English

• Breast cancer represents the most frequently diagnosed malignancy and ranks as the second leading cause of cancer-related mortality in women globally^[1]. Approximately 15%-20% of breast cancer cases are classified as triple-negative breast cancer (TNBC), a subtype characterized by heightened invasiveness and an elevated risk of metastasis^[2]. Owing to its lack of estrogen receptor, progesterone receptor, and HER2, TNBC is refractory to hormonal therapy and therefore relies predominantly on chemotherapy^[3]. Platinum-based agents, such as cisplatin and carboplatin, demonstrate moderate efficacy in suppressing TNBC cell proliferation; however, their clinical utility is often limited by intrinsic or acquired resistance, as well as associated toxicities^[4-6]. These limitations have prompted increased research interest in developing non-platinum metal-based chemotherapeutic agents.

  Copper complexes are attracting attention as next-generation chemotherapeutics, not only because copper is an essential trace element involved in key physiological processes^[7-9], but also because its ions can form complexes with various ligands through diverse coordination modes, creating a synergistic effect that inhibits tumor growth through multiple mechanisms^[10-13]. Thiosemicarbazones are a class of compounds known for their broad spectrum of biological activities, including antiviral, antibacterial, and anticancer properties^[14-15]. Several thiosemicarbazones, such as Triapine, DpC, and COTI-2, have advanced into clinical trials^[16-17]. Furthermore, thiosemicarbazones can function as ligands and exhibit strong chelating ability towards various biologically relevant metal ions^[18-19]. Compared to the free ligands, their coordination to metal ions often enhances anticancer efficacy^[19-21]. Notably, thiosemicarbazone metal complexes act through diverse mechanisms: they can target DNA^[22-24], induce lysosome damage^{[22, 25]}, activate immunotherapy^[26-27], trigger endoplasmic reticulum (ER) stress^[28-29], and suppress protein disulfide isomerase (PDI) activity^[30-31], etc. Accordingly, developing a Cu(Ⅱ) complex with multiple targets holds promise as a therapeutic strategy against cancer.

  Herein, we report the synthesis and characterization of three copper(Ⅱ) complexes (C1-C3) derived from 6, 7-dihydro-5H-quinoline-8-one thiosemicarbazones (Scheme 1). The in vitro antitumor activities of these Cu(Ⅱ) complexes were investigated against various cancer cell lines. Among them, C3 exhibited the strongest cytotoxicity against MDA-MB-231 cells. Further studies revealed that C3 targeted the ER stress response and induced mitochondrial dysfunction.

  Scheme 1

  

  Scheme 1.  Synthesis routes of the Cu(Ⅱ) complexes C1-C3

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  1.   Experimental

  1.1   Reagents and chemicals

  Thiosemicarbazide, 4-(2-methylphenyl)-3-thiosemicarbazide, 4-methyl-3-thiosemicarbazide, 6, 7-dihydro-5H-quinoline-8-one, CuBr₂, and all other organic solvents were purchased from Bidepharm (Shanghai, China) or Innochem (Beijing, China), and used without further purification.

  1.2   Synthesis of Cu(Ⅱ) complexes

  The Cu(Ⅱ) complexes were synthesized by mixing 6, 7-dihydro-5H-quinoline-8-one (0.1 mmol), the different substituents-thiosemicarbazide (0.1 mmol), CuBr₂ (0.1 mmol), and CH₃OH (2 mL) in a 10 mL Pyrex glass tube. Then, the glass tubes were heated in an oven at 65 ℃ for 48 h. After cooling the tubes to room temperature slowly, single crystals suitable for single-crystal X-ray diffraction were obtained. The structure of crystals was characterized by X-ray crystallography, elemental analysis, and HRMS.

  C1:   80.2% of yield. Anal. Calcd. for C₁₀H₁₂Br₂CuN₄S·CH₃OH(%): C, 27.77; H, 3.39; N, 11.78. Found(%): C, 27.85; H, 3.36; N, 11.71. HRMS(m/z): 282.000 9 [M-CH₃OH-2Br-H]⁺.

