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Transduction effect of antisense K-ras on malignant phenotypes in gastric cancer cells
1Institute for Cancer Research, Yonsei University College of Medicine, Yonsei Cancer Center, Seoul, South Korea.
Abstract:
The antitumoral effects of antisense RNA to K-ras were investigated in gastric cancer cell lines by examining the level of K-ras expression and the tumorigenicity in vitro and in vivo. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), DNA sequencing, and immunoblotting analysis revealed that YCC-1 gastric cancer cells overexpressed wild type K-ras, whereas YCC-2 cells had a homozygous mutation in codon 12 from GGT (glycine) to AGT (serine), while SNU-1 cells had a heterozygous mutation to GAT (asparagine) in the identical position. Both YCC-1 and YCC-2 cells were transduced by LNC-AS/K-ras containing the antisense 2.2 kb genomic K-ras DNA fragment covering exon 2 and exon 3 specific for K-ras. The application of antisense K-ras significantly downregulated the expression of K-ras and had no influence on the expression of either H-ras or N-ras. The antisense-transduced YCC-2 cells grew considerably slower than the control group transduced by LNCX, whereas the growth inhibition of antisense-transduced YCC-1 cells was less prominent than that of transduced YCC-2 cells. In addition, the tumorigenicity of YCC-2 cells transduced by LNC-AS/K-ras was totally lost. Therefore, our results imply that the specific inhibition of K-ras p21 protein can be accomplished by introducing the antisense covering the K-ras- specific region to gastric cancer cells with aberrant K-ras expression, resulting in a reduction of the growth rate and suppression of tumorigenicity.
Insights
Antisense RNA targeting K-ras significantly reduced K-ras expression in gastric cancer cells. This inhibition suppressed tumor growth and tumorigenicity, particularly in cells with K-ras mutations.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- K-ras is a key oncogene frequently implicated in various cancers, including gastric cancer.
- Aberrant K-ras expression and mutations are critical drivers of gastric tumor development and progression.
- Targeting K-ras offers a potential therapeutic strategy for gastric cancer.
Purpose of the Study:
- To investigate the antitumoral effects of antisense RNA targeting K-ras in gastric cancer cell lines.
- To evaluate the impact of K-ras inhibition on cancer cell growth and tumorigenicity in vitro and in vivo.
- To assess the specificity of antisense K-ras in downregulating K-ras expression without affecting other ras family members.
Main Methods:
- Gastric cancer cell lines (YCC-1, YCC-2, SNU-1) were characterized for K-ras gene status (wild-type, mutations).
- Cells were transduced with LNC-AS/K-ras containing antisense K-ras DNA or a control vector (LNCX).
- K-ras expression levels were analyzed using Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), DNA sequencing, and immunoblotting. Tumorigenicity was assessed in vivo.
Main Results:
- YCC-1 cells overexpressed wild-type K-ras, while YCC-2 and SNU-1 cells harbored K-ras mutations at codon 12.
- Antisense K-ras transduction significantly downregulated K-ras expression in both YCC-1 and YCC-2 cells, with no effect on H-ras or N-ras.
- Antisense K-ras treatment led to slower growth in YCC-2 cells and complete loss of tumorigenicity, while YCC-1 cells showed less prominent growth inhibition.
Conclusions:
- Specific inhibition of K-ras p21 protein is achievable using antisense RNA targeting the K-ras-specific region.
- Antisense K-ras therapy can reduce growth rate and suppress tumorigenicity in gastric cancer cells with aberrant K-ras.
- This approach holds promise for developing targeted therapies against K-ras-driven gastric cancers.