Wosiak Agnieszka, Szmajda-Krygier Dagmara, Pietrzak Jacek, Boncela Joanna, Balcerczak Ewa
Laboratory of Molecular Diagnostics and Pharmacogenomics, Department of Pharmaceutical Biochemistry and Molecular Diagnostics, Medical University of Lodz, 1 Muszynskiego, 90-151 Lodz, Poland.
Institute of Medical Biology, Polish Academy of Science, 106 Lodowa, 93-232 Lodz, Poland.
Bioengineering (Basel). 2023 May 9;10(5):570. doi: 10.3390/bioengineering10050570.
Suppressor of mothers against decapentaplegic homolog 4 (SMAD family member 4, ) 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 gene expression, as patients with decreased 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 and genes.
The effect of 5-FU on the expression of and 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.
Significant changes in the level of and 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 gene in all cell lines at both exposure times, while the concentration of 100 µmol/L increased the expression of the gene in CACO-2 cells. The level of expression of the gene was higher for all cells treated with 5-FU at the highest concentrations, while the exposure time was extended to 48 h.
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 gene expression, which may increase the effectiveness of therapy.
母亲对脱磷酸化蛋白同源物4的抑制因子（SMAD家族成员4， ）参与腺瘤 - 癌途径，导致结肠癌的发生。编码的蛋白质是转化生长因子β（TGFβ）途径中的关键下游信号传导介质。该途径具有肿瘤抑制功能，包括细胞周期停滞和细胞凋亡。其在晚期癌症中的激活可促进肿瘤发生，包括转移和化疗耐药。大多数结直肠癌患者接受以5-氟尿嘧啶（5-FU）为基础的化疗作为辅助治疗。然而，肿瘤细胞的多药耐药性阻碍了治疗的成功。在结直肠癌中，对基于5-FU治疗的耐药性受 基因表达的影响，因为 基因表达降低的患者可能有更高的发生5-FU诱导耐药的风险。导致这种现象发生的机制尚未完全了解。因此，本研究评估5-FU对 基因和 基因表达变化的可能影响。
使用实时荧光定量PCR评估5-FU对源自CACO-2、SW480和SW620细胞系的结肠癌细胞中 和 表达的影响。采用MTT法评估5-FU对结肠癌细胞的细胞毒性，并使用流式细胞仪评估其对细胞凋亡诱导和DNA损伤起始的影响。
在24小时和48小时暴露期间，用不同浓度的5-FU处理的CACO-2、SW480和SW620细胞中， 和 基因表达水平发生了显著变化。在两个暴露时间点，5 μmol/L浓度的5-FU导致所有细胞系中 基因表达降低，而100 μmol/L浓度增加了CACO-2细胞中 基因的表达。在最高浓度的5-FU处理所有细胞且暴露时间延长至48小时时， 基因的表达水平更高。
5-FU在体外对CACO-2细胞产生的观察到的变化在为结直肠癌患者选择药物浓度时可能具有临床相关性。有可能5-FU在较高浓度时对结肠癌细胞有更强的作用。低浓度的5-FU可能没有治疗效果，还可能影响癌细胞的耐药性。较高浓度和延长暴露时间可能会影响 基因表达，这可能会提高治疗效果。
