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G1 arrest and apoptosis
Biomedicine & Pharmacotherapy 121 (2020) 109598
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Penfluridol: An antipsychotic agent suppresses lung cancer cell growth and metastasis by inducing G0/G1 arrest and apoptosis
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Qiang Xuea,1, Zhihao Liua,1, Zhanzhan Fenga, Ying Xua, Weiqiong Zuoa, Qianqian Wanga, Tiantao Gaoa, Jun Zenga, Xi Hua, Fanfan Jiaa, Yongxia Zhub, Yong Xiaa,*, Luoting Yua,* a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biothrapy, Chengdu, 610041, China b Department of Obstetrics and Gynecology, Henan Provincial People’s Hospital, People's Hospital of Zhengzhou University, People's Hospital of Henan University, Zhengzhou 450003, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Penfluridol Lung cancer Cell cycle arrest Apoptosis Metastasis
Lung cancer remains the leading cause of cancer mortality because of highly malignant and metastatic potential. The current status of lung cancer treatment is limited, and more treatment options are needed. Interesting, antipsychotic drugs have been reported to show anti-cancer effects. In this present study, we investigated the anticancer potential of penfluridol (PF), an anti-schizophrenic drug, in lung cancer and its underlying mechanism in vitro and in vivo. In vitro, it could inhibit the viability of various lung cancer cells with G0/G1 phase arrest via increasing the expression level of p21/p27 and decreasing the expression levels of cyclin-CDK complex. Meanwhile, cell-cycle arrest causes DNA repair in the nucleus, which was associated with the upregulation of H2A.X and p-H2A.X. Moreover, PF could also decrease mitochondrial membrane potential and increase reactive oxygen species levels in the lung cancer cells. These results implied that PF might induce the mitochondriamediated intrinsic apoptosis. In addition, PF inhibits the migration and invasion of lung cancer cells via downregulation of FAK-MMP signaling. In vivo, oral administration of PF at concentration of 10 mg/kg inhibited tumor growth in A549 xenograft model. Notably, PF is an approved drug and the price is exceedingly cheap, so this study demonstrates the potential of PF to treat lung cancer.
1. Introduction Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer mortality [1]. It is divided into two types. One is the small cell lung cancer (SCLC), which accounts for about 15% of all lung cancers, and the other one is non-small cell lung cancer (NSCLC), which accounts for about 85% [2–5]. Although there have been advances in the treatment of lung cancer, such as the targeted therapies with EGFR inhibitors and ALK inhibitors, and immunotherapy using PD1/PD-L1 antibodies at present, many patients do not respond to the treatment. Therefore, finding new therapeutic drugs for lung cancer patients is still necessary [6–8]. Because of some major problems including high failure rates and withdrawal risks in new anticancer drug discovery and development, drug repurposing has been drawn lots of attentions from both academic institution and pharmaceutical industry and become a new option to
find new therapeutic drugs. The studies recently showed that schizophrenia patients were tended to have lower risk at cancer after antipsychotic treatment [9], such as chlorpromazine and trifluoperazine [10–12], indicating that antipsychotic can be used in the treatment of cancer patients. Notably, pentafluridol (PF), an antipsychotic agent, showed antitumor activity in breast cancer [13,14]. However, it has been reported to have a relatively limited amount of anticancer activities. Restricted growth of every cancer lies that the tumor cell and its progeny into controlled expansion and invasion. One of suppressed neoplastic progression is that deregulated cell proliferation and cellcycle arrest. Disrupting tumor cell cycle can suppress cell proliferation and induce cell apoptosis [15]. Cell apoptosis is a programmed cell death, which is the response of somatic cells to many forms of stress and damage, especially the DNA damage [16]. Cell-cycle arrest causes DNA repair, repairing unsuccessful cells enter the apoptosis program. H2A.X
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Corresponding authors at: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biothrapy, Chengdu 610041, China. E-mail addresses: [email protected] (Y. Xia), [email protected] (L. Yu). 1 These authors have contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109598 Received 18 September 2019; Received in revised form 16 October 2019; Accepted 26 October 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Fig. 1. The anti-proliferation effects of penfluridol against lung cancer cells. (A) Chemical structure of PF. (B) Several solid tumor cell lines were treated with PF for 72 h, cell viability was tested by MTT assay. Values were plotted as mean ± SEM. (C) Inhibitory effects of various concentrations PF on the three lung cancer cells after (24, 48 and 72 h) the treatment (IC50 values, μM). Values were plotted as mean ± SEM from 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. (D) The effects of PF on colony formation in A549 and LL2 cells for 6–8 days (20×). Values were plotted as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
metastasis of tumor cells. PF treatment also suppressed the migration and invasion of lung cancer cells via downregulation of FAK-MMP signaling [22,23]. In vivo, we found that PF suppressed tumor growth in A549 cells xenograft mouse model. We obtained that penfluridol may offer treatment goal against lung cancer. This is the first study to introduce lung cancer after penfluridol treatment, which provides a potential drug candidate for the treatment of lung cancer.
is a variant of the histone H2A family and is thought to be involved in DNA repair in the nucleus [17,18]. A growing number of published data indicate that H2A.X is dependent on its C-terminal phosphorylation to regulate apoptosis of cancer cells [19–21]. Targeting cell cycle and apoptosis pathways is promising, and there are already some targeted drugs. The aim of this study is repurposing the PF for treating lung cancer. Our data provided evidence that PF against lung cancer cells growth and the possible underlying mechanism of cell death. PF treatment could inhibit lung cancer cells’ growth by induce G0/G1 cell cycle arrest and cell apoptosis. Mitochondria-mediated apoptosis pathway may be the way of PF-induced apoptosis. Moreover, the decreasing protein expression levels of signaling pathways were found after PF treatment, such as AKT and NF-kB. Through elevated protein levels of H2A.X and p-H2A.X, we found that cell death also through DNA repair pathway [20,21]. Focal Adhesion Kinase (FAK) has been shown to play an important role in the progression of tumors to a malignant phenotype. Therefore, blocking the expression of FAK can inhibit the invasion and
2. Materials and methods 2.1. Materials Penfluridol (PF) was obtained from the AstaTech BioPharmaceutical Corp (Chengdu, China). And for in vitro experiments, PF dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and stored at -20℃. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and the Annexin V-FITC apoptosis detection kits 2
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Fig. 2. Induction of G0/G1 phase arrest by PF. (A, B and E, F) A549 and H446 were treated with different concentrations of PF (0, 2.5, 5 and 10 μM) for 36 h and 12 h, respectively. Then cell-cycle distribution analyzed by FCM. Values were plotted as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (C–D and G–H) PF treatment (24 h) on A549 and LL2 cells influenced the expression of cell cycle-related proteins, such as p21, p27, CKD2, cyclin D1, cyclinE and β-actin were employed as a standard. Values are mean ± SEM. *p < 0.05. Figs. 3 and 4. Induction of apoptosis by PF.
against MMP9, FAK and p-FAK were purchased from Abcam (Cambridge, England). The other antibodies were purchased from Cell Signaling Technology Company (Beverly, MA, USA).
were purchased from KeyGEN Biology Co. Ltd (Nanjing, China). Rhodamine-123 (Rh123), propidium iodide(PI) and Hoechst 33342 were purchased from Sigma (St Louis, MO, USA). The antibodies against cyclinE, Bcl2 and Bax were purchased from BD Bioscience (Franklin, NJ, USA), the antibodies against p21, IKBa and p-IKBa were purchased from Wanlei Bio (Shenyang, China), and the antibodies 3
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Fig. 3. (A) A549 and H446 cells were treated with 10 μM PF for 24 h and analyzed by light microscopy (scale bars represent 500 μM). (B) Percentages of the number of viable cells compared to vehicle after PF treatment. ***p < 0.001. (C) Two lung cancer cells were treated with various concentration of PF (0, 5 and 10 μM) for 24 h, the cells nuclei were stained with Hoechst 33,342 and observed by fluorescence microscope (scale bars represent 20 μM). (D) Percentages of intact nucleus compared to vehicle. *p < 0.05; **p < 0.01; ***p < 0.001.
measured by ACEA NovoCyte and analyzed with NovoExpress software (Agilent Biosciences, CA, USA). For the apoptosis assay, lung cancer cells apoptosis induced by PF and were quantified with the apoptosis detection kit. Cells were harvested and incubated with Annexin V-FITC for 15 min in the dark and incubated with PI for 15 min, then was detected and analysed.
2.2. Cell lines Human lung cancer cell line A549, H446, H1993 and SPC-A1, mouse cell line LL2 were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM or RPMI 1640 media supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin and streptomycin) under condition with 5% CO2 at 37 °C.
2.6. Measurement of ΔΨm and ROS levels in cells
2.3. Cell viability and colony formation assay
Cell mitochondrial membrane potential (ΔΨm) was measured by FCM with Rh123 (5 μg/ml) staining. After treatment with PF, cells were stained with Rh123 for 30 min in the incubator. Cells were harvested and washed with PBS. Then it was measured by ACEA NovoCyte. Reactive oxygen species(ROS) was measured with DCFH-DA (10 μM) staining in cells. After treatment with PF for different hours, we obtain cells and measured using FCM.