  C2:   76.5% of yield. Anal. Calcd. for C₁₁H₁₄Br₂CuN₄S(%): C, 28.87; H, 3.08; N, 12.24. Found(%): C, 28.93; H, 3.07; N, 12.27. HRMS(m/z): 296.017 3 [M-2Br-H]⁺.

  C3:   77.9% of yield. Anal. Calcd. for C₁₇H₁₈Br₂CuN₄S(%): C, 38.25; H, 3.40; N, 10.50. Found(%): C, 38.31; H, 3.38; N, 10.47. HRMS(m/z): 372.044 9 [M-2Br-H]⁺.

  1.3   X-ray crystallography of Cu(Ⅱ) complexes

  X-ray diffraction data for single crystals of Cu(Ⅱ) complexes C1-C3 were collected at 296.15 K on a Bruker SMART Apex Ⅱ CCD diffractometer. Structural solution and refinement were achieved using Olex 2. Non-hydrogen atoms were refined for anisotropy and atomic coordinates through the full matrix least squares method F², while hydrogen atoms were placed in geometrically ideal positions and constrained to ride on their parent atoms^[32]. Crystallographic data, refinement parameters, and selected bond angles/lengths for C1-C3 are summarized in Table S1-S3 (Supporting information).

  1.4   Cell cytotoxicity assays

  The cytotoxicity of the Cu(Ⅱ) complexes (C1-C3) against various cell lines (MDA-MB-231, HepG2, A549, and WI-38) was examined by MTT assay. In brief, the cell suspension was seeded in 96-well plates at a density of 5 000 cells per well, and then incubated overnight at 37 ℃ under 5% of CO₂ volume fraction in medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution. Subsequently, the cells were treated with the Cu(Ⅱ) complexes of different concentrations (0, 1, 2, 5, 10, and 20 μmol·L^-1), and clinical cisplatin was employed as the control. Following a 48-h incubation, 10 μL of MTT solution (5 mg·mL^-1) was added per well, followed by a further 4-h incubation. The medium was then replaced with 100 μL DMSO, and the plate was agitated to solubilize the formazan crystals. Absorbance of the resulting solution was measured at 570 nm using a microplate reader. Finally, the half maximal inhibitory concentration (IC₅₀) values for the Cu(Ⅱ) complexes were determined from the absorbance data using GraphPad Prism software.

  1.5   Acridine orange/ethidium bromide double staining

  MDA-MB-231 cells were plated in 6-well plates at a density of 5×10⁴ cells per well and incubated overnight. Subsequently, the cancer cells were treated with C3 at concentrations of 0, 1.5, and 3.0 μmol·L^-1. After 24 h of incubation, the cells were washed with phosphate-buffered saline (PBS), stained with acridine orange (AO)/ethidium bromide (EB) for 20 min, and then visualized under a fluorescence microscope.

  1.6   Cell colony study

  A colony assay was performed to evaluate the inhibitory effect of C3 on colony formation. In brief, MDA-MB-231 cells were seeded into 6-well plates overnight. Then, the cells were treated with C3 at concentrations of 0, 1.5, and 3.0 μmol·L^-1. After 3 d of drug exposure, the cells were fixed with 100% cold methanol for 20 min, stained with 0.1% crystal violet for 10 min at room temperature. The samples were washed with water and captured using an inverted microscope.

  1.7   Reactive oxygen species (ROS) detection

  The effects of Cu(Ⅱ) complex on intracellular levels of reactive oxygen species (ROS) were investigated using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. MDA-MB-231 cells were transplanted into six-well plates and treated with C3 at concentrations of 0, 1.5, and 3.0 μmol·L^-1. Following a 24-h incubation, the cells were washed with PBS and incubated with 10 μmol·L^-1 DCFH-DA solution for 30 min. The cells were washed with serum-free medium and observed under a fluorescence microscope.

  1.8   Mitochondrial membrane potential detection

  Mitochondrial membrane potential was assessed using the fluorescent probe JC-1. MDA-MB-231 cells were seeded in 24-well plates and incubated for 24 h under standard conditions (37 ℃, 5% of CO₂ volume fraction). The medium was then replaced with fresh medium containing C3 (0, 1.5, and 3.0 μmol·L^-1), and incubated for another 24 h. Subsequently, the cells were stained with JC-1 in the dark at 37 ℃ for 20 min and immediately imaged under an inverted fluorescence microscope.

