Research ArticleExperimental Studies
Open Access

Imipramine-induced Apoptosis and Metastasis Inhibition in Human Bladder Cancer T24 Cells Through EGFR/ERK/NF-κB Pathway Suppression

WEI-SHU WANG, YU-CHANG LIU, TSAI-LIN LO, FEI-TING HSU and CHIH-HUNG CHIANG
In Vivo March 2025, 39 (2) 669-682; DOI: https://doi.org/10.21873/invivo.13871

Abstract

Background/Aim: Bladder cancer is a prevalent malignancy, ranging from superficial forms to more aggressive types that invade the muscle and require extensive treatment. Imipramine, traditionally used as an antidepressant, has shown potential as an anti-cancer agent.

Materials and Methods: In this study, human bladder cancer T24 cells were treated with varying concentrations of imipramine to evaluate its cytotoxic and apoptotic effects.

Results: Imipramine induced cytotoxicity in a dose-dependent manner, significantly increasing apoptosis as shown by Annexin-V/PI staining and TUNEL assay. The drug also up-regulated cleaved caspase-3 and down-regulated the anti-apoptotic factor XIAP. Moreover, imipramine activated both extrinsic/intrinsic apoptotic pathways, evidenced by the increased expression of Fas, FasL, cleaved caspase-8, and cleaved caspase-9, along with mitochondrial dysfunction and ROS production. Imipramine inhibited the migration and invasion of bladder cancer cells, likely through the down-regulation of metastasis-related proteins and suppression of the EGFR/ERK/NF-Embedded ImageB signaling pathway.

Conclusion: Imipramine could be a promising therapeutic agent for bladder cancer.

Keywords:

Introduction

Bladder cancer is a common malignant tumor among women and ranks as the fourth most common malignancy in men. This type of cancer varies widely, ranging from non-invasive tumors that are typically low-grade and may recur, requiring patients to undergo long-term invasive monitoring, to highly aggressive and invasive tumors that are associated with a high disease-specific mortality rate (1-3). Currently, the standard treatment options for bladder cancer include surgery, chemotherapy, and radiotherapy (1, 4). Bladder cancer patients frequently face the challenges of depression, and severe depression can significantly impact their willingness to pursue standard treatment, ultimately leading to a poorer prognosis (5).

When cancer patients experience depression that adversely impacts their quality of life or interferes with their adherence to cancer treatment, prescribing antidepressants is a viable therapeutic option. Furthermore, research has demonstrated that antidepressants can effectively relieve a range of cancer-related symptoms, thus contributing to an improved quality of life for these patients (6, 7). Recently, antidepressants have gained attention for their potential anti-cancer properties, in addition to their ability to enhance quality of life. These medications not only alleviate depressive symptoms but may also promote tumor suppression by inducing apoptosis, halting cell cycle progression, inactivating oncogenic kinase pathways, and up-regulating anti-tumor immune responses (8-10).

Imipramine, a tricyclic antidepressant (TCA), has been shown to inhibit tumor growth in several types of cancer, including hepatocellular carcinoma, triple-negative breast cancer, prostate cancer, and glioblastoma (11-14). The anticancer mechanism of imipramine may be associated with the inhibition of several oncogenic kinases and transcription factors, including protein kinase B (PKB/AKT), extracellular signal-regulated kinase (ERK), nuclear factor-kappaB (NF-Embedded ImageB), and signal transducer and activator of transcription 3 (STAT3) (15). Therefore, the primary objective of the present study was to investigate the anticancer effects and underlying mechanisms of imipramine in bladder cancer cells.

Materials and Methods

Cell culture. The human bladder carcinoma cell line T24 was obtained from Professor Jing-Gung Chung’s lab at China Medical University, Taiwan, and was used in this study (16). This cell line was cultured in its respective medium, McCoy’s 5A, supplemented with 5% FBS, 5% FCS, and 1% penicillin and streptomycin. Cell maintenance was carried out in a humidified incubator at 37°C with an atmosphere of 95% air and 5% CO2.

Cell viability analysis. The MTT assay was used to assess the viability of T24 cells. T24 cells were initially seeded into 96-well plates at a density of 5×103 cells per well and incubated overnight. The cells were then treated with varying concentrations of imipramine (0, 10, 30, 50, 70, and 90 μM) for 48 h. After the treatment period, the culture medium was replaced with 100 μl of 5 mg/ml MTT reagent solution, and the cells were incubated for an additional 2 h. The MTT solution was then removed, and the resulting formazan crystals were dissolved in 100 μl of DMSO. Absorbance at 570 nm was measured using a SpectraMax iD3 multifunction microplate reader (17).

