Abstract
Background/Aim: In hepatocellular carcinoma (HCC) treatment, radiotherapy (RT) stands as a pivotal approach, yet the emergence of radioresistance poses a formidable challenge. This study aimed to explore the potential synergy between quetiapine and RT for HCC treatment. Materials and Methods: A Hep3B xenograft mouse model was used, the investigation tracked tumor progression, safety parameters, and molecular mechanisms. Results: The findings revealed a synergistic anti-HCC effect when quetiapine was coupled with RT that prolonged tumor growth time and a significantly higher growth inhibition rate compared to the control group. Safety assessments indicated minimal pathological changes, suggesting potential of quetiapine in mitigating RT-induced alterations in liver and kidney functions. Mechanistically, the combination suppressed metastasis and angiogenesis-related proteins, while triggering the activation of apoptosis-related proteins via targeting Epidermal growth factor receptor (EGFR)-mediated signaling. Conclusion: The potential of the quetiapine and RT combination is emphasized, offering enhanced anti-HCC efficacy, a safety profile, and positioning quetiapine as a radiosensitizer for HCC treatment.
Among individuals diagnosed with cancer, the prevalence of psychiatric disorders is increasing, especially in those at advanced stages of the disease. Psychiatric disorders generally have an impact on the quality of life of cancer patients. Commonly observed psychiatric disorders include delirium, depression, anxiety, delusions, hallucinations, and disorganized behaviors. Antipsychotic medications are often prescribed to alleviate psychotic symptoms in this population (1-4).
In addition to their antipsychotic effects, the potential of antipsychotic medications to exhibit anticancer effects has been an ongoing area of research (5-7). While the current approval status from the U.S. Food and Drug Administration does not include use of any specific antipsychotic for cancer treatment, several studies have provided evidence that long-term use of antipsychotic medications may be associated with a decreased risk of certain cancers, including hepatocellular carcinoma, gastric cancer, and colorectal cancer. It is important to note that the risk does not decrease for all cancers, as there are indications pointing to an elevated risk for breast cancer (8-12).
Preclinical studies have also demonstrated that antipsychotics induce tumor suppression by promoting apoptosis, inhibiting cell cycle progression, downregulating the tumor microenvironment, and disrupting crucial signaling pathways involved in tumor growth, survival, angiogenesis, and metabolism (8, 13). For instance, trifluoperazine, a typical antipsychotic, has been indicated to elicit apoptosis and induce cell-cycle arrest, leading to the growth inhibition of glioblastoma cells (14). Moreover, antipsychotics have been recognized as potential adjuvants to enhance the anti-cancer efficacy of therapeutic agents or radiation (8, 15). Olanzapine, an atypical antipsychotic, has been shown to increase the sensitivity of lung and pancreatic cancer cells to chemotherapeutic agents, including 5-fluorouracil, gemcitabine, and cisplatin (16). Aripiprazole, an atypical antipsychotic, has been identified as a radiosensitizer that boosts the therapeutic efficacy of radiation by upregulating the generation of reactive oxygen species (ROS) in head and neck cancer cells (17).
Quetiapine, an atypical antipsychotic, exhibited an inverse association with the incidence of hepatocellular carcinoma (HCC) in individuals diagnosed with schizophrenia (9). Our previous studies also presented evidence that quetiapine halts the progression of HCC by inactivating nuclear factor-kB (NF-κB) signaling (18). Inhibition of NF-κB signaling alleviate the chemo-radioresistance of tumor cells (19). While radiotherapy (RT) is utilized in the treatment of HCC, chemotherapy is not commonly employed (20-22). It is worthwhile to investigate whether quetiapine can enhance the effectiveness of radiotherapy against HCC by targeting EGFR-mediated signaling and acting as a radiosensitizer to reduce EGFR-mediated signaling. The objective of the present study was to assess the inhibitory efficacy and mechanism of quetiapine in combination with radiation in HCC in vivo.
Materials and Methods
Cell culture. In this investigation, human hepatoma cell lines, specifically Hep3B, were chosen for experimentation. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS). Following this, the cells underwent incubation in a controlled environment, maintained at 37°C with a 5% carbon dioxide (CO2) concentration, using a humidified incubator (23). The information of reagents that were used is presented in Table I.
Reagents used in the study.
