Breast cancer (BC) is a malignancy commonly detected in women, characterized by a significant global mortality rate. The global incidence of breast cancer reached 2.3 million cases in 2022 (Ferlay J et al., 2024). As the most often diagnosed cancer in females, it represents around 23% of all cancer cases (Trapani et al., 2022). The molecular differences observed in breast tumors necessitate the use of multiple drugs to treat this illness. In addition to surgery, chemotherapy, and radiotherapy, which form the foundation of breast cancer treatment, targeted therapy using drugs that specifically target molecular receptors appears to be a promising approach in many conditions (Dodelzon et al., 2021). One example of this is endocrine therapy, which uses the selective estrogen receptor modulator tamoxifen to target and restrict the proliferation of breast cancer cells that are positive for the estrogen receptor (ER), ultimately leading to their cell death by apoptosis (Li et al., 2017). In contrast, the triple-negative breast cancer (TNBC) subtype has the highest probability of recurrence and the most unfavorable prognosis for survival (Yin et al., 2020).

Anthracycline drugs are frequently used as chemotherapeutic agents for malignant breast cancer. People who are resistant to endocrine therapy or have metastatic cancer extensively use doxorubicin (DOX) a commonly prescribed anthracycline drug, to treat their breast cancer (Shen and Liu, 2023). Although the specific mechanisms underlying its chemotherapeutic efficacy are not fully understood, DOX primarily promotes apoptotic cell death in cancer cells (Kciuk et al., 2023). When administered as a solitary treatment, Dox generally achieves response rates ranging from 40% to 60%, with the possibility of reaching as high as 80% (Rahman et al., 1999). Despite the effectiveness of initial cancer treatment, the emergence of DOX (doxorubicin) resistance poses a significant challenge in medical practice and significantly contributes to treatment failure (Rahman et al., 1999). Researchers have proposed various mechanisms to develop multidrug resistance (MDR) to DOX in breast cancer. It could be an intrinsic or acquired DOX resistance. Secondary resistance develops when breast cancer cells that were previously responsive to DOX become resistant during treatment (Sadida et al., 2024).

MDR represents acquired resistance to numerous anticancer drugs with different structures or mechanisms. Furthermore, MDR cells can gain tolerance to new toxic substances. Several biological pathways can produce multidrug resistance. The most common mechanism is transmembrane protein overexpression, which causes typical MDR (Assaraf et al., 2019). These proteins eliminate chemicals from the cell in one direction, lowering the concentrations of cytotoxic agents below the effective level. The most abundantly generated transport proteins in MDR tumor cells are ATP-binding cassette transporters (ABCTs). ATP hydrolysis powers these proteins’ active transport of chemotherapeutic drugs against the concentration gradient (Teodori et al., 2022). Overexpression of these proteins increases chemotherapy efflux outside cancer cells and therapy failure, indicating MDR (Nobili et al., 2020). However, these proteins are abundant in different healthy tissues and perform various physiological and pharmacological functions (El-Readi et al., 2021). Their presence protects tissues from xenobiotics while also affecting absorption, distribution, metabolism, excretion, and toxicity (ADMET) as well as drug bioavailability. P-glycoprotein (P-gp/ABCB1/MDR1), multidrug resistance protein 1 (MRP1/ABCC1), and breast cancer-resistant protein (BCRP/ABCG2/MXR) are the most important members of this family (Ashique et al., 2024).

As a result, the effectiveness of existing therapeutic methods in BC treatment is insufficient, creating a significant need for more research in this area. Regardless of some unsuccessful outcomes, natural products are still being considered as a potential solution to the difficulties that may arise with MDR in breast cancer (Malla and Kiran, 2022). Reports indicate that they can reverse its MDR by modulating ABCT, inhibiting cell cycle progression, inducing cell death, reducing recurrence, and stopping metastasis while causing few side effects (El-Readi et al., 2024; Wink et al., 2012; El-Readi et al., 2021).

Coptisine (COP) is one of the major secondary metabolites present in Rhizoma coptidis. It is a natural protoberberine alkaloid commonly found in the Ranunculaceae and Papaveraceae plant families (Figure 1) (Cao et al., 2012).