The effect of PF on cell viability was measured by MTT assay and colony formation assay assays [24,25]. In the cell viability test, cancer cells (1.5–8 × 103 cells/well) were seeded in 96-well plates and cultured for 24 h, medium containing various concentrations of PF were added and incubated separately for 24, 48 and 72 h. Then the absorbance was measured with Spectra MAX M5 microplate spectrophotometer (Molecular Devices, CA, USA). In the colony formation test, the lung cancer cells (600–800 cells/ well) were inoculated in 6-well plates and cultured for 24 h. Then various concentrations of the PF with 2 mL medium were added and incubated 8 days. After fixed with 4% paraformldehyde and stained with crystal violet solution, the cells were observed and counted under the microscope.
2.7. Western blotting analysis The western blotting analysis was performed as described previously [26,27]. After exposure to various concentrations of PF for the indicated time, cells were washed with PBS and lysed in RIPA buffer (Beyotime, Beijing, China). The protein concentration was measured by the BCA method [28]. Equal amounts of total protein from each sample were subjected to SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Massachusetts, USA). After blocking with 5% fat-free milk in TBS/T for 1.5 h at room temperature, the membranes were incubated with primary and secondary antibodies. Then the immunoreactive protein bands were detected.
2.4. Nuclear morphology analysis The lung cancer cells (1–2 × 105 cells/well) were inoculated in 6well plates and cultured for 24 h. Then added various concentrations of the PF with 2 mL and incubated 24 h. After the cells were washed with PBS and fixed with 4% paraformldehyde, the cells were stained by the Hoechst 33342 (10 μg/mL) for 15 min in the dark and then was contained by fluorescence quencher for 12 h in dark.
2.8. Scratch assay in vitro Lung cancer cells were seeded in a 24-well plate. When cells grew to 80% confluence, we scraped the monolayer cells by 10 μL sterile tips, then added different concentrations of PF. Wound width was measured at 0 h, 12 h and 24 h after the PF treatment by photography. Images were acquired using a general microscope.
2.5. Cell cycle and apoptosis analysis by flow cytometry (FCM) After treated with PF for the indicated time, the cells were harvested, stained with PI for 15 min in dark. Cell cycle distribution was 4
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Fig. 4. (A) FCM analysis of apoptosis cells stained with Annexin V- FITC/PI after treatment PF and DMSO for 24/48 h. Quantified values are shown on the right. Values were plotted as mean ± SEM. **p < 0.01; ***p < 0.001. (B-G) PF treated of the three lung cancer cells for the expression of apoptotic-related proteins, including procaspase-3, cleaved caspase-3, Bax and Bcl-2. Values were plotted as mean ± SEM. *p < 0.05; **p < 0.01.
2.9. Tanswell migration and invasion analysis
2.10. Xenograft mouse model
In the migration analysis, cells (3 × 104 -1 × 105) in serum-free medium were added in the upper chamber, and medium containing 10% FBS was added at the bottom with different concentrations of PF. After 24 h, cells were fixed with 4% paraformldehyde and washed with PBS. Then cells were stained with crystal violet solution for 20 min, viewed under a microscope and counted. In the invasion assay, the upper surface of the transwell was coated with matrigel (BD Biosciences, NJ, USA). The transwell was placed in the incubator and the matrigel was solidified. After cells were hatched with different concentrations of PF for 24 h, cells were fixed and stained, washed twice with cold PBS. At the end, cells were counted by microscope.
The animal experiments were approved and conducted in strict by the Institutional Animal Care and Treatment Committee of Sichuan University, China. And Female BALB/c nude mice (6–8 weeks old) were purchased from HFK bioscience CO, LTD, Beijing, China. Exponentially growing the lung cancer cell of A549 were harvested, a suspension of 100 μL containing 5 × 107 cells was injected in the right flank. The mice were divided into 2 groups (6 mice per group) at random when the tumor volume was about 100–200 mm³. PF (10 mg/kg) or vehicle (water / ethanol / PEG 400 / 2% acetic acid in 8:3:3:1) was administered at the indicated doses once a day by oral gavage. Tumor volume measured and body weight of the mice were recorded every three days during PF treatment. The tumor volume was calculated using the formula [length×(width)2/2]. Experiment was terminated at day 26 by 5
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Fig. 5. PF-induced apoptosis via mitochondria-mediated apoptotic and blocks cell survival signaling pathways. (A) Effects of PF reduced the mitochondrial membrane potential (ΔΨm) in A549, H446 and LL2 cells. Flow cytometry analysis was performed after treatment with PF (24 h). Values were plotted as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (B) ROS levels increased in A549 and H446 cells after treatment with different dose of PF (24 h). Values were plotted as mean ± SEM. **p < 0.01; ***p < 0.001. (C and E) A549 and LL2 cells were treated with PF for 24 h and the expression of related proteins was analyzed by western blot analysis. They were p-IKBa, IKBa, p-AKT, AKT, p-H2A.X, H2A.X and GAPDH as internal control. (D and F) Relative proteins expressions were quantified by Image J software. Values are mean ± SEM. *p < 0.05; **p < 0.01.
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Fig. 6. PF impaired cellular migration and invasion in vitro. (A)Compared with the control group, PF inhibited obviously wound healing of A549 cells. Quantified dates is shown on the right. Experiments were repeated twice. Values were plotted as mean ± SEM. *p < 0.05; ***p < 0.001. (B) Western blot analysed the migration and invasion proteins for FAK, p-FAK, MMP 9 and GAPDH following the treatment of PF in A549 cells. The graphic data shown are mean ± SEM. *p < 0.05 (C) PF inhibited A549 cells migration in the transwell migration assay. 3 × 104 A549 cell were seeded in the top chamber of transwell with serum-free medium and treated with DMSO or PF. After 24 h, migrated cells were stained, then pictures were taken with a microscope. Values were plotted as mean ± SEM. **p < 0.01; ***p < 0.001. (D) PF inhibited A549 cell invasion. 1 × 105 (invasion) A549 cells were treated with PF and could cross the matrigel as well as the transwell membrancege. Values were plotted as mean ± SEM. ***p < 0.001.
72 h obviously with the IC50 (half-maximal inhibitory concentration) values less than 12 μM (Fig. 1B). Then we chose human lung cancer cell lines A549, H446 and mouse cell line LL2 for further studies. We researched the time- and concentration- dependent effects of PF on three cells viability, respectively. As a result, increasing PF concentration and longer time could suppressed cell viabilities (Fig. 1C). It was indicated that PF could suppress lung cancer cells viability in a time- and dose- dependent manner. Nest, we performed colony formation assay to further studied the effects of PF on lung cancer cell proliferation. It can be seen that PF could suppress cells viability in a dose dependent manner (Fig. 1D). And quantity and group of the colony formation of lung cancer cells were significantly inhibited by PF.
euthanizing mice. The mice tumors and organs were extracted. Tumors were used for Western blot analysis and IHC analysis. 2.11. Toxicity evaluation BALB/c nude mice were randomly divided into two groups (n = 6). Animals were given PF (10 mg/kg) or vehicle by oral gavage. During PF treatment, the mice were observed for general signs of toxicity every day. On the 26th day, we extract the eyeball blood, heart, liver, spleen, lung and kidney from all mice. Then they were determined with biochemical analysis. 2.12. Statistical analyses All datas were analyzed by GraphPad Prism 5.01 software. The data was shown as mean ± SEM. The statistical comparisons were made by Student’s t-test There is a statistically significant p values, *p < 0.05; **p < 0.01; ***p < 0.001.
3.2. Induction of G0/G1 phase arrest by PF We next assessed whether PF could effect the cell cycle distribution by flow cytometry analysis. As shown in Fig. 2A/B, after incubation with concentration- dependent manner of PF, G0/G1 phase arrest was induced. When the A549 cells were treated with vehicle for 36 h, the G0/G1 phase arrest rate was 52.21%, whereas the arrest rate increased to 54.45%, 58.70% and 71.10% when cells were treated with 2.5 μM, 5 μM and 10 μM of PF. Similar results were obtained in H446 cells (Fig. 2E/F) and A549 cells for 24 h (Supplementary Figures A). As we all know, p21 and p27 are important members of the family of cyclin-dependent kinase inhibitors, and p21 is the downstream signal molecule of p53. p21/p27 mediates inhibition of cyclin-CDK complex
3. Results 3.1. The anti-proliferation effects of penfluridol against lung cancer cells Recently, several drugs repurposing were discovered in lung cancer, and it is a way to treat lung cancer [14]. We investigated the proliferation inhibition caused by PF treatment on several lung cancer cell lines. The results indicated that PF could increased tumor cell death for 7
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Fig. 7. Anti-tumor efficacy of PF in xenograft mouse model and safety profile. (A) Tumor pictures from control and penfluridol-treated mice. (B and C) Tumor volume of the mice were measured every 3 days and tumors were weighed once in the control and PF group. Values were plotted as mean ± SEM. (n = 6). *p < 0.05. (D) Tumor tissues from A549 xenograft treated with control or PF-treated for 26 days were immunohistochemically analyzed with anti-Ki-67, anticleaved caspase-3 and anti-p-AKT antibodies. (E) The tumor tissue extracts proteins from control and PF-treated mice, and the expression of related proteins was analyzed by western blot analysis.They were p-H2A.X, cleaved caspase-3, cyclin E, p-AKT and MMP 9. GAPDH was used to verify equivalent loading of protein. Quantitative analysis is below. Values were plotted as mean ± SEM.