  1.9   Western blot assay

  MDA-MB-231 cells were seeded in 10 cm dishes and incubated with C3 (0, 1.5, and 3.0 μmol·L^-1) for 48 h. Subsequently, total proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer and quantified via bicinchoninic acid (BCA) assay. Proteins were separated by 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with 5% non-fat milk for 30 min. The membranes were then incubated with primary antibodies overnight at 4 ℃, followed by incubation with secondary antibodies for 1 h at room temperature after thorough washing with TBST (tris-buffered saline with Tween-20).

  1.10   Three-dimensional spheroid culture

  The inhibitory effect of the Cu(Ⅱ) complex on 3D multicellular tumor spheroids was evaluated in vitro. MDA-MB-231 spheroids were established in agarose-coated 96-well plates over 5 d. Subsequently, the 3D spheroids were then exposed to C3 (0, 3, and 6 μmol·L^-1). The culture medium containing C3 was refreshed every two days, and spheroid morphology was monitored using an inverted microscope.

  1.11   PDI inhibition

  The activity of PDI was quantified with the Proteostat PDI assay kit (Enzo Life Sciences, Lausanne, Switzerland), which monitors the insulin-reduction reaction catalyzed by PDI. Assays were performed in strict accordance with the manufacturer′s protocol. The different concentrations of the test Cu(Ⅱ) complexes—or bacitracin as a reference—were incubated with insulin and PDI. The reaction was initiated by the addition of dithiothreitol (DTT) and allowed to proceed for 0.5 h at 37 ℃, then quenched with the provided stop solution. The fluorescence was recorded on a microplate reader. The IC₅₀ values were then calculated.

  1.12   Molecular docking

  The interaction mechanism between the Cu(Ⅱ) complex and the protein disulfide isomerase (PDI; PDB ID: 4EKZ) was investigated through molecular docking simulations performed with AutoDock Tool 4.2. The protein structure was prepared by removing crystallographic water molecules. Docking procedures were validated before conducting docking experiments with C3 and PDI. The resulting docking poses were visualized and analyzed using PyMOL software.

  1.13   Wound-healing assay

  MDA-MB-231 cells were seeded into 6-well plates and incubated overnight. A tip of a sterile toothpick was then used to introduce a vertical scratch through the monolayer. The cells were washed with PBS and exposed to C3 (0.75 or 1.5 μmol·L^-1) for 24 h. Subsequently, the medium was aspirated, and the wells were gently rinsed with 1 mL PBS. Migration into the wound area was imaged using an inverted microscope.

  2.   Results and discussion

  2.1   Design and structure of Cu(Ⅱ) complexes

  The Cu(Ⅱ) complexes were synthesized by reacting 6, 7-dihydro-5H-quinoline-8-one, thiosemicarbazide, and CuBr₂ in a 10 mL Pyrex glass tube at 65 ℃ for 48 h, as illustrated in Scheme 1. The dark green crystals were examined by single-crystal X-ray diffraction, and their structures are shown in Fig. 1. The crystal data for Cu(Ⅱ) complexes are shown in Table S1-S3. Complexes C1-C3 crystallize in the monoclinic crystal system, space group P2₁/c, P2₁/n, and P2₁/c, respectively (Table S1). All these mononuclear Cu(Ⅱ) complexes have similar coordination geometries, which are a five-coordinated distorted trigonal bipyramid geometry with a center Cu²⁺ ion. C1-C3 consist of mononuclear units comprising one Cu(Ⅱ) ion, one ligand, and two bromide ions. The bond angles of these Cu(Ⅱ) complexes for N1—Cu—N2 are in a range of 79.0°-79.34°, for N1—Cu1—S1 are in a range of 158.89°-159.40°, and for Br2—Cu1—Br1 are in a range of 99.39°-104.34° (Table S2). The Cu—N [0.197 8(5)-0.204 5(5) nm] and Cu—S [0.228 59(12)-0.230 14(17) nm] bond lengths (Table S3) are consistent with those reported for related Cu(Ⅱ) complexes^[33-34]. As shown in Fig.S1-S3, the UV-Vis absorption peaks of C1-C3 exhibited no significant change, indicating that all copper complexes maintained good stability in PBS. The purity of complexes C1-C3 was greater than 95% (Fig.S4-S6).

