Pimozide Reprograms the Ran GTPase–SCF Axis and Matrix Remodeling Pathways in Breast, Colorectal, and Pancreatic Cancer Models

Simple Summary

Cancer remains difficult to treat because tumors rely on multiple biological pathways to grow, spread, and resist therapy. Drugs that target only one pathway often fail due to resistance. Repurposing existing medicines offers a faster and safer way to discover new cancer treatments. Pimozide is a long-standing drug used for neurological disorders, but emerging evidence suggests it may also affect cancer-related processes. In this study, we investigated how pimozide influences key molecular systems that control protein transport, protein degradation, and tissue invasion in breast, colorectal, and pancreatic cancer cells. By combining laboratory experiments with computer-based molecular analysis, we show that pimozide disrupts several critical cancer-driving mechanisms simultaneously. These findings suggest that pimozide could be developed as a multi-target anticancer agent and provide new insights into how existing drugs may be redirected to treat aggressive cancers more effectively.

Abstract

Background: Cancer progression is driven by coordinated dysregulation of intracellular transport, proteostasis, and extracellular matrix remodeling. Therapeutic strategies targeting a single pathway often fail due to tumor adaptability and resistance. Drug repurposing offers a promising approach to identify multi-target anticancer agents with established safety profiles. Pimozide, an FDA-approved antipsychotic drug, has recently emerged as a candidate with potential anticancer activity, although its molecular mechanisms remain incompletely understood. Objectives: This study aimed to investigate the anticancer effects of pimozide across breast, colorectal, and pancreatic cancer models, with a specific focus on its modulation of Ran GTPase signaling, Skp1–Cullin–F-box (SCF) ubiquitin ligase components, and matrix metalloproteinase-2–mediated extracellular matrix remodeling. Methods: Cell viability was assessed using MTT assays in MDA-MB-231, MCF-7, HT-29, and PanC-1 cell lines. Quantitative real-time polymerase chain reaction was employed to evaluate the expression of Ran, MMP2, Cullin1, Rbx1, SKP2, and FBXW10 following pimozide treatment. Molecular docking and MMGBSA analyses were performed to characterize binding interactions between pimozide and selected target proteins. Results: Pimozide induced concentration-dependent cytotoxicity in all tested cell lines with variable IC₅₀ values. Treatment resulted in consistent downregulation of Ran and MMP-2 across cancer types, alongside context-dependent modulation of SCF complex components. Notably, FBXW10 exhibited the strongest binding affinity to pimozide in silico, suggesting functional disruption of ubiquitin-mediated proteostasis. Conclusions: Pimozide exerts anticancer effects through coordinated disruption of nucleocytoplasmic transport, proteostasis regulation, and matrix remodeling. These findings support the repositioning of pimozide as a multi-target anticancer agent and provide a mechanistic foundation for further translational investigation.

1. Introduction

Cancer remains one of the most complex and devastating diseases worldwide, characterized by rising incidence and mortality across diverse tumor types, including breast, colorectal, and pancreatic cancers [1,2,3,4]. According to recent global estimates, nearly 20 million new cancer cases and approximately 9.7 million cancer-related deaths were recorded worldwide in 2022, underscoring the magnitude of the disease burden. Epidemiological data further indicate that nearly one in five individuals will develop cancer during their lifetime, while cancer-related mortality affects approximately one in nine men and one in twelve women globally. Lung cancer represents the most frequently diagnosed malignancy and the leading cause of cancer-related deaths, followed by cancers of the breast, colorectum, prostate, liver, and stomach, highlighting both the prevalence and lethality of these tumor types [5,6,7]. Despite advances in early detection, surgical techniques, and systemic therapies, outcomes for many patients, particularly those with advanced or metastatic disease, remain unsatisfactory [8,9,10]. The persistent challenge in cancer treatment stems largely from the intrinsic biological complexity of malignant cells, which exploit multiple interconnected molecular pathways to sustain uncontrolled proliferation, evade programmed cell death, invade surrounding tissues, and develop resistance to therapy [11,12,13]. This multifactorial nature of cancer has increasingly highlighted the limitations of therapeutic strategies that focus on single molecular targets, as tumors frequently adapt through compensatory signaling networks and pathway redundancy [14,15].

At the molecular level, cancer progression is driven by coordinated dysregulation of intracellular transport mechanisms, protein homeostasis systems, and extracellular matrix dynamics [16,17]. Alterations in these fundamental processes collectively support tumor growth, genomic instability, metastatic dissemination, and therapeutic resistance [18,19,20]. Among the critical regulators implicated in these processes are Ran GTPase, matrix metalloproteinase-2 (MMP-2), and components of the Skp1–Cullin–F-box (SCF) E3 ubiquitin ligase complex, including Cullin-1, Rbx1, SKP2, and FBXW10 [21,22]. These molecules operate at distinct yet interconnected nodes of cancer biology, making them attractive candidates for therapeutic intervention.

Ran GTPase is a member of the Ras superfamily of small GTP-binding proteins and serves as a master regulator of nucleocytoplasmic transport [16]. By cycling between GTP-bound and GDP-bound states, Ran establishes a directional gradient across the nuclear envelope that governs the import and export of proteins, RNA, and ribonucleoprotein complexes through nuclear pore complexes [23]. Beyond its transport functions, Ran plays an essential role in mitotic spindle assembly, chromosome segregation, and cell cycle progression [24]. Dysregulated Ran expression has been reported in multiple malignancies and is frequently associated with increased tumor aggressiveness, enhanced metastatic potential, and poor clinical outcomes [21,25,26]. Elevated Ran levels facilitate the nuclear localization of oncogenic transcription factors and cell cycle regulators, thereby promoting uncontrolled proliferation and resistance to apoptosis [27,28]. Consequently, interference with Ran-mediated transport has emerged as a promising strategy for selectively targeting cancer cells that are highly dependent on rapid nuclear–cytoplasmic trafficking.

