Document Type : Original Research Article
Introduction
Breast cancer is one of the most commonly diagnosed cancers worldwide, accounting for a significant proportion of cancer-related mortality among women(1). Despite advancements in early detection and treatment, breast cancer remains a major health burden, with hormone receptor-positive subtypes, such as MCF-7, exhibiting variable responses to chemotherapy(2). Conventional chemotherapeutic agents, including anthracyclines like Bleomycin, have been widely used to manage breast cancer due to their potent cytotoxic effects(3). However, the clinical utility of Bleomycin is often limited by multiple factors, including the development of multidrug resistance (MDR), systemic toxicity, and reduced therapeutic efficacy over time. These limitations necessitate the exploration of novel strategies to enhance the effectiveness of Bleomycin while minimizing its adverse effects(4, 5).
Bleomycin exerts its anticancer activity through multiple mechanisms, including DNA intercalation, inhibition of topoisomerase II, and the generation of reactive oxygen species (ROS), ultimately leading to apoptosis in cancer cells(6). However, breast cancer cells, particularly MCF-7, have been shown to develop resistance to Bleomycin through various mechanisms, such as increased expression of ATP-binding cassette (ABC) transporters, elevated antioxidant defense mechanisms, and enhanced DNA repair pathways(7, 8). These adaptive responses reduce drug accumulation in cancer cells, neutralize ROS-mediated cytotoxicity, and enable tumor cells to evade apoptosis. Consequently, overcoming Bleomycin resistance remains a critical challenge in breast cancer therapy(9).
Nanotechnology has emerged as a promising approach for enhancing the efficacy of chemotherapeutic agents by improving drug delivery, increasing intracellular drug retention, and modulating tumor microenvironmental factors(10). Among the various nanoparticles explored for cancer therapy, metal-based nanoparticles have attracted considerable attention due to their unique physicochemical properties and ability to induce oxidative stress selectively in cancer cells. Copper nanoparticles (CuNPs) have demonstrated significant potential in this regard, as they can generate ROS, disrupt mitochondrial function, and interfere with cellular redox homeostasis(11). These properties make CuNPs an attractive candidate for use as a chemosensitizer to enhance the therapeutic effects of conventional anticancer drugs(12, 13).
Copper is an essential trace element that plays a crucial role in various biological processes, including cellular respiration, antioxidant defense, and enzyme activation(14). However, dysregulated copper homeostasis has been implicated in cancer progression, with elevated intracellular copper levels observed in several malignancies. Cancer cells exhibit increased demand for copper due to its involvement in angiogenesis, metastasis, and redox regulation(15). This differential copper metabolism provides an opportunity to exploit CuNPs as a targeted therapeutic agent that selectively enhances oxidative stress in cancer cells while sparing normal tissues. Previous studies have demonstrated that CuNPs can potentiate the effects of chemotherapy by promoting ROS generation, impairing DNA repair mechanisms, and sensitizing cancer cells to apoptosis(16).
Oxidative stress plays a dual role in cancer therapy, as moderate levels of ROS can promote tumor survival and drug resistance, whereas excessive ROS accumulation can trigger cell death. Many cancer cells, including MCF-7, maintain a delicate balance between ROS production and antioxidant defense mechanisms to sustain their survival(17, 18). The primary intracellular antioxidant, glutathione (GSH), serves as a critical defense mechanism against chemotherapy-induced oxidative damage(19). However, the depletion of GSH disrupts this balance, rendering cancer cells more vulnerable to oxidative stress and apoptosis. CuNPs has been shown to deplete GSH levels while simultaneously increasing lipid peroxidation markers such as malondialdehyde (MDA), leading to enhanced chemosensitivity(20, 21).
Furthermore, mitochondrial dysfunction is a key determinant of chemotherapy response, as mitochondria play a central role in energy metabolism, ROS production, and apoptosis regulation(22). CuNPs has been reported to induce mitochondrial membrane depolarization, leading to cytochrome c release and activation of caspase-dependent apoptotic pathways(23). By impairing mitochondrial function, CuNPs can further amplify the cytotoxic effects of Bleomycin, thereby enhancing treatment efficacy in breast cancer cells(24).