  Figure 1

  

  Figure 1.  Crystal structures of C1-C3 with thermal ellipsoids plotted at the 30% probability level

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  2.2   Anticancer activities of Cu(Ⅱ) complexes

  To evaluate the effect of these Cu(Ⅱ) complexes on the activity of cancer cells (MDA-MB-231, HepG2, and A549), the IC₅₀ values were examined using the MTT assay after treatment with various concentrations of C1-C3. As shown in Table 1, the IC₅₀ values of C1-C3 against cancer cells ranged from (1.42±0.22) μmol·L^-1 to (7.14±0.35) μmol·L^-1. Among the cancer cell lines examined, the Cu(Ⅱ) complexes exhibited the greatest potency toward MDA-MB-231 cells [IC₅₀=(1.42±0.22) μmol·L^-1-(4.89±0.21) μmol·L^-1]. Interestingly, their cytotoxic efficacy remained markedly higher than that of cisplatin (rank order: C3 > C2 > C1 > cisplatin). Replacing the H atom at the N-4 position with a methyl (C2) or o-Tol (C3) group enhances the anticancer activity compared to the unmodified thiosemicarbazone (C1). This indicates that the N4-substituent plays a pivotal role in determining cytotoxicity, highlighting the potential to develop a diverse library of such Cu(Ⅱ) thiosemicarbazone complexes for systematic anticancer screening. Among all tested complexes, C3 exhibited the highest cytotoxicity against triple-negative breast cancer cells.

  Table 1

  

  Table 1.  IC₅₀ values of C1-C3 toward various cell lines for 48 h^* μmol·L^-1

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  Compound	MDA-MB-231	HepG2	A549	WI-38
  C1	4.89±0.21	4.75±0.25	6.91±0.41	11.33±0.28
  C2	2.54±0.19	3.65±0.26	7.14±0.35	8.85±0.51
  C3	1.42±0.22	3.08±0.23	4.26±0.29	7.44±0.32
  CuBr2	> 100	> 100	> 100	> 100
  Cisplatin	15.22±0.78	17.47±0.74	15.32±0.80	10.03±0.54
  *Data are shown as mean±standard deviation (SD, n=3).

  2.3   Cell colony study

  The colony formation assay was employed to assess the capacity of single cells to survive drug treatment and develop into colonies, thereby serving as an indicator of proliferative ability^[35]. Following a 24 h treatment with complex C3, MDA-MB-231 cells were cultured for an additional 72 h. The resulting colonies were then stained with crystal violet to visualize and quantify cell viability and proliferation. As shown in Fig.2, the treatment groups exhibited a significant reduction in colony number compared to the control, indicating that C3 effectively suppressed cell proliferation.

  Figure 2

  

  Figure 2.  Anti-proliferation activity of C3 assessed by cell colony study

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  2.4   Increasing intracellular ROS

  Prior research has demonstrated that cancer cells exhibit greater sensitivity to elevated ROS levels than normal cells, and that such excess ROS dysregulates the redox balance, triggering apoptotic cell death^[36-37]. Then, the changes in intracellular ROS were determined using the DCHF-DA as a fluorescent probe^[38]. As depicted in Fig.3, the blank control group exhibited negligible green fluorescence. In contrast, following C3 treatment, a pronounced increase in green fluorescence intensity was observed, signifying a substantial elevation of intracellular ROS levels in MDA-MB-231 cells relative to the control. In addition, as the concentration of C3 increased, the intensity of the green fluorescence became higher. These results indicated that C3 can increase the ROS level in MDA-MB-231 cells, and this effect is concentration-dependent.