treatment, we investigated the levels of cell morphology by microscope. As shown in Fig. 3A, both A549 and H446 cells showed cell shrinkage which represent features of apoptosis after PF treatment. Fig. 3B shows percentages of the number of viable cells compared to vehicle after PF treatment. Then as Fig. 3C indicated, Hoechst 33342 staining assay also showed that PF treatment induced apoptosis in A549 and H446 cells. Fig. 3D shows percentages of intact nucleus compared to vehicle. We can see that condensed nuclei of bright blue fluorescent, reduction of cell volume and nuclear fragmentation after PF treatment, demonstrating the features of cell. We further studied whether PF treatment could induce apoptosis in A549, H446 and LL2 cells, the AnnexinV-FITC/PI dual-labeling was
expression levels, resulting in cell-cycle arrest [29,30]. Moreover, we examined protein expression levels in three lung cancer cells after PF treated by western blotting analysis. As shown in Fig. 2C/D, the expression of cyclin D1 and cyclin E decreased while that of p27 increased in A549 with a concentration-dependent manner. Similar results were obtained in LL2 and H446 cells (Fig. 2G/H and Supplementary Figures B). We did that G0/G1 phase arrest was induced with PF treated by protein expression levels.
3.3. Induction of apoptosis by PF To confirm the induction of apoptosis in lung cancer cells with PF 8
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is consistent with its effects in vitro, a xenograft mouse model was established. A549 cells were injected into the right flank of BALB/c nude mice. The mice received the following treatments: vehicle and penfluridol at 10 mg/kg. Four weeks after oral administration in mice, the tumors of the mice in PF-treated group were significantly smaller than those in the control group (Fig. 7A and B). Furthermore, a significant tumor weight slide was also observed (Fig. 7C). To validate the results of the in vivo assays, we investigated the expression of proliferation, apoptosis and migration markers using immunoblotting and IHC analyses of the A549 tumor samples. The tumor sections were shown Ki-67-positive cells declined, whereas the cleaved caspase-3 positive cells increased. Besides, the number of pAKT-positive cells in PF treatment was less compared with vehicle group (Fig. 7D). Furthermore, the immunoblotting results showed that the expression of p-H2A.X and cleaved caspase-3 were increased, cyclin E and p-AKT were markedly inhibited in the PF-treated group (Fig. 7E). Overall, these findings suggested that PF blocked proliferation and metastasis of lung cancer by regulating AKT and MMP signaling. Besides, during the treatment of A549 xenograft in BALB/c nude mice, we did not observe side effect. there were no significant changes in hematological, serum biochemical parameters (Supplementary Figures F) and pathological changes of main organs (Supplementary Figures G), suggesting the mice were tolerant to PF treatment.
performed. As shown in Fig. 4A, the effect of apoptosis was apparently exhibited. When the A549 cells were treated with vehicle, the apoptosis rate was 4.74%, whereas the apoptosis cells increased to 17.64%, 26.16% and 44.54% when cells were treated with 2.5 μM, 5 μM and 10 μM PF, respectively. Similar results were obtained in H446 and LL2 cells in a concentration-dependent manner. Moreover, we studied protein expression levels of apoptosis by western blotting analysis, such as Bcl-2, cleaved caspase-3, procaspase-3 and Bax (Figs. 4B/C, D/E and 3 F/G). And these proteins play important roles in apoptosis. 3.4. PF-induced apoptosis via mitochondria-mediated apoptotic and blocks cells survival signaling pathways As shown in Fig. 4, with the protein expression levels of apoptosis, we suggesting that PF-induced apoptosis might via the mitochondriamediated apoptotic pathway. In order to verify the hypothesis, A549 and H446 cells was colored with a green fluorochrome Rh123 to measure the changes in the mitochondrial membrane potential (ΔΨm). We observed that a significant loss of ΔΨm after PF treatment (Fig. 5A). Moreover, the major source of ROS is mitochondria and redundant ROS could lead to apoptosis of cells [13]. If increasing ROS level in cancer cells after drugs treatment, we could come to a conclusion that druginduced apoptosis via be the mitochondrial apoptotic pathway. As shown in Fig. 5B, ROS was significantly higher in the PF treated group than in the control group, and higher trend is related to a concentration-dependent manner. These results confirmed that PF induced the lung cancer cells apoptosis via mitochondria-mediated apoptotic pathway. We then investigated signaling pathways involved following PF treatments, mainly including PI3K-AKT-mTOR pathway and repair of DNA damage. PI3K-AKT-mTOR is one of the signaling pathways involved in tumor cell proliferation and apoptosis. AKT exerts antiapoptotic effects by phosphorylating target proteins through various downstream pathways. As shown in Fig. 5C/E (and Supplementary Figure C), the expression of phosphorylated AKT/IKBa was decreased without affect its total protein after PF treatment. Furthermore, cellcycle arrest causes DNA repair, H2A.X is considered a marker of activation of DNA damage response, and we analyzed the extent of DNA damage by inspecting the level of H2A.X [18]. we examined protein expression levels in A549 and LL2 cells after PF treated and the expression of H2A.X and phosphorylated H2A.X were increased, was changed protein expression levels of apoptotic. These contribute to PF’s effects on lung cancer cells survival/apoptotic.
4. Discussion Lung cancer show a rapid incidence rate and hardly therapeutic effect in both men and women. Although there were targeted therapies (EGFR inhibitors and ALK inhibitor) and immunotherapy (PD1/PD-L1 antibodies) at present, the benefit of their treatments was gradually weakened due to many lung cancer patients did not respond. Therefore, it is necessary to find new therapeutic drugs. Fortunately, scientific research proves that schizophrenia could reduce the risk of cancer after anti-psychotic drugs treatment [31,32]. The studies also suggested that anti-psychotic drugs such as chlorpromazine, trifluoperazine and thioridazine showed anticancer activities [10–12,33]. Penfluridol (PF) is an anti-psychotic drug while has not been reported about its activities to treat lung cancer. Our results elucidated PF’s activity suppresses lung cancer cell growth and metastasis. Our data firstly showed that PF has the potential activities in inhibiting the proliferation of lung cancer cells by MTT assay. It prompted us to further study PF’s anticancer potential and possible mechanisms of impact. The tumor cell cycle is disordered and apoptosis is not normal after PF treatment by flow cytometry analysis. So we tried to study from these two aspects. Cell cycle disturbance is one of the important markers of cancer. There are numerous proteins play a critical role in cell cycle progression from recently studies, such as cyclins and cyclin-dependent kinase (CDKs) complex [34,35]. Some proteins suppress the cyclin-CDK complexes, and it has an adverse adjustment function of cell cycle arrest, including p21 and p27. Our results showed that PF could increase p21 and p27 expression and the expression of cyclin-CDK complex are down-regulated in lung cancer cells. So, it is suggested that PF could induce the relevant cyclin-CDK complexes disorder of G0/G1 cell cycle in lung cancer cells. Another feature of tumor cell is abnormal balance of apoptosis. Apoptosis is divided into intrinsic apoptosis and extrinsic apoptotic pathway. Intrinsic apoptosis is also called mitochondrial apoptosis, which Bcl-2 and Bax play pivotal role. Our results showed that PF induced the loss of mitochondrial membrane potential (ΔΨm), which is related with the down-regulation of Bcl-2 and procaspase-3 and upregulation of Bax and cleaved caspase-3. Reactive oxygen species (ROS) plays an important role in the initiation and regulation of apoptosis. Excessive amount of ROS induce apoptosis through cellular oxidative stress response [36]. PF treatment could increase the level of ROS. At last, we suggested that there is specific pathway inducing cells apoptosis after PF treatment. There are signaling pathways of PI3K-AKT-
3.5. PF impaired cellular migration and invasion in vitro Lung cancer metastasis poses a primary threat to patient survival. Tumor cell migration and invasion is one of the committed steps in cancer metastasis. As shown in Fig. 6A, the wound healing course was suppressed after 3 μM PF treatment. Then we used transwell means to test the effects of PF on cell migration and invasion. As shown in Fig. 6C, 10 μM PF-treated groups showed reduced significantly migrated cell numbers on A549 and H446 cells (Supplementary Figures E), similar results were observed in invasion assay (Fig. 6D). We further confirm the effects of PF on cell migration and invasion related protein pathway. As shown in Fig. 6B (and Supplementary Figures D), phosphorylated FAK were decreased without affecting FAK expression level after PF treatment in A549 and LL2 cells. And the results exhibited that the treatment with drug significantly inhibited the expression of MMP-9 in LL2 cells. This finding suggested that PF suppressed migration and invasion of lung cancer cells by blocking the FAK-related migration signaling pathway. 3.6. Anti-tumor efficacy of PF in vivo To investigate whether the antitumor activity of penfluridol in vivo 9
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mTOR pathway following PF treatments. PF treatment could decrease the level of phosphorylated AKT/IKBa, which will change protein expression levels of apoptotic. Moreover, elevated protein levels of H2A.X and p-H2A.X after PF treatment, suggested that PF also induced cell death through DNA repair. In A549 xenograft mouse model, PF suppressed tumor growth at 10 mg/kg with once daily dosing for 26 days. The tumor sections were shown reduced expression of Ki-67 and increased expression of Cleaved caspase-3 when PF treatment compared with the vehicle treated groups. Importantly, obvious side effect did not observed. The metastasis of lung cancer is the leading cause of death in patients. Matrix metalloproteinases (MMPs) are associated with tumor angiogenesis invasion and metastasis [37]. In this study, PF significantly inhibited lung cancer cells migration and invasion. Furthermore, after PF treatment, the related protein expression levels were decreased both in vitro and in vivo, including MMP 9 and p-FAK. In summary, the results of the study showed that PF has potential anti-lung cancer activity in vitro and in vivo. Considering that the safety of PF is already conformed and the cost is lower, it provides the basis for the development of PF into an anti-lung cancer drug.