Given these promising properties of Bleomycin, the present study aims to investigate its potential role as a chemosensitizer in enhancing the cytotoxicity of Bleomycin against MCF-7 breast cancer cells. Specifically, we seek to evaluate the combined effects of CuNPs and Bleomycin on cell viability, oxidative stress markers, and apoptotic pathways. By elucidating the underlying mechanisms of action, this study aims to provide valuable insights into the potential application of CuNPs in overcoming chemoresistance and improving breast cancer treatment strategies.
CuNPs with a size range of 30–10 nm and a purity of 99.99% were sourced from US Research Nanomaterials Inc. and obtained through the local representative, Pishgaman Nano Materials Iranian Company in Mashhad. Bleomycin was purchased from Pfizer (New York, NY, USA). Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA, and phosphate-buffered saline (PBS) were supplied by Gibco (Thermo Fisher Scientific, Waltham, MA, USA). The MTT reagent, 2’,7’-dichlorofluorescin diacetate (DCFH-DA), thiobarbituric acid (TBA), and 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich. All other chemicals used were of analytical grade and were acquired from standard suppliers.
MCF-7 human breast cancer cells were obtained from National Cell Bank of Iran (Pasteur Institute, Tehran, Iran) and cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% L-glutamine. The cells were maintained in a humidified atmosphere at 37°C with 5% CO₂. The culture medium was replaced every two to three days, and the cells were passaged using trypsin-EDTA (0.25%) when they reached 80–90% confluence. For experimental procedures, the cells were plated in 96-well plates for viability assays. All experiments were performed at least in triplicate to ensure reproducibility and accuracy of results(25, 26).
MCF-7 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and allowed to attach overnight. Cells were treated with various concentrations of CuNPs (10, 15, 20, 25 and 30 µM) in combination with Bleomycin (10 µg/mL) for 48 hours. Untreated cells served as controls.
The cytotoxic effects of the treatments on MCF-7 cells were evaluated using the MTT assay. Following treatment, 20 µL of MTT reagent (5 mg/mL) was added to each well, and the cells were incubated for 4 hours at 37°C. The formazan crystals produced during the reaction were dissolved in 150 µL of dimethyl sulfoxide (DMSO). The absorbance was then measured at 570 nm using a microplate reader (BioTek, Winooski, VT, USA). The cell viability percentage was calculated by comparing the absorbance of treated cells with that of the untreated control cells(27, 28).
Assessment of Intracellular ROS Levels
The intracellular reactive oxygen species (ROS) levels were evaluated using the DCFH-DA probe. Cells were incubated with 10 µM DCFH-DA for 30 minutes at 37°C, followed by exposure to CuNPs and Bleomycin. Fluorescence intensity was measured using a fluorescence microplate reader (excitation: 485 nm; emission: 530 nm). ROS levels were expressed as a fold change relative to the untreated control group(29, 30).
Determination of Lipid Peroxidation
Lipid peroxidation was assessed by quantifying malondialdehyde (MDA) levels using the thiobarbituric acid-reactive substances (TBARS) assay. Cell lysates were combined with TBA reagent and incubated at 95°C for 30 minutes. After cooling, the samples were centrifuged at 10,000 × g for 10 minutes, and the absorbance of the supernatant was measured at 532 nm. The MDA levels were expressed in nmol/mg protein(31, 32).
Quantification of Glutathione (GSH) Levels
Glutathione levels were measured using the DTNB reagent. Cell lysates were prepared, and 50 µL of each sample was mixed with an equal volume of 10 mM DTNB reagent in PBS. Absorbance was recorded at 412 nm, and GSH concentrations were determined using a standard curve(33, 34).
Statistical Analysis
All experiments were performed in triplicate, and results are presented as the mean ± standard deviation (SD). Statistical analysis was carried out using GraphPad Prism v8.0 (GraphPad Software, San Diego, CA, USA). Comparisons between groups were made using one-way ANOVA followed by Tukey’s post hoc test. A p-value of less than 0.05 was considered statistically significant(35, 36).
