Thioridazine Sensitizes Esophageal Carcinoma Cell Lines to Radiotherapy-Induced Apoptosis In Vitro and In Vivo

Abstract


Abstract Background
Radiotherapy is one of the primary treatments for esophageal squamous cell carcinoma (ESCC). Identification of novel radio-sensitizing agents will improve the therapeutic outcome of radiotherapy. This study aimed to determine the radio-sensitizing effect of the antipsychotic agent thioridazine in ESCC and explored the underlying mechanisms.


Abstract Material/Methods
ECA-109 and TE-1 ESCC cells were treated with thioridazine and radiotherapy alone and in combination. Cell survival was measured by MTT assay. Cell cycle and apoptosis were monitored by flow cytometry. Western blot analysis was used to analyze the expression of phospho-PI3K, phosphor-AKT, phospho-mTOR, Caspase-3, Caspase-9, Bax, Bcl-2, Bal-xl, Bak, and p53. The xenograft mouse model was used to study the in vivo anticancer effect of thioridazine and irradiation.


Abstract Results
Combined treatment with thioridazine and irradiation significantly reduced viability of ESCC cells compared with thioridazine or irradiation treatment alone. Thioridazine and irradiation treatment induced G0/G1 phases cell cycle arrest through down-regulation of CDK4 and cyclinD1. In addition, thioridazine and irradiation treatment induced apoptosis through up-regulation of cleaved capase-3 and 9, as well as an increase in the expression of Bax and Bak and a decrease in the expression of Bcl-2 and Bcl-xl. Furthermore, thioridazine and irradiation treatment inhibited the PI3K-AKT-mTOR pathway and up-regulated the expression of p53. In xenograft mice, thioridazine and irradiation reduced ESCC tumor growth.


Abstract Conclusions
Thioridazine sensitizes ESCC cells to radiotherapy. Thioridazine may play a role in ESCC radiation therapy as a promising radiosensitizer.


Background
Background Esophageal cancer is the eighth most common cancer and the sixth most common cause of cancer deaths worldwide [ 1 ]. Esophageal squamous cell carcinoma (ESCC) is the major histopathological subtype of esophageal cancer [ 2 ]. Radiotherapy is an important form of nonsurgical management of ESCC [ 3 , 4 ]. Unfortunately, systemic radiotherapy alone is ineffective in patients with unresectable, recurrent, or metastatic ESCC [ 5 ]. Radiation-induced apoptosis is the major cell death mechanism involved in cancer radiotherapy. The evasion of apoptosis is a prominent hallmark of cancer, and induces resistance to radiotherapy [ 6 ]. Therefore, agents that inhibit apoptosis may be a novel strategy for antagonizing cancer resistance to radiotherapy. Thioridazine (10-[2-(1-methyl-2-piperidyl) ethyl]-2-(methylthio)phenothiazine) is a first-generation antipsychotic drug that has been used to treat psychotic disorders such as psychosis and schizophrenia [ 7 ]. Thioridazine has antimicrobial activity, and is thus used to treat antibiotic-resistant organisms such as Mycobacterium tuberculosis [ 8 ]. In addition, thioridazine has anticancer effects via its anti-proliferation and anti-survival activities [ 9 ]. Thioridazine also induces cell apoptosis in cervical cancer, endometrial cancer [ 10 ], ovarian cancer [ 11 ], activated B-cell subtype of diffuse B-cell lymphoma [ 12 ], neuroblastoma and glioma [ 13 ], gastric cancer [ 14 ], leukemia [ 15 ], and melanoma cells [ 16 ]. It has been reported that thioridazine induces apoptosis by targeting the PI3K-Akt-mTOR pathway [ 17 ]. Activation of the PI3K-Akt-mTOR pathway has been reported to contribute to resistance of esophageal cancer to several commonly used classes of chemotherapeutic agents [ 18 ]. Therefore, thioridazine is currently considered as a potential anticancer drug in chemotherapy or radiotherapy. Since high concentrations of thioridazine cause adverse effects such as dysrhythmia and sudden death, low concentrations of thioridazine may be favorable for thioridazine-based combination cancer therapy by reducing the occurrence of adverse effects and improving the anticancer effects. However, the role and mechanisms of thioridazine in radiation-induced apoptosis in ESCC remains unknown. In the current study, we explored the anticancer and radio-sensitizing effects of thioridazine in ESCC in vitro and in vivo and investigated the underlying molecular mechanisms.


