The Proapoptotic Action of Pyrrolidinedione–Thiazolidinone Hybrids towards Human Breast Carcinoma Cells Does Not Depend on Their Genotype

Simple Summary

Breast cancer is one of the most frequent tumors worldwide, based on the number of new cases and deaths. Unfortunately, the low selectivity of action and the rapid development of multiple drug resistances remain the main disadvantages of anticancer compounds. The search for new agents with pronounced antitumor activity is an urgent task in modern biology and medicine. We focused on the investigation of the antitumor potential of novel hybrid pyrrolidinedione–thiazolidinone derivatives. The synthesized derivatives are effective and selective agents that exhibit their antitumor effects in breast carcinoma cells via (1) inhibiting viability, proliferation, and the ability to form colonies; (2) inducing extrinsic and intrinsic apoptotic pathways; and (3) decreasing the level of proteins associated with autophagy, invasion, and metastasis. Our results indicate that synthesized derivatives are potential candidates for deeper exploration of their therapeutic efficiency.

Abstract

The development of new, effective agents for the treatment of breast cancer remains a high-priority task in oncology. A strategy of treatment for this pathology depends significantly on the genotype and phenotype of human breast cancer cells. We aimed to investigate the antitumor activity of new pyrrolidinedione–thiazolidinone hybrid molecules Les-6287, Les-6294, and Les-6328 towards different types of human breast cancer cells of MDA-MB-231, MCF-7, T-47D, and HCC1954 lines and murine breast cancer 4T1 cells by using the MTT, clonogenic and [³H]-Thymidine incorporation assays, flow cytometry, ELISA, and qPCR. The studied hybrids possessed toxicity towards the mentioned tumor cells, with the IC₅₀ ranging from 1.37 to 21.85 µM. Simultaneously, these derivatives showed low toxicity towards the pseudonormal human breast epithelial cells of the MCF-10A line (IC₅₀ > 93.01 µM). Les-6287 at 1 µM fully inhibited the formation of colonies of the MCF-7, MDA-MB-231, and HCC1954 cells, while Les-6294 and Les-6328 did that at 2.5 and 5 µM, respectively. Les-6287 suppressed DNA biosynthesis in the MCF-7, MDA-MB-231, and HCC1954 cells. At the same time, such an effect on the MCF-10A cells was significantly lower. Les-6287 induces apoptosis using extrinsic and intrinsic pathways via a decrease in the mitochondrial membrane potential, increasing the activity of caspases 3/7, 8, 9, and 10 in all immunohistochemically different human breast cancer cells. Les-6287 decreased the concentration of the metastasis- and invasion-related proteins MMP-2, MMP-9, and ICAM-1. It did not induce autophagy in treated cells. In conclusion, the results of our study suggest that the synthesized hybrid pyrrolidinedione–thiazolidinones might be promising agents for treating breast tumors of different types.

1. Introduction

Cancer ranks as the second-leading cause of death [1]. Despite advancements in cancer treatment approaches (immune-, hormone-, cytokine-, and RNA-based therapies), chemotherapy is still an important therapeutic option [2,3]. However, its use is often limited by significant side effects, systemic toxicity, and drug resistance [4,5].

Breast cancer is the major cause of morbidity and mortality among other malignant tumors in the world. This oncological disease is one of the serious public health challenges worldwide that kills more European women than any other type of cancer [1,6]. Breast cancer can be classified into four molecular subgroups according to how the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2) are expressed: luminal A (ER+ and/or PR+, and HER2−), luminal B (ER+ and/or PR+, and HER2+), HER2+ (ER−, PR−, and HER2+), and triple-negative (TNBC: ER−, PR−, and HER2−) [7].

Immunohistochemical determination of breast cancer subtypes, in addition to their diagnostic and prognostic value, plays a crucial role in choosing the most effective treatment strategy [8]. In recent years, targeted therapy has been used for breast cancer treatment, which includes HER2-targeted therapy (trastuzumab, pertuzumab), ER/PR-targeted therapy (tamoxifen, letrozole, exemestane), as well as tyrosine kinase, PARP, CDK4/6, and mTOR inhibitors (lapatinib, neratinib, ribociclib, tucatinib, and others) [9,10,11,12]. Unfortunately, the rapid development of drug resistance is the main problem with modern targeted drugs, which leads to the rapid progression of cancer [13]. Moreover, TNBC does not respond to traditional treatments, and most targeted drugs are useless. Therefore, chemotherapy remains one of the main systematic methods of breast cancer treatment, but its effectiveness in many cases is low. For enhancing effectiveness, chemotherapy drugs can be used in combination with other therapeutic approaches, such as targeted immunotherapy [14]. In addition, the novel, highly effective compound can be used for the synthesis of antibody–drug conjugates. With the approval of trastuzumab emtansine and trastuzumab deruxtecan, this approach is the focus of world scientists [15]. So, the development of new, highly effective chemotherapeutic agents for the treatment of immunohistochemically different subtypes of breast cancer is an acute task of modern pharmacology and medicine.

Applying the pharmacophore/molecular hybridization approaches has proven to be an effective strategy for designing and searching for anticancer-hit compounds with 4-thiazolidinone scaffolds [16,17,18,19]. Ciminalum and pyrrolidinedione-containing molecules possess a privileged place in the design of different chemotypes of 4-thiazolidinone hybrids with anticancer activity (Figure 1). So, a series of pyrrolidinedione–thiazolidinone hybrid molecules with potential anticancer properties have been reported [20,21,22,23,24]. Buzun et al. described a Ciminalum-4-thiazolidinone hybrid with an impact on leukemia, melanoma, colon, glioma, gastric, and breast tumor NCI cell lines with a GI₅₀ value of 1.57 μM [21]. The same authors also reported the cytotoxic effect of one more Ciminalum-4-thiazolidinone hybrid on breast carcinoma MCF-7 and MDA-MB-231 cells with IC₅₀ values of 5–15 μM at 24 h. It induced mitochondria-dependent apoptosis and decreased the topoisomerase II concentration [22]. Finiuk et al. described a series of the Ciminalum-bearing pyrrolidinedione-4-thiazolidinone hybrid molecules Les-6294, Les-6287, and Les-6328 (Figure 1) that inhibited the metabolic activity of Jurkat, KB3-1, HeLa, HCT116, A549, and U251 cells with the IC₅₀ values of 1–9 µM following 72 h of incubation [23,24].

With the aim of in-depth studies and as a part of our ongoing systematic research of the anticancer properties of Ciminalum–pyrrolidinedione–4-thiazolidinone hybrids, herein we report on the evaluation of the impact of the hybrid molecules Les-6287, Les-6294, and Les-6328 (Figure 1) [23,24] on the viability, metabolic activity, DNA synthesis, apoptosis, and autophagy induction in breast carcinoma cells.

