Preliminary study of cyto-impedance: Molecular profiling of functional impedance in motility-promoting treatment of normal cells

Abstract

Aims

Cell motility is critical for physiological processes including wound healing. However, high concentrations of motility-promoting agents may suppress cellular migration; this newly observed phenomenon warrants further characterization.

Methods

EH-P002A, a small molecule that enhances cellular motility in vitro and wound healing in vivo, was used to treat two human cell lines HFF-1 and Beas-2B. Whole genome expression profiling was applied.

Results

Elevating the EH-P002A concentration to a level far below a cytotoxic concentration resulted in suppression of the migratory abilities of the cells. During this suppression, the expression of WWC2, SMARCA1, PIAS1, PHF8, SDHAF2, and RP11-676M6.1 (a pseudogene) were down-regulated, and MALAT1, NEAT1, MSH6, RN7SL1, AARS, LCP1, APP, ERLIN2, MIEF1, MEGF8, RPS2, PLK1, ENO1, DOCK8, TNRC18, DCAF7, TPT1, RPL3, CAP1, and PLK4 were upregulated. More importantly, two genes, namely PDPK2 and COMMD2, were consistently up-regulated upon EH-P002A treatment regardless of the suppression; and 11 genes consistently down-regulated upon EH-P002A treatment, including RP11-490H24.5, RNF43, MT-ND5, FTL, MT-CO1, RPS6, MT-ND1, MT-ND4, RNU2-2P, MT-CO3, and STT3A.

Conclusions

We introduce the term 'cyto-impedance' to describe the phenomenon in which an increase in motility-promoting agent concentration might suppress the drug's effect without detectable cytotoxicity. The gene expression signatures of cyto-impedance have also been preliminary revealed.

Highlights

• •Cyto-impedance: high-dose motility drug suppresses migration without cytotoxicity.
• •EH-P002A promotes wound healing in vitro and in vivo.
• •Mitochondrial genes consistently down-regulated in cyto-impedance.
• •New gene signatures revealed for functional impairment in motility therapy.

1. Introduction

Cellular motility is critically important for wound healing because it is essential for the coordinated movement of various cell types to the injury site, enabling tissue repair and the restoration of the skin's barrier function [[1], [2], [3]]. It is a key process throughout the healing cascade, from the initial inflammatory response to the final stages of re-epithelialization, especially when involving the spatial translocation of stem cells [4]. Impaired motility can lead to delayed or chronic, non-healing wounds.

Globally, nearly 18.6 million people are affected by diabetic foot ulcers (DFU) each year, which lead to over 1.6 million amputations annually [5,6]. Diabetic wounds represent one of the most common types of chronic wounds and constitute a major clinical hurdle in modern medical practice [6,7]. Impaired cellular migration is a central mechanism contributing to the challenges in diabetic wound healing, affecting various cell types such as neutrophils, macrophages, keratinocytes, endothelial cells, and fibroblasts [8,9]. Metabolic disruptions in diabetes can hinder cellular motility, leading to delayed tissue repair. For instance, hyperglycemia and associated inflammation impair fibroblast and endothelial cell migration, exacerbating non-healing wounds [10].

Classical drugs that promote cellular motility are prokinetic agents used for gastrointestinal issues, which work by enhancing or stimulating muscle contractions. Common examples include the cholinergic agonist bethanechol [11], the dopamine antagonist metoclopramide [12], the serotonin agonist prucalopride [13], and the motilin agonist erythromycin [14]. Recently, phosphodiesterase 5 inhibitor sildenafil has been found to promote the amoeboid migration of dendritic cells, resulting in an antitumor effect [15]. However, the substantial clinical need to enhance the migration of various differentiated cells and stem cells for wound healing and tissue repair remains unmet. Consequently, further efforts are required to develop novel drugs that effectively promote wound healing and tissue repair across diverse clinical scenarios.

For years, researchers have proposed using living cells to screen for lead compounds that modulate cellular motility [16]. By developing a living cell-screening platform to identify compounds that inhibit or promote cellular movement, we successfully identified several candidates that enhance cellular migration in vitro and accelerate wound healing in vivo, including EH-P002A as a motility-promoting small molecule.

