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

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.


Introduction
Breast cancer (BC) is responsible for the highest mortality rate among women worldwide, making it the deadliest cancer [1,2]. The incidence of BC in women is rapidly increasing, with 2.3 million new cases diagnosed each year, and over 680,000 deaths from BC recorded in 2020, according to the World Health Organization (WHO) [3]. Abnormalities and imbalances in cell proliferation and apoptosis signaling pathways have been identified as fundamental factors in BC progression [2,4,5].
One of the significant biomarkers for the onset of BC is considered to be the estrogen receptor beta (ER). Over 80% of BCs are known to be "ER-positive," indicating that the growth of BC cells is dependent on estrogen. Additionally, approximately 65% of these BC cells are also dependent on progesterone, referred to as "PR-positive" [6][7][8]. BC is a heterogeneous disease classified into four primary subtypes, including ER/PR+ and Her2−

Cell Proliferation Assay
The MCF-7/Dox (Doxorubicin resistant) cells were seeded in a 96-well plate at a density of 2 × 10 4 cells/100 µL per well. The cells were allowed to attach overnight and then treated with different concentrations of DHA, doxorubicin, alone, or in combination in fresh media. After 48 h of incubation, the culture medium was removed, and the cells were washed twice with PBS. The adherent cells were then stained with crystal violet staining solution (Cell Viability Assay Kit (ab232855), abcam, Boston, MA, USA) for 20 min, followed by washing and air-drying. The solubilization solution was added, and the absorbance was measured at 570 nm using a microplate reader as per the manufacturer's instructions.

Invasion Assay
Approximately 2 × 10 4 MCF-7/Dox cells were suspended in 200 µL of serum-free DMEM and added to the upper chamber of a 24-well BioCoat Matrigel invasion chamber with an 8 µm pore size membrane, which was then placed in the corresponding assay plate from BD Biosciences (Bedford, MA, USA)). The lower chamber was filled with 700 µL of DMEM containing 10% fetal calf serum (FCS) and varying concentrations of DHA, doxorubicin, alone or in combination. Following a 48 h incubation period, cells remaining in the upper chamber were removed, and cells that had passed through the membrane pores and reached the lower side were fixed with 10% formalin and stained with 0.1% crystal violet blue solution. The stained cells were manually counted to determine the number of cells that had migrated through the pores, as previously described [22].

Doxorubicin Accumulation
To evaluate intracellular doxorubicin levels, MCF-7/Dox, and parental MCF-7 cells (1 × 10 5 ) were cultured with various concentrations of DHA overnight. The cells were then exposed to 2 µM doxorubicin in complete PBS media supplemented with 0.5 mM MgCl2, 0.7 mM CaCl2, and 0.1% glucose, and incubated at 37 • C for 1 h. After washing with PBS, the cells were treated with lysis buffer containing 0.1% Triton X-100. Fluorescence was measured using a Perkin Elmer LS-50B fluorescence spectrometer with an excitation wavelength of 470 nm and an emission wavelength of 595 nm, as previously described [57].

Cell Cycle Analysis by Flow Cytometry
Cell cycle analysis was performed using flow cytometry. Briefly, 1 × 10 5 cells were cultured in a 6-well plate overnight, and the media were removed after 24 h. The cells were then treated as described above with DOX or DHA, individually or in combination for 24 h. After trypsinization and centrifugation at 3000× g for 5 min, the pellet was dissolved in ice-cold ethanol and stored at −20 • C until use. Prior to cell cycle analysis, the cell pellet was washed trice with PBS and incubated at 37 • C in the presence of RNase A. The cells were then stained with 0.5 µg/mL of propidium iodide for 30 min prior to FACS analysis (BD Biosciences-Allschwil, Switzerland).

Annexin V Apoptosis Assay
An Annexin V-FITC staining kit (BD Bioscience, Franklin Lakes, NJ, USA) was used to analyze apoptotic cell death. MCF-7/Dox cells grown in 60 mm diameter dishes were treated with either DOX or DHA alone or in combination for 24 h. After trypsinization and centrifugation, the cell pellets were washed with PBS and suspended in 100 µL of binding buffer containing 5 µL of Annexin V-Fluorescein isothiocyanate (FITC) and 5 µL of propidium iodide (PI). The stained cells were then incubated for 15 min at room temperature and analyzed using a FACSCalibur flow cytometry system (BD Bioscience, Franklin Lakes, NJ, USA).