[8] T. Shukuya, D.P. Carbone, Predictive markers for the efficacy of anti-PD-1/PD-L1 antibodies in lung cancer, J. Thorac. Oncol. (2016) S155608641600438X. [9] Y. Barak, A. Achiron, M. Mandel, I. Mirecki, D. Aizenberg, Reduced cancer incidence among patients with schizophrenia, Cancer 104 (2005) 2817–2821, https://doi.org/10.1002/cncr.21574. [10] S.Y. Shin, et al., The antipsychotic agent chlorpromazine induces autophagic cell death by inhibiting the Akt/mTOR pathway in human U-87MG glioma cells, Carcinogenesis 34 (2013) 2080–2089, https://doi.org/10.1093/carcin/bgt169. [11] C.T. Yeh, A.T.H. Wu, P.M.H. Chang, et al., Trifluoperazine, an antipsychotic agent, inhibits cancer stem cell growth and overcomes drug resistance of lung cancer, Am. J. Respir. Crit. Care Med. 186 (11) (2012) 1180–1188. [12] M.H. Van Woert, S.H. Palmer, Inhibition of the growth of mouse melanoma by chlorpromazine, Cancer Res. 29 (11) (1969) 1952. [13] E. Hedrick, X. Li, S. Safe, Penfluridol represses integrin expression in breast cancer through induction of reactive oxygen species and downregulation of sp transcription factors, Mol. Cancer Ther. (2016) 1535–7163 MCT-16-0451. [14] A. Ranjan, P. Gupta, S.K. Srivastava, Penfluridol: an antipsychotic agent suppresses metastatic tumor growth in triple negative breast cancer by inhibiting integrin signaling axis, Cancer Res. 76 (4) (2016) 877–890. [15] Y. Xia, et al., SKLB316, a novel small-molecule inhibitor of cell-cycle progression, induces G2/M phase arrest and apoptosis in vitro and inhibits tumor growth in vivo, Cancer Lett. 355 (2014) 297–309. [16] G.I. Evan, K.H. Vousden, Proliferation, cell cycle and apoptosis in cancer, Nature 411 (6835) (2001) 342–348. [17] O. Fernandez-Capetillo, A. Lee, M. Nussenzweig, H2AX: the histone guardian of the genome, DNA Repair 3 (8–9) (2004) 0–967. [18] A. Sharma, K. Singh, A. Almasan, Histone H2AX phosphorylation: a marker for DNA damage, Methods Mol. Biol. 920 (2012) 613–626. [19] P.J. Cook, B.G. Ju, F. Telese, X. Wang, C.K. Glass, M.G. Rosenfeld, Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions, Nature 458 (2009) 591–596. [20] W.M. Bonner, C.E. Redon, J.S. Dickey, et al., Gamma H2AX and cancer, Nat. Rev. Cancer 8 (2008) 957–967. [21] A. Nagamachi, N. Yamasaki, K. Miyazaki, et al., Haploinsufficiency and acquired loss of Bcl11b and H2AX induces blast crisis of chronic myelogenous leukemia in a transgenic mouse model, Cancer Sci. 100 (2009) 1219–1226. [22] C.H. Hsin, Y.E. Chou, S.F. Yang, MMP-11 promoted the oral cancer migration and FAK/Src activation, Oncotarget 8 (20) (2017). [23] H. Yoon, J.P. Dehart, J.M. Murphy, Understanding the roles of FAK in Cancer: inhibitors, genetic models, and new insights, J. Histochem. Cytochem. 63 (2) (2015) 114–128. [24] D. Gerlier, N. Thomasset, Use of MTT colorimetric assay to measure cell activation, J. Immunol. Methods 94 (1) (1986) 57–63. [25] J.A. Plumb, Cell sensitivity assays: the MTT assay, Methods Mol. Biol. 88 (88) (2011) 237–245. [26] P. Hossenlopp, D. Seurin, B. Segoviaquinson, et al., Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies, Anal. Biochem. 154 (1) (1986) 138–143. [27] E. Shacter, J.A. Williams, M. Lim, et al., Differential susceptibility of plasma proteins to oxidative modification: examination by western blot immunoassay, Free Radic. Biol. Med. 17 (5) (1994) 429. [28] J.M. Walker, The Bicinchoninic Acid (BCA) Assay for Protein Quantitation[M]// The Protein Protocols Handbook, (1996), pp. 11–14. [29] O. Coqueret, New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol. 13 (2) (2003) 65–70. [30] K. Somasundaram, H. Zhang, Y.X. Zeng, et al., Arrest of the cell cycle by the tumorsuppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiP1, Nature 389 (6647) (1997) 187–190. [31] S.O. Dalton, C. Johansen, A.H. Poulsen, M. Norgaard, H.T. Sorensen, J.K. McLaughlin, et al., Cancer risk among users of neuroleptic medication: a population-based cohort study, Br. J. Cancer 95 (7) (2006) 934–939. [32] Y. Barak, A. Achiron, M. Mandel, I. Mirecki, D. Aizenberg, Reduced cancer incidence amongpatients with schizophrenia, Cancer 104 (12) (2005) 2817–2821. [33] J. Mu, H. Xu, Y. Yang, W. Huang, J. Xiao, M. Li, et al., Thioridazine, an antipsychotic drug, elicits potent antitumor effects in gastric cancer, Oncol. Rep. 31 (5) (2014) 2107–2114. [34] K. Vermeulen, D.R. Van Bockstaele, Z.N. Berneman, The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer, Cell Proliferat 36 (2003) 131–149. [35] G. Delsal, M. Loda, M. Pagano, Cell cycle and cancer: critical events at the G1 restriction point, Crit. 615 Rev. Oncog. 7 (1996) 127–142. [36] H.W. Yang, K.J. Hwang, H.C. Kwon, et al., Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos, Hum. Reprod. 13 (4) (1998) 998–1002. [37] L.M. Coussens, Z. Werb, Matrix metal loproteinases and the development of cancer, Chem. Biol. 3 (11) (1996) 895–904.
Fundings This research was supported by the National Key R&D Program (Grant Number: 2017YFA0505601), the Central Universities (2017SCU12046, the Postdoctoral Foundation of Sichuan University), China Postdoctoral Science Foundation (2018T110981 and 2017M612977), the National Natural Science Foundation of China (81702898 and 81602950), and Post-Doctor Research Project, West China Hospital, Sichuan University (2019HXBH017) Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgements None. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109598. References [1] F. Bray, J. Ferlay, I. Soerjomataram, et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA: Cancer J. Clin. 68 (6) (2018). [2] M.L. Janssen-Heijnen, J.W. Coebergh, The changing epidemiology of lung cancer in Europe, Lung Cancer 41 (2013) 245–258. [3] J. Ferlay, G. Randi, C. Bosetti, F. Levi, E. Negri, P. Boyle, Declining mortality from bladder cancer in Europe, BJU Int. 101 (2008) 11–19. [4] D.M. Jackman, B.E. Johnson, Small-cell lung cancer, Cancer Chemother. Pharmacol. 315 (8162) (1980) 252-252. [5] E. Rodriguez, R.C. Lilenbaum, Small cell lung cancer: past, present, and future, Curr. Oncol. Rep. 12 (2010) 327–334. [6] J.P. Koivunen, C. Mermel, K. Zejnullahu, et al., EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer, Clin. Cancer Res. 14 (13) (2008) 4275–4283. [7] D.A. Haber, D.W. Bell, R. Sordella, et al., Molecular targeted therapy of lung cancer: egfr mutations and response to EGFR inhibitors, Cold Spring Harb. Symp. Quant. Biol. 70 (2005) 419–426.