Different experimental sets have examined COP’s in vitro anti-cancer effects in various cancer cell lines, targeting apoptosis and cell cycle arrest. COP (0–25 μM, 24 h) reduced HCT116 cell viability, adhesion, and migration (Cao et al., 2018). COP inhibits apoptosis by modulating ROS, Bcl-2, Bid, Bax, cytochrome c, Apaf-a, AIF, XIAP, caspase-3, and caspase-9 (Zhang et al., 2018). COP (0–28.11 μM, 24 h; 0–75 μM, 48 h) inhibited HCT-116 cells in G1 phase and reduced expression of key genes like CDK4, CDK2, cyclin E, and cyclin C during the G1/S (Han et al., 2018). COP showed the same anticancer effects on breast cancer cells (Li et al., 2014), non-small cell lung cancer cells (Rao et al., 2017), liver cancer cells (Zhou et al., 2018), and pancreatic cancer cells (Zhang et al., 2019). COP (12.5–100 μM for 24 h) reduced HepG2 cell viability and growth by reducing miR-122 expression. Human hepatoma cells showed increased 67kD laminin receptor/cGMP pathway transduction and apoptosis (Zhou et al., 2018). A subsequent study found that COP (0–64 μM, 24 h) reduced MMP-9 mRNA expression in MDA-MB-231 cells by increasing TIMP-1 mRNA, a known MMP inhibitor (Li et al., 2014). COP dose-dependently inhibited PANC-1 cell metastasis, causing G1 cell cycle arrest and S phase reduction (25–150 μM, 48 h) (Zhang et al., 2019). In non-small cell lung cancer cell line A549, COP (12.5–50 μM, 48 h) caused cell cycle arrest at G0/G1 and G2/M phases, as well as effects on G1/S phase and downregulated Cyclins D/E, CDK2/4/6, Cdc2/25C, and p21 (Rao et al., 2017). COP (12.5–50 μM, 48 h) increased pH2AX protein levels, indicating DNA damage, in A594 cells. Upregulation of ROS, caspase-3/9, Bax/Bcl-2, and PARP cleavage caused apoptosis (Han et al., 2019). Large doses (150 mg/kg) of COP have been shown to hinder in vivo tumor development and reduce the probability of tumor cell proliferation. Furthermore, it inhibits the cell cycle’s progress in colorectal cancer cells and induces apoptosis (Huang et al., 2017). Previous studies have documented the potential of COP as an anti-cancer, anti-inflammatory, and antibacterial agent because of its ability to modulate the signaling transduction pathways (Wu et al., 2019; Chai et al., 2024). Virtual screenings, molecular docking of binding affinity, and in silico toxicity assessments have previously been used to analyze 375 phytochemicals. An additional round of in vitro testing was conducted on COP and five other compounds after these in silico results (Schäfer et al., 2023). The multidrug-resistant CEM/ADR5000 cells exhibited cross-resistance to COP, the IC₅₀ values were 9.6 µM for CCRF-CEM cells and greater than 250 µM for CEM/ADR5000 cells and moderate P-glycoprotein inhibition and markedly enhanced intracellular DOX accumulation (Schäfer et al., 2023). COP derivative (8-oxocoptisine) exhibits substantial P-glycoprotein multidrug resistance inhibition activity, with an ED50 value of 0.018 μg/mL in MES-SA/DX5 cells and 0.0005 μg/mL in HCT15 cells, respectively. On the other hand, our understanding of how COP and chemotherapy interact to alter ABCT resistance and its effects on breast cancer that has developed resistance is incomplete.

Combining COP with DOX, according to our hypothesis, would increase DOX’s efficacy by facilitating its accumulation within resistant breast cancer cells. Combining COP with DOX is an innovative approach that could protect against the dose-limiting toxicity of DOX while simultaneously increasing its efficacy in cancer treatment.

The occurrence of MDR during chemotherapy is a significant factor that limits clinical treatment and results in chemotherapy ineffectiveness. P-gp/MDR1, BCRP, and MRP1 are members of the ABC transporter family. They can interact with chemotherapeutic medicines such as DOX and expel intracellular pharmaceuticals to extracellular cells through ATP hydrolysis, causing MDR. Studies have demonstrated the efficacy of Traditional Chinese Medicine (TCM) as a supplementary medication for chemotherapy in counteracting MDR. TCM achieves this by reducing the function and expression of ABC transporters in cancer cells. BC patients have a favorable outcome and can achieve a complete recovery through timely and suitable medical intervention during the initial stages of the illness (Waks et al., 2016). The primary cause of death from BC is MDR, which can lead to metastatic disease. The main obstacle to finding a cure for the disease is the spread of cancer throughout the body. Therefore, the reverse of MDR is an important goal in the BC investigation. Because of this, there has been significant interest in a new medicine or phytochemical product that could inhibit MDR in breast cancer (El-Readi et al., 2024). Particularly, compounds that have minimal adverse effects have attracted attention (Eslami et al., 2024). For this study, MCF7 and MDA-MB-231 cells were used as parent cells to demonstrate a typical scenario. Despite being both invasive ductal/BC cells, they exhibit numerous phenotypic/genotypic distinctions. MCF7 cells depend on hormones, showing positive expression of estrogen, progesterone, and endothelial growth factor receptors (ER, PR, and EGF). MDA-MB-231 cells, on the other hand, are known for being triple-negative (Theodossiou et al., 2019). The lack of estrogen receptor (ER) in MDA-MB-231 has made it unresponsive to antiestrogen therapy, including the commonly used selective estrogen receptor modulator tamoxifen in BC prevention and treatment (Radmacher and Simon, 2000). MCF7 cells are primarily based on oxidative phosphorylation for ATP production under normal oxygen conditions but increase their glycolytic activity when oxygen is limited. In contrast, MDA-MB-231 cells are predominantly based on glycolysis for ATP production regardless of oxygen availability, indicating a Warburg-type metabolism (Theodossiou et al., 2019). While MDA-MB-231 cells show a more mesenchymal phenotype and have been found to have MDR, MCF7 cells have an epithelial phenotype (Theodossiou et al., 2019). To examine the effect of COP on sensitive and resistant cell lines, this study created DOX-resistant cell lines as in vitro models of acquired MDR through continuous treatment with a low dosage of DOX (1.8 μM). This model was successful because resistant cell lines had strong expressions of the ABCTs genes and proteins. The study aimed to investigate the effects of COP on MDR in various BC cell lines and their commonly used resistant models. It also examined the synergistic interaction between COP and DOX in modulating ABCTs in resistant BC.