Cytotoxicity refers to the ability of a substance, agent, or process to be toxic to cells, meaning it can cause cellular damage, dysfunction, or death. In contexts like drug development, immunology (e.g., cytotoxic T cells), or toxicology, “cytotoxicity” is often used to imply cell-killing potential, especially when screening for anti-cancer agents.

Drugs designed to enhance other specific cellular functions can induce toxicity at higher doses, with distinct toxicity profiles depending on concentration [17]. Drug repurposing leverages these concentration-dependent effects to target different pathways [18]. Traditionally, drug toxicities are classified as mechanism-based (on-target), immune-mediated, off-target, bioactivation-related, or idiosyncratic [19].

Cellular dysfunction typically refers to a loss of normal physiological function in cells. For example, hepatocellular dysfunction indicates that hepatocytes are not functioning properly, resulting in impaired overall liver function. Myocardial dysfunction means that myocytes are not contracting effectively, leading to reduced cardiac performance. Therefore, the term “cellular dysfunction” generally encompasses a broader concept than drug-induced toxicity alone, as it can arise from various causes, including ischemia, inflammation, genetic abnormalities, or metabolic disturbances.

Here, we introduce cyto-impedance, a novel and unique on-target dysfunction observed in drugs promoting cellular motility for wound healing. Unlike irreversible cytotoxicity, cyto-impedance reflects a reversible functional impairment of cellular motility without involving cell death. In other word, overdosing with motility-promoting agents can paradoxically reduce therapeutic efficacy, resulting in an outcome contrary to the intended goal.

Using EH-P002A, we preliminarily characterized the molecular profile of cyto-impedance induced by excessive dosing.

2. Methods

2.1. Cellular motility-promoting agent

EH-P002A (N-phenyl-2,3-dihydrobenzo[b] [1,4]dioxine-6-carboxamide), a patented small molecule (China patent number: ZL202410532596.3), promotes cellular motility in vitro and wound healing in vivo. Its molecular formula is C15H13NO3, with a molecular weight of 255.1 g/mol (structure shown in Fig. 1A). EH-P002A was synthesized by Beijing Sciencebiochem Co., Ltd. (Room 1164, Floor 1, Zone E, Xianning Industrial Park, No. 8 Shuangqiao Road, Guanzhuang, Chaoyang District, Beijing, China).

Fig. 1.

EH-P002A promotes wound healing in a dose-dependent manner. A. Molecular structure of EH-P002A. B. No detectable cytotoxicity was observed in HFF-1 or Beas-2B cells upon EH-P002A treatment. C. Wound images from mice treated with EH-P002A at two doses, captured at different time points. D. Wound healing rates on Days 7 and 9. Dose-dependent efficacy on Day 7 was observed.

2.2. Cell culture and reagents

Human fibroblast cell line HFF-1 (Cat. No. SCRC-1041, ATCC) and bronchial epithelial cell line Beas-2B (Cat. No. CRL-3588, ATCC) were cultured in Dulbecco's Modified Eagle Medium supplemented with 10 % fetal bovine serum in a humidified 5 % CO2 incubator at 37 °C, and confirmed to be free of mycoplasma contamination.

2.3. Cytotoxicity assay

HFF-1 and Beas-2B cells were seeded at 5 × 10³ cells/well in 96-well plates and incubated overnight at 37 °C with 5 % CO₂. EH-P002A was prepared as a 20 mM stock in DMSO (Sigma, #D8418) and diluted in complete medium to different concentrations. After 48 h, 20 μl of CCK8 solution (Invigentech, #IV08-1000) was added per well, incubated for 1.5 h, and absorbance was measured at 450 nm. Five replicates were performed per concentration.

2.4. In vitro wound-healing assays

As reported previously [20,21], cells were seeded into 6-well plates and cultured to a 90 % confluence, followed by 24-h starvation in a serum-free medium. The sterile 200-μL tips were used to create artificial wounds in the cell monolayer and the floating cells were rinsed away with PBS. An inverted microscope was used to capture respective images of the wound at different time points.