2. Materials and Methods

2.1. Materials

All chemicals and solvents were purchased from commercial suppliers. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 98%, Product No. M2128), dimethyl sulfoxide (DMSO, ReagentPlus^®, ≥99.5%, Product No. D5879), β-mercaptoethanol (Product No. M3148), methanol (≥99.9%, Product No. 34860), crystal violet (Product No. C0775), sodium hydroxide (Product No. 484024), sodium dodecyl sulfate (SDS, ≥98.5%, Product No. L3771), sodium chloride (ReagentPlus^®, ≥99%, Product No. S9625), doxorubicin (Product No. PHR1789), and Tris (2-amino-2-(hydroxymethyl)-1,3-propanediol, ≥99.8%, Product No. 93362) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (96%) was obtained from Avantor Performance Materials (Gliwice, Poland). The trichloroacetic acid (TCA, pure p.a. ≥ 98%, Cat. No. 76-03-09) was from Chempur (Piekary Slaskie, Poland). The human breast carcinoma MCF-7 (HTB-22), MDA-MB-231 (HTB-26), T-47D (HTB-133), and HCC1954 (CRL-2338) cells, normal human breast epithelial MCF-10A (CRL-10317) cells, and murine breast cancer 4T1 (CRL-2539) cells were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s Minimal Eagle Medium (DMEM, Cat. No. 11995065), RPMI-1640 medium (Cat. No. 11875093), fetal bovine serum (FBS, Cat. No. 10091148), horse serum (Cat. No. 16050122), phosphate-buffered saline (PBS, Cat. No. J61196.AP), 0.05% trypsin with 0.02% EDTA (Cat. No. 25300054), glutamine (Cat. No. 25030081), and penicillin/streptomycin solution (Cat. No. 15140122) were from Gibco (San Diego, CA, USA). An MEGM Mammary Epithelial Cell Growth Medium BulletKit (Product No. CC-3150) was from Lonza Bioscience (Basel, Switzerland). [³H]-thymidine (7 Ci/mmol, Cat. No. MT6031) was received from Moravek Biochemicals (Brea, CA, USA), and Scintillation Cocktail Ultima Gold XR (Product No. 6013119) was received from PerkinElmer (Waltham, MA, USA). DNase-free RNase A Solution (Cat. No. M6101) was from Promega (Madison, WI, USA). FITC Annexin V Apoptosis Detection Kit II (Cat. No. 556547) and JC-1 MitoScreen Kit (Cat. No. 551302) were purchased from BD Pharmingen (San Diego, CA, USA). The propidium iodide (Cat. No. 638), FAM-FLICA^® Caspase-3/7 Assay Kit (Cat. No. 93), FAM-FLICA^® Caspase-8 Assay Kit (Cat. No. 99), FAM-FLICA^® Caspase-9 Assay Kit (Cat. No. 912), and FAM-FLICA^® Caspase-10 Assay Kit (Cat. No. 922) were purchased from ImmunoChemistry Technologies (Bloomington, MN, USA). High-sensitivity human Beclin-1 SimpleStep ELISA Kit (Cat. No. ab254511), and high-sensitivity human SimpleStep ELISA Kits for MMP-2 (Cat. No. ab267813), MMP-9 (Cat. No. ab246539), and ICAM-1 (CD54, Cat. No. ab174445) were from Abcam (Cambridge, UK); LC3B ELISA Kit (Cat. No. E4774Hu) was from BT LAB (Shanghai, China). RLT buffer (Cat. No. 79216) and the RNeasy Mini Kit (Cat. No. 74104) were from Qiagen (Wrocław, Poland). The High-Capacity cDNA Reverse Transcription Kit (Cat. No. 4368814) was purchased from Thermo Fisher Scientific Inc., Waltham, MA, USA.

2.2. Studied Compounds

The synthesis and characterization of pyrrolidinedione–thiazolidinone hybrid molecules Les-6287 (1-(4-hydroxyphenyl)-3-[5-[2-chloro-3-(4-nitrophenyl)prop-2-enylidene]-4-oxo-2-thioxothiazolidine-3-yl]pyrrolidine-2,5-dione) and Les-6294 (1-(4-chlorophenyl)-3-[5-[2-chloro-3-(4-nitrophenyl)prop-2-enylidene]-4-oxo-2-thioxothiazolidine-3-yl]pyrrolidine-2,5-dione) was reported in [23] and Les-6328 (1-(4-bromophenyl)-3-[5-[2-chloro-3-(4-nitrophenyl)prop-2-enylidene]-4-oxo-2-thioxothiazolidine-3-yl]pyrrolidine-2,5-dione)—in [24]. The 100 mM stock solutions of pyrrolidinedione–thiazolidinones were prepared in DMSO.

2.3. Culture of MCF-7, MDA-MB-231, HCC1954, T-47D, 4T1, and MCF-10A Cells

The MCF-7 and MDA-MB-231 cells were cultured in the DMEM medium containing 10% of the FBS, HCC1954, T-47D, and 4T1 cells in the RPMI-1640 medium with the addition of 10% of the FBS, and MCF-10A cells in MEGM Mammary Epithelial Cell Growth Medium BulletKit supplemented with penicillin/streptomycin cocktail and 5% of horse serum. The cells were cultured in a humanized atmosphere at 5% CO₂ and 37 °C. After trypsinization, cells were counted using a Scepter 3.0 handheld automated cell counter (Millipore, Burlington, MA, USA) [25].

2.4. The MTT Assay

The MTT test was used to assess the metabolic activity of cells treated with the investigated substances following the Sigma-Aldrich manufacturer’s instructions [23]. Briefly, compounds were added in 100 µL of the cultural medium, and cells were incubated for the next 24 and 48 h. Doxorubicin was used as a reference drug. The DMSO was added to dissolve the crystals of formazan. We used the ThermoScientific Evolution 201 UV–VIS spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA) to measure the absorbance of formazan at 570 nm. The initial value of the relative number of cells in control (under 0.1% of the DMSO) was accepted as 100%. We used GraphPad Prism Version 9 to calculate the half-maximal inhibition concentration (IC₅₀) of the studied compounds (the concentration of the drug that reduced cell viability by 50%).

2.5. The Clonogenic Assay

The clonogenic assay was carried out utilizing the previously published methodology [26]. The MCF-7, MDA-MB-231, HCC1954, and MCF-10A cells (500 cells/2 mL) were seeded in a 12-well plate overnight. Afterward, studied derivatives or doxorubicin (1, 2.5, 5, 10, and 50 µM) were added to cells. The media was changed after 72 h to a compound-free one, and the cells were cultured for the following 14 days. Then, the cells were washed, fixed, and stained with 0.1% crystal violet. After washing, the plates were dried at room temperature. The colonies were recorded by ImageJ software (Version 1.30). The value of colonies in control (non-treated cells) was accepted as 100%. The effect of compounds on the colony-forming ability of cells was expressed as the percentage of colonies compared to the control.