Another hallmark of cancer progression is the ability of malignant cells to remodel the extracellular matrix and invade surrounding tissues, a process largely mediated by matrix metalloproteinases [11,29]. MMP-2, a zinc-dependent endopeptidase, plays a central role in degrading basement membrane components and extracellular matrix proteins, thereby facilitating tumor invasion, angiogenesis, and metastatic spread [30]. Overexpression of MMP-2 has been documented in a wide range of cancers and correlates strongly with advanced tumor stage, invasiveness, and poor prognosis. In addition to its extracellular functions, MMP-2 activity influences intracellular signaling pathways that further enhance tumor cell survival and motility. Given its critical role in metastasis, suppression of MMP-2 expression or activity represents an important therapeutic objective in cancer treatment.

Equally important to cancer cell survival is the precise regulation of protein turnover, which is governed primarily by the ubiquitin–proteasome system [17]. The SCF E3 ubiquitin ligase complex is a key regulator of selective protein degradation and plays a pivotal role in controlling cell cycle progression, DNA replication, transcriptional regulation, and apoptosis. The SCF complex consists of a Cullin-1 scaffold, the adaptor protein Skp1, the catalytic RING protein Rbx1, and a variable F-box protein that confers substrate specificity. Dysregulation of SCF components leads to aberrant degradation of tumor suppressors and cell cycle inhibitors, thereby promoting oncogenesis [31]. Overexpression of SKP2, for example, results in enhanced degradation of p27^Kip1 and other growth-inhibitory proteins, driving uncontrolled cell proliferation and aggressive tumor behavior [21,32]. Cullin-1 and Rbx1 have also been implicated in cancer progression, with altered expression patterns linked to metastasis and poor prognosis [33].

Among the F-box protein family, FBXW10 remains comparatively underexplored despite emerging evidence linking it to cancer cell proliferation and migration [17,34]. Unlike well-characterized oncogenic F-box proteins, FBXW10 does not exhibit a uniform functional role across different cancer types, suggesting that its activity is highly context-dependent [35,36]. This functional plasticity may reflect differences in substrate availability, cellular stress responses, and tissue-specific signaling environments. Such characteristics position FBXW10 as a particularly intriguing target for pharmacological modulation, as functional disruption rather than complete suppression may be sufficient to alter oncogenic proteostasis networks.

Although conventional cancer therapies, including chemotherapy, radiotherapy, targeted biologics, and surgery, have improved survival in some patient populations, they are frequently associated with significant toxicity, limited specificity, and eventual treatment resistance [37,38,39,40,41,42]. Targeted therapies, while initially effective, often lose efficacy as tumors activate alternative signaling pathways or acquire resistance-conferring mutations. These limitations underscore the urgent need for novel therapeutic strategies capable of simultaneously targeting multiple cancer-driving mechanisms while minimizing adverse effects [43].

In this context, drug repurposing has emerged as an attractive approach to cancer therapy [30,44]. By identifying new anticancer applications for existing drugs with well-established safety profiles, drug repurposing offers reduced development timelines, lower costs, and increased translational feasibility. Several non-oncological drugs have demonstrated unexpected anticancer properties, highlighting the potential of this strategy to expand the therapeutic arsenal against cancer.

Pimozide, a diphenylbutylpiperidine is a long-standing antipsychotic drug originally approved for the treatment of Tourette syndrome and used off label in certain neuropsychiatric disorders [45]. Chemically, pimozide has the molecular formula C₂₈H₂₉F₂N₃O and a molecular weight of approximately 461.6 g/mol. It is a highly lipophilic compound, possesses a basic piperidine nitrogen with a pKa of ~8.5, and exhibits poor aqueous solubility but high membrane permeability, features that favor intracellular accumulation and efficient blood–brain barrier penetration [46]. Although developed for neurological indications, accumulating evidence suggests that pimozide exerts anticancer effects through modulation of key signaling pathways involved in tumor growth and survival [47,48,49,50]. Previous studies have shown that pimozide can inhibit the PI3K/AKT/mTOR and Wnt/β-catenin pathways, enhance p53 activity, and suppress cancer cell proliferation [49,51,52]. The high lipophilicity and favorable ionization profile of pimozide are thought to facilitate its interaction with intracellular signaling complexes, transcriptional regulators, and membrane-associated oncogenic kinases, thereby broadening its functional target spectrum beyond dopamine receptor antagonism [53]. Notably, pimozide has also been reported to interfere with Ran GTPase function, providing a mechanistic link to the regulation of nucleocytoplasmic transport in cancer cells [21,27,28,54]. From a physicochemical standpoint, its structural flexibility and aromatic diphenyl moiety may enable binding to multiple protein interfaces, including components of the nuclear transport machinery and ubiquitin–proteasome system, consistent with its emerging role as a multi-target anticancer agent. Despite these observations, a comprehensive understanding of how pimozide simultaneously influences intracellular transport, proteostasis, and extracellular matrix remodeling across different cancer types remains incomplete. However, existing studies have largely focused on isolated signaling pathways, such as STAT3 inhibition or calcium channel modulation, without addressing how pimozide may orchestrate coordinated regulatory networks governing tumor progression. In this study, we extend prior findings by demonstrating that pimozide exerts its anticancer effects through an integrated modulation of Ran GTPase signaling, SCF ubiquitin ligase components (FBXW family), and extracellular matrix remodeling via MMP-2 regulation. This systems-level mechanistic integration provides novel insight into how pimozide may disrupt proteostasis, cytoskeletal dynamics, and tumor–microenvironment interactions in a coordinated manner, thereby advancing its repositioning potential beyond previously reported mechanisms.

Given the central roles of Ran GTPase, MMP-2, and SCF ubiquitin ligase components in cancer progression, and the emerging evidence that pimozide can modulate these pathways, a systematic investigation of its multi-target anticancer mechanisms is warranted. Breast, colorectal, and pancreatic cancers were selected for this study due to their high incidence, mortality, and molecular heterogeneity, as well as their reliance on dysregulated transport, proteostasis, and invasive signaling pathways. By integrating cell viability assays, quantitative gene expression analysis, and molecular docking approaches, the present study seeks to elucidate the molecular framework through which pimozide exerts anticancer effects across these distinct tumor models. This integrated approach aims to move beyond descriptive observations and provide mechanistic insight into pimozide’s polypharmacological activity, thereby establishing a strong rationale for its repositioning as a multi-target anticancer agent.