2.5. Transwell assays for migration

Transwell assays have been described previously [20,21]. Briefly, cells were suspended in 200 μl of serum-free medium containing EH-P002A in the upper chamber of a Transwell. The lower chamber contained 800 μl of 20 % FBS-containing medium. After 48 h, cells were fixed with 600 μl methanol (Fisher, #A452-4) and stained with 0.1 % (w/v) crystal violet (Solarbio, #G1063-10). Migrated cells were counted in five random microscopic fields (100 × magnification) per experiment, averaged, and compared to controls. Experiments were performed in triplicate.

2.6. Whole genome expression profiling

Total RNA from HFF-1 cells was extracted using TRIzol reagent per manufacturer's instructions. RNA concentration was measured with a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Transcriptome analysis was conducted by Sequanta Technologies Co., Ltd. (Shanghai, China) using next-generation sequencing (Illumina NovaSeq 6000, PE 150, 2 x 150 bp paired-end reads). Quality control was performed using FastQC (version 0.11.9) with default parameters.

2.7. In vivo murine wound healing model

The wound healing model was applied by following a well-established procedure [22]. Briefly, male BALB/c mice (13 weeks, 25–30 g) were anesthetized with 5 % chloral hydrate (100 μl/20 g body weight, intraperitoneal). Two 15-mm-diameter wounds were created on either side of the spine using a biopsy punch. EH-P002A (1 or 10 mg/kg/day) or vehicle (100 μl) was administered intraperitoneally on days 1, 3, 5, 7, and 9. Mice were euthanized by CO2 inhalation on day 11. Wound healing rate was calculated as: (original wound area – remaining wound area)/original wound area × 100 %.

Animal experiments were approved by the Laboratory Animal Management and Use Committee of Guangdong Huawei Testing Co., Ltd. (Approval No. 202507004). All procedures were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animals and local regulations, adhering to the 3Rs principles (Replacement, Reduction, Refinement).

2.8. Signaling pathway analyses

To investigate the biological roles of differentially expressed genes (DEGs), we performed Gene Ontology (GO) enrichment analysis (focusing on biological process) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. These analyses were conducted using the clusterProfiler package (v3.14.3) in R, with DEGs as input and significance defined by adjusted p < 0.05.

To visualize relationships between key DEGs and significantly enriched GO terms or KEGG pathways, we generated Sankey diagrams using the GseaVis package (v0.0.9) in R. Nodes represent genes or enriched terms/pathways, with flow lines indicating membership. Up-regulated genes are shown in red; down-regulated genes in blue.

2.9. Statistical analysis

Wound healing rates were compared using the Mann-Whitney U test. Migrated cell numbers were analyzed with Dunnett's test.

3. Results

3.1. EH-P002A promotes wound healing without cytotoxicity

EH-P002A exhibited no detectable cytotoxicity in HFF-1 or Beas-2B cells (Fig. 1B), supporting its potential for clinical use. In a murine wound healing model, EH-P002A enhanced healing in a dose-dependent manner on days 7 without body weight loss at the doses of 1 and 10 mg/kg body weight (Fig. 1C and D), confirming its efficacy in promoting cellular motility in vivo.

3.2. Dose-dependent cyto-impedance

Following our initial observation that EH-P002A exhibited no cytotoxicity in vitro (as shown in Fig. 1B), we hypothesized that higher doses might enhance therapeutic efficacy by promoting greater cell migration. Accordingly, we tested progressively higher concentrations of EH-P002A. Unexpectedly, as shown in Fig. 2, relatively higher doses of EH-P002A reduced the migratory ability of HFF-1 and Beas-2B cells in a dose-dependent manner, demonstrating cyto-impedance. This suggests that excessive dosing of motility-promoting agents may impair therapeutic efficacy, a critical consideration for clinical development.

Fig. 2.