Material and Methods
Material and Methods


Cell culture
Cell culture The ECA-109 and TE-1 ESCC cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 100 mg/mL of penicillin and streptomycin (Invitrogen, USA) in 5% CO2 at 37°C.


MTT assay
MTT assay MTT assay was performed to determine cell survival. Cells were seeded in 96-well plates at a density of 3000 cells per well. After culturing for 24 h, cells were treated with 0, 1, 5, 10, 15, 20, 25, and 30 μM thioridazine for 12 h. To investigate the effect of thioridazine and irradiation on cell proliferation, cells received X-ray irradiation for 12 h at a single dose of 2, 6, and 8 Gy after thioridazine treatment. Control dishes were sham-irradiated under the same conditions. MTT (3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma, USA) was added to each well at a final concentration of 0.5 mg per milliliter, and incubated for 4 h at 37°C. The supernatant was then removed and formazan precipitates were dissolved using 150 μl dimethyl sulfoxide. Absorbance was read at 570 nm wavelength. All experiments were repeated 3 times.


Flow cytometry
Flow cytometry Cell cycle analysis and quantification of cell apoptosis was performed by flow cytometry, as previously reported [ 19 ]. Briefly, cells were seeded in 96well plates at a density of 3000 cells per well and treated with 15 μM thioridazine, followed by 4-Gy irradiation. Cells were fixed in 2% paraformaldehyde, and then stained with an Annexin V-FITC Apoptosis Kit (Keygene Biotechnology). Data were acquired on a FACSCalibur flow cytometer (BD, USA) using Cell-Quest software (BD, Bioscience). For each experiment, 10 000 events per sample were recorded. All experiments were repeated 3 times.


Western blot
Western blot Cells were lysed with RIPA lysis buffer. Protein lysates (10 μL) were subjected to electrophoresis on 6–15% SDS-polyacrylamide gel (Beyotime Biotechnology) and transferred to Polyvinylidene fluoride Membranes (Millipore, Billerica, USA). The membranes were blocked in the solution containing 5% BSA and 1×PBS-0.1% Tween20. The membranes were incubated with primary antibodies overnight at 4°C followed by incubation with secondary antibodies at room temperature for 2 h. Primary antibodies used in this study were antibodies against Caspase-3, Caspase-9, Bax, Bcl-2, Bcl-xl, Bak, phospho-PI3K, phospho-AKT, phospho-mTOR, P53 (Cell Signaling Technology), or β-actin (Sigma-Aldrich, USA). Goat polyclonal anti-mouse IgG-horseradish peroxidase (HRP) or goat polyclonal anti-rabbit IgG-HRP were used as secondary antibodies. Bands were visualized by chemiluminescence detection kit (Pierce, USA). All experiments were repeated 3 times.


Mouse xenograft model
Mouse xenograft model Animal experiments were approved by the Ethics Committee of Anhui Medical University. BALB/c-nu/nu mice (female, 5–6 weeks old) were subcutaneously inoculated with 0.1 milliliter of ECA109 cells (1×107 cells per mL) at the right flank of the abdomen. When the tumors reached a volume of 100 mm3 after inoculation on day 10, the animals were randomly assigned into 4 groups (n=12 per group): the control group, the thioridazine group (THZ group), the irradiation (IR) group, and the THZ + IR group treated with THZ and IR. The mice in the control group were treated with PBS orally, whereas the mice in the THZ and THZ+IR groups were treated with oral administration of 25 mg per kg thioridazine every 3 days for 27 days. For mice in the IR and THZ+IR group, tumors were irradiated with an RS-2000 biological irradiator using X-rays at a single dose of 4 Gys at 2 Gee/min on day 11. Six mice were randomly selected from each group and used for measuring tumor volume. Tumors were measured using a caliper, and tumor volume (in mm3 ) was calculated using the following formula: tumor volume (mm3 )=(A×B2 )/2, where A is the larger diameter and Bis the smaller diameter of the tumor. The other 6 mice were used to observe the survival period. Tumors were then excised, frozen, and stored.