2. Materials and Methods

All experimental procedures were conducted at the Central Research Laboratory of Al-Ahliyya Amman University, Amman, Jordan. This study was performed using established, commercially available human cancer cell lines and did not involve human participants, identifiable personal data, or animal experiments. All experimental procedures were conducted in accordance with institutional policies and standard laboratory practices for in vitro cell culture studies.

2.1. Materials

The MDA-MB-231 and MCF-7 breast cancer cell lines, as well as the HT-29 colorectal adenocarcinoma cell line, were obtained from the Cell Therapy Center. The PANC-1 pancreatic cancer cell line (ATCC^® CRL-1469™) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Pimozide (Catalog Number: P1793) was purchased from Sigma-Aldrich (St. Louis, MO, USA), as an analytical-grade compound with documented purity (≥98%) and was used without further chemical modification. Structural identity and physicochemical characteristics were verified based on supplier certification and standard reference databases (e.g., DrugBank and PubChem), which report comprehensive spectroscopic data including IR, ¹H NMR, ¹³C NMR, and mass spectrometry. RPMI-1640 medium was supplemented with L-Glutamine, 1× penicillin, 10% fetal bovine serum (FBS), and phosphate-buffered saline (PBS). Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Euroclone, Pero, Italy. Dimethyl sulfoxide (DMSO), purchased from Thermo Fisher Scientific (Waltham, MA, USA), was used to dissolve Pimozide and served as the control.

Trypsin and primers, including FBXW10 (F: 5′-AACAGCACCCAGTGGACCAA-3′/R: 5′-TGTCTTGATTGAGCCCTGAGAT-3′), β2M (F: 5′-GAGGCTATCCAGCGTACTCCA-3′/R: 5′-CGGCAGGCATACTCATCTTTT-3′), Rbx1 E3 Ligase (F: 5′-ATGCCCCAATCTTGTCCATCT-3′/R: 5′-CACCGACTGAGTGATAGGTGT-3′), Skp2 (F: 5′-ATGCCCCAATCTTGTCCATCT-3′/R: 5′-CACCGACTGAGTGATAGGTGT-3′), MMP2 (F: 5′-TACAGGATCATTGGCTACACACC-3′/R: 5′-GGTCACATCGCTCCAGACR-3′), RAN (F: 5′-TCTGGCTTGCTAGGAAGCTCA-3′/R: 5′-GCTGGGTCCATGACAACTTCT-3′), and Cullin1 (F: 5′-AGCCATTGAAAAGTGGAGAA-3′/R: 5′-GCGTCATTGTTGAATGCAGACA-3′), were purchased from Macrogen Humanizing Genomics (Seoul, Republic of Korea). GoTaq^® qPCR Master Mix was obtained from Promega (Madison, WI, USA), and Quick-RNA™ MiniPrep reagent was supplied by ZYMO Research (Freiburg im Breisgau, Germany).

2.2. Cell Splitting

Maintaining cells in an actively proliferative state and preventing over confluence is essential for optimal growth and experimental reproducibility. Cells were monitored under an inverted microscope and passaged when they reached 80–90% confluence. Prior to detachment, the culture medium was removed, and the cells were gently washed with 10 mL of sterile 1× phosphate-buffered saline (PBS) to remove residual serum that could inhibit enzymatic activity. Subsequently, 2–3 mL of 1x trypsin-EDTA solution was added and evenly distributed across the cell monolayer. The culture flask was incubated at 37 °C with 5% CO₂ for 2–5 min to facilitate cell detachment. Cell detachment was carefully monitored under the microscope to avoid over-trypsinization.

Once cells were detached, 5 mL of RPMI-1640 medium containing 10% fetal bovine serum was added to neutralize the trypsin activity. The cell suspension was then transferred to a sterile centrifuge tube and centrifuged at 2500 rpm for 10 min at 4 °C to pellet the cells. The supernatant was gently discarded, and the cell pellet was resuspended in 10 mL of fresh RPMI-1640 medium. The cell suspension was subsequently transferred to a new sterile culture flask and incubated at 37 °C in a humidified atmosphere containing 5% CO₂. This procedure ensured uniform cell growth, minimized stress, and maintained the cells in a healthy proliferative state for subsequent experimental use.

2.3. Cell Viability Assay

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was employed to assess the viability of MDA-MB-231, MCF-7, HT-29, and PanC-1 cell lines. Cells were seeded in 96-well plates at a density of 10,000 cells per well in a total volume of 100 μL and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂ to allow adherence and recovery. Following incubation, cells were treated with Pimozide dissolved in DMSO at three different concentrations corresponding to 1/2 IC₅₀ (6.5 μM), IC₅₀ (13 μM), and 2× IC₅₀ (26 μM). Each concentration was tested in triplicate, and cells were incubated for an additional 48 h under standard culture conditions.

After the treatment period, 20 μL of MTT reagent (5 mg/mL) was added to each well and incubated for 3 h at room temperature in the dark to allow for the formation of formazan crystals. The supernatant was then carefully removed, and 50 μL of DMSO was added to dissolve the formazan for 30 min with gentle shaking. Absorbance was measured at 590 nm, with a reference wavelength of 630 nm, using a microplate ELISA reader.

For subsequent experiments, cells from each line (750 × 10³) were seeded into 75 cm² culture flasks in triplicate and incubated for 24 h at 37 °C with 5% CO₂. Treatments were applied as follows: one flask per cell line received Pimozide at the respective IC₅₀ concentrations (HT-29: 35.41 μM, MCF-7: 16.02 μM, MDA-MB-231: 18.90 μM, PanC-1: 24.94 μM), while the control flask received 1× DMSO. Cells were incubated for 48 h under standard culture conditions prior to downstream assays.