Flow cytometry. T24 cells were seeded into 6-well plates (2×105 cells/well) and incubated overnight. The cells were then treated with 0, 30, and 50 μM of imipramine for 48 h. After treatment, the cells were harvested and stained using the Annexin V-FITC Apoptosis Detection Kit (Vazyme Biotech Co., Ltd., Nanjing, PR China) (18), APO-BRDUTM Kit (TUNEL), Anti-Fas-FITC, Anti-FasL-PE, FITC-DEVD-FMK (cleaved caspase-3), Red-IETD-FMK (cleaved caspase-8), FITC-VAD-FMK (cleaved caspase-9), 3, 3′-Dihexyloxacarbocyanine Iodide (DiOC6) (Enzo Life Sciences, Farmingdale, NY, USA), fluo-3-acetomethoxyester (Fluo-3/AM, 2.5 μg/μl), and ROS peroxide-sensitive fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA, 500 μl at 10 μM, Molecular Probes) to detect apoptosis (17, 19, 20). After staining, the cells were resuspended in 300 μl of PBS, and the signal intensity of each marker was detected using NovoCyte flow cytometry (Agilent Technologies Inc., Santa Clara, CA, USA). Fluorescence intensity quantification was performed using FlowJo software version 7.6 (FlowJo LLC, Ashland, OR, USA).

Western blotting assay. T24 cells were plated in 10 cm dishes at a density of 1×106 cells per dish and allowed to grow overnight. The cells were then treated with varying concentrations of imipramine (0, 30, and 50 μM) for 48 h. After treatment, total cell proteins were extracted using RIPA protein lysis buffer, separated on 6-15% SDS-PAGE gels, and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies overnight at 4°C, followed by a one-hour incubation with secondary antibodies at room temperature (Table I). The protein bands were visualized using the Immobilon Western chemiluminescent HRP substrate and detected with a UVP ChemiDoc-It™ imaging system. Protein expression levels were quantified using ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA) (21).

View this table:
Table I.

Antibodies for western blotting.

Transwell assay (cell migration and invasion assay). Cell invasion and migration abilities were analyzed using transwell cell culture chambers with or without matrigel (30 μl matrigel in 70 μl serum-free McCoy’s 5A or DMEM). T24 cells treated with 0, 30, and 50 μM imipramine for 48 h were harvested and seeded into the upper chamber of the transwell at a density of 1×106 cells/well. The lower chamber contained culture medium with 10% serum. After 48 h, the migrated and invaded cells on the transwell membrane were fixed using a fixative buffer (methanol and acetic acid in a 3:1 ratio) and stained with 0.3% crystal violet solution for 10 min. The stained cells were photographed at ×100 magnification using a light microscope and quantified using ImageJ software version 1.50 (22).

Wound healing assay. T24 cells were seeded into 6-well plates with ibidi culture-inserts (Cat: 80241, ibidi GmbH, Gräfelfing, Germany) and allowed to adhere overnight. After removing the two-well insert, different doses of imipramine (0, 30, and 50 μM) were added to the medium. Migration patterns were observed under a microscope at 0 and 9 h (for T24 cells). The gap area was quantified using ImageJ software version 1.50 (23).

Statistical analyses. The results of the experiments were expressed as the mean±standard deviation. Statistical analysis was performed using Student’s t-test and one-way ANOVA independent analysis of variance. A p-value of less than 0.05 was considered statistically significant.

Results

Imipramine significantly induced cytotoxicity and apoptosis in bladder cancer cells. To assess whether imipramine induces cytotoxicity and apoptosis in human bladder cancer T24 cells, we treated the cells with varying concentrations of imipramine (ranging from 0 to 90 μM) for 48 h and monitored the effects on cell viability and apoptotic markers. Cell viability was evaluated using the MTT assay, which revealed a dose-dependent decrease in cell viability following imipramine treatment, as depicted in Figure 1A. This reduction in viability suggests that imipramine exerts cytotoxic effects on T24 cells. To further investigate the induction of apoptosis, we performed flow cytometry using Annexin V staining, which measures early apoptotic events. The proportion of Annexin V-positive cells increased in a dose-dependent manner, confirming that imipramine promoted apoptosis in T24 cells (Figure 1B and C). These findings were corroborated by the TUNEL assay, a method that specifically detects DNA fragmentation associated with late-stage apoptosis, which also showed a significant increase in apoptotic cells after imipramine treatment (Figure 1B and C). Additionally, a dose-dependent up-regulation of cleaved caspase-3 was observed (Figure 1D). Imipramine also induced the protein expression of cleaved caspase-3 in a dose-dependent manner, while the anti-apoptotic factor XIAP (X-linked inhibitor of apoptosis protein) (24) was reduced by imipramine (Figure 1E). Quantification analysis indicated a significant increase in cleaved caspase-3 and a reduction in XIAP (Figure 1F). These results confirm that imipramine induces cytotoxicity and apoptosis in bladder cancer cells through the up-regulation of apoptotic markers such as cleaved caspase-3 and the down-regulation of the anti-apoptotic protein XIAP. These findings suggest that imipramine promotes apoptosis by shifting the balance between pro-apoptotic and anti-apoptotic factors, further underscoring its potential as a therapeutic agent for bladder cancer treatment.

