Silencing the wild-type and mutant K-ras increases the resistance to 5-flurouracil in HCT-116 as a colorectal cancer cell line

Abstract

Colon cancer is the second to third common cancer worldwide. Several efforts have been made to reveal the pathways responsible for drug resistance in this type of cancer. We aimed to investigate the effect of silencing both mutant and wild-type Kristen Rous sarcoma (k-ras) on the response of human colorectal tumor 116 (HCT-116) as a colon cancer cell line to the cytotoxic effect of 5-flurouracil (5-FU). One oligonucelotide against mutant k-ras (12th codon, namely 207) and two against wild-type k-ras (namely 535 and 689) were cloned into pSilencer neo2.1. The linearized vectors besides the negative control plasmid were stably transfected into HCT-116. The proliferation rates of these cells in different concentrations of 5-FU and the apoptosis rates of the cells after treatment with lethal doses of 5-FU were studied. Moreover, the cell cycle in these cells was also analyzed by staining the cells with propidium iodide. Stably transfected cells were named HCT207ks, HCT535ks, HCT689ks, and HCT-Sc (transfected with the negative control plasmid). Decreased expression of k-ras in HCT207ks, HCT535ks, and to a lesser extent in HCT689ks was proved by quantitative real-time PCR. Although in HCT207ks the cells were mostly in G0/G1 and G2/M phases, in HCT535ks and HCT689ks, the cells in the S phase were higher in comparison with nontransfected HCT-116. Lethal doses of 5-FU in HCT-116 and HCT-Sc were 2.5-3 and 3-3.5 µmol/l, whereas in HCT207ks, HCT535ks, and HCT689ks, they were 35-40, 37.5-40, and 22.5-25 µmol/l. In conclusion, silencing mutant and wild-type k-ras would increase the resistance of HCT-116 cell line as a model of colorectal cancer to 5-FU. The degree of resistance was related directly to the k-ras mRNA level. Therefore, both mutant and wild-type k-ras may play a role in sensitizing colorectal cancer cells to 5-FU as a common chemotherapeutic drug.

Down-regulation of let-7 microRNA increased K-ras expression in lung damage induced by radon

“…Since the K-ras gene was reported to be inhibited by let-7 family in various cancers (Büssing et al, 2008;Johnson et al, 2005), it could be used as a potential target gene in tumor therapy (Teimoori-Toolabi et al, 2015) to slow proliferation and tumor growth of lung cancer cells (Esquela-Kerscher et al, 2008;Kumar et al, 2008;Johnson et al, 2007).…”

Eupatilin Impacts on the Progression of Colon Cancer by Mitochondria Dysfunction and Oxidative Stress

“…Importantly, mutations in APC contribute to 5-FU resistance in colon cancer cells [25]. Furthermore, silencing wild-type and mutant KRAS enhances the resistance to 5-FU in colon cancer cell lines [26]. Therefore, the further validation of the effect of eupatilin in colon cancer cells with genetic mutation will lead to conclusive evidence suggesting that eupatilin can exert a therapeutic effect in combination treatment with conventional chemotherapy in colon cancer.…”

Strategies for Improving siRNA-Induced Gene Silencing Efficiency

“…Recent experiences showed that siRNAs closer to the start codon are more efficient than farther ones. 22 Based on the algorithm by Reynolds et al, the sequence preferences of the sense strand are influential on the siRNA efficiency. Reynolds studies on the sense strand showed that the presence of A at the third position and U at the tenth position, A at the nineteenth position, besides the absence of G or C at the nineteenth and G at the thirteenth nucleotide potentiate the silencing effect of siRNA.…”