2.6. [³H]-Thymidine Incorporation Assay

Following a 24 h cell treatment, the incorporation of [³H]-thymidine into the DNA of the breast tumor and normal cells was used to measure the antiproliferative activity of Les-6287. The MCF-7, MDA-MB-231, HCC1954, and MCF-10A cells were seeded into six-well plates (density 3 × 10⁵ cells/well) and cultured for 24 h. Then, they were incubated under the same conditions for the next 24 h with a growth medium containing the tested compounds and reference drug (doxorubicin) at different concentrations (1, 2.5, 5, 10, 50 µM). After 24 h, the medium was changed to a fresh one without FBS. A total of 0.5 µCi of tritium-labeled thymidine (specific activity: 7 Ci/mmol) was added to cells for 4 h. The cells were washed with 1 mL of 0.05 M Tris-HCl buffer pH 7.4 containing 0.11 M NaCl and 1 mL of 5% trichloroacetic acid (TCA). Each well was filled with 1 mL of a 0.1 M NaOH solution containing 1% SDS to complete cell lysis. After 5 min, the acquired cell lysates were put into scintillation vials and pre-filled with two milliliters of scintillation liquid. The Scintillation Counter 1900 TR, TRI-CARB (Packard, Perkin Elmer, Inc., San Jose, CA, USA) was used to record the results [25]. The effect of compounds on DNA synthesis was expressed as % of the control (non-treated cells).

2.7. Flow Cytometry of Apoptosis Induction in Breast Tumor Cells

The FITC Annexin V Apoptosis Detection Kit II and a flow cytometer (BD FACSCanto II, BD Biosciences Systems, San Jose, CA, USA) were used to detect the effect of studied derivatives on apoptosis induction in MCF-7 and MDA-MB-231 lines incubated for 24 h with Les-6287 (1 and 1.5 µM), reference drug–doxorubicin (1 µM), and 0.15% of solvent DMSO (dose that corresponds to its content at 1.5 µM of compound Les-6287). The assay was performed following the manufacturer’s instructions and the previously described methodology [25]. Cells were washed twice with cold PBS and resuspended in the Binding Buffer from the Kit. The FITC Annexin V and propidium iodide (PI) were added to 100 µL of cell suspension and incubated for 15 min at room temperature, protected from light. Then, 300 µL of Binding Buffer was added, and samples were analyzed using a flow cytometer. A total of 10,000 events were measured. The FACSDiva software (Version 6.1.3, BD Biosciences Systems, San Jose, CA, USA) was applied to analyze all flow cytometry data. The BD Cytometer Setup and Tracking Beads (BD Biosciences, San Diego, CA, USA) were used for the calibration of the flow cytometer [25].

2.8. Assessment of Changes in Mitochondrial Membrane Potential

The JC-1 MitoScreen kit (BD Pharmigen, San Diego, CA, USA) and a flow cytometer (BD FACSCanto II) were used to detect the decrease in mitochondrial membrane potential (MMP, ΔΨm) under the action of the studied compounds. The assay was performed following the manufacturer’s instructions and the previously described methodology [25,27,28]. The MCF-7 and MDA-MB-231 cells were treated for 24 h with Les-6287 (1 and 1.5 µM), reference drug–doxorubicin (1 µM), and 0.15% of solvent DMSO (dose that corresponds to its content at 1.5 µM of compound Les-6287). Following washing, the cells were further suspended in 0.5 mL of buffer with 10 µg/mL JC-1 and incubated for 15 min at room temperature in the dark. Then, cells were washed twice with buffer, resuspended in 300 µL PBS, and analyzed [25].

2.9. Caspases 3/7, 8, 9, and 10 Enzymatic Activity Assays

The assay was performed following the manufacturer’s instructions and the previously described methodology [25]. The MCF-7 and MDA-MB-231 cells were treated for 24 h with Les-6287 (1 and 1.5 µM), reference drug–doxorubicin (1 µM), and 0.15% of solvent DMSO (dose that corresponds to its content at 1.5 µM of compound Les-6287). The cells were gathered, rinsed twice with chilled PBS, and reconstituted in an Apoptosis Wash Buffer. Then, 10 µL of freshly diluted FLICA solution was added to 290 µL of cell suspension and incubated for an hour at 37 °C. Following two rounds of washing with 2 mL of Apoptosis Wash Buffer, the cells were centrifuged, resuspended in 300 µL of the buffer, and then analyzed [25].

2.10. RNA Isolation and Quantitative PCR (qPCR)

The MCF-7 and MDA-MB-231 cells were treated for 24 h with Les-6287 and reference drug–doxorubicin (both at 1 µM concentration), harvested, and lysed in RLT buffer (Qiagen, Venlo, The Netherlands) supplemented with 1% of β-mercaptoethanol. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s protocol and quantified on a NanoDrop spectrophotometer (NanoDrop™ One/OneC, ThermoFisher, Waltham, MA, USA). Next, 1 μg of RNA from every sample was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher, Waltham, MA, USA) according to the standard manufacturer’s instructions. The expression of genes, namely MAP1LC3b (Bio-Rad, Hercules, CA, USA) and BECN1 (Bio-Rad, Hercules, CA, USA), was evaluated using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on the StepOne Plus system (ThermoFisher, Waltham, MA, USA). StepOne Software v2.3 (Thermo Fisher Scientific, Waltham, MA, USA) was used for results calculations. The results were normalized to GAPDH (Bio-Rad, Hercules, CA, USA) and untreated cells (control). The primers used to investigate the expression of the mentioned genes are listed in

Table 1. The list of PrimePCR SYBR Green assays used in the study.

Assay	Target Species	Catalog Number	Manufacturer
MAP1LC3B	Human	qHsaCEP0041298	Bio-Rad
BECN1	Human	qHsaCID0016032	Bio-Rad
GAPDH	Human	qHsaCEP0041396	Bio-Rad

. Data were presented as a relative expression using the 2^−ΔΔCt method.

2.11. ELISA Measurement of Beclin-1, LC3B, MMP-2, MMP-9, and ICAM-1

The MCF-7 and MDA-MB-231 cells were treated for 24 h with Les-6287 (1, 1.5, and 2 μM), as well as a reference drug–doxorubicin (1, 1.5, and 2 μM). In order to determine the Beclin-1 concentration in the cell lysates, the SimpleStep ELISA (Abcam, Cambridge, UK) Kit was used. MMP-2, MMP-9, and ICAM-1 concentrations were measured in culture supernatants using high-sensitivity human SimpleStep ELISA kits (Abcam, Cambridge, UK) according to the manufacturer’s protocols. The LC3B ELISA kit (BT LAB, Shanghai, China) was used to check the protein concentration in cell lysates from analyzed breast cancer cells. The procedure for preparing the samples was shown previously [29].

2.12. Statistical Analysis

Results were presented as mean ± standard deviation (M ± SD). Data analysis of gene expression studies was performed with GraphPad Prism Version 10 software; data from ELISA’s measurements were analyzed with GraphPad Prism Version 6.0; and other results were analyzed and illustrated with GraphPad Prism Version 9 (GraphPad Software, San Diego, CA, USA). The differences between the control and tested groups were evaluated by means of ANOVA tests (p < 0.05).

3. Results

3.1. Cytotoxicity of Derivatives towards Breast Cancer Cells

Continuing screening of the antitumor activity of pyrrolidinedione–thiazolidinone hybrid molecules, we studied in vitro cytotoxic activities towards human breast carcinoma MCF-7, T-47D, MDA-MB-231, and HCC1954 cells, murine breast carcinoma 4T1 cells, and normal human breast epithelial MCF-10A cells using the MTT test, assays on the incorporation of [³H]-thymidine into DNA, and clonogenic assays.