Figure 1.
Figure 1.
Figure 1.

Cytotoxicity and apoptosis induction by imipramine in T24 cells. (A) T24 cells were treated with different concentrations of imipramine (0, 10, 30, 50, 70, and 90 μM) for 48 h. T24 cell viability after imipramine treatment was assessed using the MTT assay. (B) Flow cytometry was used to evaluate Annexin-V and BrdU staining in imipramine-treated T24 cells. (C) Quantification of Annexin-V and BrdU signaling was performed using FlowJo software. (D) Cleaved caspase-3 activation in T24 cells after imipramine treatment was measured and quantified by flow cytometry. (E) Protein levels of cleaved caspase-3 and XIAP in T24 cells post-imipramine treatment were analyzed via western blot. (F) Quantification of cleaved caspase-3 and XIAP expression was done using ImageJ.

Imipramine triggered extrinsic and intrinsic apoptosis signaling in bladder cancer cells. To investigate whether imipramine influences both the extrinsic and intrinsic apoptotic pathways, we evaluated the activation of key markers involved in these pathways using flow cytometry and western blot analysis. Our findings indicated that imipramine significantly enhanced the activation of several components of the extrinsic apoptotic pathway. Specifically, Fas, Fas ligand (FasL), and cleaved caspase-8 expression levels were markedly elevated following imipramine treatment, as illustrated in Figure 2A-C. These results suggest that imipramine engages the extrinsic apoptosis pathway, likely through the Fas/FasL signaling axis, which is known to trigger caspase-8 activation and subsequent cell death (25). In addition to its effects on the extrinsic pathway, imipramine also activated the intrinsic apoptotic pathway. We observed that imipramine induced a loss of mitochondrial membrane potential (MMP, ΔΨm), a hallmark of intrinsic apoptosis (26). Moreover, imipramine treatment led to an increase in intracellular calcium (Ca2+) accumulation and elevated production of reactive oxygen species (ROS), as shown in Figure 2D-F. These cellular changes are critical in initiating intrinsic apoptosis, ultimately leading to mitochondrial dysfunction and cell death. Consistently, we found a dose-dependent up-regulation of cleaved caspase-9, an essential mediator of the intrinsic pathway (27), as demonstrated in Figure 2G. Western blot analysis further supported these observations, showing that higher doses of imipramine corresponded with increased protein expression of Fas, FasL, and cleaved caspase-8, reinforcing its role in activating the extrinsic pathway (Figure 2H). Concurrently, imipramine induced the expression of cleaved caspase-9, further confirming the involvement of the intrinsic pathway in imipramine-mediated apoptosis (Figure 2I). Collectively, these findings suggest that imipramine effectively triggers both extrinsic and intrinsic apoptotic pathways in bladder cancer cells. By simultaneously activating Fas/FasL signaling and inducing mitochondrial dysfunction, imipramine promotes a comprehensive apoptotic response, highlighting its potential as a powerful therapeutic agent against bladder cancer.

Figure 2.
Figure 2.
Figure 2.

Extrinsic and intrinsic apoptosis pathways induction by imipramine in T24 cells. (A-C) Flow cytometry was utilized to assess Fas, FasL, and cleaved caspase-8 staining in T24 cells treated with imipramine. (D) Mitochondrial membrane potential (MMP) loss in imipramine-treated T24 cells was evaluated by flow cytometry. (E-F) Flow cytometry was employed to measure the accumulation of Ca2+ and reactive oxygen species (ROS) in imipramine-treated T24 cells. (G) Cleaved caspase-9 activation in imipramine-treated T24 cells was examined using flow cytometry. (H-I) Protein levels of Fas, Fas-L, cleaved caspase-8, and cleaved caspase-9 in T24 cells following imipramine treatment were analyzed by western blot.

