Related Experiment Video
Updated: Aug 16, 2026
In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice
Published on: August 23, 2019
Inhibition of thymidylate synthase activity by antisense oligodeoxynucleotide and possible role in thymineless
1School of Medical Technology, National Taiwan University, Taipei, Taiwan, Republic of China.
Abstract:
Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer. However, efficacy of TS-targeted anticancer drugs is limited by the development of drug resistance as a result of TS gene amplification. In this work, a phosphorothioated antisense oligonucleotide (ODN), designated ATS-2, was used to suppress cellular synthesis of TS. ATS-2 at 0.2 microM concentration was mixed with lipofectin in a charge ratio of 1:1 and was used to treat the human embryonic kidney (HEK) cell line. A reduction of TS mRNA and protein was achieved. Furthermore, a dose-dependent reduction of cumulative viable cells of up to 98% was observed. Flow cytometer analysis of cell cycle progression indicates that ATS-2-treated cells were arrested and went into apoptosis at the S phase, possibly because of thymidine shortage, suggesting that ATS-2 is specifically effective for dividing cells. When used in combination with the anticancer drug FdUrd, ATS-2 exerted a additive inhibitory effect on cellular proliferation. To elucidate the possible role of cellular thymidine kinase (TdR kinase) in ATS-2 treatment, a second cell line, HeLa, was used. Both HEK and HeLa have similar rates of cell division and ODN uptake. In contrast to HEK, which was shown to have very low levels of TdR kinase activity in [(3)H]thymidine incorporation experiments, [(3)H]thymidine incorporation in HeLa was 15-fold greater than that of HEK. We found that HeLa cells were sensitive to FdUrd but were rather resistant to ATS-2. On the contrary, HEK cells were sensitive to ATS-2 but insensitive to FdUrd. Effects of ATS-2 and FdUrd are, therefore, complementary in thymineless treatment too.
Insights
A novel antisense oligonucleotide (ATS-2) effectively suppresses thymidylate synthase (TS) synthesis, inhibiting cancer cell growth and inducing apoptosis. ATS-2 shows complementary effects with FdUrd, offering a potential new strategy for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Thymidylate synthase (TS) is a critical target in cancer chemotherapy.
- Drug resistance due to TS gene amplification limits the efficacy of current TS-targeted drugs.
- Antisense oligonucleotides (ODNs) offer a potential strategy to overcome resistance by directly targeting TS synthesis.
Purpose of the Study:
- To investigate the efficacy of a phosphorothioated antisense oligonucleotide (ATS-2) in suppressing cellular TS synthesis.
- To evaluate the impact of ATS-2 on cancer cell viability, cell cycle progression, and apoptosis.
- To explore the combined effects of ATS-2 and the anticancer drug FdUrd and the role of thymidine kinase (TdR kinase) in treatment response.
Main Methods:
- Treatment of human embryonic kidney (HEK) and HeLa cell lines with ATS-2 and/or FdUrd.
- Quantification of TS mRNA and protein levels.
- Assessment of cell viability and proliferation using cell counting.
- Flow cytometry analysis for cell cycle progression and apoptosis.
- Measurement of TdR kinase activity via [(3)H]thymidine incorporation.
Main Results:
- ATS-2 significantly reduced TS mRNA and protein levels in HEK cells.
- A dose-dependent reduction in viable cells (up to 98%) was observed in ATS-2 treated cells.
- ATS-2 induced S-phase arrest and apoptosis, suggesting specificity for dividing cells.
- ATS-2 and FdUrd exhibited complementary effects, with HEK cells sensitive to ATS-2 and resistant to FdUrd, while HeLa cells showed the opposite sensitivity profile.
Conclusions:
- ATS-2 effectively suppresses TS synthesis and inhibits cancer cell proliferation, inducing apoptosis.
- The efficacy of ATS-2 is linked to cellular TdR kinase activity, with low activity favoring ATS-2 response.
- Combined treatment with ATS-2 and FdUrd demonstrates additive inhibitory effects, offering a potentially synergistic approach for cancer therapy.
More Related Videos
Related Concept Videos
02:30siRNA - Small Interfering RNAs
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the ATP-dependent...
02:15Experimental RNAi
01:19Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
01:11Pharmacogenetics of Drug Targets: β₂-Adrenergic Receptors, Apo E, Thymidylate Synthase
01:28Biosynthesis of Nucleic Acids
01:22Antiviral Nucleoside Inhibitors