Dose-dependent cyto-impedance induced by EH-P002A. A. Wound-healing assay images of HFF-1 cells treated with indicated concentrations of EH-P002A, followed by quantification in the right panel. A promoting effect on wound healing was observed at 8 μM, while overdosage of EH-P002A suppressed the migratory ability. B. Images of migrated HFF-1 cells in transwell assays treated with varying EH-P002A concentrations followed by quantification in the right panel, showing promotion at 8 μM and suppression at higher doses. C. The data of wound-healing assay of Beas-2B cells were quantified, showing a promoting effect at 8–20 μM concentrations, and a suppressive effect at a higher concentration of 80 μM. D. Quantification of the transwell assays of Beas-2B cells confirmed dose-dependent cyto-impedance at 40 and 80 μM concentrations. ∗, P < 0.05; ∗∗, P = 0.001; ∗∗∗, P < 0.001.

3.3. Genes downregulated in cyto-impedance

To peer into the molecular mechanisms underlying the EH-P002A-induced migratory cyto-impedance, we performed whole-genome expression profiling on HFF-1 cells treated for 48 h with low (8 μM) or high (12 μM) doses of EH-P002A. This analysis identified six downregulated genes associated with cyto-impedance: WWC2, SMARCA1, PIAS1, PHF8, SDHAF2, and the pseudogene RP11-676M6.1 (Fig. 3A).

Fig. 3.

Differentially expressed genes in cyto-impedance (6 downregulated, 20 upregulated). HFF-1 cells were treated with EH-P002 for 48 h. The cellular mRNA levels were assessed via whole-genome expression profiling. A. The downregulated genes during cyto-impedance. B. The upregulated genes associated with cyto-impedance.

3.4. Genes upregulated in cyto-impedance

As expected, 20 genes showed upregulated expression associated with cyto-impedance, including MALAT1, NEAT1, MSH6, RN7SL1, AARS, LCP1, APP, ERLIN2, MIEF1, MEGF8, RPS2, PLK1, ENO1, DOCK8, TNRC18, DCAF7, TPT1, RPL3, CAP1, and PLK4 (Fig. 3B).

3.5. Genes with persistent dose-dependent changes

We also considered the possibility that the genes with persistently regulated expression under cyto-impedance condition play more important biological roles. We reported here that 2 genes, PDPK2 and COMMD2, were persistently upregulated in a dose-dependent manner (Fig. 4A). Eleven genes were persistently downregulated, including RP11-490H24.5, RNF43, MT-ND5, FTL, MT-CO1, RPS6, MT-ND1, MT-ND4, RNU2-2P, MT-CO3, and STT3A (Fig. 4B).

Fig. 4.

Altered gene expressions in a dose-dependent manner. A. Two genes with dose-dependent upregulation associated with cyto-impedance. B. Eleven genes with dose-dependent downregulation during cyto-impedance.

3.6. Altered signaling pathways upon cyto-impedance

Functional enrichment analysis was performed to interpret gene expression changes associated with EH-P002A-induced cyto-impedance in HFF-1 cells. GO biological process analysis revealed significant enrichment in terms related to cell migration, signal transduction, and mitochondrial energy metabolism (Supplementary Figure 1A). Notable terms included regulation of cell motility and ATP synthesis coupled proton transport. KEGG pathway analysis highlighted alterations in pathways critical for energy production and cellular response, including oxidative phosphorylation and thermogenesis (Supplementary Figure 1B).

Sankey diagrams illustrated direct links between key DEGs and enriched functional categories (Supplementary Fig. 1C and 1D). Notably, a cluster of consistently down-regulated mitochondrial genes (MT-ND1, MT-CO1, MT-ND4, MT-ND5, MT-CO3) was strongly associated with the GO term mitochondrial electron transport and the KEGG pathway oxidative phosphorylation.

Together, these results demonstrate that overdosage of EH-P002A suppresses mitochondrial energy metabolism while disrupting cytoskeletal dynamics and cell-cycle checkpoints, providing the first comprehensive functional annotation of the candidate molecular mechanisms underlying cyto-impedance. However, the distinct gene expression profiles observed between therapeutic doses of EH-P002A (which promote motility) and overdoses (which induce cyto-impedance; Supplementary Figure 2) warrant further investigation to better characterize and mitigate this cyto-impedance effect.