Xenograft HCC model. Male CAnN Cg-Foxn1nu/CrlNarl mice, aged 4 to 6 weeks, were obtained from the National Laboratory Animal Center for the purposes of this study. Hep3B cells (n=6/group), each at a concentration of 5×106 cells in 100 μl phosphate-buffered saline (PBS) per mouse, were subcutaneously administered into the right flank of the mice. All experiments were repeated twice. Subsequently, a 10 days period was allowed for the progression and development of HCC tumors in the experimental subjects (24). Upon reaching a tumor volume of 80 to 100 mm3, mice were randomly allocated into four groups. The subsequent treatments were administered as follows: the control group received treatment with 0.1% DMSO in 100 μl double-distilled water; quetiapine-treated mice underwent gavage administration of 20 mg/kg/d in 100 μl double-distilled water; the RT group received a single dose of 6 Gy radiation on day 0 using a linear accelerator, with only the tumor exposed to radiation; and the combination treatment group underwent both quetiapine and radiation, as previously described. Tumor volume assessments were conducted at 4-day intervals through meticulous caliper measurements. The calculation employed the formula: tumor volume=length×width2×0.523. Humane euthanasia of mice on the 16th day post-treatment was performed using an isoflurane overdose, ensuring compliance with ethical considerations. This facilitated subsequent comprehensive analyses for a thorough understanding of the experimental outcomes. The synergistic effect of combination was evaluated by Chou-Talalay method (25, 26).
Immunohistochemistry (IHC) staining. Resected tumors were fixed in 4% paraformaldehyde (PFA) at 4°C. The subsequent processing involved sectioning the paraffin-embedded tumor specimens into 5-μm thickness, performed by Bio-Check Laboratories Ltd (New Taipei City, Taiwan, ROC). Immunohistochemical (IHC) staining was applied for the comprehensive assessment of protein expression within the tumors, following the manufacturer’s instructions meticulously (27). For this purpose, tumor tissues affixed to slides underwent staining with a panel of antibodies [anti-EGFR, anti-FGFR2, anti-pPKCδ, anti-NF-κB p65, anti-pP38, anti-cleaved caspase 3, anti-cleaved caspase 8, anti-cleaved caspase 9, anti-BAX, anti-BAK, anti-MMP-9, anti-VEGF, anti-b-FGF, anti-C-FLIP, and anti-XIAP]. The subsequent scanning process employed the EVOS M5000 Imaging System (Invitrogen, Waltham, MA, USA) at a magnification of 100× to capture detailed images. To quantify the IHC staining, Image J version 1.50 (National Institutes of Health, Bethesda, MD, USA) was utilized. At least three fields acquired from light microscope images were used for quantification. This methodological approach facilitated a meticulous and systematic examination of protein expression patterns in the tumor samples, contributing to a comprehensive understanding of the experimental outcomes. The information of primary antibodies is listed in Table II.
Primary antibodies used in this study for IHC staining.
Hematoxylin and eosin (H&E) staining. After a 16-day treatment period, mice were euthanized on day 16. Organs, including the liver, heart, spleen, small intestine, and kidney, were harvested and fixed in 4% paraformaldehyde at 4°C. Following fixation, the tissues underwent paraffin embedding, and 5 μm-thick tumor tissue sections were prepared by Bio-Check Laboratories Ltd. (New Taipei City, Taiwan, ROC). Subsequently, the sections were subjected to H&E staining (28). H&E staining, known for providing detailed visualization of cellular structures, was employed for comprehensive histopathological analysis. Two samples from each group, each assessed from four different perspectives, were evaluated by an experienced veterinary surgeon to assign severity scores.
Biochemistry analysis. After administering anesthesia, blood samples (n=3) were obtained from the inferior vena cava of mice on day 10. All experiments were repeated twice. The collected blood was allowed to clot, followed by centrifugation at 1,400×g for 20 min. The resulting supernatant serum was carefully collected and stored at −80°C for subsequent analysis. Alamine aminotransferase (AST), alanine aminotransferease (ALT), creatinine (CREA), and gamma-glutamyl transferase (γ-GT) levels in each serum sample were quantified using analytical services provided by Axel Biotech. Inc. (Taichung, Taiwan, ROC) (29).
Radiosensitivity index (RSI) analysis. A total of 426 patients’ RNA sequence data from TCGA-LIHC were utilized for RSI analysis. For this analysis, the patients’ expression levels of PKC-δ and P38 were categorized. The first quartile (lowest 50% of numbers, n=213) was defined as low expression, while the fourth quartile (highest 50% of numbers, n=213) was defined as high expression levels. The RSI was calculated using the formula present in Mohammadi et al. study (30).