The study found that COP had a moderate cytotoxic effect on the tested cell lines, although this effect was not as strong as the cytotoxic medication DOX. However, the combination of non-toxic concentration of COP 31 μM (10 μg/mL) and DOX significantly enhanced the cytotoxicity of DOX in the resistant cell lines. Figures 2, 3 demonstrate this synergy in both resistant and sensitive breast cancer cell lines. The results suggest that COP could serve as an adjuvant drug in the treatment of BC. Thus, the combination of DOX demonstrates a potent cytotoxic impact at low dosages with few adverse effects, particularly in drug-resistant BC.

Furthermore, the COP inhibited ABCT’s function and expression, as evidenced by a notable increase in Rho123 accumulation (Figure 5) and downregulation of ABCT gene (Figure 6) and protein expression (Figure 7). Both DOX and VER increased Rho123 accumulation, indicating ABCT inhibition at the dose used. DOX and P-gp interaction depends on cancer type, DOX dose, and cancer cell P-gp expression. DOX can increase chemotherapy sensitivity by inhibiting P-gp. In other cases, it may not overcome MDR. Normally a substrate for ABCT transporters, DOX can also inhibit them under certain conditions. To overcome drug resistance, optimizing its use in cancer treatment requires understanding this dual role (Robey et al., 2018). Research has demonstrated that doxorubicin inhibits ABC transporter efflux activity at concentration-dependent rates. At high doses, it can bind to P-gp and other transporters, making them less effective at effluxing other substrates. The drug concentration and cellular context are major factors in this dual role. This dose-dependent inhibition lends credence to the idea that doxorubicin can play a dual role as substrate and inhibitor, depending on the concentration and saturation of the transporter. DOX is effectively refluxed by ABC transporters at therapeutic doses typically used in clinical settings, resulting in diminished efficacy. On the other hand, when doxorubicin concentrations are increased in an experimental setting, the transporters become saturated, and their efflux function is inhibited (Gottesman et al., 2016).

The data indicated that COP effectively reversed multidrug resistance (MDR) in BC and restored the sensitivity of DOX-resistant cells to its cytotoxic effects. Previously, COP reduced MDA-MB-231 adhesion, migration, and invasion in vitro by downregulating MMP-9 and increasing TIMP-1, presumably due to its anti-metastatic effect on BC (Li et al., 2014). Previously, IQA demonstrated concentration-dependent inhibition of P-gp/MDR1 activity in both Caco-2 and CEM/ADR5000 cells, effectively reversing their resistance to doxorubicin (El-Readi et al., 2013). The expression analysis revealed a common set of key genes associated with apoptosis, cell cycle, and drug metabolism. Exposure of Caco-2 cells to isoquinoline alkaloid (IQA) led to a notable reduction in mRNA levels of MDR1, MRP1, BCRP, CYP3A4, GST, and hPXR (El-Readi et al., 2013). The postulated mechanism for the chemosensitizing effect of IQA involves the modification of the ATPase activity of MDR1 (Ativui et al., 2021). Understanding the mechanism by which IQA reduces MDR in BC is important. In line with the current findings, researchers have used the MTT assay to assess the drug resistance and cytotoxicity of berberine (Ber) and doxorubicin (DOX), either separately or in combination, in MCF-7/DOX^Fluc, which stably expresses the firefly luciferase reporter gene. Ber had a synergistic impact in enhancing the inhibitory effect of DOX on cell growth. In vivo, Ber significantly decreased the release of d-luciferin potassium salt in the MCF-7/DOX^Fluc cell line, suppressing the activity of P-gp/MDR1 and MRP1 in these cells. Additionally, it enhanced the uptake of DOX in tumor tissues by reversing multidrug resistance (MDR) by inhibiting the efflux of ABCTs and reducing their expression (Qian et al., 2021). Figure 8 summarizes the MDR reversal mechanism of COP that boosted DOX concentration in resistant cells and improved its cytotoxicity in MCF-7/ADR and MDB-MB-231/ADR cells, and that it could overcome MDR by inhibiting ABCT function and expression in a resistant BC.

This study demonstrated that COP could overcome MDR by inhibiting ABCT function and expression in a resistant BC. COP increases DOX concentration in resistant cells and enhances its cytotoxicity in MCF-7/ADR and MDB-MB-231/ADR cells. More in vitro studies are required to validate the effect of COP on ABCT targets of ABCTs, such as ATPase activities and drug accumulation studies. In vivo studies are needed to make sure that the current results are correct and to see how well COP and DOX combinations work to reverse multidrug resistance (MDR) in breast cancer models in animals.