Abstract
Background/Aim: Oral squamous cell carcinoma (OSCC) has limited treatment options. This study investigated imipramine, a tricyclic antidepressant, as a potential therapy for OSCC using a SAS-bearing xenograft animal model. Materials and Methods: The SAS-bearing xenograft model evaluated imipramine’s impact on tumor growth. The control group received no treatment, while the imipramine-treated group received regular doses. Tumor growth, confirmed by imaging, and histological analysis assessed size and weight. Imipramine’s effects on apoptosis, epithelial-to-mesenchymal transition (EMT), and transcription factors (AKT, ERK, STAT3) were analyzed. Results: Imipramine significantly suppressed tumor growth within 6 days of treatment, with sustained activity. Computer tomography (CT) scans and histology confirmed reduced size and weight by imipramine. Imipramine induced apoptosis via caspase-dependent/-independent pathways, inhibited EMT, and down-regulated phosphorylated AKT, ERK, and STAT3. Conclusion: Imipramine shows promise as an effective OSCC therapy, inhibiting tumor growth, inducing apoptosis, and inhibiting EMT. Its impact on transcription factors and modulation of the AKT/ERK/STAT3 pathway suggest a multifaceted approach.
Depression is common among patients with head and neck cancer and can have a negative impact on their survival rate. Nonetheless, for patients who are diagnosed with depression after cancer, receiving ongoing psychiatric outpatient treatment following cancer diagnosis could result in improved overall survival outcomes (1). Oral squamous cell carcinoma (OSCC) is a common malignancy that typically appears in the head and neck region. Its aggressive invasiveness and propensity to spread to the lymph nodes in the neck make it a challenging cancer to treat effectively (2). Furthermore, a study showed that the diagnosis of oral cancer is significantly linked to a heightened risk of developing depression (3).
Depression generally has a negative impact on the quality of life of cancer patients and may also impede their treatment and recovery. The use of antidepressants has not only reduced the risk of developing depression but also improved the quality of life of patients with head and neck cancer (4). Antidepressants are commonly prescribed to alleviate a range of prevalent symptoms, such as depression, fatigue, anxiety, chronic pain, and insomnia. They are widely used by both the general population and cancer patients (5-7). For example, doxepin, a tricyclic antidepressant (TCA), can relieve radiotherapy-induced mucositis pain during the treatment of head and neck cancer (8).
In addition, several recent studies have shown that antidepressants can potentially exert anticancer effects through various mechanisms, such as inducing apoptosis, interfering with cell cycle progression, and inhibiting the signaling transduction pathways implicated in tumor progression (9, 10). Notably, long-term use of TCAs or selective serotonin reuptake inhibitors (SSRIs) has been associated with a reduced risk of oral cancer occurrence (5). Therefore, investigating the potential anti-oral cancer effects of antidepressant medications is an important research area.
Imipramine, a TCA, has been shown to suppress the progression of non-small cell lung cancer and glioblastoma by inhibiting the epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) signaling, and inducing apoptosis (11, 12). However, it is unclear whether imipramine possesses anti-oral cancer potential. Therefore, the main aim of this study was to evaluate the inhibitory efficacy and underlying mechanisms of imipramine in OSCC in vivo.
Materials and Methods
Cell culture of OSCC cells. The SAS human oral cancer cell line was cultured in DMEM (Dulbecco’s Modified Eagle Medium, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Thermo Fisher Scientific), 1% penicillin and streptomycin, and 2 mM L-glutamine (13). The cells were maintained at a temperature of 37°C in a humidified incubator with 5% CO2.