Les-6287, Les-6294, and Les-6328 compounds demonstrated high metabolic inhibitory activity towards breast carcinoma MCF-7, T-47D, MDA-MB-231, 4T1, and HCC1954 cells of different types (estrogen and progesterone receptor-positive, triple-negative, and HER2-positive, respectively; Figure 2,

Table 2. The IC₅₀ values of studied compounds targeting cells in MTT assay.

Cell Line	Timepoint, h	IC50, µM (M ± SD)
		Les-6287	Les-6294	Les-6328	Dox
MCF-7	24	2.34 ± 0.16	6.74 ± 0.64	3.26 ± 0.40	3.47 ± 0.49
	48	1.43 ± 0.18	3.54 ± 0.14	2.18 ± 0.19	0.18 ± 0.07
T-47D	24	3.11 ± 0.19	4.48 ± 0.51	4.08 ± 0.56	3.66 ± 0.33
	48	1.74 ± 0.25	2.66 ± 0.21	1.97 ± 0.58	2.31 ± 0.24
MDA-MB-231	24	3.86 ± 0.24	21.85 ± 9.92	6.09 ± 0.33	6.18 ± 0.08
	48	1.37 ± 0.15	3.72 ± 0.22	2.01 ± 0.12	1.09 ± 0.09
4T1	24	1.60 ± 0.14	2.20 ± 0.19	2.25 ± 0.34	2.37 ± 0.25
	48	1.62 ± 0.21	2.22 ± 0.25	1.94 ± 0.15	1.98 ± 0.21
HCC1954	24	2.52 ± 0.65	4.53 ± 0.18	9.91 ± 0.17	1.90 ± 0.11
	48	2.25 ± 0.64	5.01 ± 0.23	6.40 ± 0.25	1.15 ± 0.16
MCF-10A	24	93.01 ± 2.29	>100	>100	15.91 ± 0.91
	48	64.58 ± 0.68	>100	>100	0.23 ± 0.05

). After 24 h of exposure, the IC₅₀ of Les-6287 was 2.34 ± 0.16 µM in MCF-7 cells, 3.86 ± 0.24 µM in MDA-MB-231 cells, and 2.52 ± 0.65 µM in HCC1954 cells. After 48 h of exposure, the IC₅₀ of Les-6287 was 1.43 ± 0.18 μM for MCF-7 cells, 1.37 ± 0.15 μM for MDA-MB-231 cells, and 2.25 ± 0.64 μM in HCC1954 cells. The IC₅₀ of compound Les-6294 was 6.74 ± 0.64 μM after 24 h of MCF-7 cell treatment and 3.54 ± 0.14 μM after 48 h of cell treatment. Following 24 h of exposure, the IC₅₀ of compound Les-6294 was 21.85 ± 9.92 μM for MDA-MB-231 cells and 3.72 ± 0.22 μM following 48 h of cell treatment. The Les-6294 compound demonstrated IC₅₀ values of 4.53 ± 0.18 μM and 5.01 ± 0.23 μM after 24 and 48 h of HCC1954 cell treatment, respectively. After 24 h of exposure, the IC₅₀ was 3.26 ± 0.40 μM under the Les-6328 toward MCF-7 cells, 6.09 ± 0.33 μM toward MDA-MB-231 cells, and 9.91 ± 0.17 μM toward HCC1954 cells. After 48 h of exposure, the IC₅₀ of 2.18 ± 0.19 μM was found under the Les-6328 treatment of MCF-7 cells, 2.01 ± 0.12 μM under its treatment of MDA-MB-231 cells, and 6.40 ± 0.25 μM under its treatment of HCC1954 cells. The IC₅₀ of the Les-6287 compound was 3.11 ± 0.19 μM after 24 h of the T-47D cell treatment and 1.74 ± 0.25 μM after 48 h of cell treatment. Following 24 h of exposure, the IC₅₀ of the Les-6294 compound was 4.48 ± 0.51 μM for these cells and 2.66 ± 0.21 μM following 48 h of cell treatment. The IC₅₀ of compound Les-6328 was 4.08 ± 0.56 μM after 24 h of the T-47D cell treatment and 1.97 ± 0.58 μM after 48 h of cell treatment. After 24 h of exposure, the IC₅₀ of Les-6287 was 1.60 ± 0.14 µM in 4T1 cells, the IC₅₀ of Les-6294 was 2.20 ± 0.19 µM, and the IC₅₀ of Les-6328 was 2.25 ± 0.34 μM. After 48 h of exposure, the IC₅₀ of Les-6287 was 1.62 ± 0.21 μM for 4T1 cells, 2.22 ± 0.25 μM for Les-6294, and 1.94 ± 0.15 µM for Les-6328. It should be noted that the IC₅₀ values for the studied hybrid molecules and doxorubicin were similar for the MCF-7, MDA-MB-231, and HCC1954 cells.

Thus, MDA-MB-231 and 4T1 triple-negative breast carcinoma cells, MCF-7 and T-47D estrogen and progesterone receptors positive, HER2 negative, and HCC1954 HER2 positive breast carcinoma cells were sensitive to the action of Les-6287, Les-6294, and Les-6328 with IC₅₀ levels of 1.37–21.85 µM at their treatment for 24 and 48 h. The hydroxy-substituted hybrid Les-6287 was more active in the studied breast carcinoma cells. At the same time, related halogen-substituted molecules Les-6328 and Les-6294 had a lower inhibitory effect on studied breast carcinoma cells compared with Les-6287.

In the case of normal breast cells of the MCF-10A line, Les-6287, Les-6294, and Les-6328 possessed lower cytotoxicity than in the case of breast carcinoma cells. The IC₅₀ values for Les-6287 were 93.01 ± 2.29 µM and 64.58 ± 0.68 µM after 24 and 48 h of exposure, respectively. The compounds Les-6294 and Les-6328 did not reach their IC₅₀ at 100 μM (Figure 2, Table 2). In turn, the IC₅₀ of Dox was 15.91 ± 0.91 µM after 24 h of MCF-10A cell treatment and 0.23 ± 0.05 μM after 48 h of cell treatment.

3.2. Studied Derivatives Inhibit the Formation of Colonies of Breast Cancer Cells

The biological efficiency of the studied compounds was determined using the clonogenic assay. An important factor in gauging an agent’s anticancer efficacy is its capacity to create colonies of new cells. This procedure is used to estimate the percentage of tumor cells that can still form colonies, a crucial phenotypic trait of tumor cells [26].

The Les-6287 at 1 µM totally inhibited the formation of colonies of MCF-7, MDA-MB-231, and HCC1954 cells (Figure 3,

Table 3. The IC₅₀ values of studied compounds in clonogenic assay.

