 or TS, was downregulated after COLO320DM cells were transfected by the KRASG12V mutant vector. **: p<0.01. (D) Increased percentage of apoptosis, from 22.5%±0.2% to 39.1%±0.2% of apoptosis (P<0.001), has been demonstrated when KRASwt-DDK-myc-COLO320DM cells, were compared to KRASG12V-DDK-myc-COLO320DM cells, in which, both were treated by the same concentration of oxaliplatin in 5 µM. *: p<0.001.
Figure 5.
Validating ERCC1 as the predictor of oxaliplatin sensitivity in CRC cells.
(A) ERCC1-knocked-down COLO320DM cells were more sensitive to oxaliplatin than parental COLO320DM cells, as demonstrated by MTT assay. (B) Protein and mRNA levels of ERCC1 were downregulated when COLO320DM cells were knocked-down by ERCC1 siRNA. *: p<0.05 (C) ERCC1-GFP-SW480G12V cells were more resistant to oxaliplatin than parental SW480G12V cells. (D) Ectopic ERCC1 was upregulated after SW480G12V cells were transfected by the ERCC1-GFP expression vector.
Figure 6.
Downregulation of ERCC1 expression in KRAS-mutant CRC cells might be related to hypermethylation of ERCC1gene, which possibly induced by up-regulation of DNMT3B (DNA methyltransferase 3B).
(A) Protein expression of ERCC1 in DLD-1(KRASG13D mutation) cells is up-regulated after 5′-azacitidine (de-methylating agent) treatment for 96 hours, which implied that the downregulation of ERCC1 in KRAS-mutant CRC cells might be partly through ERCC1 hypermethylation. (B) Downregulation of ERCC1 in COLO320DM (KRAS wild-type) cells transfected by KRASG13D-mutant-vector for 24 and 96 hours may not only be restored by 5′-azacitidine in 10 µM, but also caused up-regulation of DNMT3B.