Effect of CuNPs and Bleomycin on Breast Cancer Cell Viability
The cytotoxic effects of CuNPs in combination with Bleomycin on breast cancer cells (MCF-7) were assessed using the MTT assay. As illustrated in Figure 1, CuNPs at concentrations of 10, 15, 20, 25, and 30 µM, combined with Bleomycin (10 µg/mL), resulted in a dose-dependent decline in cell viability following 48 hours of treatment. The combination therapy exhibited a synergistic effect, leading to significantly greater cytotoxicity compared to either treatment alone (p<0.05).
To evaluate oxidative stress, ROS levels were measured following treatment with CuNPs in combination with Bleomycin. Treatment with CuNPs (10, 15, 20, 25, and 30 µM) and Bleomycin (10 µg/mL) resulted in a significant elevation of ROS levels, surpassing those observed with Bleomycin alone (p<0.01), suggesting a synergistic enhancement of oxidative stress.
Assessment of Lipid Peroxidation
MDA levels, a marker of lipid peroxidation, were significantly elevated in MCF-7 cells treated with CuNPs in combination with Bleomycin. The co-administration of CuNPs (10, 15, 20, 25, and 30 µM) and Bleomycin (10 µg/mL) resulted in a notable increase in MDA levels compared to individual treatments , indicating greater oxidative damage to cellular membranes.
Reduction of Intracellular Glutathione (GSH) Levels
GSH depletion was observed following treatment with CuNPs and Bleomycin. CuNPs (10, 15, 20, 25, and 30 µM) combined with Bleomycin (10 µg/mL) significantly reduced GSH levels compared to individual treatments, emphasizing the role of oxidative stress in the cytotoxic mechanism.
Synergistic Cytotoxicity Mediated by Oxidative Stress
Overall, the findings demonstrate that CuNPs combined with Bleomycin exert a synergistic cytotoxic effect on MCF-7 breast cancer cells. This effect is likely mediated through enhanced oxidative stress, as evidenced by increased ROS production, lipid peroxidation, and depletion of intracellular GSH levels.
Breast cancer remains one of the most prevalent malignancies worldwide, necessitating the development of novel therapeutic strategies that enhance efficacy while minimizing adverse effects(37). Bleomycin is a widely used chemotherapeutic agent; however, its long-term clinical application is limited due to dose-dependent pulmonary toxicity and drug resistance(38). Recent advancements in nanotechnology have explored the potential of metal-based nanoparticles, including CuNPs, as adjuvant therapies to potentiate the cytotoxic effects of chemotherapeutic agents while mitigating their limitations(39). This study investigated the synergistic cytotoxicity of CuNPs in combination with Bleomycin in MCF-7 breast cancer cells, emphasizing oxidative stress-induced cell death mechanisms.
The MTT assay demonstrated a dose-dependent reduction in MCF-7 cell viability upon treatment with Bleomycin alone, as well as an enhanced cytotoxic effect when combined with CuNPs. This enhanced cytotoxicity suggests a potential interaction between CuNPs and Bleomycin, leading to increased cell death. Several mechanisms may account for this synergistic interaction, including the ability of CuNPs to disrupt redox homeostasis, enhance ROS production, and potentiate DNA damage, thereby amplifying Bleomycin’s cytotoxic effects(40).
Oxidative stress plays a critical role in the cytotoxicity of both CuNPs and Bleomycin. The generation of ROS is one of the primary mechanisms by which CuNPs exert their toxic effects on cancer cells(41). Copper ions can undergo redox cycling via Fenton-like reactions, leading to excessive ROS production. These reactions result in the accumulation of hydroxyl radicals and superoxide anions, which contribute to oxidative damage in cellular components(42). Bleomycin, on the other hand, is known to induce ROS production via redox cycling of its quinone moiety(43). The combination of CuNPs and Bleomycin likely exacerbates ROS generation beyond the threshold of cellular antioxidant defenses, overwhelming the intrinsic antioxidant response and leading to oxidative damage-induced cell death(44, 45).
The increase in MDA levels in MCF-7 cells treated with CuNPs and Bleomycin suggests a significant degree of lipid peroxidation. Lipid peroxidation is a hallmark of oxidative stress-mediated cytotoxicity, in which free radicals attack polyunsaturated fatty acids within the plasma membrane, leading to the formation of lipid peroxides and aldehyde byproducts such as MDA(46, 47). The peroxidation of membrane lipids results in loss of membrane integrity, increased permeability, and subsequent cell death via necrosis or apoptosis(48). The observed increase in MDA levels in the combination treatment group strongly suggests that CuNPs enhance Bleomycin -induced oxidative membrane damage, thereby amplifying its cytotoxic effects.