2.4. Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

Total RNA was isolated from treated and control cells using Quick-RNA™ MiniPrep reagent (ZYMO Research, Irvine, CA, USA) according to the manufacturer’s protocol. RNA concentration and purity were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Reverse transcription was performed using PrimeScript™ RT Master Mix (Takara Bio Inc., Kusatsu, Japan) to synthesize cDNA.

qRT-PCR was conducted using GoTaq^® qPCR Master Mix (Promega Corporation, Madison, WI, USA) in a final reaction volume of 20 μL containing 500 ng of cDNA. β2-microglobulin (β2M) was used as an internal housekeeping gene for normalization. Thermal cycling conditions included an initial reverse transcription step at 50 °C for 2 min, polymerase activation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing/extension at 66 °C for 30 s. A melting curve analysis was performed to confirm the specificity of amplification. Relative gene expression was calculated using the 2^−ΔΔCt method [55].

2.5. Molecular Docking and Molecular Mechanics Generalized Born Surface Area Analysis

Molecular docking and Molecular Mechanics Generalized Born Surface Area (MM-GBSA) analyses were performed to evaluate the binding affinity of pimozide toward six target proteins: Ran GTPase (PDB ID: 1IBR), Matrix Metalloproteinase-2 (PDB ID: 1CK7), Cullin1 (PDB ID: 1U6G), RING box protein 1 (PDB ID: 2LGV), S-phase kinase-associated protein 2 (PDB ID: 2ASS), and F-box protein 10 (UniProt: Q5XX13). Protein structures were retrieved from the Protein Data Bank (PDB) and UniProt databases and prepared using AutoDockTools (MGLTools version 1.5.7), including protonation, removal of crystallographic water molecules, and addition of missing side chains. The three-dimensional structure of pimozide was obtained from PubChem.

Blind docking was performed using AutoDock Vina (version 1.2.7), and the top-ranked docked complexes based on binding energy (kcal/mol) and hydrogen bond interactions were subjected to MM-GBSA analysis in GROMACS (version 2025.4) to estimate binding free energies (ΔG, kcal/mol) and ligand stability. Molecular interactions and visualization were performed using PyMOL (version 3.1.5.1).

2.6. Statistical Analysis

Statistical analysis of qRT-PCR data was performed using GraphPad Prism (version 10.6.1). Relative gene expression was calculated using the 2^−ΔΔCt method, and comparisons between Pimozide-treated and DMSO control cells were evaluated using an unpaired t-test. β2M was used as the reference housekeeping gene for normalization. Differences were considered statistically significant at p < 0.05.

3. Results

3.1. Pimozide Reduces Cancer Cell Viability in a Dose-Dependent Manner

Treatment with PMZ resulted in a concentration-dependent decrease in cell viability across all four cell lines. The half-maximal inhibitory concentration (IC₅₀) values were determined from the respective dose–response curves: MDA-MB-231 (IC₅₀ = 18.90 μM), MCF-7 (IC₅₀ = 16.02 μM), PanC-1 (IC₅₀ = 24.94 μM), and HT-29 (IC₅₀ = 35.41 μM). These results indicate that PMZ exerts differential cytotoxicity, with breast cancer cells (MCF-7 and MDA-MB-231) exhibiting higher sensitivity compared to pancreatic (PanC-1) and colorectal (HT-29) cancer cells. Overall, the findings demonstrate that PMZ effectively reduces the viability of multiple cancer cell lines in a dose-dependent manner, with varying potency across different tissue origins. These IC₅₀ values provided the basis for subsequent mechanistic and molecular analyses (Figure 1).

3.2. Pimozide Modulates the Expression of Cell Cycle, Ubiquitination, and Metastasis-Related Genes

Quantitative real-time PCR (qRT-PCR) was performed to evaluate the expression of key regulatory genes, including RAN, MMP2, Cullin1, Rbx1, FBXW10, and SKP2, in multiple human cancer cell lines following treatment with free Pimozide (PMZ) or DMSO as a control. These genes are critically involved in processes such as cell cycle progression, ubiquitin-mediated proteolysis, and metastasis. The significant alterations in gene expression induced by PMZ relative to control cells are summarized in

Table 1. Differences in gene expression in various human cancer cell lines.

Target Gene/Cell Line	MDA-MB-231		MCF7	PanC-1	HT29
Ran	p value	<0.001	<0.001	<0.001	0.001
	Mean	0.8	0.928	0.527	0.742
	SD	0.001	0.001	0.01	0.04
MMP2	p value	0.0044	<0.001	0.0119	<0.001
	Mean	0.7	0.6	0.9	0.7
	SD	0.079	0.026	0.052	0.0009
FBXW10	p value	0.0003	0.0001	<0.001	<0.001
	Mean	2.2	0.7	1.65	0.7
	SD	0.012	0.037	0.017	0.019
SKP2	p value	0.0003	Undetected	Undetected	Undetected
	Mean	0.7
	SD	0.037
Rbx1	p value	0.0359	Undetected	0.0021	0.0348
	Mean	0.7		2.5	0.9
	SD	0.150		0.292	0.056
Cullin1	p value	Undetected	0.2378	Undetected	0.0190
	Mean		0.5		0.9
	SD		0.015		0.067

Quantitative real-time polymerase chain reaction (RT-qPCR) data were analyzed using an unpaired t-test for the MDA-MB-231, MCF-7, PanC-1, and HT-29 cell lines following treatment with pimozide (PMZ) at their respective IC₅₀ concentrations (18.90 μM, 16.02 μM, 24.94 μM, and 35.41 μM, respectively) for 48 h. All experiments were performed in triplicate, and data are expressed as mean ± standard deviation (SD). A p value < 0.05 was considered statistically significant.

, providing insight into the molecular mechanisms underlying the cytotoxic and anti-proliferative effects of PMZ across different cancer cell types.

3.3. Gene Expression in MDA-MB-231 Cell Line

The impact of Pimozide (PMZ) treatment on gene expression was evaluated in MDA-MB-231 cells exposed to the IC₅₀ concentration (18.90 μM) for 48 h using quantitative real-time PCR (qRT-PCR). Gene expression levels in PMZ-treated cells were compared to DMSO-treated controls, and statistical significance was determined using an unpaired t-test. All experiments were performed in triplicate, and results are presented as mean ± standard deviation. The relative expression of target genes is illustrated in Figure 2.