Data analysis and statistics
Data analysis and statistics Dose-response curves, combination indices, and dose reduction indices were generated using CompuSyn 1.0 software (Paramus, USA). Concentrations and corresponding effect levels for all data points were input to generate a complete report of analytic results. The effect of a treatment was expressed as a decimal between 0 (no effect) and 1 (100% effect). The combination index (CI) was used to determine whether the interaction of 2 or more drugs is synergistic, additive, or antagonistic as follows: CI=1, additive; CI < 1, synergistic; and CI > 1, antagonistic. The CI was calculated using the following equation: CI=((D1/Dx1)+(D2/Dx2)), D1 and D2 were the dose of drug 1 and drug 2 in combination treatment, respectively, and Dx1 and Dx2 were the dose of drug 1 and drug 2 used alone, respectively. Statistical analysis was performed using SSPS 13.0 software (SPSS Inc.) and Prism 5.0 software (GraphPad Prism). Data are presented as mean ±SD. Statistical comparisons between groups were performed using Student’s t-tests, and a P value of < 0.05 was considered to indicate statistical significance.


Results
Results


Thioridazine enhances radio-sensitivity of ESCC cells
Thioridazine enhances radio-sensitivity of ESCC cells MTT assay was used to evaluate the effects of thioridazine and irradiation on cell survival in ECA-109 and TE-1 cells. Thioridazine treatment inhibited cell viability of ECA-109 and TE-1 cells in a dose-dependent manner ( Figure 1A ) (P < 0.01). Irradiation alone inhibited cell viability of ECA-109 and TE-1 cells in a dose-dependent manner ( Figure 1B ). Combination treatment with both thioridazine and irradiation further reduced the percentage of cell viability of both cells ( Figure 1B, 1C ). The CI of thioridazine (15μM) and irradiation (4 Gy) were 0.71 in ECA-109 cells and 0.91 in TE-1 cells (CI≤1), respectively ( Figure 1D, 1E ), indicating that thioridazine and irradiation synergistically inhibited cell viability in ECA-109 and TE-1 cells.


Thioridazine and irradiation induces cell cycle arrest at the G0/G1 phases in ECA-109 and TE-1 cells
Thioridazine and irradiation induces cell cycle arrest at the G0/G1 phases in ECA-109 and TE-1 cells We further investigated the effect of thioridazine and irradiation on cell cycle progression in ECA-109 and TE-1 cells using flow cytometry. The percentage of ECA-109 cells and TE-1 cells at the G0/G1 phases was reduced after the treatment with thioridazine and irradiation alone compared with the control group ( Figure 2A–2D ). Combination treatment with thioridazine and irradiation significantly increased the percentage of cells at the G0/G1 phases compared with thioridazine and irradiation treatment alone ( Figure 2A–2D ). Western blot analysis showed that thioridazine and irradiation treatment alone significantly reduced the expression of CDK4 and cyclinD1 in ECA-109 and TE-1 cells. Combination treatment with thioridazine and irradiation significantly decreased the expression of CDK4 and cyclinD1 compared with thioridazine and irradiation treatment alone ( Figure 2E, 2F ).


Thioridazine and irradiation induces apoptosis in ECA-109 and TE-1 cells
Thioridazine and irradiation induces apoptosis in ECA-109 and TE-1 cells We investigated the effect of thioridazine and irradiation on cell apoptosis in ECA-109 and TE-1 cells using flow cytometry. Thioridazine and irradiation treatment alone significantly increased the percentage of apoptotic cells in ECA-109 and TE-1 cells compared with the control group ( Figure 3A–3D ). Combination treatment with thioridazine and irradiation significantly increased the percentage of apoptotic cells in ECA-109 and TE-1 cells compared with thioridazine and irradiation treatment alone ( Figure 3A–3D ). In addition, combination treatment with thioridazine and irradiation increased the expression of cleaved capase-3 and -9 in ECA-109 and TE-1 cells ( Figure 3E ). Furthermore, combination treatment with thioridazine and irradiation increased the expression of Bax and Bak, the pro-apoptotic proteins, and decreased the expression of Bcl-2 and Bcl-xl, the anti-apoptotic proteins, in ECA-109 and TE-1 cells ( Figure 3F ).


