Caffeic Acid Phenethyl Ester as a DHODH Inhibitor and Its Synergistic Anticancer Properties in Combination with 5-Fluorouracil in a Breast Cancer Cell Line

Abstract

Introduction: A combination of chemotherapy agents is the best choice in breast cancer treatment to increase the patient survival rate. 5-fluorouracil (5-FU) is one of the drugs applied in combination with other drugs to control and delay development of cancer cells. Nevertheless, the occurrence of multidrug resistance and dose-limiting cytotoxicity have limited the efficacy of 5-FU treatment. Therefore, the discovery of new anti-breast cancer drugs should be pursued.

Objective: To study potency of a promising naturally derived compound, caffeic acid phenethyl ester (CAPE), for breast cancer treatment in single and combination with 5-FU.

Methods: Cytotoxicity of CAPE, 5-FU, and 5-FU+CAPE was studied by in vitro MTT experiment in MCF-7 cell line, and RT-PCR analysis was used to evaluate the change in gene expression due to the treatment. Moreover, an enzymatic assay and molecular docking analysis were applied to evaluate the possible mechanism of substance-induced apoptosis.

Results: The study revealed that a single treatment of CAPE showed cytotoxicity with IC₅₀ 6.6 ± 1.0 µM and 6.5 ± 2.9 µM at 24 h and 48 h, respectively. Meanwhile, 5-FU showed cytostatic activity. The 5-FU + CAPE has a synergistic effect at 24 h treatment with a CI = 0.5 and an additive effect at 48 h treatment with CI = 1.0. CAPE was also found to enhances the mRNA expression of caspase-8 and BAX within 6 hours in combination with 5-FU compared to 5-FU treatment alone. Our study reveals a new mechanism of CAPE which is related to the inhibition of human dihydroorotate dehydrogenase (HsDHODH) with an IC₅₀ of 120.7 ± 6.8 µM, by bound to the ubiquinone-binding site of the enzyme and could be responsible for inducing extrinsic and intrinsic apoptosis.

Conclusion: This study demonstrated the cytotoxicity of CAPE potential to induce apoptosis of breast cancer MCF-7 cell line single and cytotoxic-cytostatic combination with 5-FU. Therefore, further studies to develop CAPE and its derivatives will be required to discover new candidates for breast cancer agents.

Keywords: 5-fluorouracil; DHODH; MCF-7; breast cancer; caffeic acid phenethyl ester; dihydroorotate dehydrogenase.

Figure 1

Cytotoxic activity of 5-FU and CAPE on the MCF-7 breast cancer cell line. There was a significant difference in the cytotoxic effect of 5-FU treatment between 24h and 48h, ****P<0.0001 (A). CAPE shows similar cytotoxicity to MCF-7 for 24h and 48h treatment (P=0.948) (B).

Figure 2

Cytotoxic activity of the combination 5-FU+CAPE on the MCF-7 breast cancer cell line for 24h and 48h treatment. Treatment with 5-FU+CAPE for 24 h had a synergistic effect (CI=0.5), whereby cell death was mostly induced by CAPE treatment (A), an additive effect at 48 h (CI = 1.0), whereby cell death was mostly affected by 5-FU treatment (B). ****P<0.0001.

Figure 3

Evaluation of pro-apoptotic mRNA expression after 6 h treatment with 5-FU, CAPE and the combination of 5-FU+CAPE in the MCF-7 cell line.

Figure 4

Profile of CAPE inhibition of HsDHODH activity. The effect of CAPE treatment on human cell growth was evaluated by in vitro HsDHODH assay. The result showed an IC₅₀ of 120.7±6.8 µM.

Figure 5

Molecular docking analysis of CAPE-HsDHODH. Molecular docking analysis revealed that CAPE binds to the ubiquinone binding site of HsDHODH, adjacent to FMN and orotate (A). A hydrogen bond formed between the O atom of CAPE with the arginine A136 (green colour). There was a Pi-alkyl bond interaction between aromatic 1 with ValA143 and FMN, and the interaction between the second aromatic with Leu 359 is shown in pink. The Pi-sigma interaction between aromatic 1 with Val 134 and second aromatic with Ala 59 is shown in purple (B). The interaction allows CAPE to bind to the ubiquinone binding site (C) with ΔG –11.13 kcal/mol and an inhibition constant Ki of 6.95 nM.

Figure 6

CAPE induces extrinsic apoptosis through inhibition of HsDHODH. Inhibition of HsDHODH by CAPE could be one of the mechanisms of action of CAPE to disrupt mitochondrial membrane potential, leading to the accumulation of mitochondrial superoxidase (O₂^•−), thus induces the accumulation of p53 and activation of caspase-8 for extrinsic apoptosis.