The inhibitory effect and mechanism of quetiapine on tumor progression in hepatocellular carcinoma in vivo

Abstract Hepatocellular carcinoma (HCC) is the primary tumor of the liver and the fourth leading cause of cancer‐related death. Recently, several studies indicated the anti‐tumor potential of antipsychotic medicine. Quetiapine, an atypical antipsychotic, is used to treat schizophrenia, bipolar disorder, and major depressive disorder since 1997. However, whether quetiapine may show potential to suppress HCC progression and its underlying mechanism is persisting unclear. Quetiapine has been shown to induce apoptosis and inhibit invasion ability in HCC in vitro. Here, we established two different HCC (Hep3B, SK‐Hep1) bearing animals to identify the treatment efficacy of quetiapine. Tumor growth, signaling transduction, and normal tissue pathology after quetiapine treatment were validated by caliper, bioluminescence image, immunohistochemistry (IHC), and hematoxylin and eosin staining, respectively. Quetiapine suppressed HCC progression in a dose‐dependent manner. Extracellular signal‐regulated kinases (ERKs) and Nuclear factor‐κB (NF‐κB) mediated downstream proteins, such as myeloid leukemia cell differentiation protein (MCL‐1), cellular FLICE‐inhibitory protein (C‐FLIP), X‐linked inhibitor of apoptosis protein (XIAP), Cyclin‐D1, matrix metallopeptidase 9 (MMP‐9), vascular endothelial growth factor‐A (VEGF‐A) and indoleamine 2,3‐dioxygenase (IDO) which involved in proliferation, survival, angiogenesis, invasion and anti‐tumor immunity were all decreased by quetiapine. In addition, extrinsic/intrinsic caspase‐dependent and caspase‐independent pathways, including cleaved caspase‐3, −8, and − 9 were increased by quetiapine. In sum, the tumor inhibition that results from quetiapine may associate with ERK and NF‐κB inactivation.

cells to death through targeting autophagy 7 as well as sensitizing bladder cancer to chemotherapy by inhibition of B-cell lymphomaextra-large (Bcl-xL) expression, respectively. 8 In addition, atypical antipsychotics such as risperidone and olanzapine have been demonstrated to trigger growth inhibition of colorectal and pancreatic cancer cells through induction of apoptosis and suppression of survivin expression. 3,9 Although numerous studies presented that antipsychotics show encouraging antitumor efficacy in cells and animal models, others provide tumor growth conflict evidence. 8,10,11 Antipsychotics such as olanzapine (3 mg/kg/day) and quetiapine (25 mg/kg/day) have been found to correlate with increased risk of liver cancer and thyroid adenoma in mice. 11 Previous studies indicated that suppression of extracellular signal-regulated kinases (ERK)/protein kinase B (AKT) signaling and induction of apoptosis were associated with quetiapine-inhibited cell survival and invasion in HCC in vitro. 4 However, whether quetiapine influences the progression of HCC in vivo has not yet been elucidated. Therefore, the main purpose of the present study was to verify the effect and mechanism of quetiapine in HCC in vivo.

| Hematoxylin and eosin stain of liver, spleen, kidney organs
Liver, spleen, kidney organs were extracted from mice and fixed by 10% neutral buffered formalin. Then, the tissue was embedded in paraffin and sliced for hematoxylin and eosin (H&E) and IHC staining. The embedded, sliced, and staining procedure of tissue was performed by Bond biotech, Inc. (Taichung, Taiwan). Finally, the pathology images of each group were captured by a light microscope (Nikon ECLIPSE Ti-U, Minato City, Tokyo, Japan) at Â100. 16

| Immunohistochemistry stain of Hep3B and SK-Hep1 tumor tissue
Tumor tissues were extracted from mice and fixed by 10% neutral buffered formalin. Embedded and sliced were conducted by Bond biotech, Inc. as well. Immunohistochemistry (IHC) staining procedure was followed by Tsai et al.'s study. 17 Quantification of IHC images were performed by using ImageJ with IHC Image Analysis Toolbox. At least five views from each group of IHC images were calculated.

| Statistical analysis
All quantitative data were presented as the mean ± SD from three independent experiments. One-way analysis of variance was used to compare the control and quetiapine-treated groups. The p-value smaller than .05 was defined as a significant difference between the control and quetiapine-treated groups.

| Quetiapine effectively suppressed Hep3B and SK-Hep1 tumor progression
To investigate whether quetiapine may have the potential to suppress HCC progression, we established Hep3B and SK-Hep1 bearing model. Figure 1A,B, the smallest Hep3B tumor on day 10 was found in the 20 mg/kg quetiapine treated group. Hep3B tumor inhibition potential was found in the 20 mg/kg quetiapine group since day2 after treatment ( Figure 1C). Moreover, tumor inhibition results of quetiapine were also validated in another SK-Hep1 bearing model.  3.2 | Quetiapine suppressed anti-apoptosis, proliferation, and metastasis-related proteins were associated with inactivation of ERK/NF-κB In Figure 2A Figure 2D). Quetiapine may suppress 0.5-0.9 times of MCL-1, C-FLIP, XIAP, and cyclinD1 protein expression level, the 20 mg/kg of quetiapine showed greater inhibition ability ( Figure 2E,F). Additionally, metastasisrelated proteins; including MMP-9 and VEGF were also decreased by 20 mg/kg quetiapine treatment about 0.6-0.9 times (Figure 2G-I). In sum, tumor anti-apoptosis, proliferation, and metastasis-related proteins were decreased by quetiapine.