RNA Isolation and Quantitative Real-Time RT-PCR
MCF-7/Dox cells were treated with 2 uM of DOX or 50 uM of DHA, individually or in combination for 48 h. Total RNA was isolated using Trizol reagent (Invitrogen) as described by the manufacturer. After DNase treatment, the RNA was transformed into cDNA using SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen, Waltham, MA, USA). Relative gene expression was determined using QuantStudio™ 12 K Flex System (Applied Biosystems, Carlsbad, CA, USA) and GoTaq qPCR Master Mix (Promega, Madison, WI, USA). The primers required for RT-PCR analysis were obtained from Integrated DNA Technologies (Coralville, CA, USA). β-Actin gene expression analysis was used as an endogenous control and normalization was performed using the 2 −∆∆Ct method to set the values of control as one. The relative gene expressions of P-gp, TG-2, and apoptotic markers, in particular, Bak1 and caspase 3 were investigated in this study.

Lipid Peroxidation Assay
The effect of DHA in the presence or absence of DOX was examined in this study using Lipid Peroxidation (MDA) Assay Kit (ab118970). MCF-7/Dox cells (2 × 10 4 cells per well) were cultured in a black 96-well plate and incubated at 37 • C in a CO 2 incubator. The next day, the cells were treated with various concentrations of DHA (25,50, and 100 uM) in the presence or absence of 2 uM of DOX. The cells were then incubated at 37 • C for 24 h. After removing the media, the cells were washed with PBS, incubated in TBA solution at 95 • C for 60 min, cooled in an ice bath for 10 min, and then analyzed with a microplate reader (Ex/Em = 532/553 nm) as described in the manufacturer's protocol.

Statistical Analysis
The mean ± SE was used to express the values obtained from statistical analysis. A value of p < 0.05 was considered statistically significant by one-way ANOVA. The comparison between the means of groups was carried out using Newman-Keuls and Dunnett's multiple-comparison tests.

Results
Anti-inflammatory bioactive fatty acids are well-known for their anti-cancer effect. To find an effective adjuvant bioactive compound to treat doxorubicin-resistant MCF-7 cells, we first checked for the anti-cancer effect of several known bioactive fatty acids against MCF-7 parental cells. Interestingly, among all tested compounds, DHA showed the best anti-breast cancer proliferative effect (Supplemental Figure S1). Based on this preliminary screening, we chose DHA to combine with doxorubicin to treat MCF-7/Dox. Further, to determine if the anti-breast cancer effect of DHA is cancer-specific, we also performed cytotoxicity experiments against normal breast epithelial cells, MCF-10A. We found that up to 100 µM of DHA is non-toxic to normal cells (Supplemental Figure S2). Our finding is also supported by other investigators' findings [17,58].
Effect of DHA treatment on proliferation and invasion: Cell viability assay was used to determine the effect of DHA co-treatment on the MCF-7/Dox breast cancer cell line. Our data demonstrated that DHA alone, dose-dependently, inhibited the MCF-7/Dox cell proliferation ( Figure 1A). Doxorubicin treatment alone did not reasonably inhibit MCF-7/Dox. However, when doxorubicin was combined with DHA, these treatments significantly increased the inhibitory effect of Dox or DHA, using MCF-7/Dox cell proliferation of either treated culture ( Figure 1A).
Metastasis is the ultimate fate of any advanced state cancer; once cancer is in its metastatic phase, it is manageable but not curable anymore. So, any treatment that can prevent metastasis would be an ideal candidate as a drug. Metastatic cancer cells require local intravasation, which involves passing through the extracellular matrix.
Moreover, to analyze the effect of doxorubicin and DHA alone or in combination, in the context of metastatic cell ability, we performed the BioCoat Matrigel invasion assay. Since the variant of MCF-7 cell is doxorubicin-resistant, we did not see any significant reduction in cancer cell invasion when treated with doxorubicin alone ( Figure 1B). We treated the cells with another chemotherapeutic drug docetaxel as a positive control to see the comparative effect of DHA alone or in combination with doxorubicin. DHA in combination with doxorubicin dose dependently inhibited the MCF-7/Dox cell invasion as compared to either treatment alone ( Figure 1B). These results indicate that a natural compound DHA could be a potential co-treatment strategy to minimize, in vitro, tumoral cell growth and migration of drug-resistant cancer cells. DHA co-treatment significantly reduced the invasion capacity of breast cancer tumor cells. 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.