  Figure 3

  

  Figure 3.  Fluorescence imaging of intracellular ROS levels in MDA-MB-231 cells after 24 h treatment with C3 (1.5 and 3.0 μmol·L^-1) and subsequent DCFH-DA staining

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  2.5   Inducing apoptosis

  Targeting cancer cells through the apoptotic pathway and inducing their death represents one of the potential therapeutic objectives of antitumor drugs^[39]. Dual staining using nucleic acid-binding dyes such as AO and EB^[40], based on their fluorescent emission, can indicate changes in cellular morphological characteristics and thereby reveal apoptotic cell death. As shown in Fig.4, the control cells appeared bright and emitted steady green fluorescence, which is attributable to the well-ordered structure of viable cells. After 24 h of treatment with C3, EB (red fluorescence) penetrated the MDA-MB-231 cells, overshadowing the green fluorescence from AO, indicating that C3 induces cancer cells' apoptosis. In addition, the cells exposed to C3 at 3.0 μmol·L^-1 exhibited a lower number of viable cells and a higher number of apoptotic cells compared to those treated with C3 at 1.5 μmol·L^-1.

  Figure 4

  

  Figure 4.  Dual AO/EB fluorescent staining assay of MDA-MB-231 cells after incubation with C3 (1.5 and 3.0 μmol·L^-1) for 24 h

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  2.6   Mitochondrial dysfunction

  A well-maintained mitochondrial membrane potential (ΔΨ_m) is essential for mitochondrial integrity and bioenergetic function^[41]. The change of ΔΨ_m in cancer cells is a recognized hallmark event, signifying the initiation of the apoptotic cascade^[42]. The JC-1 staining was determined to evaluate the effect of the Cu(Ⅱ) complex on ΔΨ_m in MDA-MB-231 cells. As shown in Fig.5, complex C3 increased bright green fluorescence and diminished red fluorescence, indicating ΔΨ_m loss. These results suggested that C3 triggers apoptosis by inducing mitochondrial dysfunction.

  Figure 5

  

  Figure 5.  Concentration-dependent disruption of ΔΨ_m induced by C3

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  2.7   Induction of ER stress

  The ER is responsible for the biosynthesis, folding, maturation, stability, and transport of membrane proteins and secretory proteins, and it constitutes an interconnected intracellular structure^[43]. Studies have shown that ROS can disrupt the folding capacity of the ER, thereby leading to the accumulation of unfolded or misfolded proteins within the ER—a phenomenon known as ER stress^[44-46]. Persistent and severe ER stress is closely associated with cell death. As shown in Fig.6A, the expression levels of ER stress-related proteins (CHOP, ATF-4, and p-eIF2α) were increased after treatment with C3 (1.5 and 3.0 μmol·L^-1) for 48 h, indicating that C3 mediates the ER stress response.

  Figure 6

  

  Figure 6.  (A) Western blot analysis of the expression levels of CHOP, eIF2α, p-eIF2α, and ATF-4 proteins in MDA-MB-231 cells after treatment with C3 (1.5 and 3.0 μmol·L^-1) for 48 h; (B) Inhibition of PDI activity by C3, bacitracin as a positive control; (C) Interaction between C3 with PDI (4EKZ) using molecular docking simulation

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  2.8   PDI inhibition

  The PDI family, widely recognized as a group of oxidoreductases abundantly present in the endoplasmic reticulum, plays a critical role in disulfide bond formation and is essential for protein folding^[47-48]. Subsequently, we used the Proteostat PDI assay kit to detect the reductase activity of PDI in the presence of C3. As shown in Fig.6B, C3 was able to effectively inhibit PDI reductase activity. Moreover, the inhibition was significantly enhanced with increasing concentrations of C3, and its inhibitory effect was much stronger than that of the well-known PDI inhibitor, bacitracin.

  Next, to better understand the binding mode of Cu(Ⅱ) complex with PDI (PDB ID: 4EKZ), the molecular docking was performed. As shown in Fig.6C, C3 can directly interact with the active site of PDI and form hydrogen bonds with the surrounding amino acid residues, which may interfere with the function of PDI. These results indicated that the induction of cancer cell death is strongly associated with the activation of ER stress and the unfolded protein response.

  2.9   Tumor sphere growth inhibition

  After assessing cytotoxicity in 2D cancer cell cultures, the investigation was extended to 3D multicellular tumor spheroids to evaluate therapeutic efficacy^[49-50]. Tumor spheroids represent an in vitro model system that recapitulates key histological and pathological features of solid tumors, such as gradients in proliferation and nutrient availability, along with the development of a hypoxic core^[51]. To further evaluate the therapeutic effects, the size and morphology of 3D MDA-MB-231 spheroids after treatment with the Cu(Ⅱ) complex were observed. Compared to the untreated control, C3 treatment significantly reduced spheroid size and caused obvious morphological damage (Fig.7). These results demonstrated that the Cu(Ⅱ) complex effectively eradicates multicellular tumor spheroids.

