Evaluation of toxicity and apoptotic effects of copper nanoparticles in combination with imatinib on chronic myeloid leukemia cells

Evaluation of toxicity and apoptotic effects of copper nanoparticles in combination with imatinib on chronic myeloid leukemia cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of toxicity and apoptotic effects of copper nanoparticles in combination with imatinib on chronic myeloid leukemia cells Mohammad Ebrahimi Torkamani, Mahshid Mohammadian, Farzaneh Kefayatirad, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7627097/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Imatinib, a first-generation tyrosine kinase inhibitor, is the mainstay of treatment for chronic myeloid leukemia (CML). However, long-term use can lead to cellular resistance, highlighting the need for more effective therapeutic strategies. This study investigated the cytotoxic and apoptotic effects of the combination of imatinib with copper nanoparticles (CuNPs) on the K562 cell line. K562 cells were treated with imatinib (optimal dose 0.5 µM) and non-toxic CuNPs (0.005 mg/mL), as single agents and in combination, in nine experimental groups. Cell survival was measured by MTT assay, intracellular H₂O₂ levels were measured as an indicator of oxidative stress, and the expression of apoptosis and tumor-related genes (Bax, Bcl-2, MMP2, MMP9, and IL-1β) was examined using real-time PCR. The CuNPs were spherical with an average size of approximately 50 nm. Both imatinib and CuNPs dose-dependently decreased cell survival, while their combination produced a significant cytotoxic effect even at suboptimal doses of the drug. The combination treatment resulted in a significant increase in intracellular H₂O₂ levels, indicating increased oxidative stress. Gene expression analysis revealed an increase in the pro-apoptotic gene Bax and a decrease in the anti-apoptotic genes Bcl-2 as well as MMP2, MMP9, and IL-1β, indicating synergistic induction of apoptosis and regulation of tumor-related pathways. The combination of imatinib and CuNPs produces enhanced cytotoxic and apoptotic effects in K562 cells, even at lower drug doses, indicating that CuNPs can be used as an effective adjuvant to enhance the efficacy of imatinib and delay the development of resistance in the treatment of CML. Cytotoxic Apoptosis Copper Nanoparticles Imatinib Chronic Myeloid Leukemia Figures Figure 1 Figure 2 Introduction Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by uncontrolled proliferation of myeloid cells in the bone marrow and their accumulation in the peripheral blood [ 1 ]. The disease is mainly associated with the presence of the Philadelphia chromosome, which results from a reciprocal translocation between chromosomes 9 and 22, resulting in the formation of a BCR-ABL fusion gene with persistent tyrosine kinase activity [ 2 ]. Although targeted therapies with tyrosine kinase inhibitors (TKIs) such as imatinib have dramatically improved the prognosis of patients with CML [ 3 ], drug resistance and persistence of leukemic stem cells (LSCs) remain major challenges [ 4 ]. LSCs can survive despite treatment and are recognized as a key factor in disease relapse [ 5 ]. Therefore, there is an urgent need for novel therapeutic approaches that can eliminate or sensitize these resistant cells. Nanotechnology has emerged as a promising area in cancer research due to the unique physicochemical properties of nanoparticles, including high surface-to-volume ratio, tunable size, and the ability to be functionalized for targeted delivery [ 6 ]. Among metal nanoparticles, copper nanoparticles (CuNPs) have shown significant anticancer potential, which is mainly attributed to their ability to generate reactive oxygen species (ROS) and induce oxidative stress in cancer cells [ 7 ]. ROS include superoxide anions, hydroxyl radicals, hydrogen peroxide, and singlet oxygen, which are produced under physiological and pathological conditions [ 8 ]. While low levels of ROS can act as messenger molecules in processes such as cell differentiation, proliferation, and immune responses [ 9 ], their excessive production disrupts the redox balance, causing oxidative damage to lipids, proteins, and DNA, ultimately activating apoptosis [ 10 ]. ROS production occurs in cells through various mechanisms. Mitochondria, especially complexes I and III of the respiratory chain, are considered the main source [ 11 ], although other organelles such as peroxisomes, microsomes, and the plasma membrane also play a role in this process [ 12 ]. Nanoparticles can enhance ROS production through light-induced electron transfer processes, generating electron–hole pairs that oxidize water to hydroxyl radicals or reduce oxygen to superoxide and hydrogen peroxide [ 13 ]. This ROS-induced damage can activate intrinsic apoptotic pathways through mitochondrial membrane permeabilization, cytochrome c release, and caspases activation as well as extrinsic pathways through interaction with death receptors [ 14 ]. In leukemic cells, including CML, high levels of ROS are a common feature that contribute to both oncogenic signaling and therapeutic resistance [ 15 ]. Targeting ROS in cancer therapy can be approached from two perspectives: antioxidant therapy, which seeks to reduce ROS levels to limit tumor progression [ 16 ], and prooxidant therapy, which induces cell death by increasing them above the cell’s tolerance threshold. The latter approach is particularly attractive for the selective destruction of cancer cells with intrinsically high levels of ROS, such as LSCs, while leaving normal cells largely intact. Prooxidant compounds such as arsenic trioxide and isothiocyanates have shown antileukemic properties, although their clinical use may be limited due to off-target toxicity[ 17 , 18 ]. In addition to oxidative stress, inflammatory mediators such as interleukin-1 beta (IL-1β) play an important role in the progression of cancer, including hematological malignancies. IL-1β is a proinflammatory cytokine produced by immune cells, fibroblasts, and cancer cells, and promotes tumor growth, angiogenesis, and metastasis by activating intracellular signaling pathways such as PI3K/Rac and β-catenin[ 19 ]. In CML, IL-1β can contribute to the inflammatory tumor microenvironment and affect the survival and proliferation of malignant cells [ 20 ]. Given these considerations, the combination of CuNPs with imatinib could be proposed as a synergistic therapeutic strategy for CML. CuNPs could act by inducing ROS-dependent apoptosis, sensitizing resistant cells, and possibly modulating inflammatory signals, while imatinib targets the oncogenic tyrosine kinase BCR-ABL [ 21 , 22 ]. Evaluation of their combined effects on cell viability, ROS production, expression of apoptotic genes (Bax, Bcl-2), matrix metalloproteinases (MMP-2, MMP-9), and IL-1β expression in CML cells could provide valuable insights into novel strategies to overcome drug resistance and improve treatment outcomes. Materials and Methods Study Design This in vitro study investigated the effects of CuNPs and imatinib, individually and in combination, on viability, oxidative stress, and gene expression in the CML cell line K562. Synthesis and Characterization CuNPs CuNPs were synthesized according to standard protocols: First, 104 mg of copper (II) 2,4-pentanedionate was mixed with 8 mL of 1-octadecene and 2 mL of hexadecylamine under nitrogen conditions. Then, 2 mL of tri-n-octylphosphine was added to the mixture and the temperature was increased to 200°C. Next, a formic acid–sulfuric acid mixture was injected to generate CO gas, and heating was continued to 220°C until a color change to dark red was observed. The reaction was continued for 20 min and then cooled to ambient temperature. The product was precipitated with ethanol/toluene, centrifuged at 6000 rpm for 20 min, redispersed in toluene, and PEGylated using HOOC-PEG-COOH in chloroform for 12 h. Finally, the PEG-coated CuNPs were collected, washed, and resuspended in molten polyethylene glycol. To confirm the physicochemical properties of the synthesized CuNPs, several analytical techniques were used as follows. Transmission Electron Microscopy (TEM): The morphology and size of the CuNPs were examined using TEM. In this method, an electron beam is passed through a thin sample and the interaction of the electrons with the sample creates an image that is detected and magnified on a fluorescent screen, photographic film or digital sensor. Dynamic Light Scattering (DLS) and Zeta Potential: The hydrodynamic diameter and surface charge of the nanoparticles were measured using DLS and zeta potential analysis. DLS provides information about the particle size distribution in the nanometer to micrometer range by analyzing the fluctuations in the intensity of laser light scattered from Brownian moving particles. For this analysis, the nanoparticle samples were dispersed in distilled water at a final concentration of 0.5% (w/v) of dry weight. The suspensions were homogenized using an ultrasonic bath before measurement. The particle size distribution and polydispersity index (PDI) were recorded in at least 30 replicates. Also, the electrophoretic mobility of the particles was measured in PALS mode to calculate the zeta potential. UV–Vis spectroscopy: The optical properties of the nanoparticles were investigated using a spectrophotometer (Shimadzu, Japan). The UV–Vis absorption spectrum of the colloidal CuNPs was recorded in the wavelength range of 400–800 nm and at different time intervals after synthesis. X-ray diffraction (XRD): The crystal structure of the CuNPs was determined using an X’Pert MDP XRD instrument. The measurements were performed with Cu Kα radiation (λ = 1.54056 Å) at a voltage of 40 kV and a step of 0.02°. Cell culture K562 cell line was obtained from the Pasteur Institute, Tehran. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C, 5% CO₂, and 90% humidity. Cultures were checked daily for confluency and contamination. For long-term storage, cells were cryopreserved in FBS/DMSO (1:10) at -180°C. Frozen cells were thawed rapidly at 37°C under sterile conditions, diluted with culture medium, and centrifuged at 1400 rpm for 5 min at 4°C. Cells were then resuspended and counted using a Neubauer hemocytometer using trypan blue staining. Cell viability assay K562 cells were seeded in 96-well plates at a density of 1 × 10⁴ cells per well in 100 µl of culture medium and incubated for 24 hours at 37°C in a humidified atmosphere with 5% CO₂. After incubation, the culture medium was replaced with fresh medium containing imatinib at different concentrations (0.1, 0.21, 0.25, 0.35, 0.42, 0.5, 1, 2, 4, 8, 16, 32, and 64 µM) or CuNPs at concentrations of 0.25, 0.125, 0.1, 0.075, 0.05, 0.025, 0.005, and 0.0025 mg/mL. Imatinib was prepared as capsules (100 mg per capsule as per the manufacturer’s instructions). The contents of the capsules were accurately weighed and the purity of the drug was calculated before preparing the working concentrations to ensure accurate dosing. The half-maximal inhibitory concentration (IC₅₀) of each compound was determined using dose-response analysis. Based on the IC₅₀ value, the treatment groups were divided as follows: Control group: untreated cells. Imatinib groups: 0.5 µM (optimal IC₅₀), 0.25 µM (½ IC₅₀), 0.1 µM (⅕ IC₅₀) and 0.05 µM (¹⁄₁₀ IC₅₀). CuNPs group: only CuNPs at a concentration of 0.005 mg/mL. Combination groups: CuNPs (0.005 mg/mL) combined with imatinib at concentrations of 0.5, 0.25, 0.1 or 0.05 µM. All treatments were performed in triplicate and cell viability was assessed after 24 hours by MTT assay. MTT assay Cell viability was assessed using the MTT kit (Kia Zist, Iran) according to the standard protocol. K562 cells were cultured in 96-well plates at the same density and treated with different concentrations of imatinib (0.5, 0.25, 0.1 and 0.05 µM), CuNPs (0.005 mg/mL) or their combination (NP + Ima 0.25, NP + Ima 0.1, NP + Ima 0.05) for 24 h. Untreated cells were considered as controls. After 24 h of treatment, 10 µL of MTT solution was added to each well and incubated for 3 h at 37°C in the dark. Then, the medium was removed and 100 µL of DMSO was added to dissolve the formazan crystals. The optical absorbance at 570 nm was measured by a microplate reader. The percentage of cell viability was calculated using the following formula: Cell viability = (Absorbance of control group / Absorbance of treated group) × 100 All experiments were performed in triplicate. Measurement of intracellular hydrogen peroxide (H₂O₂) The level of intracellular H₂O₂ as an indicator of oxidative stress was measured using a fluorometric Hydrogen Peroxide Assay kit (Sigma-Aldrich, Germany) according to the manufacturer's instructions. K562 cells (1 × 10⁴ cells/well) were seeded in 96-well plates and treated with imatinib, CuNPs, or their combination for 24 h. After treatment, the cells were washed with PBS, centrifuged, and incubated with 50 µL of freshly prepared Master Mix for 15–30 min at room