Effect of DHA co-treatment on doxorubicin accumulation in MCF-7/Dox cells:
Drug sensitivity is known to increase with drug accumulation in tumor cells, while reduction in drug incorporation in cells is a key mechanism of drug resistance in tumors. When doxorubicin-resistant (MCF-7/Dox) and sensitive (MCF-7) cells were treated with doxorubicin alone, the drug accumulation in MCF-7/Dox was significantly lower than in MCF-7 cells (Figure 2). To investigate whether DHA co-treatment enhances cell killing by increasing doxorubicin in MCF-7/Dox cells, we analyzed the cellular doxorubicin content. Interestingly, DHA co-treatment significantly increased doxorubicin accumulation in MCF-7/Dox cells in a dose-dependent manner, possibly by altering the cell membrane composition (Figure 2). We also examined whether DHA co-treatment could enhance doxorubicin accumulation in parental MCF-7 cells. The doxorubicin accumulation was increased by 2-fold and 3-fold in the presence of 50 µM and 100 µM of DHA, respectively. Although DHA co-treatment significantly enhanced doxorubicin accumulation in both doxorubicin-resistant and sensitive MCF-7 cells, the doxorubicin accumulation was still lower in MCF-7/Dox cells than in parental MCF-7 cells (Figure 2). 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.

Effect of DHA co-treatment on cell cycle arrest in MCF-7/Dox cells:
To investigate whether DHA co-treatment arrests MCF-7/Dox cells on a specific cell cycle phase, cell cycle analysis was performed using FACS. Treatment with doxorubicin alone did not significantly alter cell cycle distribution compared to the untreated control. However, treatment with DHA alone or in combination with doxorubicin resulted in a significant decrease in cells in the G1 phase and an increase in the G2/M phase compared to doxorubicin alone treated or untreated control. Additionally, DHA and DHA+Dox treatments increased the number of cells in S phase compared to doxorubicin alone treated or untreated control. These results suggest that DHA co-treatment increases the percentage of G2/M and S phase cells in MCF-7/Dox cells compared to untreated and doxorubicin alone treated cultures (Figure 3).
Effect of DHA co-treatment on the P-gp and TG−2 gene expression: P-glycoprotein (P-gp) is an ATP-binding cassette transporter that is strongly associated with multi-drug resistance in cancer cells. Transglutaminase 2 (TG−2), on the other hand, plays a critical role in shifting glucose metabolism, enabling cancer cells to survive in stressful conditions and enhancing their metastatic potential. To investigate whether DHA co-treatment can improve drug sensitivity by reducing the expression of these genes, we examined their mRNA levels using real-time RT-PCR. Interestingly, we found that DHA significantly down-regulated the expression of both P-gp and TG−2 genes (Figure 4).

Effect of DHA co-treatment in inducing apoptosis in MCF-7/Dox cells:
Apoptosis induction is the main mechanism of action for most anti-cancer drugs. In this study, we aimed to investigate if DHA co-treatment can enhance apoptosis in doxorubicin-treated MCF-7/Dox cells, by Annexin V apoptosis assay using FACS analysis ( Figure 5A). Our results showed that the combination of DHA and doxorubicin significantly increased apoptotic cell death, especially after 48 and 72 h of treatment. Furthermore, we examined the expression levels of pro-apoptotic genes, Caspase 3 and Bak1, by real-time RT-PCR ( Figure 5B). Our data indicated that both genes were significantly up-regulated in MCF-7/Dox cells treated with doxorubicin in the presence of DHA, compared to cells treated with doxorubicin alone. 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.
In addition, we investigated the role of lipid peroxidation in regulating cell death and also whether DHA co-treatment can enhance lipid peroxidation in doxorubicin-treated cells. We tested the levels of lipid peroxidation in parental (MCF-7/WT) and doxorubicinresistant MCF-7 (MCF-7/Dox) cells. Our data showed that doxorubicin treatment alone increased lipid peroxidation in MCF-7/WT but not in MCF-7/Dox cells ( Figure 5C). However, when combined with DHA, lipid peroxidation levels were significantly enhanced both in parental and doxorubicin-resistant MCF-7 cells ( Figure 5C). Therefore, the combination of doxorubicin and DHA effectively enhances lipid peroxidation, not only in parental MCF-7 cells but also in doxorubicin-resistant MCF-7 cells, in a dose-dependent manner.

