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. 2026 Mar 31;29(1):84–86. doi: 10.3831/KPI.2026.29.1.84

Reversing ABCB1-Mediated Multidrug Resistance in Colorectal Cancer: electroacupuncture shows therapeutic potential in vivo

Yuqin Qiu 1, Zhenjia Fan 2, Xuewei Qi 3, Honglin Jiang 2, Weipeng Zhao 4, Yuxiang Wan 2,*, Jinchang Huang 2,*
PMCID: PMC13054849  PMID: 41953550

Abstract

Objectives

ATP-binding cassette sub-family B member 1 (ABCB1) is closely associated with multidrug resistance (MDR) in colorectal cancer; however, safe and effective clinical treatment options remain limited. This study aimed to evaluate electroacupuncture, a traditional Chinese medicine technique, for its potential to overcome ABCB1-mediated MDR in colorectal cancer and investigate the underlying mechanisms.

Methods

The tumor-inhibitory effects of electroacupuncture were investigated in nude mice with ABCB1 overexpression-induced MDR. Chemotherapy drug metabolism in tumor tissues and blood was assessed using pharmacokinetic analysis. In vivo fluorescence imaging was employed to monitor the accumulation of the ABCB1 substrate Rhodamine 123 within tumors. ABCB1 expression was analyzed using Western blotting, polymerase chain reaction (PCR), and immunofluorescence. Proteomic and bioinformatic analyses were conducted to explore the mechanisms through which electroacupuncture overcomes ABCB1-mediated drug resistance.

Results

Electroacupuncture significantly enhanced the sensitivity of ABCB1-mediated MDR nude mice to paclitaxel, markedly inhibiting tumor growth and promoting apoptosis. Tumor drug concentrations increased, while the expression of hypoxia-inducible factor 1-alpha (HIF1A) and its downstream target, ABCB1, decreased. Proteomic analyses suggested that extracellular matrix (ECM) remodeling plays a key role, indicated by decreased TIMP-1 and increased collagen expressions (collagen alpha-1(I) chain [COL1A1], collagen alpha-2(I) chain [COL1A2], and collagen alpha-1 (XVIII) chain [COL18A1]). Collectively, these findings indicate that electroacupuncture may influence tumor mechanical properties and the hypoxic microenvironment by modulating the balance between ECM degradation and deposition, providing a potential mechanistic explanation for the reversal of ABCB1-mediated MDR.

Conclusion

Electroacupuncture may reverse ABCB1-mediated tumor MDR, potentially through ECM remodeling and improvement of the tumor mechanical microenvironment. These insights provide a basis for developing new strategies to combat clinical drug resistance.

Keywords: colorectal cancer, multidrug resistance, electroacupuncture, ATP-binding cassette sub-family B member 1 (ABCB1), extracellular matrix

INTRODUCTION

Colorectal cancer (CRC) remains a highly prevalent malignancy globally, ranking as the second leading cause of cancer-related deaths. Over the past decade, the incidence of late-stage cases has increased substantially, particularly among younger populations [1]. Chemotherapy is a crucial treatment modality for advanced CRC. However, tumor resistance to multiple chemotherapy agents, referred to as multidrug resistance (MDR), substantially contributes to CRC’s high mortality rate by compromising chemotherapy efficacy. This finding represents a critical clinical challenge that requires effective intervention.

MDR arises from multiple mechanisms, including increased drug efflux, enhanced activity of drug-metabolizing enzymes, alterations in chemotherapy drug targets, augmented cellular DNA repair, epithelial-mesenchymal transition, and genetic factors [2, 3]. Among these, ATP-binding cassette (ABC) transporters-mediated drug efflux constitutes the principal mechanism inducing MDR. The ABC family includes 49 genes associated with MDR, with ABC sub-family B member 1 (ABCB1) being the first human ABC transmembrane protein identified, encoded by the multidrug resistance 1 (MDR1) gene. ABCB1 consists of two homologous hydrophobic transmembrane domains (TMDs), each containing six TMDs and a highly conserved nucleotide-binding domain (NBD). The TMDs are primarily responsible for substrate recognition and transport, whereas the NBD hydrolyzes ATP to release energy, inducing conformational changes in the TMDs that enable substrate drug efflux [4-6]. ABCB1 exhibits broad substrate specificity; high-affinity substrates are efficiently expelled from cells, whereas low-affinity substrates tend to accumulate intracellularly. Inhibition of ABCB1 efflux activity sensitizes drug-resistant tumor cells to chemotherapeutic agents, either through competitive binding to TMDs or by interfering with ATP hydrolysis [7]. ABCB1 is typically expressed in tissues such as the liver, kidney, and gastrointestinal tract epithelium, but is overexpressed in various malignancies, including CRC. Elevated ABCB1 expression is closely associated with MDR to commonly used chemotherapy agents, including taxanes, anthracyclines, vincristine, and etoposide [8]. Several approaches, including small-molecule leucine kinase inhibitors, nanomedicines, and natural compounds [9-11], have demonstrated efficacy in reversing MDR in ABCB1-overexpressing tumor cells. Nevertheless, these strategies have not yet translated into clinically approved treatments.