4. Discussion

Over one million diabetic patients undergo lower extremity amputations annually due to non-healing foot ulcers [23]. In cancer patients receiving radiation therapy, which is one of the mainstays of anticancer treatment [24,25], chronic skin injuries with delayed healing ulcer are a complex challenge [[26], [27], [28]]. While proton therapy reduces skin injury from radiation, conventional photon therapy will remain standard in many countries for decades [29,30], meaning skin damage will continue to be a common side effect of radiotherapy. Adipose tissue-derived mesenchymal stem cells and stem cell-derived exosomes have been proposed to treat these chronic lesions [[31], [32], [33]]. Enhancing cellular motility of epithelial cells and stem cells is therefore believed to be a promising strategy to improve outcomes for diabetic ulcers and radiation-induced ulcers.

Similarly, promoting vascular reconstruction after myocardial infarction (MI) may reduce infarct size and improve heart function [[34], [35], [36]]. Following an MI, endothelial cells (EC) must migrate, proliferate, and fuse to form new blood vessels (angiogenesis) to restore blood and nutrient supply to the damaged heart muscle [37]. Impaired EC movement after MI severely hinders vascular regeneration because cell migration is a critical step in the formation of new blood vessels. Without proper EC movement, the new vasculature needed to supply the injured heart with oxygen cannot be established, leading to a failure in repairing the microcirculation and ultimately worsening the long-term damage and potential for heart failure [[38], [39], [40]].

Cellular motility is also essential for neurological repair. It enables key processes such as the migration of neural stem cells to injury sites, where they can differentiate and replace lost neurons, and the directed outgrowth of axons toward their synaptic targets—a fundamental form of cellular movement critical for functional recovery [[41], [42], [43], [44]].

Taken together, application of cellular motility-promoting drugs is essential for accelerating diabetic wound healing, MI repair, and neurological repair. Currently, few drugs are available to address these clinical challenges by promoting normal cell motility. Through our proprietary drug screening system, we have identified several promising lead compounds including EH-P002A that enhance normal cell motility.

In the present study, we reported a phenomenon that higher EH-P002A concentrations could result in suppressed migratory ability of the cells, and we termed this phenomenon as cyto-impedance. Whole-genome profiling revealed distinct molecular signatures during cyto-impedance, with both upregulated and downregulated genes, providing initial insights into its underlying molecular mechanisms.

Interestingly, genes responsible for mitochondrial energy production [45]—namely MT-CO1, MT-CO3, MT-ND1, MT-ND4, and MT-ND5—were downregulated following motility-promoting treatment in the present study. This observation aligns with the established understanding that highly motile cells are typically arrested in the G0/G1 phase, during which they are unable to proliferate [46]. Given that cell division is highly energy-intensive [47,48], the reduced expression of these genes likely reflects the lower energy demands of non-dividing, migratory cells compared to their proliferating counterparts.

The discovery of cyto-impedance—where high doses of motility-promoting drugs reduce efficacy without cytotoxicity—presents a challenge for the clinical application of motility-promoting treatments. Our preliminary understanding of cyto-impedance necessitates further investigation, as the underlying molecular mechanisms and clinical implications remain poorly understood. Moreover, effective strategies must be developed to address this newly identified functional toxicity. To achieve this, we need to establish robust methods for assessing and mitigating this toxicity effectively.

Motility-promoting drugs are anticipated to play a pivotal role in the future treatment of chronic, non-healing wounds. In this evolving field, proposed strategies to maximize therapeutic efficacy while minimizing adverse effects include the development of cell-specific agents tailored to target particular cell types (e.g., epithelial cells or fibroblasts) under varying pathological conditions, dynamic dose adjustment of these drugs to avoid cyto-impedance that could impede cellular migration in affected tissues, and the development of targeted organ- or wound-specific drug delivery systems.

In conclusion, our study is the first to describe cyto-impedance (Supplementary Figure 2), offering a novel perspective on the challenges of motility-promoting therapies. The gene expression signatures of cyto-impedance were also preliminarily revealed. These findings may guide future efforts to enhance wound healing and organ repair. Further studies are needed to fully elucidate the underlying molecular mechanisms for optimizing therapeutic strategies.