Animal model establishment and treatment procedure. The animal experiment was conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) at the China Medical University, under the reference ID CMU-IACUC-2022-301. Six-week-old male BALB/c nude mice were obtained from the National Laboratory Animal Center in Taipei, Taiwan. The mice weighed between 20-25 g and were subcutaneously injected with SAS cells (5×106) into the right leg. Treatment initiation occurred when the tumor size reached approximately 100 mm3, as measured using a digital caliper (14). Tumor volume was measured every three days and calculated using the formula: Volume=Height × Weight2 × 0.523. Once the tumors reached the desired size, the mice were randomly divided into three groups (N=6/each group): Control group (received 0.1% DMSO daily gavage treatment), imipramine group (received 20 mg/kg imipramine daily gavage treatment), and high-dose imipramine group (received 40 mg/kg imipramine daily gavage treatment). On day 21, post-treatment, the mice were humanely sacrificed, and both the tumors and organs were collected for further experimentation.
Computer tomography (CT) scan. At the end of the 21-day treatment period, the mice underwent tumor size evaluation using a CT scan (Mediso Ltd., Budapest, Hungary). The mice bearing SAS tumors were sedated with 1-3% isoflurane for the entire-body CT examination. The scanning parameters used were as follows: tube energy=55 kVp × 145 μA, direction=360°, and voxel size= 145×145×145 μm (15).
Hematoxylin and eosin (H&E) staining of SAS tumor. On day 18, the mice were humanely sacrificed, and their heart, lung, kidney, liver, spleen, and small intestine were collected for further analysis. The collected organs were prepared for H&E staining using the same fixation, paraffin embedding, and slicing procedure as for IHC staining. The H&E staining procedure was conducted by Bio-Check Laboratories Ltd (New Taipei, Taiwan, ROC), following previously established protocols. Microscopic images of the H&E-stained sections were captured (16). The scouring material was followed by our previous publication (17).
Immunohistochemistry (IHC) staining of SAS tumor. At the end of the 21-day treatment period, the mice were sacrificed, and tumor tissues were obtained for immunohistochemical (IHC) staining. The formalin-fixed, paraffin-embedded tumor tissues were cut into 5 μm sections, and these sections were subjected to IHC staining. Antibodies specific to the target of interest were utilized for immunohistochemical staining. Subsequently, the IHC stained sections were observed and imaged using a microscope (18). Three different tumor samples were isolated for IHC staining. We used four fields from each slice for quantification of protein levels. The antibodies used in this experiment are listed in Table I.
Antibodies used in the present study for IHC.
Statistical analysis. Statistical analysis was performed using Microsoft Excel 2017 version (Redmond, WA, USA). The significance of the results within the different dose groups of imipramine treatment was evaluated using the conventional Student’s t-test and one-way analysis of variance (ANOVA). A p-value of less than 0.05 was considered statistically significant.
Results
Imipramine demonstrated a significant inhibition of OSCC progression in an SAS bearing animal model. In order to investigate the impact of imipramine on tumor bearing animal model, we established a SAS-bearing xenograft animal model. As shown in Figure 1A, mice with tumors reaching 100 mm3 after SAS inoculation were randomly assigned to the control group (treated with 0.1% DMSO), imipramine 20 mg/kg group, or imipramine 40 mg/kg group. Whole-body CT scans were performed on SAS-bearing mice to assess tumor progression on day 0 and day 14 after treatment initiation. The treatment duration was set at 21 days, and mice were euthanized on day 21. Tissue samples including the heart, liver, spleen, kidney, and small intestine were collected from the mice for H&E staining, while tumor samples were subjected to IHC staining. The results demonstrated significant inhibition of SAS tumor growth in mice treated with imipramine. Notably, a substantial inhibition effect was observed as early as 6 days into the treatment, as shown in Figure 1B and Table II. Additionally, it was observed that the time taken for the tumor size to reach 500 mm3 was only 5.81 days in the imipramine 0 mg/kg (control) group, while it required 25.17 days in the imipramine 40 mg/kg group. Therefore, the mean tumor growth time in the imipramine 40 mg/kg group is 4.33 times longer compared to the imipramine 0 mg/kg group (Table III). The anti-tumor effect of imipramine was also evident from monitoring of tumor area using CT scans (Figure 1C). The extracted tumor samples further confirmed the suppressive efficacy of imipramine on SAS tumors (Figure 1D). Additionally, the weight of isolated tumors revealed a consistent reduction pattern in the imipramine-treated groups (Figure 1E). Moreover, higher doses of imipramine exhibited superior tumor inhibition efficacy compared to lower doses. Following imipramine treatment, we conducted validation of normal tissue pathology and mice body weight to confirm that these dosages of imipramine do not induce general toxicity. As shown in Figure 1F, there were no noticeable alterations in the heart, liver, spleen, kidney, and small intestine across all imipramine treatment groups. The alteration of tissue pathology was also validated by experienced pathologist and score by Shackelford’s (2002) four-scale method (Table IV) (17). Furthermore, Figure 1G demonstrates that the body weight remained unchanged throughout the entire treatment process. In conclusion, imipramine exhibited the ability to effectively suppress OSCC progression based on the findings from our study.