Acupuncture, a traditional Chinese medical treatment, is widely used to alleviate tumor-related symptoms such as cancer-associated pain, fatigue, insomnia, nausea, vomiting, bone marrow suppression, menopausal symptoms, and arthralgia [12-14]. Previous animal studies have shown that acupuncture administered around subcutaneous tumors in mice increases the local concentration of the chemotherapeutic agent paclitaxel [15]. Given the strong correlation between drug concentration and chemotherapy sensitivity, peri-tumor acupuncture may represent a viable strategy to overcome MDR when used in combination with chemotherapy. This study aimed to investigate whether electroacupuncture can mitigate ABCB1-mediated MDR in CRC and explore the underlying mechanisms.

MATERIALS AND METHODS

1. Cell culture

The human CRC cell line SW620 and its doxorubicin-selected ABCB1-overexpressing subline SW620/Ad300 were generously provided by Professor Zhe-Sheng Chen from St. John’s University. Both cell lines were cultured at 37℃, 5% carbon dioxide (CO2) using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. SW620/Ad300 cells were maintained in 300 μg/L doxorubicin, which was removed 2 weeks before the experiments.

2. Cytotoxicity assay

Cell drug sensitivity was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cells were seeded in a 96-well plate and incubated with various concentrations of paclitaxel and doxorubicin. After 48 h, 20 μL of CCK-8 solution was added to each well and incubated for an additional 4 h. The optical density at 450 nm was measured using a microplate reader, and cell survival curves were generated to determine the half-maximal inhibitory concentration.

3. Animals and establishment of nude mouse subcutaneous tumor model

Specific pathogen-free BALB/c nude mice, male, aged 6-8 weeks, weighing 18-20 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were housed under standard laboratory conditions: temperature of 22-25℃, humidity of 50-60%, 12-hour light-dark cycle, and free access to water. A 0.2 mL suspension containing 5 × 106 SW620/Ad300 cells was subcutaneously injected into the lateral side of the left thigh. Tumors were considered successfully established when the average diameter reached 5 mm [16]. All procedures were approved by the Medical and Laboratory Animal Ethics Committee of Beijing University of Traditional Chinese Medicine (No. BUCM 2024082001-3156).

4. In vivo drug resistance reversal experiment

Tumor-bearing nude mice were randomly assigned to four groups (n = 6 per group) using a computer-generated sequence. Outcome assessors were blinded to group assignments to minimize bias during data collection and analysis. The TG group served as the control. The paclitaxel alone (PTG) group received intraperitoneal paclitaxel (2 mg/kg) every 3 days. In the electroacupuncture group (EATG), mice were anesthetized with 2% pentobarbital sodium (60 mg/kg, intraperitoneal), and electroacupuncture was applied at four points located 5 mm from the tumor margin using sterile disposable needles (0.18 × 15 mm; Suzhou Dongbang Medical Co., Ltd.), with a penetration depth of approximately 5 mm. Electroacupuncture was performed using the SDZ-V series Huatuo electronic low-frequency acupuncture apparatus, with alternating scattered and dense waves: scattered wave at 3-4 Hz for 5 s and dense wave at 15-20 Hz for 10 s. The intensity was adjusted to prevent noticeable limb tremors. Each electroacupuncture session lasted 30 min and was performed every 3 days. In the EAPTG group, mice received electroacupuncture as described above, followed by intraperitoneal paclitaxel injections 1 hour later, every 3 days. Tumor sizes were measured using calipers, and body weights were recorded every 3 days. At the end of the study, all mice were euthanized under anesthesia, and tumor tissues were excised and stored at –80℃ for subsequent analysis.