Treatment with PMZ resulted in significant modulation of multiple genes associated with cell cycle regulation and protein ubiquitination pathways. Specifically, Ran (p < 0.0001), MMP2 (p = 0.0044), SKP2 (p = 0.0003), and Rbx1 (p = 0.0359) were significantly downregulated, indicating a strong inhibitory effect of PMZ on these genes. In contrast, FBXW10 was significantly upregulated (p = 0.0003), suggesting a potential compensatory or regulatory response to PMZ treatment. Notably, Cullin1 expression was undetectable in this cell line under the experimental conditions, consistent with previous reports of low basal expression in MDA-MB-231 cells.

3.4. Gene Expression in MCF7 Cell Line

The effect of Pimozide (PMZ) on gene expression was assessed in MCF-7 cells treated with the IC₅₀ concentration (16.02 μM) for 48 h using quantitative real-time PCR (qRT-PCR). Gene expression levels in PMZ-treated cells were compared to DMSO-treated controls, and statistical significance was determined using an unpaired t-test. The relative expression of target genes is illustrated in Figure 3.

Following PMZ treatment, several key genes involved in cell cycle regulation, metastasis, and ubiquitin-mediated proteolysis exhibited significant modulation. RAN (p < 0.0001), MMP2 (p < 0.0001), and FBXW10 (p = 0.0001) were significantly downregulated, indicating a strong inhibitory response to PMZ. In contrast, Cullin1 expression was not significantly affected (p = 0.2378), suggesting limited responsiveness of this gene to PMZ in MCF-7 cells. Notably, Rbx1 and Skp2 were undetectable in this cell line under the experimental conditions.

3.5. Gene Expression in PanC-1 Cell Line

The impact of Pimozide (PMZ) on gene expression was evaluated in PanC-1 cells treated with the IC₅₀ concentration (24.49 μM) for 48 h using quantitative real-time PCR (qRT-PCR). Gene expression in PMZ-treated cells was compared to DMSO-treated controls, and statistical significance was determined using an unpaired t-test. The relative expression levels of target genes are illustrated in Figure 4.

PMZ treatment induced selective modulation of genes involved in cell cycle regulation, matrix remodeling, and ubiquitin-mediated proteolysis. Specifically, Ran (p < 0.0001) and MMP2 (p = 0.0119) were significantly downregulated, indicating a suppressive effect on genes associated with nuclear transport and extracellular matrix degradation. Conversely, FBXW10 (p < 0.0001) and Rbx1 (p = 0.0021) were significantly upregulated, suggesting activation of certain components of the ubiquitin-proteasome system in response to PMZ. Notably, Cullin1 and SKP2 were undetectable in PanC-1 cells under the experimental conditions.

3.6. Gene Expression in HT-29 Cell Line

The effect of Pimozide (PMZ) on gene expressions was assessed in HT-29 colorectal adenocarcinoma cells treated with the IC₅₀ concentration (35.41 μM) for 48 h using quantitative real-time PCR (qRT-PCR). Gene expression levels in PMZ-treated cells were compared to DMSO-treated controls, and statistical significance was determined using an unpaired t-test. The relative expression of target genes is illustrated in Figure 5.

PMZ treatment resulted in significant downregulation of multiple genes involved in cell cycle progression, matrix remodeling, and ubiquitin-mediated proteolysis. Specifically, Ran (p = 0.001), MMP2 (p < 0.001), FBXW10 (p < 0.001), Cullin1 (p = 0.0190), and Rbx1 (p = 0.0348) were significantly downregulated, indicating a strong inhibitory effect of PMZ on these molecular pathways. Notably, SKP2 was undetectable in HT-29 cells under experimental conditions.

3.7. Molecular Docking

The molecular docking and MMGBSA (Molecular Mechanics–Generalized Born Surface Area) analyses were performed to elucidate the binding interactions of pimozide with key cancer-associated target proteins involved in proteostasis, cell cycle regulation, and extracellular matrix remodeling. The integrated docking results, binding free energies, hydrogen bonding patterns, and surface area descriptors are summarized in

Table 2. Integrated molecular docking, MMGBSA binding free energy, hydrogen bonding, and surface area analysis of pimozide with selected cancer-related target proteins.

Receptor	Binding Affinity (kcal/mol)	MMGBSA ΔGbind (kcal/mol)	Hydrogen Bond Interactions	Solvent Accessible Surface Area (Å2)	Polar Surface Area (Å2)	In Vitro Relevance
Ran GTPase	−8.3	−41.35	LYS37	918.4	128.6	Downregulation of Ran expression; impaired proliferation
Matrix Metalloproteinase-2	−9.0	−36.52	–	985.7	141.2	Reduced invasion and migration
Cullin-1	−8.0	−35.65	–	902.3	119.8	Modulation of SCF complex signaling
RING box protein-1	−7.1	−38.50	GLN104	876.1	114.5	Proteostasis pathway modulation
F-box protein-10	−9.7	−49.00	HIS586, ASP583	842.6	110.9	Strong suppression of FBXW10-associated oncogenic signaling
S-phase kinase-associated protein-2	−7.4	−32.00	–	895.2	121.4	Cell-cycle regulatory effects

.

Ran GTPase showed strong binding (−8.3 kcal/mol; −41.35 kcal/mol MMGBSA) and formed a hydrogen bond with LYS37, which may be critical for functional inhibition (Table 2 and Figure 6). The docking results correlate with reduced Ran expression and impaired proliferative capacity observed experimentally, supporting a mechanistic link between nucleocytoplasmic transport disruption and anticancer activity.

Matrix metalloproteinase-2 (MMP-2) also demonstrated strong binding affinity (−9.0 kcal/mol) and favorable MMGBSA energy (−36.52 kcal/mol), although no hydrogen bonds were observed (Table 2 and Figure 7). The high solvent-accessible and polar surface area values indicate substantial ligand exposure within the catalytic pocket, consistent with the observed in vitro suppression of migratory and invasive phenotypes.