Glutathione is a key antioxidant that plays a pivotal role in cellular defense against oxidative stress(49). The significant depletion of intracellular GSH levels in cells treated with CuNPs and Bleomycin indicates an overwhelmed antioxidant defense system. The depletion of GSH suggests that the increased ROS levels induced by the combination therapy may have exhausted the cellular antioxidant capacity, rendering the cells more susceptible to oxidative damage. Additionally, the oxidation of GSH to glutathione disulfide (GSSG) further contributes to redox imbalance and initiates apoptotic pathways through activation of pro-apoptotic proteins(50).
One of the major mechanisms of Bleomycin -induced cytotoxicity is its ability to intercalate into DNA and inhibit topoisomerase II, leading to DNA strand breaks and apoptosis(51). The presence of CuNPs may exacerbate DNA damage through ROS-induced oxidative stress, causing additional DNA fragmentation and activation of apoptosis. This combination therapy is likely to enhance the intrinsic (mitochondrial) apoptotic pathway, as excessive ROS levels disrupt mitochondrial integrity, leading to cytochrome c release(52). Cytochrome c subsequently interacts with apoptotic protease-activating factor 1 (Apaf-1), triggering the activation of caspase-9 and ultimately caspase-3, which executes the apoptotic program. Additionally, poly (ADP-ribose) polymerase (PARP) cleavage, a hallmark of apoptosis, further confirms the activation of cell death pathways(53, 54).
Moreover, the interplay between Bleomycin and CuNPs may contribute to p53-dependent apoptotic signaling. The excessive oxidative DNA damage caused by ROS can activate p53, leading to cell cycle arrest and programmed cell death(55). This suggests that CuNPs not only enhance the cytotoxic effects of Bleomycin but also reinforce apoptotic pathways through both mitochondrial and p53-mediated mechanisms. Given the central role of oxidative stress in this process, the extent of ROS production and the subsequent cellular antioxidant response will be critical determinants of the overall therapeutic efficacy and toxicity of this combination therapy.
Comparing these results with previous studies on metal-based nanoparticles in cancer therapy, similar trends have been observed with other nanoparticles, such as silver nanoparticles (AgNPs) and iron oxide nanoparticles (Fe3O4 NPs), which also exhibit ROS-mediated cytotoxicity(56, 57). For instance, AgNPs have been shown to induce mitochondrial dysfunction and oxidative DNA damage, leading to apoptosis in breast cancer cells(58). However, the advantage of CuNPs lies in their dual role as both a catalytic enhancer of ROS generation and a redox-active metal capable of interfering with essential cellular antioxidant systems, making them a particularly effective candidate for combination therapy(59). Moreover, unlike gold nanoparticles (AuNPs), which primarily serve as drug carriers with minimal intrinsic toxicity, CuNPs exhibit intrinsic cytotoxic effects, further strengthening their potential as a standalone or adjunct therapeutic agent(60).
A key mechanistic insight from this study is the role of CuNPs in exacerbating oxidative stress by interfering with antioxidant defense mechanisms, particularly by depleting intracellular GSH levels. The depletion of GSH, a critical intracellular antioxidant, makes cells more vulnerable to ROS-induced damage, leading to increased lipid peroxidation and subsequent membrane instability(61). Similar effects have been reported with other transition metal nanoparticles, such as ZnO NPs, which also induce oxidative stress-related apoptosis in various cancer cell lines(62). However, CuNPs exhibit unique redox cycling properties that contribute to their higher efficacy in disrupting redox balance, making them an attractive alternative to other metal-based nanotherapeutics(42).
The observed synergy between CuNPs and Bleomycin in this study suggests a potential strategy to overcome chemoresistance in breast cancer cells. Bleomycin, a widely used anthracycline chemotherapeutic, exerts its cytotoxic effects primarily through DNA intercalation and topoisomerase II inhibition, leading to apoptotic cell death. However, its clinical application is often limited by resistance mechanisms, including enhanced DNA repair, drug efflux, and increased antioxidant defenses(63). By introducing CuNPs into the treatment regimen, these resistance mechanisms can be mitigated through heightened oxidative stress, thereby sensitizing cancer cells to Bleomycin -induced cytotoxicity. Previous studies on metal nanoparticle-drug combinations have reported similar findings, with metal-based nanocarriers effectively overcoming multidrug resistance by disrupting cellular redox equilibrium and promoting apoptosis(64).