5. Liquid chromatography–mass spectrometry (LC-MS) pharmacokinetics

The SW620/Ad300 nude mice were assigned to either a paclitaxel chemotherapy group or a combination group receiving electroacupuncture and paclitaxel. Following the interventions, mice were anesthetized, and blood samples were collected via the retro-orbital route at 10, 30, 60, 90, 120, and 240 min post-paclitaxel administration. Tumor tissues were excised, rapidly frozen in liquid nitrogen, and stored at –80℃. Plasma samples (100 μL) were mixed with 300 μL methanol, vortexed for 30 min, and centrifuged at 12,000 rpm for 10 min at 4℃, and 10 μL of the resulting supernatant was used for analysis. Tumor tissues (100 mg) were homogenized in 300 μL methanol, vortexed, centrifuged under the same conditions, and 10 μL of supernatant was analyzed. Standard solutions were prepared in methanol, and 10 μL of each was injected for detection.

Chromatographic analysis was conducted using a Thermo Scientific U3000 ultra-high-performance liquid chromatography system coupled to an SCIEX 5600 mass spectrometer. Separation was achieved using an ACQUITY UPLC BEH C18 Column (1.7 μm, 2.1 mm × 100 mm) maintained at 40℃. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), delivered at a flow rate of 0.3 mL/min. Mass spectrometry was performed in positive ion mode using electrospray ionization.

6. Small animal in vivo fluorescence imaging

The accumulation of the ABCB1-specific fluorescent substrate Rhodamine 123 (Rho-123) in tumors was assessed using the Molecular Devices small animal in vivo imaging system (MIIS XFP-STD). Anesthetized mice received a tail vein injection of Rho-123 solution (20 mg/kg) and were positioned to ensure complete exposure of the subcutaneous graft tumors. Images were captured using the system’s MetaMorph-MIIS software in multi-channel automatic capture mode. Channel 1 (W1) served as a reference with an exposure time of 15 ms, and channel 2 (W2, 488Ex-630Em) was used for fluorescence detection with an exposure time of 12 s. Regions of interest (ROI) were delineated, and the fluorescence intensities were quantified.

7. Immunohistochemistry/immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay

Tumor tissues were fixed, paraffin-embedded, sectioned at 3 μm, and rehydrated. Immunohistochemistry, immunofluorescence, and TUNEL assay were performed following previously described protocols [15] for the TG, PTG, EATG, and EAPTG groups.

8. Western blot analysis

Tumor tissues were lysed using radioimmunoprecipitation assay (RIPA) buffer, and protein concentration was determined using the bicinchoninic acid (BCA) method. Western blotting was performed according to established methods [15]. Grayscale values of protein bands were quantified using ImageJ software.

9. Quantitative polymerase chain reaction (qPCR) analysis

Primers for qPCR were designed and synthesized (Supplementary Table S1). Total RNA was extracted using TRIzol reagent (Vazyme). qPCR was performed using a CFX96 real-time q-PCR system (Bio-Rad, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control. The CT difference between the internal control and the target gene is presented as -ΔCT. ΔΔCT is the difference between the ΔCT values of paired specimens. 2ΔΔCT indicates the exponential value of ΔCT, and this value indicates the change in expression.

10. Four-dimensional data independent acquisition (4D-DIA) proteomics

Data-independent acquisition (DIA) proteomic analysis was performed on tumor tissues. Detailed protocols are provided in the supplemental materials. Briefly, equal aliquots of the samples were pooled to generate a data-dependent acquisition (DDA) database and quality control. Peptides were fractionated and separated using high-performance liquid chromatography and subsequently analyzed by mass spectrometry in DIA mode. Bioinformatics analysis included clustering analysis, Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, and enrichment analysis. Specific analytical methods are described in the supplementary materials.

11. Statistical analysis

All experiments were performed in triplicate. Data were analyzed using SPSS 20.0 software (IBM, USA). Student’s t-test was applied for pairwise comparisons, and one-way analysis of variance (ANOVA) followed by Bonferroni’s post-test was applied for multiple comparisons. Differences were considered statistically significant at p < 0.05.

RESULTS

1. Electroacupuncture enhances the sensitivity of ABCB1-mediated CRC MDR nude mice to paclitaxel

Before formal experiments, the MDR of SW620/Ad300 cells was confirmed, demonstrating ABCB1 expression (Supplementary Figs. S1, S2, Table S2). In vivo studies demonstrated that the combination of electroacupuncture and paclitaxel most effectively inhibited tumor growth compared with the control, electroacupuncture alone, and paclitaxel alone groups (Fig. 1a, b). At the conclusion of the experiment, tumors were excised and weighed. The EAPTG group exhibited a significantly lower average tumor weight, corresponding to an inhibition rate of 84.58% (Fig. 1c, Supplementary Table S3).

