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. 2023 Mar 29;15(7):1658.
doi: 10.3390/nu15071658.

Docosahexaenoic Acid, a Key Compound for Enhancing Sensitization to Drug in Doxorubicin-Resistant MCF-7 Cell Line

Sergio Crovella  1 , Allal Ouhtit  1 , Shaikh Mizanoor Rahman  2 , Md Mizanur Rahman  1
Affiliations

Docosahexaenoic Acid, a Key Compound for Enhancing Sensitization to Drug in Doxorubicin-Resistant MCF-7 Cell Line

Sergio Crovella et al. Nutrients. .

Abstract

Drug resistance is a well-known and significant obstacle in the battle against cancer, rendering chemotherapy treatments often ineffective. To improve the effectiveness of chemotherapy, researchers are exploring the use of natural molecules that can enhance its ability to kill cancer cells and limit their spread. Docosahexaenoic acid (DHA), a lipid found in marine fish, has been shown to enhance the cytotoxicity of various anti-cancer drugs in vitro and in vivo. While the combined use of chemotherapeutic drugs with DHA demonstrated promising preliminary results in clinical trials, there is still a significant amount of information to be discovered regarding the precise mechanism of action of DHA. As the biological pathways involved in the chemosensitization of already chemoresistant MCF-7 cells are still not entirely unraveled, in this study, we aimed to investigate whether DHA co-treatment could enhance the ability of the chemotherapy drug doxorubicin to inhibit the growth and invasion of MCF-7 breast cancer cells (MCF-7/Dox) that had become resistant to the drug. Upon treating MCF-7/Dox cells with DHA or DHA-doxorubicin, it was observed that the DHA-doxorubicin combination effectively enhanced cancer cell death by impeding in vitro propagation and invasive ability. In addition, it led to an increase in doxorubicin accumulation and triggered apoptosis by arresting the cell cycle at the G2/M phase. Other observed effects included a decrease in the multi-drug resistance (MDR) carrier P-glycoprotein (P-gp) and TG2, a tumor survival factor. Augmented quantities of molecules promoting apoptosis such as Bak1 and caspase-3 and enhanced lipid peroxidation were also detected. Our findings in the cell model suggest that DHA can be further investigated as a natural compound to be used alongside doxorubicin in the treatment of breast cancer that is unresponsive to chemotherapy.

Keywords: apoptosis; breast cancer; chemoresistance; chemosensitization; docosahexaenoic acid; drug accumulation; natural bioactive compound.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The impact of DHA on the proliferation, and invasion of doxorubicin-resistant MCF-7 breast cancer cells (MCF-7/Dox). (A) For the cell proliferation experiment, the MCF-7/Dox cells were treated with various concentrations of DHA, doxorubicin (2 µM) alone or in combination for 48 h, and stained using a crystal violet staining solution and measured the absorbance to determine the percent survival of cells. (B) For the invasion experiment, MCF-7/Dox cells in serum free medium were placed in the upper chamber of a 24-well BioCoat Matrigel invasion chamber and the lower chamber was filled with varying concentrations of DHA with or without doxorubicin (2 µM) in serum containing medium or with 2 µM of docetaxel alone as a positive control. After 48 h of incubation, the cells that traversed the membrane pores to the lower side were fixed with 10% formalin, stained with 0.1% crystal violet blue, and counted manually. The values shown represent the average ± standard error of the mean, obtained from three separate experiments. A p-value of <0.05 is considered significant by a Newman–Keuls one-way ANOVA with multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to untreated control.
Figure 2
Figure 2
Effect of DHA on doxorubicin accumulation in doxorubicin-resistant and parental MCF-7 cells. Both Doxorubicin-resistant MCF-7/Dox cells and the parental Doxorubicin-sensitive MCF-7 cells were treated with various concentrations of DHA overnight, followed by treatment with 2 µM of doxorubicin for 1 h at 37 °C. After washing, cells were lysed using a lysis buffer, and fluorescence intensity was measured using a Perkin Elmer LS-50B spectrometer with an excitation wavelength of 470 nm and emission wavelength of 595 nm. The values presented are the mean ± standard error of the mean, based on three independent experiments. A p-Value of <0.05 is considered significant by a Newman–Keuls one-way ANOVA with multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to respective untreated control.
Figure 3
Figure 3
Effect of DHA co-treatment on cell cycle arrest in MCF-7/Dox cells. MCF-7/Dox cells were treated with either DHA, Dox, or a combination of both for 24 h, followed by isolation, treatment with ethanol, staining with propidium iodide, and FACS analysis to determine cell cycle distribution. The reported values represent the average ± standard error of the mean from three independent experiments. A p-value of <0.05 is considered significant by a Newman–Keuls one-way ANOVA with multiple comparisons test. * p < 0.05, ** p < 0.01 compared to respective untreated control. G1, Gap 1 phase for cell growth; S, synthesis phase for DNA synthesis; G2, Gap 2 phase for cell growth; M, mitosis phase for cell multiplication.
Figure 4
Figure 4
Effect of DHA on P-gp and TG−2 gene expression in MCF-7/Dox cells. MCF-7/Dox cells were treated with doxorubicin (Dox) (2 µM), DHA 50 µM, alone or in combination for 48 h. RNA was extracted and evaluated for gene expression through real-time RT-PCR. * A p-Value of less than 0.05 is considered significant when compared to the control group treated only with Dox, as determined by a one-way ANOVA.
Figure 5
Figure 5
Effect of DHA on induction of apoptosis of MCF-7/Dox cells. (A) Percent apoptotic cell analysis by Annexin V staining and FACS, (B) Pro-apoptotic genes Bak1 and Caspase 3 measurement by real-time RT-PCR and (C) Assessment of lipid peroxidation using lipid peroxidation assay kit. The level of lipid peroxidation was studied in doxorubicin-resistant MCF-7 (MCF-7/Dox) as well as parental MCF-7 (MCF-7/WT) cells exposed to Dox only or a combination of Dox and DHA. Each value represents the mean ± SEM of independent triplicate cultures. A value of p < 0.05 is considered significant by a one-way ANOVA. ** p < 0.001, † p < 0.001, * p < 0.05 compared to respective Dox alone treated culture.
Figure 6
Figure 6
The effect of DHA administration on MCF-7/Dox cells.
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