Cell Line	IC50 (M ± SD)
	Les-6287	Les-6294	Les-6328	Dox
MCF-7	0.42 ± 0.08	1.33 ± 0.10	1.57 ± 0.12	0.42 ± 0.08
MDA-MB-231	0.42 ± 0.11	0.67 ± 0.08	1.19 ± 0.03	0.42 ± 0.11
HCC1954	0.43 ± 0.11	3.05 ± 0.03	1.54 ± 0.02	0.43 ± 0.11
MCF-10A	>50	>50	>50	0.47 ± 0.11

). Doxorubicin demonstrated a similar effect on the formation of colonies of MCF-7, MDA-MB-231, and HCC1954 cells as Les-6287 did. Les-6294 and Les-6328 demonstrated a less prominent ability to inhibit the formation of colonies of these cells. Les-6294 and Les-6328 at 5 µM totally inhibited the formation of colonies of MCF-7 cells. We did not observe the growth of MDA-MB-231 colonies after their exposure to Les-6294 at 5 µM and Les-6328 at 2.5 µM. The IC₅₀ of Les-6294 was 0.67 ± 0.08 µM, and the IC₅₀ of Les-6328 was 1.19 ± 0.03 µM for these cells. Les-6294 and Les-6328, at a concentration of 2.5 µM, practically completely inhibited the formation of colonies of HCC1954 cells. The IC₅₀ of Les-6294 was 3.05 ± 0.03 µM, and the IC₅₀ of Les-6328 was 1.54 ± 0.02 µM for this cell line (Figure 3, Table 3).

Three studied derivatives slightly inhibited colony formation in normal human breast epithelial MCF-10A cells without reaching the IC₅₀ at 50 µM (Figure 3, Table 3). Les-6328 at a concentration of 50 µM inhibited the formation of colonies in these cells by 25.87%, Les-6287 by 32.57%, and Les-6294 by 37.12%.

3.3. The Effect of Studied Derivatives on the DNA Biosynthesis Process in Breast Carcinoma and Normal Breast Epithelial Cells

It should be noted that compound Les-6287 was the most active among the studied derivatives. It was used further for in-depth evaluation of the anticancer activity towards human breast carcinoma MCF-7, MDA-MB-231, and HCC1954 cells.

The impact of the most active compound in MTT and clonogenic assays was therefore examined on DNA biosynthesis to identify the mechanism underlying their growth inhibition effect on human breast carcinoma cells of the MCF-7, MDA-MB-231, and HCC1954 lines and normal human breast epithelial cells of the MCF-10A line. The tested derivative inhibited DNA synthesis in breast cancer cell lines in a dose-dependent manner (Figure 4,

Table 4. The IC₅₀ values of studied compounds targeting cells in [³H]-thymidine assay.

Cell Line	IC50 (M ± SD)
	Les-6287	Dox
MCF-7	2.37 ± 0.02	0.66 ± 0.13
MDA-MB-231	2.32 ± 0.04	0.63 ± 0.11
HCC1954	3.67 ± 0.09	1.07 ± 0.04
MCF-10A	43.54 ± 1.16	3.19 ± 0.43

). In MCF-7 and MDA-MB-231 cells, Les-6287 exhibited similar DNA synthesis inhibitory effects, with IC₅₀ values of 2.37 ± 0.02 µM and 2.32 ± 0.04 µM, respectively. This compound had a lower effect on DNA synthesis in HCC1954 cells, with an IC₅₀ value of 3.67 ± 0.09 µM. For MCF-10A cells, Les-6287 possessed a less pronounced impact on DNA biosynthesis, and the IC₅₀ value for this cell line was higher than that observed for breast cancer cells. The IC₅₀ value of Les-6287 was 43.54 ± 1.16 µM for MCF-10A cells. The doxorubicin exhibited a higher effect on DNA biosynthesis in breast carcinoma and normal breast epithelial cells (Figure 4, Table 4).

3.4. Les-6287 Induces Apoptosis by Extrinsic and Intrinsic Pathways

Les-6287 in 1 and 1.5 µM doses induced apoptosis in MCF-7 and MDA-MB-231 cell lines (Figure 5). In MCF-7 cells, 3.85 ± 1.20% of early apoptotic, 21.60 ± 0.78% of late apoptotic, and 0.50 ± 0.24% of necrotic cells were observed under Les-6287 at 1 µM treatment, 2.30 ± 0.42% of early apoptotic, 29.35 ± 6.43% of late apoptotic, and 0.30 ± 0.11% of necrotic cells under Les-6287 at 1.5 µM treatment. It should be noted that Les-6287 did not affect the necrotic cell level in MCF-7 cells. We detected 2.10 ± 0.14% of early apoptotic, 5.10 ± 0.49% of late apoptotic, and 0.90 ± 0.02% of necrotic MDA-MB-231 cells after 24 h of incubation with a 1 µM concentration of Les-6287. In contrast, after cell incubation with a 1.5 µM concentration of Les-6287, 5.65 ± 0.92% of early apoptotic, 29.00 ± 9.76% of late, and 4.50 ± 1.82% of necrotic cells were detected. In the case of the control MDA-MB-231 cells, 1.10 ± 0.14% of early apoptotic, 3.95 ± 0.85% of late apoptotic, and 0.85 ± 0.21% of necrotic cells were detected. The doxorubicin-induced appearance of 0.55 ± 0.07% and 0.10 ± 0.01% of early apoptotic cells, 60.60 ± 7.35% and 42.20 ± 17.25% of late apoptotic cells, and 31.75 ± 5.59% and 51.25 ± 3.36% of necrotic cells in MCF-7 and MDA-MB-231 cells, respectively. As for the DMSO (0.15%), it did not cause changes in the number of early, late apoptotic, and necrotic cells, whose values were 2.35 ± 0.07%, 3.6 ± 0.99%, and 6.0 ± 1.2% for MCF-7 and 1.25 ± 0.21%, 3.25 ± 0.07%, and 0.60 ± 0.28% for MDA-MB-231 cells, respectively (Figure 5).

A reduction in mitochondrial membrane potential (ΔΨm) due to increased membrane permeability is associated with the early stages of apoptotic death that occur via the intrinsic pathway [30]. JC-1 fluorescent dye labeling was used to examine the impact of Les-6287 on the intrinsic apoptotic pathway in breast carcinoma cells.

As shown in Figure 6, Les-6287 caused an increase in the percentage of cells with decreased mitochondrial membrane potential in MCF-7 and MDA-MB-231 cells. However, it was greater for MCF-7 cells: 21.00 ± 1.20% (Les-6287, 1.0 μM) and 38.85 ± 3.46% (Les-6287, 1.5 μM). For MDA-MB-231 cells, the values were as follows: 3.75 ± 1.34% Les-6287 (1.0 μM) and 17.85 ± 3.46% Les-6287 (1.5 μM).

An appearance of pores and a related increase in mitochondrial membrane permeability result in the release of cytochrome c into the cytosol and apoptosome formation that triggers the activation of caspase 9 [28]. The assumption of the present assay was to evaluate caspase-9 activity in MCF-7 and MDA-MB-231 cancer cells after 24 h of incubation with the tested compounds. Les-6287 increased caspase-9 activity in both line cells (Figure 7). The level of active caspase 9 in the MCF-7 cells increased to 24.40 ± 0.98%. (Les-6287, 1.0 μM) and 36.45 ± 1.05% (Les-6287, 1.5 μM) compared to the control group. In contrast, an increase in caspase-9 activity to 15.30 ± 0.80% (Les-6287, 1.0 μM) and 21.88 ± 2.00% (Les-6287, 1.5 μM) was observed in the MDA-MB-231 cell line. The data from the present experiment correlate well with changes in ΔΨm, confirming that apoptosis is induced via the intrinsic pathway.