Imipramine significantly down-regulated the metastatic capacity of bladder cancer cells. To evaluate whether imipramine impedes the metastatic potential of bladder cancer cells, we performed a series of functional assays, including wound healing, transwell migration, and invasion assays. The transwell migration assay revealed a significant reduction in the number of migrated cells following imipramine treatment, as shown in Figure 3A. This suggests that imipramine exerts a notable inhibitory effect on cell migration. In parallel, the invasion assay demonstrated that imipramine substantially decreased the invasive capabilities of bladder cancer cells (Figure 3B). Consistent with these findings, the wound healing assay indicated that imipramine-treated cells exhibited a larger gap area than untreated cells, indicating impaired cellular migration and delayed wound closure (Figure 3C). Furthermore, western blot analysis was conducted to assess the expression levels of metastasis-related proteins. The results revealed that imipramine treatment led to the down-regulation of key metastasis-associated proteins, including VEGF, MMP2, and MMP9 (Figure 3D). These proteins play critical roles in promoting cancer cell migration, invasion, and angiogenesis, and their reduction further supports the notion that imipramine suppresses the metastatic behavior of bladder cancer cells. In summary, the results from these assays collectively suggest that imipramine effectively inhibits both the migration and invasion abilities of bladder cancer cells, which could contribute to its potential as a therapeutic agent in limiting bladder cancer metastasis.

Figure 3.
Figure 3.
Figure 3.

Metastasis inhibition by imipramine in T24 cells. Transwell migration (A) and invasion (B) assays were performed on T24 cells following imipramine treatment. (C) The wound healing assay was used to evaluate the effect of imipramine on cell migration in T24 cells. (D) Protein levels of VEGF, MMP-2, and MMP-9 in T24 cells after imipramine treatment, along with their quantification, were analyzed by western blot.

Imipramine suppressed bladder cancer progression via the inactivation of EGFR/ERK/NF-Embedded ImageB. To investigate the potential regulatory effects of imipramine on the EGFR/ERK/NF-Embedded ImageB signaling pathway in bladder cancer, we utilized both immunofluorescence staining and western blotting techniques. Our results demonstrated that imipramine exerted a dose-dependent inhibitory effect on the membrane expression of EGFR in bladder cancer cells, as shown in Figure 4A. Furthermore, imipramine significantly decreased the nuclear translocation of NF-Embedded ImageB, a key regulator in cancer progression, as depicted in Figure 4B. Western blot analysis provided additional evidence, showing that imipramine reduced the phosphorylation levels of EGFR, ERK, and NF-Embedded ImageB in T24 cells (Figure 4C). These findings strongly indicate that imipramine disrupts the progression of bladder cancer by targeting and inhibiting key components of the EGFR/ERK/NF-Embedded ImageB signaling pathway. This highlights the therapeutic potential of imipramine in mitigating bladder cancer by blocking this critical signaling axis.

Figure 4.
Figure 4.
Figure 4.

EGFR/ERK/NF-Embedded ImageB inactivation by imipramine in T24 cells. (A-B) Surface expression of EGFR and nuclear translocation of NF-Embedded ImageB were assayed by IF staining on T24 cells following imipramine treatment. (C) Protein levels of EGFR (Tyr1068), EGFR, ERK (Thr202/Tyr204), ERK, NF-Embedded ImageB (Ser536) and NF-Embedded ImageB in T24 cells after imipramine treatment, along with their quantification, were analyzed by western blot.

Potential regulation of imipramine on a bladder cancer model. Imipramine demonstrates significant anti-cancer activity against bladder cancer cells through various mechanisms. It induces dose-dependent cytotoxicity and apoptosis in T24 cells, evidenced by decreased cell viability, increased Annexin-V and TUNEL staining, and up-regulation of cleaved caspase-3, along with down-regulation of the anti-apoptotic protein XIAP. Imipramine activates both extrinsic (up-regulating Fas, FasL, and cleaved caspase-8) and intrinsic (loss of mitochondrial membrane potential and increased ROS) apoptotic pathways, showcasing its comprehensive approach to promoting apoptosis. Additionally, it inhibits the metastatic potential of bladder cancer cells by reducing migration and invasion and down-regulating key proteins like VEGF, MMP2, and MMP9. Imipramine also suppresses the EGFR/ERK/NF-Embedded ImageB signaling pathway by reducing EGFR expression and inhibiting its phosphorylation. These findings highlight imipramine’s potential as a therapeutic agent against bladder cancer by inducing apoptosis, inhibiting metastasis, and disrupting critical signaling pathways (Figure 5).