Figure 1.

Figure 1

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Effect of paclitaxel and electroacupuncture on the growth of SW620/Ad300 tumors in nude athymic mice. (a) Subcutaneous tumor images of nude mice after 18 days of intervention in the control group (TG), paclitaxel group (PTG), electroacupuncture group (EATG), and electroacupuncture combined with paclitaxel group (EAPTG) (n = 6). (b) Tumor growth curves. Error bars represent the standard error of the mean (SEM). *p < 0.05 versus TG; #p < 0.05 versus PTG (one-way ANOVA with Bonferroni post-test). (c) Tumor weights. Error bars represent SEM. *p < 0.05 versus TG; #p < 0.05 versus PTG (one-way ANOVA with Bonferroni post-test). (d) Average body weight of the four groups over time. (e) Average body weight changes before and after the intervention. *p < 0.05; ns, not significant (p > 0.05). (f) Body weight changes in nude mice treated with 2% pentobarbital sodium combined with paclitaxel (NPTG), paclitaxel alone (PTG), and electroacupuncture combined with paclitaxel (EAPTG) over time (n = 6). (g) Subcutaneous tumor volume changes in NPTG, PTG, and EAPTG groups over time. *p < 0.05 versus NPTG/PTG; ns, not significant, p > 0.05 (one-way ANOVA with Bonferroni post-test).

During treatment, nude mice in the PTG and EAPTG groups experienced gradual body weight loss, with a marked difference observed before and after treatment in the EAPTG group. No significant weight changes were observed in the EATG and TG groups (Fig. 1d, e). This phenomenon is likely attributable to the combination of chemotherapy and anesthesia. To further investigate, body weight and tumor growth were assessed in the combined paclitaxel-anesthesia group (NPTG). The NPTG group exhibited weight loss (Figs. 1f, Supplementary Fig. S3); however, no significant differences in tumor growth were observed compared with the PTG group (Fig. 1g). Spearman correlation analysis revealed no significant association between weight loss and tumor volume in the NPTG group (r = –0.2, p = 0.7139, Supplementary Fig. S4).

2. Electroacupuncture combined with paclitaxel inhibits tumor growth and promotes apoptosis in ABCB1-mediated MDR CRC cells

Hematoxylin and eosin (HE) staining revealed increased areas of degeneration and necrosis in tumors from mice treated with the combination of electroacupuncture and paclitaxel, accompanied by reduced nuclear division. Tumor cell proliferation, as indicated by Ki67 immunostaining, was significantly decreased. Consistently, TUNEL assay results demonstrated enhanced apoptosis, further supporting the substantial suppression of tumor growth by the combination therapy. Compared with the TG group, all treatment groups exhibited increased apoptosis, with the EAPTG group showing the highest level (Fig. 2).

Figure 2.

Figure 2

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Histopathology, Ki67 staining, and TUNEL detection of tumor tissues in SW620/Ad300 MDR nude mice. Scale bar 50 μm.

3. Electroacupuncture enhances the accumulation of ABCB1 substrate drugs in MDR CRC

To evaluate the effect of peri-tumoral electroacupuncture on paclitaxel pharmacokinetics, drug levels were measured in the plasma and tumor tissues of MDR nude mice in the PTG and EAPTG groups. The EAPTG group reached peak plasma drug concentration earlier than the PTG group (Fig. 3a). The area under the concentration-time curve (AUC) for plasma was 352,911 ng·mL–1·h for EAPTG and 342,480 ng·mL–1·h for PTG. In tumor tissues, paclitaxel concentrations were markedly higher than in the combination group at all time points, with tumor AUCs of 110,222 ng·g–1·h (EAPTG) compared with 53542 ng·g–1·h in the PTG group (Fig. 3b). The tumor-to-plasma concentration ratio (TPR) was calculated to assess selective drug distribution. Initially, PTG exhibited a slightly higher TPR than the combination group. However, from 90 min to 240 min, the TPR in EAPTG exceeded that of the PTG, indicating enhanced drug accumulation in drug-resistant tumor tissues with combination therapy (Fig. 3c).

Figure 3.