Activation of caspases 8 and 10 is a key process required to induce apoptosis via the extrinsic pathway [31]. Thus, we measured the caspase 8 and 10 activity in the MCF-7 and MDA-MB-231 cells after 24 h of treatment with the tested compounds (Figure 8 and Figure 9). After treatment with Les-6287, activation of the caspase 8 and 10 was observed in 11.08 ± 1.30% and 11.58 ± 2.30% (caspase 8 and 10, Les-6287, 1.0 μM) and 17.78 ± 5.19% and 20.50 ± 1.49% (caspase 8 and 10, Les-6287, 1.5 μM) of the MCF-7 cell population, respectively, while for MDA-MB-231, an increase in the active form of the caspase 8 and 10 was up to 18.23 ± 0.62% and 14.48 ± 2.53% (Les-6287, 1.0 μM) and 23.50 ± 1.03% and 19.78 ± 0.80% (Les-6287, 1.5 μM), respectively. These observations indicate that the results correspond well with those of the Annexin V/PI assay, demonstrating that the apoptosis process can be induced by an extrinsic pathway mediated by death receptors.

During apoptosis, the fusion of the extrinsic and intrinsic pathways into one common pathway takes place. At this point, the cascade of executioner caspases (caspases 3, 6, 7) begins, and the transition into the final phase of programmed cell death [32]. We assessed the activity of caspase 3/7 in MCF-7 and MDA-MB-231 cells after treatment with tested compounds (Figure 10). The percentage of the MCF-7 cells with active caspase 3/7 reached 21.38 ± 1.50% (Les-6287, 1.0 μM) and 47.08 ± 3.40% (Les-6287, 1.5 μM) of the tested cell population. For the MDA-MB-231 cancer cells, the value oscillated between 17.20 ± 1.58% (Les-6287, 1.0 μM) and 26.63 ± 8.32% (Les-6287, 1.5 μM). Doxorubicin activated caspases 8, 9, 10, and 3/7 in a more pronounced manner than the studied derivatives did (data are not presented). This investigation shows that all previous results were consistent with each other, indicating the ability of Les-6287 to induce apoptosis, and this process follows two pathways.

3.5. Compounds Inhibit Autophagy in Breast Cancer Cells

Due to the correlation between the degree of autophagy and the posttranslational alterations of LC3A/B and the regulation of Beclin-1, we used PCR and ELISA to examine these proteins.

As demonstrated in Figure 11, in MCF-7 and MDA-MB-1 cells treated for 24 h with doxorubicin (1 µM), the expression of both MAP1LC3b and BECN1 genes was significantly increased compared to control cells. In contrast, incubation of MCF-7 and MDA-MB-1 with Les-6287 did not affect MAP1LC3B and BECN1 expression.

Figure 12 demonstrates a dose-dependent inhibitory effect of Les-6287 and the reference drug, doxorubicin, on the concentration of Beclin-1 in MCF-7 and MDA-MB-231 cells. In the untreated control MCF-7 and MDA-MB-231 cells, the concentration of Beclin-1 was 4.08 ng/mL and 11.79 ng/mL, respectively. The most significant decrease in Beclin-1 concentration was observed in doxorubicin—1.89 ng/mL at 2 μM in MCF-7 cells. Additionally, 24 h incubation with Les-6287 resulted in a reduction in Beclin-1 concentration to 2.78 ng/mL at 2 μM in the MCF-7 cells and 9.83 ng/mL in the MDA-MB-231 cells.

As demonstrated in Figure 13, the inhibitory activity towards microtubule-associated protein 1A/1B light chain 3B (LC3B) was observed for all three doses of Les-6287 (1.0, 1.5, and 2.0 μM) in both analyzed breast cancer cell lines. For Les-6287, the concentration of this biomarker of autophagy was reduced from 574.84 ng/mL in the control MCF-7 cells to 537.74 ng/mL. In the MDA-MB-231 breast cancer cells, the concentration of LC3B in the non-treated control was 698.25 ng/mL. Les-6287 at concentrations of 2 μM reduced the level of LC3B to 483.50 ng/mL.

Thus, autophagy is reduced in breast carcinoma MCF-7 and MDA-MB-231 cells under the action of Les-6287.

3.6. Les-6287 Decreases the Concentration of MMP-2, MMP-9, and ICAM-1 Proteins, Which Are Involved in Metastasis and Invasion

MMP-2 is considered a prognostic marker in many carcinomas. Talvensaari-Mattila et al. proved that MMP-2 is associated with survival in breast carcinoma and correlates to shortened survival [33].

An inhibitory effect on MMP-2 was observed in all the tested samples in both examined breast cancer cell lines. As demonstrated in Figure 14, the most significant decrease was observed for Les-6287, from 610.57 pg/mL in untreated MCF-7 control cells to 395.91 pg/mL. The doxorubicin at 2 μM decreased the MMP-2 concentration to 425.40 pg/mL in MCF-7 cells. After incubation with Les-6287 at 2 μM, the concentration of MMP-2 was lowered to 711.74 pg/mL in the MDA-MB-231 breast cancer cells, in comparison with the control, where the MMP-2 concentration was 1055.21 pg/mL. Doxorubicin (2 μM) caused a decline in the MMP-2 concentration to 470.57 pg/mL, respectively.

Additionally, the concentrations of MMP-9 and ICAM-1 were measured in the MDA-MB-231 breast cancer cells after 24 h of incubation with Les-6287, as well as doxorubicin (Figure 15). We found that the MMP-9 and ICAM-1 concentrations in MCF-7 breast cancer cells were below the detection range. In the case of MMP-9, we proved that all examined doses of Les-6287 and doxorubicin decreased MMP-9 concentrations. The most significant decrease in the ICAM-1 concentration (157.25 pg/mL) was observed after 24 h of incubation with Les-6287 (2 µM). The doxorubicin at concentrations of 1.0 and 1.5 µM increased the ICAM-1 concentration, but the highest dose of this reference drug decreased the ICAM-1 concentration (583.04 pg/mL) compared to the control (841.55 pg/mL).

Thus, one can assume that Les-6287 may inhibit the metastasis and invasion of breast carcinoma cells. It should be noted that these processes are key factors in cancer progression and are often associated with a poor prognosis [34,35].