Therapeutic effect of imipramine on a SAS-bearing animal model. (A) Flow chart displaying the animal experiment. (B) Tumor volume measurements. (C) CT images showing the tumor at different time points. (D) Extracted tumor images. (E) Tumor weight under different imipramine dosages on day 21. (F) Validation of pathology changes in the heart, liver, spleen, kidney, and small intestine through H&E staining. (G) Recording of mice body weight during the treatment process.
Statistical analysis of tumor volume in different imipramine treatment groups and dates.
Average duration of tumor growth, delay time, inhibition rate, and enhancement ratio observed in SAS-bearing mice following imipramine treatment.
Severity scores for pathological alterations after imipramine treatment.
Imipramine mediated tumor inhibition is confirmed by the induction of apoptosis and inactivation EMT effect. To investigate the potential anti-tumor effect of imipramine on oral squamous cell carcinoma (OSCC), we conducted protein expression analysis on tumor sections treated with imipramine. In Figure 2A, we observed a significant increase in the cleavage of caspase-3, a marker indicative of caspase-dependent apoptosis, in response to imipramine treatment at a dosage of 40 mg/kg. The cleavage of caspase-3 was found to be approximately 3-4 times higher compared to the untreated group. This difference was statistically significant, as demonstrated in Table V. Moreover, imipramine treatment also activated the intrinsic apoptosis marker, cleaved caspase-9, indicating the induction of intrinsic apoptosis pathways. Furthermore, imipramine effectively triggered the activation of the extrinsic apoptosis factor, cleaved caspase-8. Moreover, epithelial-to-mesenchymal transition (EMT) is a crucial characteristic associated with the progression of OSCC, but the impact of imipramine on EMT remains unclear. In Figure 2B, we observed that imipramine treatment led to an increase in the endothelial factor E-cadherin, while the mesenchymal factor N-cadherin was decreased. Imipramine also effectively suppressed other EMT-related factors such as Twist, Snail-1, ZEB-1, and ZEB-2, as illustrated in Figure 2B. The statistical analysis comparing the imipramine-treated and untreated groups (Table VI, Table VII) confirmed the significant regulation of these proteins. Overall, these findings suggest that imipramine induces apoptosis in the OSCC model while simultaneously suppressing EMT. These regulatory effects may contribute to the anti-tumor effect of imipramine in the context of OSCC.
Protein alteration by imipramine on SAS-bearing animal tumor tissues. The change of (A) apoptosis-related factors, (B) EMT related factors, and (C) signaling transduction-related factors after imipramine treatment are presented. **p<0.01, ***p<0.005, ****p<0.0001 vs. 0 mg/kg imipramine.
Statistical analysis of apoptosis-associated protein in different imipramine treatment groups.
Statistical analysis of signaling transduction-associated protein in different imipramine treatment groups.
Statistical analysis of signal transduction-associated protein in different imipramine treated groups.