Figure 3

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Distribution of ABCB1 substrate drugs in SW620/Ad300 MDR nude mice in vivo. (a) Plasma concentrations of paclitaxel at 10, 30, 60, 120, and 240 min after intraperitoneal injection of paclitaxel or electroacupuncture combined with paclitaxel (n = 6). (b) Tumor tissue concentrations of paclitaxel at 10, 30, 60, 120, and 240 min after intraperitoneal injection of paclitaxel or electroacupuncture combined with paclitaxel (n = 6). (c) TPR at 10, 30, 60, 120, and 240 min after intraperitoneal injection of paclitaxel or electroacupuncture combined with paclitaxel (n = 6). (d) Fluorescence intensity of Rho123 in tumor tissues 60 min after intravenous injection (n = 6). *p < 0.05; ns, not significant, p > 0.05 (one-way ANOVA with Bonferroni post-test). (e) In vivo fluorescence imaging of Rho123 in tumor tissues 60 min after intravenous injection (n = 6).

To further investigate the effect of electroacupuncture on the efflux function of ABCB1, in vivo small-animal fluorescence imaging was employed to assess the accumulation of the ABCB1-specific fluorescent substrate Rho123 in tumor tissues of MDR nude mice following tail vein injection. Fluorescence intensity in the EATG and EAPTG groups was significantly higher in the TG and PTG groups, with the combined therapy group showing the greatest increase. No significant difference was observed between the PTG and TG groups (Fig. 3d, e). These results suggest that electroacupuncture enhances the chemosensitivity of MDR CRC by inhibiting ABCB1-mediated drug efflux, thereby promoting the accumulation of chemotherapy agents in drug-resistant tumors.

4. Electroacupuncture reduces the expression levels of ABCB1 in MDR tumor tissues of CRC

ABCB1 overexpression is a major contributor to MDR. The effects of electroacupuncture on ABCB1 expression were examined in tumor tissues. Immunofluorescence analysis demonstrated that electroacupuncture reduced ABCB1 expression on the cell membrane. Both the EATG and EAPTG groups exhibited significantly lower ABCB1 levels compared with the TG and PTG (Fig. 4a). Consistent with these results, Western blot analysis confirmed that downregulation of ABCB1 protein following electroacupuncture treatment (Fig. 4b). qPCR analysis revealed significantly reduced MDR1 mRNA expression in both EATG and EAPTG groups (Fig. 4c). The MDR1 gene contains a hypoxia-inducible factor 1 (HIF-1) binding site and is transcriptionally regulated by HIF-1. HIF1A mRNA levels were markedly decreased in the electroacupuncture-treated cohorts (Fig. 4d). Previous studies demonstrated that electroacupuncture normalizes tumor vasculature, improves the hypoxic tumor microenvironment, and reduces HIF1 mRNA expression [17]. These findings suggest that electroacupuncture may further suppress ABCB1 expression through the downregulation of HIF1A.

Figure 4.

Figure 4

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Electroacupuncture downregulates ABCB1 expression in tumor tissues of SW620/Ad300 MDR nude mice. (a) Immunofluorescence staining of ABCB1 expression. (b) Western blotting of ABCB1 protein expression. (c) qPCR of ABCB1 mRNA expression. (d) qPCR of HIF1A mRNA expression. *p < 0.05 versus TG; ns, not significant (p > 0.05).

5. Proteomic analysis suggests an association between electroacupuncture and extracellular matrix remodeling

To investigate the molecular mechanisms underlying electroacupuncture-mediated reversal of ABCB1-associated MDR, proteomic analysis of tumor tissues was performed. Comparison between the EAPTG and PTG groups identified 216 differentially expressed proteins (DEPs), including 142 upregulated and 74 downregulated proteins (Fig. 5a, b). KEGG pathway enrichment analysis revealed that the extracellular matrix (ECM)-receptor interaction was the most significantly enriched pathway among the DEPs (Fig. 5c). GO analysis indicated that the molecular functions of DEPs were primarily associated with ECM structural constituent, with cellular components predominantly localized in collagen-containing ECM (Fig. 5d). Notably, following combined electroacupuncture intervention, the expression of metalloproteinase inhibitor 1 (TIMP-1), an enzyme involved in ECM degradation, was significantly decreased (Fig. 5e). In contrast, the expressions of collagen alpha-1(I) chain (COL1A1), collagen alpha-2(I) chain (COL1A2), and collagen alpha-1(XVIII) chain (COL18A1) were upregulated (Figs. 5f-h), consistent with ECM remodeling. A protein–protein interaction (PPI) network of DEPs with a composite score > 0.4 was constructed using Cytoscape software, and the top 10 hub genes were identified using the cytoHubba plugin. GO and KEGG enrichment analyses of these hub genes confirmed their involvement in collagen fibril organization, ECM organization, and ECM-receptor interaction (Supplementary Fig. S5). Furthermore, bioinformatics analysis of The Cancer Genome Atlas (TCGA) database showed that high TIMP1 expression was associated with poor prognosis in patients with CRC (Fig. 5i). Collectively, these findings suggest that peri-tumoral electroacupuncture may facilitate ECM collagen remodeling within the tumor microenvironment, contributing to the reversal of ABCB1-mediated MDR.