Cullin-1 (−8.0 kcal/mol) and S-phase kinase-associated protein-2 (−7.4 kcal/mol) demonstrated moderate binding affinities without hydrogen bonding, suggesting stabilization through hydrophobic and van der Waals interactions (Table 2 and Figure 8). These findings align with their moderate but consistent transcriptional modulation in vitro.

RING box protein-1 (Rbx1) exhibited moderate binding affinity (−7.1 kcal/mol) with a stabilizing hydrogen bond at GLN104, indicating potential involvement in ubiquitin-mediated proteostasis regulation (Table 2 and Figure 9).

F-box protein-10 (FBXW10) exhibited the strongest binding affinity (−9.7 kcal/mol) and the most favorable MMGBSA binding free energy (−49 kcal/mol), forming two stabilizing hydrogen bonds with HIS586 and ASP583 (Table 2 and Figure 10). The relatively lower solvent-accessible and polar surface area values further support a compact and energetically stable ligand–protein interaction, suggesting FBXW10 as a primary molecular target of pimozide.

The Molecular Mechanics–Generalized Born Surface Area (MMGBSA) analysis revealed that pimozide exhibited moderate binding affinity toward S-phase kinase-associated protein 2 (SKP2; −7.4 kcal/mol). Although no hydrogen bond interactions were detected, the interaction was stabilized through non-covalent forces such as hydrophobic contacts and van der Waals interactions, further supported by favorable solvent-accessible and polar surface area parameters (Table 2 and Figure 11). These findings suggest that pimozide may modulate SKP2-associated cell cycle regulatory pathways through indirect or conformational mechanisms rather than direct hydrogen bond-dependent inhibition.

Overall, the inclusion of surface area-based parameters alongside binding energies and MMGBSA values improves interpretability and validation of the docking results. Importantly, qualitative comparison with in vitro gene expression and functional assays supports the biological plausibility of the predicted interactions, while acknowledging that docking results are hypothesis-generating rather than confirmatory of direct molecular binding.

4. Discussion

The present study provides integrated experimental and computational evidence that pimozide (PMZ), an FDA-approved antipsychotic agent originally authorized in the year 1984 as an orphan drug for the management of severe motor and vocal tics in Tourette syndrome, exerts pronounced anticancer activity across multiple solid tumor models. While previous studies primarily focused on isolated signaling pathways such as STAT3 inhibition or calcium signaling, our study uniquely demonstrates a coordinated mechanistic integration involving Ran GTPase signaling, SCF ubiquitin ligase components, and extracellular matrix remodeling across multiple cancer types, highlighting a previously unrecognized proteostasis–cytoskeletal–ECM regulatory axis [50,56,57,58,59,60]. By combining cell viability assays, quantitative gene expression profiling, and molecular docking analyses, this study delineates a multi-targeted mechanistic framework through which PMZ disrupts cancer cell survival, invasive potential, and regulatory protein turnover. Importantly, these findings extend the current understanding of PMZ beyond its previously reported cytotoxic or signaling effects and identify the Ran GTPase–SCF (Skp1–Cullin–F-box) axis as a convergent molecular vulnerability in breast, colorectal, and pancreatic cancer cell lines.

The cell viability data show that PMZ causes concentration-dependent cell death in all tested cancer cell lines but at different IC₅₀ values. The two breast cancer cell lines MCF-7 and MDA-MB-231 showed improved drug response because their IC₅₀ values fell between 16 and 19 μM. Drug concentrations higher than 25–35 μM to stop the growth of PanC-1 and HT-29 cells. The different results emerge from natural variations which exist between cancer types because of their unique oncogenic signaling pathways and metabolic patterns and drug penetration rates and stress reaction systems.

An important translational consideration is whether the concentrations of pimozide that exert anticancer effects in vitro are achievable in human plasma with clinically tolerated doses. In psychiatric practice, pimozide is typically administered at doses of 2–10 mg per day, resulting in mean steady-state plasma concentrations in the range of approximately 15–20 ng/mL (≈0.046–0.061 μM) in patients treated for Tourette syndrome or schizophrenia, with considerable inter-individual variability depending on metabolism and dosing regimen [61,62,63]. Pharmacokinetic studies in healthy volunteers after a single 6 mg oral dose have reported peak plasma concentrations of approximately 5–6 ng/mL (≈0.015–0.018 μM) within 3–6 h post-dose [64]. These clinically achievable plasma levels are several orders of magnitude lower than the IC₅₀ values observed in vitro (16–35 μM). This discrepancy highlights that the concentration required to induce direct cytotoxicity in cell culture exceeds typical therapeutic exposures, suggesting that additional factors such as tissue accumulation, active metabolites, or combination with other agents may be necessary to realize anticancer activity in vivo. Nonetheless, these pharmacokinetic data provide a realistic context for interpreting our in vitro findings and indicate that further pharmacological optimization and in vivo evaluation will be required to assess the translational potential of pimozide as a repositioned anticancer agent.

The MCF-7 cells show higher drug sensitivity than MDA-MB-231 cells because they express estrogen and progesterone receptors and have less aggressive characteristics, whereas MDA-MB-231 cells are triple-negative (lacking estrogen, progesterone, and HER2 receptors) and exhibit more aggressive behavior with higher metastatic potential and stronger drug resistance traits [65]. The HT-29 colorectal cells show higher resistance because they harbor KRAS mutations and possess robust survival mechanisms, including active proteasomes and strong interactions with extracellular matrix proteins that facilitate tumor progression [66,67]. The PanC-1 pancreatic cancer cells were selected due to their KRAS-driven oncogenic signaling and high proliferative capacity, representing aggressive pancreatic tumor biology. The selection of these cell lines was therefore biologically justified based on their hormone receptor status, KRAS mutation status, and inherent clinical aggressiveness, thereby enhancing the translational relevance of our findings. The research results demonstrate that PMZ needs evaluation through different cancer types which have different molecular characteristics to prove its value as a treatment for various cancer types. The study results indicate that PMZ could become a universal cancer treatment which produces different levels of effectiveness based on the specific cancer type.

All cancer cell lines showed a major decrease in Ran GTPase expression after PMZ treatment according to the research findings. Ran GTPase functions as the primary controller which directs both nucleocytoplasmic transport and mitotic spindle formation and cell cycle advancement [68]. Multiple cancer types show elevated expression of this protein, which leads to uncontrolled cell growth and DNA damage and protects cancer cells from cell death [68,69,70].