temperature in the dark. Fluorescence intensity at excitation/emission wavelengths of 540/590 nm was recorded using a microplate reader, and intracellular H₂O₂ concentration was calculated from the standard curve. Gene Expression Analysis via Real-Time PCR Treated and control K562 cells (approximately 1×10 6 ) were collected for RNA extraction using a Trizol-based kit (DnaZist, Iran). RNA integrity and concentration were assessed using NanoDrop (OD₂₆₀/₂₈₀ ratio ≈ 1.8). cDNA synthesis was performed using a Pars Toos kit, Iran. A 20 µL quantitative PCR reaction was performed with SYBR Green dye in a real-time thermocycler. The cycling conditions were: initial denaturation at 95°C for 4 min, followed by 35 cycles of denaturation at 94°C, annealing at 57–60°C, and extension at 72°C (each step 30 s), and finally a final extension step at 72°C for 5 min. The target genes included Bax, Bcl-2, MMP2, MMP9, and IL-1β, and β-actin was used as a reference gene. The primer sequences are presented in Table 1 . The relative expression of the genes was calculated using the 2^ –ΔΔCT method. Table 1 Sequences of forward and reverse primers. Gene Primer ACTB 5´-GGAACGGTGAAGGTGACAG-3´(forward) 5´-GTGGGGTGGCTTTTAGGATG-3´(reverse) Bcl-2 5´-ATGTGAAACTGAATTGGAGAGTG-3´(forward) 5´-TGTTGTTGATAGGATGTTTGCTT-3´(reverse) Bax 5´-CTCACCGCCTCACTCACCC-3´ (forward) 5´-CCCACACCCCCCAATAATTAC-3´ (reverse) MMP2 5´-TACGATGGAGGCGCTAATGG-3´ (forward) 5´-GAAGGTGTTCAGGTATTGCACTG-3´ (reverse) MMP9 5´-TTCCAGTACCGSGSSSGCC-3´ (forward) 5´-CCTTTCCTCCAGAACAATACC-3´ (reverse) IL 1 B 5´-GGCTTATTACAGTGGCAATG-3´ (forward) 5´-TAGTGGTGGTCGGAGATT-3´ (reverse) ACTB, Beta Actin; BCL2, B-Cell Lymphoma 2; BAX, BCL2 Associated X, Apoptosis Regulator; MMP2, Matrix Metallopeptidase 2; MMP9, Matrix Metallopeptidase 9; IL-1B, Interleukin 1 Beta. Statistical Analysis Data were presented as mean ± standard deviation. Synergism analysis was performed in the MTT assay using CompuSyn software. Statistical significance was assessed using one-way ANOVA and appropriate post-hoc tests in Prism and SPSS software; p < 0.05 was considered as the significance level. Results Characterization of CuNPs TEM analysis was performed to investigate the morphology and size distribution of CuNPs. Micrographs showed that the nanoparticles were mostly spherical in shape. However, the particles were seen to be discrete and their size distribution was not uniform (Supplementary Figure S1 A). DLS results showed that the average size of the CuNPs was in the range of 20–50 nm and their average diameter was about 40 nm (Supplementary Figure S1 B). The optical properties of the nanoparticles were evaluated using UV–VIS spectroscopy. The absorption spectrum showed a distinct peak at around 600 nm, which corresponds to the surface plasmon resonance (SPR) band and is characteristic of metallic CuNPs. This finding confirms the successful reduction of copper ions to CuNPs (Supplementary Figure S1 C). XRD analysis was also performed to determine the crystallinity and phase purity of the nanoparticles. The recorded diffraction patterns showed that the synthesized nanoparticles were highly pure and selectively produced, and corresponded to the crystal structures of copper (Supplementary Figure S1 D). Evaluation of Cell Viability and Estimation of Hydrogen Peroxide (H₂O₂) in K562 Cells The cytotoxic effects of imatinib and CuNPs on K562 cells were first evaluated separately using the MTT assay. As shown in Fig. 1 A, imatinib caused a concentration-dependent decrease in cell viability. At concentrations above 16 µM, almost complete loss of viability was observed; while, the calculated IC₅₀ value was about 0.5 µM, which was consistent with the dose-response curve. Similarly, CuNPs also showed a dose-dependent cytotoxic effect (Fig. 1 B). At lower concentrations (0.0025–0.025 mg/mL), a slight decrease in viability was observed; however, increasing the dose to 0.25 mg/mL resulted in a significant decrease, such that the viability reached about 30%. These findings indicated that both agents independently had cytotoxic effects against K562 cells. To investigate potential synergy, cells were treated with imatinib alone (0.5, 0.25, 0.1, and 0.05 µM) or in combination with CuNPs (0.005 mg/mL). As seen in Fig. 1 C, imatinib alone significantly reduced viability at concentrations of 0.5 µM (P < 0.001) and 0.25 µM (P < 0.01), while lower concentrations (0.1 and 0.05 µM) showed no significant difference compared to the control group. In contrast, when combined with CuNPs, even lower doses of imatinib (0.1 and 0.05 µM) significantly reduced viability (P < 0.001), indicating increased synergistic cytotoxic effects. Notably, the combination of CuNPs with imatinib at a concentration of 0.25 µM produced the greatest reduction in viability, which was even greater than monotherapy with imatinib or CuNPs alone (P < 0.001). Taken together, these results indicate that CuNPs enhance the anticancer efficacy of imatinib, especially at concentrations below the IC₅₀, where the drug alone is less effective. As shown in Fig. 1 D, treatment with imatinib at concentrations of 0.5 and 0.25 µM, as well as CuNPs alone (0.005 mg/mL), resulted in a significant increase in H₂O₂ levels compared to the untreated control group (P < 0.001). In contrast, imatinib at lower concentrations (0.1 and 0.05 µM) did not significantly increase H₂O₂ levels. Importantly, all combination groups (CuNP + imatinib) showed a significantly greater increase in H₂O₂ levels than imatinib at the same concentration (P < 0.001). This synergistic increase was most pronounced in the CuNP + imatinib (0.25 µM) group, with the highest fluorescence intensity observed. Interestingly, the combination of CuNP with imatinib at a concentration of 0.05 µM also showed a significant increase in H₂O₂ production compared to imatinib at the optimal dose of 0.5 µM (P = 0.015). Gene Expression BAX (proapoptotic) gene expression was examined in K562 cells after 24 h of treatment using Real-time PCR and 2⁻ ΔΔCT method. As shown in Fig. 2 A, BAX expression was significantly increased in all treatment groups compared to control, except in the CuNP alone group (P < 0.001). Imatinib at the optimal concentration (0.5 µM) induced BAX expression significantly more than suboptimal doses (0.25, 0.1 and 0.05 µM) or CuNP alone (P < 0.001). Importantly, while lower doses of imatinib were not significantly different from control, the combination of imatinib with CuNPs caused a concentration-dependent and significant increase in BAX expression compared to imatinib monotherapy and control (P < 0.001). Bcl2 (anti-apoptotic) gene expression was similarly examined by Real-time PCR. As shown in Fig. 2 B, Bcl2 expression was significantly reduced in all combination groups (imatinib + CuNPs) compared to control (P < 0.001). Imatinib alone at concentrations of 0.5 and 0.25 µM also reduced BCL2 levels compared to control (P < 0.001). Furthermore, combination treatments consistently showed greater suppression than either agent alone, indicating a concentration-dependent effect (P < 0.001). To further evaluate the apoptotic response, the Bax/Bcl-2 ratio was calculated (Fig. 2 C). This ratio was significantly increased in all combination groups (imatinib + CuNPs) compared to control and single-agent treatments (P < 0.001). Although imatinib alone increased this ratio in a concentration-dependent manner, only the optimal dose (0.5 µM) showed a significant increase compared to the control (P < 0.001). These results indicate that CuNPs enhance imatinib-induced apoptosis mainly by enhancing the proapoptotic Bax/Bcl-2 balance. Treatment with imatinib or CuNP alone significantly increased MMP9 gene expression compared to the control (P < 0.001), except at the optimal dose of imatinib (0.5 µM) where the expression remained close to the basal level. In contrast, combined treatment with imatinib and CuNP significantly decreased MMP9 expression in a concentration-dependent manner. The greatest suppression was observed at 0.25 and 0.1 µM imatinib plus CuNP concentrations, where MMP9 levels were significantly lower than in the control and single agent groups (P < 0.001) (Fig. 2 D). MMP2 gene expression showed an inverse pattern to MMP9. While imatinib alone induced a concentration-dependent decrease in MMP2 compared to control, the addition of CuNP further enhanced this effect. Specifically, at 0.25 and 0.1 µM imatinib plus CuNP concentrations, MMP2 expression was significantly reduced compared to control and imatinib alone groups (P < 0.001). Interestingly, treatment with imatinib or CuNP alone increased MMP2 expression compared to control (P < 0.001), while their combination caused strong suppression of expression, highlighting a synergistic inhibitory effect (Fig. 2 E). IL-1β gene expression was examined after 24 h of treatment with different concentrations of imatinib, alone or in combination with CuNP. As shown in Fig. 2 F, IL-1β expression was not significantly different in the control groups (with or without CuNP). Imatinib alone caused a concentration-dependent decrease in IL-1β expression, such that at doses of 0.25, 0.1 and 0.05 µM, a significant decrease was observed compared to control (P < 0.001). At the optimal dose (0.5 µM), IL-1β expression was also reduced compared to control (P < 0.001). Importantly, the combined treatment of imatinib and CuNP significantly reduced IL-1β expression compared to imatinib alone at all concentrations (P < 0.001). This synergistic effect was most pronounced at the concentration of 0.25 µM, where IL-1β expression was reduced to approximately half the level of the imatinib alone group. Even at the lowest concentration of imatinib (0.05 µM), the addition of CuNP significantly reduced IL-1β compared to the control and single-agent treatments (P < 0.001). Discussion Cancer is essentially a disease of uncontrolled cell growth and survival caused by genetic and epigenetic alterations. These alterations lead to the activation of oncogenes and the inactivation of tumor suppressor genes, resulting in uncontrolled proliferation, reduced apoptosis, and metastasis. According to the World Health Organization (WHO), hematological malignancies are predicted to surpass cardiovascular diseases in prevalence in the near future. More than 5000 hematological disorders have been identified, many of which are associated with functional or genetic abnormalities of hematopoietic cells [ 23 ]. Among them, leukemia is recognized as a hallmark malignancy characterized by the uncontrolled proliferation of white blood cells and their precursors in the bone marrow and peripheral blood [ 1 ]. CML accounts for approximately 15–20% of all leukemias and is closely associated with the Philadelphia chromosome. This chromosomal rearrangement results in the production of the BCR-ABL fusion protein, which has a permanent tyrosine kinase activity independent of upstream regulatory signals. Activation of downstream signaling pathways by this protein results in increased proliferation, decreased apoptosis, and genomic instability. The discovery of this fusion oncogene was a turning point in the treatment of CML and led to the development of tyrosine kinase inhibitors (TKIs) such as imatinib [ 1 , 24 ]. Imatinib, introduced in 2000 and approved by the FDA, revolutionized the treatment of this disease and remains the first-line therapy for many patients [ 25 ]. However, resistance to imatinib is common, especially in chronic phase CML. Mutations in the BCR-ABL tyrosine kinase domain, particularly at amino acids 315–253, impair drug binding and are responsible for approximately 60% of resistance cases [ 26 ]. To overcome this resistance, second- and third-generation drugs such as dasatinib, nilotinib, bosutinib, and ponatinib have been developed. Although these drugs inhibit specific resistances, they cause severe side effects, including hyperglycemia, pancreatitis, hepatotoxicity, hypertension, and cardiovascular complications [ 27 – 29 ]. On the other hand, access to these new drugs is limited in many resource-poor countries, highlighting the need to find alternative approaches. A major challenge in cancer therapy is the design of drugs that simultaneously exhibit selectivity, high efficacy, and minimal systemic toxicity [ 30 ]. Classical chemotherapy is often associated with the development of drug resistance; In particular, the antiapoptotic pathways induced by BCR-ABL in myeloid cells complicate treatment [ 31 , 32 ]. Therefore, attention has been paid to novel anticancer agents. Among them, metal compounds such as vanadium, titanium, copper, ruthenium, and rhodium have attracted attention due to their cytotoxic potential, often leading to apoptosis induction through disruption of nucleic acids or the cell cycle [ 4 , 33 ]. Nanotechnology has opened up new horizons in cancer therapy. Nanoparticles (NPs), which are typically less than 100 nm in size, can accumulate in tumor tissue through enhanced permeability and accumulation (EPR) [ 34 ]. In leukemia, one of the major obstacles to treatment is the survival of leukemic stem cells (LSCs), which are resistant to chemotherapy and lead to relapse. These cells have low levels of reactive oxygen species (ROS) and thus escape ROS-induced apoptosis [ 35 ]. Therefore, manipulating ROS levels has been considered a novel approach to selectively target these cells [ 36 ]. CuNPs are a promising candidate due to their ability to increase oxidative stress in malignant cells. There is increasing evidence that ROS production by nanoparticles can inhibit drug resistance and enhance the efficacy of chemotherapy. In the present study, CuNPs were investigated as ROS enhancers in combination with imatinib in K562 cells. The MTT assay determined the non-toxic concentrations of CuNPs (0.005 mg/mL) and imatinib (0.5 µM). Then, the cells were divided into different groups including drug alone, nanoparticles alone, and their combination at suboptimal concentrations. The results showed that the combination of CuNPs–imatinib significantly decreased survival and increased the Bax/Bcl-2 ratio. Also, the intracellular H₂O₂ level was increased, indicating that CuNPs enhanced oxidative stress and, together with imatinib, increased apoptosis. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, which are associated with invasion, angiogenesis, and metastasis [ 37 ], were also investigated. Increased expression of these genes was observed in single treatments; however, the CuNPs–imatinib combination showed complex expression changes that likely reflect remodeling of the tumor microenvironment and require further study. Our findings are consistent with previous studies. Hassan Doost et al. (2016) showed that polybutyl cyanoacrylate nanoparticles loaded with imatinib increased drug stability and toxicity in K562 cells [ 38 ]. Elderdery et al. (2022) also observed that the combination of berbamine with CuO–TiO–chitosan nanoparticles increased the expression of proapoptotic genes (caspases, Bax, Bad, P53) and decreased the expression of Bcl2 [ 39 ]. Also, Shirangi et al. (2022) showed that sericin–iron oxide nanoparticles were able to deliver siRNA against ROR1 in breast cancer cells and significantly increased apoptosis [ 40 ]. Silver nanoparticles also showed high selectivity; Hojat Afshar et al. (2023) reported that AgNPs loaded with LukS-PV protein induced apoptosis in K562 cells while sparing normal cells [ 41 ]. Zheng et al. (2016) also enhanced the delivery of SNX-2112 using chitosan-loaded carbon nanotubes [ 42 ]. Moreover, Li et al. (2010) showed that loading doxorubicin onto antibody-bound carbon nanotubes produced stronger cytotoxicity than the free drug [ 43 ]. ROS generation plays a key role in these studies. Azizi et al. (2017) demonstrated that albumin-coated CuNPs increased ROS in breast cells [ 44 ]. Similarly, Guo et al. showed that ZnO nanoparticles increased daunorubicin toxicity via the ROS pathway. Our findings also support that CuNPs in combination with imatinib increased ROS levels and enhanced cytotoxicity [ 45 ]. In addition, nanoparticles can improve drug delivery. Gossai et al. (2016) reported that gold nanoparticles can selectively release dasatinib in CML cells and reduce off-target toxicity[ 46 ]. Khan et al. (2018) also showed that silver nanoparticles induce apoptosis in mammary cells through increased ROS and mitochondrial dysfunction [ 47 ]. Overall, CuNPs enhance the efficacy of imatinib through two pathways: (1) induction of apoptosis via ROS and (2) improved drug stability and delivery. Increased Bax/Bcl-2 ratio, increased H₂O₂, and decreased cell survival demonstrate this synergy. While imatinib acts primarily through inhibition of BCR-ABL, CuNPs activate a complementary pathway through oxidative stress. Overall, these findings support a model in which CuNPs (CuNPs) enhance the efficacy of imatinib through two mechanisms: induction of apoptosis via ROS-dependent pathways, improved drug stability and delivery, and increased duration of intracellular activity. The synergistic increase in Bax/Bcl-2 ratio, increased H₂O₂ levels, and decreased cell viability collectively indicate the therapeutic potential of this compound. Interestingly, imatinib alone induced limited ROS production, except at optimal doses, suggesting that its primary mechanism remains BCR-ABL inhibition, while CuNPs provide a complementary pathway by inducing oxidative stress. Our data on interleukin-1β (IL-1β) expression also confirm the antiproliferative effects of the CuNP–imatinib combination, consistent with enhanced BCR-ABL inhibition. Meanwhile, the expression patterns of MMPs (matrix metalloproteinases) highlight the complex relationship between apoptosis, proliferation, and tumor microenvironment remodeling. While moderate apoptosis was associated with increased MMPs, extensive apoptosis induced by the combination treatment suppressed their expression, consistent with reduced proliferation and angiogenesis. Despite these promising in vitro results, there are still limitations. Further experiments are needed to confirm apoptosis via extrinsic pathways. In vivo studies are also needed to evaluate pharmacokinetics, safety, and optimal dosing strategies. More extensive analyses at different time points could clarify whether CuNPs enhance the stability and sustained activity of imatinib. Addressing these gaps will be critical to moving CuNP–imatinib combination therapy into clinical use. Conclusion This study showed that the combination of imatinib with CuNPs produced significant cytotoxic and apoptotic effects on K562 cells, even at doses lower than the optimal dose of imatinib. By increasing oxidative stress and ROS production, CuNPs shifted the Bax/Bcl-2 ratio in favor of apoptosis and reduced the expression of genes related to invasion and inflammation, such as MMP2, MMP9, and IL-1β. These results indicate that CuNPs can provide complementary pathways to induce cell death in drug-resistant cells and enhance the efficacy of imatinib. The use of this combination could help reduce the drug dose, decrease systemic toxicity, and delay the development of resistance. Despite the promising results in the cell model, further studies in animal models and pharmacology and safety evaluation are needed to determine the transferability of this approach to the clinical treatment of CML. Declarations Acknowledgments The authors sincerely thank Islamic Azad University, Shahrood Branch for their support in this study. This research did not receive any specific funding from public, commercial, or not-for-profit organizations. Author Contributions Conceptualization, M.E-T, M.M. and .M-T.G.; methodology, M.E-T., F.K., M.M. and .M-T.G.; validation, M.M. and .M-T.G.; formal analysis, S.A., M.M. and .M-T.G.; investigation, M.M. and .M-T.G.; resources, M.E-T., F.K., M.M. and .M-T.G.; data curation, M.E-T., F.K., S.A., M.M. and .M-T.G.; writing—original draft preparation, M.E-T., M.M. and .M-T.G.; writing—review and editing, M.M. and .M-T.G.; visualization, M.M. and .M-T.G.; supervision, M.M. and .M-T.G.; project administration, M-T.G.; funding acquisition, M.E-T, M.M. and .M-T.G. All authors have read and agreed to the published version of the manuscript. Funding No funds, grants, or other support were received during the preparation of this manuscript. Data availability The datasets of the current study are available from the corresponding authors upon reasonable request. Competing Interests The authors declare that they have no competing interests. Ethics Approval Not applicable References Suttorp M, Millot F, Sembill S, Deutsch H, Metzler M. Definition, epidemiology, pathophysiology, and essential criteria for diagnosis of pediatric chronic myeloid leukemia. Cancers. 2021;13(4):798. https://doi.org/10.3390/cancers13040798 Haider MZ, Anwer F. Genetics, philadelphia chromosome. StatPearls [Internet]: StatPearls Publishing; 2023. Ramanarayanan J. Advancements in Tyrosine Kinase Inhibitors Therapy: Role of Imatinib, the First Targeted Treatment for Chronic Myeloid Leukemia. Medicine and Medical Research: New Perspectives Vol 3. 2024:30-44. https://doi.org/10.9734/bpi/mmrnp/v3/1238 Alves R, Gonçalves AC, Rutella S, Almeida AM, De Las Rivas J, Trougakos IP, et al. Resistance to tyrosine kinase inhibitors in chronic myeloid leukemia—from molecular mechanisms to clinical relevance. Cancers. 2021;13(19):4820. https://doi.org/10.3390/cancers13194820 Dessie G, Derbew Molla M, Shibabaw T, Ayelign B. Role of stem-cell transplantation in leukemia treatment. Stem cells and cloning: advances and applications. 2020:67-77. https://doi.org/10.2147/sccaa.s262880 Giri PM, Banerjee A, Layek B. A recent review on cancer nanomedicine. Cancers. 2023;15(8):2256. https://doi.org/10.3390/cancers15082256 Al-Zharani M, Qurtam AA, Daoush WM, Eisa MH, Aljarba NH, Alkahtani S, et al. Antitumor effect of copper nanoparticles on human breast and colon malignancies. Environmental Science and Pollution Research. 2021;28(2):1587-95. https://doi.org/10.1007/s11356-020-09843-5 Ozcan A, Ogun M. Biochemistry of reactive oxygen and nitrogen species. Basic principles and clinical significance of oxidative stress. 2015;3:37-58. https://doi.org/10.5772/61193 Schieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Current biology. 2014;24(10):R453-R62. https://doi.org/10.1016/j.cub.2014.03.034 Sinha K, Das J, Pal PB, Sil PC. Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. Archives of toxicology. 2013;87(7):1157-80. https://doi.org/10.1007/s00204-013-1034-4 Zhao RZ, Jiang S, Zhang L, Yu ZB. Mitochondrial electron transport chain, ROS generation and uncoupling. International journal of molecular medicine. 2019;44(1):3-15. https://doi.org/10.3892/ijmm.2019.4188 Del Río LA, López-Huertas E. ROS generation in peroxisomes and its role in cell signaling. Plant and Cell Physiology. 2016;57(7):1364-76. https://doi.org/10.1093/pcp/pcw076 Qiu TA, Gallagher MJ, Hudson-Smith NV, Wu J, Krause MO, Fortner JD, et al. Research highlights: unveiling the mechanisms underlying nanoparticle-induced ROS generation and oxidative stress. Environmental Science: Nano. 2016;3(5):940-5. https://doi.org/10.1039/c6en90021f Lossi L. The concept of intrinsic versus extrinsic apoptosis. Biochemical Journal. 2022;479(3):357-84. https://doi.org/10.1042/bcj20210854 Chen Y-F, Liu H, Luo X-J, Zhao Z, Zou Z-Y, Li J, et al. The roles of reactive oxygen species (ROS) and autophagy in the survival and death of leukemia cells. Critical Reviews in Oncology/Hematology. 2017;112:21-30. https://doi.org/10.1016/j.critrevonc.2017.02.004 Raza MH, Siraj S, Arshad A, Waheed U, Aldakheel F, Alduraywish S, et al. ROS-modulated therapeutic approaches in cancer treatment. Journal of cancer research and clinical oncology. 2017;143(9):1789-809. https://doi.org/10.1007/s00432-017-2464-9 Irwin ME, Rivera-Del Valle N, Chandra J. Redox control of leukemia: from molecular mechanisms to therapeutic opportunities. Antioxidants & redox signaling. 2013;18(11):1349-83. https://doi.org/10.1089/ars.2011.4258 Luo M, Zhou L, Huang Z, Li B, Nice EC, Xu J, et al. Antioxidant therapy in cancer: rationale and progress. Antioxidants. 2022;11(6):1128. https://doi.org/10.3390/antiox11061128 Rébé C, Ghiringhelli F. Interleukin-1β and cancer. Cancers. 2020;12(7):1791. https://doi.org/10.3390/cancers12071791 Okabe S, Tauchi T, Tanaka Y, Ohyashiki K. Interleukin 1 Beta As a Promising Therapeutic Target in ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells. Blood. 2018;132:4249. https://doi.org/10.1182/blood-2018-99-114030 El-Tanani M, Nsairat H, Matalka II, Lee YF, Rizzo M, Aljabali AA, et al. The impact of the BCR-ABL oncogene in the pathology and treatment of chronic myeloid leukemia. Pathology-Research and Practice. 2024;254:155161. https://doi.org/10.1016/j.prp.2024.155161 Vo TTT, Peng T-Y, Nguyen TH, Bui TNH, Wang C-S, Lee W-J, et al. The crosstalk between copper-induced oxidative stress and cuproptosis: a novel potential anticancer paradigm. Cell Communication and Signaling. 2024;22(1):353. https://doi.org/10.1186/s12964-024-01726-3 Anand P, Kunnumakara AB, Sundaram C, Harikumar KB, Tharakan ST, Lai OS, et al. Cancer is a preventable disease that requires major lifestyle changes. Pharmaceutical research. 2008;25(9):2097-116. https://doi.org/10.1007/s11095-008-9661-9 Jabbour E, Kantarjian H. Chronic myeloid leukemia: a review. JAMA. 2025. https://doi.org/10.1001/jama.2025.0220 Cohen P, Cross D, Jänne PA. Kinase drug discovery 20 years after imatinib: progress and future directions. Nature reviews drug discovery. 2021;20(7):551-69. https://doi.org/10.1038/s41573-021-00195-4 Wei Y, Stockelberg D, Hullberg S, Ricksten A, Wadenvik H. Changes in expression of apoptosis-related genes are linked to the molecular response to imatinib treatment in chronic-phase chronic myeloid leukemia patients. Acta Haematologica. 2007;117(2):83-90. https://doi.org/10.1159/000096858 Cortes JE, Jones D, O'Brien S, Jabbour E, Konopleva M, Ferrajoli A, et al. Nilotinib as front-line treatment for patients with chronic myeloid leukemia in early chronic phase. Journal of Clinical Oncology. 