4. Discussion

Heterocyclic compounds represent the chemical basis of >85% of the FDA-approved medicines [36]. Approximately 50% of those heterocycles have a hybrid structure composed of two-parent molecules (pharmacophores) that may affect different pharmacological targets [37]. Such a synergistic action of a hybrid molecule leads to enhanced pharmacological potential. In this study, we used the pyrrolidinedione–thiazolidinone hybrids, namely Les-6287, Les-6294, and Les-6328. Both components of these hybrids demonstrated medicinal effects and are used in various drugs and drug-like substances [38]. Les-6287, Les-6294, and Les-6328 compounds were evaluated for their impact on the metabolic activity and survivability of breast carcinoma cells of MCF-7 and T-47D (ER+, PR+, HER2-), MDA-MB-231 and 4T1 (ER-, PR-, HER2-), and HCC1954 (ER-, PR-, HER2+) lines, and normal breast cells of the MCF-10A line.

The TNBC and receptor-positive breast cancer cell lines under study (MDA-MB-231, 4T1, MCF-7, T-47D, and HCC1954) were found to be sensitive to the toxic action of hybrid heterocyclic molecules (Les-6287, Les-6294, and Les-6328), with the half-maximal inhibitory concentration (IC₅₀) ranging from 1.37 to 21.85 µM depending on the incubation time (24 or 48 h). We have shown that the hydroxy-substituted hybrid compound Les-6287 exhibited the most pronounced cytotoxic activity (24 h) in MCF-7, T-47D, HCC1954, MDA-MB-231, and 4T1 cells with IC₅₀ values of 2.34, 3.11, 2.52, 3.86, and 1.60 µM, respectively, compared to halogen-substituted derivatives Les-6294 with IC₅₀ values of 6.74, 4.48, 4.53, 21.85, and 2.20 μM, and Les-6328 with IC₅₀ values of 3.26, 4.08, 9.91, 6.09, and 2.25 μM, respectively. Both halogen-substituted derivatives, Les-6294 (with chloro-substituent) and Les-6328 (with bromo-substituent), showed almost the same activity. In our opinion, the presence of a hydroxy group in position 4 of the phenyl ring leads to a higher activity level. Thus, breast carcinoma cells of MCF-7, T-47D (ER+, PR+, HER2-), MDA-MB-231, 4T1 (ER-, PR-, HER2-), and HCC1954 (ER-, PR-, HER2+) lines were likewise sensitive to the action of Les-6287 and Les-6328 compounds. Les-6294 demonstrated varying toxicity towards studied breast carcinoma cells at 24 h of treatment and comparable toxicity at the 48 h mark. It should be stressed that the cytotoxic activity of Les-6287, Les-6294, and Les-6328 compounds was significantly higher for studied breast carcinoma cells than for normal human breast epithelial MCF-10A cells (IC₅₀ > 64.58 μM).

The studied derivatives demonstrated selectivity toward the analyzed breast cancer cells, and the selectivity index (SI = IC₅₀ for MCF-10A/IC₅₀ for human breast tumor cells) for Les-6287 was 24.10–47.13, Les-6294—4.57–37.59, and for Les-6328—10.09–50.76. More detailed dose–response studies are being planned to determine the therapeutic windows of the compounds.

In earlier research, Finiuk et al. (2022) discovered that the pyrrolidinedione–thiazolidinone hybrids Les-6287 and Les-6294 inhibited the metabolic activity of the MDA-231, MCF-7, cervical (HeLa), colon (HCT-116), and glioma (U251) cells, as well as of human leukemia (Jurkat) cells. The IC₅₀ value of these derivatives ranged from roughly 1.3 to 9.2 µM [23]. The IC₅₀ values of these derivatives were higher than 73.6 µM for normal human keratinocytes (HaCaT) and 29–67 µM for mitogen-activated lymphocytes collected from the peripheral blood of a healthy donor [24].

One important metric for estimating the anticancer activity of investigated drugs is the colony-forming capacity of tumor cells. The ability of tumor cells to form colonies may also be associated with their potential to metastasize or spread to other parts of the body [26]. The Les-6287 compound at 1 µM effectively inhibited the formation of colonies of the MCF-7, MDA-MB-231, and HCC1954 breast cancer cells at a similar level as the well-known anticancer drug doxorubicin did. In this case, the Les-6294 and Les-6328 derivatives at a 1 µM dose had a weaker effect on the colony-forming ability of breast cells versus the Les-6287. Although at a 2.5–5 µM dose, the Les-6294 and Les-6328 heterocycles fully inhibited the formation of breast cancer colonies. It is important to note that the studied hybrid heterocyclic molecules did not show cytotoxic activity towards normal MCF-10A breast epithelial cells, and the IC₅₀ value of the most effective Les-6287 compound was only 93.01 ± 2.29 µM under 24 h of treatment. In addition, the studied derivatives very weakly inhibited the ability of normal human breast epithelial cells to form colonies, and a significant number of MCF-10A colonies were observed under treatment with Les-6287, Les-6294, and Les-6328 (50 μM).

DNA biosynthesis is a vital process involved in the proliferation of all mammalian cells. An enhanced metabolism of cancer cells causes them to progress more quickly and uncontrollably, leading to the rapid proliferation of diseased tissues. Because of this, substances with anticancer potential should exhibit antiproliferative properties [39]. The Les-6287 compound inhibited DNA synthesis in the MCF-7, MDA-MB-231, and HCC1954 cell lines, with IC₅₀ in the range of 2.32–3.67 µM. A considerably decreased effect of Les-6287 on the suppression of DNA synthesis was observed in the treated MCF-10A cells (IC₅₀ equaled 43.54 µM).

Two cell lines, namely the hormone-receptor-positive MCF-7 cells, which were more susceptible to the chemicals, and triple-negative MDA-MB-231 breast cancer cells, which were more resistant, were used to examine the mechanisms of action of the Les-6287.

It is known that TNBC is refractory to targeted hormone receptor-dependent therapies and demonstrates a more aggressive clinical course [14]. Thus, the revealed cytotoxicity of the Les-6287 compound against MDA-MB-231 cells belonging to the TNBC type displaces the potential of this agent in targeted chemotherapy. In addition, the role of the immune system realized via programmed death ligand 1 (PD-L1) should be taken into consideration. PD-L1 participates in an inhibitory immune checkpoint axis in TNBC [40]. Its expression in the TNBC allows one to propose the application of PD-L1 inhibitors in combination with novel heterocyclic compounds to enhance anticancer chemotherapy through simultaneous targeting of cancer cell viability and the immune microenvironment.

Apoptosis plays a crucial role in the cell death of cancerous cells. A series of caspase activation events during apoptosis facilitate signal transduction and cellular death [41]. A reduction in mitochondrial membrane potential, the release of cytochrome c, and an increase in caspase-9 and caspase-3 activity are all seen in the intrinsic pathway of apoptosis. The early indicator of apoptosis is thought to be the loss of mitochondrial transmembrane potential (ΔΨm). Conversely, executive caspase 3 and caspase 7 are triggered by a signal from activated caspases 8, 9, and 10 in both the intrinsic and extrinsic pathways of apoptosis [42].