Thioridazine and irradiation treatment resulted in inhibition of the PI3K-AKT-mTOR pathway and up-regulation of P53 expression in ECA-109 and TE-1 cells
Thioridazine and irradiation treatment resulted in inhibition of the PI3K-AKT-mTOR pathway and up-regulation of P53 expression in ECA-109 and TE-1 cells Western blot analysis was used to evaluate the effect of thioridazine and irradiation on activation of the PI3K-AKT-mTOR pathway and the expression of P53 in ECA-109 and TE-1 cells ( Figure 4A–4D ). Compared with the control, the expression of phosphorylated PI3K, AKT and mTOR was significantly lower in ECA-109 and TE-1 cells treated with thioridazine ( Figure 4 ). Irradiation did not significantly alter the expression of phosphorylated PI3K, AKT, and mTOR ( Figure 4 ). Combination treatment with thioridazine and irradiation significantly inhibited the expression of phosphorylated PI3K, AKT, and mTOR compared with the treatment with thioridazine or irradiation alone ( Figure 4 ). In addition, compared with the control, the expression of P53 was significantly increased in cells treated with thioridazine or irradiation ( Figure 4 ). Combination treatment with thioridazine and irradiation significantly increased the expression of p53 compared with the treatment with thioridazine or irradiation alone ( Figure 4 ).


Thioridazine and irradiation reduced ESCC tumor growth in xenograft mice
Thioridazine and irradiation reduced ESCC tumor growth in xenograft mice We further studied the effect of thioridazine and irradiation on tumor growth in nude mice transplanted with ECA-109 cells. The tumor volume was significantly less in the THZ and RT group than in the control group. The tumor volume was significantly less in the THZ + RT group than in the THZ group and the RT group ( Figure 5A ). More mice survived in the THZ and RT groups than in the control group. More mice survived in the THZ + RT group than in the THZ or RT groups ( Figure 5B ).