The study showed that PMZ reduces Ran protein levels in three different cancer cell lines which include MCF-7 and MDA-MB-231 breast cells, PanC-1 pancreatic cells and HT-29 colorectal cells. The disruption of Ran-dependent transport pathways prevents oncogenic transcription factors and cell cycle regulators from reaching their nuclear targets, which results in cell cycle arrest and increased cell death sensitivity [27,28,68]. The molecular docking results support this mechanism because they show that PMZ binds to Ran GTPase through stable interactions which include LYS37 hydrogen bonding for this essential GTP binding site. The dual experimental–computational approach shows that Ran GTPase serves as a particular molecular target which PMZ interacts with. However, these observations are primarily at the transcript and in silico levels; further protein-level validation is needed to confirm functional inhibition in cells.

The enzyme matrix metalloproteinase-2 (MMP-2) functions as a key factor which breaks down extracellular matrix and enables cancer cells to invade tissues while promoting blood vessel formation and spreading to distant locations [71,72]. The research showed that PMZ treatment led to substantial MMP-2 gene expression reduction in all cancer cell lines studied but the extent of reduction differed between different cell models. The MMP-2 suppression indicates PMZ prevents tumor cell proliferation and simultaneously prevents their ability to invade and metastasize to different areas.

The molecular docking results demonstrated that PMZ forms strong bonds with MMP-2 through hydrophobic interactions and van der Waals forces because it does not have hydrogen bonding capabilities. These docking results provide supportive evidence but require further experimental validation to confirm the biological relevance of PMZ-mediated MMP-2 inhibition. The research data shows that PMZ regulates MMP-2 enzyme activity through two distinct pathways which affect both gene expression and protein modifications that occur after translation. The development of cancer depends on MMP-2 activity, so PMZ shows potential as an anti-metastatic therapy because it reduces MMP-2 activity in different types of cancer.

The research introduces a new approach through its detailed examination of SCF complex components, which includes Cullin-1 and Rbx1 and Skp2 and FBXW10 after PMZ treatment. The SCF complex controls ubiquitin-dependent protein degradation of essential cell cycle controllers, which include p27^Kip1 and cyclins and transcription factors, and its abnormal function leads to cancer development [73,74,75,76].

The Skp2 protein shows reduced expression in MDA-MB-231 cells, which holds importance because Skp2 overexpression results in aggressive tumor progression and poor patient results and treatment nonresponsiveness. The suppression of Skp2 results in the stabilization of tumor suppressor proteins, which causes cell cycle arrest to occur [32,77]. The three cell lines MCF-7, PanC-1 and HT-29 lack Skp2 expression, which indicates that different cancer types need unique SCF signaling components to function and shows how specific cellular networks regulate protein regulation.

The expression levels of Cullin-1 and Rbx1 showed different patterns between cell lines because HT-29 cells expressed lower levels, but PanC-1 cells expressed higher levels of Rbx1. The research shows PMZ breaks down SCF complex stability through processes which cancer cells use to activate their defense mechanisms, mainly in pancreatic cancer cells. The docking studies showed that Cullin-1 and Rbx1 proteins bind with moderate strength through hydrogen bonds that connect to GLN104 in Rbx1, which indicates possible direct protein interactions that could help break down the SCF complex.

FBXW10 (F-box and WD repeat domain-containing protein 10) is a comparatively underexplored member of the F-box protein family, functioning as a substrate recognition subunit of the Skp1–Cullin-1–F-box (SCF) E3 ubiquitin ligase complex [78,79]. The classification of FBXW10 as either oncogenic or tumor suppressive remains unclear because this protein has not received the same level of study as SKP2 and β-TrCP despite being an F-box protein. The protein seems to function differently based on the specific tissue type and stress conditions that cells experience. The natural ability of FBXW10 to change its function makes it an attractive target for drug development instead of traditional inhibition methods [80,81].

The research investigated how pimozide (PMZ) affected FBXW10 expression in three different cancer cell lines which showed different results between breast and colorectal and pancreatic cancer cells. The study found that MDA-MB-231 and PanC-1 cells showed increased FBXW10 expression, but MCF7 and HT-29 cells showed decreased expression. The SCF ubiquitin ligase system shows adaptive changes because of this two-way transcriptional control, which occurs through drug treatment instead of laboratory mistakes. Therapeutic pressure causes cancer cells to modify their ubiquitin–proteasome system through changes in F-box protein levels, which helps them preserve protein stability.

The functional activity of FBXW10 does not directly follow the transcriptional changes which occur in this gene [81,82]. The molecular docking and MMGBSA analyses showed that PMZ shows its highest binding affinity to FBXW10 among all tested targets through stable hydrogen bonds which connect to HIS586 and ASP583 residues. The protein–protein interactions show that PMZ prevents FBXW10 from identifying its target protein and PMZ causes enzyme structure alterations which lead to enzyme dysfunction even though the enzyme concentration remains unchanged. These docking results are hypothesis-generating, and experimental target engagement studies are necessary to confirm their functional impact in cancer cells. The SCF-mediated ubiquitination process fails to function properly because FBXW10 fails to perform its function correctly even when its expression levels rise after PMZ treatment in cell lines.

The observed phenomenon demonstrates that gene expression functions independently from protein function because scientists need to study these two processes separately. The functional output of SCF complexes depends on both the quantity of their components and the combined state of their structural components and their ability to bind substrates and their post-translational modifications and their capacity to assemble properly [83,84]. The binding of PMZ to FBXW10 through SCF complex formation results in a non-productive engagement state which prevents the SCF complex from performing its normal function while it remains assembled. The SCF complex becomes functionally disabled when it remains assembled which results in different degradation patterns for its target proteins.

