Synergistic Effects of Oxaliplatin, 5-Fluorouracil, and Novel Synthetic Uracil Analog U-359 on Breast Cancer Cell Carcinogenesis

Abstract

Breast cancer presents significant global challenges, necessitating effective treatments to combat drug resistance and minimize chemotherapy side effects. This study evaluated the cytotoxic effects of U-359, Oxaliplatin (Ox), and 5-Fluorouracil (5-FU) in MCF-7 and MCF-10A cells using MTT and RealTime-GLO assays. Morphological changes were assessed by light microscopy following Wright-Giemsa staining. Apoptosis induction was studied using qPCR for apoptotic markers, the RealTime-Glo™ Annexin V assay, and the cleaved PARP1 ELISA assay. Caspase 8 and 9 activities, ABCB1, ABCG2, and NF-κB protein levels were quantified using ELISA. Synergy was analyzed using the Bliss Independence Model. The results indicated that combining U-359 with Ox and 5-FU enhanced cytotoxicity compared to individual treatments. U-359 induced apoptosis-associated morphological changes in MCF-7 cells, which were augmented with the Ox and 5-FU treatment. Apoptosis assays confirmed the up-regulation of pro-apoptotic markers and the down-regulation of anti-apoptotic markers with U-359 alone or in combination. Elevated cleaved PARP1 levels suggested robust apoptosis induction with U-359 and Ox or 5-FU. Caspase activity assays demonstrated a significant activation of caspase 8 and 9, implicating both apoptotic pathways. Furthermore, U-359 down-regulated ABCB1, ABCG2, and NF-κB in MCF-7 cells, which were up-regulated by Ox and 5-FU alone. The Bliss Independence Model revealed strong synergistic interactions (SI < 1) between U-359 and Ox or 5-FU, particularly in reducing ABCB1 and NF-κB levels. U-359 combined with Ox and 5-FU shows potential for overcoming chemotherapy resistance in breast cancer by enhancing apoptosis and modulating drug resistance. Further clinical studies are needed to optimize treatment and improve outcomes.

Keywords: 5-fluorouracil; MCF-7; U-359; anticancer compounds; apoptosis; multidrug resistance; oxalipaltin.

Figure 1

Chemical structures of U-359, 5-FU, and Ox.

Figure 2

The cytotoxic effect of U-359 (A), Ox (B), and 5-FU (C) in MCF-7 and MCF-7-10A cells, analyzed by MTT assay. Each data point represents the mean of three replicates, and the error bars indicate SEM.

Figure 3

Effect of U-359, Ox, 5-FU, U-359+Ox, and U-359+5-FU on the morphology of MCF-7 and MCF-10A cells after 24 h of incubation. After staining with Giemsa dye, the cells were photographed under a light microscope with a built-in camera (magnification 100×).

Figure 4

Effect of U-359 (3.8 μM for MCF-7 and 13 μM for MCF-10A), Ox (34 μM for MCF-7 and 35 μM for MCF-10A), 5-FU (25 μM for both cell lines), U-359+Ox, and U-359+5-FU on MCF-7 and MCF-10A cell viability, expressed as the percentage change in luminescence signal (%), after 24 h of incubation (A,B). Data are expressed as mean ± SEM. Statistical significance was assessed using one-way ANOVA and a post hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001, in comparison with control; ^### p < 0.001, in comparison with Ox; ⁺⁺⁺ p < 0.001, in comparison with 5-FU; NS, not significant.

Figure 5

Effect of U-359, Ox, 5-FU, U-359+Ox, and U-359+5-FU on induction of apoptosis and/or necrosis in MCF-7 after 24 h of treatment, measured by the changes in luminescence (A) and fluorescence (B) signals. Data are expressed as mean ± SEM. Statistical significance was assessed using one-way ANOVA and a post hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001, * p < 0.05 in comparison with control; NS, not significance.

Figure 6

Effect of U-359, Ox, 5-FU, U-359+Ox, and U-359+5-FU on concentration of cleaved PARP1 in MCF-7 cells after 24 h of treatment, measured by ELISA-based method using Cleaved PARP1 Human SimpleStep ELISA^® Kit. Data are expressed as mean ± SEM. Statistical significance was assessed using one-way ANOVA and a post hoc multiple comparison Student–Newman–Keuls test. * p < 0.05 and *** p < 0.001, in comparison with control; ^### p < 0.001, in comparison with Ox; ⁺⁺⁺ p < 0.001, in comparison with 5-FU. NS, not significant.

Figure 7

Effect of U-359, Ox, 5-FU, U-359+Ox, and U-359+5-FU on Caspase 9 (A) and 8 (B) activity in MCF-7 cells after 24 h of treatment, measured by the percentage change in luminescence signal (%). Data are expressed as mean ± SEM. Statistical significance was assessed using one-way ANOVA and a post hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001, in comparison with control; ^# p < 0.05, in comparison with Ox; ⁺⁺⁺ p < 0.001, in comparison with 5-FU. NS, not significant.

Figure 8

Effect of U-359, Ox, 5-FU, Ox+U-359, and 5-FU+U-359 on ABCB1 (A) and ABCG2 (B) and NF-κB (C) protein levels in MCF-7 cells after 24 h of incubation, measured by ELISA-based method. Data are presented as mean ± SEM. In each experiment, three replicates were used. The control consisted of untreated MCF-7 cells. Statistical significance was assessed by one-way ANOVA and a post hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001, ** p < 0.01 and * p < 0.05, in comparison with control; ^### p < 0.001, in comparison with Ox; ⁺⁺⁺ p < 0.001, in comparison with 5-FU. NS, not significant changes.