  Figure 7

  

  Figure 7.  Microscopic observation of the changes in MDA-MB-231 multicellular tumor spheroids after a 7-day treatment with C3

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  2.10   Anti-migration ability

  A major factor underlying the therapeutic recalcitrance of triple-negative breast cancer is the exceptionally aggressive biological behavior exhibited by these tumors^{[2, 52]}. Therefore, the suppression of cancer cell metastasis constitutes a critical indicator in drug assessment. To determine whether C3 impedes the migration of MDA-MB-231 cells, an in vitro wound-healing assay was performed. As illustrated in Fig.8, C3 exposure significantly suppressed cell migration in a dose-dependent manner; the anti-migratory potency of 1.5 μmol·L^-1 C3 was markedly greater than that of 0.75 μmol·L^-1. These results strongly suggested that C3 exhibits potent anti-migratory activity against MDA-MB-231 cells.

  Figure 8

  

  Figure 8.  Effect of C3 (0.75 and 1.5 μmol·L^-1) on MDA-MB-231 cell migration for 24 h

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  3.   Conclusions

  In summary, we have designed and synthesized a series of Cu(Ⅱ) complexes (C1-C3) exhibiting pronounced antitumor activity, with C3 emerging as the most potent. C3 displayed potent activity against MDA-MB-231 cells and their 3D spheroids. Mechanistic studies revealed that its potent activity is mediated through a dual-pathway mechanism: C3 induces substantial ROS generation, which concurrently triggers ER stress, inhibits PDI activity, and induces mitochondrial dysfunction. The convergence of these cellular stresses ultimately drives apoptotic cell death. These findings indicate C3 as a promising lead compound for further anticancer development.

  Supporting information is available at http://www.wjhxxb.cn

  
  
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  Scheme 1  Synthesis routes of the Cu(Ⅱ) complexes C1-C3

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  Figure 1  Crystal structures of C1-C3 with thermal ellipsoids plotted at the 30% probability level

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  Figure 2  Anti-proliferation activity of C3 assessed by cell colony study

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  Figure 3  Fluorescence imaging of intracellular ROS levels in MDA-MB-231 cells after 24 h treatment with C3 (1.5 and 3.0 μmol·L^-1) and subsequent DCFH-DA staining

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  Figure 4  Dual AO/EB fluorescent staining assay of MDA-MB-231 cells after incubation with C3 (1.5 and 3.0 μmol·L^-1) for 24 h

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  Figure 5  Concentration-dependent disruption of ΔΨ_m induced by C3

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  Figure 6  (A) Western blot analysis of the expression levels of CHOP, eIF2α, p-eIF2α, and ATF-4 proteins in MDA-MB-231 cells after treatment with C3 (1.5 and 3.0 μmol·L^-1) for 48 h; (B) Inhibition of PDI activity by C3, bacitracin as a positive control; (C) Interaction between C3 with PDI (4EKZ) using molecular docking simulation

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  Figure 7  Microscopic observation of the changes in MDA-MB-231 multicellular tumor spheroids after a 7-day treatment with C3

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  Figure 8  Effect of C3 (0.75 and 1.5 μmol·L^-1) on MDA-MB-231 cell migration for 24 h

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  Table 1.  IC₅₀ values of C1-C3 toward various cell lines for 48 h^* μmol·L^-1

  Compound	MDA-MB-231	HepG2	A549	WI-38
  C1	4.89±0.21	4.75±0.25	6.91±0.41	11.33±0.28
  C2	2.54±0.19	3.65±0.26	7.14±0.35	8.85±0.51
  C3	1.42±0.22	3.08±0.23	4.26±0.29	7.44±0.32
  CuBr2	> 100	> 100	> 100	> 100
  Cisplatin	15.22±0.78	17.47±0.74	15.32±0.80	10.03±0.54
  *Data are shown as mean±standard deviation (SD, n=3).

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