Disclosure of AI-assisted technologies

AI-assisted technology was used to check for grammatical errors and typos.

Funding

This study was partially supported by Pingdingshan University Talent Startup Fund (No. PXY-BSQD-2023021).

CRediT authorship contribution statement

Chang-Zhi Li: Formal analysis, Funding acquisition, Investigation, Methodology, Writing – review & editing. Hong-Juan Zhou: Formal analysis, Investigation, Methodology, Writing – review & editing. Jin-Dong Chen: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. Jie Huang: Data curation, Formal analysis, Investigation, Writing – review & editing. Yi-Xiu Liu: Data curation, Investigation, Project administration. Chao-Nan Qian: Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

List of abbreviation

AARS

Aminoacyl-tRNA synthetase

APP

Amyloid precursor protein

CAP1

Cyclase-associated protein 1

COMMD2

Copper metabolism MURR1 domain containing 2

DCAF7

DDB1- and CUL4-associated factor 7

DEG

Differentially expressed gene

DFU

Diabetic foot ulcers

DOCK8

Dedicator of cytokine 8

EC

Endothelial cell

ENO1

Alpha-enolase 1

ERLIN2

ER lipid raft-associated protein 2

FTL:

Ferritin light chain

GO

Gene oncology

KEGG

Kyoto encyclopedia of genes and genomes

LCP1

Lymphocyte cytosolic protein 1

MALAT1

Metastasis-associated lung adenocarcinoma transcript 1

MEGF8

Multiple epidermal growth factor-like domains 8

MI

Myocardial infarction

MIEF1

Mitochondrial elongation factor 1

MSH6

MutS homolog 6

MT-CO1

Mitochondrial cytochrome c oxidase subunit-1

MT-CO3

Mitochondrial cytochrome c oxidase subunit-3

MT-ND1

Mitochondrially encoded NADH dehydrogenase subunit 1

MT-ND4

Mitochondrially encoded NADH dehydrogenase subunit 4

MT-ND5

Mitochondrially encoded NADH dehydrogenase subunit 5

NEAT1

Nuclear paraspeckle assembly transcript 1

PDPK2

3-phosphoinositide-dependent protein kinase 2

PHF8

Plant homeodomain finger-containing protein 8

PIAS1

Protein inhibitor of activated STAT-1

PLK1

Polo like kinase 1

PLK4

Polo like kinase 4

RN7SL1

RNA component of signal recognition particle 7SL1

RNF43

E3 ubiquitin ligase ring finger protein 43

RNU2-2P

RNA, U2 small nuclear 2

RPL3

Ribosomal protein L3

RPS2

Ribosomal protein S2

RPS6

Ribosomal protein S6

SDHAF2

Succinate dehydrogenase assembly factor 2

SMARCA1

SNF2 related chromatin remodeling ATPase 1

STT3A

STT3 oligosaccharyltransferase complex catalytic subunit A

TNRC18

Trinucleotide repeat containing 18

TPT1

Tumor protein, translationally-controlled 1

WWC2

WW and C2 domain containing 2

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Figure 1.

Pathway analysis of EH-P002A-induced cyto-impedance in HFF-1 cells. A. Gene ontology (GO) enrichment analyses of differentially expressed genes (DEGs), covering biological process (BP), cellular component (CC), and molecular function (MF). The top 10 terms with adjusted p < 0.05 are shown. B. KEGG pathway enrichment of DEGs. The top 10 pathways ranked by adjusted p < 0.05 are displayed. C. Sankey diagram linking key DEGs to enriched GO biological process terms. Red nodes: up-regulated genes; blue nodes: down-regulated genes. D. Sankey diagram linking key DEGs to enriched KEGG pathways. Node colors as in C.

Supplementary Figure 2.

Graphical abstract of cyto-impedance upon overdose treatment of the motility-promoting agent EH-P002A. Two heatmaps illustrate the distinct expression profiles of 39 genes identified in the study. These genes were categorized as either up-regulated (red) or down-regulated (blue) following treatment with EH-P002A at a therapeutic dose or an overdosage.

Data availability

Data will be made available on request.