2010;28(3):392-7. https://doi.org/10.1200/jco.2009.25.4896 Cortes JE, Kantarjian HM, Brümmendorf TH, Kim D-W, Turkina AG, Shen Z-X, et al. Safety and efficacy of bosutinib (SKI-606) in chronic phase Philadelphia chromosome–positive chronic myeloid leukemia patients with resistance or intolerance to imatinib. Blood, The Journal of the American Society of Hematology. 2011;118(17):4567-76. https://doi.org/10.1182/blood-2013-08-524256 O'Hare T, Shakespeare WC, Zhu X, Eide CA, Rivera VM, Wang F, et al. AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer cell. 2009;16(5):401-12. https://doi.org/10.2210/pdb3ik3/pdb Mottier A, Mouchet F, Pinelli E, Gauthier L, Flahaut E. Environmental impact of engineered carbon nanoparticles: from releases to effects on the aquatic biota. Current Opinion in Biotechnology. 2017;46:1-6. https://doi.org/10.1016/j.copbio.2016.11.024 Almuhanna HNB. Elucidating BCR-mediated regulation of FOXO1 in chronic lymphocytic leukaemia: a role for deubiquitinase proteins? : University of Glasgow; 2024. https://doi.org/10.5525/gla.thesis.84781 Baranwal J, Barse B, Di Petrillo A, Gatto G, Pilia L, Kumar A. Nanoparticles in cancer diagnosis and treatment. Materials. 2023;16(15):5354. https://doi.org/10.3390/ma16155354 Ott I, Gust R. Non platinum metal complexes as anti‐cancer drugs. Archiv der Pharmazie: An International Journal Pharmaceutical and Medicinal Chemistry. 2007;340(3):117-26. https://doi.org/10.1002/ardp.200600151 Ko W, Kang T, Lee S, Kim Y, Lee B. Effects of luteolin on the inhibition of proliferation and induction of apoptosis in human myeloid leukaemia cells. Phytotherapy research. 2002;16(3):295-8. https://doi.org/10.1002/ptr.871 Stelmach P, Trumpp A. Leukemic stem cells and therapy resistance in acute myeloid leukemia. Haematologica. 2023;108(2):353-66. https://doi.org/10.3324/haematol.2022.280800 Dayem AA, Choi H-Y, Kim J-H, Cho S-G. Role of oxidative stress in stem, cancer, and cancer stem cells. Cancers. 2010;2(2):859-84. https://doi.org/10.3390/cancers2020859 Simões G, Pereira T, Caseiro A. Matrix metaloproteinases in vascular pathology. Microvascular research. 2022;143:104398. https://doi.org/10.1016/j.mvr.2022.104398 Hasandoost L, Akbarzadeh A, Attar H, Heydarinasab A. In vitro effect of imatinib mesylate loaded on polybutylcyanoacrylate nanoparticles on leukemia cell line K562. Artificial cells, nanomedicine, and biotechnology. 2017;45(3):665-9. https://doi.org/10.1080/21691401.2016.1175444 Elderdery AY, Alzahrani B, Hamza SMA, Mostafa-Hedeab G, Mok PL, Subbiah SK. [Retracted] CuO‐TiO2‐Chitosan‐Berbamine Nanocomposites Induce Apoptosis through the Mitochondrial Pathway with the Expression of P53, BAX, and BCL‐2 in the Human K562 Cancer Cell Line. Bioinorganic Chemistry and Applications. 2022;2022(1):9602725. https://doi.org/10.1155/2022/9602725 Shirangi A, Mottaghitalab F, Dinarvand S, Atyabi F. Theranostic silk sericin/SPION nanoparticles for targeted delivery of ROR1 siRNA: Synthesis, characterization, diagnosis and anticancer effect on triple-negative breast cancer. International journal of biological macromolecules. 2022;221:604-12. https://doi.org/10.1016/j.ijbiomac.2022.09.020 Haghighatafshar H, Eimani BG, Moazamian E, Amani J. Cytotoxic and apoptotic effects of chemically synthesized silver nanoparticles loaded with recombinant Staphylococcus LukS-PV toxin. Journal of Biotechnology. 2023;373:42-8. https://doi.org/10.1016/j.jbiotec.2023.07.001 Zheng L, Wu S, Tan L, Tan H, Yu B. Chitosan-functionalised single-walled carbon nanotube-mediated drug delivery of SNX-2112 in cancer cells. Journal of biomaterials applications. 2016;31(3):379-86. https://doi.org/10.1177/0885328216651183 Li R, Wu Ra, Zhao L, Wu M, Yang L, Zou H. P-glycoprotein antibody functionalized carbon nanotube overcomes the multidrug resistance of human leukemia cells. ACS nano. 2010;4(3):1399-408. https://doi.org/10.1021/nn9011225 Azizi M, Ghourchian H, Yazdian F, Dashtestani F, AlizadehZeinabad H. Cytotoxic effect of albumin coated copper nanoparticle on human breast cancer cells of MDA-MB 231. PloS one. 2017;12(11):e0188639. https://doi.org/10.1371/journal.pone.0188639 Guo D, Wu C, Jiang H, Li Q, Wang X, Chen B. Synergistic cytotoxic effect of different sized ZnO nanoparticles and daunorubicin against leukemia cancer cells under UV irradiation. Journal of Photochemistry and Photobiology B: Biology. 2008;93(3):119-26. https://doi.org/10.1016/j.jphotobiol.2008.07.009 Gossai NP, Naumann JA, Li N-S, Zamora EA, Gordon DJ, Piccirilli JA, et al. Drug conjugated nanoparticles activated by cancer cell specific mRNA. Oncotarget. 2016;7(25):38243. https://doi.org/10.18632/oncotarget.9430 Khan AU, Yuan Q, Khan ZUH, Ahmad A, Khan FU, Tahir K, et al. An eco-benign synthesis of AgNPs using aqueous extract of Longan fruit peel: Antiproliferative response against human breast cancer cell line MCF-7, antioxidant and photocatalytic deprivation of methylene blue. Journal of Photochemistry and Photobiology B: Biology. 2018;183:367-73. https://doi.org/10.1016/j.jphotobiol.2018.05.007 Additional Declarations No competing interests reported. Supplementary Files suplementaryfile.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7627097","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":523660744,"identity":"7bc12343-0f26-449e-89f0-b36c1045e654","order_by":0,"name":"Mohammad Ebrahimi Torkamani","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Ebrahimi","lastName":"Torkamani","suffix":""},{"id":523660745,"identity":"b37619fb-ea3d-462b-8df9-975976793947","order_by":1,"name":"Mahshid Mohammadian","email":"","orcid":"","institution":"Urmia University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mahshid","middleName":"","lastName":"Mohammadian","suffix":""},{"id":523660746,"identity":"ead5dec7-d64d-42b9-b54b-a692bbf9c72c","order_by":2,"name":"Farzaneh Kefayatirad","email":"","orcid":"","institution":"Dorevitch Pathology","correspondingAuthor":false,"prefix":"","firstName":"Farzaneh","middleName":"","lastName":"Kefayatirad","suffix":""},{"id":523660747,"identity":"b1a40978-b0bb-46a6-8fdc-afb5c87799ef","order_by":3,"name":"Safiyeh Aghazadeh","email":"","orcid":"","institution":"Urmia University","correspondingAuthor":false,"prefix":"","firstName":"Safiyeh","middleName":"","lastName":"Aghazadeh","suffix":""},{"id":523660748,"identity":"02a36e66-04c7-415f-bbab-021e6e77c7c9","order_by":4,"name":"Mohammad Taghi Goodarzi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3RPQrCMBiA4S8EMlVcO9UTCIqgSBGv0uDQ1bGDQ0shbrpmELxKywd1yQE6Vlwc20WcxAyCuKR1E8wLGRLyQH4AbLbfjepBkip4TTPjZvYm6ehbAsztdKLhMabXOvIHsz2KqNog9LcZwchApgWwuVTh+JDlogwKBFcFkKsWMukJJJIkmjAEKAHyuANZSkrEOnggDDoQetGES0YEcIEwaidcEH2XlXRI6vJd6IwVj80EEWv9YgvZP52b+833vBNiYyL6B8XHdzh6xQx0tG7bYbPZbH/eExrkT3ePYtSwAAAAAElFTkSuQmCC","orcid":"","institution":"Islamic Azad University","correspondingAuthor":true,"prefix":"","firstName":"Mohammad","middleName":"Taghi","lastName":"Goodarzi","suffix":""}],"badges":[],"createdAt":"2025-09-16 07:23:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7627097/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7627097/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92726585,"identity":"23ad5b29-8184-43fa-8da1-1d6c0813b8b9","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":570310,"visible":true,"origin":"","legend":"","description":"","filename":"EbrahimiManuscript16Sep.docx","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/7ba92848d691f2cc66a0a347.docx"},{"id":92726582,"identity":"3b1da31c-5aea-4a70-87c4-ce440ab3630b","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7284,"visible":true,"origin":"","legend":"","description":"","filename":"9df049db8e1948a089e6481f03db73c3.json","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/4eaf77bc76469b16cd8a23bd.json"},{"id":92726590,"identity":"c1882fe1-0a57-4abd-8b6e-7bf7133758b7","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":799835,"visible":true,"origin":"","legend":"","description":"","filename":"suplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/8e02dee9c6d806739c2ffce4.docx"},{"id":92726592,"identity":"5f2a94d2-2bc6-4e63-826d-31305ae0c72c","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":123168,"visible":true,"origin":"","legend":"","description":"","filename":"9df049db8e1948a089e6481f03db73c31enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/11d141d3fa4d4db7e319e8f1.xml"},{"id":92726589,"identity":"cdceb893-9906-4fed-b110-17d59f5f4cb2","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":202272,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/f48ed5aa692b9bda9a748935.png"},{"id":92727647,"identity":"27cf275b-43fc-4a0d-8482-01c44e14685c","added_by":"auto","created_at":"2025-10-03 14:57:03","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":287751,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/e6eb506411cd7be0f8110a70.png"},{"id":92726588,"identity":"59d41fd3-9383-4e6f-9f8b-2a7e13f5b3e0","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":37281,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/a2f1395ebf6f16974f8ef586.png"},{"id":92726595,"identity":"6efc56b8-aee2-4521-889f-c29f46295dba","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":77869,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/2bb92245de470dabe94ff5fe.png"},{"id":92726593,"identity":"beb225c6-25ce-4742-96a0-5303b7c01d1c","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122102,"visible":true,"origin":"","legend":"","description":"","filename":"9df049db8e1948a089e6481f03db73c31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/501184ed60249cc2bf960f05.xml"},{"id":92726594,"identity":"f4f5269a-72ba-411b-a7e6-ccd4c5d617c8","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":133000,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/e40b3a141a060ebb3cf21e22.html"},{"id":92728095,"identity":"ca6aaeb9-ed9d-4127-9a90-7b32a76aeaf6","added_by":"auto","created_at":"2025-10-03 15:05:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":229076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of imatinib (IMA) and CuNPs (CuNPs) on K562 cell viability and oxidative stress.\u003c/strong\u003e (A) Dose–response curve of IMA after 24 h, showing concentration-dependent cytotoxicity with an IC₅₀ of ~0.5 μM. (B) Dose–response curve of CuNPs, demonstrating reduced viability to ~30% at 0.25 mg/mL. (C) Comparative analysis of IMA alone (yellow bars) versus IMA + CuNPs (green bars). CuNPs markedly enhanced cytotoxicity at suboptimal IMA doses (0.1 and 0.05 μM, ***P\u0026lt;0.001). (D) Intracellular H₂O₂ levels were significantly elevated by IMA (0.5 and 0.25 μM) and CuNPs alone, while combination treatment produced a synergistic increase across all doses. The strongest effect was observed with IMA (0.25 μM) + CuNPs, whereas even IMA 0.05 μM + CuNPs exceeded H₂O₂ levels of IMA at 0.5 μM (P=0.015). Data are presented as mean ± SD of three independent experiments. ns = not significant; **P\u0026lt;0.01; ***P\u0026lt;0.001. CuNPs, copper nanoparticles; IMA, imatinib; H₂O₂, hydrogen peroxide\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/fcb0a261b4492f72b33e3a8d.png"},{"id":92727645,"identity":"3b2c811d-b76d-4229-80e7-7b2fb487008c","added_by":"auto","created_at":"2025-10-03 14:57:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":344587,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of imatinib (IMA), CuNPs (CuNPs), and their combinations on gene expression in K562 cells.\u003c/strong\u003e(A–C) IMA upregulated BAX and downregulated BCL2, with CuNP co-treatment further enhancing apoptosis as shown by the increased Bax/Bcl-2 ratio. (D–E) MMP9 and MMP2 expression showed variable changes with single agents, but combination therapy consistently suppressed both in a dose-dependent manner. (F) IL1B expression was significantly reduced in all CuNP–IMA groups compared with IMA alone. Data are presented as fold change ± SD of three independent experiments. ns = not significant; ***P \u0026lt; 0.001. CuNPs, copper nanoparticles; IMA, imatinib; IL1B, interleukin-1β; MMP, matrix metalloproteinase\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/bca1f004b28466a789a8d27e.png"},{"id":92909318,"identity":"acb65ee3-b21f-483e-99e1-3684cbc9a805","added_by":"auto","created_at":"2025-10-07 02:46:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1230875,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/0ba12e44-6ba5-4faa-83ab-574ff92cca77.pdf"},{"id":92726586,"identity":"97fd8c51-03a3-4b65-aade-e8b41c1cf8ca","added_by":"auto","created_at":"2025-10-03 14:49:03","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":799835,"visible":true,"origin":"","legend":"","description":"","filename":"suplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7627097/v1/93edc6ad1dd1fece607e77a2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of toxicity and apoptotic effects of copper nanoparticles in combination with imatinib on chronic myeloid leukemia cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by uncontrolled proliferation of myeloid cells in the bone marrow and their accumulation in the peripheral blood [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The disease is mainly associated with the presence of the Philadelphia chromosome, which results from a reciprocal translocation between chromosomes 9 and 22, resulting in the formation of a BCR-ABL fusion gene with persistent tyrosine kinase activity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Although targeted therapies with tyrosine kinase inhibitors (TKIs) such as imatinib have dramatically improved the prognosis of patients with CML [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], drug resistance and persistence of leukemic stem cells (LSCs) remain major challenges [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. LSCs can survive despite treatment and are recognized as a key factor in disease relapse [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore, there is an urgent need for novel therapeutic approaches that can eliminate or sensitize these resistant cells.