Statistical analysis. The results are reported as mean±standard deviation. Statistical significance was determined using one-way analysis of variance (ANOVA) with GraphPad Prism version 7.0 (San Diego, CA, USA), where a significance level of p<0.05 was applied. To enhance robustness and reproducibility, all experiments were independently repeated at least two to three times.
Results
Quetiapine significantly enhanced the efficacy of radiotherapy in inhibiting the progression of HCC in vivo. In Figure 1A, we established a Hep3B xenograft mouse model, and intervention was divided into four groups: control, quetiapine (QUE), RT, and combination (Comb) (n=6/group). Tumors were inoculated into the right flank of the mice, and treatment commenced when the average tumor size reached 80-100 mm3. On day 0, defined as the treatment initiation date, a single radiotherapy (11) session was administered, with adequate whole-body shielding to limit exposure to the tumor region only. Quetiapine, dissolved in 0.1% DMSO, was administered via gavage once daily. As shown in Figure 1B, an image of an extracted tumor from day 16 demonstrated that the combination of quetiapine and RT effectively suppressed tumor progression. Figure 1C revealed the average volume of HCC progression during treatment, highlighting the superior inhibitory effect of quetiapine in combination with RT. Statistical analysis revealed a difference between the CTRL and combination groups starting from day 8, and a difference between monotherapy and combination therapy from day 12 onward (Table III). In Table IV, we observed that the combination group had the longest mean tumor growth Time (MTGT). The mean growth inhibition rate in the combination group was 4.61 times greater than that in the CTRL group. Furthermore, Figures 1D, E, F and G depicted the progression of HCC in individual mice under different conditions: CTRL, quetiapine, RT, and combination. Furthermore, we assessed the potential combination efficacy based on tumor volume in Table V. The observed growth inhibition rate exceeded our expectations, suggesting that the combination of quetiapine and RT exhibited a synergistic effect when compared to monotherapy (Combination index <1). The weight of the extracted tumors also confirmed the enhanced anti-HCC efficacy achieved with the combination of quetiapine and RT (Figure 1H). In summary, these results strongly suggest the enhanced efficacy of quetiapine when combined with RT in the HCC model. In other words, quetiapine can be utilized as a radiosensitizing agent to increase the sensitivity of HCC to RT.
Anti-HCC effects of quetiapine and radiotherapy (RT) in the Hep3B xenograft model. (A) The experimental procedure for the combination of quetiapine and RT in the Hep3B xenograft model is outlined. (B) Images of tumors taken on day 16, and (C) tumor volume during treatment are shown (D) Tumor progression patterns in the control (CTRL) group, (E) Quetiapine (QUE) treatment group, (F) RT treatment group, and (G) combination (Comb) treatment group are shown. (H) Quantitative analysis of the tumor weights in each group on day 16.
Statistical analysis of tumor volume in different treatment groups and dates.
Mean tumor growth time, delay time, and inhibition rate in Hep3B tumor bearing mice after treatment with different condition are showed.
Mean tumor growth inhibition rate and combination index in Hep3B tumor bearing mice after treatment with quetiapine, RT, the combination of both.
Combination of quetiapine and RT may not induce general toxicity in HCC model. To confirm whether the combination of quetiapine and RT is a safe strategy for HCC treatment, we initially assessed the pathological changes in each group using H&E staining. In Figure 2A, we observed only slight damage in the RT-treated group. Severity scores recorded by an experienced veterinary surgeon indicated that the combination group did not exhibit significant pathological alterations in the heart, liver, kidney, spleen, and small intestine (Table VI). Furthermore, liver function markers, such as AST and ALT levels in mice serum, were elevated in the RT group but decreased when combined with quetiapine (Figure 2B). Table VII shows that the γGT level, which represents liver function in mice serum, increased after RT treatment but decreased when combined with quetiapine. Additionally, the marker of kidney function (CREA) exhibited a similar induction pattern in the RT group but decreased in the combination treatment group. Notably, the body weight of the mice did not exhibit significant differences in all treatment groups (Figure 2C). In summary, while RT may induce minor pathological changes, combining it with quetiapine appears to mitigate these effects. These results suggest the potential safety of combining quetiapine with RT for HCC treatment.
Safety assessment of quetiapine in combination with radiotherapy (RT) in on Hep3B bearing mice. (A) Tissue pathology assessed by hematoxylin and eosin (H&E) staining (scale bar = 100 μm). (B) Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) in all treatment groups. (C) Changes in body weight of mice in each group during the treatment period.
Severity scores for pathological alterations after different treatment.
Serum levels of gamma glutamyl transpeptidase (γGT) and creatinine (CREA) from Hep3B tumor bearing mice is displayed.