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Penfluridol: An antipsychotic agent suppresses lung cancer cell growth and metastasis by inducing G0/G1 arrest and apoptosis
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Qiang Xuea,1, Zhihao Liua,1, Zhanzhan Fenga, Ying Xua, Weiqiong Zuoa, Qianqian Wanga, Tiantao Gaoa, Jun Zenga, Xi Hua, Fanfan Jiaa, Yongxia Zhub, Yong Xiaa,*, Luoting Yua,* a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biothrapy, Chengdu, 610041, China b Department of Obstetrics and Gynecology, Henan Provincial People’s Hospital, People's Hospital of Zhengzhou University, People's Hospital of Henan University, Zhengzhou 450003, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Penfluridol Lung cancer Cell cycle arrest Apoptosis Metastasis
Lung cancer remains the leading cause of cancer mortality because of highly malignant and metastatic potential. The current status of lung cancer treatment is limited, and more treatment options are needed. Interesting, antipsychotic drugs have been reported to show anti-cancer effects. In this present study, we investigated the anticancer potential of penfluridol (PF), an anti-schizophrenic drug, in lung cancer and its underlying mechanism in vitro and in vivo. In vitro, it could inhibit the viability of various lung cancer cells with G0/G1 phase arrest via increasing the expression level of p21/p27 and decreasing the expression levels of cyclin-CDK complex. Meanwhile, cell-cycle arrest causes DNA repair in the nucleus, which was associated with the upregulation of H2A.X and p-H2A.X. Moreover, PF could also decrease mitochondrial membrane potential and increase reactive oxygen species levels in the lung cancer cells. These results implied that PF might induce the mitochondriamediated intrinsic apoptosis. In addition, PF inhibits the migration and invasion of lung cancer cells via downregulation of FAK-MMP signaling. In vivo, oral administration of PF at concentration of 10 mg/kg inhibited tumor growth in A549 xenograft model. Notably, PF is an approved drug and the price is exceedingly cheap, so this study demonstrates the potential of PF to treat lung cancer.
1. Introduction Lung cancer is the most commonly diagnosed cancer and the leading cause of cancer mortality [1]. It is divided into two types. One is the small cell lung cancer (SCLC), which accounts for about 15% of all lung cancers, and the other one is non-small cell lung cancer (NSCLC), which accounts for about 85% [2–5]. Although there have been advances in the treatment of lung cancer, such as the targeted therapies with EGFR inhibitors and ALK inhibitors, and immunotherapy using PD1/PD-L1 antibodies at present, many patients do not respond to the treatment. Therefore, finding new therapeutic drugs for lung cancer patients is still necessary [6–8]. Because of some major problems including high failure rates and withdrawal risks in new anticancer drug discovery and development, drug repurposing has been drawn lots of attentions from both academic institution and pharmaceutical industry and become a new option to
find new therapeutic drugs. The studies recently showed that schizophrenia patients were tended to have lower risk at cancer after antipsychotic treatment [9], such as chlorpromazine and trifluoperazine [10–12], indicating that antipsychotic can be used in the treatment of cancer patients. Notably, pentafluridol (PF), an antipsychotic agent, showed antitumor activity in breast cancer [13,14]. However, it has been reported to have a relatively limited amount of anticancer activities. Restricted growth of every cancer lies that the tumor cell and its progeny into controlled expansion and invasion. One of suppressed neoplastic progression is that deregulated cell proliferation and cellcycle arrest. Disrupting tumor cell cycle can suppress cell proliferation and induce cell apoptosis [15]. Cell apoptosis is a programmed cell death, which is the response of somatic cells to many forms of stress and damage, especially the DNA damage [16]. Cell-cycle arrest causes DNA repair, repairing unsuccessful cells enter the apoptosis program. H2A.X
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Corresponding authors at: State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation Center for Biothrapy, Chengdu 610041, China. E-mail addresses: [email protected] (Y. Xia), [email protected] (L. Yu). 1 These authors have contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109598 Received 18 September 2019; Received in revised form 16 October 2019; Accepted 26 October 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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Fig. 1. The anti-proliferation effects of penfluridol against lung cancer cells. (A) Chemical structure of PF. (B) Several solid tumor cell lines were treated with PF for 72 h, cell viability was tested by MTT assay. Values were plotted as mean ± SEM. (C) Inhibitory effects of various concentrations PF on the three lung cancer cells after (24, 48 and 72 h) the treatment (IC50 values, μM). Values were plotted as mean ± SEM from 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. (D) The effects of PF on colony formation in A549 and LL2 cells for 6–8 days (20×). Values were plotted as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.
metastasis of tumor cells. PF treatment also suppressed the migration and invasion of lung cancer cells via downregulation of FAK-MMP signaling [22,23]. In vivo, we found that PF suppressed tumor growth in A549 cells xenograft mouse model. We obtained that penfluridol may offer treatment goal against lung cancer. This is the first study to introduce lung cancer after penfluridol treatment, which provides a potential drug candidate for the treatment of lung cancer.
is a variant of the histone H2A family and is thought to be involved in DNA repair in the nucleus [17,18]. A growing number of published data indicate that H2A.X is dependent on its C-terminal phosphorylation to regulate apoptosis of cancer cells [19–21]. Targeting cell cycle and apoptosis pathways is promising, and there are already some targeted drugs. The aim of this study is repurposing the PF for treating lung cancer. Our data provided evidence that PF against lung cancer cells growth and the possible underlying mechanism of cell death. PF treatment could inhibit lung cancer cells’ growth by induce G0/G1 cell cycle arrest and cell apoptosis. Mitochondria-mediated apoptosis pathway may be the way of PF-induced apoptosis. Moreover, the decreasing protein expression levels of signaling pathways were found after PF treatment, such as AKT and NF-kB. Through elevated protein levels of H2A.X and p-H2A.X, we found that cell death also through DNA repair pathway [20,21]. Focal Adhesion Kinase (FAK) has been shown to play an important role in the progression of tumors to a malignant phenotype. Therefore, blocking the expression of FAK can inhibit the invasion and
2. Materials and methods 2.1. Materials Penfluridol (PF) was obtained from the AstaTech BioPharmaceutical Corp (Chengdu, China). And for in vitro experiments, PF dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM and stored at -20℃. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and the Annexin V-FITC apoptosis detection kits 2
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Fig. 2. Induction of G0/G1 phase arrest by PF. (A, B and E, F) A549 and H446 were treated with different concentrations of PF (0, 2.5, 5 and 10 μM) for 36 h and 12 h, respectively. Then cell-cycle distribution analyzed by FCM. Values were plotted as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (C–D and G–H) PF treatment (24 h) on A549 and LL2 cells influenced the expression of cell cycle-related proteins, such as p21, p27, CKD2, cyclin D1, cyclinE and β-actin were employed as a standard. Values are mean ± SEM. *p < 0.05. Figs. 3 and 4. Induction of apoptosis by PF.
against MMP9, FAK and p-FAK were purchased from Abcam (Cambridge, England). The other antibodies were purchased from Cell Signaling Technology Company (Beverly, MA, USA).
were purchased from KeyGEN Biology Co. Ltd (Nanjing, China). Rhodamine-123 (Rh123), propidium iodide(PI) and Hoechst 33342 were purchased from Sigma (St Louis, MO, USA). The antibodies against cyclinE, Bcl2 and Bax were purchased from BD Bioscience (Franklin, NJ, USA), the antibodies against p21, IKBa and p-IKBa were purchased from Wanlei Bio (Shenyang, China), and the antibodies 3
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Fig. 3. (A) A549 and H446 cells were treated with 10 μM PF for 24 h and analyzed by light microscopy (scale bars represent 500 μM). (B) Percentages of the number of viable cells compared to vehicle after PF treatment. ***p < 0.001. (C) Two lung cancer cells were treated with various concentration of PF (0, 5 and 10 μM) for 24 h, the cells nuclei were stained with Hoechst 33,342 and observed by fluorescence microscope (scale bars represent 20 μM). (D) Percentages of intact nucleus compared to vehicle. *p < 0.05; **p < 0.01; ***p < 0.001.
measured by ACEA NovoCyte and analyzed with NovoExpress software (Agilent Biosciences, CA, USA). For the apoptosis assay, lung cancer cells apoptosis induced by PF and were quantified with the apoptosis detection kit. Cells were harvested and incubated with Annexin V-FITC for 15 min in the dark and incubated with PI for 15 min, then was detected and analysed.
2.2. Cell lines Human lung cancer cell line A549, H446, H1993 and SPC-A1, mouse cell line LL2 were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM or RPMI 1640 media supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin and streptomycin) under condition with 5% CO2 at 37 °C.
2.6. Measurement of ΔΨm and ROS levels in cells
2.3. Cell viability and colony formation assay
Cell mitochondrial membrane potential (ΔΨm) was measured by FCM with Rh123 (5 μg/ml) staining. After treatment with PF, cells were stained with Rh123 for 30 min in the incubator. Cells were harvested and washed with PBS. Then it was measured by ACEA NovoCyte. Reactive oxygen species(ROS) was measured with DCFH-DA (10 μM) staining in cells. After treatment with PF for different hours, we obtain cells and measured using FCM.