\u003c/p\u003e\u003cp\u003eNanotechnology has emerged as a promising area in cancer research due to the unique physicochemical properties of nanoparticles, including high surface-to-volume ratio, tunable size, and the ability to be functionalized for targeted delivery [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among metal nanoparticles, copper nanoparticles (CuNPs) have shown significant anticancer potential, which is mainly attributed to their ability to generate reactive oxygen species (ROS) and induce oxidative stress in cancer cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. ROS include superoxide anions, hydroxyl radicals, hydrogen peroxide, and singlet oxygen, which are produced under physiological and pathological conditions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While low levels of ROS can act as messenger molecules in processes such as cell differentiation, proliferation, and immune responses [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], their excessive production disrupts the redox balance, causing oxidative damage to lipids, proteins, and DNA, ultimately activating apoptosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eROS production occurs in cells through various mechanisms. Mitochondria, especially complexes I and III of the respiratory chain, are considered the main source [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], although other organelles such as peroxisomes, microsomes, and the plasma membrane also play a role in this process [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Nanoparticles can enhance ROS production through light-induced electron transfer processes, generating electron\u0026ndash;hole pairs that oxidize water to hydroxyl radicals or reduce oxygen to superoxide and hydrogen peroxide [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This ROS-induced damage can activate intrinsic apoptotic pathways through mitochondrial membrane permeabilization, cytochrome c release, and caspases activation as well as extrinsic pathways through interaction with death receptors [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In leukemic cells, including CML, high levels of ROS are a common feature that contribute to both oncogenic signaling and therapeutic resistance [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTargeting ROS in cancer therapy can be approached from two perspectives: antioxidant therapy, which seeks to reduce ROS levels to limit tumor progression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and prooxidant therapy, which induces cell death by increasing them above the cell\u0026rsquo;s tolerance threshold. The latter approach is particularly attractive for the selective destruction of cancer cells with intrinsically high levels of ROS, such as LSCs, while leaving normal cells largely intact. Prooxidant compounds such as arsenic trioxide and isothiocyanates have shown antileukemic properties, although their clinical use may be limited due to off-target toxicity[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to oxidative stress, inflammatory mediators such as interleukin-1 beta (IL-1β) play an important role in the progression of cancer, including hematological malignancies. IL-1β is a proinflammatory cytokine produced by immune cells, fibroblasts, and cancer cells, and promotes tumor growth, angiogenesis, and metastasis by activating intracellular signaling pathways such as PI3K/Rac and β-catenin[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In CML, IL-1β can contribute to the inflammatory tumor microenvironment and affect the survival and proliferation of malignant cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGiven these considerations, the combination of CuNPs with imatinib could be proposed as a synergistic therapeutic strategy for CML. CuNPs could act by inducing ROS-dependent apoptosis, sensitizing resistant cells, and possibly modulating inflammatory signals, while imatinib targets the oncogenic tyrosine kinase BCR-ABL [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Evaluation of their combined effects on cell viability, ROS production, expression of apoptotic genes (Bax, Bcl-2), matrix metalloproteinases (MMP-2, MMP-9), and IL-1β expression in CML cells could provide valuable insights into novel strategies to overcome drug resistance and improve treatment outcomes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design\u003c/h2\u003e\u003cp\u003eThis \u003cem\u003ein vitro\u003c/em\u003e study investigated the effects of CuNPs and imatinib, individually and in combination, on viability, oxidative stress, and gene expression in the CML cell line K562.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSynthesis and Characterization CuNPs\u003c/h3\u003e\n\u003cp\u003eCuNPs were synthesized according to standard protocols: First, 104 mg of copper (II) 2,4-pentanedionate was mixed with 8 mL of 1-octadecene and 2 mL of hexadecylamine under nitrogen conditions. Then, 2 mL of tri-n-octylphosphine was added to the mixture and the temperature was increased to 200\u0026deg;C. Next, a formic acid\u0026ndash;sulfuric acid mixture was injected to generate CO gas, and heating was continued to 220\u0026deg;C until a color change to dark red was observed. The reaction was continued for 20 min and then cooled to ambient temperature. The product was precipitated with ethanol/toluene, centrifuged at 6000 rpm for 20 min, redispersed in toluene, and PEGylated using HOOC-PEG-COOH in chloroform for 12 h. Finally, the PEG-coated CuNPs were collected, washed, and resuspended in molten polyethylene glycol.\u003c/p\u003e\u003cp\u003eTo confirm the physicochemical properties of the synthesized CuNPs, several analytical techniques were used as follows. Transmission Electron Microscopy (TEM): The morphology and size of the CuNPs were examined using TEM. In this method, an electron beam is passed through a thin sample and the interaction of the electrons with the sample creates an image that is detected and magnified on a fluorescent screen, photographic film or digital sensor. Dynamic Light Scattering (DLS) and Zeta Potential: The hydrodynamic diameter and surface charge of the nanoparticles were measured using DLS and zeta potential analysis. DLS provides information about the particle size distribution in the nanometer to micrometer range by analyzing the fluctuations in the intensity of laser light scattered from Brownian moving particles. For this analysis, the nanoparticle samples were dispersed in distilled water at a final concentration of 0.5% (w/v) of dry weight. The suspensions were homogenized using an ultrasonic bath before measurement. The particle size distribution and polydispersity index (PDI) were recorded in at least 30 replicates. Also, the electrophoretic mobility of the particles was measured in PALS mode to calculate the zeta potential. UV\u0026ndash;Vis spectroscopy: The optical properties of the nanoparticles were investigated using a spectrophotometer (Shimadzu, Japan). The UV\u0026ndash;Vis absorption spectrum of the colloidal CuNPs was recorded in the wavelength range of 400\u0026ndash;800 nm and at different time intervals after synthesis. X-ray diffraction (XRD): The crystal structure of the CuNPs was determined using an X\u0026rsquo;Pert MDP XRD instrument. The measurements were performed with Cu Kα radiation (λ\u0026thinsp;=\u0026thinsp;1.54056 \u0026Aring;) at a voltage of 40 kV and a step of 0.02\u0026deg;.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eK562 cell line was obtained from the Pasteur Institute, Tehran. Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37\u0026deg;C, 5% CO₂, and 90% humidity. Cultures were checked daily for confluency and contamination. For long-term storage, cells were cryopreserved in FBS/DMSO (1:10) at -180\u0026deg;C. Frozen cells were thawed rapidly at 37\u0026deg;C under sterile conditions, diluted with culture medium, and centrifuged at 1400 rpm for 5 min at 4\u0026deg;C. Cells were then resuspended and counted using a Neubauer hemocytometer using trypan blue staining.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eK562 cells were seeded in 96-well plates at a density of 1 \u0026times; 10⁴ cells per well in 100 \u0026micro;l of culture medium and incubated for 24 hours at 37\u0026deg;C in a humidified atmosphere with 5% CO₂. After incubation, the culture medium was replaced with fresh medium containing imatinib at different concentrations (0.1, 0.21, 0.25, 0.35, 0.42, 0.5, 1, 2, 4, 8, 16, 32, and 64 \u0026micro;M) or CuNPs at concentrations of 0.25, 0.125, 0.1, 0.075, 0.05, 0.025, 0.005, and 0.0025 mg/mL. Imatinib was prepared as capsules (100 mg per capsule as per the manufacturer\u0026rsquo;s instructions). The contents of the capsules were accurately weighed and the purity of the drug was calculated before preparing the working concentrations to ensure accurate dosing. The half-maximal inhibitory concentration (IC₅₀) of each compound was determined using dose-response analysis. Based on the IC₅₀ value, the treatment groups were divided as follows: Control group: untreated cells. Imatinib groups: 0.5 \u0026micro;M (optimal IC₅₀), 0.25 \u0026micro;M (\u0026frac12; IC₅₀), 0.1 \u0026micro;M (⅕ IC₅₀) and 0.05 \u0026micro;M (\u0026sup1;\u0026frasl;₁₀ IC₅₀). CuNPs group: only CuNPs at a concentration of 0.005 mg/mL. Combination groups: CuNPs (0.005 mg/mL) combined with imatinib at concentrations of 0.5, 0.25, 0.1 or 0.05 \u0026micro;M. All treatments were performed in triplicate and cell viability was assessed after 24 hours by MTT assay.\u003c/p\u003e\n\u003ch3\u003eMTT assay\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed using the MTT kit (Kia Zist, Iran) according to the standard protocol. K562 cells were cultured in 96-well plates at the same density and treated with different concentrations of imatinib (0.5, 0.25, 0.1 and 0.05 \u0026micro;M), CuNPs (0.005 mg/mL) or their combination (NP\u0026thinsp;+\u0026thinsp;Ima 0.25, NP\u0026thinsp;+\u0026thinsp;Ima 0.1, NP\u0026thinsp;+\u0026thinsp;Ima 0.05) for 24 h. Untreated cells were considered as controls. After 24 h of treatment, 10 \u0026micro;L of MTT solution was added to each well and incubated for 3 h at 37\u0026deg;C in the dark. Then, the medium was removed and 100 \u0026micro;L of DMSO was added to dissolve the formazan crystals. The optical absorbance at 570 nm was measured by a microplate reader. The percentage of cell viability was calculated using the following formula:\u003c/p\u003e\u003cp\u003eCell viability = (Absorbance of control group / Absorbance of treated group) \u0026times; 100\u003c/p\u003e\u003cp\u003eAll experiments were performed in triplicate.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of intracellular hydrogen peroxide (H₂O₂)\u003c/h2\u003e\u003cp\u003eThe level of intracellular H₂O₂ as an indicator of oxidative stress was measured using a fluorometric Hydrogen Peroxide Assay kit (Sigma-Aldrich, Germany) according to the manufacturer's instructions. K562 cells (1 \u0026times; 10⁴ cells/well) were seeded in 96-well plates and treated with imatinib, CuNPs, or their combination for 24 h. After treatment, the cells were washed with PBS, centrifuged, and incubated with 50 \u0026micro;L of freshly prepared Master Mix for 15\u0026ndash;30 min at room temperature in the dark. Fluorescence intensity at excitation/emission wavelengths of 540/590 nm was recorded using a microplate reader, and intracellular H₂O₂ concentration was calculated from the standard curve.