Figure 5.
Figure 5.

Schematic representation of imipramine’s effects on bladder cancer.

Discussion

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a crucial role in controlling tumor growth, survival, angiogenesis, invasion, and metastasis by activating downstream kinase pathways involved in tumor progression. Its over-expression has been observed in bladder cancer and is linked to an increased risk of recurrence (28-30). The activity of the ERK/NF-Embedded ImageB axis can be up-regulated through EGFR signaling, which is linked to the expression of anti-apoptotic, angiogenic, and invasion-related proteins, including XIAP, VEGF, MMP2, and MMP9 (20, 31-33). Our findings indicate that treatment with imipramine significantly reduces the phosphorylation levels of EGFR, ERK, and NF-Embedded ImageB (Figure 4C). Furthermore, imipramine inhibits both the membrane expression of EGFR and the nuclear translocation of NF-Embedded ImageB (Figure 4A and B).

Muscle-invasive bladder cancer (MIBC) has a poorer prognosis than non-muscle-invasive bladder cancer (NMIBC) due to its higher invasiveness. MMP2, MMP9, and VEGF play crucial roles in degrading the extracellular matrix and facilitating new blood vessel formation, which contribute to tumor invasion and metastasis. Elevated levels of these proteins have been linked to bladder cancer, indicating a greater likelihood of metastasis and a worse prognosis (34-36). Our data demonstrate that treatment with imipramine significantly reduces migration and invasion capacities, as well as the expression of these proteins (Figure 3).

Apoptosis is an important target in anticancer therapy, as it promotes the death of tumor cells. Anticancer drugs can activate apoptosis by either triggering death receptors on the cell membrane or increasing internal stresses such as DNA damage and endoplasmic reticulum (ER) stress. These actions initiate both the extrinsic and intrinsic pathways, leading to the activation of caspases, which ultimately induce apoptosis (17, 37, 38). Our results reveal that imipramine not only effectively induces apoptosis but also suppresses the expression of the anti-apoptotic protein XIAP (Figure 1). Treatment with imipramine significantly enhances extrinsic signaling (Fas, FasL, and cleaved Caspase-8) as well as intrinsic signaling (loss of ΔΨm and cleaved Caspase-9) (Figure 2D and G). In addition, we observed an accumulation of both Ca2+ and ROS in the cytosol (Figure 2E and F). The production of ROS may lead to DNA damage, while the accumulation of Ca2+ in the cytosol is a key feature of ER stress (39, 40).

In conclusion, this study demonstrates that imipramine exerts anti-bladder cancer properties, which include the induction of apoptosis through both extrinsic and intrinsic pathways, a reduction in invasion potential, and the suppression of the EGFR/ERK/NF—Embedded ImageB pathway. We propose that both the induction of apoptosis and the suppression of the EGFR/ERK/NF-Embedded ImageB pathway significantly contribute to the inhibitory effectiveness of imipramine on the growth and invasion of bladder cancer cells.

Acknowledgements

Some experiments and data analyses were conducted at the Medical Research Core Facilities Center, Office of Research & Development, China Medical University in Taichung, Taiwan.

Footnotes

  • Authors’ Contributions

    WSW, YCL, and TLL conducted all experiments, performed statistical analyses, and compiled the data. They also drafted the initial manuscript. FTH and CHC conceptualized the study, supervised the research, conducted the literature review, and prepared the final manuscript.

  • Funding

    This study is finically supported by Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan, R.O.C. (Funding number: BRD-110012), Taoyuan General Hospital, Ministry of Health and Welfare, Taoyuan, Taiwan, R.O.C. (Funding number: PTH-113057), and National Science and Technology Council (Funding number: NSTC 113-2314-B-087-001), Taipei, Taiwan, R.O.C.

  • Conflicts of Interest

    The Authors confirm that they have no financial interests that could be perceived as conflicts with the findings or conclusions presented in this study.

  • Received November 1, 2024.
  • Revision received November 5, 2024.
  • Accepted November 6, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Imipramine-induced Apoptosis and Metastasis Inhibition in Human Bladder Cancer T24 Cells Through EGFR/ERK/NF-κB Pathway Suppression
WEI-SHU WANG, YU-CHANG LIU, TSAI-LIN LO, FEI-TING HSU, CHIH-HUNG CHIANG
In Vivo Mar 2025, 39 (2) 669-682; DOI: 10.21873/invivo.13871
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