Discussion
Drug resistance is a significant problem in cancer treatment, which can develop through various mechanisms, including decreased drug uptake by cancer cells, increased drug efflux from cancer cells, and changes in drug target expression or function. DHA is a bioactive natural compound that has shown promising potential as a therapeutic agent for drug-resistant breast cancer. Several studies have demonstrated that DHA can sensitize breast cancer cells to chemotherapy drugs and overcome drug resistance [59][60][61][62]. However, the exact mechanisms by which DHA exerts its drug sensitization effects are still not entirely unraveled. In this study, we explored the possible mechanisms by which DHA exerts its drug sensitization potential. We found that co-treatment with DHA enhanced the drug sensitization of doxorubicin in already doxorubicin-resistant MCF-7 breast cancer cells by inhibiting cell proliferation and invasion, inducing G2/M phase cell cycle arrest, enhancing drug accumulation in cancer cells, inducing the expression of apoptotic genes, inducing lipid peroxidation, and down-regulating drug efflux-regulating genes such as P-gp and TG2. Figure 6 presents an overview of the potential mechanisms through which DHA may exert its anti-breast cancer effects. N-3 long-chain polyunsaturated fatty acids (PUFAs), such as DHA, are recognized as potential agents to counteract invasive breast cancer and prevent cancer in animal models [50]. In rat models with a DHA-rich diet, DHA has been shown to act synergistically with doxorubicin and enhance its effectiveness against drug-resistant cancer cells. Several trials have investigated the effects of dietary supplementation with PUFAs in breast cancer patients (see https://clinicaltrials.gov/ct2/results?cond=breast+cancer&term=DHA& cntry=&state=&city=&dist= accessed on 26 December 2022), but few have reported positive outcomes. Therefore, additional preclinical studies are necessary to confirm the potential benefits of DHA and support its use in human trials.
Cell cycle progression is a tightly regulated process that is essential for the growth and division of cells. Recent studies have suggested that inducing G2/M phase cell cycle arrest can lead to the inhibition of cell proliferation and the induction of apoptosis [63,64]. In our study, we also found that DHA treatment, alone or in combination with doxorubicin, arrested the cell cycle of MCF-7/Dox cells at the G2/M phase. G2/M phase cell cycle arrest may be an effective strategy for sensitizing drug-resistant breast cancer cells to chemotherapy.
Cancer cells can become resistant to chemotherapy by various mechanisms including decreased drug uptake, increased drug efflux, altered drug targets, and increased DNA damage repair mechanisms. One of the key mechanisms of drug resistance is the reduction in drug accumulation within the cell [43]. Enhancing drug accumulation can overcome drug resistance and promote cancer cell killing. This can be achieved through various approaches such as co-treatment with agents that can inhibit drug efflux transporters, mod-ification of the drug to improve its uptake, or alteration of the tumor microenvironment to increase drug penetration [65]. In our study, we found that doxorubicin accumulation was enhanced in both doxorubicin sensitive and resistant MCF-7 cells when treated with DHA. Downregulation of P-glycoprotein (P-gp) and Transglutaminase 2 (TG2) has been identified as a potential mechanism to sensitize drug-resistant breast cancer cells to chemotherapy [66]. These two proteins are known to be overexpressed in many drugresistant cancer cells, leading to increased drug efflux and decreased drug accumulation, thereby reducing the effectiveness of chemotherapy [66][67][68][69][70]. One mechanism by which drug accumulation can be enhanced is by inhibiting drug efflux transporters, which are proteins that pump drugs out of cancer cells and decrease drug accumulation [70][71][72][73]. DHA was shown to increase the intracellular accumulation of drugs in cancer cells by altering the cell membrane composition [50] or inhibiting drug efflux transporters [22,74]. Our previous findings were supported by this study, which demonstrated that treatment with DHA resulted in down-regulation of P-gp protein expression in MCF-7/Dox cells [22]. However, the downregulation of these proteins may also have potential side effects and affect normal cell functions; thus, further studies are needed to fully understand the mechanisms and determine the optimal strategies for downregulating P-gp and TG2 in drug-resistant breast cancer cells.