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGene Expression Analysis via Real-Time PCR\u003c/h3\u003e\n\u003cp\u003eTreated and control K562 cells (approximately 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e) were collected for RNA extraction using a Trizol-based kit (DnaZist, Iran). RNA integrity and concentration were assessed using NanoDrop (OD₂₆₀/₂₈₀ ratio\u0026thinsp;\u0026asymp;\u0026thinsp;1.8). cDNA synthesis was performed using a Pars Toos kit, Iran. A 20 \u0026micro;L quantitative PCR reaction was performed with SYBR Green dye in a real-time thermocycler. The cycling conditions were: initial denaturation at 95\u0026deg;C for 4 min, followed by 35 cycles of denaturation at 94\u0026deg;C, annealing at 57\u0026ndash;60\u0026deg;C, and extension at 72\u0026deg;C (each step 30 s), and finally a final extension step at 72\u0026deg;C for 5 min. The target genes included Bax, Bcl-2, MMP2, MMP9, and IL-1β, and β-actin was used as a reference gene. The primer sequences are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The relative expression of the genes was calculated using the 2^\u003csup\u003e\u0026ndash;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSequences of forward and reverse primers.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eACTB\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-GGAACGGTGAAGGTGACAG-3\u0026acute;(forward)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-GTGGGGTGGCTTTTAGGATG-3\u0026acute;(reverse)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eBcl-2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-ATGTGAAACTGAATTGGAGAGTG-3\u0026acute;(forward)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-TGTTGTTGATAGGATGTTTGCTT-3\u0026acute;(reverse)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eBax\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-CTCACCGCCTCACTCACCC-3\u0026acute; (forward)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-CCCACACCCCCCAATAATTAC-3\u0026acute; (reverse)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eMMP2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-TACGATGGAGGCGCTAATGG-3\u0026acute; (forward)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-GAAGGTGTTCAGGTATTGCACTG-3\u0026acute; (reverse)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eMMP9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-TTCCAGTACCGSGSSSGCC-3\u0026acute; (forward)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-CCTTTCCTCCAGAACAATACC-3\u0026acute; (reverse)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eIL 1 B\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-GGCTTATTACAGTGGCAATG-3\u0026acute; (forward)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026acute;-TAGTGGTGGTCGGAGATT-3\u0026acute; (reverse)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eACTB, Beta Actin; BCL2, B-Cell Lymphoma 2; BAX, BCL2 Associated X, Apoptosis Regulator; MMP2, Matrix Metallopeptidase 2; MMP9, Matrix Metallopeptidase 9; IL-1B, Interleukin 1 Beta.\u003c/p\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eData were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Synergism analysis was performed in the MTT assay using CompuSyn software. Statistical significance was assessed using one-way ANOVA and appropriate post-hoc tests in Prism and SPSS software; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered as the significance level.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCharacterization of CuNPs\u003c/h2\u003e\u003cp\u003eTEM analysis was performed to investigate the morphology and size distribution of CuNPs. Micrographs showed that the nanoparticles were mostly spherical in shape. However, the particles were seen to be discrete and their size distribution was not uniform (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). DLS results showed that the average size of the CuNPs was in the range of 20\u0026ndash;50 nm and their average diameter was about 40 nm (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). The optical properties of the nanoparticles were evaluated using UV\u0026ndash;VIS spectroscopy. The absorption spectrum showed a distinct peak at around 600 nm, which corresponds to the surface plasmon resonance (SPR) band and is characteristic of metallic CuNPs. This finding confirms the successful reduction of copper ions to CuNPs (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). XRD analysis was also performed to determine the crystallinity and phase purity of the nanoparticles. The recorded diffraction patterns showed that the synthesized nanoparticles were highly pure and selectively produced, and corresponded to the crystal structures of copper (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of Cell Viability and Estimation of Hydrogen Peroxide (H₂O₂) in K562 Cells\u003c/h2\u003e\u003cp\u003eThe cytotoxic effects of imatinib and CuNPs on K562 cells were first evaluated separately using the MTT assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, imatinib caused a concentration-dependent decrease in cell viability. At concentrations above 16 \u0026micro;M, almost complete loss of viability was observed; while, the calculated IC₅₀ value was about 0.5 \u0026micro;M, which was consistent with the dose-response curve. Similarly, CuNPs also showed a dose-dependent cytotoxic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). At lower concentrations (0.0025\u0026ndash;0.025 mg/mL), a slight decrease in viability was observed; however, increasing the dose to 0.25 mg/mL resulted in a significant decrease, such that the viability reached about 30%. These findings indicated that both agents independently had cytotoxic effects against K562 cells.\u003c/p\u003e\u003cp\u003eTo investigate potential synergy, cells were treated with imatinib alone (0.5, 0.25, 0.1, and 0.05 \u0026micro;M) or in combination with CuNPs (0.005 mg/mL). As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, imatinib alone significantly reduced viability at concentrations of 0.5 \u0026micro;M (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and 0.25 \u0026micro;M (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while lower concentrations (0.1 and 0.05 \u0026micro;M) showed no significant difference compared to the control group. In contrast, when combined with CuNPs, even lower doses of imatinib (0.1 and 0.05 \u0026micro;M) significantly reduced viability (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating increased synergistic cytotoxic effects. Notably, the combination of CuNPs with imatinib at a concentration of 0.25 \u0026micro;M produced the greatest reduction in viability, which was even greater than monotherapy with imatinib or CuNPs alone (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Taken together, these results indicate that CuNPs enhance the anticancer efficacy of imatinib, especially at concentrations below the IC₅₀, where the drug alone is less effective.\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, treatment with imatinib at concentrations of 0.5 and 0.25 \u0026micro;M, as well as CuNPs alone (0.005 mg/mL), resulted in a significant increase in H₂O₂ levels compared to the untreated control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, imatinib at lower concentrations (0.1 and 0.05 \u0026micro;M) did not significantly increase H₂O₂ levels. Importantly, all combination groups (CuNP\u0026thinsp;+\u0026thinsp;imatinib) showed a significantly greater increase in H₂O₂ levels than imatinib at the same concentration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This synergistic increase was most pronounced in the CuNP\u0026thinsp;+\u0026thinsp;imatinib (0.25 \u0026micro;M) group, with the highest fluorescence intensity observed. Interestingly, the combination of CuNP with imatinib at a concentration of 0.05 \u0026micro;M also showed a significant increase in H₂O₂ production compared to imatinib at the optimal dose of 0.5 \u0026micro;M (P\u0026thinsp;=\u0026thinsp;0.015).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eGene Expression\u003c/h2\u003e\u003cp\u003eBAX (proapoptotic) gene expression was examined in K562 cells after 24 h of treatment using Real-time PCR and 2⁻\u003csup\u003eΔΔCT\u003c/sup\u003e method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, BAX expression was significantly increased in all treatment groups compared to control, except in the CuNP alone group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Imatinib at the optimal concentration (0.5 \u0026micro;M) induced BAX expression significantly more than suboptimal doses (0.25, 0.1 and 0.05 \u0026micro;M) or CuNP alone (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Importantly, while lower doses of imatinib were not significantly different from control, the combination of imatinib with CuNPs caused a concentration-dependent and significant increase in BAX expression compared to imatinib monotherapy and control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eBcl2 (anti-apoptotic) gene expression was similarly examined by Real-time PCR. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Bcl2 expression was significantly reduced in all combination groups (imatinib\u0026thinsp;+\u0026thinsp;CuNPs) compared to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Imatinib alone at concentrations of 0.5 and 0.25 \u0026micro;M also reduced BCL2 levels compared to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Furthermore, combination treatments consistently showed greater suppression than either agent alone, indicating a concentration-dependent effect (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003eTo further evaluate the apoptotic response, the Bax/Bcl-2 ratio was calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This ratio was significantly increased in all combination groups (imatinib\u0026thinsp;+\u0026thinsp;CuNPs) compared to control and single-agent treatments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Although imatinib alone increased this ratio in a concentration-dependent manner, only the optimal dose (0.5 \u0026micro;M) showed a significant increase compared to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These results indicate that CuNPs enhance imatinib-induced apoptosis mainly by enhancing the proapoptotic Bax/Bcl-2 balance.\u003c/p\u003e\u003cp\u003eTreatment with imatinib or CuNP alone significantly increased MMP9 gene expression compared to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), except at the optimal dose of imatinib (0.5 \u0026micro;M) where the expression remained close to the basal level. In contrast, combined treatment with imatinib and CuNP significantly decreased MMP9 expression in a concentration-dependent manner. The greatest suppression was observed at 0.25 and 0.1 \u0026micro;M imatinib plus CuNP concentrations, where MMP9 levels were significantly lower than in the control and single agent groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eMMP2 gene expression showed an inverse pattern to MMP9. While imatinib alone induced a concentration-dependent decrease in MMP2 compared to control, the addition of CuNP further enhanced this effect. Specifically, at 0.25 and 0.1 \u0026micro;M imatinib plus CuNP concentrations, MMP2 expression was significantly reduced compared to control and imatinib alone groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Interestingly, treatment with imatinib or CuNP alone increased MMP2 expression compared to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while their combination caused strong suppression of expression, highlighting a synergistic inhibitory effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eIL-1β gene expression was examined after 24 h of treatment with different concentrations of imatinib, alone or in combination with CuNP. