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Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines

Purpose: Suppressor of mothers against decapentaplegic homolog 4 (SMAD family member 4, SMAD4) is involved in the adenoma–carcinoma pathway, leading to the development of colon cancer. The encoded protein is a key downstream signaling mediator in the TGFβ pathway. This pathway has tumor-suppressor f...

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Autores principales: Wosiak, Agnieszka, Szmajda-Krygier, Dagmara, Pietrzak, Jacek, Boncela, Joanna, Balcerczak, Ewa
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10215742/
https://www.ncbi.nlm.nih.gov/pubmed/37237640
http://dx.doi.org/10.3390/bioengineering10050570
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author Wosiak, Agnieszka
Szmajda-Krygier, Dagmara
Pietrzak, Jacek
Boncela, Joanna
Balcerczak, Ewa
author_facet Wosiak, Agnieszka
Szmajda-Krygier, Dagmara
Pietrzak, Jacek
Boncela, Joanna
Balcerczak, Ewa
author_sort Wosiak, Agnieszka
collection PubMed
description Purpose: Suppressor of mothers against decapentaplegic homolog 4 (SMAD family member 4, SMAD4) is involved in the adenoma–carcinoma pathway, leading to the development of colon cancer. The encoded protein is a key downstream signaling mediator in the TGFβ pathway. This pathway has tumor-suppressor functions, including cell-cycle arrest and apoptosis. Its activation in late-stage cancer can promote tumorigenesis, including metastasis and chemoresistance. Most colorectal cancer patients receive chemotherapy based on 5-FU as an adjuvant treatment. However, the success of therapy is hampered by multidrug resistance by neoplastic cells. In colorectal cancer, resistance to 5-FU-based therapy is influenced by SMAD4 gene expression, as patients with decreased SMAD4 gene expression probably have a higher risk of developing 5-FU-induced resistance. The mechanism leading to the development of this phenomenon is not fully understood. Therefore, the present study assesses the possible influence of 5-FU on changes in the expression of the SMAD4 and TGFB1 genes. Patients and methods: The effect of 5-FU on the expression of SMAD4 and TGFB1 in colorectal cancer cells derived from the CACO-2, SW480 and SW620 cell lines was evaluated using real-time PCR. The cytotoxicity of 5-FU on colon cancer cells was assessed by the MTT method, and its effect on the induction of cell apoptosis and the initiation of DNA damage using a flow cytometer. Results: Significant changes in the level of SMAD4 and TGFB1 gene expression were noted in the CACO-2, SW480 and SW620 cells treated with 5-FU at various concentrations during 24 h and 48 h exposure. The use of 5-FU at a concentration of 5 µmol/L resulted in a decrease in the expression of the SMAD4 gene in all cell lines at both exposure times, while the concentration of 100 µmol/L increased the expression of the SMAD4 gene in CACO-2 cells. The level of expression of the TGFB1 gene was higher for all cells treated with 5-FU at the highest concentrations, while the exposure time was extended to 48 h. Conclusion: The observed in vitro changes in CACO-2 cells caused by 5-FU may be of clinical relevance when choosing the drug concentration for treating patients with colorectal cancer. It is possible that 5-FU has a stronger effect on colorectal cancer cells at the higher concentrations. Low concentrations of 5-FU may not have a therapeutic effect and may also influence drug resistance in cancer cells. Higher concentrations and prolonged exposure time may affect SMAD4 gene expression, which may increase the effectiveness of therapy.
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spelling pubmed-102157422023-05-27 Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines Wosiak, Agnieszka Szmajda-Krygier, Dagmara Pietrzak, Jacek Boncela, Joanna Balcerczak, Ewa Bioengineering (Basel) Article Purpose: Suppressor of mothers against decapentaplegic homolog 4 (SMAD family member 4, SMAD4) is involved in the adenoma–carcinoma pathway, leading to the development of colon cancer. The encoded protein is a key downstream signaling mediator in the TGFβ pathway. This pathway has tumor-suppressor functions, including cell-cycle arrest and apoptosis. Its activation in late-stage cancer can promote tumorigenesis, including metastasis and chemoresistance. Most colorectal cancer patients receive chemotherapy based on 5-FU as an adjuvant treatment. However, the success of therapy is hampered by multidrug resistance by neoplastic cells. In colorectal cancer, resistance to 5-FU-based therapy is influenced by SMAD4 gene expression, as patients with decreased SMAD4 gene expression probably have a higher risk of developing 5-FU-induced resistance. The mechanism leading to the development of this phenomenon is not fully understood. Therefore, the present study assesses the possible influence of 5-FU on changes in the expression of the SMAD4 and TGFB1 genes. Patients and methods: The effect of 5-FU on the expression of SMAD4 and TGFB1 in colorectal cancer cells derived from the CACO-2, SW480 and SW620 cell lines was evaluated using real-time PCR. The cytotoxicity of 5-FU on colon cancer cells was assessed by the MTT method, and its effect on the induction of cell apoptosis and the initiation of DNA damage using a flow cytometer. Results: Significant changes in the level of SMAD4 and TGFB1 gene expression were noted in the CACO-2, SW480 and SW620 cells treated with 5-FU at various concentrations during 24 h and 48 h exposure. The use of 5-FU at a concentration of 5 µmol/L resulted in a decrease in the expression of the SMAD4 gene in all cell lines at both exposure times, while the concentration of 100 µmol/L increased the expression of the SMAD4 gene in CACO-2 cells. The level of expression of the TGFB1 gene was higher for all cells treated with 5-FU at the highest concentrations, while the exposure time was extended to 48 h. Conclusion: The observed in vitro changes in CACO-2 cells caused by 5-FU may be of clinical relevance when choosing the drug concentration for treating patients with colorectal cancer. It is possible that 5-FU has a stronger effect on colorectal cancer cells at the higher concentrations. Low concentrations of 5-FU may not have a therapeutic effect and may also influence drug resistance in cancer cells. Higher concentrations and prolonged exposure time may affect SMAD4 gene expression, which may increase the effectiveness of therapy. MDPI 2023-05-09 /pmc/articles/PMC10215742/ /pubmed/37237640 http://dx.doi.org/10.3390/bioengineering10050570 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wosiak, Agnieszka
Szmajda-Krygier, Dagmara
Pietrzak, Jacek
Boncela, Joanna
Balcerczak, Ewa
Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines
title Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines
title_full Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines
title_fullStr Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines
title_full_unstemmed Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines
title_short Assessment of the Influence of 5-Fluorouracil on SMAD4 and TGFB1 Gene Expression, Apoptosis Induction and DNA Damage in Human Cell Lines
title_sort assessment of the influence of 5-fluorouracil on smad4 and tgfb1 gene expression, apoptosis induction and dna damage in human cell lines
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10215742/
https://www.ncbi.nlm.nih.gov/pubmed/37237640
http://dx.doi.org/10.3390/bioengineering10050570
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