The effect of PF on cell viability was measured by MTT assay and colony formation assay assays [24,25]. In the cell viability test, cancer cells (1.5–8 × 103 cells/well) were seeded in 96-well plates and cultured for 24 h, medium containing various concentrations of PF were added and incubated separately for 24, 48 and 72 h. Then the absorbance was measured with Spectra MAX M5 microplate spectrophotometer (Molecular Devices, CA, USA). In the colony formation test, the lung cancer cells (600–800 cells/ well) were inoculated in 6-well plates and cultured for 24 h. Then various concentrations of the PF with 2 mL medium were added and incubated 8 days. After fixed with 4% paraformldehyde and stained with crystal violet solution, the cells were observed and counted under the microscope.
2.7. Western blotting analysis The western blotting analysis was performed as described previously [26,27]. After exposure to various concentrations of PF for the indicated time, cells were washed with PBS and lysed in RIPA buffer (Beyotime, Beijing, China). The protein concentration was measured by the BCA method [28]. Equal amounts of total protein from each sample were subjected to SDS-PAGE gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Massachusetts, USA). After blocking with 5% fat-free milk in TBS/T for 1.5 h at room temperature, the membranes were incubated with primary and secondary antibodies. Then the immunoreactive protein bands were detected.
2.4. Nuclear morphology analysis The lung cancer cells (1–2 × 105 cells/well) were inoculated in 6well plates and cultured for 24 h. Then added various concentrations of the PF with 2 mL and incubated 24 h. After the cells were washed with PBS and fixed with 4% paraformldehyde, the cells were stained by the Hoechst 33342 (10 μg/mL) for 15 min in the dark and then was contained by fluorescence quencher for 12 h in dark.
2.8. Scratch assay in vitro Lung cancer cells were seeded in a 24-well plate. When cells grew to 80% confluence, we scraped the monolayer cells by 10 μL sterile tips, then added different concentrations of PF. Wound width was measured at 0 h, 12 h and 24 h after the PF treatment by photography. Images were acquired using a general microscope.
2.5. Cell cycle and apoptosis analysis by flow cytometry (FCM) After treated with PF for the indicated time, the cells were harvested, stained with PI for 15 min in dark. Cell cycle distribution was 4
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Fig. 4. (A) FCM analysis of apoptosis cells stained with Annexin V- FITC/PI after treatment PF and DMSO for 24/48 h. Quantified values are shown on the right. Values were plotted as mean ± SEM. **p < 0.01; ***p < 0.001. (B-G) PF treated of the three lung cancer cells for the expression of apoptotic-related proteins, including procaspase-3, cleaved caspase-3, Bax and Bcl-2. Values were plotted as mean ± SEM. *p < 0.05; **p < 0.01.
2.9. Tanswell migration and invasion analysis
2.10. Xenograft mouse model
In the migration analysis, cells (3 × 104 -1 × 105) in serum-free medium were added in the upper chamber, and medium containing 10% FBS was added at the bottom with different concentrations of PF. After 24 h, cells were fixed with 4% paraformldehyde and washed with PBS. Then cells were stained with crystal violet solution for 20 min, viewed under a microscope and counted. In the invasion assay, the upper surface of the transwell was coated with matrigel (BD Biosciences, NJ, USA). The transwell was placed in the incubator and the matrigel was solidified. After cells were hatched with different concentrations of PF for 24 h, cells were fixed and stained, washed twice with cold PBS. At the end, cells were counted by microscope.
The animal experiments were approved and conducted in strict by the Institutional Animal Care and Treatment Committee of Sichuan University, China. And Female BALB/c nude mice (6–8 weeks old) were purchased from HFK bioscience CO, LTD, Beijing, China. Exponentially growing the lung cancer cell of A549 were harvested, a suspension of 100 μL containing 5 × 107 cells was injected in the right flank. The mice were divided into 2 groups (6 mice per group) at random when the tumor volume was about 100–200 mm³. PF (10 mg/kg) or vehicle (water / ethanol / PEG 400 / 2% acetic acid in 8:3:3:1) was administered at the indicated doses once a day by oral gavage. Tumor volume measured and body weight of the mice were recorded every three days during PF treatment. The tumor volume was calculated using the formula [length×(width)2/2]. Experiment was terminated at day 26 by 5
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Fig. 5. PF-induced apoptosis via mitochondria-mediated apoptotic and blocks cell survival signaling pathways. (A) Effects of PF reduced the mitochondrial membrane potential (ΔΨm) in A549, H446 and LL2 cells. Flow cytometry analysis was performed after treatment with PF (24 h). Values were plotted as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (B) ROS levels increased in A549 and H446 cells after treatment with different dose of PF (24 h). Values were plotted as mean ± SEM. **p < 0.01; ***p < 0.001. (C and E) A549 and LL2 cells were treated with PF for 24 h and the expression of related proteins was analyzed by western blot analysis. They were p-IKBa, IKBa, p-AKT, AKT, p-H2A.X, H2A.X and GAPDH as internal control. (D and F) Relative proteins expressions were quantified by Image J software. Values are mean ± SEM. *p < 0.05; **p < 0.01.
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Fig. 6. PF impaired cellular migration and invasion in vitro. (A)Compared with the control group, PF inhibited obviously wound healing of A549 cells. Quantified dates is shown on the right. Experiments were repeated twice. Values were plotted as mean ± SEM. *p < 0.05; ***p < 0.001. (B) Western blot analysed the migration and invasion proteins for FAK, p-FAK, MMP 9 and GAPDH following the treatment of PF in A549 cells. The graphic data shown are mean ± SEM. *p < 0.05 (C) PF inhibited A549 cells migration in the transwell migration assay. 3 × 104 A549 cell were seeded in the top chamber of transwell with serum-free medium and treated with DMSO or PF. After 24 h, migrated cells were stained, then pictures were taken with a microscope. Values were plotted as mean ± SEM. **p < 0.01; ***p < 0.001. (D) PF inhibited A549 cell invasion. 1 × 105 (invasion) A549 cells were treated with PF and could cross the matrigel as well as the transwell membrancege. Values were plotted as mean ± SEM. ***p < 0.001.
72 h obviously with the IC50 (half-maximal inhibitory concentration) values less than 12 μM (Fig. 1B). Then we chose human lung cancer cell lines A549, H446 and mouse cell line LL2 for further studies. We researched the time- and concentration- dependent effects of PF on three cells viability, respectively. As a result, increasing PF concentration and longer time could suppressed cell viabilities (Fig. 1C). It was indicated that PF could suppress lung cancer cells viability in a time- and dose- dependent manner. Nest, we performed colony formation assay to further studied the effects of PF on lung cancer cell proliferation. It can be seen that PF could suppress cells viability in a dose dependent manner (Fig. 1D). And quantity and group of the colony formation of lung cancer cells were significantly inhibited by PF.
euthanizing mice. The mice tumors and organs were extracted. Tumors were used for Western blot analysis and IHC analysis. 2.11. Toxicity evaluation BALB/c nude mice were randomly divided into two groups (n = 6). Animals were given PF (10 mg/kg) or vehicle by oral gavage. During PF treatment, the mice were observed for general signs of toxicity every day. On the 26th day, we extract the eyeball blood, heart, liver, spleen, lung and kidney from all mice. Then they were determined with biochemical analysis. 2.12. Statistical analyses All datas were analyzed by GraphPad Prism 5.01 software. The data was shown as mean ± SEM. The statistical comparisons were made by Student’s t-test There is a statistically significant p values, *p < 0.05; **p < 0.01; ***p < 0.001.
3.2. Induction of G0/G1 phase arrest by PF We next assessed whether PF could effect the cell cycle distribution by flow cytometry analysis. As shown in Fig. 2A/B, after incubation with concentration- dependent manner of PF, G0/G1 phase arrest was induced. When the A549 cells were treated with vehicle for 36 h, the G0/G1 phase arrest rate was 52.21%, whereas the arrest rate increased to 54.45%, 58.70% and 71.10% when cells were treated with 2.5 μM, 5 μM and 10 μM of PF. Similar results were obtained in H446 cells (Fig. 2E/F) and A549 cells for 24 h (Supplementary Figures A). As we all know, p21 and p27 are important members of the family of cyclin-dependent kinase inhibitors, and p21 is the downstream signal molecule of p53. p21/p27 mediates inhibition of cyclin-CDK complex
3. Results 3.1. The anti-proliferation effects of penfluridol against lung cancer cells Recently, several drugs repurposing were discovered in lung cancer, and it is a way to treat lung cancer [14]. We investigated the proliferation inhibition caused by PF treatment on several lung cancer cell lines. The results indicated that PF could increased tumor cell death for 7
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Fig. 7. Anti-tumor efficacy of PF in xenograft mouse model and safety profile. (A) Tumor pictures from control and penfluridol-treated mice. (B and C) Tumor volume of the mice were measured every 3 days and tumors were weighed once in the control and PF group. Values were plotted as mean ± SEM. (n = 6). *p < 0.05. (D) Tumor tissues from A549 xenograft treated with control or PF-treated for 26 days were immunohistochemically analyzed with anti-Ki-67, anticleaved caspase-3 and anti-p-AKT antibodies. (E) The tumor tissue extracts proteins from control and PF-treated mice, and the expression of related proteins was analyzed by western blot analysis.They were p-H2A.X, cleaved caspase-3, cyclin E, p-AKT and MMP 9. GAPDH was used to verify equivalent loading of protein. Quantitative analysis is below. Values were plotted as mean ± SEM.