Quetiapine combined with RT is associated with the inhibition of EGFR/FGFR-2/PKC-δ/P38 mediated metastasis proteins. After confirming the tumor inhibition effect of quetiapine combined with RT, our focus shifted to elucidate the potential mechanism underlying the anti-HCC activity. As depicted in Figure 3A, we observed a significant suppression in the expression of b-FGF, associated with FGFR-2 activation (31), in the combination of quetiapine and RT. Additionally, proteins linked to metastasis and angiogenesis, such as MMP-9 and VEGF-A (32), were markedly reduced in the combination group compared to the control or monotherapy. Furthermore, the phosphorylation of EGFR, FGFR-2, PKC-δ, and P38, which are upstream regulators of the aforementioned proteins, was effectively suppressed in the quetiapine and RT combination. The crucial transcription factor NF-κB, downstream of EGFR/FGFR-2/PKC-δ/P38 signaling (33), was also diminished by the combination of quetiapine and RT (Figure 3B). Interestingly, RT was found to up-regulate the expression of these proteins, a phenomenon that was mitigated by quetiapine. Quantification of these proteins is presented in the right panel of Figure 3A-B. The statistical analysis of each protein between different treatment group was presented in Table VIII and Table IX. Furthermore, our earlier studies demonstrated an increased RSI value in HCC patients with high expression of EGFR, ERK, and NF-κB (24). In Figure 3C, we also showed that HCC samples with high expression of PKC-δ, P38 and VEGF-A exhibit resistance to radiotherapy. In summary, the quetiapine-sensitized anti-HCC effect, when combined with RT, is associated with the inactivation of EGFR/FGFR-2/PKC-δ/P38/NF-κB-mediated signal transduction.
Attenuating effect of quetiapine on radiotherapy (RT)-induced epidermal growth factor receptor (EGFR)/fibroblast growth factor receptor 2 (FGFR-2)/protein kinase C delta (PKC-δ)/P38/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in hepatocellular carcinoma (HCC). (A) The expression pattern and quantification result of b-FGF, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor A (VEGF-A) after different treatments on Hep3B tumor tissue. (B) The expression pattern and quantification results of EGFR, FGFR-2, PKC-δ, P38 and NF-κB after different treatments on Hep3B tumor tissue. (C) The radiosensitivity index (RSI) analysis results from TCGA-LIHC dataset is showed.
Statistical analysis of angiogenesis related proteins in different treated groups.
Statistical analysis of signaling transduction-associated proteins in different treated groups.
Quetiapine combined with RT is associated with the inhibition of anti-apoptosis and the induction of apoptosis related proteins. Additionally, we evaluated whether the toxicity induced by the combination of quetiapine and RT is associated with the regulation of apoptosis-related mechanisms. As indicated in Figure 4A, the anti-apoptosis proteins, including C-FLIP and XIAP (34, 35), are effectively suppressed in combination of quetiapine and RT. In Figure 4B, a superior activation of caspase-3, -8, and -9 was found in the combination group compared to the monotherapy one. Moreover, mitochondria-mediated apoptosis factors, such as BAX and BAK (36), were also observed to be activated in the combination of quetiapine and RT. The statistical analysis of each protein between different treatment group was presented in Table X and Table XI. In summary, not only was the anti-apoptotic effect diminished by quetiapine combined with RT, but the accumulation of apoptosis induction factors was also increased (Figure 5).
The enhancing effect of quetiapine on radiotherapy (RT)-induced caspase-dependent apoptosis signaling in hepatocellular carcinoma (HCC). (A) The expression pattern and quantification result of cellular FLICE inhibitory protein (C-FLIP) and X-linked inhibitor of apoptosis protein (XIAP) after different treatments on Hep3B tumor tissue. (B) The expression pattern and quantification result of cleaved caspase-3, -8, -9, Bcl-2 associated X protein (BAX) and Bcl-2 homologous antagonist killer (BAK) after different treatments on Hep3B tumor tissue.
Statistical analysis of anti-apoptosis related proteins in different treated groups.
Statistical analysis of apoptosis related proteins in different treated groups.
Proposed mechanism of quetiapine sensitizing hepatocellular carcinoma (HCC) to radiotherapy (RT). Quetiapine diminishes RT-induced epidermal growth factor receptor (EGFR)/fibroblast growth factor receptor 2 (FGFR-2)-mediated signaling, leading to a decrease in metastasis, angiogenesis, and anti-apoptosis related proteins. Additionally, quetiapine enhances RT-mediated apoptosis induction through the activation of the caspase-dependent pathway.