Apoptosis and lipid peroxidation have been identified as potential mechanisms to sensitize drug-resistant breast cancer cells to chemotherapy. Apoptosis is a programmed cell death process that can be activated by chemotherapy, leading to the elimination of cancer cells. However, drug-resistant cancer cells often have defects in the apoptotic pathway, which allows them to survive despite exposure to chemotherapy. Our study demonstrated that treatment with a combination of DHA and doxorubicin resulted in increased expression of pro-apoptotic genes, leading to greater apoptotic cell death in MCF-7/Dox cells. Similar findings have been reported by others, who have shown that DHA can enhance the cytotoxicity of drugs against cancer cells by inducing the expression of apoptotic genes and activating apoptotic pathways [41]. Lipid peroxidation is a process where free radicals attack the polyunsaturated fatty acids (PUFAs) in cell membranes, resulting in the formation of lipid peroxides and other reactive oxygen species (ROS). This process has been linked to the development and progression of breast cancer and other cancer types. Studies have suggested that DHA may have an anti-breast cancer effect by inducing lipid peroxidation [75]. Recent research has indicated that ferroptosis may be a potential mechanism for DHA's anti-cancer effects, particularly in breast cancer [76,77]. Ferroptosis is a regulated form of cell death characterized by the accumulation of irondependent lipid peroxides in the cell membrane, leading to oxidative damage and cell death. Studies have demonstrated that DHA can induce ferroptosis in cancer cells by elevating lipid peroxide levels [78,79]. DHA-induced ferroptosis is dependent on the iron-dependent lipoxygenase (LOX) pathway, which is involved in lipid peroxide production. Our study showed a significant increase in lipid peroxidation in MCF-7/Dox cells treated with DHA, suggesting that DHA may activate ferroptosis as a mechanism to sensitize drug-resistant breast cancer cells to the drug.
In the recent past, pyroptosis has emerged as a prospective mechanism by which DHA exerts its anti-cancer properties, particularly in breast cancer. Pyroptosis is a form of programmed cell death that is triggered by inflammatory signals, leading to the release of proinflammatory cytokines and the activation of caspase-1.  [80,81]. They identified genetic variants involved in inflammation blockade, such as COX-2 activity, PTGS, and CCL2, as well as genes involved in oxidative stress pathways and apoptosis, such as myeloperoxidase genes and NF-kB. A recent study by Pizato, Luzete et al. (2018) demonstrated that DHA can trigger pyroptosis in breast cancer cells [17]. This process is dependent on the NLRP3 inflammasome and the production of reactive oxygen species (ROS). Our research found that DHA treatment led to a significant increase in lipid peroxidation, and pro-apoptotic caspase expression in MCF-7/Dox cells, indicating that DHA may activate pyroptosis to sensitize drug-resistant breast cancer cells to the drug. Additional research is necessary to fully comprehend the activation of ferroptosis and pyroptosis when DHA is used in combination with other treatments for drug-resistant breast cancer cells.
In conclusion, docosahexaenoic acid (DHA) has shown promise as an adjuvant drug to sensitize drug-resistant breast cancer cells to chemotherapy. Based on our findings and those of others, DHA is believed to enhance drug sensitivity through various mechanisms, including modulation of drug efflux pumps; alteration of membrane properties; activation of apoptotic pathways; inhibition of signaling pathways; modulation of epigenetic mechanisms; induction of programmed cell death, including pyroptosis and ferroptosis, in breast cancer cells; and can enhance the effectiveness of chemotherapy drugs such as doxorubicin, docetaxel, and cisplatin. However, further studies are necessary to fully understand the mechanisms of DHA-induced cell death and its potential interactions with other chemotherapy drugs. Clinical trials are also needed to determine the safety and effectiveness of using DHA as a supplement to standard breast cancer treatments. Overall, DHA holds potential as an adjuvant bioactive compound to improve the treatment of drug-resistant breast cancer, but more research is required to establish its efficacy.