treatment, we investigated the levels of cell morphology by microscope. As shown in Fig. 3A, both A549 and H446 cells showed cell shrinkage which represent features of apoptosis after PF treatment. Fig. 3B shows percentages of the number of viable cells compared to vehicle after PF treatment. Then as Fig. 3C indicated, Hoechst 33342 staining assay also showed that PF treatment induced apoptosis in A549 and H446 cells. Fig. 3D shows percentages of intact nucleus compared to vehicle. We can see that condensed nuclei of bright blue fluorescent, reduction of cell volume and nuclear fragmentation after PF treatment, demonstrating the features of cell. We further studied whether PF treatment could induce apoptosis in A549, H446 and LL2 cells, the AnnexinV-FITC/PI dual-labeling was
expression levels, resulting in cell-cycle arrest [29,30]. Moreover, we examined protein expression levels in three lung cancer cells after PF treated by western blotting analysis. As shown in Fig. 2C/D, the expression of cyclin D1 and cyclin E decreased while that of p27 increased in A549 with a concentration-dependent manner. Similar results were obtained in LL2 and H446 cells (Fig. 2G/H and Supplementary Figures B). We did that G0/G1 phase arrest was induced with PF treated by protein expression levels.
3.3. Induction of apoptosis by PF To confirm the induction of apoptosis in lung cancer cells with PF 8
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is consistent with its effects in vitro, a xenograft mouse model was established. A549 cells were injected into the right flank of BALB/c nude mice. The mice received the following treatments: vehicle and penfluridol at 10 mg/kg. Four weeks after oral administration in mice, the tumors of the mice in PF-treated group were significantly smaller than those in the control group (Fig. 7A and B). Furthermore, a significant tumor weight slide was also observed (Fig. 7C). To validate the results of the in vivo assays, we investigated the expression of proliferation, apoptosis and migration markers using immunoblotting and IHC analyses of the A549 tumor samples. The tumor sections were shown Ki-67-positive cells declined, whereas the cleaved caspase-3 positive cells increased. Besides, the number of pAKT-positive cells in PF treatment was less compared with vehicle group (Fig. 7D). Furthermore, the immunoblotting results showed that the expression of p-H2A.X and cleaved caspase-3 were increased, cyclin E and p-AKT were markedly inhibited in the PF-treated group (Fig. 7E). Overall, these findings suggested that PF blocked proliferation and metastasis of lung cancer by regulating AKT and MMP signaling. Besides, during the treatment of A549 xenograft in BALB/c nude mice, we did not observe side effect. there were no significant changes in hematological, serum biochemical parameters (Supplementary Figures F) and pathological changes of main organs (Supplementary Figures G), suggesting the mice were tolerant to PF treatment.
performed. As shown in Fig. 4A, the effect of apoptosis was apparently exhibited. When the A549 cells were treated with vehicle, the apoptosis rate was 4.74%, whereas the apoptosis cells increased to 17.64%, 26.16% and 44.54% when cells were treated with 2.5 μM, 5 μM and 10 μM PF, respectively. Similar results were obtained in H446 and LL2 cells in a concentration-dependent manner. Moreover, we studied protein expression levels of apoptosis by western blotting analysis, such as Bcl-2, cleaved caspase-3, procaspase-3 and Bax (Figs. 4B/C, D/E and 3 F/G). And these proteins play important roles in apoptosis. 3.4. PF-induced apoptosis via mitochondria-mediated apoptotic and blocks cells survival signaling pathways As shown in Fig. 4, with the protein expression levels of apoptosis, we suggesting that PF-induced apoptosis might via the mitochondriamediated apoptotic pathway. In order to verify the hypothesis, A549 and H446 cells was colored with a green fluorochrome Rh123 to measure the changes in the mitochondrial membrane potential (ΔΨm). We observed that a significant loss of ΔΨm after PF treatment (Fig. 5A). Moreover, the major source of ROS is mitochondria and redundant ROS could lead to apoptosis of cells [13]. If increasing ROS level in cancer cells after drugs treatment, we could come to a conclusion that druginduced apoptosis via be the mitochondrial apoptotic pathway. As shown in Fig. 5B, ROS was significantly higher in the PF treated group than in the control group, and higher trend is related to a concentration-dependent manner. These results confirmed that PF induced the lung cancer cells apoptosis via mitochondria-mediated apoptotic pathway. We then investigated signaling pathways involved following PF treatments, mainly including PI3K-AKT-mTOR pathway and repair of DNA damage. PI3K-AKT-mTOR is one of the signaling pathways involved in tumor cell proliferation and apoptosis. AKT exerts antiapoptotic effects by phosphorylating target proteins through various downstream pathways. As shown in Fig. 5C/E (and Supplementary Figure C), the expression of phosphorylated AKT/IKBa was decreased without affect its total protein after PF treatment. Furthermore, cellcycle arrest causes DNA repair, H2A.X is considered a marker of activation of DNA damage response, and we analyzed the extent of DNA damage by inspecting the level of H2A.X [18]. we examined protein expression levels in A549 and LL2 cells after PF treated and the expression of H2A.X and phosphorylated H2A.X were increased, was changed protein expression levels of apoptotic. These contribute to PF’s effects on lung cancer cells survival/apoptotic.
4. Discussion Lung cancer show a rapid incidence rate and hardly therapeutic effect in both men and women. Although there were targeted therapies (EGFR inhibitors and ALK inhibitor) and immunotherapy (PD1/PD-L1 antibodies) at present, the benefit of their treatments was gradually weakened due to many lung cancer patients did not respond. Therefore, it is necessary to find new therapeutic drugs. Fortunately, scientific research proves that schizophrenia could reduce the risk of cancer after anti-psychotic drugs treatment [31,32]. The studies also suggested that anti-psychotic drugs such as chlorpromazine, trifluoperazine and thioridazine showed anticancer activities [10–12,33]. Penfluridol (PF) is an anti-psychotic drug while has not been reported about its activities to treat lung cancer. Our results elucidated PF’s activity suppresses lung cancer cell growth and metastasis. Our data firstly showed that PF has the potential activities in inhibiting the proliferation of lung cancer cells by MTT assay. It prompted us to further study PF’s anticancer potential and possible mechanisms of impact. The tumor cell cycle is disordered and apoptosis is not normal after PF treatment by flow cytometry analysis. So we tried to study from these two aspects. Cell cycle disturbance is one of the important markers of cancer. There are numerous proteins play a critical role in cell cycle progression from recently studies, such as cyclins and cyclin-dependent kinase (CDKs) complex [34,35]. Some proteins suppress the cyclin-CDK complexes, and it has an adverse adjustment function of cell cycle arrest, including p21 and p27. Our results showed that PF could increase p21 and p27 expression and the expression of cyclin-CDK complex are down-regulated in lung cancer cells. So, it is suggested that PF could induce the relevant cyclin-CDK complexes disorder of G0/G1 cell cycle in lung cancer cells. Another feature of tumor cell is abnormal balance of apoptosis. Apoptosis is divided into intrinsic apoptosis and extrinsic apoptotic pathway. Intrinsic apoptosis is also called mitochondrial apoptosis, which Bcl-2 and Bax play pivotal role. Our results showed that PF induced the loss of mitochondrial membrane potential (ΔΨm), which is related with the down-regulation of Bcl-2 and procaspase-3 and upregulation of Bax and cleaved caspase-3. Reactive oxygen species (ROS) plays an important role in the initiation and regulation of apoptosis. Excessive amount of ROS induce apoptosis through cellular oxidative stress response [36]. PF treatment could increase the level of ROS. At last, we suggested that there is specific pathway inducing cells apoptosis after PF treatment. There are signaling pathways of PI3K-AKT-
3.5. PF impaired cellular migration and invasion in vitro Lung cancer metastasis poses a primary threat to patient survival. Tumor cell migration and invasion is one of the committed steps in cancer metastasis. As shown in Fig. 6A, the wound healing course was suppressed after 3 μM PF treatment. Then we used transwell means to test the effects of PF on cell migration and invasion. As shown in Fig. 6C, 10 μM PF-treated groups showed reduced significantly migrated cell numbers on A549 and H446 cells (Supplementary Figures E), similar results were observed in invasion assay (Fig. 6D). We further confirm the effects of PF on cell migration and invasion related protein pathway. As shown in Fig. 6B (and Supplementary Figures D), phosphorylated FAK were decreased without affecting FAK expression level after PF treatment in A549 and LL2 cells. And the results exhibited that the treatment with drug significantly inhibited the expression of MMP-9 in LL2 cells. This finding suggested that PF suppressed migration and invasion of lung cancer cells by blocking the FAK-related migration signaling pathway. 3.6. Anti-tumor efficacy of PF in vivo To investigate whether the antitumor activity of penfluridol in vivo 9
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mTOR pathway following PF treatments. PF treatment could decrease the level of phosphorylated AKT/IKBa, which will change protein expression levels of apoptotic. Moreover, elevated protein levels of H2A.X and p-H2A.X after PF treatment, suggested that PF also induced cell death through DNA repair. In A549 xenograft mouse model, PF suppressed tumor growth at 10 mg/kg with once daily dosing for 26 days. The tumor sections were shown reduced expression of Ki-67 and increased expression of Cleaved caspase-3 when PF treatment compared with the vehicle treated groups. Importantly, obvious side effect did not observed. The metastasis of lung cancer is the leading cause of death in patients. Matrix metalloproteinases (MMPs) are associated with tumor angiogenesis invasion and metastasis [37]. In this study, PF significantly inhibited lung cancer cells migration and invasion. Furthermore, after PF treatment, the related protein expression levels were decreased both in vitro and in vivo, including MMP 9 and p-FAK. In summary, the results of the study showed that PF has potential anti-lung cancer activity in vitro and in vivo. Considering that the safety of PF is already conformed and the cost is lower, it provides the basis for the development of PF into an anti-lung cancer drug.