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, IL-1β expression was not significantly different in the control groups (with or without CuNP). Imatinib alone caused a concentration-dependent decrease in IL-1β expression, such that at doses of 0.25, 0.1 and 0.05 \u0026micro;M, a significant decrease was observed compared to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At the optimal dose (0.5 \u0026micro;M), IL-1β expression was also reduced compared to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Importantly, the combined treatment of imatinib and CuNP significantly reduced IL-1β expression compared to imatinib alone at all concentrations (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This synergistic effect was most pronounced at the concentration of 0.25 \u0026micro;M, where IL-1β expression was reduced to approximately half the level of the imatinib alone group. Even at the lowest concentration of imatinib (0.05 \u0026micro;M), the addition of CuNP significantly reduced IL-1β compared to the control and single-agent treatments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCancer is essentially a disease of uncontrolled cell growth and survival caused by genetic and epigenetic alterations. These alterations lead to the activation of oncogenes and the inactivation of tumor suppressor genes, resulting in uncontrolled proliferation, reduced apoptosis, and metastasis. According to the World Health Organization (WHO), hematological malignancies are predicted to surpass cardiovascular diseases in prevalence in the near future. More than 5000 hematological disorders have been identified, many of which are associated with functional or genetic abnormalities of hematopoietic cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Among them, leukemia is recognized as a hallmark malignancy characterized by the uncontrolled proliferation of white blood cells and their precursors in the bone marrow and peripheral blood [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCML accounts for approximately 15\u0026ndash;20% of all leukemias and is closely associated with the Philadelphia chromosome. This chromosomal rearrangement results in the production of the BCR-ABL fusion protein, which has a permanent tyrosine kinase activity independent of upstream regulatory signals. Activation of downstream signaling pathways by this protein results in increased proliferation, decreased apoptosis, and genomic instability. The discovery of this fusion oncogene was a turning point in the treatment of CML and led to the development of tyrosine kinase inhibitors (TKIs) such as imatinib [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Imatinib, introduced in 2000 and approved by the FDA, revolutionized the treatment of this disease and remains the first-line therapy for many patients [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, resistance to imatinib is common, especially in chronic phase CML. Mutations in the BCR-ABL tyrosine kinase domain, particularly at amino acids 315\u0026ndash;253, impair drug binding and are responsible for approximately 60% of resistance cases [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. To overcome this resistance, second- and third-generation drugs such as dasatinib, nilotinib, bosutinib, and ponatinib have been developed. Although these drugs inhibit specific resistances, they cause severe side effects, including hyperglycemia, pancreatitis, hepatotoxicity, hypertension, and cardiovascular complications [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. On the other hand, access to these new drugs is limited in many resource-poor countries, highlighting the need to find alternative approaches.\u003c/p\u003e\u003cp\u003eA major challenge in cancer therapy is the design of drugs that simultaneously exhibit selectivity, high efficacy, and minimal systemic toxicity [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Classical chemotherapy is often associated with the development of drug resistance; In particular, the antiapoptotic pathways induced by BCR-ABL in myeloid cells complicate treatment [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, attention has been paid to novel anticancer agents. Among them, metal compounds such as vanadium, titanium, copper, ruthenium, and rhodium have attracted attention due to their cytotoxic potential, often leading to apoptosis induction through disruption of nucleic acids or the cell cycle [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNanotechnology has opened up new horizons in cancer therapy. Nanoparticles (NPs), which are typically less than 100 nm in size, can accumulate in tumor tissue through enhanced permeability and accumulation (EPR) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In leukemia, one of the major obstacles to treatment is the survival of leukemic stem cells (LSCs), which are resistant to chemotherapy and lead to relapse. These cells have low levels of reactive oxygen species (ROS) and thus escape ROS-induced apoptosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, manipulating ROS levels has been considered a novel approach to selectively target these cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. CuNPs are a promising candidate due to their ability to increase oxidative stress in malignant cells. There is increasing evidence that ROS production by nanoparticles can inhibit drug resistance and enhance the efficacy of chemotherapy.\u003c/p\u003e\u003cp\u003eIn the present study, CuNPs were investigated as ROS enhancers in combination with imatinib in K562 cells. The MTT assay determined the non-toxic concentrations of CuNPs (0.005 mg/mL) and imatinib (0.5 \u0026micro;M). Then, the cells were divided into different groups including drug alone, nanoparticles alone, and their combination at suboptimal concentrations. The results showed that the combination of CuNPs\u0026ndash;imatinib significantly decreased survival and increased the Bax/Bcl-2 ratio. Also, the intracellular H₂O₂ level was increased, indicating that CuNPs enhanced oxidative stress and, together with imatinib, increased apoptosis. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, which are associated with invasion, angiogenesis, and metastasis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], were also investigated. Increased expression of these genes was observed in single treatments; however, the CuNPs\u0026ndash;imatinib combination showed complex expression changes that likely reflect remodeling of the tumor microenvironment and require further study.\u003c/p\u003e\u003cp\u003eOur findings are consistent with previous studies. Hassan Doost et al. (2016) showed that polybutyl cyanoacrylate nanoparticles loaded with imatinib increased drug stability and toxicity in K562 cells [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Elderdery et al. (2022) also observed that the combination of berbamine with CuO\u0026ndash;TiO\u0026ndash;chitosan nanoparticles increased the expression of proapoptotic genes (caspases, Bax, Bad, P53) and decreased the expression of Bcl2 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Also, Shirangi et al. (2022) showed that sericin\u0026ndash;iron oxide nanoparticles were able to deliver siRNA against ROR1 in breast cancer cells and significantly increased apoptosis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Silver nanoparticles also showed high selectivity; Hojat Afshar et al. (2023) reported that AgNPs loaded with LukS-PV protein induced apoptosis in K562 cells while sparing normal cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Zheng et al. (2016) also enhanced the delivery of SNX-2112 using chitosan-loaded carbon nanotubes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, Li et al. (2010) showed that loading doxorubicin onto antibody-bound carbon nanotubes produced stronger cytotoxicity than the free drug [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eROS generation plays a key role in these studies. Azizi et al. (2017) demonstrated that albumin-coated CuNPs increased ROS in breast cells [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Similarly, Guo et al. showed that ZnO nanoparticles increased daunorubicin toxicity via the ROS pathway. Our findings also support that CuNPs in combination with imatinib increased ROS levels and enhanced cytotoxicity [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition, nanoparticles can improve drug delivery. Gossai et al. (2016) reported that gold nanoparticles can selectively release dasatinib in CML cells and reduce off-target toxicity[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Khan et al. (2018) also showed that silver nanoparticles induce apoptosis in mammary cells through increased ROS and mitochondrial dysfunction [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOverall, CuNPs enhance the efficacy of imatinib through two pathways: (1) induction of apoptosis via ROS and (2) improved drug stability and delivery. Increased Bax/Bcl-2 ratio, increased H₂O₂, and decreased cell survival demonstrate this synergy. While imatinib acts primarily through inhibition of BCR-ABL, CuNPs activate a complementary pathway through oxidative stress.\u003c/p\u003e\u003cp\u003eOverall, these findings support a model in which CuNPs (CuNPs) enhance the efficacy of imatinib through two mechanisms: induction of apoptosis via ROS-dependent pathways, improved drug stability and delivery, and increased duration of intracellular activity. The synergistic increase in Bax/Bcl-2 ratio, increased H₂O₂ levels, and decreased cell viability collectively indicate the therapeutic potential of this compound. Interestingly, imatinib alone induced limited ROS production, except at optimal doses, suggesting that its primary mechanism remains BCR-ABL inhibition, while CuNPs provide a complementary pathway by inducing oxidative stress.\u003c/p\u003e\u003cp\u003eOur data on interleukin-1β (IL-1β) expression also confirm the antiproliferative effects of the CuNP\u0026ndash;imatinib combination, consistent with enhanced BCR-ABL inhibition. Meanwhile, the expression patterns of MMPs (matrix metalloproteinases) highlight the complex relationship between apoptosis, proliferation, and tumor microenvironment remodeling. While moderate apoptosis was associated with increased MMPs, extensive apoptosis induced by the combination treatment suppressed their expression, consistent with reduced proliferation and angiogenesis.\u003c/p\u003e\u003cp\u003eDespite these promising \u003cem\u003ein vitro\u003c/em\u003e results, there are still limitations. Further experiments are needed to confirm apoptosis via extrinsic pathways. In vivo studies are also needed to evaluate pharmacokinetics, safety, and optimal dosing strategies. More extensive analyses at different time points could clarify whether CuNPs enhance the stability and sustained activity of imatinib. Addressing these gaps will be critical to moving CuNP\u0026ndash;imatinib combination therapy into clinical use.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study showed that the combination of imatinib with CuNPs produced significant cytotoxic and apoptotic effects on K562 cells, even at doses lower than the optimal dose of imatinib. By increasing oxidative stress and ROS production, CuNPs shifted the Bax/Bcl-2 ratio in favor of apoptosis and reduced the expression of genes related to invasion and inflammation, such as MMP2, MMP9, and IL-1β. These results indicate that CuNPs can provide complementary pathways to induce cell death in drug-resistant cells and enhance the efficacy of imatinib. The use of this combination could help reduce the drug dose, decrease systemic toxicity, and delay the development of resistance. Despite the promising results in the cell model, further studies in animal models and pharmacology and safety evaluation are needed to determine the transferability of this approach to the clinical treatment of CML.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank Islamic Azad University, Shahrood Branch for their support in this study. This research did not receive any specific funding from public, commercial, or not-for-profit organizations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, M.E-T, M.M. and .M-T.G.; methodology, M.E-T., F.K., M.M. and .M-T.G.; validation, M.M. and .M-T.G.; formal analysis, S.A., M.M. and .M-T.G.; investigation, M.M. and .M-T.G.; resources, M.E-T., F.K., M.M. and .M-T.G.; data curation, M.E-T., F.K., S.A., M.M. and .M-T.G.; writing\u0026mdash;original draft preparation, M.E-T., M.M. and .M-T.G.; writing\u0026mdash;review and editing, M.M. and .M-T.G.; visualization, M.M. and .M-T.G.; supervision, M.M. and .M-T.G.; project administration, M-T.G.; funding acquisition, M.E-T, M.M. and .M-T.G. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe datasets of the current study are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSuttorp M, Millot F, Sembill S, Deutsch H, Metzler M. Definition, epidemiology, pathophysiology, and essential criteria for diagnosis of pediatric chronic myeloid leukemia. Cancers. 2021;13(4):798. https://doi.org/10.3390/cancers13040798\u003c/li\u003e\n\u003cli\u003eHaider MZ, Anwer F. Genetics, philadelphia chromosome. StatPearls [Internet]: StatPearls Publishing; 2023.\u003c/li\u003e\n\u003cli\u003eRamanarayanan J. Advancements in Tyrosine Kinase Inhibitors Therapy: Role of Imatinib, the First Targeted Treatment for Chronic Myeloid Leukemia. Medicine and Medical Research: New Perspectives Vol 3. 2024:30-44. https://doi.org/10.9734/bpi/mmrnp/v3/1238\u003c/li\u003e\n\u003cli\u003eAlves R, Gon\u0026ccedil;alves AC, Rutella S, Almeida AM, De Las Rivas J, Trougakos IP, et al. Resistance to tyrosine kinase inhibitors in chronic myeloid leukemia\u0026mdash;from molecular mechanisms to clinical relevance. Cancers. 2021;13(19):4820. https://doi.org/10.3390/cancers13194820\u003c/li\u003e\n\u003cli\u003eDessie G, Derbew Molla M, Shibabaw T, Ayelign B. Role of stem-cell transplantation in leukemia treatment. Stem cells and cloning: advances and applications. 2020:67-77. https://doi.org/10.2147/sccaa.s262880\u003c/li\u003e\n\u003cli\u003eGiri PM, Banerjee A, Layek B. A recent review on cancer nanomedicine. Cancers. 2023;15(8):2256. https://doi.org/10.3390/cancers15082256\u003c/li\u003e\n\u003cli\u003eAl-Zharani M, Qurtam AA, Daoush WM, Eisa MH, Aljarba NH, Alkahtani S, et al. Antitumor effect of copper nanoparticles on human breast and colon malignancies. Environmental Science and Pollution Research. 2021;28(2):1587-95. https://doi.org/10.1007/s11356-020-09843-5\u003c/li\u003e\n\u003cli\u003eOzcan A, Ogun M. Biochemistry of reactive oxygen and nitrogen species. Basic principles and clinical significance of oxidative stress. 