Figure 5.

Figure 5

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DIA proteomics comparison between the PTG and EAPTG groups. (a) Volcano plot of DEPs. (b) Heatmaps of DEPs in PTG and EAPTG groups. (c) KEGG enrichment analysis of DEPs. (d) GO enrichment analysis of DEPs. (e-h) Differential expression of TIMP1, COL1A1, COL1A2, and COL18A1 between PTG and EAPTG groups. (i) Kaplan-Meier curves of overall survival in patients with CRC stratified by high versus low TIMP1 expression from the TCGA database.

DISCUSSION

The development of MDR in CRC chemotherapy is closely associated with ABCB1. Beyond mediating tumor cell resistance to its substrate drugs, such as doxorubicin and paclitaxel, ABCB1 also contributes to resistance against commonly used chemotherapy agents in CRC, including non-substrate drugs such as 5-fluorouracil, oxaliplatin, and irinotecan. Research on ABCB1 reversal agents has been ongoing for decades. First-generation reversal agents, such as verapamil, quinidine, and cyclosporine A, were not specifically designed to target ABCB1. They competitively bound to ABCB1 as high-affinity substrates and exhibited in vitro reversal of drug resistance. However, their clinical application was limited due to low affinity and unacceptable toxicity at the doses required for effective ABCB1 inhibition [4, 18]. Second-generation reversal agents, including dexverapamil, dexniguldipine, and biricodar, were structurally modified to increase affinity and reduce toxicity [19]. Nevertheless, competition between chemotherapy drugs and these inhibitors for cytochrome P450 activity caused unpredictable pharmacokinetic interactions, rendering them unsuitable for clinical use [4, 18]. Third-generation ABCB1 inhibitors, such as zosuquidar, elacridar, tariquidar, and laniquidar, demonstrated high selectivity and potency against ABCB1 with no significant pharmacokinetic interactions with chemotherapy. However, clinical trials were prematurely discontinued due to excessive toxic side effects when combined with chemotherapy [20, 21]. Currently, no drugs are approved specifically for treating MDR in tumors, making it a significant obstacle to the success of chemotherapy. The development of ABCB1 transporter-specific inhibitors for clinical application remains a considerable challenge.

In this study, we provide preliminary evidence that peritumoral electroacupuncture may reverse ABCB1-mediated MDR in CRC. Acupuncture, a traditional Chinese medical intervention, has a history of thousands of years and has gained global recognition due to its ease of application, precise efficacy, minimal adverse reactions, and high patient acceptance. Nevertheless, research on acupuncture in antitumor therapy is limited. Most studies focus on management, treating acupuncture as an adjuvant or alternative therapy, while its potential role in enhancing sensitivity to radiotherapy and chemotherapy is largely overlooked. Previous studies have shown that acupuncture in mice can influence the distribution of chemotherapy drugs [22]. Our research results indicate that combined electroacupuncture treatment significantly increased the sensitivity of drug-resistant nude mice to paclitaxel, with tumor growth inhibition rates in the combination group 2.02 and 1.68 times higher than those in the chemotherapy-only and electroacupuncture-only groups, respectively. Although nude mice receiving combined treatment experienced notable weight loss, safety evaluations suggest that this effect was attributable to the concurrent administration of chemotherapy drugs and anesthetics. Weight loss induced by anesthesia combined with chemotherapy did not significantly correlate with tumor progression, indicating a toxic response independent of therapeutic effects. In contrast, electroacupuncture alone demonstrated a favorable safety profile. It should be noted that toxicity assessment in this study primarily relied on changes in body weight. Future investigations should incorporate comprehensive toxicity evaluations, including hematological parameters, organ function assessments, and observations of feeding behavior and activity levels, to substantiate these findings.