[8] T. Shukuya, D.P. Carbone, Predictive markers for the efficacy of anti-PD-1/PD-L1 antibodies in lung cancer, J. Thorac. Oncol. (2016) S155608641600438X. [9] Y. Barak, A. Achiron, M. Mandel, I. Mirecki, D. Aizenberg, Reduced cancer incidence among patients with schizophrenia, Cancer 104 (2005) 2817–2821, https://doi.org/10.1002/cncr.21574. [10] S.Y. Shin, et al., The antipsychotic agent chlorpromazine induces autophagic cell death by inhibiting the Akt/mTOR pathway in human U-87MG glioma cells, Carcinogenesis 34 (2013) 2080–2089, https://doi.org/10.1093/carcin/bgt169. [11] C.T. Yeh, A.T.H. Wu, P.M.H. Chang, et al., Trifluoperazine, an antipsychotic agent, inhibits cancer stem cell growth and overcomes drug resistance of lung cancer, Am. J. Respir. Crit. Care Med. 186 (11) (2012) 1180–1188. [12] M.H. Van Woert, S.H. Palmer, Inhibition of the growth of mouse melanoma by chlorpromazine, Cancer Res. 29 (11) (1969) 1952. [13] E. Hedrick, X. Li, S. Safe, Penfluridol represses integrin expression in breast cancer through induction of reactive oxygen species and downregulation of sp transcription factors, Mol. Cancer Ther. (2016) 1535–7163 MCT-16-0451. [14] A. Ranjan, P. Gupta, S.K. Srivastava, Penfluridol: an antipsychotic agent suppresses metastatic tumor growth in triple negative breast cancer by inhibiting integrin signaling axis, Cancer Res. 76 (4) (2016) 877–890. [15] Y. Xia, et al., SKLB316, a novel small-molecule inhibitor of cell-cycle progression, induces G2/M phase arrest and apoptosis in vitro and inhibits tumor growth in vivo, Cancer Lett. 355 (2014) 297–309. [16] G.I. Evan, K.H. Vousden, Proliferation, cell cycle and apoptosis in cancer, Nature 411 (6835) (2001) 342–348. [17] O. Fernandez-Capetillo, A. Lee, M. Nussenzweig, H2AX: the histone guardian of the genome, DNA Repair 3 (8–9) (2004) 0–967. [18] A. Sharma, K. Singh, A. Almasan, Histone H2AX phosphorylation: a marker for DNA damage, Methods Mol. Biol. 920 (2012) 613–626. [19] P.J. Cook, B.G. Ju, F. Telese, X. Wang, C.K. Glass, M.G. Rosenfeld, Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions, Nature 458 (2009) 591–596. [20] W.M. Bonner, C.E. Redon, J.S. Dickey, et al., Gamma H2AX and cancer, Nat. Rev. Cancer 8 (2008) 957–967. [21] A. Nagamachi, N. Yamasaki, K. Miyazaki, et al., Haploinsufficiency and acquired loss of Bcl11b and H2AX induces blast crisis of chronic myelogenous leukemia in a transgenic mouse model, Cancer Sci. 100 (2009) 1219–1226. [22] C.H. Hsin, Y.E. Chou, S.F. Yang, MMP-11 promoted the oral cancer migration and FAK/Src activation, Oncotarget 8 (20) (2017). [23] H. Yoon, J.P. Dehart, J.M. Murphy, Understanding the roles of FAK in Cancer: inhibitors, genetic models, and new insights, J. Histochem. Cytochem. 63 (2) (2015) 114–128. [24] D. Gerlier, N. Thomasset, Use of MTT colorimetric assay to measure cell activation, J. Immunol. Methods 94 (1) (1986) 57–63. [25] J.A. Plumb, Cell sensitivity assays: the MTT assay, Methods Mol. Biol. 88 (88) (2011) 237–245. [26] P. Hossenlopp, D. Seurin, B. Segoviaquinson, et al., Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies, Anal. Biochem. 154 (1) (1986) 138–143. [27] E. Shacter, J.A. Williams, M. Lim, et al., Differential susceptibility of plasma proteins to oxidative modification: examination by western blot immunoassay, Free Radic. Biol. Med. 17 (5) (1994) 429. [28] J.M. Walker, The Bicinchoninic Acid (BCA) Assay for Protein Quantitation[M]// The Protein Protocols Handbook, (1996), pp. 11–14. [29] O. Coqueret, New roles for p21 and p27 cell-cycle inhibitors: a function for each cell compartment? Trends Cell Biol. 13 (2) (2003) 65–70. [30] K. Somasundaram, H. Zhang, Y.X. Zeng, et al., Arrest of the cell cycle by the tumorsuppressor BRCA1 requires the CDK-inhibitor p21WAF1/CiP1, Nature 389 (6647) (1997) 187–190. [31] S.O. Dalton, C. Johansen, A.H. Poulsen, M. Norgaard, H.T. Sorensen, J.K. McLaughlin, et al., Cancer risk among users of neuroleptic medication: a population-based cohort study, Br. J. Cancer 95 (7) (2006) 934–939. [32] Y. Barak, A. Achiron, M. Mandel, I. Mirecki, D. Aizenberg, Reduced cancer incidence amongpatients with schizophrenia, Cancer 104 (12) (2005) 2817–2821. [33] J. Mu, H. Xu, Y. Yang, W. Huang, J. Xiao, M. Li, et al., Thioridazine, an antipsychotic drug, elicits potent antitumor effects in gastric cancer, Oncol. Rep. 31 (5) (2014) 2107–2114. [34] K. Vermeulen, D.R. Van Bockstaele, Z.N. Berneman, The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer, Cell Proliferat 36 (2003) 131–149. [35] G. Delsal, M. Loda, M. Pagano, Cell cycle and cancer: critical events at the G1 restriction point, Crit. 615 Rev. Oncog. 7 (1996) 127–142. [36] H.W. Yang, K.J. Hwang, H.C. Kwon, et al., Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos, Hum. Reprod. 13 (4) (1998) 998–1002. [37] L.M. Coussens, Z. Werb, Matrix metal loproteinases and the development of cancer, Chem. Biol. 3 (11) (1996) 895–904.
Fundings This research was supported by the National Key R&D Program (Grant Number: 2017YFA0505601), the Central Universities (2017SCU12046, the Postdoctoral Foundation of Sichuan University), China Postdoctoral Science Foundation (2018T110981 and 2017M612977), the National Natural Science Foundation of China (81702898 and 81602950), and Post-Doctor Research Project, West China Hospital, Sichuan University (2019HXBH017) Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgements None. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109598. References [1] F. Bray, J. Ferlay, I. Soerjomataram, et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA: Cancer J. Clin. 68 (6) (2018). [2] M.L. Janssen-Heijnen, J.W. Coebergh, The changing epidemiology of lung cancer in Europe, Lung Cancer 41 (2013) 245–258. [3] J. Ferlay, G. Randi, C. Bosetti, F. Levi, E. Negri, P. Boyle, Declining mortality from bladder cancer in Europe, BJU Int. 101 (2008) 11–19. [4] D.M. Jackman, B.E. Johnson, Small-cell lung cancer, Cancer Chemother. Pharmacol. 315 (8162) (1980) 252-252. [5] E. Rodriguez, R.C. Lilenbaum, Small cell lung cancer: past, present, and future, Curr. Oncol. Rep. 12 (2010) 327–334. [6] J.P. Koivunen, C. Mermel, K. Zejnullahu, et al., EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer, Clin. Cancer Res. 14 (13) (2008) 4275–4283. [7] D.A. Haber, D.W. Bell, R. Sordella, et al., Molecular targeted therapy of lung cancer: egfr mutations and response to EGFR inhibitors, Cold Spring Harb. Symp. Quant. Biol. 70 (2005) 419–426.
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