2015;3:37-58. https://doi.org/10.5772/61193\u003c/li\u003e\n\u003cli\u003eSchieber M, Chandel NS. ROS function in redox signaling and oxidative stress. Current biology. 2014;24(10):R453-R62. https://doi.org/10.1016/j.cub.2014.03.034\u003c/li\u003e\n\u003cli\u003eSinha K, Das J, Pal PB, Sil PC. Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. Archives of toxicology. 2013;87(7):1157-80. https://doi.org/10.1007/s00204-013-1034-4\u003c/li\u003e\n\u003cli\u003eZhao RZ, Jiang S, Zhang L, Yu ZB. Mitochondrial electron transport chain, ROS generation and uncoupling. International journal of molecular medicine. 2019;44(1):3-15. https://doi.org/10.3892/ijmm.2019.4188\u003c/li\u003e\n\u003cli\u003eDel R\u0026iacute;o LA, L\u0026oacute;pez-Huertas E. ROS generation in peroxisomes and its role in cell signaling. Plant and Cell Physiology. 2016;57(7):1364-76. https://doi.org/10.1093/pcp/pcw076\u003c/li\u003e\n\u003cli\u003eQiu TA, Gallagher MJ, Hudson-Smith NV, Wu J, Krause MO, Fortner JD, et al. Research highlights: unveiling the mechanisms underlying nanoparticle-induced ROS generation and oxidative stress. Environmental Science: Nano. 2016;3(5):940-5. https://doi.org/10.1039/c6en90021f\u003c/li\u003e\n\u003cli\u003eLossi L. The concept of intrinsic versus extrinsic apoptosis. Biochemical Journal. 2022;479(3):357-84. https://doi.org/10.1042/bcj20210854\u003c/li\u003e\n\u003cli\u003eChen Y-F, Liu H, Luo X-J, Zhao Z, Zou Z-Y, Li J, et al. The roles of reactive oxygen species (ROS) and autophagy in the survival and death of leukemia cells. Critical Reviews in Oncology/Hematology. 2017;112:21-30. https://doi.org/10.1016/j.critrevonc.2017.02.004\u003c/li\u003e\n\u003cli\u003eRaza MH, Siraj S, Arshad A, Waheed U, Aldakheel F, Alduraywish S, et al. ROS-modulated therapeutic approaches in cancer treatment. Journal of cancer research and clinical oncology. 2017;143(9):1789-809. https://doi.org/10.1007/s00432-017-2464-9\u003c/li\u003e\n\u003cli\u003eIrwin ME, Rivera-Del Valle N, Chandra J. Redox control of leukemia: from molecular mechanisms to therapeutic opportunities. Antioxidants \u0026amp; redox signaling. 2013;18(11):1349-83. https://doi.org/10.1089/ars.2011.4258\u003c/li\u003e\n\u003cli\u003eLuo M, Zhou L, Huang Z, Li B, Nice EC, Xu J, et al. Antioxidant therapy in cancer: rationale and progress. Antioxidants. 2022;11(6):1128. https://doi.org/10.3390/antiox11061128\u003c/li\u003e\n\u003cli\u003eR\u0026eacute;b\u0026eacute; C, Ghiringhelli F. Interleukin-1\u0026beta; and cancer. Cancers. 2020;12(7):1791. https://doi.org/10.3390/cancers12071791\u003c/li\u003e\n\u003cli\u003eOkabe S, Tauchi T, Tanaka Y, Ohyashiki K. Interleukin 1 Beta As a Promising Therapeutic Target in ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells. Blood. 2018;132:4249. https://doi.org/10.1182/blood-2018-99-114030\u003c/li\u003e\n\u003cli\u003eEl-Tanani M, Nsairat H, Matalka II, Lee YF, Rizzo M, Aljabali AA, et al. The impact of the BCR-ABL oncogene in the pathology and treatment of chronic myeloid leukemia. Pathology-Research and Practice. 2024;254:155161. https://doi.org/10.1016/j.prp.2024.155161\u003c/li\u003e\n\u003cli\u003eVo TTT, Peng T-Y, Nguyen TH, Bui TNH, Wang C-S, Lee W-J, et al. The crosstalk between copper-induced oxidative stress and cuproptosis: a novel potential anticancer paradigm. Cell Communication and Signaling. 2024;22(1):353. https://doi.org/10.1186/s12964-024-01726-3\u003c/li\u003e\n\u003cli\u003eAnand P, Kunnumakara AB, Sundaram C, Harikumar KB, Tharakan ST, Lai OS, et al. Cancer is a preventable disease that requires major lifestyle changes. Pharmaceutical research. 2008;25(9):2097-116. https://doi.org/10.1007/s11095-008-9661-9\u003c/li\u003e\n\u003cli\u003eJabbour E, Kantarjian H. Chronic myeloid leukemia: a review. JAMA. 2025. https://doi.org/10.1001/jama.2025.0220\u003c/li\u003e\n\u003cli\u003eCohen P, Cross D, J\u0026auml;nne PA. Kinase drug discovery 20 years after imatinib: progress and future directions. Nature reviews drug discovery. 2021;20(7):551-69. https://doi.org/10.1038/s41573-021-00195-4\u003c/li\u003e\n\u003cli\u003eWei Y, Stockelberg D, Hullberg S, Ricksten A, Wadenvik H. Changes in expression of apoptosis-related genes are linked to the molecular response to imatinib treatment in chronic-phase chronic myeloid leukemia patients. Acta Haematologica. 2007;117(2):83-90. https://doi.org/10.1159/000096858\u003c/li\u003e\n\u003cli\u003eCortes JE, Jones D, O\u0026apos;Brien S, Jabbour E, Konopleva M, Ferrajoli A, et al. Nilotinib as front-line treatment for patients with chronic myeloid leukemia in early chronic phase. Journal of Clinical Oncology. 2010;28(3):392-7. https://doi.org/10.1200/jco.2009.25.4896\u003c/li\u003e\n\u003cli\u003eCortes JE, Kantarjian HM, Br\u0026uuml;mmendorf TH, Kim D-W, Turkina AG, Shen Z-X, et al. Safety and efficacy of bosutinib (SKI-606) in chronic phase Philadelphia chromosome\u0026ndash;positive chronic myeloid leukemia patients with resistance or intolerance to imatinib. Blood, The Journal of the American Society of Hematology. 2011;118(17):4567-76. https://doi.org/10.1182/blood-2013-08-524256\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Hare T, Shakespeare WC, Zhu X, Eide CA, Rivera VM, Wang F, et al. AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance. Cancer cell. 2009;16(5):401-12. https://doi.org/10.2210/pdb3ik3/pdb\u003c/li\u003e\n\u003cli\u003eMottier A, Mouchet F, Pinelli E, Gauthier L, Flahaut E. Environmental impact of engineered carbon nanoparticles: from releases to effects on the aquatic biota. Current Opinion in Biotechnology. 2017;46:1-6. https://doi.org/10.1016/j.copbio.2016.11.024\u003c/li\u003e\n\u003cli\u003eAlmuhanna HNB. Elucidating BCR-mediated regulation of FOXO1 in chronic lymphocytic leukaemia: a role for deubiquitinase proteins? : University of Glasgow; 2024. https://doi.org/10.5525/gla.thesis.84781\u003c/li\u003e\n\u003cli\u003eBaranwal J, Barse B, Di Petrillo A, Gatto G, Pilia L, Kumar A. Nanoparticles in cancer diagnosis and treatment. Materials. 2023;16(15):5354. https://doi.org/10.3390/ma16155354\u003c/li\u003e\n\u003cli\u003eOtt I, Gust R. Non platinum metal complexes as anti‐cancer drugs. Archiv der Pharmazie: An International Journal Pharmaceutical and Medicinal Chemistry. 2007;340(3):117-26. https://doi.org/10.1002/ardp.200600151\u003c/li\u003e\n\u003cli\u003eKo W, Kang T, Lee S, Kim Y, Lee B. Effects of luteolin on the inhibition of proliferation and induction of apoptosis in human myeloid leukaemia cells. Phytotherapy research. 2002;16(3):295-8. https://doi.org/10.1002/ptr.871\u003c/li\u003e\n\u003cli\u003eStelmach P, Trumpp A. Leukemic stem cells and therapy resistance in acute myeloid leukemia. Haematologica. 2023;108(2):353-66. https://doi.org/10.3324/haematol.2022.280800\u003c/li\u003e\n\u003cli\u003eDayem AA, Choi H-Y, Kim J-H, Cho S-G. Role of oxidative stress in stem, cancer, and cancer stem cells. Cancers. 2010;2(2):859-84. https://doi.org/10.3390/cancers2020859\u003c/li\u003e\n\u003cli\u003eSim\u0026otilde;es G, Pereira T, Caseiro A. Matrix metaloproteinases in vascular pathology. Microvascular research. 2022;143:104398. https://doi.org/10.1016/j.mvr.2022.104398\u003c/li\u003e\n\u003cli\u003eHasandoost L, Akbarzadeh A, Attar H, Heydarinasab A. In vitro effect of imatinib mesylate loaded on polybutylcyanoacrylate nanoparticles on leukemia cell line K562. Artificial cells, nanomedicine, and biotechnology. 2017;45(3):665-9. https://doi.org/10.1080/21691401.2016.1175444\u003c/li\u003e\n\u003cli\u003eElderdery AY, Alzahrani B, Hamza SMA, Mostafa-Hedeab G, Mok PL, Subbiah SK. [Retracted] CuO‐TiO2‐Chitosan‐Berbamine Nanocomposites Induce Apoptosis through the Mitochondrial Pathway with the Expression of P53, BAX, and BCL‐2 in the Human K562 Cancer Cell Line. Bioinorganic Chemistry and Applications. 2022;2022(1):9602725. https://doi.org/10.1155/2022/9602725\u003c/li\u003e\n\u003cli\u003eShirangi A, Mottaghitalab F, Dinarvand S, Atyabi F. Theranostic silk sericin/SPION nanoparticles for targeted delivery of ROR1 siRNA: Synthesis, characterization, diagnosis and anticancer effect on triple-negative breast cancer. International journal of biological macromolecules. 2022;221:604-12. https://doi.org/10.1016/j.ijbiomac.2022.09.020\u003c/li\u003e\n\u003cli\u003eHaghighatafshar H, Eimani BG, Moazamian E, Amani J. Cytotoxic and apoptotic effects of chemically synthesized silver nanoparticles loaded with recombinant Staphylococcus LukS-PV toxin. Journal of Biotechnology. 2023;373:42-8. https://doi.org/10.1016/j.jbiotec.2023.07.001\u003c/li\u003e\n\u003cli\u003eZheng L, Wu S, Tan L, Tan H, Yu B. Chitosan-functionalised single-walled carbon nanotube-mediated drug delivery of SNX-2112 in cancer cells. Journal of biomaterials applications. 2016;31(3):379-86. https://doi.org/10.1177/0885328216651183\u003c/li\u003e\n\u003cli\u003eLi R, Wu Ra, Zhao L, Wu M, Yang L, Zou H. P-glycoprotein antibody functionalized carbon nanotube overcomes the multidrug resistance of human leukemia cells. ACS nano. 2010;4(3):1399-408. https://doi.org/10.1021/nn9011225\u003c/li\u003e\n\u003cli\u003eAzizi M, Ghourchian H, Yazdian F, Dashtestani F, AlizadehZeinabad H. Cytotoxic effect of albumin coated copper nanoparticle on human breast cancer cells of MDA-MB 231. PloS one. 2017;12(11):e0188639. https://doi.org/10.1371/journal.pone.0188639\u003c/li\u003e\n\u003cli\u003eGuo D, Wu C, Jiang H, Li Q, Wang X, Chen B. Synergistic cytotoxic effect of different sized ZnO nanoparticles and daunorubicin against leukemia cancer cells under UV irradiation. Journal of Photochemistry and Photobiology B: Biology. 2008;93(3):119-26. https://doi.org/10.1016/j.jphotobiol.2008.07.009\u003c/li\u003e\n\u003cli\u003eGossai NP, Naumann JA, Li N-S, Zamora EA, Gordon DJ, Piccirilli JA, et al. Drug conjugated nanoparticles activated by cancer cell specific mRNA. Oncotarget. 2016;7(25):38243. https://doi.org/10.18632/oncotarget.9430\u003c/li\u003e\n\u003cli\u003eKhan AU, Yuan Q, Khan ZUH, Ahmad A, Khan FU, Tahir K, et al. An eco-benign synthesis of AgNPs using aqueous extract of Longan fruit peel: Antiproliferative response against human breast cancer cell line MCF-7, antioxidant and photocatalytic deprivation of methylene blue. Journal of Photochemistry and Photobiology B: Biology. 2018;183:367-73. https://doi.org/10.1016/j.jphotobiol.2018.05.007\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cytotoxic, Apoptosis, Copper Nanoparticles, Imatinib, Chronic Myeloid Leukemia","lastPublishedDoi":"10.21203/rs.3.rs-7627097/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7627097/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImatinib, a first-generation tyrosine kinase inhibitor, is the mainstay of treatment for chronic myeloid leukemia (CML). However, long-term use can lead to cellular resistance, highlighting the need for more effective therapeutic strategies. This study investigated the cytotoxic and apoptotic effects of the combination of imatinib with copper nanoparticles (CuNPs) on the K562 cell line. K562 cells were treated with imatinib (optimal dose 0.5 \u0026micro;M) and non-toxic CuNPs (0.005 mg/mL), as single agents and in combination, in nine experimental groups. Cell survival was measured by MTT assay, intracellular H₂O₂ levels were measured as an indicator of oxidative stress, and the expression of apoptosis and tumor-related genes (Bax, Bcl-2, MMP2, MMP9, and IL-1β) was examined using real-time PCR. The CuNPs were spherical with an average size of approximately 50 nm. Both imatinib and CuNPs dose-dependently decreased cell survival, while their combination produced a significant cytotoxic effect even at suboptimal doses of the drug. The combination treatment resulted in a significant increase in intracellular H₂O₂ levels, indicating increased oxidative stress. Gene expression analysis revealed an increase in the pro-apoptotic gene Bax and a decrease in the anti-apoptotic genes Bcl-2 as well as MMP2, MMP9, and IL-1β, indicating synergistic induction of apoptosis and regulation of tumor-related pathways. The combination of imatinib and CuNPs produces enhanced cytotoxic and apoptotic effects in K562 cells, even at lower drug doses, indicating that CuNPs can be used as an effective adjuvant to enhance the efficacy of imatinib and delay the development of resistance in the treatment of CML.\u003c/p\u003e","manuscriptTitle":"Evaluation of toxicity and apoptotic effects of copper nanoparticles in combination with imatinib on chronic myeloid leukemia cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 14:48:58","doi":"10.21203/rs.3.rs-7627097/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1603ca10-ebf2-464f-b95d-3daf91356d06","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-07T02:38:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 14:48:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7627097","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7627097","identity":"rs-7627097","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}