As a drug efflux transporter, ABCB1 actively expels chemotherapy agents such as paclitaxel, reducing intracellular drug concentration and retention in tumor tissues. In this study, electroacupuncture not only increased the plasma AUC of paclitaxel but also enhanced local drug accumulation in drug-resistant tumors, evidenced by higher intratumoral concentrations at all evaluated time points. Notably, during later stages of administration, when plasma concentrations in the combination group were lower than in the chemotherapy-only group, the TPR continued to rise significantly. This phenomenon cannot be explained solely by systemic exposure. We hypothesize that electroacupuncture inhibits the efflux function of overexpressed ABCB1 on tumor cell membranes, thereby retaining paclitaxel within the tumor. This mechanism is crucial for overcoming drug resistance. In vivo fluorescence imaging further supports the notion that electroacupuncture may enhance the accumulation of ABCB1 substrate drugs in drug-resistant tumors.

ABCB1 expression is regulated by multiple signaling pathways [23-25]. The MDR1 gene, which encodes ABCB1, contains HIF-1 binding sites and is transcriptionally regulated by HIF-1. Inhibition of HIF-1 expression significantly reduces hypoxia-induced MDR1 expression [26]. Tumor hypoxia is closely associated with rapid proliferation, abnormal angiogenesis, and ECM remodeling. Abnormal vasculature and uneven proliferation lead to heterogeneous oxygen supply, generating hypoxic regions. Continuous deposition, remodeling, and cross-linking increase tumor stiffness, compress blood vessels, reduce blood perfusion, and exacerbate hypoxia by activating mechanosensitive signaling pathways, promoting epithelial-mesenchymal transition [27, 28]. Our previous research demonstrated that electroacupuncture reduces microvascular density in subcutaneous transplanted tumors, improves vascular structure and function, and modulates the hypoxic microenvironment [17]. In this study, the mRNA levels of ABCB1 and HIF1A were significantly decreased in tumor tissues following electroacupuncture intervention. Given that HIF1A is a well-established hypoxia marker and positively regulates ABCB1 transcription, this down-regulation suggests that electroacupuncture may influence tumor hypoxia and associated downstream signaling pathways. However, it should be noted that the amelioration of hypoxia is a plausible hypothesis rather than a directly measured outcome. To explore potential mechanisms, significant alterations in ECM-related proteins identified through proteomics were integrated. These changes may result from ECM remodeling induced by electroacupuncture, which could modulate hypoxia by altering the tumor’s physical microenvironment.

As a critical component of the tumor microenvironment, the ECM not only provides structural support to tumor cells but also actively regulates their proliferation, migration, therapeutic resistance, and angiogenesis. Collagen, the most abundant structural protein within the ECM, undergoes continuous synthesis, degradation, and cross-linking during ECM remodeling. This dynamic activity significantly impacts tissue mechanical properties, regulates cellular behavior, and influences tumor progression. Analysis of proteomics data revealed that electroacupuncture induced significant alterations in proteins associated with ECM structure and composition, particularly those related to collagen fibers. Specifically, the electroacupuncture combination intervention significantly reduced TIMP-1 expression. TIMP-1, a member of the TIMP family, inhibits the proteolytic activity of matrix metalloproteinases (MMPs) by forming noncovalent 1:1 stoichiometric complexes, thereby impeding ECM protein degradation and increasing matrix rigidity [29]. Furthermore, TIMP-1 attenuates tumor cell sensitivity to various anticancer agents by activating downstream pro-survival pathways and exhibiting anti-apoptotic activity [30]. Elevated TIMP-1 expression is associated with poor prognosis in multiple malignancies, including CRC. The reduction in TIMP1 expression following electroacupuncture intervention may relieve MMP inhibition, potentially facilitating ECM degradation. Conversely, electroacupuncture intervention significantly upregulated the expression of specific collagen subtypes, including COL1A1, COL1A2, and COL18A1. COL18A1 is a key basement membrane proteoglycan; cleavage of its C-terminus generates endostatin, a potent inhibitor of endothelial cell proliferation and tumor angiogenesis [31]. Upregulation of COL18A1 expression by electroacupuncture may contribute to its observed effects on inhibiting tumor angiogenesis and promoting vascular normalization, consistent with our previous research. Type I collagen, the major ECM component, is formed by a heterotrimer consisting of two α1 chains encoded by COL1A1 and one α2 chain encoded by COL1A2. The upregulation of COL1A1 and COL1A2 following electroacupuncture intervention may indicate the synthesis and deposition of new collagen fibers. During tumor progression, the ECM undergoes continuous, disordered remodeling rather than unidirectional degradation or deposition. The seemingly paradoxical changes induced by electroacupuncture suggest that the intervention may facilitate dynamic reconstruction of ECM components and structure, a hypothesis warranting further investigation in contemporary research. As previously discussed, alterations in matrix stiffness are critical physical factors influencing tumor hypoxia. We hypothesize that ECM remodeling driven by these molecular changes may link electroacupuncture treatment to the observed reduction in HIF1A expression, ultimately leading to downregulation of ABCB1. Recent studies indicate that ECM stiffness significantly impacts ABCB1. Excessive deposition of collagen type I enhances ABCB1 activity and protein expression [32]. Moreover, ABCB1 expression is elevated in ovarian, liver, and pancreatic cancer cells cultured in high-stiffness hydrogels [33, 34]. Increased tumor stiffness induces epithelial-to-mesenchymal transition in tumor cells, upregulating ABCB1 expression and contributing to CRC resistance to 5-fluorouracil (5-FU) and oxaliplatin [35, 36]. However, other studies report that ABCB1 expression and activity peak at moderate stiffness, with both excessively hard and soft matrix stiffness reducing drug efflux [37].