Our analysis of caspase-9 activity revealed a marked rise in the activity of this protein under the Les-6287 treatment of the MCF-7 and MDA-MB-231 cells. Caspase-3/7 activity increased in correlation with its rise. Notably, the MCF-7 breast carcinoma cells lack caspase 3 [43], indicating that the activation of caspase 7 is linked to the functioning of the executioner caspases. Thus, Les-6287 induced the intrinsic route of apoptosis. The activation of caspases 8 and 10 takes place in the extrinsic pathway [41]. Les-6287 activated caspases 8 and 10 in MCF-7 and MDA-MB-231 cells. This might indicate that it also caused the extrinsic pathway of programmed cell death. These findings can also be linked to our outcomes, which are that Les-6287 influences the upregulation of active caspase 3/7 in studied cells. Thus, Les-6287 induced the extrinsic and intrinsic apoptotic pathways in breast tumor cells (Figure 16).

We did not reveal significant values of necrosis in breast cancer cells treated with the Les-6287 compound—0.30–0.50% of the necrotic MCF-7 cells and 0.90–4.50% of the necrotic MDA-MB-231 cells, while the doxorubicin-induced necrotic cells were 31.75 and 51.25% in the MCF-7 and MDA-MB-231 lines, respectively.

Autophagy is a process that can be dysregulated in many diseases, including cancer, which plays a dichotomous role by inhibiting or initiating tumor progression. Understanding the molecular mechanism of this self-degradation system is important to developing new targeted therapies. More evidence supports the idea that inhibiting autophagy is a promising strategy to fight advanced cancer [44]. We demonstrated that the tested compound decreased Beclin-1 and LC3B concentrations in MCF-7 and MDA-MB-231 breast cancer cells. Doxorubicin increased the expression of the MAP1LC3B and BECN1 genes and elevated the concentrations of Beclin-1 and LC3B in the MCF-7 and MDA-MB-231 breast cancer cells.

It has been demonstrated that caspase-3 activation inhibits autophagy by cleaving Beclin-1 [45]. Thus, the cleavage of this protein under the action of Les-6287 may have a role in autophagy suppression during apoptosis in breast carcinoma cells.

Similar results were obtained by Buzun et al. (2022), who reported that the Ciminalum–4-thiazolidinone hybrid induced the intrinsic and extrinsic apoptotic pathways (decreased ΔΨm, increase in caspase-9 and caspase-8 concentration) in the MCF-7 and MDA-MB-231 cells. This derivative caused a decrease in concentrations of Beclin-1 and LC3B and inhibited autophagy in both analyzed breast carcinoma cells [22]. Campos et al. (2022) demonstrated that 2-imino-4-thiazolidinones have proapoptotic activity due to a significant stimulation of caspases-3/7 in C6 glioblastoma cells [16].

In many studies, it has been confirmed that matrix metalloproteinases (MMPs) play pivotal roles in metastasis and are associated with survival in several cancers [46]. Matrix metalloproteinase-9 (MMP-9) represents a major member of the MMP family, and its overexpression was confirmed in many human malignant tumors such as gastric, liver, lung, bone, skin, and breast [47]. Jiang and co-authors reported that the overexpression of MMP-2 and MMP-9 in cancer cells was linked to poor survival, lymph node metastasis, and larger tumor size [46]. The concentrations of MMP-2 and MMP-9 in breast cancer cells declined under treatment with the Les-6287, as well as the doxorubicin.

Chen et al. demonstrated that ICAM-1 in TNBC is strongly involved in tumorigenesis and metastasis. Its high expression was found to correlate with a poor prognosis in TNBC. ICAM-1 promotes EMT via integrin-mediated TGF-β/EMT signaling in TNBC by regulating the expression of genes such as MMPs. The authors proved that silencing ICAM-1 expression significantly inhibited bone metastasis in tumor-bearing mice [48]. These findings support the hypothesis that ICAM-1 targeted therapy in triple-negative breast cancer patients is rational.

In our study, we confirmed the inhibitory effect of Les-6287 on the ICAM-1 concentration in the MDA-MB-231 breast cancer cell line belonging to TNBC. The inhibitory effect was almost four times stronger than that of the reference compound—doxorubicin.

The results obtained at the ELISA measurements proved that the Les-6287 compound can decrease the concentration of MMP-2, MMP-9, and ICAM-1 proteins involved in metastasis and poor prognosis in breast cancer patients (Figure 16).

Therefore, our preclinical study indicates the prospect of further research into the hybrid pyrrolidinedione–thiazolidinone molecules as promising cancer-targeted compounds for the treatment of immunohistochemically different subtypes of breast cancer. We plan long-term studies to reveal cell responses for a better understanding of the temporal effects of treatments with these heterocyclic agents. These hybrid molecules will be combined with standard and new chemotherapeutic agents that could enhance efficacy and reduce drug resistance. Further work should include a study of the therapeutic potential and safety profile in vivo of the most effective hybrid heterocyclic derivative, particularly Les-6287, in breast cancer-bearing animals, followed by a detailed study of the pharmacokinetics and pharmacodynamics of this compound in the organisms of treated animals. If animal studies are successful, the most effective hybrid heterocyclic molecule (i.e., Les-6287) might be used as a candidate for the synthesis of conjugates of antibodies with pyrrolidinedione–thiazolidinone agents. The carriers (i.e., biomimetic or nanoscale carriers) or integrating miRNA-based gene therapy could be combined with the antitumor effects of the Les-6287 compound to improve the therapeutic index and reduce off-target effects. In the distant future, personalized medical techniques, such as single-cell sequencing, may be used to identify patients who would benefit the most from such combination therapies.

5. Conclusions

We have shown that novel hybrid pyrrolidinedione–thiazolidinone molecules Les-6287, Les-6294, and Les-6328 possessed cytotoxicity towards human breast carcinoma cells of MCF-7, T-47D, MDA-MB-231, and HCC1954 lines and murine breast cancer 4T1 cells that differ in their genotype (depending on ER, PR, and HER2 expression). They demonstrated low toxicity towards normal human breast cells in MCF-10A. Les-6287 completely prevented MCF-7 (ER+, PR+, HER2-), MDA-MB-231 (ER-, PR-, HER2-), and HCC1954 (ER-, PR-, HER2+) cells from forming colonies at 1 µM; the Les-6294 and Les-6328 compounds did the same at 2.5 or 5 µM doses. Conversely, at 50 µM, none of the investigated derivatives reached the IC₅₀ and only mildly hindered the formation of colonies of normal human breast epithelial MCF-10A cells. Les-6287 caused apoptosis in the MCF-7 and MDA-MB-231 cells. Both an intrinsic pathway—shown as a rise in caspase-9 activity and a decrease in the mitochondrial potential—and an extrinsic pathway with the activation of caspases 8 and 10 are followed in this process. Furthermore, elevated activity of common executioner caspases 3/7 for both apoptotic pathways was found. In addition, the derivative under investigation reduced the concentrations of Beclin-1 and LC3B and hindered autophagy. Les-6287 reduced the concentration of MMP-2, MMP-9, and ICAM-1, all of which are linked to metastasis and a poor prognosis in breast cancer patients. Thus, the developed hybrid pyrrolidinedione–thiazolidinones are promising substances for targeting human breast carcinoma cells. The obtained outcomes serve as the basis for precise research into the mechanisms underlying the therapeutic activity of Les-6287, Les-6294, and Les-6328 in vivo for breast cancer of different subtypes.