Discussion
Discussion Currently, although chemo-radiotherapy has significantly improved the treatment effects for unresectable esophageal cancer, radio-resistance limits the utilization of radiotherapy [ 20 , 21 ]. This unfavorable therapeutic outcomes is due to radio-resistance related to various biological factors, while increasing radiation may induce the increasing occurrence of adverse events, including bleeding, perforation, radiation esophagitis and pneumonitis, and other adverse effects [ 22 ]. Complicated molecular mechanisms could include high activity of Akt, overexpression of Bcl-2 and Bcl-xL, and defects in the release of mitochondrial proteins, and unclear mechanisms are involved in radio-resistance in various cancer cell types [ 23 25 ]. Therefore, a novel radiosensitizer interrupting the tumor signal pathway followed by synchronous radiotherapy is urgently required to improve the curative effect of radiotherapy, with minimal adverse effects. Thioridazine is an antipsychotic drug widely used to treat schizophrenia and psychosis. Recently, it has been reported that thioridazine suppresses cancer cell growth by inhibiting proliferation, inducing apoptosis, and regulating the activity of the anti-apoptosis signaling pathway [ 10 14 ]. Thioridazine can significantly enhance the anti-proliferative activity of doxorubicin in treating breast cancer cells and cancer stem cell-like cells, which shows that thioridazine has significant chemo-sensitizing ability [ 26 , 27 ]. Moreover, thioridazine can increase the TRAIL-mediated apoptosis via the ROS mediated inhibition of Akt signaling and down-regulation of Mcl-1 and c-FLIP(L) [ 28 ]. Based on these studies, we propose that thioridazine should be developed as an effective radiosensitizer and used for cancer radiotherapy. The radiotherapy reaction of cancer cells may depend on the phase in the cell cycle [ 29 , 30 ]. Tumor cells are least sensitive in S phase. In the present study, we found that thioridazine combined irradiation inhibited proliferation of ECA-109 and TE-1 cells by inducing a G0/G1 phase cell cycle arrest. Cyclin D1 and cyclin-dependent kinase 4 (CDK4) have been reported to be involved in the regulation of cell proliferation by mediating the G1 to S phase transition [ 31 ]. We found that combination treatment decreased the expression of CDK4 and cyclinD1 in both esophagus cancer cells compared with thioridazine and irradiation treatment alone, indicating that thioridazine can enhance the irradiation-mediated cell suppression via cell cycle arrest. Radiotherapy plays a paramount role in the treatment of solid tumors, largely by inducing cell apoptosis, mainly through the intrinsic pathway [ 32 ]. However, radiotherapy resistance in various cancers present a huge challenge in clinical treatment. Research has demonstrated that activation of PI3K-Akt-mTOR and overexpression of Bcl-2 and Bcl-XL, important members of Bcl-2 family involving in intrinsic apoptosis pathway and frequently overexpressed in human tumors, are vital molecular mechanisms in radiotherapy resistance [ 33 , 34 ]. The intrinsic pathway of apoptosis starts with BH3-only protein activation, which inhibits the pro-survival Bcl-2-like proteins, followed by activation of pro-apoptotic proteins BAX and BAK. BAX and BAK begin to form the oligomers, destroy the mitochondrial outer membrane, and promote cytochrome c release, which activate caspase-9 to cleaved caspase-9, depending on activated APAF1. Therefore, caspase-3 is activated and followed by apoptosis [ 35 , 36 ]. Recent research suggests that in head and neck squamous cell cancer (HNSCC) and small-cell lung cancer(SCLC), the combination of radiation and inhibitor of anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL induced more apoptosis than the summation of their separate effects, which means down-regulation of Bcl-2 and Bcl-xL possess the capacity to radio-sensitize cancer cells [ 24 , 37 , 38 ]. In the current study, compared with thioridazine and radiation treatment alone, the combined treatment with thioridazine and irradiation dramatically induced the cleavage of caspase-9 and caspase-3, which was consistent with the annexin V-FITC/PI double-labeled flow cytometry analysis, suggesting that thioridazine and radiation induce apoptosis via the mitochondrial-mediated intrinsic pathway. We found that thioridazine and radiation co-treatment reduced the level of Bcl-2/Bcl-xL with concomitantly elevated expression of pro-apoptotic Bax/Bak in both TE-1 and ECA-109 cells, suggesting that cell apoptosis induced by thioridazine and radiation may be partly facilitated by regulating the proportion of Bax, Bcl-2, and Bcl-xL proteins. The PI3K-Akt-mTOR pathway plays a pivotal role in cell growth, proliferation, and metabolism, which is dysregulated in a variety of cancers [ 39 , 40 ]. Recent advances have also demonstrated that PI3K-Akt activity contributes to cancer radiotherapy resistance, mainly through 3 mechanisms: intrinsic radio-resistance, hypoxia, and tumor-cell proliferation [ 41 , 42 ]. PI3K-Akt-mTOR inhibitors, including single and dual inhibitors, sensitize various cancer types to radiotherapy, which is a promising approach for cancer treatments. LY294002 and wortmannin, well-studied specific PI3K inhibitors, respectively sensitize cancer cells to radiation through inactivation of PKB and lead to inhibition of cellular DNA-PK. Palomid 529 (Akt inhibitor), RAD001 (mTOR inhibitor), and BEZ235 (dual PI3K/mTOR inhibitor) lead to more apoptosis and DNA double-strand breaks, increased apoptosis, and decreased number of tumor blood vessels, cell death in a PTEN-independent manner, and autophagic cell death [ 42 44 ]. Thioridazine has been shown to have pro-apoptosis effects on cervical and endometrial cells, possibly by inhibition of PI3K-Akt-mTOR-p70S6K signaling, accompanied by the inhibition of tumor growth and metastasis in vivo by cervical, endometrial, and melanoma tumor xenografts in mice [ 45 , 46 ]. Moreover, evidence shows that thioridazine exerts anti-angiogenic activity by inhibiting VEGFR2-PI3K-mTOR signal transduction, and induced G0/G1 arrest and cancer cell apoptosis [ 47 , 48 ]. In the present study, we show that thioridazine can inhibit the PI3K/Akt/mTOR pathway, and, combined with radiation, can significantly increase the expression of p53 compared with the treatment of thioridazine. Our results suggest that thioridazine may increase sensitivity of esophagus cancer cells to radiation via inhibiting the PI3K/mTOR pathway.


Conclusions
Conclusions To confirm the anti-tumor effect of combination therapy with thioridazine and irradiation in vitro , we found this higher activity after combination therapy was also supported in a tumor xenograft model. Tumor volume was significantly reduced in the combination group after the treatment, and survival time was much longer than in the control group. Our results suggest that thioridazine is a promising candidate for radiotherapy sensitization in ESCC treatment.


Source of support: Departmental sources


Metadata
Authors
Hongxia Li, Li Juan, Leiming Xia, Yi Wang, Yangyi Bao, Guoping Sun
Journal
Medical Science Monitor : International Medical Journal of Experimental and Clinical Research
Publisher
International Scientific Literature, Inc.
Date
Med_Sci_Monit_2016_Jul_25_4f87
PM Id
27453171
PMC Id
4970441
Images
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