In contrast to studies utilizing non-metallic nanoparticles such as polymeric or lipid-based nanocarriers, which primarily function as drug delivery systems, CuNPs contribute to cancer therapy beyond passive drug transport(65). Their intrinsic cytotoxic properties, coupled with their ability to amplify chemotherapy-induced oxidative stress, set them apart from conventional nanocarriers(66). Additionally, while iron-based nanoparticles such as Fe3O4 have been explored for their potential in cancer therapy, their effects are often mediated through magnetic hyperthermia rather than direct cytotoxicity(67). In comparison, CuNPs provide a more direct and aggressive approach by actively generating ROS and disrupting cellular homeostasis(68).
Overall, this study provides strong evidence that CuNPs, when combined with Bleomycin, significantly enhance therapeutic efficacy through oxidative stress-mediated pathways. The findings suggest that CuNPs can be used to potentiate the effects of conventional chemotherapeutics, offering a promising strategy for improving treatment outcomes in breast cancer. Future studies should explore the long-term effects of CuNPs - Bleomycin combination therapy, particularly in in vivo models, to assess potential toxicity and systemic biodistribution. Additionally, investigating the molecular pathways involved in CuNPs mediated ROS generation and apoptosis induction could further elucidate their role as an emerging nanotherapeutic in oncology.
Potential Clinical Implications and Future Directions
The findings of this study suggest that CuNPs can significantly enhance the cytotoxic effects of Bleomycin in breast cancer cells through oxidative stress-mediated mechanisms. The observed increase in ROS generation, lipid peroxidation, and depletion of intracellular glutathione highlights the potential of CuNPs as an adjuvant in breast cancer therapy. However, before clinical translation, several crucial aspects need to be addressed. One major concern is the potential for off-target toxicity, as systemic administration of CuNPs may lead to damage in normal tissues. Future studies should focus on developing tumor-targeted CuNPs by functionalizing them with tumor-specific ligands to enhance selective accumulation in cancer cells. Additionally, while in vitro findings provide strong evidence of synergistic cytotoxicity, in vivo studies are essential to assess pharmacokinetics, biodistribution, and systemic toxicity. Investigating these parameters will help determine the therapeutic index and safety profile of CuNPs based combination therapy.
Moreover, optimization of the CuNPs to Bleomycin ratio is critical to achieving maximum therapeutic efficacy while minimizing potential adverse effects. Excessive ROS production could lead to unintended toxicity, necessitating precise dose adjustments for clinical application. Another important consideration is the stability and clearance pathways of CuNPs, as long-term retention and aggregation could pose toxicity risks. Therefore, understanding their biodegradation and elimination from the body is essential for clinical feasibility. Comparatively, other studies exploring metal-based nanoparticles, such as gold and silver nanoparticles, have demonstrated similar ROS-mediated cytotoxic effects, though CuNPs exhibit distinct redox properties that may provide a stronger synergistic interaction with Bleomycin. Further research into nanoparticle engineering, including coating strategies and controlled-release mechanisms, may enhance the therapeutic potential of CuNPs while mitigating toxicity concerns. This study lays the foundation for future investigations into CuNPs based combinational chemotherapy and underscores the necessity of translational research to bring this promising approach to clinical application.
This study provides compelling evidence that CuNPs enhance Bleomycin -induced cytotoxicity in MCF-7 breast cancer cells by increasing oxidative stress, inducing lipid peroxidation, depleting intracellular antioxidants, and promoting apoptotic cell death. These findings open new avenues for using CuNPs as a potential adjuvant to conventional chemotherapy, paving the way for novel combination therapies in breast cancer treatment. Future research should focus on optimizing nanoparticle formulations to maximize therapeutic benefits while minimizing adverse effects, ultimately advancing the clinical application of CuNPs based combination therapies.
The author(s) declare that they have no competing interests.