Imipramine mediated tumor inhibition is associated with AKT/ERK/STAT3 signaling. To investigate the potential signaling regulation of imipramine, we conducted further analysis on the expression of several transcription factors. Figure 2C illustrates that imipramine effectively down-regulated the phosphorylation of AKT and ERK. Furthermore, statistical analysis (p<0.0001) revealed a significant difference between the imipramine-treated and untreated groups (Table VIII). Imipramine also suppressed the phosphorylation of their downstream regulator, STAT3. These findings suggest that the potential anti-tumor effect of imipramine may be attributed to inactivation of the AKT/ERK/STAT3 signaling pathway.
Statistical analysis of signal transduction-associated protein in different imipramine treated groups.
Discussion
The spread of cancer cells to lymph nodes is a critical factor that affects prognosis of patients with oral and oropharyngeal carcinomas. In particular, metastasis to the cervical lymph nodes is the most common occurrence in OSCC patients, leading to a significant 50% decrease in survival rate (19, 20). Epithelial-Mesenchymal Transition (EMT), facilitated by EMT-related proteins, is a process in which non-invasive epithelial cells convert into an invasive mesenchymal phenotype characterized by an up-regulation of N-cadherin and a down-regulation of E-cadherin (21-23). This process is involved in the metastasis of cancer and also contributes to the development of drug resistance in cancer cells (24). It is important to highlight that imipramine demonstrated significant efficacy in up-regulating the expression of E-cadherin, while down-regulating protein levels of N-cadherin, Twist, Snail-1, ZEB1, and ZEB2 in OSCC ex vivo (Figure 2B).
The activation of both AKT and ERK, which are critical oncogenic kinases, contributes to tumor progression by up-regulating the activation and function of multiple transcription factors. For instance, both AKT and ERK have the ability to trigger the activation of STAT-3, leading to the promotion of tumor cell growth, metastasis, and EMT (25, 26). The elevated activation of AKT, ERK, and STAT-3 have been demonstrated to correlate with tumor metastasis and poor survival in patients with OSCC (27-29). Notably, our findings reveal a significant reduction in the activation of AKT, ERK, and STAT-3 following treatment with imipramine (Figure 2E).
The induction of apoptosis by therapeutic agents effectively halts tumor growth. The extrinsic apoptotic pathway, often called the death receptor pathway, and the intrinsic apoptotic pathway, known as the mitochondrial pathway, have the ability to initiate the activation of effector caspases. Caspase-8 and caspase-9 are specifically activated through the extrinsic and intrinsic apoptotic pathway, respectively. Once activated, these caspases play an essential role in mediating the process of apoptosis by subsequently activating major executioner caspase-3 (30, 31). Additionally, our data demonstrates that imipramine effectively triggered the activation of caspase-3, caspase-8, and caspase-9, it also effectively halted the growth of OSCC in vivo (Figure 2A).
In conclusion, the study provided compelling evidence that imipramine demonstrates remarkable effectiveness in inhibiting OSCC growth, inducing apoptosis, and suppressing the activation of oncogenic kinases. Our findings suggest that the induction of apoptosis and the suppression of the AKT/ERK axis may be linked to the inhibitory effects of imipramine on OSCC progression in vivo.
Acknowledgements
The Authors would like to thank the National Yang-Ming Chiao-Tung University Hospital, Yilan, Taiwan, ROC for the financial support (ID: RD2023-011 and RD2023-002), the Show Chwan Memorial Hospital, Changhua, Taiwan, ROC (ID: SRD-110035) and the Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan, ROC (ID: BRD-110028) and the National Science and Technology Council, Taipei, Taiwan (ID: MOST 111-2314-B-A49-026-MY3).
Footnotes
Authors’ Contributions
Li-Cho Hsu, Ching Ni Lin, Fei-Ting Hsu Ying-Tzu Chen, and Po-Lung Chang carried out the experiment. Fei-Ting Hsu, Ling-Ling Hsieh, and Hsiao-Yu Wang wrote the manuscript. Kuang-Hsuan Lin, Hsin-Chang Hsiao, and Hsi-Feng Tu conceived the original idea, supervised the project.
Conflicts of Interest
None to be declared.
- Received June 17, 2023.
- Revision received July 22, 2023.
- Accepted July 25, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