Discussion
The correlation between the presence of psychiatric disorders and adverse outcomes in cancer patients undergoing radiotherapy was demonstrated. Interventions for psychiatric disorders were conducive to improving the quality of life (37, 38). In addition to addressing psychiatric disorders, antipsychotic medications have been investigated for their potential anti-cancer properties. The primary objective of our current investigation was to ascertain whether the antipsychotic quetiapine exhibits a radiosensitizing effect for HCC in vivo. The results showed that quetiapine treatment significantly enhanced the inhibitory effectiveness of radiation on the growth of HCC (Figure 1, Table III, Table IV, Table V).
Both EGFR and FGFR2 act as transmembrane receptor tyrosine kinases, facilitating the progression of HCC by activating downstream oncogenic pathways (31, 39). NF-κB, a critical transcription factor, plays a crucial role in driving effector molecules involved in tumor progression (40). The signaling from PKC-δ/P38 pathway contributes to the overall upregulation of NF-κB activity (41). The activation of the PKC-δ/P38 pathway may be associated with the phosphorylation of EGFR and FGFR-2 (33, 42).
High expression of both EGFR and NF-κB correlated with a diminished survival advantage of radiotherapy in HCC patients (24). The information retrieved from the TCGA databank revealed that heightened levels of both PKC-δ and P38 were linked to a reduced effectiveness of radiotherapy in improving survival outcomes for HCC patients (Figure 3C). FGFR2 was also reported to trigger the radioresistance of tumor cells (43). Our findings indicated that the administration of quetiapine resulted in a significant reduction in the radiation-augmented activation of the EGFR/FGFR-2/PKC-™δ/P38/NF-κB pathway (Figure 3A-B).
VEGF-A, MMP-9, C-FLIP, and XIAP are downstream molecules of NF-κB that promote tumor angiogenesis, metastasis, and inhibit apoptosis. Furthermore, the heightened expression of these molecules contributes to the radioresistance of tumor cells. Suppressing these molecules enhances the radiosensitivity of tumor cells (44-46). Our results demonstrated that quetiapine effectively decreased the protein expression of radiation-induced VEGF-A, MMP-9, C-FLIP, and XIAP (Figure 3A and Figure 4A).
Apoptosis is one of the mechanisms through which radiation induces the death of tumor cells. Radiosensitizers can enhance anticancer efficacy of radiation by inducing apoptosis (47-49). Our investigation revealed that the combined treatment group exhibited markedly elevated protein levels of cleaved caspase-3, -8, -9, BAX, and BAK in HCC tumor tissues compared to those in the groups treated with quetiapine or radiation alone (Figure 4B). Based on these results, it is suggested that the induction of apoptosis by quetiapine in combination with radiation involves both extrinsic and intrinsic pathways. Additionally, quetiapine has been reported to induce apoptosis in chemotherapy resistant tumor cells (50).
In conclusion, quetiapine, when used as a radiosensitizer, enhances the effectiveness of radiation in inhibiting tumor growth and inducing apoptosis in HCC in vivo (Figure 5). The anti-HCC efficacy of quetiapine in combination with radiation is associated with the inhibition of the EGFR/FGFR-2/PKC-δ/P38/NF-κB pathway and the induction of apoptosis.
Acknowledgements
Experiments and data analysis were partially conducted at the Medical Research Core Facilities Center, Office of Research & Development, China Medical University, Taichung, Taiwan, ROC.
Footnotes
Authors’ Contributions
JDY, YCL, HCW, FTH, TLL conducted all experiments, FTH, TLL performed statistical analyses, and summarized the data. MCH and JHC drafted the initial version of the paper. MCH and JHC conceptualized the presented idea, supervised the findings of this work, conducted the literature review, and prepared the final version of the paper.
Funding
The study received support from the Chang Bing Show Chwan Memorial Hospital, Changhua, Taiwan (ID: SRD-111012), National Science and Technology Council, Taipei, Taiwan (ID: NSTC 112-2314-B-758-001-MY3), National Yang-Ming Chiao Tung University Hospital, Yilan County, Taiwan (ID: RD2021-007 and RD2024-008), Cathay General Hospital, Taipei, Taiwan (ID: CGH-MR-A10916).
Conflicts of Interest
The Authors affirm that they have no financial interests that might be construed as conflicting with the findings or conclusions presented in this study.
- Received December 6, 2023.
- Revision received January 15, 2024.
- Accepted January 26, 2024.
- Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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).