As indicated in
3.3 | Quetiapine activated the protein expression of apoptosis related proteins and suppressed the protein expression of immunosuppressive proteins in Hep3B and SK-Hep1 bearing model As showed in Figure 3A, the apoptosis-related protein included caspase-dependent and caspase-independent molecules were all increased by quetiapine treatment. Cleaved caspase-3, caspase-8, and caspase-9 that involved in extrinsic and intrinsic caspase-dependent apoptotic pathway 22,23 were increased 1.2-1.8 times by quetiapine ( Figure 3B,C). Moreover, caspase-independent apoptosis-related protein EndoG 24 was also activated by quetiapine ( Figure 3A-C). We then identified whether tumor microenvironment-associated proteins such as IDO and PD-L1 may be altered by quetiapine. As illustrated in Figure 3D-F, though PD-L1 may be induced by quetiapine, IDO protein which suppresses activation of cytotoxicity T cells, was suppressed by quetiapine.

| Quetiapine did not induce tissue damage and body weight loss of Hep3B and SK-Hep1 bearing mice
As indicated in Figure 4A,B, the bodyweight of Hep3B and SK-Hep1 bearing mice were not found to be altered by quetiapine treatment. In addition, the pathology pattern of liver, spleen, and kidney of each treatment group in Hep3B and SK-Hep1 bearing mice had not been influencing by quetiapine treatment ( Figure 4C). In addition, we performed cleaved caspase-3 and ki-67 staining on mice liver, spleen, and kidney tissue. As indicated in Figure 4D-F, no obvious induction of apoptosis and proliferation effect were found in all treatment groups.
Overall results indicated that quetiapine had not induced tissue toxicity and body weight loss in HCC bearing mice.

| DISCUSSION
Use of quetiapine has been found to decrease the incidence of HCC with a dose-dependent trend when daily dose ≥30 mg. 3 In this study, we demonstrated the inhibitory effect of quetiapine on tumor progression in HCC in vivo. In addition, we also showed a potential anti-HCC mechanism of quetiapine.
For improvement of survival benefit of patients with HCC, more effective strategies such as immune checkpoint inhibitors therapy and tyrosine kinase inhibitors therapy have been developing. 25 Pembrolizumab, an anti-programmed death-1 (PD-1) antibody, In addition to induction of angiogenesis, VEGF and IDO contribute to tumor progression by attenuating anti-tumor immunity. 28,29 High VEGF expression evokes proliferation and expansion of immunosuppressive cells. Reduced VEGF level has been shown to enhance the anti-cancer efficacy of anti-PD-1 antibodies. 30 IDO mediates the conversion of tryptophan to kynurenine leading to suppression of CD8 + T cells activation. 31  Our data presented that quetiapine effectively inhibited the growth of HCC in vivo ( Figure 1). Furthermore, liver, kidney, spleen pathology alteration, and general toxicity were not induced by quetiapine treatment on Hep3B and SK-Hep1 bearing animals ( Figure 4). We also found protein levels of NF-κB p65 (Sre536), ERK1/2 (Thr202, Tyr204), MCL-1, C-FLIP, XIAP, Cyclin-D1, and MMP-9 were significantly reduced by quetiapine treatment (Figure 2A-F). Inhibition of anti-apoptotic proteins and induction of apoptosis by complementary agents have been presented to boost the anti-HCC efficacy of therapeutic agents. 38,39 In addition to the reduction of anti-apoptotic proteins, expression of apoptotic proteins cleaved-caspase-3, À8, À9, and Endo-G was significantly increased by quetiapine treatment (Figure 3A-C). Endo-G is a caspase-independent death effector to trigger nucleosome DNA fragmentation leading to tumor cell death. 40 Expression of Endo-G was deceased in human HCC tissues. 41 Our results in figure 3A-C indicated that quetiapine may induce Endo-G expression in HCC bearing mice tumor.
In conclusion, suppression of ERK/NF-κB signaling and induction of apoptosis through extrinsic/intrinsic caspase-dependent and caspase-independent pathways are involved in the quetiapineinhibited progression of HCC in vivo. According to the therapeutic efficacy and potential mechanism, we suggest that quetiapine as a complementary agent may provide therapeutic benefits to patients with HCC. Further clinical trial is warranty on the base of our preclinical and published retrospective national health database studies.