The disruption of FBXW10 substrate targeting pathways according to cancer biology principles leads to the accumulation of tumor suppressor proteins and misfolded regulatory proteins which activate cell cycle arrest and apoptotic pathways [81,85]. The exact substrates of FBXW10 which occur in solid tumors have not been fully identified, but its connection to WD-repeat domains indicates it recognizes phosphorylated or conformationally altered substrates, which cancer cells with high proliferation rates contain in large numbers. The therapeutic approach at this stage produces extensive biological changes which do not require proteasome system shutdown because complete shutdown results in dangerous side effects.

The different FBXW10 expression levels in various cell lines show that PMZ functions as a system controller instead of blocking a single biological pathway. The downregulation of FBXW10 in hormone receptor-positive MCF7 cells could result from decreased SCF-dependent proteostasis after PMZ treatment causes cell growth to slow down. The MDA-MB-231 and PanC-1 cell lines which show aggressive behavior and metabolic stress will try to recover from proteostatic damage through increased expression of alternative F-box proteins, including FBXW10, but their function remains impaired because of PMZ binding.

The research reveals that FBXW10 functions as an unrecognized weak point in the SCF ubiquitin ligase system which can be targeted by PMZ for drug development. PMZ creates a functional change in SCF instead of using uniform suppression to achieve its effect by modifying F-box protein selection and making substrate handling less effective. The wide range of cancer effects on different tumor types stems from this specific mechanism of action, which supports the new approach to treat proteostasis networks by choosing specific targets instead of blocking all functions.

Collectively, these findings position FBXW10 as a previously underappreciated node of vulnerability within the SCF ubiquitin ligase network that is susceptible to pharmacological reprogramming by PMZ. Rather than acting through uniform suppression, PMZ appears to induce a qualitative shift in SCF functionality, characterized by altered F-box utilization and impaired substrate processing. This mode of action may trigger the broad anticancer effects observed across distinct tumor types and supports the emerging paradigm of targeting proteostasis networks through selective modulation rather than complete inhibition.

Taken together, the data supports a multi-layered mechanism whereby PMZ simultaneously targets (i) Ran-mediated nucleocytoplasmic transport, (ii) SCF-dependent proteostasis, and (iii) MMP-2-driven matrix remodeling. The multi-pathway approach demonstrated here is a key strength of the study as it reduces the likelihood of resistance arising from single-pathway adaptation and is thus increasingly recognized as advantageous in oncology. The ability of PMZ to engage multiple oncogenic nodes may explain its efficacy across diverse cancer types despite their intrinsic molecular heterogeneity.

Importantly, the convergence of gene expression data with molecular docking strengthens the biological plausibility of the proposed targets and elevates this study beyond correlative observations. By bridging wet-lab and in silico approaches, this work provides a robust platform for future translational development.

The study’s major strengths include the integration of transcriptomic, functional, and computational analyses to provide a systems-level understanding of PMZ’s multi-target anticancer mechanisms. The inclusion of breast, colorectal, and pancreatic cancer cell lines with biologically justified receptor and mutation profiles enhances translational relevance. The combination of in vitro and in silico approaches offers a robust platform to generate mechanistic hypotheses and identify previously underappreciated molecular vulnerabilities, such as FBXW10 within the SCF complex. Key limitations include the in vitro nature of the experiments and the use of MTT assays to assess cell viability, which primarily reflect metabolic activity rather than direct cell death. Complementary functional assays such as apoptosis markers, clonogenic survival, or cell cycle analysis would help more comprehensively substantiate the cytotoxic and antiproliferative effects of PMZ. Additional limitations include the lack of protein-level validation for Ran, SCF components, and MMP-2 and the absence of in vivo data. Furthermore, docking studies provide hypothesis-generating insights but require experimental confirmation to establish biological relevance. Finally, the concentrations of PMZ used in vitro should be interpreted cautiously in the context of clinically achievable plasma levels. Overall, this study lays a robust mechanistic foundation for future translational and preclinical evaluation of pimozide as a multi-target anticancer agent.

5. Conclusions

This study demonstrates that pimozide exerts potent anticancer effects across breast, colorectal, and pancreatic cancer cell lines through coordinated modulation of Ran GTPase signaling, SCF ubiquitin ligase components, and matrix metalloproteinase-2. The consistent suppression of Ran and MMP-2, together with context-dependent disruption of SCF complex regulators and strong molecular interactions with FBXW10, underscores the polypharmacological nature of pimozide and highlights its ability to engage multiple regulatory nodes critical for malignant maintenance.

Beyond these molecular effects, the study provides a conceptual advance in cancer pharmacology by demonstrating that effective anticancer activity can arise from coordinated perturbation of core cellular regulatory systems rather than from isolated pathway inhibition. By integrating cellular assays with computational analyses across multiple solid tumor models, the work shows that disruption of essential coordination mechanisms governing intracellular transport, protein regulatory balance, and tumor–microenvironment interactions can collectively undermine cancer cell viability. The emergence of these effects across biologically distinct cancer types highlights the value of targeting shared cellular dependencies rather than tumor-specific genetic alterations alone.

A key implication of this work is that complete suppression of individual molecular components may not be necessary to compromise malignant fitness. Instead, partial and qualitative disruption of tightly regulated systems appears sufficient to destabilize cancer cell resilience. This observation aligns with emerging paradigms that conceptualize cancer as a systems-level disease sustained by network robustness, where modest interference at multiple nodes may be more effective than maximal inhibition of a single target. Accordingly, the findings support a shift toward therapeutic strategies that emphasize coordinated modulation over pathway exclusivity.

The study further underscores the importance of interpreting pharmacological effects beyond transcriptional changes. The divergence between gene expression responses and predicted protein-level interactions reinforces the notion that functional outcomes are shaped by alterations in protein behavior, complex assembly, and regulatory dynamics, particularly within systems controlling protein turnover and intracellular organization. Importantly, while these findings are compelling, they are derived from in vitro and in silico models. Protein-level validation and in vivo studies will be essential to confirm the mechanistic insights and translational potential of pimozide as a multi-target anticancer agent. From a translational perspective, these results illustrate the potential of repurposed drugs to act as mechanistic probes that reveal exploitable vulnerabilities in cancer cells. Although limited to preclinical models, this work establishes a robust framework for further investigation and supports the repositioning of pimozide as a promising multi-target anticancer agent.