This study has some limitations. Proteomics data provided critical evidence of correlation, but did not directly confirm alterations in ECM mechanical properties or ultrastructure. Future research should directly assess the effects of electroacupuncture on ECM remodeling, tissue hardness, and hypoxia to clarify the mechanism by which it reverses ABCB1-mediated resistance. Furthermore, conditional overexpression or knockout models of ABCB1, TIPM1, COL1A1, and COL1A2 are needed to validate the effects of electroacupuncture on ABCB1-mediated MDR. Finally, a detailed and comprehensive toxicity assessment remains indispensable.

CONCLUSION

In summary, our study demonstrates that peri-tumor electroacupuncture effectively suppresses ABCB1 expression and function in an in vivo model. This suppression facilitates the accumulation of chemotherapeutic agents in drug-resistant CRC tissues, thereby reversing ABCB1-mediated MDR. Further analysis suggests that this effect may be associated with the regulation of ECM remodeling. Electroacupuncture modulates ECM degradation and collagen deposition through key molecules, including TIMP1, COL1A1, and COL1A2, potentially altering tumor stiffness and the hypoxic microenvironment. These changes may inhibit the HIF1A/ABCB1 resistance signaling axis. Collectively, these findings not only highlight the potential of electroacupuncture to counteract drug resistance but also provide a novel strategic framework for targeting the ECM stiffness-hypoxia axis to overcome ABCB1-mediated drug resistance. Future research should aim to validate this hypothesis at both mechanical and microenvironmental levels and systematically assess the safety and translational potential of this intervention.

SUPPLEMENTARY MATERIALS

Supplementary data is available at https://doi.org/10.3831/KPI.2026.29.1.84.

jop-29-1-84-supple.pdf (26.8MB, pdf)

ACKNOWLEDGEMENTS

The authors would like to thank Professor Zhe-sheng Chen from St. John's University, USA, for his gift of sensitive and resistant cells SW620 and SW620/Ad300, and for his suggestions on the safety evaluation of animal experiments.

Footnotes

AUTHORS’ CONTRIBUTIONS

Conceptualization: Yuqin Qiu, Jinchang Huang; Methodology: Yuqin Qiu, Yuxiang Wan; Validation: Yuqin Qiu, Zhenjia Fan, Xuewei Qi; Formal analysis: Honglin Jiang, Weipeng Zhao; Investigation: Yuqin Qiu, Zhenjia Fan, Xuewei Qi; Resources: Jinchang Huang, Yuxiang Wan; Data curation: Yuqin Qiu, Yuxiang Wan; Writing - Original Draft: Yuqin Qiu; Writing - Review & Editing: Jinchang Huang, Yuxiang Wan, Yuqin Qiu; Visualization: Yuqin Qiu, Zhenjia Fan; Supervision: Jinchang Huang, Yuxiang Wan; Funding acquisition: Jinchang Huang, Yuxiang Wan.

ETHICAL APPROVAL

This research was approved by the Medical and Laboratory Animal Ethics Committee of Beijing University of Traditional Chinese Medicine (No. BUCM 2024082001-3156, approval date September 19, 2024).

DATA AVAILABILITY

The data that support the findings of this study are available within the article and its supplementary material.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

FUNDING

This research was supported by the Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN202500478), National Natural Science Foundation of China (No. 82074545 and 82205292), Project of the China Association of Chinese Medicine (CACM-2022-QNRC2-B02), and Project of Beijing University of Chinese Medicine (2022-JYB-JBZR-042 and 2024-JYB-JBZD-041).

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